EP1144444A2 - Adn complementaires codant pour des proteines secretees avec des peptides signaux - Google Patents
Adn complementaires codant pour des proteines secretees avec des peptides signauxInfo
- Publication number
- EP1144444A2 EP1144444A2 EP99959633A EP99959633A EP1144444A2 EP 1144444 A2 EP1144444 A2 EP 1144444A2 EP 99959633 A EP99959633 A EP 99959633A EP 99959633 A EP99959633 A EP 99959633A EP 1144444 A2 EP1144444 A2 EP 1144444A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- sequence
- sequences
- seq
- protein
- cdna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
Definitions
- cDNAs complementary DNAs
- mRNAs messenger RNAs
- sequencing is only performed on DNA which is derived from protein coding fragments of the genome.
- ESTs expressed sequence tags
- the ESTs may then be used to isolate or purify cDNAs which include sequences adjacent to the EST sequences.
- the cDNAs may contain all of the sequence of the EST which was used to obtain them or only a fragment of the sequence of the EST which was used to obtain them.
- the cDNAs may contain the full coding sequence of the gene from which the EST was derived or, alternatively, the cDNAs may include fragments of the coding sequence of the gene from which the EST was derived. It will be appreciated that there may be several cDNAs which include the EST sequence as a result of alternate splicing or the activity of alternative promoters. In the past, these short EST sequences were often obtained from oligo-dT primed cDNA libraries.
- 5'UTRs have been shown to affect either the stability or translation of mRNAs.
- regulation of gene expression may be achieved through the use of alternative 5'UTRs as shown, for instance, for the translation of the tissue inhibitor of metalloprotease mRNA in mitogenically activated cells (Waterhouse ef al, J Biol Chem. 265:5585-9. 1990).
- modification of 5'UTR through mutation, insertion or translocation events may even be implied in pathogenesis.
- the 5 fragile X syndrome the most common cause of inherited mental retardation, is partly due to an insertion of multiple CGG trinucleotides in the 5'UTR of the fragile X mRNA resulting in the inhibition of protein synthesis via ribosome stalling (Feng ef al., Science 268:731-4, 1995).
- An aberrant mutation in regions of the 5'UTR known to inhibit translation of the proto-oncogene c-myc was shown to result in upregulation of c-myc protein levels in cells derived from patients with multiple myelomas (Willis ef al., Curr Top Microbiol Immunol
- oligo-dT primed cDNA libraries does not allow the isolation of complete 5'UTRs since such incomplete sequences obtained by this process may not include the first exon of the mRNA, particularly in situations where the first exon is short. Furthermore, they may not include some exons, often short ones, which are located upstream of splicing sites. Thus, there is a need to obtain sequences derived from the 5' ends of mRNAs.
- proteins which are secreted from the cell in which they are synthesized, as well as the secreted proteins themselves, are particularly valuable as potential therapeutic agents. Such proteins are often involved in cell to cell communication and may be responsible for producing a clinically relevant response in their target cells.
- tissue plasminogen activator G-CSF
- GM-CSF GM-CSF
- erythropoietin human growth hormone
- insulin interferon-D
- interferon-D interferon-D
- interferon-O, and interleukin-2 are currently in clinical use. These proteins are used to treat a wide range of conditions, including acute myocardial infarction, acute ischemic stroke, anemia, diabetes, growth hormone deficiency, hepatitis, kidney carcinoma, chemotherapy induced neutropenia and multiple sclerosis. For these reasons, cDNAs encoding secreted proteins or fragments thereof represent a particularly valuable source of therapeutic agents. Thus, there is a need for the identification and characterization of secreted proteins and the nucleic acids encoding them.
- secretory proteins include short peptides, called signal peptides, at their amino termini which direct their secretion.
- signal peptides are encoded by the signal sequences located at the 5' ends of the coding sequences of genes encoding secreted proteins. Because these signal peptides will direct the extracellular secretion of any protein to which they are operably linked, the signal sequences may be exploited to direct the efficient secretion of any protein by operably linking the signal sequences to a gene encoding the protein for which secretion is desired.
- fragments of the signal peptides called membrane-translocating sequences, may also be used to direct the intracellular import of a peptide or protein of interest.
- signal sequences encoding signal peptides also find application in simplifying protein purification techniques. In such applications, the extracellular secretion of the desired protein greatly facilitates purification by reducing the number of undesired proteins from which the desired protein must be selected. Thus, there exists a need to identify and characterize the 5' fragments of the genes for secretory proteins which encode signal peptides.
- Sequences coding for secreted proteins may also find application as therapeutics or diagnostics.
- such sequences may be used to determine whether an individual is likely to express a detectable phenotype, such as a disease, as a consequence of a mutation in the coding sequence for a secreted protein.
