WO2001004636A1 - Mammalian cells expressing desaturases and elongases - Google Patents
Mammalian cells expressing desaturases and elongases Download PDFInfo
- Publication number
- WO2001004636A1 WO2001004636A1 PCT/US2000/019011 US0019011W WO0104636A1 WO 2001004636 A1 WO2001004636 A1 WO 2001004636A1 US 0019011 W US0019011 W US 0019011W WO 0104636 A1 WO0104636 A1 WO 0104636A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- leu
- desaturase
- phe
- val
- Prior art date
Links
- 210000004962 mammalian cell Anatomy 0.000 title description 26
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 131
- 241001465754 Metazoa Species 0.000 claims abstract description 79
- 235000020978 long-chain polyunsaturated fatty acids Nutrition 0.000 claims abstract description 63
- 210000004027 cell Anatomy 0.000 claims description 235
- 239000000203 mixture Substances 0.000 claims description 45
- 241000282414 Homo sapiens Species 0.000 claims description 37
- 239000013598 vector Substances 0.000 claims description 35
- 210000004102 animal cell Anatomy 0.000 claims description 32
- 241000907999 Mortierella alpina Species 0.000 claims description 16
- 238000009472 formulation Methods 0.000 claims description 16
- 238000001890 transfection Methods 0.000 claims description 15
- 101000848239 Homo sapiens Acyl-CoA (8-3)-desaturase Proteins 0.000 claims description 13
- 235000016709 nutrition Nutrition 0.000 claims description 7
- 239000002537 cosmetic Substances 0.000 claims description 5
- 230000009261 transgenic effect Effects 0.000 abstract description 62
- 235000004626 essential fatty acids Nutrition 0.000 abstract description 34
- 230000015572 biosynthetic process Effects 0.000 abstract description 14
- 238000003786 synthesis reaction Methods 0.000 abstract description 11
- 150000002066 eicosanoids Chemical class 0.000 abstract description 9
- 150000007523 nucleic acids Chemical class 0.000 description 72
- 238000000034 method Methods 0.000 description 66
- 102000053602 DNA Human genes 0.000 description 53
- 108020004414 DNA Proteins 0.000 description 50
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 50
- 230000014509 gene expression Effects 0.000 description 49
- 108091028043 Nucleic acid sequence Proteins 0.000 description 43
- 239000000047 product Substances 0.000 description 42
- 210000001519 tissue Anatomy 0.000 description 35
- 108700019146 Transgenes Proteins 0.000 description 33
- 102000039446 nucleic acids Human genes 0.000 description 33
- 108020004707 nucleic acids Proteins 0.000 description 33
- 235000014113 dietary fatty acids Nutrition 0.000 description 31
- 229930195729 fatty acid Natural products 0.000 description 31
- 239000000194 fatty acid Substances 0.000 description 31
- 150000004665 fatty acids Chemical class 0.000 description 31
- 239000013612 plasmid Substances 0.000 description 31
- 235000013336 milk Nutrition 0.000 description 29
- 239000008267 milk Substances 0.000 description 29
- 210000004080 milk Anatomy 0.000 description 29
- 108010037138 Linoleoyl-CoA Desaturase Proteins 0.000 description 28
- HOBAELRKJCKHQD-QNEBEIHSSA-N dihomo-γ-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCCCC(O)=O HOBAELRKJCKHQD-QNEBEIHSSA-N 0.000 description 27
- 125000003729 nucleotide group Chemical group 0.000 description 27
- 238000009396 hybridization Methods 0.000 description 26
- 239000002773 nucleotide Substances 0.000 description 26
- 102100034544 Acyl-CoA 6-desaturase Human genes 0.000 description 25
- 235000021342 arachidonic acid Nutrition 0.000 description 22
- 229940114079 arachidonic acid Drugs 0.000 description 22
- 235000020778 linoleic acid Nutrition 0.000 description 22
- 235000020660 omega-3 fatty acid Nutrition 0.000 description 22
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 21
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 21
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 20
- 241000699666 Mus <mouse, genus> Species 0.000 description 19
- 239000013604 expression vector Substances 0.000 description 19
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 19
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 16
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 description 16
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 239000002299 complementary DNA Substances 0.000 description 15
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 15
- 229960002733 gamolenic acid Drugs 0.000 description 15
- 230000037361 pathway Effects 0.000 description 14
- 230000008488 polyadenylation Effects 0.000 description 14
- 229920002477 rna polymer Polymers 0.000 description 14
- 239000000523 sample Substances 0.000 description 14
- 230000000295 complement effect Effects 0.000 description 13
- 230000000694 effects Effects 0.000 description 13
- 239000012634 fragment Substances 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 13
- 238000013518 transcription Methods 0.000 description 13
- 230000035897 transcription Effects 0.000 description 13
- TWSWSIQAPQLDBP-CGRWFSSPSA-N (7e,10e,13e,16e)-docosa-7,10,13,16-tetraenoic acid Chemical compound CCCCC\C=C\C\C=C\C\C=C\C\C=C\CCCCCC(O)=O TWSWSIQAPQLDBP-CGRWFSSPSA-N 0.000 description 12
- HOBAELRKJCKHQD-UHFFFAOYSA-N (8Z,11Z,14Z)-8,11,14-eicosatrienoic acid Natural products CCCCCC=CCC=CCC=CCCCCCCC(O)=O HOBAELRKJCKHQD-UHFFFAOYSA-N 0.000 description 12
- 235000021298 Dihomo-γ-linolenic acid Nutrition 0.000 description 12
- 108091026890 Coding region Proteins 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 11
- 239000002253 acid Substances 0.000 description 11
- 230000001105 regulatory effect Effects 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 108091034117 Oligonucleotide Proteins 0.000 description 10
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 10
- 238000010276 construction Methods 0.000 description 10
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 10
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 10
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- WTTJVINHCBCLGX-UHFFFAOYSA-N (9trans,12cis)-methyl linoleate Natural products CCCCCC=CCC=CCCCCCCCC(=O)OC WTTJVINHCBCLGX-UHFFFAOYSA-N 0.000 description 9
- 108010073542 Delta-5 Fatty Acid Desaturase Proteins 0.000 description 9
- 241000124008 Mammalia Species 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 150000002617 leukotrienes Chemical class 0.000 description 9
- 229960004488 linolenic acid Drugs 0.000 description 9
- 235000020665 omega-6 fatty acid Nutrition 0.000 description 9
- 150000003180 prostaglandins Chemical class 0.000 description 9
- 239000012679 serum free medium Substances 0.000 description 9
- 102100034542 Acyl-CoA (8-3)-desaturase Human genes 0.000 description 8
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 8
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 8
- 235000021292 Docosatetraenoic acid Nutrition 0.000 description 8
- TWSWSIQAPQLDBP-UHFFFAOYSA-N adrenic acid Natural products CCCCCC=CCC=CCC=CCC=CCCCCCC(O)=O TWSWSIQAPQLDBP-UHFFFAOYSA-N 0.000 description 8
- 239000003623 enhancer Substances 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 7
- 230000027455 binding Effects 0.000 description 7
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 7
- 229940090949 docosahexaenoic acid Drugs 0.000 description 7
- 230000002538 fungal effect Effects 0.000 description 7
- 210000004602 germ cell Anatomy 0.000 description 7
- 238000003780 insertion Methods 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 210000005075 mammary gland Anatomy 0.000 description 7
- 229940012843 omega-3 fatty acid Drugs 0.000 description 7
- 229940033080 omega-6 fatty acid Drugs 0.000 description 7
- JIWBIWFOSCKQMA-UHFFFAOYSA-N stearidonic acid Natural products CCC=CCC=CCC=CCC=CCCCCC(O)=O JIWBIWFOSCKQMA-UHFFFAOYSA-N 0.000 description 7
- 235000019737 Animal fat Nutrition 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- 241001529936 Murinae Species 0.000 description 6
- 206010038997 Retroviral infections Diseases 0.000 description 6
- 101710087237 Whey acidic protein Proteins 0.000 description 6
- 108010011713 delta-15 desaturase Proteins 0.000 description 6
- -1 dextran sulfate Substances 0.000 description 6
- 235000013601 eggs Nutrition 0.000 description 6
- 235000013350 formula milk Nutrition 0.000 description 6
- 238000000520 microinjection Methods 0.000 description 6
- 239000006014 omega-3 oil Substances 0.000 description 6
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 150000003595 thromboxanes Chemical class 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 5
- 108010024223 Adenine phosphoribosyltransferase Proteins 0.000 description 5
- 102100029457 Adenine phosphoribosyltransferase Human genes 0.000 description 5
- 229930193140 Neomycin Natural products 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 238000002509 fluorescent in situ hybridization Methods 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 235000021243 milk fat Nutrition 0.000 description 5
- 229960004927 neomycin Drugs 0.000 description 5
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000011830 transgenic mouse model Methods 0.000 description 5
- YUFFSWGQGVEMMI-JLNKQSITSA-N (7Z,10Z,13Z,16Z,19Z)-docosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCCCC(O)=O YUFFSWGQGVEMMI-JLNKQSITSA-N 0.000 description 4
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 4
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 4
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 4
- 235000021294 Docosapentaenoic acid Nutrition 0.000 description 4
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- OPGOLNDOMSBSCW-CLNHMMGSSA-N Fursultiamine hydrochloride Chemical compound Cl.C1CCOC1CSSC(\CCO)=C(/C)N(C=O)CC1=CN=C(C)N=C1N OPGOLNDOMSBSCW-CLNHMMGSSA-N 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 239000005642 Oleic acid Substances 0.000 description 4
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 108020004518 RNA Probes Proteins 0.000 description 4
- 239000003391 RNA probe Substances 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 108020004440 Thymidine kinase Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 210000004748 cultured cell Anatomy 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 235000005911 diet Nutrition 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
- 108010066198 glycyl-leucyl-phenylalanine Proteins 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 4
- 210000001161 mammalian embryo Anatomy 0.000 description 4
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 108010012581 phenylalanylglutamate Proteins 0.000 description 4
- 102000040430 polynucleotide Human genes 0.000 description 4
- 108091033319 polynucleotide Proteins 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- 230000002103 transcriptional effect Effects 0.000 description 4
- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 description 3
- 101150094949 APRT gene Proteins 0.000 description 3
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 3
- RVKIPWVMZANZLI-UHFFFAOYSA-N H-Lys-Trp-OH Natural products C1=CC=C2C(CC(NC(=O)C(N)CCCCN)C(O)=O)=CNC2=C1 RVKIPWVMZANZLI-UHFFFAOYSA-N 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- FYPWFNKQVVEELI-ULQDDVLXSA-N Leu-Phe-Val Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=CC=C1 FYPWFNKQVVEELI-ULQDDVLXSA-N 0.000 description 3
- 108010079364 N-glycylalanine Proteins 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108010064997 VPY tripeptide Proteins 0.000 description 3
- QWCZXKIFPWPQHR-JYJNAYRXSA-N Val-Pro-Tyr Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QWCZXKIFPWPQHR-JYJNAYRXSA-N 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 235000015872 dietary supplement Nutrition 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 239000003925 fat Substances 0.000 description 3
- 235000019197 fats Nutrition 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 3
- 108010028295 histidylhistidine Proteins 0.000 description 3
- 108010085325 histidylproline Proteins 0.000 description 3
- 235000020256 human milk Nutrition 0.000 description 3
- 210000004251 human milk Anatomy 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 108010034529 leucyl-lysine Proteins 0.000 description 3
- 108010030617 leucyl-phenylalanyl-valine Proteins 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 108010009298 lysylglutamic acid Proteins 0.000 description 3
- 230000009871 nonspecific binding Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- HMFHBZSHGGEWLO-UHFFFAOYSA-N pentofuranose Chemical group OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 3
- 108010051242 phenylalanylserine Proteins 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000004017 serum-free culture medium Substances 0.000 description 3
- 108010048818 seryl-histidine Proteins 0.000 description 3
- 210000001082 somatic cell Anatomy 0.000 description 3
- 230000000392 somatic effect Effects 0.000 description 3
- 108010003137 tyrosyltyrosine Proteins 0.000 description 3
- 108010011667 Ala-Phe-Ala Proteins 0.000 description 2
- VQAVBBCZFQAAED-FXQIFTODSA-N Ala-Pro-Asn Chemical compound C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)N)C(=O)O)N VQAVBBCZFQAAED-FXQIFTODSA-N 0.000 description 2
- BYLSYQASFJJBCL-DCAQKATOSA-N Asn-Pro-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O BYLSYQASFJJBCL-DCAQKATOSA-N 0.000 description 2
- RKXVTTIQNKPCHU-KKHAAJSZSA-N Asp-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O RKXVTTIQNKPCHU-KKHAAJSZSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- LNAVIIOBBICBIS-NBRVCOCJSA-N CCCCCCCCCCCCCCC\C=C\C=C\C(O)=O Chemical compound CCCCCCCCCCCCCCC\C=C\C=C\C(O)=O LNAVIIOBBICBIS-NBRVCOCJSA-N 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 108020003215 DNA Probes Proteins 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- MUSGDMDGNGXULI-DCAQKATOSA-N Glu-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O MUSGDMDGNGXULI-DCAQKATOSA-N 0.000 description 2
- LERGJIVJIIODPZ-ZANVPECISA-N Gly-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)CN)C)C(O)=O)=CNC2=C1 LERGJIVJIIODPZ-ZANVPECISA-N 0.000 description 2
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 2
- FSPVILZGHUJOHS-QWRGUYRKSA-N Gly-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CNC=N1 FSPVILZGHUJOHS-QWRGUYRKSA-N 0.000 description 2
- TVUWMSBGMVAHSJ-KBPBESRZSA-N Gly-Leu-Phe Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 TVUWMSBGMVAHSJ-KBPBESRZSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- DFHVLUKTTVTCKY-PBCZWWQYSA-N His-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CN=CN1)N)O DFHVLUKTTVTCKY-PBCZWWQYSA-N 0.000 description 2
- KWBISLAEQZUYIC-UWJYBYFXSA-N His-His-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CN=CN2)N KWBISLAEQZUYIC-UWJYBYFXSA-N 0.000 description 2
- PMWSGVRIMIFXQH-KKUMJFAQSA-N His-His-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1NC=NC=1)C1=CN=CN1 PMWSGVRIMIFXQH-KKUMJFAQSA-N 0.000 description 2
- LQGCNWWLGGMTJO-ULQDDVLXSA-N His-Met-Phe Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N LQGCNWWLGGMTJO-ULQDDVLXSA-N 0.000 description 2
- KFQDSSNYWKZFOO-LSJOCFKGSA-N His-Val-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O KFQDSSNYWKZFOO-LSJOCFKGSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 108010065920 Insulin Lispro Proteins 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- PBCHMHROGNUXMK-DLOVCJGASA-N Leu-Ala-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 PBCHMHROGNUXMK-DLOVCJGASA-N 0.000 description 2
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 2
- INCJJHQRZGQLFC-KBPBESRZSA-N Leu-Phe-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O INCJJHQRZGQLFC-KBPBESRZSA-N 0.000 description 2
- YEIYAQQKADPIBJ-GARJFASQSA-N Lys-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)N)C(=O)O YEIYAQQKADPIBJ-GARJFASQSA-N 0.000 description 2
- YXPJCVNIDDKGOE-MELADBBJSA-N Lys-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N)C(=O)O YXPJCVNIDDKGOE-MELADBBJSA-N 0.000 description 2
- OBZHNHBAAVEWKI-DCAQKATOSA-N Lys-Pro-Asn Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O OBZHNHBAAVEWKI-DCAQKATOSA-N 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- ULNXMMYXQKGNPG-LPEHRKFASA-N Met-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCSC)N ULNXMMYXQKGNPG-LPEHRKFASA-N 0.000 description 2
- PHKBGZKVOJCIMZ-SRVKXCTJSA-N Met-Pro-Arg Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PHKBGZKVOJCIMZ-SRVKXCTJSA-N 0.000 description 2
- QAVZUKIPOMBLMC-AVGNSLFASA-N Met-Val-Leu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(C)C QAVZUKIPOMBLMC-AVGNSLFASA-N 0.000 description 2
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 2
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 2
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 2
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 2
- 238000000636 Northern blotting Methods 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- ABEFOXGAIIJDCL-SFJXLCSZSA-N Phe-Thr-Trp Chemical compound C([C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 ABEFOXGAIIJDCL-SFJXLCSZSA-N 0.000 description 2
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 2
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 2
- 108010079005 RDV peptide Proteins 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 2
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 101150003725 TK gene Proteins 0.000 description 2
- IMDMLDSVUSMAEJ-HJGDQZAQSA-N Thr-Leu-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IMDMLDSVUSMAEJ-HJGDQZAQSA-N 0.000 description 2
- 108091036066 Three prime untranslated region Proteins 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000006601 Thymidine Kinase Human genes 0.000 description 2
- WURLIFOWSMBUAR-SLFFLAALSA-N Tyr-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC3=CC=C(C=C3)O)N)C(=O)O WURLIFOWSMBUAR-SLFFLAALSA-N 0.000 description 2
- BRPKEERLGYNCNC-NHCYSSNCSA-N Val-Glu-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N BRPKEERLGYNCNC-NHCYSSNCSA-N 0.000 description 2
- URIRWLJVWHYLET-ONGXEEELSA-N Val-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C URIRWLJVWHYLET-ONGXEEELSA-N 0.000 description 2
- VTIAEOKFUJJBTC-YDHLFZDLSA-N Val-Tyr-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N VTIAEOKFUJJBTC-YDHLFZDLSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 108010005233 alanylglutamic acid Proteins 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- AHANXAKGNAKFSK-PDBXOOCHSA-N all-cis-icosa-11,14,17-trienoic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCCCC(O)=O AHANXAKGNAKFSK-PDBXOOCHSA-N 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 235000020244 animal milk Nutrition 0.000 description 2
- 230000003466 anti-cipated effect Effects 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000004952 blastocoel Anatomy 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 230000008568 cell cell communication Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229960002086 dextran Drugs 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 230000000378 dietary effect Effects 0.000 description 2
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- PRHHYVQTPBEDFE-UHFFFAOYSA-N eicosatrienoic acid Natural products CCCCCC=CCC=CCCCCC=CCCCC(O)=O PRHHYVQTPBEDFE-UHFFFAOYSA-N 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 108010084389 glycyltryptophan Proteins 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 239000012510 hollow fiber Substances 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 238000007901 in situ hybridization Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 2
- 108010017391 lysylvaline Proteins 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108010053725 prolylvaline Proteins 0.000 description 2
- 150000003815 prostacyclins Chemical class 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 150000004671 saturated fatty acids Chemical class 0.000 description 2
- 235000003441 saturated fatty acids Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 108010080629 tryptophan-leucine Proteins 0.000 description 2
- 108010045269 tryptophyltryptophan Proteins 0.000 description 2
- 108010044292 tryptophyltyrosine Proteins 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 210000004340 zona pellucida Anatomy 0.000 description 2
- UHEPSJJJMTWUCP-DHDYTCSHSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-[(1r)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;sulfuric acid Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N UHEPSJJJMTWUCP-DHDYTCSHSA-N 0.000 description 1
- AGNGYMCLFWQVGX-AGFFZDDWSA-N (e)-1-[(2s)-2-amino-2-carboxyethoxy]-2-diazonioethenolate Chemical compound OC(=O)[C@@H](N)CO\C([O-])=C\[N+]#N AGNGYMCLFWQVGX-AGFFZDDWSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 description 1
- DQVAZKGVGKHQDS-UHFFFAOYSA-N 2-[[1-[2-[(2-amino-4-methylpentanoyl)amino]-4-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-4-methylpentanoic acid Chemical compound CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(=O)NC(CC(C)C)C(O)=O DQVAZKGVGKHQDS-UHFFFAOYSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- DKJPOZOEBONHFS-ZLUOBGJFSA-N Ala-Ala-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O DKJPOZOEBONHFS-ZLUOBGJFSA-N 0.000 description 1
- YLTKNGYYPIWKHZ-ACZMJKKPSA-N Ala-Ala-Glu Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O YLTKNGYYPIWKHZ-ACZMJKKPSA-N 0.000 description 1
- FJVAQLJNTSUQPY-CIUDSAMLSA-N Ala-Ala-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN FJVAQLJNTSUQPY-CIUDSAMLSA-N 0.000 description 1
- VBDMWOKJZDCFJM-FXQIFTODSA-N Ala-Ala-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N VBDMWOKJZDCFJM-FXQIFTODSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- HGRBNYQIMKTUNT-XVYDVKMFSA-N Ala-Asn-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N HGRBNYQIMKTUNT-XVYDVKMFSA-N 0.000 description 1
- SHYYAQLDNVHPFT-DLOVCJGASA-N Ala-Asn-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SHYYAQLDNVHPFT-DLOVCJGASA-N 0.000 description 1
- MBWYUTNBYSSUIQ-HERUPUMHSA-N Ala-Asn-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N MBWYUTNBYSSUIQ-HERUPUMHSA-N 0.000 description 1
- BTYTYHBSJKQBQA-GCJQMDKQSA-N Ala-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N)O BTYTYHBSJKQBQA-GCJQMDKQSA-N 0.000 description 1
- DECCMEWNXSNSDO-ZLUOBGJFSA-N Ala-Cys-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O DECCMEWNXSNSDO-ZLUOBGJFSA-N 0.000 description 1
- WKOBSJOZRJJVRZ-FXQIFTODSA-N Ala-Glu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WKOBSJOZRJJVRZ-FXQIFTODSA-N 0.000 description 1
- OMMDTNGURYRDAC-NRPADANISA-N Ala-Glu-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OMMDTNGURYRDAC-NRPADANISA-N 0.000 description 1
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 1
- WGDNWOMKBUXFHR-BQBZGAKWSA-N Ala-Gly-Arg Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N WGDNWOMKBUXFHR-BQBZGAKWSA-N 0.000 description 1
- 108010076441 Ala-His-His Proteins 0.000 description 1
- SHKGHIFSEAGTNL-DLOVCJGASA-N Ala-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CN=CN1 SHKGHIFSEAGTNL-DLOVCJGASA-N 0.000 description 1
- HHRAXZAYZFFRAM-CIUDSAMLSA-N Ala-Leu-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O HHRAXZAYZFFRAM-CIUDSAMLSA-N 0.000 description 1
- QUIGLPSHIFPEOV-CIUDSAMLSA-N Ala-Lys-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O QUIGLPSHIFPEOV-CIUDSAMLSA-N 0.000 description 1
- LDLSENBXQNDTPB-DCAQKATOSA-N Ala-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LDLSENBXQNDTPB-DCAQKATOSA-N 0.000 description 1
- MAEQBGQTDWDSJQ-LSJOCFKGSA-N Ala-Met-His Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N MAEQBGQTDWDSJQ-LSJOCFKGSA-N 0.000 description 1
- XIWKVCDQMCNKOZ-UVBJJODRSA-N Ala-Met-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N XIWKVCDQMCNKOZ-UVBJJODRSA-N 0.000 description 1
- XRUJOVRWNMBAAA-NHCYSSNCSA-N Ala-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 XRUJOVRWNMBAAA-NHCYSSNCSA-N 0.000 description 1
- DHBKYZYFEXXUAK-ONGXEEELSA-N Ala-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 DHBKYZYFEXXUAK-ONGXEEELSA-N 0.000 description 1
- HYIDEIQUCBKIPL-CQDKDKBSSA-N Ala-Phe-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N HYIDEIQUCBKIPL-CQDKDKBSSA-N 0.000 description 1
- WEZNQZHACPSMEF-QEJZJMRPSA-N Ala-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 WEZNQZHACPSMEF-QEJZJMRPSA-N 0.000 description 1
- CYBJZLQSUJEMAS-LFSVMHDDSA-N Ala-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C)N)O CYBJZLQSUJEMAS-LFSVMHDDSA-N 0.000 description 1
- IPWKGIFRRBGCJO-IMJSIDKUSA-N Ala-Ser Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](CO)C([O-])=O IPWKGIFRRBGCJO-IMJSIDKUSA-N 0.000 description 1
- UCDOXFBTMLKASE-HERUPUMHSA-N Ala-Ser-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N UCDOXFBTMLKASE-HERUPUMHSA-N 0.000 description 1
- SYIFFFHSXBNPMC-UWJYBYFXSA-N Ala-Ser-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N SYIFFFHSXBNPMC-UWJYBYFXSA-N 0.000 description 1
- CREYEAPXISDKSB-FQPOAREZSA-N Ala-Thr-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CREYEAPXISDKSB-FQPOAREZSA-N 0.000 description 1
- RIPMDCIXRYWXSH-KNXALSJPSA-N Ala-Trp-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N3CCC[C@@H]3C(=O)O)N RIPMDCIXRYWXSH-KNXALSJPSA-N 0.000 description 1
- JPOQZCHGOTWRTM-FQPOAREZSA-N Ala-Tyr-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPOQZCHGOTWRTM-FQPOAREZSA-N 0.000 description 1
- MUGAESARFRGOTQ-IGNZVWTISA-N Ala-Tyr-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N MUGAESARFRGOTQ-IGNZVWTISA-N 0.000 description 1
- BOKLLPVAQDSLHC-FXQIFTODSA-N Ala-Val-Cys Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CS)C(=O)O)N BOKLLPVAQDSLHC-FXQIFTODSA-N 0.000 description 1
- CLOMBHBBUKAUBP-LSJOCFKGSA-N Ala-Val-His Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N CLOMBHBBUKAUBP-LSJOCFKGSA-N 0.000 description 1
- SSQHYGLFYWZWDV-UVBJJODRSA-N Ala-Val-Trp Chemical compound CC(C)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O SSQHYGLFYWZWDV-UVBJJODRSA-N 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 102000011730 Arachidonate 12-Lipoxygenase Human genes 0.000 description 1
- 108010076676 Arachidonate 12-lipoxygenase Proteins 0.000 description 1
- 102000009515 Arachidonate 15-Lipoxygenase Human genes 0.000 description 1
- 108010048907 Arachidonate 15-lipoxygenase Proteins 0.000 description 1
- 102000001381 Arachidonate 5-Lipoxygenase Human genes 0.000 description 1
- 108010093579 Arachidonate 5-lipoxygenase Proteins 0.000 description 1
- OTOXOKCIIQLMFH-KZVJFYERSA-N Arg-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N OTOXOKCIIQLMFH-KZVJFYERSA-N 0.000 description 1
- NONSEUUPKITYQT-BQBZGAKWSA-N Arg-Asn-Gly Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N)CN=C(N)N NONSEUUPKITYQT-BQBZGAKWSA-N 0.000 description 1
- KWTVWJPNHAOREN-IHRRRGAJSA-N Arg-Asn-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O KWTVWJPNHAOREN-IHRRRGAJSA-N 0.000 description 1
- NYZGVTGOMPHSJW-CIUDSAMLSA-N Arg-Glu-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N NYZGVTGOMPHSJW-CIUDSAMLSA-N 0.000 description 1
- NKBQZKVMKJJDLX-SRVKXCTJSA-N Arg-Glu-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NKBQZKVMKJJDLX-SRVKXCTJSA-N 0.000 description 1
- GOWZVQXTHUCNSQ-NHCYSSNCSA-N Arg-Glu-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O GOWZVQXTHUCNSQ-NHCYSSNCSA-N 0.000 description 1
- AQPVUEJJARLJHB-BQBZGAKWSA-N Arg-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N AQPVUEJJARLJHB-BQBZGAKWSA-N 0.000 description 1
- HQIZDMIGUJOSNI-IUCAKERBSA-N Arg-Gly-Arg Chemical compound N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQIZDMIGUJOSNI-IUCAKERBSA-N 0.000 description 1
- BNODVYXZAAXSHW-IUCAKERBSA-N Arg-His Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 BNODVYXZAAXSHW-IUCAKERBSA-N 0.000 description 1
- OTZMRMHZCMZOJZ-SRVKXCTJSA-N Arg-Leu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O OTZMRMHZCMZOJZ-SRVKXCTJSA-N 0.000 description 1
- COXMUHNBYCVVRG-DCAQKATOSA-N Arg-Leu-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O COXMUHNBYCVVRG-DCAQKATOSA-N 0.000 description 1
- BTJVOUQWFXABOI-IHRRRGAJSA-N Arg-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCNC(N)=N BTJVOUQWFXABOI-IHRRRGAJSA-N 0.000 description 1
- MNBHKGYCLBUIBC-UFYCRDLUSA-N Arg-Phe-Phe Chemical compound C([C@H](NC(=O)[C@H](CCCNC(N)=N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 MNBHKGYCLBUIBC-UFYCRDLUSA-N 0.000 description 1
- DNLQVHBBMPZUGJ-BQBZGAKWSA-N Arg-Ser-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O DNLQVHBBMPZUGJ-BQBZGAKWSA-N 0.000 description 1
- UVTGNSWSRSCPLP-UHFFFAOYSA-N Arg-Tyr Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccc(O)cc1)C(=O)O UVTGNSWSRSCPLP-UHFFFAOYSA-N 0.000 description 1
- XRLOBFSLPCHYLQ-ULQDDVLXSA-N Arg-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O XRLOBFSLPCHYLQ-ULQDDVLXSA-N 0.000 description 1
- CGWVCWFQGXOUSJ-ULQDDVLXSA-N Arg-Tyr-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O CGWVCWFQGXOUSJ-ULQDDVLXSA-N 0.000 description 1
- PSUXEQYPYZLNER-QXEWZRGKSA-N Arg-Val-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PSUXEQYPYZLNER-QXEWZRGKSA-N 0.000 description 1
- SUMJNGAMIQSNGX-TUAOUCFPSA-N Arg-Val-Pro Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)N1CCC[C@@H]1C(O)=O SUMJNGAMIQSNGX-TUAOUCFPSA-N 0.000 description 1
- LEFKSBYHUGUWLP-ACZMJKKPSA-N Asn-Ala-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LEFKSBYHUGUWLP-ACZMJKKPSA-N 0.000 description 1
- JJGRJMKUOYXZRA-LPEHRKFASA-N Asn-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)N)N)C(=O)O JJGRJMKUOYXZRA-LPEHRKFASA-N 0.000 description 1