- the undesirable phenotype may be corrected by introducing a normal coding sequence using gene therapy.
- expression of the protein may be reduced using antisense or triple helix based strategies.
- the secreted human polypeptides encoded by the coding sequences may also be used as therapeutics by administering them directly to an individual having a condition, such as a disease, resulting from a mutation in the sequence encoding the polypeptide. In such an instance, the condition can be cured or ameliorated by administering the polypeptide to the individual.
- the secreted human polypeptides or fragments thereof may be used to generate antibodies useful in determining the tissue type or species of origin of a biological sample.
- the antibodies may also be used to determine the cellular localization of the secreted human polypeptides or the cellular localization of polypeptides which have been fused to the human polypeptides.
- the antibodies may also be used in immunoaffinity chromatography techniques to isolate, purify, or enrich the human polypeptide or a target polypeptide which has been fused to the human polypeptide.
- cDNAs including the 5' ends of their corresponding mRNA may be used to efficiently identify and isolate 5'UTRs and upstream regulatory regions which control the location, developmental stage, rate, and quantity of protein synthesis, as well as the stability of the mRNA (Theil et al., BioFactors 4:87-93, (1993). Once identified and characterized, these regulatory regions may be utilized in gene therapy or protein purification schemes to obtain the desired amount and locations of protein synthesis or to inhibit, reduce, or prevent the synthesis of undesirable gene products.
- cDNAs containing the 5' ends of secretory protein genes may include sequences useful as probes for chromosome mapping and the identification of individuals. Thus, there is a need to identify and characterize the sequences upstream of the 5' coding sequences of genes encoding secretory proteins.
- the present invention relates to purified, isolated, or recombinant cDNAs which encode secreted proteins or fragments thereof.
- the purified, isolated or recombinant cDNAs contain the entire open reading frame of their corresponding mRNAs, including a start codon and a stop codon.
- the cDNAs may include nucleic acids encoding the signal peptide as well as the mature protein. Such cDNAs will be referred herein as "full-length" cDNAs.
- the cDNAs may contain a fragment of the open reading frame. Such cDNAs will be referred herein as "ESTs" or "5'ESTs".
- the fragment may encode only the sequence of the mature protein.
- the fragment may encode only a fragment of the mature protein.
- a further aspect of the present invention is a nucleic acid which encodes the signal peptide of a secreted protein.
- corresponding mRNA refers to the mRNA which was the template for the cDNA synthesis which produced the cDNA of the present invention.
- purified does not require absolute purity; rather, it is intended as a relative definition.
- Individual cDNA clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. The sequences obtained from these clones could not be obtained directly either from the library or from total human DNA.
- the cDNA clones are not naturally occurring as such, but rather are obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
- the conversion of mRNA into a cDNA library involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection.
- cDNA synthetic substance
- creating a cDNA library from messenger RNA and subsequently isolating individual clones from that library results in an approximately 10 0 6 fold purification of the native message.
- Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
- isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
- a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
- the term “recombinant” means that the cDNA is adjacent to "backbone" nucleic acid to which it is not adjacent in its natural environment. Additionally, to be “enriched” the cDNAs will represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid backbone molecules.
- Backbone molecules according to the present invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.
- the enriched cDNAs represent 15% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
- the enriched cDNAs represent 50% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. In a highly preferred embodiment, the enriched cDNAs represent 90% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
- cDNAs encoding secreted polypeptides or fragments thereof which are present in cDNA libraries in which one or more cDNAs encoding secreted polypeptides or fragments thereof make up 5% or more of the number of nucleic acid inserts in the backbone molecules are "enriched recombinant cDNAs" as defined herein.
- cDNAs encoding secreted polypeptides or fragments thereof which are in a population of plasmids in which one or more cDNAs of the present invention have been inserted such that they represent 5% or more of the number of inserts in the plasmid backbone are " enriched recombinant cDNAs" as defined herein.
- cDNAs encoding secreted polypeptides or fragments thereof which are in cDNA libraries in which the cDNAs encoding secreted polypeptides or fragments thereof constitute less than 5% of the number of nucleic acid inserts in the population of backbone molecules, such as libraries in which backbone molecules having a cDNA insert encoding a secreted polypeptide are extremely rare, are not "enriched recombinant cDNAs.”
- polypeptide refers to a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
- polypeptides which contain one or more analogs of an amino acid (including, for example, non- naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
- amino acids including, for example, non- naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.
- polypeptides include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
- nucleotide as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.
- nucleotide is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide.
- nucleotide is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO 95/04064.
- the polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.
- base paired and “Watson & Crick base paired” are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another be virtue of their sequence identities in a manner like that found in double-helical DNA with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds (See Stryer, L, Biochemistry, 4 th edition, 1995).
- complementary or “complement thereof are used herein to refer to the sequences of polynucleotides which are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region.