- KXEGPPNPXOKKHK-ZLUOBGJFSA-N Asn-Asp-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O KXEGPPNPXOKKHK-ZLUOBGJFSA-N 0.000 description 1
- XVVOVPFMILMHPX-ZLUOBGJFSA-N Asn-Asp-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O XVVOVPFMILMHPX-ZLUOBGJFSA-N 0.000 description 1
- FANGHKQYFPYDNB-UBHSHLNASA-N Asn-Asp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N FANGHKQYFPYDNB-UBHSHLNASA-N 0.000 description 1
- JREOBWLIZLXRIS-GUBZILKMSA-N Asn-Glu-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JREOBWLIZLXRIS-GUBZILKMSA-N 0.000 description 1
- CTQIOCMSIJATNX-WHFBIAKZSA-N Asn-Gly-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O CTQIOCMSIJATNX-WHFBIAKZSA-N 0.000 description 1
- OWUCNXMFJRFOFI-BQBZGAKWSA-N Asn-Gly-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O OWUCNXMFJRFOFI-BQBZGAKWSA-N 0.000 description 1
- IKLAUGBIDCDFOY-SRVKXCTJSA-N Asn-His-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O IKLAUGBIDCDFOY-SRVKXCTJSA-N 0.000 description 1
- NYGILGUOUOXGMJ-YUMQZZPRSA-N Asn-Lys-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O NYGILGUOUOXGMJ-YUMQZZPRSA-N 0.000 description 1
- VOKWBBBXJONREA-DCAQKATOSA-N Asn-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(=O)N)N VOKWBBBXJONREA-DCAQKATOSA-N 0.000 description 1
- RBOBTTLFPRSXKZ-BZSNNMDCSA-N Asn-Phe-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RBOBTTLFPRSXKZ-BZSNNMDCSA-N 0.000 description 1
- BCADFFUQHIMQAA-KKHAAJSZSA-N Asn-Thr-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O BCADFFUQHIMQAA-KKHAAJSZSA-N 0.000 description 1
- CPYHLXSGDBDULY-IHPCNDPISA-N Asn-Trp-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O CPYHLXSGDBDULY-IHPCNDPISA-N 0.000 description 1
- FYRVDDJMNISIKJ-UWVGGRQHSA-N Asn-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FYRVDDJMNISIKJ-UWVGGRQHSA-N 0.000 description 1
- ZAESWDKAMDVHLL-RCOVLWMOSA-N Asn-Val-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O ZAESWDKAMDVHLL-RCOVLWMOSA-N 0.000 description 1
- HPNDBHLITCHRSO-WHFBIAKZSA-N Asp-Ala-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)NCC(O)=O HPNDBHLITCHRSO-WHFBIAKZSA-N 0.000 description 1
- NECWUSYTYSIFNC-DLOVCJGASA-N Asp-Ala-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 NECWUSYTYSIFNC-DLOVCJGASA-N 0.000 description 1
- KVMPVNGOKHTUHZ-GCJQMDKQSA-N Asp-Ala-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KVMPVNGOKHTUHZ-GCJQMDKQSA-N 0.000 description 1
- BLQBMRNMBAYREH-UWJYBYFXSA-N Asp-Ala-Tyr Chemical compound N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O BLQBMRNMBAYREH-UWJYBYFXSA-N 0.000 description 1
- QHAJMRDEWNAIBQ-FXQIFTODSA-N Asp-Arg-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O QHAJMRDEWNAIBQ-FXQIFTODSA-N 0.000 description 1
- ZLGKHJHFYSRUBH-FXQIFTODSA-N Asp-Arg-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLGKHJHFYSRUBH-FXQIFTODSA-N 0.000 description 1
- SDHFVYLZFBDSQT-DCAQKATOSA-N Asp-Arg-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N SDHFVYLZFBDSQT-DCAQKATOSA-N 0.000 description 1
- FAEIQWHBRBWUBN-FXQIFTODSA-N Asp-Arg-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N)CN=C(N)N FAEIQWHBRBWUBN-FXQIFTODSA-N 0.000 description 1
- JDHOJQJMWBKHDB-CIUDSAMLSA-N Asp-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N JDHOJQJMWBKHDB-CIUDSAMLSA-N 0.000 description 1
- GHODABZPVZMWCE-FXQIFTODSA-N Asp-Glu-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O GHODABZPVZMWCE-FXQIFTODSA-N 0.000 description 1
- OVPHVTCDVYYTHN-AVGNSLFASA-N Asp-Glu-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OVPHVTCDVYYTHN-AVGNSLFASA-N 0.000 description 1
- DGKCOYGQLNWNCJ-ACZMJKKPSA-N Asp-Glu-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O DGKCOYGQLNWNCJ-ACZMJKKPSA-N 0.000 description 1
- YDJVIBMKAMQPPP-LAEOZQHASA-N Asp-Glu-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O YDJVIBMKAMQPPP-LAEOZQHASA-N 0.000 description 1
- OGTCOKZFOJIZFG-CIUDSAMLSA-N Asp-His-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(O)=O OGTCOKZFOJIZFG-CIUDSAMLSA-N 0.000 description 1
- UBPMOJLRVMGTOQ-GARJFASQSA-N Asp-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC(=O)O)N)C(=O)O UBPMOJLRVMGTOQ-GARJFASQSA-N 0.000 description 1
- ORRJQLIATJDMQM-HJGDQZAQSA-N Asp-Leu-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O ORRJQLIATJDMQM-HJGDQZAQSA-N 0.000 description 1
- WWOYXVBGHAHQBG-FXQIFTODSA-N Asp-Met-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O WWOYXVBGHAHQBG-FXQIFTODSA-N 0.000 description 1
- LTCKTLYKRMCFOC-KKUMJFAQSA-N Asp-Phe-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LTCKTLYKRMCFOC-KKUMJFAQSA-N 0.000 description 1
- UCHSVZYJKJLPHF-BZSNNMDCSA-N Asp-Phe-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O UCHSVZYJKJLPHF-BZSNNMDCSA-N 0.000 description 1
- KESWRFKUZRUTAH-FXQIFTODSA-N Asp-Pro-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O KESWRFKUZRUTAH-FXQIFTODSA-N 0.000 description 1
- UAXIKORUDGGIGA-DCAQKATOSA-N Asp-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CCCCN)C(=O)O UAXIKORUDGGIGA-DCAQKATOSA-N 0.000 description 1
- XUVTWGPERWIERB-IHRRRGAJSA-N Asp-Pro-Phe Chemical compound N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccccc1)C(O)=O XUVTWGPERWIERB-IHRRRGAJSA-N 0.000 description 1
- DINOVZWPTMGSRF-QXEWZRGKSA-N Asp-Pro-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O DINOVZWPTMGSRF-QXEWZRGKSA-N 0.000 description 1
- ZQFRDAZBTSFGGW-SRVKXCTJSA-N Asp-Ser-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ZQFRDAZBTSFGGW-SRVKXCTJSA-N 0.000 description 1
- XAPPCWUWHNWCPQ-PBCZWWQYSA-N Asp-Thr-His Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O XAPPCWUWHNWCPQ-PBCZWWQYSA-N 0.000 description 1
- GXHDGYOXPNQCKM-XVSYOHENSA-N Asp-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O GXHDGYOXPNQCKM-XVSYOHENSA-N 0.000 description 1
- PLNJUJGNLDSFOP-UWJYBYFXSA-N Asp-Tyr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PLNJUJGNLDSFOP-UWJYBYFXSA-N 0.000 description 1
- XQFLFQWOBXPMHW-NHCYSSNCSA-N Asp-Val-His Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O XQFLFQWOBXPMHW-NHCYSSNCSA-N 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 239000005496 Chlorsulfuron Substances 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- NDUSUIGBMZCOIL-ZKWXMUAHSA-N Cys-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CS)N NDUSUIGBMZCOIL-ZKWXMUAHSA-N 0.000 description 1
- KIHRUISMQZVCNO-ZLUOBGJFSA-N Cys-Asp-Asp Chemical compound SC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O KIHRUISMQZVCNO-ZLUOBGJFSA-N 0.000 description 1
- RWAZRMXTVSIVJR-YUMQZZPRSA-N Cys-Gly-His Chemical compound [H]N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CC1=CNC=N1)C(O)=O RWAZRMXTVSIVJR-YUMQZZPRSA-N 0.000 description 1
- LBOLGUYQEPZSKM-YUMQZZPRSA-N Cys-Gly-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CS)N LBOLGUYQEPZSKM-YUMQZZPRSA-N 0.000 description 1
- JDHMXPSXWMPYQZ-AAEUAGOBSA-N Cys-Gly-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CS)N JDHMXPSXWMPYQZ-AAEUAGOBSA-N 0.000 description 1
- KGIHMGPYGXBYJJ-SRVKXCTJSA-N Cys-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CS KGIHMGPYGXBYJJ-SRVKXCTJSA-N 0.000 description 1
- UDDITVWSXPEAIQ-IHRRRGAJSA-N Cys-Phe-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UDDITVWSXPEAIQ-IHRRRGAJSA-N 0.000 description 1
- YYLBXQJGWOQZOU-IHRRRGAJSA-N Cys-Phe-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CS)N YYLBXQJGWOQZOU-IHRRRGAJSA-N 0.000 description 1
- LKHMGNHQULEPFY-ACZMJKKPSA-N Cys-Ser-Glu Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O LKHMGNHQULEPFY-ACZMJKKPSA-N 0.000 description 1
- KFYPRIGJTICABD-XGEHTFHBSA-N Cys-Thr-Val Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N)O KFYPRIGJTICABD-XGEHTFHBSA-N 0.000 description 1
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- 108010090461 DFG peptide Proteins 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- RUFHOVYUYSNDNY-ACZMJKKPSA-N Glu-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O RUFHOVYUYSNDNY-ACZMJKKPSA-N 0.000 description 1
- IRDASPPCLZIERZ-XHNCKOQMSA-N Glu-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N IRDASPPCLZIERZ-XHNCKOQMSA-N 0.000 description 1
- NCWOMXABNYEPLY-NRPADANISA-N Glu-Ala-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O NCWOMXABNYEPLY-NRPADANISA-N 0.000 description 1
- CVPXINNKRTZBMO-CIUDSAMLSA-N Glu-Arg-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)CN=C(N)N CVPXINNKRTZBMO-CIUDSAMLSA-N 0.000 description 1
- NLKVNZUFDPWPNL-YUMQZZPRSA-N Glu-Arg-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O NLKVNZUFDPWPNL-YUMQZZPRSA-N 0.000 description 1
- WOSRKEJQESVHGA-CIUDSAMLSA-N Glu-Arg-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O WOSRKEJQESVHGA-CIUDSAMLSA-N 0.000 description 1
- SYDJILXOZNEEDK-XIRDDKMYSA-N Glu-Arg-Trp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O SYDJILXOZNEEDK-XIRDDKMYSA-N 0.000 description 1
- AKJRHDMTEJXTPV-ACZMJKKPSA-N Glu-Asn-Ala Chemical compound C[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AKJRHDMTEJXTPV-ACZMJKKPSA-N 0.000 description 1
- CKOFNWCLWRYUHK-XHNCKOQMSA-N Glu-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N)C(=O)O CKOFNWCLWRYUHK-XHNCKOQMSA-N 0.000 description 1
- NKLRYVLERDYDBI-FXQIFTODSA-N Glu-Glu-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NKLRYVLERDYDBI-FXQIFTODSA-N 0.000 description 1
- KRGZZKWSBGPLKL-IUCAKERBSA-N Glu-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)O)N KRGZZKWSBGPLKL-IUCAKERBSA-N 0.000 description 1
- HPJLZFTUUJKWAJ-JHEQGTHGSA-N Glu-Gly-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HPJLZFTUUJKWAJ-JHEQGTHGSA-N 0.000 description 1
- HILMIYALTUQTRC-XVKPBYJWSA-N Glu-Gly-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HILMIYALTUQTRC-XVKPBYJWSA-N 0.000 description 1
- VXQOONWNIWFOCS-HGNGGELXSA-N Glu-His-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCC(=O)O)N VXQOONWNIWFOCS-HGNGGELXSA-N 0.000 description 1
- XOFYVODYSNKPDK-AVGNSLFASA-N Glu-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCC(=O)O)N XOFYVODYSNKPDK-AVGNSLFASA-N 0.000 description 1
- ATVYZJGOZLVXDK-IUCAKERBSA-N Glu-Leu-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O ATVYZJGOZLVXDK-IUCAKERBSA-N 0.000 description 1
- NJCALAAIGREHDR-WDCWCFNPSA-N Glu-Leu-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NJCALAAIGREHDR-WDCWCFNPSA-N 0.000 description 1
- ZTVGZOIBLRPQNR-KKUMJFAQSA-N Glu-Met-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZTVGZOIBLRPQNR-KKUMJFAQSA-N 0.000 description 1
- UDEPRBFQTWGLCW-CIUDSAMLSA-N Glu-Pro-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O UDEPRBFQTWGLCW-CIUDSAMLSA-N 0.000 description 1
- SWDNPSMMEWRNOH-HJGDQZAQSA-N Glu-Pro-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWDNPSMMEWRNOH-HJGDQZAQSA-N 0.000 description 1
- YQAQQKPWFOBSMU-WDCWCFNPSA-N Glu-Thr-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O YQAQQKPWFOBSMU-WDCWCFNPSA-N 0.000 description 1
- YSWHPLCDIMUKFE-QWRGUYRKSA-N Glu-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 YSWHPLCDIMUKFE-QWRGUYRKSA-N 0.000 description 1
- LSYFGBRDBIQYAQ-FHWLQOOXSA-N Glu-Tyr-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LSYFGBRDBIQYAQ-FHWLQOOXSA-N 0.000 description 1
- MLILEEIVMRUYBX-NHCYSSNCSA-N Glu-Val-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O MLILEEIVMRUYBX-NHCYSSNCSA-N 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- JVWPPCWUDRJGAE-YUMQZZPRSA-N Gly-Asn-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JVWPPCWUDRJGAE-YUMQZZPRSA-N 0.000 description 1
- YYPFZVIXAVDHIK-IUCAKERBSA-N Gly-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN YYPFZVIXAVDHIK-IUCAKERBSA-N 0.000 description 1
- UUWOBINZFGTFMS-UWVGGRQHSA-N Gly-His-Met Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCSC)C(O)=O UUWOBINZFGTFMS-UWVGGRQHSA-N 0.000 description 1
- NSTUFLGQJCOCDL-UWVGGRQHSA-N Gly-Leu-Arg Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N NSTUFLGQJCOCDL-UWVGGRQHSA-N 0.000 description 1
- IUZGUFAJDBHQQV-YUMQZZPRSA-N Gly-Leu-Asn Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IUZGUFAJDBHQQV-YUMQZZPRSA-N 0.000 description 1
- YIFUFYZELCMPJP-YUMQZZPRSA-N Gly-Leu-Cys Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(O)=O YIFUFYZELCMPJP-YUMQZZPRSA-N 0.000 description 1
- VBOBNHSVQKKTOT-YUMQZZPRSA-N Gly-Lys-Ala Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O VBOBNHSVQKKTOT-YUMQZZPRSA-N 0.000 description 1
- FXGRXIATVXUAHO-WEDXCCLWSA-N Gly-Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN FXGRXIATVXUAHO-WEDXCCLWSA-N 0.000 description 1
- GGAPHLIUUTVYMX-QWRGUYRKSA-N Gly-Phe-Ser Chemical compound OC[C@@H](C([O-])=O)NC(=O)[C@@H](NC(=O)C[NH3+])CC1=CC=CC=C1 GGAPHLIUUTVYMX-QWRGUYRKSA-N 0.000 description 1
- OOCFXNOVSLSHAB-IUCAKERBSA-N Gly-Pro-Pro Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 OOCFXNOVSLSHAB-IUCAKERBSA-N 0.000 description 1
- GLACUWHUYFBSPJ-FJXKBIBVSA-N Gly-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN GLACUWHUYFBSPJ-FJXKBIBVSA-N 0.000 description 1
- YOBGUCWZPXJHTN-BQBZGAKWSA-N Gly-Ser-Arg Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YOBGUCWZPXJHTN-BQBZGAKWSA-N 0.000 description 1
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 1
- AJHCSUXXECOXOY-NSHDSACASA-N Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-NSHDSACASA-N 0.000 description 1
- COZMNNJEGNPDED-HOCLYGCPSA-N Gly-Val-Trp Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O COZMNNJEGNPDED-HOCLYGCPSA-N 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- XJQDHFMUUBRCGA-KKUMJFAQSA-N His-Asn-Phe Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XJQDHFMUUBRCGA-KKUMJFAQSA-N 0.000 description 1
- OBTMRGFRLJBSFI-GARJFASQSA-N His-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O OBTMRGFRLJBSFI-GARJFASQSA-N 0.000 description 1
- VOKCBYNCZVSILJ-KKUMJFAQSA-N His-Asn-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC2=CN=CN2)N)O VOKCBYNCZVSILJ-KKUMJFAQSA-N 0.000 description 1
- VOEGKUNRHYKYSU-XVYDVKMFSA-N His-Asp-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O VOEGKUNRHYKYSU-XVYDVKMFSA-N 0.000 description 1
- WGVPDSNCHDEDBP-KKUMJFAQSA-N His-Asp-Phe Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O WGVPDSNCHDEDBP-KKUMJFAQSA-N 0.000 description 1
- ZZLWLWSUIBSMNP-CIUDSAMLSA-N His-Asp-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZZLWLWSUIBSMNP-CIUDSAMLSA-N 0.000 description 1
- TVTIDSMADMIHEU-KKUMJFAQSA-N His-Cys-Phe Chemical compound N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CS)C(=O)N[C@@H](Cc1ccccc1)C(O)=O TVTIDSMADMIHEU-KKUMJFAQSA-N 0.000 description 1
- STOOMQFEJUVAKR-KKUMJFAQSA-N His-His-His Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)C1=CNC=N1 STOOMQFEJUVAKR-KKUMJFAQSA-N 0.000 description 1
- JENKOCSDMSVWPY-SRVKXCTJSA-N His-Leu-Asn Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O JENKOCSDMSVWPY-SRVKXCTJSA-N 0.000 description 1
- RNMNYMDTESKEAJ-KKUMJFAQSA-N His-Leu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CN=CN1 RNMNYMDTESKEAJ-KKUMJFAQSA-N 0.000 description 1
- ZSKJIISDJXJQPV-BZSNNMDCSA-N His-Leu-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CN=CN1 ZSKJIISDJXJQPV-BZSNNMDCSA-N 0.000 description 1
- VGYOLSOFODKLSP-IHPCNDPISA-N His-Leu-Trp Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CN=CN1 VGYOLSOFODKLSP-IHPCNDPISA-N 0.000 description 1
- BCZFOHDMCDXPDA-BZSNNMDCSA-N His-Lys-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CN=CN2)N)O BCZFOHDMCDXPDA-BZSNNMDCSA-N 0.000 description 1
- TVMNTHXFRSXZGR-IHRRRGAJSA-N His-Lys-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O TVMNTHXFRSXZGR-IHRRRGAJSA-N 0.000 description 1
- QCBYAHHNOHBXIH-UWVGGRQHSA-N His-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CN=CN1 QCBYAHHNOHBXIH-UWVGGRQHSA-N 0.000 description 1
- CHIAUHSHDARFBD-ULQDDVLXSA-N His-Pro-Tyr Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 CHIAUHSHDARFBD-ULQDDVLXSA-N 0.000 description 1
- PZAJPILZRFPYJJ-SRVKXCTJSA-N His-Ser-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O PZAJPILZRFPYJJ-SRVKXCTJSA-N 0.000 description 1
- RXKFKJVJVHLRIE-XIRDDKMYSA-N His-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC3=CN=CN3)N RXKFKJVJVHLRIE-XIRDDKMYSA-N 0.000 description 1
- XHQYFGPIRUHQIB-PBCZWWQYSA-N His-Thr-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC1=CN=CN1 XHQYFGPIRUHQIB-PBCZWWQYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 description 1
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- YUGVQABRIJXYNQ-CIUDSAMLSA-N Leu-Ala-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YUGVQABRIJXYNQ-CIUDSAMLSA-N 0.000 description 1
- YUGVQABRIJXYNQ-UHFFFAOYSA-N Leu-Ala-Ala Natural products CC(C)CC(N)C(=O)NC(C)C(=O)NC(C)C(O)=O YUGVQABRIJXYNQ-UHFFFAOYSA-N 0.000 description 1
- CZCSUZMIRKFFFA-CIUDSAMLSA-N Leu-Ala-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O CZCSUZMIRKFFFA-CIUDSAMLSA-N 0.000 description 1
- XBBKIIGCUMBKCO-JXUBOQSCSA-N Leu-Ala-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XBBKIIGCUMBKCO-JXUBOQSCSA-N 0.000 description 1
- HXWALXSAVBLTPK-NUTKFTJISA-N Leu-Ala-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(C)C)N HXWALXSAVBLTPK-NUTKFTJISA-N 0.000 description 1
- IBMVEYRWAWIOTN-RWMBFGLXSA-N Leu-Arg-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(O)=O IBMVEYRWAWIOTN-RWMBFGLXSA-N 0.000 description 1
- KKXDHFKZWKLYGB-GUBZILKMSA-N Leu-Asn-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKXDHFKZWKLYGB-GUBZILKMSA-N 0.000 description 1
- VIWUBXKCYJGNCL-SRVKXCTJSA-N Leu-Asn-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 VIWUBXKCYJGNCL-SRVKXCTJSA-N 0.000 description 1
- WGNOPSQMIQERPK-UHFFFAOYSA-N Leu-Asn-Pro Natural products CC(C)CC(N)C(=O)NC(CC(=O)N)C(=O)N1CCCC1C(=O)O WGNOPSQMIQERPK-UHFFFAOYSA-N 0.000 description 1
- MYGQXVYRZMKRDB-SRVKXCTJSA-N Leu-Asp-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN MYGQXVYRZMKRDB-SRVKXCTJSA-N 0.000 description 1
- IWTBYNQNAPECCS-AVGNSLFASA-N Leu-Glu-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IWTBYNQNAPECCS-AVGNSLFASA-N 0.000 description 1
- PRZVBIAOPFGAQF-SRVKXCTJSA-N Leu-Glu-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O PRZVBIAOPFGAQF-SRVKXCTJSA-N 0.000 description 1
- LAGPXKYZCCTSGQ-JYJNAYRXSA-N Leu-Glu-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LAGPXKYZCCTSGQ-JYJNAYRXSA-N 0.000 description 1
- WQWSMEOYXJTFRU-GUBZILKMSA-N Leu-Glu-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O WQWSMEOYXJTFRU-GUBZILKMSA-N 0.000 description 1
- OXRLYTYUXAQTHP-YUMQZZPRSA-N Leu-Gly-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(O)=O OXRLYTYUXAQTHP-YUMQZZPRSA-N 0.000 description 1
- VBZOAGIPCULURB-QWRGUYRKSA-N Leu-Gly-His Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N VBZOAGIPCULURB-QWRGUYRKSA-N 0.000 description 1
- HYIFFZAQXPUEAU-QWRGUYRKSA-N Leu-Gly-Leu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(C)C HYIFFZAQXPUEAU-QWRGUYRKSA-N 0.000 description 1
- APFJUBGRZGMQFF-QWRGUYRKSA-N Leu-Gly-Lys Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN APFJUBGRZGMQFF-QWRGUYRKSA-N 0.000 description 1
- KEVYYIMVELOXCT-KBPBESRZSA-N Leu-Gly-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KEVYYIMVELOXCT-KBPBESRZSA-N 0.000 description 1
- BTNXKBVLWJBTNR-SRVKXCTJSA-N Leu-His-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(O)=O BTNXKBVLWJBTNR-SRVKXCTJSA-N 0.000 description 1
- AOFYPTOHESIBFZ-KKUMJFAQSA-N Leu-His-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O AOFYPTOHESIBFZ-KKUMJFAQSA-N 0.000 description 1
- OYQUOLRTJHWVSQ-SRVKXCTJSA-N Leu-His-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O OYQUOLRTJHWVSQ-SRVKXCTJSA-N 0.000 description 1
- HMDDEJADNKQTBR-BZSNNMDCSA-N Leu-His-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O HMDDEJADNKQTBR-BZSNNMDCSA-N 0.000 description 1
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 1
- UBZGNBKMIJHOHL-BZSNNMDCSA-N Leu-Leu-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 UBZGNBKMIJHOHL-BZSNNMDCSA-N 0.000 description 1
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 1
- UCNNZELZXFXXJQ-BZSNNMDCSA-N Leu-Leu-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 UCNNZELZXFXXJQ-BZSNNMDCSA-N 0.000 description 1
- RTIRBWJPYJYTLO-MELADBBJSA-N Leu-Lys-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N RTIRBWJPYJYTLO-MELADBBJSA-N 0.000 description 1
- NTISAKGPIGTIJJ-IUCAKERBSA-N Leu-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC(C)C NTISAKGPIGTIJJ-IUCAKERBSA-N 0.000 description 1
- MVVSHHJKJRZVNY-ACRUOGEOSA-N Leu-Phe-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MVVSHHJKJRZVNY-ACRUOGEOSA-N 0.000 description 1
- RRVCZCNFXIFGRA-DCAQKATOSA-N Leu-Pro-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RRVCZCNFXIFGRA-DCAQKATOSA-N 0.000 description 1
- VULJUQZPSOASBZ-SRVKXCTJSA-N Leu-Pro-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O VULJUQZPSOASBZ-SRVKXCTJSA-N 0.000 description 1
- MUCIDQMDOYQYBR-IHRRRGAJSA-N Leu-Pro-His Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N MUCIDQMDOYQYBR-IHRRRGAJSA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 description 1
- IWMJFLJQHIDZQW-KKUMJFAQSA-N Leu-Ser-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IWMJFLJQHIDZQW-KKUMJFAQSA-N 0.000 description 1
- SIGZKCWZEBFNAK-QAETUUGQSA-N Leu-Ser-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SIGZKCWZEBFNAK-QAETUUGQSA-N 0.000 description 1
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 1
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 1
- FGZVGOAAROXFAB-IXOXFDKPSA-N Leu-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(C)C)N)O FGZVGOAAROXFAB-IXOXFDKPSA-N 0.000 description 1
- HGLKOTPFWOMPOB-MEYUZBJRSA-N Leu-Thr-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HGLKOTPFWOMPOB-MEYUZBJRSA-N 0.000 description 1
- FPFOYSCDUWTZBF-IHPCNDPISA-N Leu-Trp-Leu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H]([NH3+])CC(C)C)C(=O)N[C@@H](CC(C)C)C([O-])=O)=CNC2=C1 FPFOYSCDUWTZBF-IHPCNDPISA-N 0.000 description 1
- WFCKERTZVCQXKH-KBPBESRZSA-N Leu-Tyr-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O WFCKERTZVCQXKH-KBPBESRZSA-N 0.000 description 1
- YQFZRHYZLARWDY-IHRRRGAJSA-N Leu-Val-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN YQFZRHYZLARWDY-IHRRRGAJSA-N 0.000 description 1
- VKVDRTGWLVZJOM-DCAQKATOSA-N Leu-Val-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O VKVDRTGWLVZJOM-DCAQKATOSA-N 0.000 description 1
- MSFITIBEMPWCBD-ULQDDVLXSA-N Leu-Val-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 MSFITIBEMPWCBD-ULQDDVLXSA-N 0.000 description 1
- 229930184725 Lipoxin Natural products 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- YIBOAHAOAWACDK-QEJZJMRPSA-N Lys-Ala-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 YIBOAHAOAWACDK-QEJZJMRPSA-N 0.000 description 1
- KNKHAVVBVXKOGX-JXUBOQSCSA-N Lys-Ala-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KNKHAVVBVXKOGX-JXUBOQSCSA-N 0.000 description 1
- ZQCVMVCVPFYXHZ-SRVKXCTJSA-N Lys-Asn-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCCN ZQCVMVCVPFYXHZ-SRVKXCTJSA-N 0.000 description 1
- QUCDKEKDPYISNX-HJGDQZAQSA-N Lys-Asn-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QUCDKEKDPYISNX-HJGDQZAQSA-N 0.000 description 1
- HKCCVDWHHTVVPN-CIUDSAMLSA-N Lys-Asp-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O HKCCVDWHHTVVPN-CIUDSAMLSA-N 0.000 description 1
- QUYCUALODHJQLK-CIUDSAMLSA-N Lys-Asp-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O QUYCUALODHJQLK-CIUDSAMLSA-N 0.000 description 1
- PHHYNOUOUWYQRO-XIRDDKMYSA-N Lys-Asp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)N PHHYNOUOUWYQRO-XIRDDKMYSA-N 0.000 description 1
- ZAWOJFFMBANLGE-CIUDSAMLSA-N Lys-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCCN)N ZAWOJFFMBANLGE-CIUDSAMLSA-N 0.000 description 1
- UGTZHPSKYRIGRJ-YUMQZZPRSA-N Lys-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O UGTZHPSKYRIGRJ-YUMQZZPRSA-N 0.000 description 1
- PBIPLDMFHAICIP-DCAQKATOSA-N Lys-Glu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PBIPLDMFHAICIP-DCAQKATOSA-N 0.000 description 1
- ITWQLSZTLBKWJM-YUMQZZPRSA-N Lys-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCCCN ITWQLSZTLBKWJM-YUMQZZPRSA-N 0.000 description 1
- PRCHKVGXZVTALR-KKUMJFAQSA-N Lys-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCCN)N PRCHKVGXZVTALR-KKUMJFAQSA-N 0.000 description 1
- OVAOHZIOUBEQCJ-IHRRRGAJSA-N Lys-Leu-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O OVAOHZIOUBEQCJ-IHRRRGAJSA-N 0.000 description 1
- AIRZWUMAHCDDHR-KKUMJFAQSA-N Lys-Leu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O AIRZWUMAHCDDHR-KKUMJFAQSA-N 0.000 description 1
- WBSCNDJQPKSPII-KKUMJFAQSA-N Lys-Lys-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O WBSCNDJQPKSPII-KKUMJFAQSA-N 0.000 description 1
- DAHQKYYIXPBESV-UWVGGRQHSA-N Lys-Met-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)NCC(O)=O DAHQKYYIXPBESV-UWVGGRQHSA-N 0.000 description 1
- ZJSZPXISKMDJKQ-JYJNAYRXSA-N Lys-Phe-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(O)=O)CC1=CC=CC=C1 ZJSZPXISKMDJKQ-JYJNAYRXSA-N 0.000 description 1
- LNMKRJJLEFASGA-BZSNNMDCSA-N Lys-Phe-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LNMKRJJLEFASGA-BZSNNMDCSA-N 0.000 description 1
- HYSVGEAWTGPMOA-IHRRRGAJSA-N Lys-Pro-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O HYSVGEAWTGPMOA-IHRRRGAJSA-N 0.000 description 1
- ZOKVLMBYDSIDKG-CSMHCCOUSA-N Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN ZOKVLMBYDSIDKG-CSMHCCOUSA-N 0.000 description 1
- TVHCDSBMFQYPNA-RHYQMDGZSA-N Lys-Thr-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O TVHCDSBMFQYPNA-RHYQMDGZSA-N 0.000 description 1
- RVKIPWVMZANZLI-ZFWWWQNUSA-N Lys-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)CCCCN)C(O)=O)=CNC2=C1 RVKIPWVMZANZLI-ZFWWWQNUSA-N 0.000 description 1
- USPJSTBDIGJPFK-PMVMPFDFSA-N Lys-Tyr-Trp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O USPJSTBDIGJPFK-PMVMPFDFSA-N 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- YRAWWKUTNBILNT-FXQIFTODSA-N Met-Ala-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YRAWWKUTNBILNT-FXQIFTODSA-N 0.000 description 1
- DCHHUGLTVLJYKA-FXQIFTODSA-N Met-Asn-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O DCHHUGLTVLJYKA-FXQIFTODSA-N 0.000 description 1
- BQVJARUIXRXDKN-DCAQKATOSA-N Met-Asn-His Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 BQVJARUIXRXDKN-DCAQKATOSA-N 0.000 description 1
- CAODKDAPYGUMLK-FXQIFTODSA-N Met-Asn-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O CAODKDAPYGUMLK-FXQIFTODSA-N 0.000 description 1
- MNNKPHGAPRUKMW-BPUTZDHNSA-N Met-Asp-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCSC)C(O)=O)=CNC2=C1 MNNKPHGAPRUKMW-BPUTZDHNSA-N 0.000 description 1
- MYAPQOBHGWJZOM-UWVGGRQHSA-N Met-Gly-Leu Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(C)C MYAPQOBHGWJZOM-UWVGGRQHSA-N 0.000 description 1
- LQMHZERGCQJKAH-STQMWFEESA-N Met-Gly-Phe Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 LQMHZERGCQJKAH-STQMWFEESA-N 0.000 description 1
- SXWQMBGNFXAGAT-FJXKBIBVSA-N Met-Gly-Thr Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O SXWQMBGNFXAGAT-FJXKBIBVSA-N 0.000 description 1
- XPCLRYNQMZOOFB-ULQDDVLXSA-N Met-His-Phe Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N XPCLRYNQMZOOFB-ULQDDVLXSA-N 0.000 description 1
- SODXFJOPSCXOHE-IHRRRGAJSA-N Met-Leu-Leu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O SODXFJOPSCXOHE-IHRRRGAJSA-N 0.000 description 1
- UNPGTBHYKJOCCZ-DCAQKATOSA-N Met-Lys-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O UNPGTBHYKJOCCZ-DCAQKATOSA-N 0.000 description 1
- HAQLBBVZAGMESV-IHRRRGAJSA-N Met-Lys-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O HAQLBBVZAGMESV-IHRRRGAJSA-N 0.000 description 1
- CGUYGMFQZCYJSG-DCAQKATOSA-N Met-Lys-Ser Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O CGUYGMFQZCYJSG-DCAQKATOSA-N 0.000 description 1
- DZMGFGQBRYWJOR-YUMQZZPRSA-N Met-Pro Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O DZMGFGQBRYWJOR-YUMQZZPRSA-N 0.000 description 1
- SBFPAAPFKZPDCZ-JYJNAYRXSA-N Met-Pro-Tyr Chemical compound [H]N[C@@H](CCSC)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O SBFPAAPFKZPDCZ-JYJNAYRXSA-N 0.000 description 1
- GGXZOTSDJJTDGB-GUBZILKMSA-N Met-Ser-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O GGXZOTSDJJTDGB-GUBZILKMSA-N 0.000 description 1
- 101100261636 Methanothermobacter marburgensis (strain ATCC BAA-927 / DSM 2133 / JCM 14651 / NBRC 100331 / OCM 82 / Marburg) trpB2 gene Proteins 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 206010068052 Mosaicism Diseases 0.000 description 1
- 241000204795 Muraena helena Species 0.000 description 1
- 101000969137 Mus musculus Metallothionein-1 Proteins 0.000 description 1
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 101100438378 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) fac-1 gene Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- BJEYSVHMGIJORT-NHCYSSNCSA-N Phe-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 BJEYSVHMGIJORT-NHCYSSNCSA-N 0.000 description 1
- LSXGADJXBDFXQU-DLOVCJGASA-N Phe-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 LSXGADJXBDFXQU-DLOVCJGASA-N 0.000 description 1
- SWZKMTDPQXLQRD-XVSYOHENSA-N Phe-Asp-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWZKMTDPQXLQRD-XVSYOHENSA-N 0.000 description 1
- CSDMCMITJLKBAH-SOUVJXGZSA-N Phe-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O CSDMCMITJLKBAH-SOUVJXGZSA-N 0.000 description 1
- NAXPHWZXEXNDIW-JTQLQIEISA-N Phe-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 NAXPHWZXEXNDIW-JTQLQIEISA-N 0.000 description 1
- HBGFEEQFVBWYJQ-KBPBESRZSA-N Phe-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 HBGFEEQFVBWYJQ-KBPBESRZSA-N 0.000 description 1
- NAOVYENZCWFBDG-BZSNNMDCSA-N Phe-His-His Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CC=CC=C1 NAOVYENZCWFBDG-BZSNNMDCSA-N 0.000 description 1
- BEEVXUYVEHXWRQ-YESZJQIVSA-N Phe-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC3=CC=CC=C3)N)C(=O)O BEEVXUYVEHXWRQ-YESZJQIVSA-N 0.000 description 1
- YKUGPVXSDOOANW-KKUMJFAQSA-N Phe-Leu-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O YKUGPVXSDOOANW-KKUMJFAQSA-N 0.000 description 1
- YTILBRIUASDGBL-BZSNNMDCSA-N Phe-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 YTILBRIUASDGBL-BZSNNMDCSA-N 0.000 description 1
- INHMISZWLJZQGH-ULQDDVLXSA-N Phe-Leu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 INHMISZWLJZQGH-ULQDDVLXSA-N 0.000 description 1
- PEFJUUYFEGBXFA-BZSNNMDCSA-N Phe-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 PEFJUUYFEGBXFA-BZSNNMDCSA-N 0.000 description 1
- AXIOGMQCDYVTNY-ACRUOGEOSA-N Phe-Phe-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 AXIOGMQCDYVTNY-ACRUOGEOSA-N 0.000 description 1
- GRVMHFCZUIYNKQ-UFYCRDLUSA-N Phe-Phe-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O GRVMHFCZUIYNKQ-UFYCRDLUSA-N 0.000 description 1
- QARPMYDMYVLFMW-KKUMJFAQSA-N Phe-Pro-Glu Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=CC=C1 QARPMYDMYVLFMW-KKUMJFAQSA-N 0.000 description 1
- NJJBATPLUQHRBM-IHRRRGAJSA-N Phe-Pro-Ser Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CO)C(=O)O NJJBATPLUQHRBM-IHRRRGAJSA-N 0.000 description 1
- XOHJOMKCRLHGCY-UNQGMJICSA-N Phe-Pro-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O XOHJOMKCRLHGCY-UNQGMJICSA-N 0.000 description 1
- WEDZFLRYSIDIRX-IHRRRGAJSA-N Phe-Ser-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=CC=C1 WEDZFLRYSIDIRX-IHRRRGAJSA-N 0.000 description 1
- XDMMOISUAHXXFD-SRVKXCTJSA-N Phe-Ser-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O XDMMOISUAHXXFD-SRVKXCTJSA-N 0.000 description 1
- BPCLGWHVPVTTFM-QWRGUYRKSA-N Phe-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O BPCLGWHVPVTTFM-QWRGUYRKSA-N 0.000 description 1
- BSKMOCNNLNDIMU-CDMKHQONSA-N Phe-Thr-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O BSKMOCNNLNDIMU-CDMKHQONSA-N 0.000 description 1
- PTDAGKJHZBGDKD-OEAJRASXSA-N Phe-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O PTDAGKJHZBGDKD-OEAJRASXSA-N 0.000 description 1
- ZVJGAXNBBKPYOE-HKUYNNGSSA-N Phe-Trp-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(O)=O)C1=CC=CC=C1 ZVJGAXNBBKPYOE-HKUYNNGSSA-N 0.000 description 1
- AOKZOUGUMLBPSS-PMVMPFDFSA-N Phe-Trp-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(C)C)C(O)=O AOKZOUGUMLBPSS-PMVMPFDFSA-N 0.000 description 1
- WSAPMHXTQAOAQQ-BVSLBCMMSA-N Phe-Trp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC3=CC=CC=C3)N WSAPMHXTQAOAQQ-BVSLBCMMSA-N 0.000 description 1
- NHHZWPNMYQUNEH-ACRUOGEOSA-N Phe-Tyr-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)N NHHZWPNMYQUNEH-ACRUOGEOSA-N 0.000 description 1
- FRMKIPSIZSFTTE-HJOGWXRNSA-N Phe-Tyr-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O FRMKIPSIZSFTTE-HJOGWXRNSA-N 0.000 description 1
- GOUWCZRDTWTODO-YDHLFZDLSA-N Phe-Val-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O GOUWCZRDTWTODO-YDHLFZDLSA-N 0.000 description 1
- VIIRRNQMMIHYHQ-XHSDSOJGSA-N Phe-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N VIIRRNQMMIHYHQ-XHSDSOJGSA-N 0.000 description 1
- 101100124346 Photorhabdus laumondii subsp. laumondii (strain DSM 15139 / CIP 105565 / TT01) hisCD gene Proteins 0.000 description 1
- 108010003541 Platelet Activating Factor Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- FELJDCNGZFDUNR-WDSKDSINSA-N Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 FELJDCNGZFDUNR-WDSKDSINSA-N 0.000 description 1
- CQZNGNCAIXMAIQ-UBHSHLNASA-N Pro-Ala-Phe Chemical compound C[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O CQZNGNCAIXMAIQ-UBHSHLNASA-N 0.000 description 1
- OOLOTUZJUBOMAX-GUBZILKMSA-N Pro-Ala-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O OOLOTUZJUBOMAX-GUBZILKMSA-N 0.000 description 1
- OYEUSRAZOGIDBY-JYJNAYRXSA-N Pro-Arg-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OYEUSRAZOGIDBY-JYJNAYRXSA-N 0.000 description 1
- SWXSLPHTJVAWDF-VEVYYDQMSA-N Pro-Asn-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWXSLPHTJVAWDF-VEVYYDQMSA-N 0.000 description 1
- AHXPYZRZRMQOAU-QXEWZRGKSA-N Pro-Asn-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H]1CCCN1)C(O)=O AHXPYZRZRMQOAU-QXEWZRGKSA-N 0.000 description 1
- VJLJGKQAOQJXJG-CIUDSAMLSA-N Pro-Asp-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O VJLJGKQAOQJXJG-CIUDSAMLSA-N 0.000 description 1
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 1
- FKLSMYYLJHYPHH-UWVGGRQHSA-N Pro-Gly-Leu Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O FKLSMYYLJHYPHH-UWVGGRQHSA-N 0.000 description 1
- XYSXOCIWCPFOCG-IHRRRGAJSA-N Pro-Leu-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XYSXOCIWCPFOCG-IHRRRGAJSA-N 0.000 description 1
- SUENWIFTSTWUKD-AVGNSLFASA-N Pro-Leu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O SUENWIFTSTWUKD-AVGNSLFASA-N 0.000 description 1
- WFIVLLFYUZZWOD-RHYQMDGZSA-N Pro-Lys-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WFIVLLFYUZZWOD-RHYQMDGZSA-N 0.000 description 1
- MZNUJZBYRWXWLQ-AVGNSLFASA-N Pro-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@@H]2CCCN2 MZNUJZBYRWXWLQ-AVGNSLFASA-N 0.000 description 1
- BUEIYHBJHCDAMI-UFYCRDLUSA-N Pro-Phe-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O BUEIYHBJHCDAMI-UFYCRDLUSA-N 0.000 description 1
- XYAFCOJKICBRDU-JYJNAYRXSA-N Pro-Phe-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O XYAFCOJKICBRDU-JYJNAYRXSA-N 0.000 description 1
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 description 1
- BGWKULMLUIUPKY-BQBZGAKWSA-N Pro-Ser-Gly Chemical compound OC(=O)CNC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 BGWKULMLUIUPKY-BQBZGAKWSA-N 0.000 description 1
- QKDIHFHGHBYTKB-IHRRRGAJSA-N Pro-Ser-Phe Chemical compound N([C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C(=O)[C@@H]1CCCN1 QKDIHFHGHBYTKB-IHRRRGAJSA-N 0.000 description 1
- SNGZLPOXVRTNMB-LPEHRKFASA-N Pro-Ser-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N2CCC[C@@H]2C(=O)O SNGZLPOXVRTNMB-LPEHRKFASA-N 0.000 description 1
- CXGLFEOYCJFKPR-RCWTZXSCSA-N Pro-Thr-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O CXGLFEOYCJFKPR-RCWTZXSCSA-N 0.000 description 1
- VDHGTOHMHHQSKG-JYJNAYRXSA-N Pro-Val-Phe Chemical compound CC(C)[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O VDHGTOHMHHQSKG-JYJNAYRXSA-N 0.000 description 1
- 241000169446 Promethis Species 0.000 description 1
- 108010003201 RGH 0205 Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 101000912235 Rebecca salina Acyl-lipid (7-3)-desaturase Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- WTUJZHKANPDPIN-CIUDSAMLSA-N Ser-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N WTUJZHKANPDPIN-CIUDSAMLSA-N 0.000 description 1
- IDQFQFVEWMWRQQ-DLOVCJGASA-N Ser-Ala-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IDQFQFVEWMWRQQ-DLOVCJGASA-N 0.000 description 1
- VQBLHWSPVYYZTB-DCAQKATOSA-N Ser-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)N VQBLHWSPVYYZTB-DCAQKATOSA-N 0.000 description 1
- KAAPNMOKUUPKOE-SRVKXCTJSA-N Ser-Asn-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KAAPNMOKUUPKOE-SRVKXCTJSA-N 0.000 description 1
- BGOWRLSWJCVYAQ-CIUDSAMLSA-N Ser-Asp-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O BGOWRLSWJCVYAQ-CIUDSAMLSA-N 0.000 description 1
- DSGYZICNAMEJOC-AVGNSLFASA-N Ser-Glu-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O DSGYZICNAMEJOC-AVGNSLFASA-N 0.000 description 1
- WOUIMBGNEUWXQG-VKHMYHEASA-N Ser-Gly Chemical compound OC[C@H](N)C(=O)NCC(O)=O WOUIMBGNEUWXQG-VKHMYHEASA-N 0.000 description 1
- MOQDPPUMFSMYOM-KKUMJFAQSA-N Ser-His-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CO)N MOQDPPUMFSMYOM-KKUMJFAQSA-N 0.000 description 1
- CAOYHZOWXFFAIR-CIUDSAMLSA-N Ser-His-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O CAOYHZOWXFFAIR-CIUDSAMLSA-N 0.000 description 1
- WEQAYODCJHZSJZ-KKUMJFAQSA-N Ser-His-Tyr Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 WEQAYODCJHZSJZ-KKUMJFAQSA-N 0.000 description 1
- IAORETPTUDBBGV-CIUDSAMLSA-N Ser-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N IAORETPTUDBBGV-CIUDSAMLSA-N 0.000 description 1
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 1
- IXZHZUGGKLRHJD-DCAQKATOSA-N Ser-Leu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O IXZHZUGGKLRHJD-DCAQKATOSA-N 0.000 description 1
- GVMUJUPXFQFBBZ-GUBZILKMSA-N Ser-Lys-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GVMUJUPXFQFBBZ-GUBZILKMSA-N 0.000 description 1
- FPCGZYMRFFIYIH-CIUDSAMLSA-N Ser-Lys-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O FPCGZYMRFFIYIH-CIUDSAMLSA-N 0.000 description 1
- RXSWQCATLWVDLI-XGEHTFHBSA-N Ser-Met-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RXSWQCATLWVDLI-XGEHTFHBSA-N 0.000 description 1
- GDUZTEQRAOXYJS-SRVKXCTJSA-N Ser-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CO)N GDUZTEQRAOXYJS-SRVKXCTJSA-N 0.000 description 1
- MQUZANJDFOQOBX-SRVKXCTJSA-N Ser-Phe-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O MQUZANJDFOQOBX-SRVKXCTJSA-N 0.000 description 1
- BSXKBOUZDAZXHE-CIUDSAMLSA-N Ser-Pro-Glu Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O BSXKBOUZDAZXHE-CIUDSAMLSA-N 0.000 description 1
- OZPDGESCTGGNAD-CIUDSAMLSA-N Ser-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CO OZPDGESCTGGNAD-CIUDSAMLSA-N 0.000 description 1
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 1
- OJFFAQFRCVPHNN-JYBASQMISA-N Ser-Thr-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O OJFFAQFRCVPHNN-JYBASQMISA-N 0.000 description 1
- BDMWLJLPPUCLNV-XGEHTFHBSA-N Ser-Thr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O BDMWLJLPPUCLNV-XGEHTFHBSA-N 0.000 description 1
- PLQWGQUNUPMNOD-KKUMJFAQSA-N Ser-Tyr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O PLQWGQUNUPMNOD-KKUMJFAQSA-N 0.000 description 1
- HNDMFDBQXYZSRM-IHRRRGAJSA-N Ser-Val-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HNDMFDBQXYZSRM-IHRRRGAJSA-N 0.000 description 1
- SIEBDTCABMZCLF-XGEHTFHBSA-N Ser-Val-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SIEBDTCABMZCLF-XGEHTFHBSA-N 0.000 description 1
- 101000877236 Siganus canaliculatus Acyl-CoA Delta-4 desaturase Proteins 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- IGROJMCBGRFRGI-YTLHQDLWSA-N Thr-Ala-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O IGROJMCBGRFRGI-YTLHQDLWSA-N 0.000 description 1
- XYEXCEPTALHNEV-RCWTZXSCSA-N Thr-Arg-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O XYEXCEPTALHNEV-RCWTZXSCSA-N 0.000 description 1
- XVNZSJIKGJLQLH-RCWTZXSCSA-N Thr-Arg-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCSC)C(=O)O)N)O XVNZSJIKGJLQLH-RCWTZXSCSA-N 0.000 description 1
- ZUUDNCOCILSYAM-KKHAAJSZSA-N Thr-Asp-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZUUDNCOCILSYAM-KKHAAJSZSA-N 0.000 description 1
- SHOMROOOQBDGRL-JHEQGTHGSA-N Thr-Glu-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SHOMROOOQBDGRL-JHEQGTHGSA-N 0.000 description 1
- KCRQEJSKXAIULJ-FJXKBIBVSA-N Thr-Gly-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O KCRQEJSKXAIULJ-FJXKBIBVSA-N 0.000 description 1
- MPUMPERGHHJGRP-WEDXCCLWSA-N Thr-Gly-Lys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O MPUMPERGHHJGRP-WEDXCCLWSA-N 0.000 description 1
- DJDSEDOKJTZBAR-ZDLURKLDSA-N Thr-Gly-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O DJDSEDOKJTZBAR-ZDLURKLDSA-N 0.000 description 1
- PRNGXSILMXSWQQ-OEAJRASXSA-N Thr-Leu-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PRNGXSILMXSWQQ-OEAJRASXSA-N 0.000 description 1
- ZSPQUTWLWGWTPS-HJGDQZAQSA-N Thr-Lys-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZSPQUTWLWGWTPS-HJGDQZAQSA-N 0.000 description 1
- CGCMNOIQVAXYMA-UNQGMJICSA-N Thr-Met-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O CGCMNOIQVAXYMA-UNQGMJICSA-N 0.000 description 1
- VEIKMWOMUYMMMK-FCLVOEFKSA-N Thr-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 VEIKMWOMUYMMMK-FCLVOEFKSA-N 0.000 description 1
- MEBDIIKMUUNBSB-RPTUDFQQSA-N Thr-Phe-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MEBDIIKMUUNBSB-RPTUDFQQSA-N 0.000 description 1
- GFRIEEKFXOVPIR-RHYQMDGZSA-N Thr-Pro-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O GFRIEEKFXOVPIR-RHYQMDGZSA-N 0.000 description 1
- IQPWNQRRAJHOKV-KATARQTJSA-N Thr-Ser-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN IQPWNQRRAJHOKV-KATARQTJSA-N 0.000 description 1
- RVMNUBQWPVOUKH-HEIBUPTGSA-N Thr-Ser-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMNUBQWPVOUKH-HEIBUPTGSA-N 0.000 description 1
- UQCNIMDPYICBTR-KYNKHSRBSA-N Thr-Thr-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UQCNIMDPYICBTR-KYNKHSRBSA-N 0.000 description 1
- NLWDSYKZUPRMBJ-IEGACIPQSA-N Thr-Trp-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(C)C)C(=O)O)N)O NLWDSYKZUPRMBJ-IEGACIPQSA-N 0.000 description 1
- UMFLBPIPAJMNIM-LYARXQMPSA-N Thr-Trp-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O)N)O UMFLBPIPAJMNIM-LYARXQMPSA-N 0.000 description 1
- IJKNKFJZOJCKRR-GBALPHGKSA-N Thr-Trp-Ser Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 IJKNKFJZOJCKRR-GBALPHGKSA-N 0.000 description 1
- NJGMALCNYAMYCB-JRQIVUDYSA-N Thr-Tyr-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O NJGMALCNYAMYCB-JRQIVUDYSA-N 0.000 description 1
- XVHAUVJXBFGUPC-RPTUDFQQSA-N Thr-Tyr-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XVHAUVJXBFGUPC-RPTUDFQQSA-N 0.000 description 1
- YOPQYBJJNSIQGZ-JNPHEJMOSA-N Thr-Tyr-Tyr Chemical compound C([C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 YOPQYBJJNSIQGZ-JNPHEJMOSA-N 0.000 description 1
- CURFABYITJVKEW-QTKMDUPCSA-N Thr-Val-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O CURFABYITJVKEW-QTKMDUPCSA-N 0.000 description 1
- PWONLXBUSVIZPH-RHYQMDGZSA-N Thr-Val-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O PWONLXBUSVIZPH-RHYQMDGZSA-N 0.000 description 1
- SBYQHZCMVSPQCS-RCWTZXSCSA-N Thr-Val-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(O)=O SBYQHZCMVSPQCS-RCWTZXSCSA-N 0.000 description 1
- BTAJAOWZCWOHBU-HSHDSVGOSA-N Thr-Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)O)C(C)C)C(O)=O)=CNC2=C1 BTAJAOWZCWOHBU-HSHDSVGOSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- VZBWRZGNEPBRDE-HZUKXOBISA-N Trp-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N VZBWRZGNEPBRDE-HZUKXOBISA-N 0.000 description 1
- AVYVKJMBNLPWRX-WFBYXXMGSA-N Trp-Ala-Ser Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 AVYVKJMBNLPWRX-WFBYXXMGSA-N 0.000 description 1
- XKGZEDNYGPNJAR-XIRDDKMYSA-N Trp-Asn-His Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)N XKGZEDNYGPNJAR-XIRDDKMYSA-N 0.000 description 1
- BSSJIVIFAJKLEK-XIRDDKMYSA-N Trp-Cys-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N BSSJIVIFAJKLEK-XIRDDKMYSA-N 0.000 description 1
- SNJAPSVIPKUMCK-NWLDYVSISA-N Trp-Glu-Thr Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SNJAPSVIPKUMCK-NWLDYVSISA-N 0.000 description 1
- UYKREHOKELZSPB-JTQLQIEISA-N Trp-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(O)=O)=CNC2=C1 UYKREHOKELZSPB-JTQLQIEISA-N 0.000 description 1
- HQJOVVWAPQPYDS-ZFWWWQNUSA-N Trp-Gly-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQJOVVWAPQPYDS-ZFWWWQNUSA-N 0.000 description 1
- RPVDDQYNBOVWLR-HOCLYGCPSA-N Trp-Gly-Leu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O RPVDDQYNBOVWLR-HOCLYGCPSA-N 0.000 description 1
- KRCPXGSWDOGHAM-XIRDDKMYSA-N Trp-Lys-Asp Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O KRCPXGSWDOGHAM-XIRDDKMYSA-N 0.000 description 1
- XGFOXYJQBRTJPO-PJODQICGSA-N Trp-Pro-Ala Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O XGFOXYJQBRTJPO-PJODQICGSA-N 0.000 description 1
- ADMHZNPMMVKGJW-BPUTZDHNSA-N Trp-Ser-Arg Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N ADMHZNPMMVKGJW-BPUTZDHNSA-N 0.000 description 1
- VMXLNDRJXVAJFT-JYBASQMISA-N Trp-Thr-Ser Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O VMXLNDRJXVAJFT-JYBASQMISA-N 0.000 description 1
- SUGLEXVWEJOCGN-ONUFPDRFSA-N Trp-Thr-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC3=CNC4=CC=CC=C43)N)O SUGLEXVWEJOCGN-ONUFPDRFSA-N 0.000 description 1
- XXJDYWYVZBHELV-TUSQITKMSA-N Trp-Trp-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)N[C@@H](CCCCN)C(=O)O)N XXJDYWYVZBHELV-TUSQITKMSA-N 0.000 description 1
- TYYLDKGBCJGJGW-WMZOPIPTSA-N Trp-Tyr Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)N)C(O)=O)C1=CC=C(O)C=C1 TYYLDKGBCJGJGW-WMZOPIPTSA-N 0.000 description 1
- UIDJDMVRDUANDL-BVSLBCMMSA-N Trp-Tyr-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UIDJDMVRDUANDL-BVSLBCMMSA-N 0.000 description 1
- RKISDJMICOREEL-QRTARXTBSA-N Trp-Val-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N RKISDJMICOREEL-QRTARXTBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- TVOGEPLDNYTAHD-CQDKDKBSSA-N Tyr-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 TVOGEPLDNYTAHD-CQDKDKBSSA-N 0.000 description 1
- HTHCZRWCFXMENJ-KKUMJFAQSA-N Tyr-Arg-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HTHCZRWCFXMENJ-KKUMJFAQSA-N 0.000 description 1
- JRXKIVGWMMIIOF-YDHLFZDLSA-N Tyr-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=C(C=C1)O)N JRXKIVGWMMIIOF-YDHLFZDLSA-N 0.000 description 1
- PMDWYLVWHRTJIW-STQMWFEESA-N Tyr-Gly-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PMDWYLVWHRTJIW-STQMWFEESA-N 0.000 description 1
- GULIUBBXCYPDJU-CQDKDKBSSA-N Tyr-Leu-Ala Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CC1=CC=C(O)C=C1 GULIUBBXCYPDJU-CQDKDKBSSA-N 0.000 description 1
- MVFQLSPDMMFCMW-KKUMJFAQSA-N Tyr-Leu-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O MVFQLSPDMMFCMW-KKUMJFAQSA-N 0.000 description 1
- DMWNPLOERDAHSY-MEYUZBJRSA-N Tyr-Leu-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DMWNPLOERDAHSY-MEYUZBJRSA-N 0.000 description 1
- CDKZJGMPZHPAJC-ULQDDVLXSA-N Tyr-Leu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 CDKZJGMPZHPAJC-ULQDDVLXSA-N 0.000 description 1
- BBSPTGPYIPGTKH-JYJNAYRXSA-N Tyr-Met-Arg Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N BBSPTGPYIPGTKH-JYJNAYRXSA-N 0.000 description 1
- UPODKYBYUBTWSV-BZSNNMDCSA-N Tyr-Phe-Cys Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CS)C(O)=O)C1=CC=C(O)C=C1 UPODKYBYUBTWSV-BZSNNMDCSA-N 0.000 description 1
- VBFVQTPETKJCQW-RPTUDFQQSA-N Tyr-Phe-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VBFVQTPETKJCQW-RPTUDFQQSA-N 0.000 description 1
- XJPXTYLVMUZGNW-IHRRRGAJSA-N Tyr-Pro-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O XJPXTYLVMUZGNW-IHRRRGAJSA-N 0.000 description 1
- WQOHKVRQDLNDIL-YJRXYDGGSA-N Tyr-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O WQOHKVRQDLNDIL-YJRXYDGGSA-N 0.000 description 1
- OJCISMMNNUNNJA-BZSNNMDCSA-N Tyr-Tyr-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=C(O)C=C1 OJCISMMNNUNNJA-BZSNNMDCSA-N 0.000 description 1
- AGDDLOQMXUQPDY-BZSNNMDCSA-N Tyr-Tyr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O AGDDLOQMXUQPDY-BZSNNMDCSA-N 0.000 description 1
- RMRFSFXLFWWAJZ-HJOGWXRNSA-N Tyr-Tyr-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 RMRFSFXLFWWAJZ-HJOGWXRNSA-N 0.000 description 1
- UUJHRSTVQCFDPA-UFYCRDLUSA-N Tyr-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 UUJHRSTVQCFDPA-UFYCRDLUSA-N 0.000 description 1
- NWEGIYMHTZXVBP-JSGCOSHPSA-N Tyr-Val-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O NWEGIYMHTZXVBP-JSGCOSHPSA-N 0.000 description 1
- RUCNAYOMFXRIKJ-DCAQKATOSA-N Val-Ala-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RUCNAYOMFXRIKJ-DCAQKATOSA-N 0.000 description 1
- KKHRWGYHBZORMQ-NHCYSSNCSA-N Val-Arg-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKHRWGYHBZORMQ-NHCYSSNCSA-N 0.000 description 1
- IVXJODPZRWHCCR-JYJNAYRXSA-N Val-Arg-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N IVXJODPZRWHCCR-JYJNAYRXSA-N 0.000 description 1
- NWDOPHYLSORNEX-QXEWZRGKSA-N Val-Asn-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCSC)C(=O)O)N NWDOPHYLSORNEX-QXEWZRGKSA-N 0.000 description 1
- QGFPYRPIUXBYGR-YDHLFZDLSA-N Val-Asn-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N QGFPYRPIUXBYGR-YDHLFZDLSA-N 0.000 description 1
- OVLIFGQSBSNGHY-KKHAAJSZSA-N Val-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N)O OVLIFGQSBSNGHY-KKHAAJSZSA-N 0.000 description 1
- IRLYZKKNBFPQBW-XGEHTFHBSA-N Val-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N)O IRLYZKKNBFPQBW-XGEHTFHBSA-N 0.000 description 1
- VVZDBPBZHLQPPB-XVKPBYJWSA-N Val-Glu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VVZDBPBZHLQPPB-XVKPBYJWSA-N 0.000 description 1
- XWYUBUYQMOUFRQ-IFFSRLJSSA-N Val-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N)O XWYUBUYQMOUFRQ-IFFSRLJSSA-N 0.000 description 1
- SYOMXKPPFZRELL-ONGXEEELSA-N Val-Gly-Lys Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N SYOMXKPPFZRELL-ONGXEEELSA-N 0.000 description 1
- PTFPUAXGIKTVNN-ONGXEEELSA-N Val-His-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)NCC(=O)O)N PTFPUAXGIKTVNN-ONGXEEELSA-N 0.000 description 1
- ZIGZPYJXIWLQFC-QTKMDUPCSA-N Val-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C(C)C)N)O ZIGZPYJXIWLQFC-QTKMDUPCSA-N 0.000 description 1
- AGXGCFSECFQMKB-NHCYSSNCSA-N Val-Leu-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N AGXGCFSECFQMKB-NHCYSSNCSA-N 0.000 description 1
- UMPVMAYCLYMYGA-ONGXEEELSA-N Val-Leu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O UMPVMAYCLYMYGA-ONGXEEELSA-N 0.000 description 1
- AEMPCGRFEZTWIF-IHRRRGAJSA-N Val-Leu-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O AEMPCGRFEZTWIF-IHRRRGAJSA-N 0.000 description 1
- BTWMICVCQLKKNR-DCAQKATOSA-N Val-Leu-Ser Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C([O-])=O BTWMICVCQLKKNR-DCAQKATOSA-N 0.000 description 1
- YMTOEGGOCHVGEH-IHRRRGAJSA-N Val-Lys-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O YMTOEGGOCHVGEH-IHRRRGAJSA-N 0.000 description 1
- XPKCFQZDQGVJCX-RHYQMDGZSA-N Val-Lys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N)O XPKCFQZDQGVJCX-RHYQMDGZSA-N 0.000 description 1
- HJSLDXZAZGFPDK-ULQDDVLXSA-N Val-Phe-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C(C)C)N HJSLDXZAZGFPDK-ULQDDVLXSA-N 0.000 description 1
- ZEBRMWPTJNHXAJ-JYJNAYRXSA-N Val-Phe-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCSC)C(=O)O)N ZEBRMWPTJNHXAJ-JYJNAYRXSA-N 0.000 description 1
- KISFXYYRKKNLOP-IHRRRGAJSA-N Val-Phe-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)O)N KISFXYYRKKNLOP-IHRRRGAJSA-N 0.000 description 1
- MJOUSKQHAIARKI-JYJNAYRXSA-N Val-Phe-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=CC=C1 MJOUSKQHAIARKI-JYJNAYRXSA-N 0.000 description 1
- ZXYPHBKIZLAQTL-QXEWZRGKSA-N Val-Pro-Asp Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N ZXYPHBKIZLAQTL-QXEWZRGKSA-N 0.000 description 1
- NHXZRXLFOBFMDM-AVGNSLFASA-N Val-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C NHXZRXLFOBFMDM-AVGNSLFASA-N 0.000 description 1
- QIVPZSWBBHRNBA-JYJNAYRXSA-N Val-Pro-Phe Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccccc1)C(O)=O QIVPZSWBBHRNBA-JYJNAYRXSA-N 0.000 description 1
- QTPQHINADBYBNA-DCAQKATOSA-N Val-Ser-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN QTPQHINADBYBNA-DCAQKATOSA-N 0.000 description 1
- UJMCYJKPDFQLHX-XGEHTFHBSA-N Val-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N)O UJMCYJKPDFQLHX-XGEHTFHBSA-N 0.000 description 1
- SDHZOOIGIUEPDY-JYJNAYRXSA-N Val-Ser-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 SDHZOOIGIUEPDY-JYJNAYRXSA-N 0.000 description 1
- GVRKWABULJAONN-VQVTYTSYSA-N Val-Thr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GVRKWABULJAONN-VQVTYTSYSA-N 0.000 description 1
- LCHZBEUVGAVMKS-RHYQMDGZSA-N Val-Thr-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)[C@@H](C)O)C(O)=O LCHZBEUVGAVMKS-RHYQMDGZSA-N 0.000 description 1
- GVNLOVJNNDZUHS-RHYQMDGZSA-N Val-Thr-Lys Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(O)=O GVNLOVJNNDZUHS-RHYQMDGZSA-N 0.000 description 1
- RFZFBOQPPFCOKG-BZSNNMDCSA-N Val-Trp-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCSC)C(=O)O)N RFZFBOQPPFCOKG-BZSNNMDCSA-N 0.000 description 1
- VBTFUDNTMCHPII-UHFFFAOYSA-N Val-Trp-Tyr Natural products C=1NC2=CC=CC=C2C=1CC(NC(=O)C(N)C(C)C)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 VBTFUDNTMCHPII-UHFFFAOYSA-N 0.000 description 1
- MIAZWUMFUURQNP-YDHLFZDLSA-N Val-Tyr-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N MIAZWUMFUURQNP-YDHLFZDLSA-N 0.000 description 1
- LMVWCLDJNSBOEA-FKBYEOEOSA-N Val-Tyr-Trp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N LMVWCLDJNSBOEA-FKBYEOEOSA-N 0.000 description 1
- NLNCNKIVJPEFBC-DLOVCJGASA-N Val-Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O NLNCNKIVJPEFBC-DLOVCJGASA-N 0.000 description 1
- XNLUVJPMPAZHCY-JYJNAYRXSA-N Val-Val-Phe Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 XNLUVJPMPAZHCY-JYJNAYRXSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- 108010070783 alanyltyrosine Proteins 0.000 description 1
- 229940126575 aminoglycoside Drugs 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 229950011321 azaserine Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000004703 blastocyst inner cell mass Anatomy 0.000 description 1
- 210000001109 blastomere Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- VJYIFXVZLXQVHO-UHFFFAOYSA-N chlorsulfuron Chemical compound COC1=NC(C)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CC=2)Cl)=N1 VJYIFXVZLXQVHO-UHFFFAOYSA-N 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical class CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- 108010054813 diprotin B Proteins 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- IQLUYYHUNSSHIY-HZUMYPAESA-N eicosatetraenoic acid Chemical compound CCCCCCCCCCC\C=C\C=C\C=C\C=C\C(O)=O IQLUYYHUNSSHIY-HZUMYPAESA-N 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000001400 expression cloning Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 230000004129 fatty acid metabolism Effects 0.000 description 1
- 230000004136 fatty acid synthesis Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 102000045442 glycosyltransferase activity proteins Human genes 0.000 description 1
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 1
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 1
- 108010078326 glycyl-glycyl-valine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010020688 glycylhistidine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 101150113423 hisD gene Proteins 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 108010040030 histidinoalanine Proteins 0.000 description 1
- 108010045383 histidyl-glycyl-glutamic acid Proteins 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002535 isoprostanes Chemical class 0.000 description 1
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 1
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 1
- 108010091871 leucylmethionine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 108010012058 leucyltyrosine Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 150000002639 lipoxins Chemical class 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 108010068488 methionylphenylalanine Proteins 0.000 description 1
- 108010034507 methionyltryptophan Proteins 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000004939 midgestation embryo Anatomy 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000000472 morula Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 238000009400 out breeding Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 108010065135 phenylalanyl-phenylalanyl-phenylalanine Proteins 0.000 description 1
- 108010084572 phenylalanyl-valine Proteins 0.000 description 1
- 108010073101 phenylalanylleucine Proteins 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 108010015840 seryl-prolyl-lysyl-lysine Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000001608 teratocarcinoma Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- RZWIIPASKMUIAC-VQTJNVASSA-N thromboxane Chemical compound CCCCCCCC[C@H]1OCCC[C@@H]1CCCCCCC RZWIIPASKMUIAC-VQTJNVASSA-N 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 238000012250 transgenic expression Methods 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 101150081616 trpB gene Proteins 0.000 description 1
- 101150111232 trpB-1 gene Proteins 0.000 description 1
- 108010058119 tryptophyl-glycyl-glycine Proteins 0.000 description 1
- 108010029384 tryptophyl-histidine Proteins 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 108010005834 tyrosyl-alanyl-glycine Proteins 0.000 description 1
- 108010035534 tyrosyl-leucyl-alanine Proteins 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000004304 visual acuity Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
- A23L33/12—Fatty acids or derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/36—Carboxylic acids; Salts or anhydrides thereof
- A61K8/361—Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/67—Vitamins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9728—Fungi, e.g. yeasts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0083—Miscellaneous (1.14.99)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P7/6431—Linoleic acids [18:2[n-6]]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P7/6432—Eicosapentaenoic acids [EPA]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P7/6434—Docosahexenoic acids [DHA]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
- C12Y114/19006—DELTA12-fatty-acid desaturase (1.14.19.6), i.e. oleoyl-CoA DELTA12 desaturase
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
Definitions
- the invention generally relates to compositions and methods for the synthesis of essential fatty acids, their derivatives and downstream products, as well as altered levels of long-chain polyunsaturated fatty acids (LC-PUFAs) and eicosanoids in transfected cultured mammalian cells and in transgenic animals.