- a first polynucleotide is deemed to be complementary to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base.
- Complementary bases are, generally, A and T (or A and U), or C and G.
- “Complement” is used herein as a synonym from “complementary polynucleotide,” “complementary nucleic acid” and “complementary nucleotide sequence” . These terms are applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
- a "complementary" sequence is a sequence which an A at each position where there is a T on the opposite strand, a T at each position where there is an A on the opposite strand, a G at each position where there is a C on the opposite strand and a C at each position where there is a G on the opposite strand.
- a "secreted” protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal peptides in its amino acid sequence.
- secreted proteins include without limitation proteins secreted wholly (e.g. soluble proteins), or partially (e.g. receptors) from the cell in which they are expressed.
- Stecreted proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
- cDNAs encoding secreted proteins may include nucleic acid sequences, called signal sequences, which encode signal peptides which direct the extracellular secretion of the proteins encoded by the cDNAs. Generally, the signal peptides are located at the amino termini of secreted proteins.
- Secreted proteins are translated by ribosomes associated with the "rough" endoplasmic reticulum. Generally, secreted proteins are co-translationally transferred to the membrane of the endoplasmic reticulum. Association of the ribosome with the endoplasmic reticulum during translation of secreted proteins is mediated by the signal peptide. The signal peptide is typically cleaved following its co- translational entry into the endoplasmic reticulum. After delivery to the endoplasmic reticulum, secreted proteins may proceed through the Golgi apparatus. In the Golgi apparatus, the proteins may undergo post- translational modification before entering secretory vesicles which transport them across the cell membrane.
- the cDNAs of the present invention have several important applications. For example, they may be used to express the entire secreted protein which they encode. Alternatively, they may be used to express fragments of the secreted protein.
- the fragments may comprise the signal peptides encoded by the cDNAs or the mature proteins encoded by the cDNAs (i.e. the proteins generated when the signal peptide is cleaved off).
- the fragments may also comprise polypeptides having at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids encoded by the cDNAs.
- Antibodies which specifically recognize the entire secreted proteins encoded by the cDNAs or fragments thereof having at least 10 consecutive amino acids, at least 15 consecutive amino acids, at least 25 consecutive amino acids, or at least 40 consecutive amino acids may also be obtained as described below.
- Antibodies which specifically recognize the mature protein generated when the signal peptide is cleaved may also be obtained as described below.
- antibodies which specifically recognize the signal peptides encoded by the cDNAs may also be obtained.
- the cDNAs include the signal sequence.
- the cDNAs may include the full coding sequence for the mature protein (i.e. the protein generated when the signal polypeptide is cleaved off).
- the cDNAs may include regulatory regions upstream of the translation start site or downstream of the stop codon which control the amount, location, or developmental stage of gene expression. As discussed above, secreted proteins are therapeutically important. Thus, the proteins expressed from the cDNAs may be useful in treating or controlling a variety of human conditions.
- the cDNAs may also be used to obtain the corresponding genomic DNA.
- the term "corresponding genomic DNA” refers to the genomic DNA which encodes mRNA which includes the sequence of one of the strands of the cDNA in which thymidine residues in the sequence of the cDNA are replaced by uracil residues in the mRNA.
- the cDNAs or genomic DNAs obtained therefrom may be used in forensic procedures to identify individuals or in diagnostic procedures to identify individuals having genetic diseases resulting from abnormal expression of the genes corresponding to the cDNAs.
- the present invention is useful for constructing a high resolution map of the human chromosomes.
- the present invention also relates to secretion vectors capable of directing the secretion of a protein of interest.
- Such vectors may be used in gene therapy strategies in which it is desired to produce a gene product in one cell which is to be delivered to another location in the body.
- Secretion vectors may also facilitate the purification of desired proteins.
- the present invention also relates to expression vectors capable of directing the expression of an inserted gene in a desired spatial or temporal manner or at a desired level.
- Such vectors may include sequences upstream of the cDNAs such as promoters or upstream regulatory sequences.
- the present invention may also be used for gene therapy to control or treat genetic diseases.
- Signal peptides may also be fused to heterologous proteins to direct their extracellular secretion.
- One embodiment of the present invention is a purified or isolated nucleic acid comprising the sequence of one of SEQ ID NOs: 24-73 or a sequence complementary thereto.
- the nucleic acid is recombinant.
- Another embodiment of the present invention is a purified or isolated nucleic acid comprising at least 8 consecutive bases of the sequence of one of SEQ ID NOs: 24-73 or one of the sequences complementary thereto.
- the nucleic acid comprises at least 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 consecutive bases of one of the sequences of SEQ ID NOs: 24-73 or one of the sequences complementary thereto.
- the nucleic acid may be a recombinant nucleic acid.
- Another embodiment of the present invention is a purified or isolated nucleic acid of at least 15 bases capable of hybridizing under stringent conditions to the sequence of one of SEQ ID NOs: 24-73 or a sequence complementary to one of the sequences of SEQ ID NOs: 24-73.
- the nucleic acid is recombinant.
- Another embodiment of the present invention is a purified or isolated nucleic acid comprising the full coding sequences of one of SEQ ID NOs: 24-73, wherein the full coding sequence optionally comprises the sequence encoding signal peptide as well as the sequence encoding mature protein.
- the nucleic acid is recombinant.
- a further embodiment of the present invention is a purified or isolated nucleic acid comprising the nucleotides of one of SEQ ID NOs: 24-73 which encode a mature protein.
- the nucleic acid is recombinant.
- Yet another embodiment of the present invention is a purified or isolated nucleic acid comprising the nucleotides of one of SEQ ID NOs: 24-73 which encode the signal peptide.
- the nucleic acid is recombinant.
- Another embodiment of the present invention is a purified or isolated nucleic acid encoding a polypeptide having the sequence of one of the sequences of SEQ ID NOs: 74-123.
- Another embodiment of the present invention is a purified or isolated nucleic acid encoding a polypeptide having the sequence of a mature protein included in one of the sequences of SEQ ID NOs: 74- 123.
- Another embodiment of the present invention is a purified or isolated nucleic acid encoding a polypeptide having the sequence of a signal peptide included in one of the sequences of SEQ ID NOs: 74- 123.
- Yet another embodiment of the present invention is a purified or isolated protein comprising the sequence of one of SEQ ID NOs: 74-123.
- Another embodiment of the present invention is a purified or isolated polypeptide comprising at least 5 or 8 consecutive amino acids of one of the sequences of SEQ ID NOs: 74-123.
- the purified or isolated polypeptide comprises at least 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 consecutive amino acids of one of the sequences of SEQ ID NOs: 74-123.
- Another embodiment of the present invention is an isolated or purified polypeptide comprising a signal peptide of one of the polypeptides of SEQ ID NOs: 74-123.
- Yet another embodiment of the present invention is an isolated or purified polypeptide comprising a mature protein of one of the polypeptides of SEQ ID NOs: 74-123.
- a further embodiment of the present invention is a method of making a protein comprising one of the sequences of SEQ ID NO: 74-123, comprising the steps of obtaining a cDNA comprising one of the sequences of sequence of SEQ ID NO: 24-73, inserting the cDNA in an expression vector such that the cDNA is operably linked to a promoter, and introducing the expression vector into a host cell whereby the host cell produces the protein encoded by said cDNA.
- the method further comprises the step of isolating the protein.
- Another embodiment of the present invention is a protein obtainable by the method described in the preceding paragraph.
- Another embodiment of the present invention is a method of making a protein comprising the amino acid sequence of the mature protein contained in one of the sequences of SEQ ID NO: 74-123, comprising the steps of obtaining a cDNA comprising one of the nucleotides sequence of sequence of SEQ ID NO: 24- 73 which encode for the mature protein, inserting the cDNA in an expression vector such that the cDNA is operably linked to a promoter, and introducing the expression vector into a host cell whereby the host cell produces the mature protein encoded by the cDNA.
- the method further comprises the step of isolating the protein.
- Another embodiment of the present invention is a mature protein obtainable by the method described in the preceding paragraph.
- Another embodiment of the present invention is a host cell containing the purified or isolated nucleic acids comprising the sequence of one of SEQ ID NOs: 24-73 or a sequence complementary thereto described herein.
- Another embodiment of the present invention is a host cell containing the purified or isolated nucleic acids comprising the full coding sequences of one of SEQ ID NOs: 24-73, wherein the full coding sequence comprises the sequence encoding the signal peptide and the sequence encoding the mature protein described herein.
- Another embodiment of the present invention is a host cell containing the purified or isolated nucleic acids comprising the nucleotides of one of SEQ ID NOs: 24-73 which encode a mature protein which are described herein.
- Another embodiment of the present invention is a host cell containing the purified or isolated nucleic acids comprising the nucleotides of one of SEQ ID NOs: 24-73 which encode the signal peptide which are
- Another embodiment of the present invention is a purified or isolated antibody capable of specifically binding to a protein having the sequence of one of SEQ ID NOs: 74-123.
- the antibody is capable of binding to a polypeptide comprising at least 10 consecutive amino acids of the sequence of one of SEQ ID NOs: 74-123.
- Another embodiment of the present invention is an array of cDNAs or fragments thereof of at least
- the array includes at least two of the sequences of SEQ ID NOs: 24-73, the sequences complementary to the sequences of SEQ ID NOs: 24-73, or
- the array includes at least five of the sequences of SEQ ID NOs: 24-73, the sequences complementary to the sequences of SEQ ID NOs: 24-73, or fragments thereof of at least 15 consecutive nucleotides.