- LC-PUFAs long-chain polyunsaturated fatty acids
- Linoleic and ⁇ -linolenic acids and their derivatives are required by animals to maintain their normal physiological functions. They, as vitamins, must be taken in via the diet. To be fully useful, these fatty acids need to be metabolized. In mammalians, these two acids can be metabolized by common enzyme systems. They are first converted to ⁇ -linolenic acid (GLA, 18:3n6) and stearidonic acid (SDA, 18:4n3), respectively, by the action of delta 6-desaturase. They are then elongated by elongase to form dihomo- ⁇ -linolenic acid (DGLA, 20:3n6) and (n3) eicosatetraenoic acid
- LC-PUFAs long-chain polyunsaturated fatty acids
- DGLA, AA and EPA can also serve as precursors of 1, 2, and 3-series prostaglandin, thromboxanes and leukotriene biosynthesis, respectively, and these eicosanoids regulate a wide range of physiological functions. Adequate supply of these precursors is also vitally important for maintaining normal physiological activities.
- the invention generally relates to compositions and methods for the synthesis of essential fatty acids, their derivatives and downstream products, as well as altered levels of long-chain polyunsaturated fatty acids (LC-PUFAs) and eicosanoids in transfected cells and in transgenic animals.
- the present invention contemplates introducing nucleic acid encoding a desaturase gene and/or elongase gene into an animal cell under conditions such that said cells synthesize essential fatty acids or demonstrate altered levels of long-chain polyunsaturated fatty acids.
- the present invention contemplates introducing nucleic acid encoding a heterologous regulatory element in operable combination with either a heterologous or a homologous desaturase gene and/or elongase gene into an animal cell under conditions such that said cells display altered levels of long-chain polyunsaturated fatty acid synthesis and/or synthesize essential fatty acids.
- the present invention contemplates introducing nucleic acid encoding a non-mammalian desaturase gene into a mammalian cell under conditions such that said cells synthesize essential fatty acids and/or display altered levels of long- chain polyunsaturated fatty acids.
- the present invention contemplates vectors comprising nucleic acid encoding a desaturase gene, said vector capable of transfecting mammalian cells.
- the present invention contemplates mammalian cells (including cells in tissue culture and in bioreactors, and in transgenic animals), as a composition, which synthesize essential fatty acids (e.g., linoleic and/or linolenic fatty acids).
- the present invention contemplates introducing nucleic acid encoding a human desaturase gene (e.g., human ⁇ 5 desaturase cDNA) into a mammalian cell under conditions such that said cells synthesize essential fatty acids and/or display altered levels of long-chain polyunsaturated fatty acids.
- a human desaturase gene e.g., human ⁇ 5 desaturase cDNA
- the present invention contemplates vectors comprising nucleic acid encoding a human desaturase gene, said vector capable of transfecting mammalian cells.
- the present invention contemplates mammalian cells (including cells in tissue culture and in bioreactors, and in transgenic animals), as a composition, which are transfected with a human desaturase gene and which synthesize essential fatty acids (e.g., linoleic and/or linolenic fatty acids).
- mammalian cells including cells in tissue culture and in bioreactors, and in transgenic animals
- essential fatty acids e.g., linoleic and/or linolenic fatty acids
- the present invention contemplates introducing nucleic acid encoding an elongase gene (e.g., Mortierella Alpina elongase cDNA) into a mammalian cell under conditions such that said cells synthesize essential fatty acids and/or display altered levels of long-chain polyunsaturated fatty acids.
- an elongase gene e.g., Mortierella Alpina elongase cDNA
- vectors comprising nucleic acid encoding an elongase gene, said vector capable of transfecting mammalian cells.
- the present invention contemplates mammalian cells (including cells in tissue culture and in bioreactors, and in transgenic animals), as a composition, which are transfected with an elongase gene and which synthesize essential fatty acids (e.g., linoleic and/or linolenic fatty acids).
- mammalian cells including cells in tissue culture and in bioreactors, and in transgenic animals
- essential fatty acids e.g., linoleic and/or linolenic fatty acids
- the present invention contemplates introducing nucleic acid encoding both a desaturase gene and an elongase gene (e.g., human ⁇ 5 desaturase cDNA and Mortierella Alpina elongase cDNA) into a mammalian cell under conditions such that said cells synthesize essential fatty acids and/or display altered levels of long-chain polyunsaturated fatty acids (e.g., increased levels of 20:0, 22:0, and/or 24:0).
- a desaturase gene and an elongase gene e.g., human ⁇ 5 desaturase cDNA and Mortierella Alpina elongase cDNA
- the present invention contemplates vectors comprising nucleic acid encoding a desaturase gene and an elongase gene, said vector capable of transfecting mammalian cells.
- the present invention also contemplates two vectors, a first vector comprising nucleic acid encoding a desaturase and a second vector comprising nucleic acid encoding an elongase (as well as cells and animals transfected with both vectors).
- the present invention contemplates mammalian cells (including cells in tissue culture and in bioreactors, and in transgenic animals), as a composition, which are transfected with a desaturase gene and an elongase gene which synthesize essential fatty acids (e.g., linoleic and/or linolenic fatty acids).
- the present invention also contemplates human and non-human transgenic animals comprising heterologous desaturase genes, as well as human and non-human transgenic animals comprising heterologous regulatory elements in operable combination with either heterologous or homologous desaturase genes and/or elongase genes.
- the present invention contemplates vectors comprising a tissue-specific promoter in operable combination with nucleic acid encoding a non- mammalian desaturase gene or a non-mammalian elongase gene, said vector capable of transfecting cells of a non-human mammalian species.
- transfection of cells (or injection of fertilized eggs) with said vector comprising said tissue-specific promoter results in transgenic animals which produce altered levels of long chain polyunsaturated fatty acids or essential fatty acids in the animals milk.
- the present invention contemplates a method, comprising: a) providing: i) a non-human animal cell, ii) a vector comprising nucleic acid encoding a heterologous desaturase and/or elongase gene, and iii) a recipient non- human female animal; b) introducing said vector into said cell to create a transfected cell; c) transferring said transfected cell into said recipient female under conditions such that at least one offspring is produced, said offspring expressing said desaturase (and/or said elongase) in one or more tissues.
- the present invention contemplates a method, comprising: a) providing: i) a vector comprising a heterologous regulatory element in operable combination with a DNA sequence encoding a homologous desaturase and/or elongase gene, ii) a non-human animal cell and iii) a recipient non-human female animal; b) introducing said vector into said cell to create a transfected cell; c) transferring said transfected cell into said recipient female under conditions such that at least one offspring is produced, said offspring expressing said desaturase (and/or said elongase) in one or more tissues.
- said heterologous regulatory element comprises a tissue-specific promoter which directs expression in mammary tissue and said offspring expresses said desaturase (and/or elongase) in said offspring's mammary tissue, resulting altered levels of long-chain polyunsaturated fatty acids in the offspring's milk.
- the present invention contemplates a method, comprising: a) providing: i) a vector comprising a heterologous regulatory element in operable combination with a DNA sequence encoding a heterologous desaturase and/or elongase, ii) a non-human cell and iii) a recipient non-human female; b) introducing said vector into said cell to create a transfected cell; c) transferring said transfected cell into said recipient female under conditions such that at least one offspring is produced, said offspring expressing said desaturase (and/or elongase) in one or more tissues.
- said heterologous regulatory element comprises a tissue-specific promoter which directs expression in mammary tissue and said offspring expresses said desaturase (and/or elongase) in said offspring's mammary tissue, resulting in altered levels of long-chain polyunsaturated fatty acids in the offspring's milk.
- the present invention contemplates a method, comprising: a) providing: i) a cell to be transfected selected from the group consisting of non-human embryonic stem (ES) cells, an fertilized egg or a cell of an early embryo, ii) a vector comprising a tissue-specific promoter in operable combination with a DNA sequence encoding a desaturase and/or elongase, iii) a recipient non-human female; b) introducing said vector into said cell to create a transfected cell; c) transferring said transfected cell into said recipient female under conditions such that at least one offspring is produced, said offspring expressing said desaturase (and/or elongase) in one or more tissues.
- ES non-human embryonic stem
- said tissue-specific promoter directs expression in mammary tissue and said offspring expresses said desaturase (and/or elongase) in said offspring's mammary tissue, resulting in the secretion of long-chain polyunsaturated fatty acids in the offspring's milk.
- the present invention be limited to particular desaturase genes or particular elongase genes. A variety of such genes and sources of such genes are contemplated. With regard to particular preferred desaturase genes, the present invention contemplates genes for ⁇ 5 desaturase, ⁇ 6 desaturase, ⁇ 12 desaturase and
- sources of heterologous desaturase genes contemplates a variety of sources, including but not limited to plant and fungal sources.
- sources of elongase genes a number of elongase genes are known, including homologues of the Saccha omyces cerevisiae ELOl gene. See Oh et al. J. Biol. Chem 272:17376 (1997). Homologues of the Arabidopsis elongation gene (FAEI) are known as well. See Lassner et al. The Plant Cell 8:281 (1996).
- the present invention contemplates the use of such genes and/or homologues for transfecting cells and the generation of transgenics.
- the present invention contemplates a variety of formulations comprising such essential fatty acids, their derivatives and downstream products, including but not limited to feed formulations, nutritional formulations and cosmetic formulations.
- the present invention contemplates a nutritional formulation comprising at least one essential fatty acid produced by one of the above-named transgenic animals or transfected cells.
- the present invention be limited to the recovery of essential fatty acids, their derivatives and downstream products from a particular bodily fluid or tissue. While milk is a convenient source, other bodily fluids containing essential fatty acids, their derivatives and downsteam products are contemplated including, but not limited to, urine.
- a preferred tissue source comprises animal fat.
- the present invention also contemplates labelled essential fatty acids, derivatives and downstream products.
- the present invention contemplates an essential fatty acid produced by one of the above- named transgenic animals or transfected cells, said essential fatty acid comprising a reporter molecule.
- Suitable reporter molecules or labels include radiolabels, enzymes, fluorescent, chemiluminescent, or chromogenic agents.
- Such labelled fatty acids, derivatives and/or downstream products can be used diagnostically by introducing them to cells in culture (including but not limited to tumor cells).
- mice rats, rabbits, pigs, goats, sheep, cows and horses.
- Figure 1 is a schematic showing Fatty Acid metabolism in Mammalian Tissues.
- Figure 2 A-D schematically shows the construction of the eukaryotic desaturase expression vectors.
- Figure 2 A schematically shows the construction of the pCMV- ⁇ 6 -bGH plasmid.
- Figure 2 B schematically shows the construction of the pCMV- ⁇ 12 -bGH plasmid.
- Figure 2 C schematically shows the construction of the pWAP- ⁇ 6 -bGH plasmid.
- Figure 2 D schematically shows the construction of the pWAP- ⁇ 12 -bGH plasmid.
- Figure 3 is a bar graph depicting the %Linoleic acid (18:2n-6) levels in control and ⁇ 12-desaturase gene-transfected L cells.
- Figure 4 is a bar graph depicting the %Eicosadienic acid levels (20:2n-6) levels in control and ⁇ 12-desaturase gene-transfected L cells.
- Figure 5 is a bar graph depicting the Fatty acid profiles in control and ⁇ 6- desaturase gene-transfected L cells.
- Figure 6 depicts the nucleotide sequence of fungal ⁇ 5 desaturase (SEQ ID:1).
- Figure 7 depicts the amino acid sequence of fungal ⁇ 5 desaturase (SEQ ID:2).
- Figure 8 depicts the nucleotide sequence of fungal ⁇ 6 desaturase (SEQ ID:3).
- Figure 9 depicts the amino acid sequence of fungal ⁇ 6 desaturase (SEQ ID:4).
- Figure 10 depicts the nucleotide sequence of fungal ⁇ 12 desaturase (SEQ ID:4).
- Figure 11 depicts the amino acid sequence of fungal ⁇ 12 desaturase (SEQ ID:6).
- Figure 12 schematically depicts downstream products of arachidonic acid in the 5-lipoxygenase pathway.
- Figure 13 schematically depicts downstream products of arachidonic acid in the 12-lipoxygenase pathway.
- Figure 14 schematically depicts downstream products of arachidonic acid in the 15-lipoxygenase pathway.
- Figure 15 schematically depicts downstream products of arachidonic acid in the cytochrome P-450 pathway.
- Figure 16 schematically depicts downstream products of arachidonic acid in the cyclooxygenase pathway (pathway I).
- Figure 17 schematically depicts downstream products of arachidonic acid in the cyclooxygenase pathway (pathway II).
- Figure 18 shows the nucleic acid sequence for human ⁇ 5 desaturase cDNA.
- Figure 19 shows the amino acid sequence for the human ⁇ 5 desaturase.
- Figure 20 shows the map of plasmid pMTK-bGH-C.
- Figure 21 shows the plasmid resulting from the insertion of the human desaturase gene into plasmid pMTK-bGH-C.
- Figure 22 shows the nucleic acid sequence for Mortierella Alpina elongase cDNA.
- Figure 23 shows the amino acid sequence of the Mortierella Alpina elongase.
- Figure 24 shows the plasmid resulting from the insertion of the elongase gene into plasmid pMTK-pGH-C.
- LC-PUFAs Long-chain polyunsaturated fatty acids
- PG prostaglandins
- LT Leukotrienes
- GLA ⁇ -linolenic acid
- DGLA dihomo- ⁇ -linolenic acid
- AA Arachidonic Acid
- EPA Eicosapentaenoic acid
- DHA Docosahexaenoic acid
- SDA stearidonic acid
- LC-PUFA refers to fatty acids with chain lengths beyond 18-carbons that has two or more double bonds.
- a large number of such LC- PUFA can be derived from Linoleic Acid (18:2n-6) and ⁇ -Linolenic Acid (18:3n-3), the two so-called “essential fatty acids.”
- Nucleic acid sequence and “nucleotide sequence” as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or
- RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
- portion when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.
- recombinant DNA molecule refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological (i.e., non-naturally occurring) techniques (also known as genetic engineering).
- vector and “vehicle” are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
- expression vector or "expression cassette” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a host organism.
- Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
- Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
- in operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
- the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
- the terms “complementary” or “complementarity” are used in reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules.
- Complementarity can be “partial” or “total.”
- Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules.
- Total or “complete” complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
- nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity).
- a nucleotide sequence which is partially complementary, i.e., “substantially homologous,” to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
- a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
- the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
- Low stringency conditions comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 « H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400,
- low stringency conditions factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target ( DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
- conditions which promote hybridization under conditions of high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
- substantially homologous refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
- the term “substantially homologous” refers to any probe which can hybridize (i.e., it is the complement of) to the single-stranded nucleic acid sequence under conditions of low stringency as described above.
- hybridization is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the
- T m of the formed hybrid and the G:C ratio within the nucleic acids.
- hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
- the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
- a hybridization complex may be formed in solution (as determined, e.g., by C 0 t or R Q ?
- a solid support e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)].
- a solid support e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)].
- Stringent hybridization when used in reference to nucleic acid hybridization typically occurs in a range from about T m -5°C (5°C below the T m of the probe) to about 20°C to 25 °C below T m .
- T m is used in reference to the "melting temperature.”
- the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
- a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. Under “stringent conditions” a sequence or fragments thereof will hybridize to the sequence's exact complement and closely related sequences.
- DNA molecules are said to have "5' ends” and "3' ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring.
- an end of an oligonucleotide is referred to as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring.
- a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.
- discrete elements are referred to as being "upstream” or 5' of the "downstream” or 3' elements. This terminology reflects the fac1?that transcription proceeds in a 5' to 3' fashion along the DNA strand.
- the promoter and enhancer elements which direct transcription of a linked gene are generally located 5 ' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.
- an oligonucleotide having a nucleotide sequence encoding a gene means a nucleic acid sequence comprising the coding region of a gene, i. e. , the nucleic acid sequence which encodes a gene product.
- the coding region may be present in either a cDNA, genomic DNA or RNA form.
- the oligonucleotide may be single- stranded (i.e., the sense strand) or double-stranded.
- Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc.
- the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
- regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences.
- a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region.
- Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
- Transcriptional control signals in eukaryotes comprise "promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription
- Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest.
- promoter sequence refers to a single promoter sequence as well as to a plurality (i.e., one or more) of promoter sequences which are operably linked to each other and to at least one DNA sequence of interest.
- a double promoter sequence i.e., a DNA sequence containing two promoter sequences
- a triple promoter sequence i.e., a DNA sequence containing three promoter sequences
- Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site [Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp. 16.7-16.8].
- a commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40. Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript.
- Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.
- the term "poly A site” or "poly A sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript.
- the poly A signal utilized in an expression vector may be "heterologous" or "endogenous.”
- An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome.
- a heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene.
- cognate promoter and "cognate promoter of RNA polymerase” refer to a promoter sequence which is a naturally occurring promoter sequence in a gene encoding the RNA polymerase.
- the cognate promoter of T 7 RNA polymerase is the promoter which is derived from the gene encoding RNA polymerase in T 7 bacteriophage.
- the cognate promoter sequence may be cloned from the genome encoding the RNA polymerase.
- the location of a promoter may be identified by approaches and methods well known in the art, including DNase footprinting of the RNA polymerase-bound genome DNA, mutational analysis, etc.
- the promoter sequence may be synthesized.
- transfection refers to the introduction of a transgene into a cell.
- transgene refers to any nucleic acid sequence which is introduced into the genome of a cell by experimental manipulations.
- a transgene may be an "endogenous DNA sequence," or a “heterologous DNA sequence”
- heterologous DNA sequence refers to a nucleotide sequence which is not endogenous to the cell into which it is introduced.
- Heterologous DNA includes a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
- Heterologous DNA also includes a nucleotide sequence which is naturally found in the cell into which it is introduced and which contains some modification relative to the naturally-occurring sequence.
- Heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is introduced.
- An example of a heterologous DNA of the present invention comprises a nucleotide sequence which encodes a desaturase which is not found in the mammalian cell into which it is introduced.
- Another example of the present invention is a desaturase gene which is ligated to a promoter sequence to which it is not naturally ligated.
- Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.
- stable transfection or "stably transfected” refers to the introduction and integration of a transgene into the genome of the transfected cell.
- stable transfectant refers to a cell which has stably integrated one or more transgenes into the genomic DNA.
- transient transfection or “transiently transfected” refers to the introduction of one or more transgenes into a transfected cell in the absence of integration of the transgene into the host cell's genome.
- transient transfectant refers to a cell which has transiently integrated one or more transgenes.
- transgenic organism refers to an organism in which one or more cells has been transiently transfected or stably transfected with a transgene by experimental manipulation.
- Transgenic organisms may be produced by several methods including the introduction of a "transgene” comprising nucleic acid (usually DNA) into an embryonic target cell or a somatic target cell (such as cells of the mammary gland) of a non-human organism by way of human intervention.
- “Insertion” is used to refer to the process whereby a portion of heterologous DNA or a heterologous gene that is introduced into the genome of a host.
- the DNA which is inserted is referred to as an "insert”.
- the terms "transgenic mammal” or “transgenic host” are used to refer to a mammal or cell which has had a transgene inserted into its genome. As a result of this insertion, the transgenic host produces heterologous biological material that it would not normally synthesize. Heterologous entities are present or are produced by a transgenic host as a result of the insertion of foreign genetic material into the host cell genome.
- primary gene product refers to a biological entity which is formed directly as a result of the transcription and translation of a homologous or heterologous gene. Examples thereof include proteins, antibodies, enzymes and the like.
- secondary gene product refers to a product which is formed as a result of the biological activity of a primary gene product. An example thereof, is PUFAs which are formed as a result of the expression of specific desaturases.
- products or “biological products” refer to products produced or synthesized by a transgenic animal as a result of the insertion of a transgene into the genome of the animal. More specifically, the term means biological products which are secondary gene products or other downstream products altered by transgene expression.
- biological products which are secondary gene products or other downstream products altered by transgene expression.
- animal as used herein includes humans.
- the invention generally relates to compositions and methods of synthesis of essential fatty acids and their derivatives, long chain poly-unsaturated fatty acids and eicosanoids in transfected cells and in transgenic animals.
- the ability to produce long chain fatty acids derived from Linoleic Acid (18:2n-6) and ⁇ -Linolenic Acid (18:3n-3) in cultured cells and in transgenic animals has far reaching economic and scientific implications.
- Arachidonic Acid and ⁇ -Linolenic Acid (GLA) are important biologically active molecules in and of themselves.
- Eicosanoids such as Prostaglandins, Thromboxanes, Prostacyclins, and Leukotrienes, molecules that again have been shown to possess various biological activities.
- Eicosanoids such as Prostaglandins, Thromboxanes, Prostacyclins, and Leukotrienes
- EPA Eicosapentaenoic Acid
- DHA Docosahexaeonic Acid
- the Description of the invention involves: A) Selecting Transgenes and the Construction of Expression Vectors Comprising the Transgenes; B) Introduction of the
- the present invention contemplates introducing desaturase genes and/or elongase genes into mammalian cells. It is not intended that the present invention be limited to one source or one type of desaturase gene or elongase gene.
- a human desaturase is contemplated.
- nucleic acid encoding the human delta 5 desaturase can be introduced into a plasmid by standard techniques.
- elongase genes are also contemplated.
- Figure 22 provides the nucleic acid sequence for a representative elongase.
- elongases can be isolated using probes and/or primers designed based on regions of the nucleic acid sequence set forth in Figure 22.
- a probe having the sequence ACTACCTCAACTACCTGGTCAAGTACTGGGAGTTGGCCGA can be used to probe genomes of various organisms by hybridization under low and high stringency conditions to identify candidate elongases.
- primers can be designed using regions at each end (or near the end) of the sequence set forth in Figure 22 for PCR amplification of elongases from other organisms.
- elongases can be identified by expression cloning. This involves isolation of RNA and generation of cDNA, incorporation of the cDNA into an expression vector, followed by transfection and expression in a system that permits testing for elongase activity.
- the expression vector which contains the murine whey acidic protein transcriptional regulatory element is in operable combination with the nucleic acid sequences encoding an elongase and/or a desaturase (e.g., ⁇ 5 -desaturase, ⁇ 6- desaturase, ⁇ 12-desaturase or ⁇ 15-desaturase sequences of the invention).
- a desaturase e.g., ⁇ 5 -desaturase, ⁇ 6- desaturase, ⁇ 12-desaturase or ⁇ 15-desaturase sequences of the invention.
- the CMV promoter can be used.
- Host cells include bacterial, yeast, plant, insect, and mammalian cells. In a preferred embodiment the host cell is mammalian. In a more preferred embodiment, the host cell is a mouse cell.
- Any number of selection systems may be used to recover transfected cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22:817-23) genes which can be employed in tk “ or aprt " cells, respectively.
- antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate [Wigler M et al, (1980) Proc Natl Acad Sci 77:3567-70]; npt, which confers resistance to the aminoglycosides neomycin and G-418 [Colbere-Garapin F et al, (1981) J. Mol. Biol. 150:1-14] and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra).
- trpB which allows cells to utilize indole in place of tryptophan
- hisD which allows cells to utilize histinol in place of histidine
- reporter gene system which expresses visible markers has gained popularity with such markers as ⁇ -glucuronidase and its substrate (GUS), luciferase and its substrate (luciferin), and ⁇ -galactosidase and its substrate (X-Gal), and expression of green fluorescent protein (GFP), being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system [Rhodes CA et al (1995) Methods Mol Biol 55:121-131].
- GFP green fluorescent protein
- FISH Fluorescent In Situ Hybridization
- DNA or RNA can be isolated from cells for detection of the transgene by Southern or Northern hybridization or by amplification based assays.
- Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on sequence of the nucleic acid sequence of interest in order to detect cells and tissues which contain the DNA or RNA encoding the transgene of interest.
- oligonucleotides and “oligomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.
- Standard PCR methods useful in the present invention are described by Innis et al.
- heterologous nucleic acid sequences are by detecting the polypeptide product of transcription of the heterologous nucleotide sequence.
- a variety of protocols which employ polyclonal or monoclonal antibodies specific for the protein product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescent activated cell sorting
- a competitive binding assay may also be used.
- a two-site, monoclonal-based immunoassay which utilizes monoclonal antibodies that are reactive to two non-interfering epitopes on the protein of interest may be employed. These and other assays are described in, among other places, R. Hampton et al, Serological Methods a Laboratory Manual, APS Press, St Paul MN (1990) and D. E. Maddox et al, J. Exp. Med.
- Means for producing labeled hybridization or PCR probes for detecting related sequences include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
- the nucleic acid sequence of interest, or any portion of it may be cloned into a vector for the production of an mRNA probe.
- Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
- reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
- stably transfected L cells were generated using the calcium phosphate precipitation method as previously described [M. Wigler et al , "Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells," Cell 11:223-232 (1977); A. Pellicer et al, “Altering genotype and phenotype by DNA-mediated gene transfer," Science 209:1414-1422 (1980); B. Wold et al, "Introduction and expression of a rabbit ⁇ -globin gene in mouse fibroblasts," Proc. Natl. Acad. Sci. USA 76:5684-5688 (1979); J.M. Roberts and R. Axel, "Gene amplification and gene correction in somatic cells," Cell 29:109-119
- This method utilizes a plasmid DNA (pDLAT3) which encodes two selectable markers: an intact APRT gene and a truncated TK gene lacking transcriptional regulatory (enhancer) sequences.
- pDLAT3 plasmid DNA
- TK transcriptional regulatory
- a first step in the generation of the transgenic animals is the introduction of a construct (or constructs) containing the desired nucleic acid sequence (or sequences) into target cells.
- a construct or constructs
- Several methods are available for introducing the expression vector (or vectors) which contains the nucleic acid sequence of a desaturase and/or elongase into a target cell, including microinjection, retroviral infection, and implantation of embryonic stem cells. These methods are discussed as follows.
- the cell is implanted into the uterus of a pseudopregnant female and allowed to develop into an animal.
- 70% carry the expression vector sequence in all of their cells, including the germ cells.
- the remaining 30% of the transgenic animals are chimeric in somatic and germ cells because integration of the expression vector sequence occurs after one or more rounds of replication.
- Heterozygous and homozygous animals can then be produced by interbreeding founder transgenics. This method has been successful in producing transgenic mice, sheep, pigs, rabbits and cattle [Jaenisch (1988) supra; Hammer et al, (1986) J. Animal Sci. 63:269; Hammer et al., (1985) Nature 315:680-683; Wagner et al., (1984) Theriogenology 21:29].
- Retroviral infection of preimplantation embryos with genetically engineered retroviruses may also be used to introduce transgenes into an animal cell.
- blastomeres have been used as targets for retroviral infection [Jaenisch, (1976) Proc. Natl. Acad. Sci USA 73: 1260-1264].
- Transfection is typically achieved using a replication-defective retrovirus carrying the transgene [Jahner et al, (1985) Proc. Natl. Acad. Sci. USA 82:6927-6931 ; Van der Putten et al, (1985) Proc. Natl.
- Transfection is obtained, for example, by culturing eight-cell embryos, from which the zona pellucida has been removed with fibroblasts which produce the virus [Van der Putten (1985), supra; Stewart et al., (1987) EMBO J. 6:383-388]. The transfected embryos are then transferred to foster mothers for continued development. Alternatively, infection can be performed at a later stage.
- Virus or virus-producing cells can be injected into the blastocoele [Jahner et al, (1982) Nature 298:623-628].
- Yet another alternative method involves intrauterine retroviral infection of the midgestation embryos [Jahner et al. (1982), supra ⁇ .
- retroviral infection methods include the ease of transfection and the insertion of a single copy of the transgene, which is flanked by the retroviral long terminal repeats (LTRs), into the chromosome.
- LTRs retroviral long terminal repeats
- this method is not a preferred method because most of the founders will show mosaicism since infection occurs after cell division has begun. This necessitates outbreeding to establish homozygous and heterozygous lines suitable for analysis of gene expression.
- ES cells embryonic stem cells
- ES cells are pluripotent cells directly derived from the inner cell mass of blastocysts [Evans et al, (1981) Nature 292:154-156; Martin (1981) Proc. Natl. Acad Sci. USA 78:7634-7638;
- Expression vectors can be introduced into ES cells using any method which is suitable for gene transfer into cells, e.g., by transfection, cell fusion, electroporation, microinjection, DNA viruses, and RNA viruses [Johnson et al, (1989) Fetal Ther. 4 (Suppl. l):28-39].
- ES cells are the most pluripotent cultured animal cells known. For example, when ES cells are injected into an intact blastocyst cavity or under the zona pellucida, at the morula stage embryo, ES cells are capable of contributing to all somatic tissues including the germ line in the resulting chimeras. Once the expression vector has been introduced into an ES cell, the modified
- ES cell is then introduced back into the embryonic environment for expression and subsequent transmission to progeny animals.
- the most commonly used method is the injection of several ES cells into the blastocoel cavity of intact blastocysts [Bradley et al, (1984) Nature 309:225-256].
- a clump of ES cells may be sandwiched between two eight-cell embryos [Bradley et al, (1987) in
- the present invention provides methods for selectively expressing a nucleotide sequence of interest in a particular cell type and/or a particular tissue.
- the transfected animal cell is allowed to develop into a transgenic animal in which the nucleotide sequence of interest, e.g., the desaturase and/or elongase gene(s), is expressed selectively in a particular tissue such as the mammary glands.
- Particular expression vectors comprising particular desaturase sequences of the present invention, pWap- ⁇ 6-bGH and pWap- ⁇ 12-bGH, utilize the murine whey acidic protein transcriptional regulatory element to direct ⁇ 6 and ⁇ 12-desaturase transcription and the bGH polyadenylation signal for proper processing of the 3' terminus of desaturase mRNAs.
- these are under the control of the murine whey acidic protein transcriptional regulatory element, that directs gene expression primarily to the lactating mammary gland tissue.
- the selective expression of the gene of interest i.e., the desaturase and/or elongase transgenes
- expression of mRNA encoded by the gene of interest may be determined by using in situ hybridization. This involves synthesis of an RNA probe which is specific for a portion of or the entire gene of interest, e.g. , by using PCR.
- the PCR amplified fragment is subcloned into a plasmid (e.g., pBluescript (Stratagene)) and the RNA probe synthesized using labelled UTP (e.g., 35 S-UTP) and RNA polymerase (e.g., T3 or T7 polymerase (Promega)).
- UTP e.g., 35 S-UTP
- RNA polymerase e.g., T3 or T7 polymerase (Promega)
- Paraffin-embedded tissue sections are mounted on slides, deparaffmized, rehydrated and the protein digested (e.g. , with proteinase K), then dehydrated prior to hybridization with the RNA probe at the desired hybridization stringency. Slides are then developed for autoradiography using commercially available developers. Labelling of tissues and cells as detected on the autoradiographs indicates expression in those tissues and cells of the mRNA encoded by the gene of interest.