- a further embodiment of the invention encompasses purified polynucleotides comprising an insert from a clone deposited in an ECACC deposit, which contains the sequences of SEQ ID NOs. 25-40 and 42-
- An additional embodiment of the invention encompasses purified polypeptides which comprise, consist of, or consist essentially of an
- An additional embodiment of the invention encompasses purified polypeptides which comprise a contiguous span of at least 5, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 amino acids of SEQ ID NOs: 74-123, wherein said contiguous span comprises at least one of the amino acid positions which was not shown to be identical to a public sequence in any of Figures 10 to 13. Also encompassed by the invention are purified polynucleotides encoding said polypeptides.
- Another embodiment of the present invention is a computer readable medium having stored thereon a sequence selected from the group consisting of a cDNA code of SEQID NOs. 24-73 and a polypeptide code of SEQ ID NOs. 74-123.
- Another embodiment of the present invention is a computer system comprising a processor and a data storage device wherein the data storage device has stored thereon a sequence selected from the group consisting of a cDNA code of SEQID NOs. 24-73 and a polypeptide code of SEQ ID NOs. 74-123.
- the computer system further comprises a sequence comparer and a data storage device having reference sequences stored thereon.
- the sequence comparer may comprise a computer program which indicates polymorphisms.
- the system further comprises an identifier which identifies features in said sequence.
- Another embodiment of the present invention is a method for comparing a first sequence to a reference sequence wherein the first sequence is selected from the group consisting of a cDNA code of SEQID NOs. 24-73 and a polypeptide code of SEQ ID NOs. 74-123 comprising the steps of reading the first sequence and the reference sequence through use of a computer program which compares sequences and determining differences between the first sequence and the reference sequence with the computer program.
- said step of determining differences between the first sequence and the reference sequence comprises identifying polymorphisms.
- Another embodiment of the present invention is a method for identifying a feature in a sequence selected from the group consisting of a cDNA code of SEQID NOs. 24-73 and a polypeptide code of SEQ ID NOs. 74-123 comprising the steps of reading the sequence through the use of a computer program which identifies features in sequences and identifying features in the sequence with said computer program.
- Figure 1 is a table with all of the parameters that can be used for each step of cDNA analysis.
- Figure 2 is an analysis of the 43 amino terminal amino acids of all human SwissProt proteins to determine the frequency of false positives and false negatives using the techniques for signal peptide identification described herein.
- Figure 3 provides a diagram of a RT-PCR-based method to isolate cDNAs containing sequences adjacent to 5'ESTs used to obtain them
- Figure 4 provides a schematic description of the promoters isolated and the way they are assembled with the corresponding 5' tags.
- Figure 5 describes the transcription factor binding sites present in each of these promoters.
- Figure 6 is a block diagram of an exemplary computer system.
- Figure 7 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
- Figure 8 is a flow diagram illustrating one embodiment of a process 250 in a computer for 5 determining whether two sequences are homologous.
- Figure 9 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence.
- Figure 10 illustrates an alignment of the protein of SEQ ID NO: 76, encoded by the cDNA SEQ ID NO: 26 with the parotid HPSP protein (SEQ ID NO: 124).
- Figure 11 illustrates an alignment of the protein of SEQ ID NO: 93, encoded by the cDNA SEQ ID NO: 94, encoded by the cDNA SEQ ID NO: 94.
- Figure 12 illustrates an alignment of the protein of SEQ ID NO: 75, encoded by the cDNA SEQ ID NO: 25 with a human putative sialyltransferase (SEQ ID NO: 126), displaying 89.4% identical residues in a 15 301 amino acid overlap.
- the sialylmotifS is in bold.
- the sialylmotifL is in italics.
- the potential transmembrane segments are underlined.
- Figure 13 illustrates an alignment of the protein of SEQ ID NO: 104, encoded by the extended cDNA SEQ ID NO: 54, with the murine recombination activating gene 1 inducing protein (SEQ ID NO: 177).
- the cDNAs of the present invention may include the entire coding sequence of the protein encoded by the corresponding mRNA, including the authentic translation start site, the signal sequence, and the sequence encoding the mature protein remaining after cleavage of the signal peptide. Such cDNAs are referred to herein as "full length cDNAs.” Alternatively, the cDNAs may include only the sequence encoding 25 the mature protein remaining after cleavage of the signal peptide, or only the sequence encoding the signal peptide.
- the methods explained therein can also be used to obtain cDNAs which encode less than the entire coding sequence of the secreted proteins encoded by the genes corresponding to the cDNAs.
- the cDNAs isolated using these methods encode at least 5 amino acids of one of the 30 proteins encoded by the sequences of SEQ ID NOs: 24-73.
- the cDNAs encode at least 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 consecutive amino acids of the proteins encoded by the sequences of SEQ ID NOs: 24-73.
- the cDNAs encode a full length protein sequence, which includes the protein coding sequences of SEQ ID NOs: 24-73.
- cDNAs of the present invention were obtained from cDNA libraries derived from mRNAs having 35 intact 5' ends as described in Examples 1 to 5 using either a chemical or enzymatic approach.
- RNA samples were respectively purchased from LABIMO and CLONTECH and used to generate cDNA libraries as described below.
- the purchased RNA had been isolated from cells or tissues using acid guanidium thiocyanate-phenol-chloroform extraction (Chomczyniski and Sacchi, Analytical Biochemistry 162:156-159, 1987).
- PolyA+ RNA was isolated from total RNA (LABIMO) by two passes of oligo dT chromatography, as described by Aviv and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972) in order to eliminate ribosomal RNA.
- RNAs The quality and the integrity of the polyA+ RNAs were checked.
- Northern blots hybridized with a probe corresponding to an ubiquitous mRNA, such as elongation factor 1 or elongation factor 2, were used to confirm that the mRNAs were not degraded.
- Contamination of the polyA + mRNAs by ribosomal sequences was checked using Northern blots and a probe derived from the sequence of the 28S rRNA. Preparations of mRNAs with less than 5% of rRNAs were used in library construction. To avoid constructing libraries with RNAs contaminated by exogenous sequences (prokaryotic or fungal), the presence of bacterial 16S ribosomal sequences or of two highly expressed fungal mRNAs was examined using PCR.
- mRNAs Following preparation of the mRNAs from various tissues as described above, selection of mRNA with intact 5' ends and specific attachment of an oligonucleotide tag to the 5' end of such mRNA is performed using either a chemical or enzymatic approach. Both techniques take advantage of the presence of the "cap" structure, which characterizes the 5'end of intact mRNAs and which comprises a guanosine generally methylated once, at the 7 position.
- the chemical modification approach involves the optional elimination of the 2', 3'-cis diol of the 3' terminal ribose, the oxidation of the 2', 3', -cis diol of the ribose linked to the cap of the 5' ends of the mRNAs into a dialdehyde, and the coupling of the dialdehyde to a derivatized oligonucleotide tag. Further detail regarding the chemical approaches for obtaining mRNAs having intact 5' ends are disclosed in International Application No. W096/34981, published November 7, 1996.
- the enzymatic approach for ligating the oligonucleotide tag to the 5' ends of mRNAs with intact 5' ends involves the removal of the phosphate groups present on the 5' ends of uncapped incomplete mRNAs, the subsequent decapping of mRNAs with intact 5' ends and the ligation of the phosphate present at the 5' end of the decapped mRNA to an oligonucleotide tag. Further detail regarding the enzymatic approaches for obtaining mRNAs having intact 5' ends are disclosed in Dumas Milne Edwards J.B. (Doctoral Thesis of Paris VI University, Le clonage des ADNc complets: difficultes et perspectives Meeting. Apports pour I'etude de la regulation de I'expression de la tryptophane hydroxylase de rat, 20 Dec. 1993), EP0 625572 and Kato ef al., Gene 150:243-250 (1994).
- the oligonucleotide tag has a restriction enzyme site (e.g. EcoRI sites) therein to facilitate later cloning procedures.
- a restriction enzyme site e.g. EcoRI sites
- the integrity of the mRNA was then examined by performing a Northern blot using a probe complementary to the oligonucleotide tag.
- EXAMPLE 3 cDNA Synthesis Using mRNA Templates Having Intact 5' Ends
- first strand cDNA synthesis was performed using reverse transcriptase with an oligo-dT primer or random nonamer.
- this oligo-dT primer contained an internal tag of at least 4 nucleotides which is different from one tissue to the other.
- methylated dCTP was used for first strand synthesis. After removal of RNA by an alkaline hydrolysis, the first strand of cDNA was precipitated using isopropanol in order to eliminate residual primers. The second strand of the cDNA was then synthesized with a Klenow fragment using a primer corresponding to the 5'end of the ligated oligonucleotide.
- the primer is 20-25 bases in length.
- Methylated dCTP was also used for second strand synthesis in order to protect internal EcoRI sites in the cDNA from digestion during the cloning process.
- the cDNAs were cloned into the phagemid pBlueScript II SK- vector (Stratagene). The ends of the cDNAs were blunted with T4 DNA polymerase (Biolabs) and the cDNA was digested with EcoRI. Since methylated dCTP was used during cDNA synthesis, the EcoRI site present in the tag was the only hemi-methylated site, hence the only site susceptible to EcoRI digestion. In some instances, to facilitate subcloning, an Hind III adaptor was added to the 3' end of cDNAs.