- expression of the protein product of the gene of interest may be determined using immunohistochemical techniques. Briefly, paraffin-embedded tissue sections are dewaxed, rehydrated, treated with a first antibody which is specific for the polypeptide product of the gene of interest. Binding is visualized, for example, by using a secondary biotinylated antibody which is specific for the constant region of the primary antibody, together with immunoperoxidase and 3,3'-amiobenzidine as a substrate. Sections may then be stained with hematoxylin to visualize the cellular histology. Antibody binding of tissues and cells which is detected by antibody binding demonstrates expression of the protein product of the gene of interest in these tissues and cells.
- the present invention provides alternate methods for altering or increasing the production of LC-PUFAs and derivative products by use of mammalian cells and transgenic animals into which has been inserted genes or cDNAs encoding desaturases.
- the present invention provides a method to generate mammalian cells in which desaturase and or elongase genes are expressed.
- the transfected cells exhibited enhanced production of both n-6 and n-3 LC-PUFAs, as they had obtained the ability to synthesize essential fatty acids or utilized the exogenous essentail fatty acids (EFA) as precursor for production of LC-PUFA.
- EFA exogenous essentail fatty acids
- the desaturase genes were introduced into animals, gene expression targeted specifically to the mammary glands, generating transgenic mice expressing delta-12, delta-6 or delta-5 genes.
- the milk derived from the transgenic mice contained a significantly higher level of LC-PUFA than that from the control mice.
- the present invention provides alternative sources of EFA and LC-PUFA in commonly used commodities, such as milk, infant formula, dietary supplement and pharmaceuticals.
- the methods of the present invention provide an alternate source for the generation of eicosanoids.
- Eicosanoids prostaglandins, leukotrienes, and lipoxins
- oxygenated lipids oxygenated lipids
- Platelet Activating Factor remain the focus of rational drug design targets given their established roles in cell-cell communication and as mediators in inflammation and pathophysiologic events. Identification of key enzymes in these pathways are implicating involvement of the nuclear membrane at the functional level.
- the present invention provides mammalian cells (including but not limited to Human HeLa cells, Mouse L cells) transfected with desaturase and/or elongase genes, that exhibits enhanced production of both n6 and n3 LC-PUFAs. While an understanding of a precise mechanism is not necessary to the successful use of the invention, it is believed that this is because the transfected cells have obtained the ability to synthesize essential fatty acids (EFA), or utilize the exogenous EFA as precursors for production of LC-PUFAs.
- EFA essential fatty acids
- the minimum number of genes required for increased LC-PUFA production varied between different cell lines, e.g., human Hela cells required only ⁇ 12 desaturase.
- the present invention contemplates the use of mammalian cells in bioreactors, for the large scale production of LC-PUFAs [see U.S. Patent No. 5,459,069 to Palsson et al. and U.S. Patent No. 5.563,068 to Zhang et al, both hereby incorporated by reference].
- Some bioreactors utilize hollow fiber systems.
- Microcarriers may also incorporate an ionic charge to assist in cell attachment to the microcarrier.
- the microcarriers are porous beads that are sufficiently large to allow cells to migrate and grow in the interior of the bead [see U.S. Patent No. 5,512,474 to Clapper et al.].
- the present invention provides the first, novel transgenic animals that express specific desaturase and/or elongase transgenes.
- a ⁇ 12-desaturase enzyme
- products of the omega 6 pathway that is GLA and Arachdonic Acid
- the omega three series of LC-PUFAs in particular EPA, DP A, and DHA were obtained in the transgenic animals. According to the information available in the current literature, these products are unexpected from ⁇ 12- desaturase gene expression.
- the desaturase transgenic animals provided in the present invention are a useful alternate source for the synthesis of LC-PUFAs and can be employed as animal models (including but not limited to models of human disease) in research, including but not limited to pharmaceutical research.
- the transgenic animals of the present invention can be used as Bioreactors for the large scale production of LC-PUFAs (See “Molecular Farming: Transgenic Animals as Bioreactors” by J. Van Brunt, Biotechnology, Volume 6, page 1149-1154, 1988, describes the alteration of the genome of various large domestic milk bearing animals yielding transgenic animals capable of producing various heterologous entities. This publication suggests methods for obtaining the primary gene product).
- the present invention further expands on the above protocols and teaches the use of transgenic animals for the production of biological products.
- the present invention has reduced to practice active expression of various desaturase and/or elongase genes (cDNA) in mammalian cells and transgenic animals and provides novel transgenic animals that express specific desaturase and/or elongase genes.
- cDNA desaturase and/or elongase genes
- the present invention provides transgenic animals expressing desaturases and/or elongases, that have not been described before.
- the PUFA derivatives obtained from these transgenic animals, such as the omega-3 products were not anticipated nor obvious.
- the transgenic animals of the present invention provides products in animal milk, that were completely unanticipated.
- the present invention has reduced to practice, active expression of various desaturase genes (cDNA) into cultured cells and these transformed cultured cells provides products that are not clearly predictable from the available prior art.
- cDNA desaturase genes
- the present invention provides animal cells which express ⁇ 12-desaturase and contains altered levels of linoleic acid, DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives.
- the invention provides a method of modifying levels of linoleic acid, DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives in animal cells (using ⁇ 12-desaturase alone).
- the milk produced in ⁇ 12-desaturase transgenic animals contains altered levels of linoleic acid, DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives.
- the milk produced in ⁇ 6-desaturase transgenic animals contains altered levels of DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives.
- the present invention provides a method of modifying levels of linoleic acid, DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives in transgenic animals (using ⁇ 12-desaturase alone).
- the present invention provides a method of modifying levels of DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives in transgenic animals (using ⁇ 6-desaturase alone).
- the present invention provides animal cells that express ⁇ 6-desaturase which contain altered levels of DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives (using ⁇ 6-desaturase alone).
- the present invention provides animal cells, mammals and milk with altered levels of molecules of the prostaglandin E series.
- the present invention provides mammals with altered levels of molecules of the prostaglandin I series, thromboxane series and leukotriene series.
- the present invention provides a method of altering the levels of molecules of prostaglandin I series, thromboxane series and leukotriene series in mammals.
- the present invention provides animal cells, mammals and transgenic animal milk with altered levels of essential fatty acids and omega-3 PUFAs when expressing both ⁇ 12-desaturase and ⁇ 15-desaturase ( ⁇ 15-desaturase converts linoleic acid to ⁇ -linolenic acid).
- the present invention provides a method of modifying levels of ⁇ -linolenic acid and omega-3 PUFAs when expressing both ⁇ 12-desaturase and ⁇ 15-desaturase.
- the present invention provides a method of producing animal cells or mammals which do not require external supply of essential fatty acids.
- Fat Free Media Growth of tissue culture cells in medium lacking essential fatty acids, especially linoleic acid. This allows individuals and companies to better define media used for maintaining cultured vertebrate cells. It may also improve the ability of cultured cells to produce recombinant proteins and /or make the process more economical.
- a pharmaceutical composition comprising the milk fat or animal fat, or fraction thereof.
- a nutritional formula comprising the milk fat or animal fat, or fraction thereof.
- the nutritional formula comprise of and include infant formula, dietary supplements and dietary substitutes and, where applicable, administratable to both humans and animals.
- Cosmetics comprising the milk fat or animal fat, or fraction thereof.
- Animal feeds comprising the milk fat or animal fat, or fraction thereof.
- DNA deoxyribonucleic acid
- cDNA complementary DNA
- RNA ribonucleic acid
- mRNA messenger ribonucleic acid
- PAGE polyacrylamide gel electrophoresis
- BAP 6-benzyl aminopurine
- Tris tris (hydroxymethyl) -aminomethane
- PBS phosphate buffered saline
- 2 X SSC 0.3 M NaCl, 0.03 M Na 3 citrate, pH 7.0
- Gibco BRL Gibco BRL (Gaithersburg, MD); Sigma (St. Louis, MO).
- Mouse L cells [thymidine kinase negative (TK " ) and adenine phosphoribosyltransferase negative (APRT " )] were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco Laboratories, Grand Island, NY) containing 10% Nu-serum (Collaborative Research Inc., Bedford, MA), 50 ug/ml gentamicin sulfate (Gibco) and 50 ug/ml 2,6-di-aminopurine (Sigma,
- Stably transfected L cells were generated using the calcium phosphate precipitation method as previously described [M. Wigler et al, "Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells," Cell 11:223-232 (1977); A. Pellicer et al, "Altering genotype and phenotype by DNA-mediated gene transfer,"
- TK + clones were isolated from the HAT + pool by limited dilution of the cells into 96-well plates at a concentration of 0.5 cells/well.
- TK + clones were analyzed for the presence of integrated desaturase sequences by slot blot hybridization analysis using a [ 32 P]radiolabeled DNA probe containing sequences from bGH exon V and 3' untranslated region.
- TK + clones containing integrated CMVie- ⁇ 6-bGH or pCMVie- ⁇ 12-bGH sequences were expanded and analyzed further.
- Stably transfected Hela cells were generated using the Lipofectamine Reagent method (BRL, Gaithersburg, MD) as suggested by the manufacturer. Briefly, 300 ng of plasmid DNA encoding the neomycin phosphotransferase gene driven by the RSV-LTR TRE [C. M. Gorman et al., "The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by
- DMEM fetal calf serum
- the above DNA/Lipofectamine solution was then added to the cells and incubated for 5 hr at 37°. Following incubation, the medium containing the DNA/Lipofectamine was removed and replaced with culture medium. After 24 hr, the cells were passed into culture medium containing 300 ug/ml G-418 sulfate (Geneticin, Gibco) to select for cells exhibiting neomycin resistance. The neomycin resistant Hela cell pool was then expanded and analyzed further.
- G-418 sulfate Geneticin, Gibco
- This example describes the construction of the eukaryotic desaturase expression vectors. DNA manipulations were carried out using standard cloning techniques. A 1,382 bp EcoR ⁇ l-XhoI DNA fragment encoding the ⁇ 6-desaturase gene was isolated from plasmid pCGR5 and ligated into plasmid pCMV-BGH-C [A. Martin-Gallardo et al, "A comparison of bGH expression in mouse L cells directed by the Moloney murine leukemia virus long terminal repeat, the simian virus 40 early or cytomegalovirus immediate early promotors," Gene 70:151-156 (1988)], which had been cleaved with BgHl and Smal.
- the termini of the DNA molecules were made flush using Klenow polymerase prior to ligation.
- the resulting plasmid, pCMVie- ⁇ 6-bGH utilizes the cytomegalovirus immediate early transcriptional regulatory element to direct ⁇ -6-desaturase transcription and the bGH polyadenylation signal for proper processing of the 3' terminus of desaturase mRNA (See Figure 2A).
- a 1,209 bp Eco l-Xhol DNA fragment encoding the ⁇ 12-desaturase gene was isolated from plasmid pCGR7 and ligated into plasmid pCMV-BGH-C which had been cleaved with Bg l and Smal to generate the plasmid, pCMVie- ⁇ 12-bGH (See Figure 2B). The termini of these DNA molecules were also made flush using Klenow polymerase prior to ligation.
- the DNA fragments encoding the ⁇ 6-desaturase gene and the ⁇ 12-desaturase gene were also ligated into plasmid, pWAP-polyA [Prieto et /.(1995)] which had been cleaved with Smal (See Figures 2C and 2D).
- the resulting plasmids, pWap- ⁇ 6-bGH and pWap- ⁇ 12-bGH utilize the murine whey acidic protein transcriptional regulatory element to direct ⁇ 6 and ⁇ 12-desaturase transcription and the bGH polyadenylation signal for proper processing of the 3' terminus of desaturase mRNAs.
- the murine whey acidic protein transcriptional regulatory element has been shown to direct gene expression primarily to lactating mammary gland tissue [C. W. Pittuis et al, Proc. Natl. Acad. Sci. U.S.A. 85:5874-5878 (1988)].
- This example describes the eneration of transgenic animals, expressing the desaturase genes.
- Plasmid pWap- ⁇ 6-bGH was cleaved with restriction endonucleases EcoRl and Pstl.
- a linear DNA fragment containing sequences encoding the WAP- ⁇ 6-bGH transcriptional unit was isolated and injected into fertilized mouse (B6/SJL) eggs as described previously [M.M. McGrane et al, J. Biol. Chem. 263:11443-11451 (1988)].
- the injected eggs were transferred to pseudopregnant females which subsequently delivered pups.
- High molecular weight chromosomal DNA was isolated from tail biopsies of the pups and was analyzed for the presence of integrated transgene sequences by slot blot hybridization analysis using a [ 32 P]radiolabeled DNA probe containing sequences from bGH exon V and 3' untranslated region.
- plasmid pWap- ⁇ 12-bGH was cleaved with restriction endonucleases EcoRl and BamHl.
- a linear DNA fragment containing sequences encoding the WAP- ⁇ 12-bGH transcriptional unit was isolated, injected and transgenic animals identified. Control, ⁇ 6 and ⁇ 12 females were mated and allowed to deliver ⁇ , progeny. Milk samples were obtained from control and transgenic mothers 5-12 days postpartum and then analyzed for desaturase activity.
- linoleic acid (18:2n-6) comprised approximately 3.25%o of total fatty acid in control cells. The percentage of this fatty acid in control cells decreased from 3.25 to 1.77 and 1.13 when the cells were incubated in serum-free medium for 1 and 3 days, respectively.
- ⁇ 12 cells had significantly elevated levels of linoleic acid.
- linoleic acid comprised approximately 19.99% of total fatty acid in ⁇ 12 cells. This represented a 515% increase in ⁇ 12 cells compared to control cells. The levels of linoleic acid increased to 21.52 and 24.04% when the cells were incubated cells in serum-free medium for 1 and 3 days, respectively (See Figure 3).
- the ⁇ 12-Hela cells had slightly elevated levels of the ⁇ 12-desaturase product, linoleic acid (18:2n-6) compared to control Hela cells. However, the levels of both arachidonic acid (20:4n-6) and adrenic acid (22:4n-6) were significantly elevated in the ⁇ 12-Hela cells. Similarly, the levels of both docosapentaenoic acid (22:5n-3) and docosahexaeonic acid (22:6n-3) were elevated in the omega-3 pathway of ⁇ 12-Hela cells compared to control Hela cells.
- All L cell clones containing stably integrated ⁇ 6-desaturase sequences contained significantly elevated levels of di-homo- ⁇ -linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) compared to control L cells, with the mean increases being 133%) and 399%, respectively.
- the levels of these fatty acids in the ⁇ 6-desaturase clones were also elevated compared to L cells expressing the
- the ⁇ -linolenic acid (18:3n-6) was then converted to di-homo ⁇ -linolenic acid (20:3n-6) by elongase whereas the steridonic acid (18:4n-3) was converted to eicosatrienoic acid (20:4n-3) by elongase and further desaturated to eicosapentaenoic acid (20:5n-3) and docosahexaeonic acid (22:6n-3) by the endogenous ⁇ 5 and ⁇ 4-desaturases.
- EXAMPLE 4 EXAMPLE 4
- mice In this example, experiments have been described that show expression of ⁇ 6 and ⁇ 12-desaturase transgenes in mice.
- mice containing either the ⁇ 6 and ⁇ 12-desaturase transgene were generated as described previously in the Methodology. The expression these transgenes is directed by the murine whey acidic protein promoter and expected to be restricted mainly to the lactating mammary gland. The females were mated and milk was collected from each lactating mother 5-12 days post partum. The milk was analyzed for ⁇ 6 and ⁇ 12-desaturase activity by determining the levels of various omega-6 and omega-3 fatty acids present within the milk, as a percentage of total fatty acid, compared to those found in control mouse milk. The results are summarized in Table 5.
- the transgenic line, ⁇ 12 F 0 76 demonstrated the largest increases in ⁇ -linolenic acid (18:3n-6), di-homo ⁇ -linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) compared to control milk (0.18, 0.80 and 0.72% compared to 0.07, 0.49 and 0.34%, respectively). Milk from this animal also demonstrated significant elevations in several omega-3 fatty acids.
- This example describes the construction of a vector comprising nucleic acid encoding a human desaturase. Briefly a 1335-bp Ncol/Hind III DNA fragment encoding the human ⁇ 5-desaturase CDNA ( Figure 18) was isolated from plasmid pRAE-28-5 and ligated into plasmid pMTK-bGH-C ( Figure 20), which had been cleaved with BgHland Pvu ⁇ l. The termini of the DNA molecules were made flush using Klenow polymerase prior to ligation.
- the resulting plasmid, pMET- h ⁇ 5-desaturase-bGHpA ( Figure 21), utilizes the mMT-I TRE to direct h ⁇ 5 -desaturase transcription and the bGH polyadenylation signal for proper processing of the 3' terminus of the h ⁇ 5 mRNA.
- This example describes the construction of a vector comprising nucleic acid encoding an elongase.
- a 1425-bp EcoRl DNA fragment encoding the M. alpina elongase cDNA ( Figure 22) was isolated from plasmid pRAE-2 and ligated into plasmid pMTK-bGH-C ( Figure 20), which had been previously cleaved with Bglll and Pvwll. The termini of the DNA molecules were made flush using Klenow polymerase prior to ligation.
- the resulting plasmid, pMET-Elongase-bGHpA ( Figure 24), utilizes the mouse metallothionein-I transcriptional regulatory element (mMT-I TRE) to direct elongase transcription and the bGH polyadenylation signal for proper processing of the 3' terminus of the elongase rnRNA.
- mMT-I TRE mouse metallothionein-I transcriptional regulatory element
- This example describes the transfection of cells with the vectors of Examples 5 and 6 and the resulting alteration in FA synthesis.
- Alpina elongase (h ⁇ 5 plus elongase) were obtained. Since L cells expressing h ⁇ 5 -desaturase and elongase would be expected to still require essential fatty acids, control (L), h ⁇ 5 and the h ⁇ 5 plus elongase cells were isolated from growing cultures (serum-containing culture medium).
- the cells were analyzed for h ⁇ 5-desaturase and elongase activity by determining the levels of various saturated fatty acids as well as various omega-6 and omega-3 polyunsaturated fatty acids within the cells as a percentage of total fatty acid. The results are summarized in Table 6.
- values given for h ⁇ 5-desaturase represent the mean values of the two mouse L cell clones while those for h ⁇ 5 plus elongase represent the mean values of the three mouse L cell clones.
- the predicted substrates of human ⁇ 5-desaturase activity are 20:3n-6 and 20-4n3. This does not rule out the possibility of, as to yet, unidentified substrates of the enzyme.
- Both mouse L cell clones containing stably integrated h ⁇ 5 sequences contained significantly elevated levels of arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) compared to control L cells, with the mean increases being 254% and 68%), respectively.
- the h ⁇ 5 clones also contained elevated levels of other downstream omega-6 and omega-3 PUFAs.
- the level of 20:3n-6 is similar between control and h ⁇ 5 cells while each cell type contains undetectable levels of GLA.
- Human ⁇ 5-desaturase activity is demonstrated by the 152% and 137% increase in the levels of 20:4n-6and 22:4n-6, respectively.
- Addition of GLA to the serum-free culture medium results in the detectable accumulation of GLA within both control and h ⁇ 5 cells.
- the level of 20:3n-6 is elevated in both cell types confirming an active endogenous elongase. Human h ⁇ 5 -desaturase activity is demonstrated by the 53%) reduction in the level of 20:3n-6 and concomminant increases in the levels of
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Birds (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Animal Husbandry (AREA)
- Emergency Medicine (AREA)
- Nutrition Science (AREA)
- Dermatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention generally relates to synthesis of essential fatty acids and their derivatives, long-chain polyunsaturated fatty acids (LC-PUFAs) and eicosanoids in transfected cells and in transgenic animals. Cells are transfected with genes encoding a desaturase and/or elongase under conditions such that the transfected cells produce altered levels of long-chain polyunsaturated fatty acids relative to the untransfected cells.
Description
MAMMALIAN CELLS EXPRESSING DESATURASES AND ELONGASES
FIELD OF THE INVENTION
The invention generally relates to compositions and methods for the synthesis of essential fatty acids, their derivatives and downstream products, as well as altered levels of long-chain polyunsaturated fatty acids (LC-PUFAs) and eicosanoids in transfected cultured mammalian cells and in transgenic animals.
BACKGROUND OF THE INVENTION
Both animal and plants have the ability to synthesize fatty acids with chain lengths up to 18-carbons and to desaturate fatty acids at the 9 position. However, during the course of evolution, animals have lost the ability to insert double bonds into fatty acids beyond the 9 position, for example, to insert double bonds into 12 and 15 positions. As a result, animals cannot convert oleic acid (18:ln9) to linoleic acid (18:2n6) and linoleic acid to α-linolenic acid (18:3n3).
Linoleic and α-linolenic acids and their derivatives are required by animals to maintain their normal physiological functions. They, as vitamins, must be taken in via the diet. To be fully useful, these fatty acids need to be metabolized. In mammalians, these two acids can be metabolized by common enzyme systems. They are first converted to γ-linolenic acid (GLA, 18:3n6) and stearidonic acid (SDA, 18:4n3), respectively, by the action of delta 6-desaturase. They are then elongated by elongase to form dihomo-γ-linolenic acid (DGLA, 20:3n6) and (n3) eicosatetraenoic acid
(20:4n3), respectively, and further metabolized by delta 5-desaturase to form arachidonic acid (AA, 20:4n6) and eicosapentaenoic acid (EPA, 20:5n3), respectively. Both AA and EPA can be further metabolized to other long-chain polyunsaturated fatty acids (LC-PUFAs) [See Figure 1]. LC-PUFAs are major constituents in cell membranes. DGLA, AA and EPA can also serve as precursors of 1, 2, and 3-series prostaglandin, thromboxanes and leukotriene biosynthesis, respectively, and these eicosanoids regulate a wide range of physiological functions. Adequate supply of
these precursors is also vitally important for maintaining normal physiological activities.
Since 6-desaturation is considered to be the rate-limiting step in the synthesis of LC-PUFAs, the essential ability of animals to convert these acids to LC-PUFA is paramount. The need of LC-PUFAs is further enhanced during the period of rapid growth. There are reports that infants whose diets are not provided with LC-PUFAs, particularly AA and docosahexaenoic acid (DHA, 22:6n3) (which are derivatives of linolenic acid) display significant differences both in biochemical parameters and also in functional properties, such as visual acuity and psychomotor tests in comparison with breast-fed infants. Normally, an infant receives these acids with the mother's milk, because human milk contains both n6 and n3 LC-PUFAs. Most of infant formulas on the market do not contain LC-PUFAs. Attempts to simulate the fat mixture in human milk on the basis of animal, vegetable and microbial oils or fats has often been encountered with the very costly raw materials, which in many cases are still not available in sufficient quantity to meet the need (See U.S.Patent No. 5,709,888 incorporated herein by reference). Hence, what is required are alternative sources of EFA and LC-PUFA, that are commonly used in commodities, such as milk, infant formula, dietary supplement and pharmaceuticals.
SUMMARY OF THE INVENTION The invention generally relates to compositions and methods for the synthesis of essential fatty acids, their derivatives and downstream products, as well as altered levels of long-chain polyunsaturated fatty acids (LC-PUFAs) and eicosanoids in transfected cells and in transgenic animals. In one embodiment, the present invention contemplates introducing nucleic acid encoding a desaturase gene and/or elongase gene into an animal cell under conditions such that said cells synthesize essential fatty acids or demonstrate altered levels of long-chain polyunsaturated fatty acids. In another embodiment, the present invention contemplates introducing nucleic acid encoding a heterologous regulatory element in operable combination with either a heterologous or a homologous desaturase gene and/or elongase gene into an animal cell under
conditions such that said cells display altered levels of long-chain polyunsaturated fatty acid synthesis and/or synthesize essential fatty acids.
In one embodiment, the present invention contemplates introducing nucleic acid encoding a non-mammalian desaturase gene into a mammalian cell under conditions such that said cells synthesize essential fatty acids and/or display altered levels of long- chain polyunsaturated fatty acids. Thus, the present invention contemplates vectors comprising nucleic acid encoding a desaturase gene, said vector capable of transfecting mammalian cells. Moreover, the present invention contemplates mammalian cells (including cells in tissue culture and in bioreactors, and in transgenic animals), as a composition, which synthesize essential fatty acids (e.g., linoleic and/or linolenic fatty acids).
In another embodiment, the present invention contemplates introducing nucleic acid encoding a human desaturase gene (e.g., human Δ5 desaturase cDNA) into a mammalian cell under conditions such that said cells synthesize essential fatty acids and/or display altered levels of long-chain polyunsaturated fatty acids. Thus, the present invention contemplates vectors comprising nucleic acid encoding a human desaturase gene, said vector capable of transfecting mammalian cells. Moreover, the present invention contemplates mammalian cells (including cells in tissue culture and in bioreactors, and in transgenic animals), as a composition, which are transfected with a human desaturase gene and which synthesize essential fatty acids (e.g., linoleic and/or linolenic fatty acids).
In yet another embodiment, the present invention contemplates introducing nucleic acid encoding an elongase gene (e.g., Mortierella Alpina elongase cDNA) into a mammalian cell under conditions such that said cells synthesize essential fatty acids and/or display altered levels of long-chain polyunsaturated fatty acids. Thus, the present invention contemplates vectors comprising nucleic acid encoding an elongase gene, said vector capable of transfecting mammalian cells. Moreover, the present invention contemplates mammalian cells (including cells in tissue culture and in bioreactors, and in transgenic animals), as a composition, which are transfected with an
elongase gene and which synthesize essential fatty acids (e.g., linoleic and/or linolenic fatty acids).
In still another embodiment, the present invention contemplates introducing nucleic acid encoding both a desaturase gene and an elongase gene (e.g., human Δ5 desaturase cDNA and Mortierella Alpina elongase cDNA) into a mammalian cell under conditions such that said cells synthesize essential fatty acids and/or display altered levels of long-chain polyunsaturated fatty acids (e.g., increased levels of 20:0, 22:0, and/or 24:0). Thus, the present invention contemplates vectors comprising nucleic acid encoding a desaturase gene and an elongase gene, said vector capable of transfecting mammalian cells. The present invention also contemplates two vectors, a first vector comprising nucleic acid encoding a desaturase and a second vector comprising nucleic acid encoding an elongase (as well as cells and animals transfected with both vectors). Moreover, the present invention contemplates mammalian cells (including cells in tissue culture and in bioreactors, and in transgenic animals), as a composition, which are transfected with a desaturase gene and an elongase gene which synthesize essential fatty acids (e.g., linoleic and/or linolenic fatty acids).
The present invention also contemplates human and non-human transgenic animals comprising heterologous desaturase genes, as well as human and non-human transgenic animals comprising heterologous regulatory elements in operable combination with either heterologous or homologous desaturase genes and/or elongase genes. In particular, the present invention contemplates vectors comprising a tissue- specific promoter in operable combination with nucleic acid encoding a non- mammalian desaturase gene or a non-mammalian elongase gene, said vector capable of transfecting cells of a non-human mammalian species. In a preferred embodiment, transfection of cells (or injection of fertilized eggs) with said vector comprising said tissue-specific promoter results in transgenic animals which produce altered levels of long chain polyunsaturated fatty acids or essential fatty acids in the animals milk.
In one embodiment, the present invention contemplates a method, comprising: a) providing: i) a non-human animal cell, ii) a vector comprising nucleic acid
encoding a heterologous desaturase and/or elongase gene, and iii) a recipient non- human female animal; b) introducing said vector into said cell to create a transfected cell; c) transferring said transfected cell into said recipient female under conditions such that at least one offspring is produced, said offspring expressing said desaturase (and/or said elongase) in one or more tissues.
In another embodiment, the present invention contemplates a method, comprising: a) providing: i) a vector comprising a heterologous regulatory element in operable combination with a DNA sequence encoding a homologous desaturase and/or elongase gene, ii) a non-human animal cell and iii) a recipient non-human female animal; b) introducing said vector into said cell to create a transfected cell; c) transferring said transfected cell into said recipient female under conditions such that at least one offspring is produced, said offspring expressing said desaturase (and/or said elongase) in one or more tissues. In a preferred embodiment, said heterologous regulatory element comprises a tissue-specific promoter which directs expression in mammary tissue and said offspring expresses said desaturase (and/or elongase) in said offspring's mammary tissue, resulting altered levels of long-chain polyunsaturated fatty acids in the offspring's milk.
In one embodiment, the present invention contemplates a method, comprising: a) providing: i) a vector comprising a heterologous regulatory element in operable combination with a DNA sequence encoding a heterologous desaturase and/or elongase, ii) a non-human cell and iii) a recipient non-human female; b) introducing said vector into said cell to create a transfected cell; c) transferring said transfected cell into said recipient female under conditions such that at least one offspring is produced, said offspring expressing said desaturase (and/or elongase) in one or more tissues. In a preferred embodiment, said heterologous regulatory element comprises a tissue-specific promoter which directs expression in mammary tissue and said offspring expresses said desaturase (and/or elongase) in said offspring's mammary tissue, resulting in altered levels of long-chain polyunsaturated fatty acids in the offspring's milk.
In yet another embodiment, the present invention contemplates a method, comprising: a) providing: i) a cell to be transfected selected from the group
consisting of non-human embryonic stem (ES) cells, an fertilized egg or a cell of an early embryo, ii) a vector comprising a tissue-specific promoter in operable combination with a DNA sequence encoding a desaturase and/or elongase, iii) a recipient non-human female; b) introducing said vector into said cell to create a transfected cell; c) transferring said transfected cell into said recipient female under conditions such that at least one offspring is produced, said offspring expressing said desaturase (and/or elongase) in one or more tissues. In a preferred embodiment, said tissue-specific promoter directs expression in mammary tissue and said offspring expresses said desaturase (and/or elongase) in said offspring's mammary tissue, resulting in the secretion of long-chain polyunsaturated fatty acids in the offspring's milk.
It is not intended that the present invention be limited to particular desaturase genes or particular elongase genes. A variety of such genes and sources of such genes are contemplated. With regard to particular preferred desaturase genes, the present invention contemplates genes for Δ5 desaturase, Δ6 desaturase, Δ12 desaturase and
Δ15 desaturase. With regard to sources of heterologous desaturase genes, the present invention contemplates a variety of sources, including but not limited to plant and fungal sources. With regard to sources of elongase genes, a number of elongase genes are known, including homologues of the Saccha omyces cerevisiae ELOl gene. See Oh et al. J. Biol. Chem 272:17376 (1997). Homologues of the Arabidopsis elongation gene (FAEI) are known as well. See Lassner et al. The Plant Cell 8:281 (1996). The present invention contemplates the use of such genes and/or homologues for transfecting cells and the generation of transgenics.
It is not intended that the present invention be limited to the uses for the essential fatty acids and LC-PUFAs generated by animal cells or transgenic animals which comprise the above-named heterologous desaturase genes. The present invention contemplates a variety of formulations comprising such essential fatty acids, their derivatives and downstream products, including but not limited to feed formulations, nutritional formulations and cosmetic formulations. Thus, for example, in one embodiment, the present invention contemplates a nutritional formulation
comprising at least one essential fatty acid produced by one of the above-named transgenic animals or transfected cells.
It is not intended that, in making the above-named formulations, the present invention be limited to the recovery of essential fatty acids, their derivatives and downstream products from a particular bodily fluid or tissue. While milk is a convenient source, other bodily fluids containing essential fatty acids, their derivatives and downsteam products are contemplated including, but not limited to, urine. A preferred tissue source comprises animal fat.
The present invention also contemplates labelled essential fatty acids, derivatives and downstream products. Thus, for example, in one embodiment, the present invention contemplates an essential fatty acid produced by one of the above- named transgenic animals or transfected cells, said essential fatty acid comprising a reporter molecule. Suitable reporter molecules or labels include radiolabels, enzymes, fluorescent, chemiluminescent, or chromogenic agents. Such labelled fatty acids, derivatives and/or downstream products can be used diagnostically by introducing them to cells in culture (including but not limited to tumor cells).
It is not intended that the present invention be limted to particular non-human animals. Animals of all types are contemplated, including but not limited to insects, nematodes, fish, birds and mammals. A variety of preferred animals are contemplated, including but not limited to mice, rats, rabbits, pigs, goats, sheep, cows and horses.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a schematic showing Fatty Acid metabolism in Mammalian Tissues.
Figure 2 A-D schematically shows the construction of the eukaryotic desaturase expression vectors. Figure 2 A schematically shows the construction of the pCMV-Δ6 -bGH plasmid.
Figure 2 B schematically shows the construction of the pCMV-Δ12 -bGH plasmid.
Figure 2 C schematically shows the construction of the pWAP-Δ6 -bGH plasmid.
Figure 2 D schematically shows the construction of the pWAP-Δ12 -bGH plasmid. Figure 3 is a bar graph depicting the %Linoleic acid (18:2n-6) levels in control and Δ12-desaturase gene-transfected L cells.
Figure 4 is a bar graph depicting the %Eicosadienic acid levels (20:2n-6) levels in control and Δ12-desaturase gene-transfected L cells.
Figure 5 is a bar graph depicting the Fatty acid profiles in control and Δ6- desaturase gene-transfected L cells.
Figure 6 depicts the nucleotide sequence of fungal Δ5 desaturase (SEQ ID:1).
Figure 7 depicts the amino acid sequence of fungal Δ5 desaturase (SEQ ID:2).
Figure 8 depicts the nucleotide sequence of fungal Δ6 desaturase (SEQ ID:3).
Figure 9 depicts the amino acid sequence of fungal Δ6 desaturase (SEQ ID:4). Figure 10 depicts the nucleotide sequence of fungal Δ12 desaturase (SEQ
ID:5).
Figure 11 depicts the amino acid sequence of fungal Δ12 desaturase (SEQ ID:6).
Figure 12 schematically depicts downstream products of arachidonic acid in the 5-lipoxygenase pathway.
Figure 13 schematically depicts downstream products of arachidonic acid in the 12-lipoxygenase pathway.
Figure 14 schematically depicts downstream products of arachidonic acid in the 15-lipoxygenase pathway. Figure 15 schematically depicts downstream products of arachidonic acid in the cytochrome P-450 pathway.
Figure 16 schematically depicts downstream products of arachidonic acid in the cyclooxygenase pathway (pathway I).
Figure 17 schematically depicts downstream products of arachidonic acid in the cyclooxygenase pathway (pathway II).
Figure 18 shows the nucleic acid sequence for human Δ5 desaturase cDNA. Figure 19 shows the amino acid sequence for the human Δ5 desaturase. Figure 20 shows the map of plasmid pMTK-bGH-C. Figure 21 shows the plasmid resulting from the insertion of the human desaturase gene into plasmid pMTK-bGH-C.
Figure 22 shows the nucleic acid sequence for Mortierella Alpina elongase cDNA.
Figure 23 shows the amino acid sequence of the Mortierella Alpina elongase. Figure 24 shows the plasmid resulting from the insertion of the elongase gene into plasmid pMTK-pGH-C.
DEFINITIONS
To facilitate understanding of the invention, a number of terms are defined below.
The abbreviations used herein are: LC-PUFAs, Long-chain polyunsaturated fatty acids; PG, prostaglandins; LT, Leukotrienes; GLA, γ-linolenic acid; DGLA, dihomo-γ-linolenic acid; AA, Arachidonic Acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid; SDA, stearidonic acid; FA, Fatty acids, EFA, essential fatty acids; Δ5D, Δ5 desaturase; Δ6D, Δ6 desaturase; Δ12D, Δ12 desaturase; TX, Thromboxanes. The term "LC-PUFA" as used herein refer to fatty acids with chain lengths beyond 18-carbons that has two or more double bonds. A large number of such LC- PUFA (but not all) can be derived from Linoleic Acid (18:2n-6) and α-Linolenic Acid (18:3n-3), the two so-called "essential fatty acids."
"Nucleic acid sequence" and "nucleotide sequence" as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or
RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
The term "portion" when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.
The term "recombinant DNA molecule" as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological (i.e., non-naturally occurring) techniques (also known as genetic engineering).
As used herein, the terms "vector" and "vehicle" are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
The term "expression vector" or "expression cassette" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
The terms "in operable combination", "in operable order" and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
As used herein, the terms "complementary" or "complementarity" are used in reference to "polynucleotides" and "oligonucleotides" (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'. Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acids is where each and every nucleic
acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
The terms "homology" and "homologous" as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). A nucleotide sequence which is partially complementary, i.e., "substantially homologous," to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
Low stringency conditions comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH2PO4 «H2O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400,
Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
The art knows well that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target ( DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions which promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe which can hybridize (i.e., it is the complement of) to the single-stranded nucleic acid sequence under conditions of low stringency as described above. As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the
Tm of the formed hybrid, and the G:C ratio within the nucleic acids.
As used herein the term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases;
these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (as determined, e.g., by C0t or RQ? analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support [e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)].
"Stringency" when used in reference to nucleic acid hybridization typically occurs in a range from about Tm-5°C (5°C below the Tm of the probe) to about 20°C to 25 °C below Tm. The term "Tm" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. Under "stringent conditions" a sequence or fragments thereof will hybridize to the sequence's exact complement and closely related sequences.
DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fac1?that transcription proceeds in a 5' to 3' fashion along the DNA strand. The promoter and
enhancer elements which direct transcription of a linked gene are generally located 5 ' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.
As used herein, the term "an oligonucleotide having a nucleotide sequence encoding a gene" means a nucleic acid sequence comprising the coding region of a gene, i. e. , the nucleic acid sequence which encodes a gene product. The coding region may be present in either a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single- stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
As used herein, the term "regulatory element" refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
Transcriptional control signals in eukaryotes comprise "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription
[Maniatis, T. et al, Science 236:1237 (1987)]. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer
depends on what cell type is to be used to express the protein of interest. The term "promoter sequence" as used herein refers to a single promoter sequence as well as to a plurality (i.e., one or more) of promoter sequences which are operably linked to each other and to at least one DNA sequence of interest. For example, one of skill in the art knows that it may be desirable to use a double promoter sequence (i.e., a DNA sequence containing two promoter sequences) or a triple promoter sequence (i.e., a DNA sequence containing three promoter sequences) to control expression of a DNA sequence of interest.
The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site [Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp. 16.7-16.8]. A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40. Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term "poly A site" or "poly A sequence" as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript.
Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene.
The terms "cognate promoter" and "cognate promoter of RNA polymerase" refer to a promoter sequence which is a naturally occurring promoter sequence in a gene encoding the RNA polymerase. For example, the cognate promoter of T7 RNA polymerase is the promoter which is derived from the gene encoding RNA polymerase
in T7 bacteriophage. The cognate promoter sequence may be cloned from the genome encoding the RNA polymerase. The location of a promoter may be identified by approaches and methods well known in the art, including DNase footprinting of the RNA polymerase-bound genome DNA, mutational analysis, etc. Alternatively, where the sequence of a cognate promoter is known, the promoter sequence may be synthesized.
The term "transfection" as used herein refers to the introduction of a transgene into a cell. The term "transgene" as used herein refers to any nucleic acid sequence which is introduced into the genome of a cell by experimental manipulations. A transgene may be an "endogenous DNA sequence," or a "heterologous DNA sequence"
(i.e., "foreign DNA"). The term "endogenous DNA sequence" refers to a nucleotide sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence. The term "heterologous DNA sequence" refers to a nucleotide sequence which is not endogenous to the cell into which it is introduced. Heterologous DNA includes a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA also includes a nucleotide sequence which is naturally found in the cell into which it is introduced and which contains some modification relative to the naturally-occurring sequence. Heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is introduced. An example of a heterologous DNA of the present invention comprises a nucleotide sequence which encodes a desaturase which is not found in the mammalian cell into which it is introduced. Another example of the present invention is a desaturase gene which is ligated to a promoter sequence to which it is not naturally ligated.
Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome
fusion, lipofection, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.
The term "stable transfection" or "stably transfected" refers to the introduction and integration of a transgene into the genome of the transfected cell. The term "stable transfectant" refers to a cell which has stably integrated one or more transgenes into the genomic DNA.
The term "transient transfection" or "transiently transfected" refers to the introduction of one or more transgenes into a transfected cell in the absence of integration of the transgene into the host cell's genome. The term "transient transfectant" refers to a cell which has transiently integrated one or more transgenes.
A "transgenic organism" as used herein refers to an organism in which one or more cells has been transiently transfected or stably transfected with a transgene by experimental manipulation. Transgenic organisms may be produced by several methods including the introduction of a "transgene" comprising nucleic acid (usually DNA) into an embryonic target cell or a somatic target cell (such as cells of the mammary gland) of a non-human organism by way of human intervention.
"Insertion" is used to refer to the process whereby a portion of heterologous DNA or a heterologous gene that is introduced into the genome of a host. The DNA which is inserted is referred to as an "insert". The terms "transgenic mammal" or "transgenic host" are used to refer to a mammal or cell which has had a transgene inserted into its genome. As a result of this insertion, the transgenic host produces heterologous biological material that it would not normally synthesize. Heterologous entities are present or are produced by a transgenic host as a result of the insertion of foreign genetic material into the host cell genome.
The term "primary gene product" refers to a biological entity which is formed directly as a result of the transcription and translation of a homologous or heterologous gene. Examples thereof include proteins, antibodies, enzymes and the like.
The term "secondary gene product" refers to a product which is formed as a result of the biological activity of a primary gene product. An example thereof, is PUFAs which are formed as a result of the expression of specific desaturases.
The term "products" or "biological products" refer to products produced or synthesized by a transgenic animal as a result of the insertion of a transgene into the genome of the animal. More specifically, the term means biological products which are secondary gene products or other downstream products altered by transgene expression. One example hereof, as described below, is LC-PUFAs produced by transgenic mice.
The term "animal" as used herein includes humans.
GENERAL DESCRIPTION OF THE INVENTION
The invention generally relates to compositions and methods of synthesis of essential fatty acids and their derivatives, long chain poly-unsaturated fatty acids and eicosanoids in transfected cells and in transgenic animals. The ability to produce long chain fatty acids derived from Linoleic Acid (18:2n-6) and α-Linolenic Acid (18:3n-3) in cultured cells and in transgenic animals has far reaching economic and scientific implications. Arachidonic Acid and γ-Linolenic Acid (GLA) are important biologically active molecules in and of themselves. Additionally, they serve as precursors for the synthesis of Eicosanoids, such as Prostaglandins, Thromboxanes, Prostacyclins, and Leukotrienes, molecules that again have been shown to possess various biological activities. Also, the ability to produce Eicosapentaenoic Acid (EPA) and Docosahexaeonic Acid (DHA) in mammalian cells and transgenic animals have important implications since these molecules have been show to be potent biologically active molecules.
The Description of the invention involves: A) Selecting Transgenes and the Construction of Expression Vectors Comprising the Transgenes; B) Introduction of the
Expression Construct into a Particular Cell; C) Transgenic Animals and Methods of Introduction of Transgenes; D) Tissue-Specific Expression of Transgenes and Detection of the Expression Construct; and E) Fatty acid Production, including
Prostaglandins, Prostacyclins, Thromboxanes and Leukotrienes, in Cell Lines and Bioreactors.
A. Selecting Transgenes And The Construction Of Expression Vectors Comprising The Transgenes The present invention contemplates introducing desaturase genes and/or elongase genes into mammalian cells. It is not intended that the present invention be limited to one source or one type of desaturase gene or elongase gene. In one embodiment, a human desaturase is contemplated. For example, nucleic acid encoding the human delta 5 desaturase can be introduced into a plasmid by standard techniques. As discussed above, elongase genes are also contemplated. Figure 22 provides the nucleic acid sequence for a representative elongase. Other elongases can be isolated using probes and/or primers designed based on regions of the nucleic acid sequence set forth in Figure 22. For example, a probe having the sequence ACTACCTCAACTACCTGGTCAAGTACTGGGAGTTGGCCGA can be used to probe genomes of various organisms by hybridization under low and high stringency conditions to identify candidate elongases. Alternatively, primers can be designed using regions at each end (or near the end) of the sequence set forth in Figure 22 for PCR amplification of elongases from other organisms. Still further, elongases can be identified by expression cloning. This involves isolation of RNA and generation of cDNA, incorporation of the cDNA into an expression vector, followed by transfection and expression in a system that permits testing for elongase activity.
Other elongases are known. See C. Oh et al., J. Biol. Chem. 272:17376 (1997). See also M.W. Lassner et al, The Plant Cell 8:281 (1996).
B. Introduction Of The Expression Construct Into A Particular Cell
In order to bring about tissue specific and/or cell type specific expression in transgenic animals, the expression vector which contains the murine whey acidic protein transcriptional regulatory element is in operable combination with the nucleic acid sequences encoding an elongase and/or a desaturase (e.g., Δ5 -desaturase, Δ6- desaturase, Δ12-desaturase or Δ15-desaturase sequences of the invention). Where the desaturase is to be transfected into a host cell (such as a cell in culture), the CMV promoter can be used. Host cells include bacterial, yeast, plant, insect, and mammalian cells. In a preferred embodiment the host cell is mammalian. In a more preferred embodiment, the host cell is a mouse cell.
Any number of selection systems may be used to recover transfected cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22:817-23) genes which can be employed in tk" or aprt" cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate [Wigler M et al, (1980) Proc Natl Acad Sci 77:3567-70]; npt, which confers resistance to the aminoglycosides neomycin and G-418 [Colbere-Garapin F et al, (1981) J. Mol. Biol. 150:1-14] and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine [Hartman SC and RC Mulligan (1988) Proc Natl Acad Sci 85:8047-51]. Recently, the use of a reporter gene system which expresses visible markers has gained popularity with such markers as β-glucuronidase and its substrate (GUS), luciferase and its substrate (luciferin), and β-galactosidase and its substrate (X-Gal), and expression of green fluorescent protein (GFP), being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system [Rhodes CA et al (1995) Methods Mol Biol 55:121-131].
Although the presence or expression of the reporter gene usually indicates the presence or expression, respectively, of the tandem heterologous nucleic acid sequence as well. However, it is preferred that the presence and expression of the desired heterologous nucleic acid sequence be confirmed. This is accomplished by procedures known in the art which include DNA-DNA or DNA-RNA hybridization or amplification using probes, or fragments of the heterologous nucleic acid sequence. For example, Fluorescent In Situ Hybridization (FISH) can be used to detect the heterologous nucleic acid sequence in cells. Several guides to FISH techniques are available, e.g., Gall et al. Meth. Enzymol. 21:470-480 (1981); Angerer et al., in "Genetic Engineering: Principles and Methods," Setlow & Hollaender, Eds. Vol. 7 pp.
43-65, Plenum Press, New York (1985). Alternatively, DNA or RNA can be isolated from cells for detection of the transgene by Southern or Northern hybridization or by amplification based assays. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on sequence of the nucleic acid sequence of interest in order to detect cells and tissues which contain the DNA or RNA encoding the transgene of interest. As used herein, the terms "oligonucleotides" and "oligomers" refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer. Standard PCR methods useful in the present invention are described by Innis et al.
(Eds.), "PCR Protocols: A Guide to Methods and Applications," Academic Press, San Diego (1990)].
Yet another alternative for the detection of heterologous nucleic acid sequences is by detecting the polypeptide product of transcription of the heterologous nucleotide sequence. A variety of protocols which employ polyclonal or monoclonal antibodies specific for the protein product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A competitive binding assay may also be used. Alternatively, a two-site, monoclonal-based immunoassay which utilizes monoclonal antibodies that are reactive to two non-interfering epitopes on the protein of interest may be employed.
These and other assays are described in, among other places, R. Hampton et al, Serological Methods a Laboratory Manual, APS Press, St Paul MN (1990) and D. E. Maddox et al, J. Exp. Med. 158:1211(1983).
A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting related sequences include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the nucleic acid sequence of interest, or any portion of it, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. A number of companies such as Pharmacia Biotech (Piscataway NJ), Promega (Madison WI), and US Biochemical Corp (Cleveland OH) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
In specific embodiments of the present invention, stably transfected L cells were generated using the calcium phosphate precipitation method as previously described [M. Wigler et al , "Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells," Cell 11:223-232 (1977); A. Pellicer et al, "Altering genotype and phenotype by DNA-mediated gene transfer," Science 209:1414-1422 (1980); B. Wold et al, "Introduction and expression of a rabbit β-globin gene in mouse fibroblasts," Proc. Natl. Acad. Sci. USA 76:5684-5688 (1979); J.M. Roberts and R. Axel, "Gene amplification and gene correction in somatic cells," Cell 29:109-119
(1982)]. This method utilizes a plasmid DNA (pDLAT3) which encodes two selectable markers: an intact APRT gene and a truncated TK gene lacking transcriptional regulatory (enhancer) sequences.
In other specific embodiments, stably transfected Hela cells were generated using the Lipofectamine Reagent method (BRL, Gaithersburg, MD) and cells were selected that exhibited neomycin resistance. The neomycin resistant Hela cell pool was then expanded and analyzed further.
C. Transgenic Animals And Methods Of Introduction Of Transgenes
A first step in the generation of the transgenic animals, is the introduction of a construct (or constructs) containing the desired nucleic acid sequence (or sequences) into target cells. Several methods are available for introducing the expression vector (or vectors) which contains the nucleic acid sequence of a desaturase and/or elongase into a target cell, including microinjection, retroviral infection, and implantation of embryonic stem cells. These methods are discussed as follows.
i. Microinjection Methods
Direct microinjection of expression vectors into pronuclei of fertilized eggs is the preferred, and most prevalent, technique for introducing heterologous nucleic acid sequences into the germ line [Palmiter (1986) Ann. Rev. Genet. 20:465-499].
Technical aspects of the microinjection procedure and important parameters for optimizing integration of nucleic acid sequences are known to the art [Brinster et al., (1985) Proc. Natl. Acad. Sci. USA 82:4438-4442; Gordon et al, (1983) Meth. Enzymol. 101:411-433; Hogan et al, (1986) Manipulation of the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Lab.].
Once the expression vector has been injected into the fertilized egg cell, the cell is implanted into the uterus of a pseudopregnant female and allowed to develop into an animal. Of the founder transgenic animals born, 70% carry the expression vector sequence in all of their cells, including the germ cells. The remaining 30% of the transgenic animals are chimeric in somatic and germ cells because integration of the expression vector sequence occurs after one or more rounds of replication. Heterozygous and homozygous animals can then be produced by interbreeding founder transgenics. This method has been successful in producing transgenic mice, sheep,
pigs, rabbits and cattle [Jaenisch (1988) supra; Hammer et al, (1986) J. Animal Sci. 63:269; Hammer et al., (1985) Nature 315:680-683; Wagner et al., (1984) Theriogenology 21:29].
ii. Retroviral Methods Retroviral infection of preimplantation embryos with genetically engineered retroviruses may also be used to introduce transgenes into an animal cell. For example, blastomeres have been used as targets for retroviral infection [Jaenisch, (1976) Proc. Natl. Acad. Sci USA 73: 1260-1264]. Transfection is typically achieved using a replication-defective retrovirus carrying the transgene [Jahner et al, (1985) Proc. Natl. Acad. Sci. USA 82:6927-6931 ; Van der Putten et al, (1985) Proc. Natl.
Acad Sci USA 82:6148-6152]. Transfection is obtained, for example, by culturing eight-cell embryos, from which the zona pellucida has been removed with fibroblasts which produce the virus [Van der Putten (1985), supra; Stewart et al., (1987) EMBO J. 6:383-388]. The transfected embryos are then transferred to foster mothers for continued development. Alternatively, infection can be performed at a later stage.
Virus or virus-producing cells can be injected into the blastocoele [Jahner et al, (1982) Nature 298:623-628]. Yet another alternative method involves intrauterine retroviral infection of the midgestation embryos [Jahner et al. (1982), supra}.
The advantages of retroviral infection methods include the ease of transfection and the insertion of a single copy of the transgene, which is flanked by the retroviral long terminal repeats (LTRs), into the chromosome. However, this method is not a preferred method because most of the founders will show mosaicism since infection occurs after cell division has begun. This necessitates outbreeding to establish homozygous and heterozygous lines suitable for analysis of gene expression.
iii. Embryonic Stem Cell Implantation
Another method of introducing transgenes into the germ line involves using embryonic stem (ES) cells as recipients of the expression vector. ES cells are pluripotent cells directly derived from the inner cell mass of blastocysts [Evans et al, (1981) Nature 292:154-156; Martin (1981) Proc. Natl. Acad Sci. USA 78:7634-7638;
Magnuson et al, (1982) J. Embryo. Exp. Morph. 81:211-217; Doetchman et al, (1988) Dev. Biol. 127:224-227], from inner cell masses [Tokunaga et al, (1989) Jpn. J. Anim. Reprod. 35:113-178], from disaggregated morulae [Eistetter, (1989) Dev. Gro. Differ. 31 :275-282] or from primordial germ cells [Matsui et al, (1992) Cell 70:841-847; Resnick et al, (1992) Nature 359:550-551]. Expression vectors can be introduced into ES cells using any method which is suitable for gene transfer into cells, e.g., by transfection, cell fusion, electroporation, microinjection, DNA viruses, and RNA viruses [Johnson et al, (1989) Fetal Ther. 4 (Suppl. l):28-39].
The advantages of using ES cells include their ability to form permanent cell lines in vitro, thus providing an unlimited source of genetic material. Additionally ES cells are the most pluripotent cultured animal cells known. For example, when ES cells are injected into an intact blastocyst cavity or under the zona pellucida, at the morula stage embryo, ES cells are capable of contributing to all somatic tissues including the germ line in the resulting chimeras. Once the expression vector has been introduced into an ES cell, the modified
ES cell is then introduced back into the embryonic environment for expression and subsequent transmission to progeny animals. The most commonly used method is the injection of several ES cells into the blastocoel cavity of intact blastocysts [Bradley et al, (1984) Nature 309:225-256]. Alternatively, a clump of ES cells may be sandwiched between two eight-cell embryos [Bradley et al, (1987) in
"Teratocarcinomas and Embryonic Stem Cells: A Practical Approach," Ed. Robertson E.J. (IRL, Oxford, U.K.), pp. 113-151; Nagy et al, (1990) Development 110:815-821]. Both methods result in germ line transmission at high frequency.
Target cells which contain the heterologous nucleic acid sequences are recovered, and the presence of the heterologous nucleic acid sequence in the target cells as well as in the animal is accomplished as described supra.
D. Tissue-Specific Expression And Detection Of Transgenes The present invention provides methods for selectively expressing a nucleotide sequence of interest in a particular cell type and/or a particular tissue. The transfected animal cell is allowed to develop into a transgenic animal in which the nucleotide sequence of interest, e.g., the desaturase and/or elongase gene(s), is expressed selectively in a particular tissue such as the mammary glands. Particular expression vectors comprising particular desaturase sequences of the present invention, pWap-Δ6-bGH and pWap-Δ12-bGH, utilize the murine whey acidic protein transcriptional regulatory element to direct Δ6 and Δ12-desaturase transcription and the bGH polyadenylation signal for proper processing of the 3' terminus of desaturase mRNAs. In addition, these are under the control of the murine whey acidic protein transcriptional regulatory element, that directs gene expression primarily to the lactating mammary gland tissue.
The selective expression of the gene of interest (i.e., the desaturase and/or elongase transgenes) in tissues and cells of transgenic animals may be determined using several methods known in the art. For example, expression of mRNA encoded by the gene of interest may be determined by using in situ hybridization. This involves synthesis of an RNA probe which is specific for a portion of or the entire gene of interest, e.g. , by using PCR. The PCR amplified fragment is subcloned into a plasmid (e.g., pBluescript (Stratagene)) and the RNA probe synthesized using labelled UTP (e.g., 35S-UTP) and RNA polymerase (e.g., T3 or T7 polymerase (Promega)). Paraffin-embedded tissue sections are mounted on slides, deparaffmized, rehydrated and the protein digested (e.g. , with proteinase K), then dehydrated prior to hybridization with the RNA probe at the desired hybridization stringency. Slides are then developed for autoradiography using commercially available developers.
Labelling of tissues and cells as detected on the autoradiographs indicates expression in those tissues and cells of the mRNA encoded by the gene of interest.
Alternatively, expression of the protein product of the gene of interest may be determined using immunohistochemical techniques. Briefly, paraffin-embedded tissue sections are dewaxed, rehydrated, treated with a first antibody which is specific for the polypeptide product of the gene of interest. Binding is visualized, for example, by using a secondary biotinylated antibody which is specific for the constant region of the primary antibody, together with immunoperoxidase and 3,3'-amiobenzidine as a substrate. Sections may then be stained with hematoxylin to visualize the cellular histology. Antibody binding of tissues and cells which is detected by antibody binding demonstrates expression of the protein product of the gene of interest in these tissues and cells.
E. Fatty Acid Production, Including Prostaglandins And Leukotrienes In Cell Lines And Bioreactors i. LC-PUFA Production
The present invention provides alternate methods for altering or increasing the production of LC-PUFAs and derivative products by use of mammalian cells and transgenic animals into which has been inserted genes or cDNAs encoding desaturases.
In particular, the present invention provides a method to generate mammalian cells in which desaturase and or elongase genes are expressed. The transfected cells exhibited enhanced production of both n-6 and n-3 LC-PUFAs, as they had obtained the ability to synthesize essential fatty acids or utilized the exogenous essentail fatty acids (EFA) as precursor for production of LC-PUFA. In addition, the desaturase genes were introduced into animals, gene expression targeted specifically to the mammary glands, generating transgenic mice expressing delta-12, delta-6 or delta-5 genes. The milk derived from the transgenic mice contained a significantly higher level of LC-PUFA than that from the control mice.
Thus, the present invention provides alternative sources of EFA and LC-PUFA in commonly used commodities, such as milk, infant formula, dietary supplement and
pharmaceuticals. In addition, the methods of the present invention provide an alternate source for the generation of eicosanoids. Eicosanoids (prostaglandins, leukotrienes, and lipoxins), oxygenated lipids, and Platelet Activating Factor remain the focus of rational drug design targets given their established roles in cell-cell communication and as mediators in inflammation and pathophysiologic events. Identification of key enzymes in these pathways are implicating involvement of the nuclear membrane at the functional level. Results from transgenic animals have identified novel bioactive eicosanoids, including 15-epi-lipoxins, isoprostanes, and isoleukotrienes, that offer new concepts to consider in formation of Lipid-derived mediators (LM) and the actions of nonsteroidal anti-inflammatory drugs. These findings indicate that LM play critical and essential roles in both signal transduction and cell-cell communication and will continue to be important pathways to be considered in novel therapeutic approaches (See Serhan, C. N. et al, Lipid mediator networks in cell signaling: update and impact of cytokines, FASEB J.10:1147-1158, 1996).
ii. Cell Lines As A Source Of Essential FA's Or Altered
Levels Of LC-PUFAs
The present invention provides mammalian cells (including but not limited to Human HeLa cells, Mouse L cells) transfected with desaturase and/or elongase genes, that exhibits enhanced production of both n6 and n3 LC-PUFAs. While an understanding of a precise mechanism is not necessary to the successful use of the invention, it is believed that this is because the transfected cells have obtained the ability to synthesize essential fatty acids (EFA), or utilize the exogenous EFA as precursors for production of LC-PUFAs. The minimum number of genes required for increased LC-PUFA production varied between different cell lines, e.g., human Hela cells required only Δ12 desaturase.
ii. Cells In Bioreactors
In some embodiments, the present invention contemplates the use of mammalian cells in bioreactors, for the large scale production of LC-PUFAs [see U.S. Patent No. 5,459,069 to Palsson et al. and U.S. Patent No. 5.563,068 to Zhang et al, both hereby incorporated by reference]. Some bioreactors utilize hollow fiber systems.
Frequently, bundles of parallel fibers are enclosed in an outer compartment; cells are grown on the outside surface of the fibers, while nutrient- and gas-enriched medium flows through the center of the hollow fibers, nourishing the cells [see, e.g., U.S. Patent No. 5.512,474 to Clapper et al, hereby incorporated by reference]. In addition, bioreactors utilizing microcarriers (e.g., DEAE-derivatived dextran beads) can be used in conjunction with the present invention. In preferred embodiments, cell adhesion proteins like collagen, fibronectin, and laminin are used to anchor the cells to the solid support; collagen is the most preferred cell adhesion protein. Microcarriers may also incorporate an ionic charge to assist in cell attachment to the microcarrier. Frequently, the microcarriers are porous beads that are sufficiently large to allow cells to migrate and grow in the interior of the bead [see U.S. Patent No. 5,512,474 to Clapper et al.].
iv. Transgenic Animals As A Source Of EFAs And For Enhanced Levels Of PUFAs The present invention provides the first, novel transgenic animals that express specific desaturase and/or elongase transgenes. By expressing a Δ12-desaturase (enzyme), products of the omega 6 pathway (that is GLA and Arachdonic Acid), were hoped for. However, surprisingly, the omega three series of LC-PUFAs, in particular EPA, DP A, and DHA were obtained in the transgenic animals. According to the information available in the current literature, these products are unexpected from Δ12- desaturase gene expression.
As mentioned in US Patent no. 5,689,050, hereby incorporated by reference, for expression data concerning transgenic organisms which express desaturases, the
Δ12- and Δ6-desaturases must be co-expressed in order to obtain the desired products. For example, expression of Δ12-desaturase by plants yields Linoleic Acid from Oleic Acid. Plants cannot convert linoleic acid to other LC-PUFAs, since it lacks the enzymatic machinery to do so. Also, if one wanted to produce GLA in plants, the Δ6- desaturase enzyme needs to be inserted (to convert Linoleic acid to GLA) along with
Δ12-desaturase. Thus, in plants, one needs to insert each and every enzyme of each metabolic conversion in order to obtain the desired results. However, based on the surprising results described herein, the latter may not be true for animals. Animal cells can convert linoleic acid to various LC-PUFA intermediates and products. But the exact pathways are not specifically and completely known. One would not have anticipated obtaining the omega 3 series of products from expression of Δ12-desaturase in animal cells, and also predicted that Δ12-, and Δ15-desaturase would need to be inserted in order to obtain EPA, DP A, and DHA. However, this was not the case. Thus, it is exemplary that essentially what happens in plants does not necessarily happen in the same manner in animals.
It is contemplated that the desaturase transgenic animals provided in the present invention are a useful alternate source for the synthesis of LC-PUFAs and can be employed as animal models (including but not limited to models of human disease) in research, including but not limited to pharmaceutical research. In addition, the transgenic animals of the present invention can be used as Bioreactors for the large scale production of LC-PUFAs (See "Molecular Farming: Transgenic Animals as Bioreactors" by J. Van Brunt, Biotechnology, Volume 6, page 1149-1154, 1988, describes the alteration of the genome of various large domestic milk bearing animals yielding transgenic animals capable of producing various heterologous entities. This publication suggests methods for obtaining the primary gene product). The present invention further expands on the above protocols and teaches the the use of transgenic animals for the production of biological products.
Although, there is a considerable body of literature which describes the recombinant or transgenic expression of heterologous glycosyltransferases, the
literature does not disclose or in any other manner suggest production of LC-PUFAs in the milk of non-human transgenic mammals as claimed in the present invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS AND USES OF THE INVENTION In preferred embodiments, the present invention has reduced to practice active expression of various desaturase and/or elongase genes (cDNA) in mammalian cells and transgenic animals and provides novel transgenic animals that express specific desaturase and/or elongase genes. The present invention provides transgenic animals expressing desaturases and/or elongases, that have not been described before. In addition, the PUFA derivatives obtained from these transgenic animals, such as the omega-3 products, were not anticipated nor obvious. In yet other embodiments, the transgenic animals of the present invention provides products in animal milk, that were completely unanticipated.
In yet other preferred embodiments, the present invention has reduced to practice, active expression of various desaturase genes (cDNA) into cultured cells and these transformed cultured cells provides products that are not clearly predictable from the available prior art.
In one embodiment, the present invention provides animal cells which express Δ12-desaturase and contains altered levels of linoleic acid, DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives.
In yet another embodiment, the invention provides a method of modifying levels of linoleic acid, DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives in animal cells (using Δ12-desaturase alone).
In other embodiments, the milk produced in Δ12-desaturase transgenic animals contains altered levels of linoleic acid, DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives.
In yet other embodiments, the milk produced in Δ6-desaturase transgenic animals contains altered levels of DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives.
In other embodiments, the present invention provides a method of modifying levels of linoleic acid, DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives in transgenic animals (using Δ12-desaturase alone).
In yet other embodiments, the present invention provides a method of modifying levels of DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives in transgenic animals (using Δ6-desaturase alone).
In another embodiment, the present invention provides animal cells that express Δ6-desaturase which contain altered levels of DGLA, AA, adrenic acid, omega-3 PUFAs and their derivatives (using Δ6-desaturase alone). In other preferred embodiments the present invention provides animal cells, mammals and milk with altered levels of molecules of the prostaglandin E series. In one embodiment, the present invention provides mammals with altered levels of molecules of the prostaglandin I series, thromboxane series and leukotriene series. In yet another embodiment, the present invention provides a method of altering the levels of molecules of prostaglandin I series, thromboxane series and leukotriene series in mammals.
In other preferred embodiments the present invention provides animal cells, mammals and transgenic animal milk with altered levels of essential fatty acids and omega-3 PUFAs when expressing both Δ12-desaturase and Δ15-desaturase (Δ15-desaturase converts linoleic acid to α-linolenic acid). In one embodiment, the present invention provides a method of modifying levels of α-linolenic acid and omega-3 PUFAs when expressing both Δ12-desaturase and Δ15-desaturase.