- the cDNAs were then size fractionated using either exclusion chromatography (AcA, Biosepra) or electrophoretic separation which yields 3 or 6 different fractions.
- the cDNAs were then directionally cloned either into pBlueScript using either the EcoRI and Smal restriction sites or the EcoRI and Hind III restriction sites when the Hind III adaptator was present in the cDNAs.
- the ligation mixture was electroporated into bacteria and propagated under appropriate antibiotic selection.
- Clones containing the oligonucleotide tag attached to cDNAs were then selected as follows.
- the plasmid DNAs containing cDNA libraries made as described above were purified (Qiagen).
- a positive selection of the tagged clones was performed as follows. Briefly, in this selection procedure, the plasmid DNA was converted to single stranded DNA using gene II endonuclease of the phage F1 in combination with an exonuclease (Chang ef al., Gene 127:95-8, 1993) such as exonuclease III or T7 gene 6 exonuclease.
- the resulting single stranded DNA was then purified using paramagnetic beads as described by Fry ef al., Biotechniques, 13: 124-131, 1992.
- the single stranded DNA was hybridized with a biotinylated oligonucleotide having a sequence corresponding to the 3' end of the oligonucleotide tag described in example 2.
- the primer has a length of 20-25 bases. Clones including a sequence complementary to the biotinylated oligonucleotide were captured by incubation with streptavidin coated magnetic beads followed by magnetic selection.
- the plasmid DNA was released from the magnetic beads and converted into double stranded DNA using a DNA polymerase such as the ThermoSequenase obtained from Amersham Pharmacia Biotech. Alternatively, protocols such as the Gene Trapper kit (Gibco BRL) may be used. The double stranded DNA was then electroporated into bacteria. The percentage of positive clones having the 5' tag oligonucleotide was estimated to typically rank between 90 and 98% using dot blot analysis.
- EXAMPLE 6 Sequencing of The 5'End of cDNA Clones The 5' ends of cloned cDNAs were then sequenced as follows. Plasmid inserts were first amplified by PCR on PE 9600 thermocyclers (Perkin-Elmer, Applied Biosystems Division, Foster City, CA) using standard SETA-A and SETA-B primers (Genset SA), AmpliTaqGold (Perkin-Elmer), dNTPs (Boehringer), buffer and cycling conditions as recommended by the Perkin-Elmer Corporation.
- PCR products were then sequenced using automatic ABI Prism 377 sequencers (Perkin Elmer). Sequencing reactions were performed using PE 9600 thermocyclers with standard dye-primer chemistry and ThermoSequenase (Amersham Pharmacia Biotech). The primers used were either T7 or 21M13 (available from Genset SA) as appropriate. The primers were labeled with the JOE, FAM, ROX and TAMRA dyes. The dNTPs and ddNTPs used in the sequencing reactions were purchased from Boehringer. Sequencing buffer, reagent concentrations and cycling conditions were as recommended by Amersham.
- sequence data obtained from the sequencing of 5' ends of all cDNA libraries made as described above were transferred to a proprietary database, where quality control and validation steps were performed.
- a proprietary base-caller working using a Unix system automatically flagged suspect peaks, taking into account the shape of the peaks, the inter-peak resolution, and the noise level.
- the proprietary base-caller also performed an automatic trimming. Any stretch of 25 or fewer bases having more than 4 suspect peaks was considered unreliable and was discarded.
- Sequences corresponding to cloning vector or ligation oligonucleotides were automatically removed from the sequences. However, the resulting sequences may contain 1 to 5 nucleotides belonging to the above mentioned sequences at their 5' end.
- sequences of the cDNA clones were entered in a database for storage and manipulation as described below.
- cDNAs derived from mRNAs which were not of interest were identified and eliminated, namely, endogenous contaminants (ribosomal RNAs, transfert RNAs, mitochondrial RNAs) and exogenous contaminants (prokaryotic RNAs and fungal RNAs) using software and parameters described in Figure 1.
- endogenous contaminants ribosomal RNAs, transfert RNAs, mitochondrial RNAs
- exogenous contaminants prokaryotic RNAs and fungal RNAs
- cDNA sequences showing showing homology to repeated sequences Alu, L1.THE and MER repeats, SSTR sequences or satellite, micro-satellite, or telomeric repeats
- EXAMPLE 7 Determination of Efficiency of 5' End Selection To determine the efficiency at which the above selection procedures isolated cDNAs which include the 5' ends of their corresponding mRNAs, the sequences of 5'ESTs or cDNAs were aligned with a reference pool of complete mRNA/cDNA extracted from the EMBL release 57 using the FASTA algorithm. The reference mRNA/cDNA starting at the most 5' transcription start site was obtained, and then compared to the 5' transcription start site position of the 5'EST or cDNA. More than 75% of 5'ESTs or cDNAs had their 5' ends close to the 5' ends of the known sequence.