In other preferred embodiments the present invention provides a method of producing animal cells or mammals which do not require external supply of essential fatty acids.
USES OF THE INVENTION
A. The following broad applications or uses of the present invention are contemplated:
1. Fat Free Media: Growth of tissue culture cells in medium lacking essential fatty acids, especially linoleic acid. This allows individuals and companies to better define media used for maintaining cultured vertebrate cells. It may also improve the ability of cultured cells to produce recombinant proteins and /or make the process more economical.
2. Research Reagent: Since obtaining essential fatty acids and their derivatives, and long chain poly-unsaturated fatty acids is expensive, these molecule by and large have not been used in the laboratory. The ability to generate these molecule would open a market for their use in countless numbers of research laboratories.
B. Methods Of Treatment And Formulations In particular, the present invention provides the following methods and/or products which are useful for the applications as mentioned:
1. Method of treating or preventing malnutrition by administering milk fat or animal fat, or fraction thereof, in an amount sufficient to affect the treatment or prevention. 2. Method of treating patients which exhibit condition(s) caused by either inadequate intake or inadequate endogenous production of PUFAs. Treatment would be administration of dietary substitute or supplements containing the milk/animal fats, or fraction thereof, produced in the desaturase expression systems.
3. A pharmaceutical composition comprising the milk fat or animal fat, or fraction thereof.
4. A nutritional formula comprising the milk fat or animal fat, or fraction thereof. The nutritional formula comprise of and include infant formula, dietary supplements and dietary substitutes and, where applicable, administratable to both humans and animals.
5. Cosmetics comprising the milk fat or animal fat, or fraction thereof.
6. Animal feeds comprising the milk fat or animal fat, or fraction thereof.
EXPERIMENTAL
In the experimental disclosure which follows, the following abbreviations and methodology apply: g (gram); mg (milligrams); μg (microgram); M (molar); mM (milliMolar); μM (microMolar); nm (nanometers); L (liter); ml (milliliter); μl (microliters);°C (degrees Centigrade); m (meter); sec. (second); DNA (deoxyribonucleic acid); cDNA (complementary DNA); RNA (ribonucleic acid); mRNA (messenger ribonucleic acid); PAGE (polyacrylamide gel electrophoresis); BAP (6-benzyl aminopurine); Tris (tris (hydroxymethyl) -aminomethane); PBS (phosphate buffered saline); 2 X SSC (0.3 M NaCl, 0.03 M Na3citrate, pH 7.0); Gibco BRL (Gaithersburg, MD); Sigma (St. Louis, MO).
Methodology
Cell Culture And Generation Of Stable Cell Lines: Mouse L cells (ATCC)[thymidine kinase negative (TK") and adenine phosphoribosyltransferase negative (APRT")] were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco Laboratories, Grand Island, NY) containing 10% Nu-serum (Collaborative Research Inc., Bedford, MA), 50 ug/ml gentamicin sulfate (Gibco) and 50 ug/ml 2,6-di-aminopurine (Sigma,
St. Louis, MO).
Stably transfected L cells were generated using the calcium phosphate precipitation method as previously described [M. Wigler et al, "Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells," Cell 11:223-232 (1977); A. Pellicer et al, "Altering genotype and phenotype by DNA-mediated gene transfer,"
Science 209:1414-1422 (1980); B. Wold et al, "Introduction and expression of a rabbit b-globin gene in mouse fibroblasts," roc. Natl. Acad. Sci. USA 76:5684-5688 (1979);
J.M. Roberts and R. Axel, "Gene amplification and gene correction in somatic cells," Cell 29:109-119 (1982)]. This method utilizes a plasmid DNA (pDlAT3) which encodes two selectable markers: an intact APRT gene and a truncated TK gene lacking transcriptional regulatory (enhancer) sequences. Cells were first selected for the APRT+ phenotype in DMEM containing 10% Nu-serum, 4 ug/ml azaserine, 15 ug/ml adenine and 50 ug/ml gentamicin sulfate. The APRT+ colonies were then grown in DMEM containing 10% Nu-serum, 50 ug/ml gentamicin sulfate, 15 ug/ml hypoxanthine, 1 ug/ml aminopterin and 5.15 ug/ml thymidine (HAT medium) to select for the TK+ phenotype. Individual TK+ clones were isolated from the HAT+ pool by limited dilution of the cells into 96-well plates at a concentration of 0.5 cells/well.
Individual TK+ clones were analyzed for the presence of integrated desaturase sequences by slot blot hybridization analysis using a [32P]radiolabeled DNA probe containing sequences from bGH exon V and 3' untranslated region. TK+ clones containing integrated CMVie-Δ6-bGH or pCMVie-Δ12-bGH sequences were expanded and analyzed further.
Stably transfected Hela cells were generated using the Lipofectamine Reagent method (BRL, Gaithersburg, MD) as suggested by the manufacturer. Briefly, 300 ng of plasmid DNA encoding the neomycin phosphotransferase gene driven by the RSV-LTR TRE [C. M. Gorman et al., "The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by
DNA-mediated transfection," Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982a)] and 30 ug of pCMVie-Δ12-bGH plasmid DNA was diluted in H2O to a volume of 200 ul. This was added to 200 ul of a 25% Lipofectamine solution, mixed gently and allowed to stand for 30 min at room temperature. Approximately 1 x 106 Hela cells in a 100 mm dish were washed twice with 10ml serum- free DMEM and 4 ml serum- free
DMEM was added. The above DNA/Lipofectamine solution was then added to the cells and incubated for 5 hr at 37°. Following incubation, the medium containing the DNA/Lipofectamine was removed and replaced with culture medium. After 24 hr, the cells were passed into culture medium containing 300 ug/ml G-418 sulfate (Geneticin,
Gibco) to select for cells exhibiting neomycin resistance. The neomycin resistant Hela cell pool was then expanded and analyzed further.
The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
EXAMPLE 1
This example describes the construction of the eukaryotic desaturase expression vectors. DNA manipulations were carried out using standard cloning techniques. A 1,382 bp EcoR~ l-XhoI DNA fragment encoding the Δ6-desaturase gene was isolated from plasmid pCGR5 and ligated into plasmid pCMV-BGH-C [A. Martin-Gallardo et al, "A comparison of bGH expression in mouse L cells directed by the Moloney murine leukemia virus long terminal repeat, the simian virus 40 early or cytomegalovirus immediate early promotors," Gene 70:151-156 (1988)], which had been cleaved with BgHl and Smal. The termini of the DNA molecules were made flush using Klenow polymerase prior to ligation. The resulting plasmid, pCMVie-Δ6-bGH, utilizes the cytomegalovirus immediate early transcriptional regulatory element to direct Δ-6-desaturase transcription and the bGH polyadenylation signal for proper processing of the 3' terminus of desaturase mRNA (See Figure 2A). Similarly, a 1,209 bp Eco l-Xhol DNA fragment encoding the Δ12-desaturase gene was isolated from plasmid pCGR7 and ligated into plasmid pCMV-BGH-C which had been cleaved with Bg l and Smal to generate the plasmid, pCMVie-Δ12-bGH (See Figure 2B). The termini of these DNA molecules were also made flush using Klenow polymerase prior to ligation.
The DNA fragments encoding the Δ6-desaturase gene and the Δ12-desaturase gene were also ligated into plasmid, pWAP-polyA [Prieto et /.(1995)] which had been cleaved with Smal (See Figures 2C and 2D). The resulting plasmids, pWap-Δ6-bGH and pWap-Δ12-bGH utilize the murine whey acidic protein transcriptional regulatory element to direct Δ6 and Δ12-desaturase transcription and the bGH polyadenylation
signal for proper processing of the 3' terminus of desaturase mRNAs. The murine whey acidic protein transcriptional regulatory element has been shown to direct gene expression primarily to lactating mammary gland tissue [C. W. Pittuis et al, Proc. Natl. Acad. Sci. U.S.A. 85:5874-5878 (1988)].
EXAMPLE 2
This example describes the eneration of transgenic animals, expressing the desaturase genes.
Plasmid pWap-Δ6-bGH was cleaved with restriction endonucleases EcoRl and Pstl. A linear DNA fragment containing sequences encoding the WAP-Δ6-bGH transcriptional unit was isolated and injected into fertilized mouse (B6/SJL) eggs as described previously [M.M. McGrane et al, J. Biol. Chem. 263:11443-11451 (1988)]. The injected eggs were transferred to pseudopregnant females which subsequently delivered pups. High molecular weight chromosomal DNA was isolated from tail biopsies of the pups and was analyzed for the presence of integrated transgene sequences by slot blot hybridization analysis using a [32P]radiolabeled DNA probe containing sequences from bGH exon V and 3' untranslated region. Similarly, plasmid pWap-Δ12-bGH was cleaved with restriction endonucleases EcoRl and BamHl. A linear DNA fragment containing sequences encoding the WAP-Δ12-bGH transcriptional unit was isolated, injected and transgenic animals identified. Control, Δ6 and Δ12 females were mated and allowed to deliver Ε, progeny. Milk samples were obtained from control and transgenic mothers 5-12 days postpartum and then analyzed for desaturase activity.
EXAMPLE 3
In this example, experiments have been described that show expression of Δ6 and Δ12-desaturases in cell culture in vitro.
Nine stably transfected L cell clones containing integrated Δ12-desaturase sequences (Δ12) were obtained as described in the methodology. T-25cm2 flasks of control (L) and Δ12 cells were incubated in serum-containing medium or in serum-free medium for either one or three days. This incubation deprived the cells of essential fatty acids (linoleic acid, 18:2n-6 and α-linolenic acid, 18:3n3). Following incubation, the cells were isolated and analyzed for Δ12-desaturase activity by determining the cellular levels of various omega-6 fatty acids as a percentage of total fatty acid. The results are summarized in Table 1. The values given for Δ12-desaturase L cell clones represents the mean values for the nine clones analyzed.
Table 1
* nd = not detected (< 0.05).
In medium containing serum, linoleic acid (18:2n-6) comprised approximately 3.25%o of total fatty acid in control cells. The percentage of this fatty acid in control cells decreased from 3.25 to 1.77 and 1.13 when the cells were incubated in serum-free medium for 1 and 3 days, respectively. In contrast, Δ12 cells had significantly elevated levels of linoleic acid. In medium containing serum, linoleic acid comprised approximately 19.99% of total fatty acid in Δ12 cells. This
represented a 515% increase in Δ12 cells compared to control cells. The levels of linoleic acid increased to 21.52 and 24.04% when the cells were incubated cells in serum-free medium for 1 and 3 days, respectively (See Figure 3). The 24.04% level of linoleic acid in Δ12 cells in serum-free medium for 3 days represented a 2,027% increase from what was present in control cells treated similarly. These results demonstrated that the Δ12-desaturase was expressed in these cells and converted oleic acid (18:ln-9) to linoleic acid (18:2n-6). Analysis of other omega-6 fatty acids from cultures incubated in serum- free medium for 24 hours indicates: 1) γ-linolenic acid (GLA, 18:3n-6) was elevated in Δ12 cells compared to control cells, 0.16 verses undetectable (<0.05); and 2) di-homo-γ-linolenic acid (20:3n-6) and arachidonic acid
(20:4n-6) were both significantly elevated in Δ12 cells compared to control cells, 1.21 and 4.06% verses 0.44 and 1.72%, respectively. The percentage of another fatty acid, eicosadienic acid (20:2n-6), in control cells was approximately 0.13% in serum-containing medium and remained relatively constant (0.17 and 0.12%) when the cells were incubated cells in serum-free medium for 1 and 3 days, respectively. In contrast, 20:2n-6 levels in Δ12 cells increased from 0.67% in serum-containing medium to 1.09 and 1.48% when the cells were incubated cells in serum- free medium for 1 and 3 days, respectively (See Figure 4).
Analysis of omega-3 fatty acids indicated that both eicosapentaenoic acid (20:5n-3) and docosapentaenoic acid (22:5n-3) were significantly elevated in Δ12 cells compared to control cells. The level of docosahexaeonic acid (22:6n-3) was also elevated in Δ12 cells compared to control cells but the increase was less pronounced (less than a 100%> increase). The levels (% of total fatty acid) of various fatty acids in serum-containing and serum-free medium are summarized in Table 2.
Table 2
* nd = not detected (< 0.05).
Stably transfected Hela cells containing integrated Δ12-desaturase sequences (Δ12) were generated as described previously. Increased Δ12-desaturase activity in this human epithelial cell line was important to demonstrate that the enzyme was capable of altering the fatty acid profile in multiple species and cell types. Control Hela and Δ12-Hela cells were isolated from cultures growing in serum-containing medium as well as duplicate cultures incubated for 24 hours in serum-free medium. Table 3 summarizes the levels of various omega-6 and omega-3 fatty acids in these cells as a percentage of total fatty acid.
The Δ12-Hela cells had slightly elevated levels of the Δ12-desaturase product, linoleic acid (18:2n-6) compared to control Hela cells. However, the levels of both arachidonic acid (20:4n-6) and adrenic acid (22:4n-6) were significantly elevated in the Δ12-Hela cells. Similarly, the levels of both docosapentaenoic acid (22:5n-3) and docosahexaeonic acid (22:6n-3) were elevated in the omega-3 pathway of Δ12-Hela cells compared to control Hela cells. These results demonstrated that the Δ12-desaturase was expressed in these Hela cells and converted oleic acid (18:ln-9) to linoleic acid (18:2n-6). The results also indicated that Hela cells had highly active endogenous Δ6-desaturase, elongase and Δ5-desaturase enzymes. These enzymes rapidly elongated and desaturated the increased pool of linoleic acid though the
Table 3
Table 4
nd = not detected (< 0.05).
omega-6 (and omega-3) pathway to generate elevated levels of arachidonic acid (20:4n-6) and other fatty acids.
Seven stably transfected L cell clones containing integrated Δ6-desaturase sequences (Δ6) were obtained as described. Since L cells expressing Δ6-desaturase would be expected to still require essential fatty acids, control (L) and Δ6 cells were isolated from growing cultures (serum-containing culture medium). The cells were analyzed for Δ6-desaturase activity by determining the levels of various omega-6 and omega-3 fatty acids within the cells as a percentage of total fatty acid. The results are summarized in Table 4 and in (Figure 5). The values given for Δ6-desaturase L cell clones represent the mean values for the seven clones analyzed.
All L cell clones containing stably integrated Δ6-desaturase sequences contained significantly elevated levels of di-homo-γ-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) compared to control L cells, with the mean increases being 133%) and 399%, respectively. In addition, the levels of these fatty acids in the Δ6-desaturase clones were also elevated compared to L cells expressing the
Δ12-desaturase. In the omega-3 series, there was a 38% decrease in the level of α-linolenic acid (18:3n3) in the Δ6-desaturase cells compared to control cells. However, the levels of eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3) and docosahexaenoic acid (22:6n-3) were all elevated in the Δ6-desaturase cells compared to both control and Δ12-desaturase cells. The increases above control cells for these fatty acids were 179%, 715% and 405%, respectively. These data demonstrated that the Δ6-desaturase was expressed in these cells and converted linoleic acid (18:2n-6) to γ-linolenic acid (18:3n-6) and α-linolenic acid (18:3n3) to steridonic acid (18:4n-3). The γ-linolenic acid (18:3n-6) was then converted to di-homo γ-linolenic acid (20:3n-6) by elongase whereas the steridonic acid (18:4n-3) was converted to eicosatrienoic acid (20:4n-3) by elongase and further desaturated to eicosapentaenoic acid (20:5n-3) and docosahexaeonic acid (22:6n-3) by the endogenous Δ5 and Δ4-desaturases.
EXAMPLE 4
In this example, experiments have been described that show expression of Δ6 and Δ12-desaturase transgenes in mice.
Female mice containing either the Δ6 and Δ12-desaturase transgene were generated as described previously in the Methodology. The expression these transgenes is directed by the murine whey acidic protein promoter and expected to be restricted mainly to the lactating mammary gland. The females were mated and milk was collected from each lactating mother 5-12 days post partum. The milk was analyzed for Δ6 and Δ12-desaturase activity by determining the levels of various omega-6 and omega-3 fatty acids present within the milk, as a percentage of total fatty acid, compared to those found in control mouse milk. The results are summarized in Table 5.
Table 5
F0 = Founder line.* nd = not detected (< 0.05).
Analysis of omega-6 fatty acids from Δ6 F058 milk indicated that γ-linolenic acid (18:3n-6), di-homo-γ-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) were all significantly increased compared to control milk (0.22, 0.74 and 0.61% versus 0.07, 0.49 and 0.34%, respectively). Analysis of omega-3 fatty acids from the Δ6 F058 milk indicated that eicosapentaenoic Acid (20:5n-3) was significantly elevated in the transgenic milk compared control milk (0.39 versus 0.22%>). Similar increases were observed in milk samples obtained from Δ12 transgenic animals. The transgenic line, Δ12 F076, demonstrated the largest increases in γ-linolenic acid (18:3n-6), di-homo γ-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) compared to control milk (0.18, 0.80 and 0.72% compared to 0.07, 0.49 and 0.34%, respectively). Milk from this animal also demonstrated significant elevations in several omega-3 fatty acids. While the levels of α-linolenic acid (18:3n3), steridonic acid (18:4n-3) and eicosatrienoic acid (20:4n-3) were not elevated, significant increases were found in the levels of eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3) and docosahexaenoic acid (22:6n-3). The levels of these fatty acids compared to those found in control milk was 0.40, 0.63 and 0.75% versus 0.22, 0.44 and 0.54, respectively. These results demonstrated that transgenic mouse lines were generated which express the Δ6-desaturase and Δ12-desaturase genes. The expression of these genes in the mammary gland of the lactating animal resulted in increased levels of several omega-3 and omega-6 fatty acids compared to those found in nontransgenic mouse milk.
EXAMPLE 5
This example describes the construction of a vector comprising nucleic acid encoding a human desaturase. Briefly a 1335-bp Ncol/Hind III DNA fragment encoding the human Δ5-desaturase CDNA (Figure 18) was isolated from plasmid pRAE-28-5 and ligated into plasmid pMTK-bGH-C (Figure 20), which had been cleaved with BgHland Pvuϊl. The termini of the DNA molecules were made flush using Klenow polymerase prior to ligation. The resulting plasmid, pMET- hΔ5-desaturase-bGHpA (Figure 21), utilizes the mMT-I TRE to direct hΔ5 -desaturase
transcription and the bGH polyadenylation signal for proper processing of the 3' terminus of the hΔ5 mRNA.
EXAMPLE 6
This example describes the construction of a vector comprising nucleic acid encoding an elongase. Briefly, a 1425-bp EcoRl DNA fragment encoding the M. alpina elongase cDNA (Figure 22) was isolated from plasmid pRAE-2 and ligated into plasmid pMTK-bGH-C (Figure 20), which had been previously cleaved with Bglll and Pvwll. The termini of the DNA molecules were made flush using Klenow polymerase prior to ligation. The resulting plasmid, pMET-Elongase-bGHpA (Figure 24), utilizes the mouse metallothionein-I transcriptional regulatory element (mMT-I TRE) to direct elongase transcription and the bGH polyadenylation signal for proper processing of the 3' terminus of the elongase rnRNA.
EXAMPLE 7
This example describes the transfection of cells with the vectors of Examples 5 and 6 and the resulting alteration in FA synthesis. Two stably transfected mouse L cell clones containing integrated human Δ5-desaturase sequences (hAS) and three stably transfected L cell clones which contain integrated sequences of both human Δ5-desaturase and M. Alpina elongase (hΔ5 plus elongase) were obtained. Since L cells expressing hΔ5 -desaturase and elongase would be expected to still require essential fatty acids, control (L), hΔ5 and the hΔ5 plus elongase cells were isolated from growing cultures (serum-containing culture medium). The cells were analyzed for hΔ5-desaturase and elongase activity by determining the levels of various saturated fatty acids as well as various omega-6 and omega-3 polyunsaturated fatty acids within the cells as a percentage of total fatty acid. The results are summarized in Table 6. The
Table 6
values given for hΔ5-desaturase represent the mean values of the two mouse L cell clones while those for hΔ5 plus elongase represent the mean values of the three mouse L cell clones.
The predicted substrates of human Δ5-desaturase activity are 20:3n-6 and 20-4n3. This does not rule out the possibility of, as to yet, unidentified substrates of the enzyme. Both mouse L cell clones containing stably integrated hΔ5 sequences contained significantly elevated levels of arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) compared to control L cells, with the mean increases being 254% and 68%), respectively. In addition, the hΔ5 clones also contained elevated levels of other downstream omega-6 and omega-3 PUFAs. There was a mean increase of 381% in level of 22:4n-6 in hΔ5 compared to control cells, while the levels of 22:5n-3 and 22:6n-3 were elevated by 223%> and 258%, respectively.
All mouse L cell clones containing stably integrated hΔ5 plus elongase sequences contained significantly elevated levels of a number of saturated fatty acids. The mean levels of 20:0, 22:0 and 24:0 were elevated by 113%,, 319% and 695%, respectively, in hΔ5 plus elongase compared to control L cells.
EXAMPLE 8
To further demonstrate hΔ5 -desaturase activity in the stably transfected mouse L cell clones, we incubated control and hΔ5 clones in serum-free culture medium and in serum-free culture medium containing 100 uM γ-linolenic acid (18:3n-6; GLA). Previous data on the Δ12-desaturase L cell clones indicated that the endogenous eleongase is active. Therefore, uptake of exogenously added GLA by mouse L cells should result in increased synthesis of di-homo-γ-linoleic acid (20:3n-6), thus providing an increased level of substrate for hΔ5-desaturase. The results are summarized in Table 7. In serum-free serum, the level of 20:3n-6 is similar between control and hΔ5 cells while each cell type contains undetectable levels of GLA. Human Δ5-desaturase activity is demonstrated by the 152% and 137% increase in the levels of 20:4n-6and 22:4n-6, respectively. Addition of GLA to the serum-free culture medium results in the detectable accumulation of GLA within both control and hΔ5 cells. In addition, the level of 20:3n-6 is elevated in both cell types confirming an active endogenous elongase. Human hΔ5 -desaturase activity is demonstrated by the 53%) reduction in the level of 20:3n-6 and concomminant increases in the levels of
20:4n-6 and 22:4n-6 (79% and 270%, respectively) in the hΔ5 clones compared to control L cells.
Table 7
* nd = not detected.
The above examples describe novel results obtained in cultured animal cells and transgenic animals, which were unexpected, and have not been described previously.
SEQUENCE LISTING
<110> Kopchick, John J. Kelder, Bruce
<120> Mammalian Cells Expressing Desaturases and Elongases
<130> OHU-03765
<140> 09/351,525 <141> 1999-07-12
<160> 10
<170> Patentln Ver. 2.0
<210> 1
<211> 1341
<212> DNA
<213> Mortierella Alpina
<400> 1 atgggaacgg accaaggaaa aaccttcacc tgggaagagc tggcggccca taacaccaag 60 gacgacctac tcttggccat ccgcggcagg gtgtacgatg tcacaaagtt cttgagccgc 120 catcctggtg gagtggacac tctcctgctc ggagctggcg agatgttact ccggtctttg 180 agatgtatca cgcgtttggg gctgcagatg ccattatgaa gaagtactat gtcggtacac 240 tggtctcgaa tgagctgccc atcttcccgg agccaacggt gttccacaaa accatcaaga 300 cgagagtcga gggctacttt acggatcgga acattgatcc caagaataga ccagagatct 360 ggggacgata cgctcttatc tttggatcct tgatcgcttc ctactacgcg cagctctttg 420 tgcctttcgt tgtcgaacgc acatggcttc aggtggtgtt tgcaatcatc atgggatttg 480 cgtgcgcaca agtcggactc aaccctcttc atgatgcgtc tcacttttca gtgacccaca 540 accccactgt ctggaagatt ctgggagcca cgcacgactt tttcaacgga gcatcgtacc 600 tggtgtggat gtaccaacat atgctcggcc atcaccccta caccaacatt gctggagcag 660 atcccgacgt gtcgacgtct gagcccgatg ttcgtcgtat caagcccaac caaaagtggt 720 ttgtcaacca catcaaccag cacatgtttg ttcctttcct gtacggactg ctggcgttca 780 aggtgcgcat tcaggacatc aacattttgt actttgtcaa gaccaatgac gctattcgtg 840 tcaatcccat ctcgacatgg cacactgtga tgttctgggg cggcaaggct ttctttgtct 900 ggtatcgcct gattgttccc ctgcagtatc tgcccctggg caaggtgctg ctcttgttca 960 cggtcgcgga catggtgtcg tcttactggc tggcgctgac cttccaggcg aaccacgttg 1020 ttgaggaagt tcagtggccg ttgcctgacg agaacgggat catccaaaag gactgggcag 1080 ctatgcaggt cgagactacg caggattacg cacacgattc gcacctctgg accagcatca 1140 ctggcagctt gaactaccag gctgtgcacc atctggttcc ccaacgtgtc gcagcaccat 1200 tatcccgata ttctggccat catcaagaac acctgcagcg agtacaaggt tccatacctt 1260 gtcaaggata cgttttggca agcatttgct tcacatttgg agcacttgcg tgttcttgga 1320 ctccgtccca aggaagagta g 1341
<210> 2
<211> 446
<212> PRT
<213> Mortierella Alpina
<400> 2
Met Gly Thr Asp Gin Gly Lys Thr Phe Thr Trp Glu Glu Leu Ala Ala 1 5 10 15
His Asn Thr Lys Asp Asp Leu Leu Leu Ala lie Arg Gly Arg Val Tyr 20 25 30
Asp Val Thr Lys Phe Leu Ser Arg His Pro Gly Gly Val Asp Thr Leu 35 40 45
Leu Leu Gly Ala Gly Arg Asp Val Thr Pro Val Phe Glu Met Tyr His 50 55 60
Ala Phe Gly Ala Ala Asp Ala He Met Lys Lys Tyr Tyr Val Gly Thr 65 70 75 80
Leu Val Ser Asn Glu Leu Pro He Phe Pro Glu Pro Thr Val Phe His 85 90 95
Lys Thr He Lys Thr Arg Val Glu Gly Tyr Phe Thr Asp Arg Asn He 100 105 110
Asp Pro Lys Asn Arg Pro Glu He Trp Gly Arg Tyr Ala Leu He Phe 115 120 125
Gly Ser Leu He Ala Ser Tyr Tyr Ala Gin Leu Phe Val Pro Phe Val 130 135 140
Val Glu Arg Thr Trp Leu Gin Val Val Phe Ala He He Met Gly Phe 145 150 155 160
Ala Cys Ala Gin Val Gly Leu Asn Pro Leu His Asp Ala Ser His Phe 165 170 175
Ser Val Thr His Asn Pro Thr Val Trp Lys He Leu Gly Ala Thr His 180 185 190
Asp Phe Phe Asn Gly Ala Ser Tyr Leu Val Trp Met Tyr Gin His Met 195 200 205
Leu Gly His His Pro Tyr Thr Asn He Ala Gly Ala Asp Pro Asp Val 210 215 220
Ser Thr Ser Glu Pro Asp Val Arg Arg He Lys Pro Asn Gin Lys Trp 225 230 235 240
Phe Val Asn His He Asn Gin His Met Phe Val Pro Phe Leu Tyr Gly 245 250 255
Leu Leu Ala Phe Lys Val Arg He Gin Asp He Asn He Leu Tyr Phe 260 265 270
Val Lys Thr Asn Asp Ala He Arg Val Asn Pro He Ser Thr Trp His 275 280 285
Thr Val Met Phe Trp Gly Gly Lys Ala Phe Phe Val Trp Tyr Arg Leu 290 295 300
He Val Pro Leu Gin Tyr Leu Pro Leu Gly Lys Val Leu Leu Leu Phe 305 310 315 320
Thr Val Ala Asp Met Val Ser Ser Tyr Trp Leu Ala Leu Thr Phe Gin 325 330 335
Ala Asn His Val Val Glu Glu Val Gin Trp Pro- Leai Pro Asp Glu Asn 340 345 350
Gly He He Gin Lys Asp Trp Ala Ala Met Gin Val Glu Thr Thr Gin 355 360 365
Asp Tyr Ala His Asp Ser His Leu Trp Thr Ser He Thr Gly Ser Leu 370 375 380
Asn Tyr Gin Ala Val His His Leu Phe Pro Asn Val Ser Gin His His 385 390 395 400
Tyr Pro Asp He Leu Ala He He Lys Asn Thr Cys Ser Glu Tyr Lys 405 410 415
Val Pro Tyr Leu Val Lys Asp Thr Phe Trp Gin Ala Phe Ala Ser His 420 425 430
Leu Glu His Leu Arg Val Leu Gly Leu Arg Pro Lys Glu Glu 435 440 445
<210> 3
<211> 1374
<212> DNA
<213> Mortierella Alpina
<400> 3 atggctgctg ctcccagtgt gaggacgttt actcgggccg aggttttgaa tgccgaggct 60 ctgaatgagg gcaagaagga tgccgaggca cccttcttga tgatcatcga caacaaggtg 120 tacgatgttc gcgagttcgt ccctgatcat cccggtggaa gtgtgattct cacgcacgtt 180 ggcaaggacg gcactgacgt ctttgacact tttcaccccg aggctgcttg ggagactctt 240 gccaactttt acgttggtga tattgacgag agcgaccgcg atatcaagaa tgatgacttt 300 gcggccgagg tccgcaagct gcgtaccttg ttccagtctc ttggttacta cgattcttcc 360 aaggcatact acgccttcaa ggtctcgttc aacctctgca tctggggttt gtcgacggtc 420 attgtggcca agtggggcca gacctcgacc ctcgccaacg tgctctcggc tgcgcttttg 480 ggtctgttct ggcagcagtg cggatggttg gctcacgact ttttgcatca ccaggtcttc 540 caggaccgtt tctggggtga tcttttcggc gccttcttgg gaggtgtctg ccagggcttc 600 tcgtcctcgt ggtggaagga caagcacaac actcaccacg ccgcccccaa cgtccacggc 660 gaggatcccg acattgacac ccaccctctg ttgacctgga gtgagcatgc gttggagatg 720 ttctcggatg tcccagatga ggagctgacc cgcatgtggt cgcgtttcat ggtcctgaac 780 cagacctggt tttacttccc cattctctcg tttgcccgtc tctcctggtg cctccagtcc 840 attctctttg tgctgcctaa cggtcaggcc cacaagccct cgggcgcgcg tgtgcccatc 900 tcgttggtcg agcagctgtc gcttgcgatg cactggacct ggtacctcgc caccatgttc 960 ctgttcatca aggatcccgt caacatgctg gtgtactttt tggtgtcgca ggcggtgtgc 1020 ggaaacttgt tggcgatcgt gttctcgctc aaccacaacg gtatgcctgt gatctcgaag 1080 gaggaggcgg tcgatatgga tttcttcacg aagcagatca tcacgggtcg tgatgtccac 1140 ccgggtctat ttgccaactg gttcacgggt ggattgaact atcagatcga gcaccacttg 1200 ttcccttcga tgcctcgcca caacttttca aagatccagc ctgctgtcga gaccctgtgc 1260 aaaaagtaca atgtccgata ccacaccacc ggtatgatcg agggaactgc agaggtcttt 1320 agccgtctga acgaggtctc caaggctgcc tccaagatgg gtaaggcgca gtaa 1374
<210> 4
<211> 457
<212> PRT
<213> Mortierella Alpina
<400> 4
Met Ala Ala Ala Pro Ser Val Arg Thr Phe Thr Arg Ala Glu Val Leu 1 5 10 15
Asn Ala Glu Ala Leu Asn Glu Gly Lys Lys Asp Ala Glu Ala Pro Phe 20 25 30
Leu Met He He Asp Asn Lys Val Tyr Asp Val Arg Glu Phe Val Pro 35 40 45
Asp His Pro Gly Gly Ser Val He Leu Thr His Val Gly Lys Asp Gly 50 55 60
Thr Asp Val Phe Asp Thr Phe His Pro Glu Ala Ala Trp Glu Thr Leu 65 70 75 80
Ala Asn Phe Tyr Val Gly Asp He Asp Glu Ser Asp Arg Asp He Lys 85 90 95
Asn Asp Asp Phe Ala Ala Glu Val Arg Lys Leu Arg Thr Leu Phe Gin 100 105 110
Ser Leu Gly Tyr Tyr Asp Ser Ser Lys Ala Tyr Tyr Ala Phe Lys Val 115 120 125
Ser Phe Asn Leu Cys He Trp Gly Leu Ser Thr Val He Val Ala Lys 130 135 140
Trp Gly Gin Thr Ser Thr Leu Ala Asn Val Leu Ser Ala Ala Leu Leu 145 150 155 160
Gly Leu Phe Trp Gin Gin Cys Gly Trp Leu Ala His Asp Phe Leu His 165 170 175
His Gin Val Phe Gin Asp Arg Phe Trp Gly Asp Leu Phe Gly Ala Phe 180 185 190
Leu Gly Gly Val Cys Gin Gly Phe Ser Ser Ser Trp Trp Lys Asp Lys 195 200 205
His Asn Thr His His Ala Ala Pro Asn Val His Gly Glu Asp Pro Asp 210 215 220
He Asp Thr His Pro Leu Leu Thr Trp Ser Glu His Ala Leu Glu Met 225 230 235 240
Phe Ser Asp Val Pro Asp Glu Glu Leu Thr Arg Met Trp Ser Arg Phe 245 250 255
Met Val Leu Asn Gin Thr Trp Phe Tyr Phe Pro He Leu Ser Phe Ala 260 265 270
Arg Leu Ser Trp Cys Leu Gin Ser He Leu Phe Val Leu Pro Asn Gly 275 280 285
Gin Ala His Lys Pro Ser Gly Ala Arg Val Pro He Ser Leu Val Glu 290 295 300
Gin Leu Ser Leu Ala Met His Trp Thr Trp Tyr Leu Ala Thr Met Phe 305 310 315 320
Leu Phe He Lys Asp Pro Val Asn Met Leu Val Tyr Phe Leu Val Ser 325 330 335
Gin Ala Val Cys Gly Asn Leu Leu Ala He Val Phe Ser Leu Asn His 340 345 350
Asn Gly Met Pro Val He Ser Lys Glu Glu Ala Val Asp Met Asp Phe 355 360 365
Phe Thr Lys Gin He He Thr Gly Arg Asp Val His Pro Gly Leu Phe 370 375 380
Ala Asn Trp Phe Thr Gly Gly Leu Asn Tyr Gin He Glu His His Leu 385 390 395 400
Phe Pro Ser Met Pro Arg His Asn Phe Ser Lys He Gin Pro Ala Val 405 410 415
Glu Thr Leu Cys Lys Lys Tyr Asn Val Arg Tyr His Thr Thr Gly Met 420 425 430
He Glu Gly Thr Ala Glu Val Phe Ser Arg Leu Asn Glu Val Ser Lys 435 440 445
Ala Ala Ser Lys Met Gly Lys Ala Gin 450 455
<210> 5
<211> 1200
<212> DNA
<213> Mortierella Alpina
<400> 5 atggcacctc ccaacactat cgatgccggt ttgacccagc gtcatatcag cacctcggcc 60 ccaaactcgg ccaagcctgc cttcgagcgc aactaccagc tccccgagtt caccatcaag 120 gagatccgag agtgcatccc tgcccactgc tttgagcgct ccggtctccg tggtctctgc 180 cacgttgcca tcgatctgac ttgggcgtcg ctcttgttcc tggctgcgac ccagatcgac 240 aagtttgaga atcccttgat ccgctatttg gcctggcctg tttactggat catgcagggt 300 attgtctgca ccggtgtctg ggtgctggct cacgagtgtg gtcatcagtc cttctcgacc 360 tccaagaccc tcaacaacac agttggttgg atcttgcact cgatgctctt ggtcccctac 420 cactcctgga gaatctcgca ctcgaagcac cacaaggcca ctggccatat gaccaaggac 480 caggtctttg tgcccaagac ccgctcccag gttggcttgc ctcccaagga gaacgctgct 540 gctgccgttc aggaggagga catgtccgtg cacctggatg aggaggctcc cattgtgact 600 ttgttctgga tggtgatcca gttcttgttc ggatggcccg cgtacctgat tatgaacgcc 660 tctggccaag actacggccg ctggacctcg cacttccaca cgtactcgcc catctttgag 720 ccccgcaact ttttcgacat tattatctcg gacctcggtg tgttggctgc cctcggtgcc 780 ctgatctatg cctccatgca gttgtcgctc ttgaccgtca ccaagtacta tattgtcccc 840 tacctctttg tcaacttttg gttggtcctg atcaccttct tgcagcacac cgatcccaag 900 ctgccccatt accgcgaggg tgcctggaat ttccagcgtg gagctctttg caccgttgac 960 cgctcgtttg gcaagttctt ggaccatatg ttccacggca ttgtccacac ccatgtggcc 1020 catcacttgt tctcgcaaat gccgttctac catgctgagg aagctaccta tcatctcaag 1080 aaactgctgg gagagtacta tgtgtacgac ccatccccga tcgtcgttgc ggtctggagg 1140 tcgttccgtg agtgccgatt cgtggaggat cagggagacg tggtcttttt caagaagtaa 1200
<210> 6 <211> 403 <212> PRT
<213> Mortierella Alpina
<400> 6
Met Ala Pro Pro Asn Thr He Asp Ala Gly Leu Thr Gin Arg His He 1 5 10 15
Ser Thr Ser Ala Pro Asn Ser Ala Lys Pro Ala Phe Glu Arg Asn Tyr 20 25 30
Gin Leu Pro Glu Phe Thr He Lys Glu He Arg Glu Cys He Pro Ala 35 40 45
His Cys Phe Glu Arg Ser Gly Leu Arg Gly Leu Cys His Val Ala He 50 55 60
Asp Leu Thr Trp Ala Ser Leu Leu Phe Leu Ala- Ala Thr Gin He Asp 65 70 75 80
Lys Phe Glu Asn Pro Leu He Arg Tyr Leu Ala Trp Pro Val Tyr Trp 85 90 ' 95
Leu Met Gin Gly He Val Cys Thr Gly Val Trp Val Leu Ala His Glu 100 105 110
Cys Gly His Gin Ser Phe Ser Thr Ser Lys Thr Leu Asn Asn Thr Val 115 120 125
Gly Trp He Leu His Ser Met Leu Leu Val Pro Tyr His Ser Trp Arg 130 135 140
He Ser His Ser Lys His His Lys Ala Thr Gly His Met Thr Lys Asp 145 150 155 160
Gin Val Phe Val Pro Lys Thr Arg Ser Gin Val Gly Leu Pro Pro Lys 165 170 175
Glu Asn Ala Ala Ala Ala Val Gin Glu Glu Asp Met Ser Val His Leu 180 185 190
Asp Glu Glu Ala Pro He Val Thr Leu Phe Trp Met Val He Gin Phe 195 200 205
Leu Phe Gly Trp Pro Ala Tyr Leu He Met Asn Ala Ser Gly Gin Asp 210 215 220
Tyr Gly Arg Trp Thr Ser His His His. Thr Tyr Tyr Tyr Tyr Ser Pro 225 230 23"5 240
He Phe Glu Pro Arg Asn Phe Phe Asp He He He Ser Asp Leu Gly 245 250 255
Val Leu Ala Ala Leu Gly Ala Leu He Tyr Ala Ser Met Gin Leu Ser 260 265 270
Leu Leu Thr Val Thr Lys Tyr Tyr He Val Pro Tyr Leu Phe Val Asn 275 280 285
Phe Trp Leu Val Leu He Thr Phe Phe Leu Gin His Thr Asp Pro Lys 290 295 300
Leu Pro His Tyr Arg Glu Gly Ala Trp Asn Phe Gin Arg Gly Ala Leu 305 310 315 320
Cys Thr Val Asp Arg Ser Phe Gly Lys Phe Leu Asp His Met Phe His 325 330 335
Gly He Val His Thr His Val Ala His His Leu Phe Ser Gin Met Pro 340 345 350
Phe Tyr His Ala Glu Glu Ala Thr Tyr His Leu Lys Lys Leu Leu Gly 355 360 365
Glu Tyr Tyr Val Tyr Asp Pro Ser Pro He Val Val Ala Val Trp Arg 370 375 380
Ser Phe Arg Glu Cys Arg Phe Val Glu Asp Gin Gly Asp Val Val Phe 385 390 395 400
Phe Lys Lys
<210> 7
<211> 1335
<212> DNA
<213> Homo sapiens
<400> 7 atggcccccg acccggtggc cgccgagacc gcggctcagg gacctacccc gcgctacttc 60 acctgggacg aggtggccca gcgctcaggg tgcgaggagc ggtggctagt gatcgaccgt 120 aaggtgtaca acatcagcga gttcacccgc cggcatccag ggggctcccg ggtcatcagc 180 cactacgccg ggcaggatgc cacggatccc tttgtggcct tccacatcaa caagggcctt 240 gtgaagaagt atatgaactc tctcctgatt ggagaactgt ctccagagca gcccagcttt 300 gagcccacca agaataaaga gctgacagat gagttccggg agctgcgggc cacagtggag 360 cggatggggc tcatgaaggc caaccatgtc ttcttcctgc tgtacctgct gcacatcttg 420 ctgctggatg gtgcagcctg gctcaccctt tgggtctttg ggacgtcctt tttgcccttc 480 ctcctctgtg cggtgctgct cagtgcagtt caggcccagg ctggctggct gcagcatgac 540 tttgggcacc tgtcggtctt cagcacctca aagtggaacc atctgctaca tcattttgtg 600 attggccacc tgaagggggc ccccgccagt tggtggaacc acatgcactt ccagcaccat 660 gccaagccca actgcttccg caaagaccca gacatcaaca tgcatccctt cttctttgcc 720 ttggggaaga tcctctctgt ggagcttggg aaacagaaga aaaaatatat gccgtacaac 780 caccagcaca aatacttctt cctaattggg cccccagcct tgctgcctct ctacttccag 840 tggtatattt tctattttgt tatccagcga aagaagtggg tggacttggc ctggatgatt 900 accttctacg tccgcttctt cctcacttat gtgccactat tggggctgaa agccttcctg 960 ggccttttct tcatagtcag gttcctggaa agcaactggt ttgtgtgggt gacacagatg 1020 aaccatattc ccatgcacat tgatcatgac cggaacatgg actgggtttc cacccagctc 1080 ctggccacat gcaatgtcca caagtctgcc ttcaatgact ggttcagtgg acacctcaac 1140 ttccagattg agcaccatct ttttcccacg atgcctcgac acaattacca caaagtggct 1200 cccctggtgc agtccttgtg tgccaagcgt ggcatagagt accagtccaa gcccctgctg 1260 tcagccttcg ccgacatcat ccactcacta aaggagtcag ggcagctctg gctagatgcc 1320 tatcttcacc aataa 1335
<210> 8
<211> 444
<212> PRT
<213> Homo sapiens
<400> 8
Met Ala Pro Asp Pro Val Ala Ala Glu Thr Ala Ala Gin Gly Pro Thr 1 5 10 15
Pro Arg Tyr Phe Thr Trp Asp Glu Val Ala Gin Arg Ser Gly Cys Glu 20 25 30
Glu Arg Trp Leu Val He Asp Arg Lys Val Tyr Asn He Ser Glu Phe 35 40 45
Thr Arg Arg His Pro Gly Gly Ser Arg Val He Ser His Tyr Ala Gly 50 55 60
Gin Asp Ala Thr Asp Pro Phe Val Ala Phe His He Asn Lys Gly Leu 65 70 75 80
Val Lys Lys Tyr Met Asn Ser Leu Leu He Gly Glu Leu Ser Pro Glu 85 90 95
Gin Pro Ser Phe Glu Pro Thr Lys Asn Lys Glu Leu Thr Asp Glu Phe 100 105 110
Arg Glu Leu Arg Ala Thr Val Glu Arg Met Gly Leu Met Lys Ala Asn 115 120 125
His Val Phe Phe Leu Leu Tyr Leu Leu His He Leu Leu Leu Asp Gly 130 135 140
Ala Ala Trp Leu Thr Leu Trp Val Phe Gly Thr Ser Phe Leu Pro Phe 145 150 155 160
Leu Leu Cys Ala Val Leu Leu Ser Ala Val Gin Ala Gin Ala Gly Trp 165 170 175
Leu Gin His Asp Phe Gly His Leu Ser Val Phe Ser Thr Ser Lys Trp 180 185 190
Asn His Leu Leu His His Phe Val He Gly His Leu Lys Gly Ala Pro 195 200 205
Ala Ser Trp Trp Asn His Met His Phe Gin His His Ala Lys Pro Asn 210 215 220
Cys Phe Arg Lys Asp Pro Asp He Asn Met His Pro Phe Phe Phe Ala 225 230 235 240
Leu Gly Lys He Leu Ser Val Glu Leu Gly Lys Gin Lys Lys Lys Tyr 245 250 255
Met Pro Tyr Asn His Gin His Lys Tyr Phe Phe Leu He Gly Pro Pro 260 265 270
Ala Leu Leu Pro Leu Tyr Phe Gin Trp Tyr He Phe Tyr Phe Val He 275 280 285
Gin Arg Lys Lys Trp Val Asp Leu Ala Trp Met He Thr Phe Tyr Val 290 295 300
Arg Phe Phe Leu Thr Tyr Val Pro Leu Leu Gly Leu Lys Ala Phe Leu 305 310 315 320
Gly Leu Phe Phe He Val Arg Phe Leu Glu Ser Asn Trp Phe Val Trp 325 330 335
Val Thr Gin Met Asn His He Pro Met His He Asp His Asp Arg Asn 340 345 350
Met Asp Trp Val Ser Thr Gin Leu Leu Ala Thr Cys Asn Val His Lys 355 360 365
Ser Ala Phe Asn Asp Trp Phe Ser Gly His Leu Asn Phe Gin He Glu 370 375 380
His His Leu Phe Pro Thr Met Pro Arg His Asn Tyr His Lys Val Ala 385 390 395 400
Pro Leu Val Gin Ser Leu Cys Ala Lys Arg Gly He Glu Tyr Gin Ser 405 410 415
Lys Pro Leu Leu Ser Ala Phe Ala Asp He He His Ser Leu Lys Glu 420 425 430
Ser Gly Gin Leu Trp Leu Asp Ala Tyr Leu His Gin 435 440
<210> 9
<211> 954
<212> DNA
<213> Mortierella Alpina
<400> 9 atggccgccg caatcttgga caaggtcaac ttcggcattg atcagccctt cggaatcaag 60 ctcgacacct actttgctca ggcctatgaa ctcgtcaccg gaaagtccat cgactccttc 120 gtcttccagg agggcgtcac gcctctctcg acccagagag aggtcgccat gtggactatc 180 acttacttcg tcgtcatctt tggtggtcgc cagatcatga agagccagga cgccttcaag 240 ctcaagcccc tcttcatcct ccacaacttc ctcctgacga tcgcgtccgg atcgctgttg 300 ctcctgttca tcgagaacct ggtccccatc ctcgccagaa acggactttt ctacgccatc 360 tgcgacgacg gtgcctggac ccagcgcctc gagctcctct actacctcaa ctacctggtc 420 aagtactggg agttggccga caccgtcttt ttggtcctca agaagaagcc tcttgagttc 480 ctgcactact tccaccactc gatgaccatg gttctctgct ttgtccagct tggaggatac 540 acttcagtgt cctgggtccc tattaccctc aacttgactg tccacgtctt catgtactac 600 tactacatgc gctccgctgc cggtgttcgc atctggtgga agcagtactt gaccactctc 660 cagatcgtcc agttcgttct tgacctcgga ttcatctact tctgcgccta cacctacttc 720 gccttcacct acttcccctg ggctcccaac gtcggcaagt gcgccggtac cgagggtgct 780 gctctctttg gctgcggact cctctccagc tatctcttgc tctttatcaa cttctaccgc 840 attacctaca atgccaaggc caaggcagcc aaggagcgtg gaagcaactt tacccccaag 900 actgtcaagt ccggcggatc gcccaagaag ccctccaaga gcaagcacat ctaa 954
<210> 10
<211> 317
<212> PRT
<213> Mortierella Alpina
<400> 10
Met Ala Ala Ala He Leu Asp Lys Val Asn Phe Gly He Asp Gin Pro 1 5 10 15
Phe Gly He Lys Leu Asp Thr Tyr Phe Ala Gin Ala Tyr Glu Leu Val 20 25 30
Thr Gly Lys Ser He Asp Ser Phe Val Phe Gin Glu Gly Val Thr Pro 35 40 45
Leu Ser Thr Gin Arg Glu Val Ala Met Trp Thr He Thr Tyr Phe Val 50 55 60
Val He Phe Gly Gly Arg Gin He Met Lys Ser Gin Asp Ala Phe Lys 65 70 75 80
Leu Lys Pro Leu Phe He Leu His Asn Phe Leu Leu Thr He Ala Ser 85 90 95
Gly Ser Leu Leu Leu Leu Phe He Glu Asn Leu Val Pro He Leu Ala 100 105 110
Arg Asn Gly Leu Phe Tyr Ala He Cys Asp Asp Gly Ala Trp Thr Gin 115 120 125
Arg Leu Glu Leu Leu Tyr Tyr Leu Asn Tyr Leu Val Lys Tyr Trp Glu 130 135 140
Leu Ala Asp Thr Val Phe Leu Val Leu Lys Lys Lys Pro Leu Glu Phe 145 150 155 160
Leu His Tyr Phe His His Ser Met Thr Met Val Leu Cys Phe Val Gin 165 170 175
Leu Gly Gly Tyr Thr Ser Val Ser Trp Val Pro He Thr Leu Asn Leu 180 185 190
Thr Val His Val Phe Met Tyr Tyr Tyr Tyr Met Arg Ser Ala Ala Gly 195 200 205
Val Arg He Trp Trp Lys Gin Tyr Leu Thr Thr Leu Gin He Val Gin 210 215 220
Phe Val Leu Asp Leu Gly Phe He Tyr Phe Cys Ala Tyr Thr Tyr Phe 225 230 235 240
Ala Phe Thr Tyr Phe Pro Trp Ala Pro Asn Val Gly Lys Cys Ala Gly 245 250 255
Thr Glu Gly Ala Ala Leu Phe Gly Cys Gly Leu Leu Ser Ser Tyr Leu 260 265 270
Leu Leu Phe He Asn Phe Tyr Arg He Thr Tyr Asn Ala Lys Ala Lys 275 280 285
Ala Ala Lys Glu Arg Gly Ser Asn Phe Thr Pro Lys Thr Val Lys Ser 290 295 300
Gly Gly Ser Pro Lys Lys Pro Ser Lys Ser Lys His He 305 310 315
Claims
1. A composition, comprising animal cells transfected with a vector comprising a human desaturase gene.
2. The composition of Claim 1, wherein said cells produce altered levels of long-chain polyunsaturated fatty acids relative to the animal cells prior to transfection.
3. The composition of Claim 1, wherein said human desaturase gene comprises a human Δ5 -desaturase.
4. The composition of Claim 1, wherein said cells are in tissue culture.
5. The composition of Claim 1, wherein said cells are in a bioreactor.
6. A nutritional formulation, comprising at least one long-chain polyunsaturated fatty acid derived from the animal cells of Claim 2.
7. A cosmetic formulation, comprising at least one long-chain polyunsaturated fatty acid derived from the animal cells of Claim 2.
8. An animal feed formulation, comprising at least one long-chain polyunsaturated fatty acid derived from the animal cells of Claim 2.
9. A composition, comprising animal cells transfected with a first vector comprising a desaturase gene and a second vector comprising an elongase gene.
10. The composition of Claim 9, wherein said cells produce altered levels of long-chain polyunsaturated fatty acids relative to the animal cells prior to transfection.
1 1. The composition of Claim 9, wherein said human desaturase gene comprises a human Δ5-desaturase.
12. The composition of Claim 9, wherein said elongase gene is a non- human elongase gene.
13. The composition of Claim 12, wherein said non-human elongase gene is a Mortierella Alpina elongase gene.
14. The composition of Claim 9, wherein said cells are in tissue culture.
15. The composition of Claim 9, wherein said cells are in a bioreactor.
16. A nutritional formulation, comprising at least one long-chain polyunsaturated fatty acid derived from the animal cells of Claim 10.
17. A cosmetic formulation, comprising at least one long-chain polyunsaturated fatty acid derived from the animal cells of Claim 10.
18. An animal feed formulation, comprising at least one long-chain polyunsaturated fatty acid derived from the animal cells of Claim 10.
19. A composition, comprising animal cells transfected with a vector comprising a desaturase gene and an elongase gene.
20. The composition of Claim 19, wherein said cells produce altered levels of long-chain polyunsaturated fatty acids relative to the animal cells prior to transfection.
21. The composition of Claim 19, wherein said human desaturase gene comprises a human Δ5-desaturase.
22. The composition of Claim 19, wherein said elongase gene is a non- human elongase gene.
23. The composition of Claim 19, wherein said non-human elongase gene is a Mortierella Alpina elongase gene.
24. The composition of Claim 19, wherein said cells are in tissue culture.
25. The composition of Claim 19, wherein said cells are in a bioreactor.
26. A nutritional formulation, comprising at least one long-chain polyunsaturated fatty acid derived from the animal cells of Claim 20.
27. A cosmetic formulation, comprising at least one long-chain polyunsaturated fatty acid derived from the animal cells of Claim 20.
28. An animal feed formulation, comprising at least one long-chain polyunsaturated fatty acid derived from the animal cells of Claim 20.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU60916/00A AU6091600A (en) | 1999-07-12 | 2000-07-11 | Mammalian cells expressing desaturases and elongases |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US35152599A | 1999-07-12 | 1999-07-12 | |
US09/351,525 | 1999-07-12 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2001004636A1 true WO2001004636A1 (en) | 2001-01-18 |
Family
ID=23381283
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/019011 WO2001004636A1 (en) | 1999-07-12 | 2000-07-11 | Mammalian cells expressing desaturases and elongases |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU6091600A (en) |
WO (1) | WO2001004636A1 (en) |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002044320A2 (en) * | 2000-11-29 | 2002-06-06 | Xenon Genetics Inc. | Human elongase genes and uses thereof |
WO2002064761A2 (en) * | 2001-02-09 | 2002-08-22 | Bayer Aktiengesellschaft | Regulation of human long chain polyunsaturated fatty acid elongation enzyme-like protein |
EP1401259A2 (en) * | 2001-05-04 | 2004-03-31 | Abbott Laboratories | Delta-4 desaturase genes and uses thereof |
WO2004079844A1 (en) * | 2003-03-06 | 2004-09-16 | Toray Industries, Inc. | Polymer electrolyte material, polymer electrolyte part, membrane electrode composite and polymer electrolyte type fuel cell |
US7736884B2 (en) | 2004-06-04 | 2010-06-15 | Fluxome Sciences A/S | Metabolically engineered Saccharomyces cells for the production of polyunsaturated fatty acids |
US7807849B2 (en) | 2004-04-22 | 2010-10-05 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US7834250B2 (en) | 2004-04-22 | 2010-11-16 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US8816111B2 (en) | 2012-06-15 | 2014-08-26 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising polyunsaturated fatty acids |
US9718759B2 (en) | 2013-12-18 | 2017-08-01 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising docosapentaenoic acid |
US9938486B2 (en) | 2008-11-18 | 2018-04-10 | Commonwealth Scientific And Industrial Research Organisation | Enzymes and methods for producing omega-3 fatty acids |
US10005713B2 (en) | 2014-06-27 | 2018-06-26 | Commonwealth Scientific And Industrial Research Organisation | Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the sn-2 position |
US10513717B2 (en) | 2006-08-29 | 2019-12-24 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of fatty acids |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998046764A1 (en) * | 1997-04-11 | 1998-10-22 | Calgene Llc | Methods and compositions for synthesis of long chain polyunsaturated fatty acids in plants |
WO1998046765A1 (en) * | 1997-04-11 | 1998-10-22 | Calgene Llc | Methods and compositions for synthesis of long chain polyunsaturated fatty acids |
WO1998046763A1 (en) * | 1997-04-11 | 1998-10-22 | Calgene Llc | Methods and compositions for synthesis of long chain polyunsaturated fatty acids |
-
2000
- 2000-07-11 AU AU60916/00A patent/AU6091600A/en not_active Abandoned
- 2000-07-11 WO PCT/US2000/019011 patent/WO2001004636A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998046764A1 (en) * | 1997-04-11 | 1998-10-22 | Calgene Llc | Methods and compositions for synthesis of long chain polyunsaturated fatty acids in plants |
WO1998046765A1 (en) * | 1997-04-11 | 1998-10-22 | Calgene Llc | Methods and compositions for synthesis of long chain polyunsaturated fatty acids |
WO1998046763A1 (en) * | 1997-04-11 | 1998-10-22 | Calgene Llc | Methods and compositions for synthesis of long chain polyunsaturated fatty acids |
Non-Patent Citations (3)
Title |
---|
CHO ET AL.: "Cloning, expression and fatty acid regulation of the human alpha-5 desaturase", J. BIOL. CHEM., vol. 274, no. 52, 24 December 1999 (1999-12-24), pages 37335 - 37339, XP002933429 * |
CHO ET AL.: "Cloning, expression and nutritional regulation of the mammalian alpha-6 desaturase", J. BIOL. CHEM., vol. 274, no. 1, 1 January 1999 (1999-01-01), pages 471 - 477, XP002933430 * |
KNUTZON ET AL.: "Identification of alpha-5-desaturase from mortierella alpina by heterologous expression in bakers yeast and canola", J. BIOL. CHEM., vol. 273, no. 45, 5 November 1998 (1998-11-05), pages 29360 - 29366, XP002933431 * |
Cited By (43)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002044320A2 (en) * | 2000-11-29 | 2002-06-06 | Xenon Genetics Inc. | Human elongase genes and uses thereof |
WO2002044320A3 (en) * | 2000-11-29 | 2003-01-09 | Xenon Genetics Inc | Human elongase genes and uses thereof |
US7297523B2 (en) | 2000-11-29 | 2007-11-20 | Xenon Pharmaceuticals Inc. | Human elongase genes and uses thereof |
WO2002064761A2 (en) * | 2001-02-09 | 2002-08-22 | Bayer Aktiengesellschaft | Regulation of human long chain polyunsaturated fatty acid elongation enzyme-like protein |
WO2002064761A3 (en) * | 2001-02-09 | 2003-05-01 | Bayer Ag | Regulation of human long chain polyunsaturated fatty acid elongation enzyme-like protein |
EP1401259A2 (en) * | 2001-05-04 | 2004-03-31 | Abbott Laboratories | Delta-4 desaturase genes and uses thereof |
EP1401259A4 (en) * | 2001-05-04 | 2005-06-22 | Abbott Lab | Delta-4 desaturase genes and uses thereof |
WO2004079844A1 (en) * | 2003-03-06 | 2004-09-16 | Toray Industries, Inc. | Polymer electrolyte material, polymer electrolyte part, membrane electrode composite and polymer electrolyte type fuel cell |
US9994880B2 (en) | 2004-04-22 | 2018-06-12 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cell |
US9458410B2 (en) | 2004-04-22 | 2016-10-04 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cell |
US7834250B2 (en) | 2004-04-22 | 2010-11-16 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US7932438B2 (en) | 2004-04-22 | 2011-04-26 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US8071341B2 (en) | 2004-04-22 | 2011-12-06 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US8106226B2 (en) | 2004-04-22 | 2012-01-31 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US8158392B1 (en) | 2004-04-22 | 2012-04-17 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US11597953B2 (en) | 2004-04-22 | 2023-03-07 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US8853432B2 (en) | 2004-04-22 | 2014-10-07 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cell |
US11220698B2 (en) | 2004-04-22 | 2022-01-11 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US9453183B2 (en) | 2004-04-22 | 2016-09-27 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cell |
US9970033B2 (en) | 2004-04-22 | 2018-05-15 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cell |
US10781463B2 (en) | 2004-04-22 | 2020-09-22 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US9963723B2 (en) | 2004-04-22 | 2018-05-08 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US10443079B2 (en) | 2004-04-22 | 2019-10-15 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cell |
US9951357B2 (en) | 2004-04-22 | 2018-04-24 | Commonweatlh Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cell |
US9926579B2 (en) | 2004-04-22 | 2018-03-27 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cell |
US7807849B2 (en) | 2004-04-22 | 2010-10-05 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
US7736884B2 (en) | 2004-06-04 | 2010-06-15 | Fluxome Sciences A/S | Metabolically engineered Saccharomyces cells for the production of polyunsaturated fatty acids |
US10513717B2 (en) | 2006-08-29 | 2019-12-24 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of fatty acids |
US9938486B2 (en) | 2008-11-18 | 2018-04-10 | Commonwealth Scientific And Industrial Research Organisation | Enzymes and methods for producing omega-3 fatty acids |
US9932289B2 (en) | 2012-06-15 | 2018-04-03 | Commonwealth Scientific And Industrial Research Ogranisation | Process for producing ethyl esters of polyunsaturated fatty acids |
US9556102B2 (en) | 2012-06-15 | 2017-01-31 | Commonwealth Scientific And Industrial Research Organisation | Process for producing ethyl esters of polyunsaturated fatty acids |
US8816111B2 (en) | 2012-06-15 | 2014-08-26 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising polyunsaturated fatty acids |
US8946460B2 (en) | 2012-06-15 | 2015-02-03 | Commonwealth Scientific And Industrial Research Organisation | Process for producing polyunsaturated fatty acids in an esterified form |
US9550718B2 (en) | 2012-06-15 | 2017-01-24 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising polyunsaturated fatty acids |
US10335386B2 (en) | 2012-06-15 | 2019-07-02 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising polyunsaturated fatty acids |
US10190073B2 (en) | 2013-12-18 | 2019-01-29 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising long chain polyunsaturated fatty acids |
US9718759B2 (en) | 2013-12-18 | 2017-08-01 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising docosapentaenoic acid |
US9725399B2 (en) | 2013-12-18 | 2017-08-08 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising long chain polyunsaturated fatty acids |
US10800729B2 (en) | 2013-12-18 | 2020-10-13 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising long chain polyunsaturated fatty acids |
US10125084B2 (en) | 2013-12-18 | 2018-11-13 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising docosapentaenoic acid |
US11623911B2 (en) | 2013-12-18 | 2023-04-11 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising docosapentaenoic acid |
US10793507B2 (en) | 2014-06-27 | 2020-10-06 | Commonwealth Scientific And Industrial Research Organisation | Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the SN-2 position |
US10005713B2 (en) | 2014-06-27 | 2018-06-26 | Commonwealth Scientific And Industrial Research Organisation | Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the sn-2 position |
Also Published As
Publication number | Publication date |
---|---|
AU6091600A (en) | 2001-01-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6410288B1 (en) | Methods and compositions for synthesis of long chain poly-unsaturated fatty acids | |
US6589767B1 (en) | Methods and compositions for synthesis of long chain polyunsaturated fatty acids | |
US6051754A (en) | Methods and compositions for synthesis of long chain poly-unsaturated fatty acids in plants | |
US6075183A (en) | Methods and compositions for synthesis of long chain poly-unsaturated fatty acids in plants | |
JP5501952B2 (en) | Δ8 desaturases and their use in making polyunsaturated fatty acids | |
US6459018B1 (en) | Polyunsaturated fatty acids in plants | |
US5589369A (en) | Cells homozygous for disrupted target loci | |
KR101138631B1 (en) | Fatty acid desaturases from fungi | |
US8221819B2 (en) | Fatty acid desaturases from primula | |
JP4997544B2 (en) | Mammalian artificial chromosome vector containing human cytochrome P450 gene (cluster) and non-human mammal having the same | |
US20030159164A1 (en) | Compositions and methods for the synthesis of fatty acids, their derivatives and downstream products | |
WO1999064616A2 (en) | Polyunsaturated fatty acids in plants | |
WO1995003402A1 (en) | EXPRESSION OF HUMAN INTERLEUKIN-1β IN A TRANSGENIC ANIMAL | |
WO2001004636A1 (en) | Mammalian cells expressing desaturases and elongases | |
EP1507855B2 (en) | Elongase genes and uses thereof | |
EP0527063A1 (en) | Production of protein of interest in the milk of transgenic mammal | |
WO2005094570A1 (en) | Transgenic fish with increased unsaturated fatty acid content | |
US7238851B2 (en) | Non-human transgenic mammals expressing an n-3 desaturase gene | |
Lake-Bullock et al. | Biphasic ordered induction of heme synthesis in differentiating murine erythroleukemia cells: role of erythroid 5-aminolevulinate synthase | |
US10182560B2 (en) | Transgene construct encoding delta 12 fatty acid | |
JP2009055876A (en) | Fatty acid desaturase gene, recombinant expression vector, non-human animal cell and non-human animal transferred with the gene | |
Indo et al. | Functional expression of a humanized gene for an ω-3 fatty acid desaturase from scarlet flax in transfected bovine adipocytes and bovine embryos cloned from the cells | |
WO2006085673A1 (en) | Hairless transgenic animal | |
JP2009510997A (en) | Delta-6 desaturase from Thraustochytrid and uses thereof | |
CA2242382C (en) | Compositions and methods for mediating cell cycle progression |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: JP |