- nucleic acid sequences were then screened to identify those having uninterrupted open reading frames (ORF) with a good coding probability using proprietary software.
- ORF open reading frames
- the full-length cDNA was obtained, only complete ORFs, namely nucleic acid sequences beginning with a start codon and ending with a stop codon, longer than 150 nucleotides were considered.
- both complete ORFS longer than 150 nucleotides and incomplete ORFs namely nucleic acid sequences beginning with a start codon and extending up to the end of the 5'EST, longer than 60 nucleotides were considered.
- EXAMPLE 9 Confirmation of Accuracy of Identification of Potential Signal Seguences in 5' ESTs The accuracy of the above procedure for identifying signal sequences encoding signal peptides was evaluated by applying the method to the 43 amino acids located at the N terminus of all human SwissProt proteins. The computed Von Heijne score for each protein was compared with the known characterization of the protein as being a secreted protein or a non-secreted protein. In this manner, the number of non-secreted proteins having a score higher than 3.5 (false positives) and the number of secreted proteins having a score lower than 3.5 (false negatives) could be calculated.
- the signal sequences from the 5' ESTs or cDNAs may be cloned into a vector designed for the identification of signal peptides.
- a vector designed for the identification of signal peptides are designed to confer the ability to grow in selective medium only to host cells containing a vector with an operably linked signal sequence.
- the signal sequence of the 5' EST or cDNA may be inserted upstream and in frame with a non-secreted form of the yeast invertase gene in signal peptide selection vectors such as those described in U.S. Patent No. 5,536,637. Growth of host cells containing signal sequence selection vectors with the correctly inserted 5' EST or cDNA signal sequence confirms that the 5' EST or cDNA encodes a genuine signal peptide.
- the presence of a signal peptide may be confirmed by cloning the 5'ESTs or cDNAs into expression vectors such as pXT1 as described below, or by constructing promoter-signal sequence- reporter gene vectors which encode fusion proteins between the signal peptide and an assayable reporter protein. After introduction of these vectors into a suitable host cell, such as COS cells or NIH 3T3 cells, the growth medium may be harvested and analyzed for the presence of the secreted protein. The medium from these cells is compared to the medium from control cells containing vectors lacking the signal sequence or cDNA insert to identify vectors which encode a functional signal peptide or an authentic secreted protein.
- the spatial and temporal expression patterns of the mRNAs corresponding to the 5' ESTs or cDNAs, as well as their expression levels, may be determined. Characterization of the spatial and temporal expression patterns and expression levels of these mRNAs is useful for constructing expression vectors capable of producing a desired level of gene product in a desired spatial or temporal manner, as will be discussed in more detail below.
- cDNAs or 5' ESTs whose corresponding mRNAs are associated with disease states may also be identified.
- a particular disease may result from lack of expression, over expression, or under expression of an mRNA corresponding to a cDNA or 5' EST.
- cDNAs and 5' ESTs responsible for the disease may be identified.
- Expression levels and patterns of mRNAs corresponding to 5' ESTs or cDNAs may be analyzed by solution hybridization with long probes as described in International Patent Application No. WO 97/05277. Briefly, a 5' EST, cDNA, or fragment thereof corresponding to the gene encoding the mRNA to be characterized is inserted at a cloning site immediately downstream of a bacteriophage (T3, T7 or SP6) RNA polymerase promoter to produce antisense RNA.
- the 5' EST or cDNA is 100 or more nucleotides in length.
- the plasmid is linearized and transcribed in the presence of ribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP and DIG-UTP).
- ribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP and DIG-UTP).
- An excess of this doubly labeled RNA is hybridized in solution with mRNA isolated from cells or tissues of interest.
- the hybridizations are performed under standard stringent conditions (40-50°C for 16 hours in an 80% formamide, 0.4 M NaCI buffer, pH 7-8).
- the unhybridized probe is removed by digestion with ribonucleases specific for single-stranded RNA (i.e.
- RNases CL3, T1, Phy M, U2 or A RNases CL3, T1, Phy M, U2 or A.
- the presence of the biotin-UTP modification enables capture of the hybrid on a microtitration plate coated with streptavidin.
- the presence of the DIG modification enables the hybrid to be detected and quantified by ELISA using an anti-DIG antibody coupled to alkaline phosphatase.
- the 5' ESTs, cDNAs, or fragments thereof may also be tagged with nucleotide sequences for the serial analysis of gene expression (SAGE) as disclosed in UK Patent Application No. 2 305 241 A.
- SAGE serial analysis of gene expression
- cDNAs are prepared from a cell, tissue, organism or other source of nucleic acid for which it is desired to determine gene expression patterns.
- the resulting cDNAs are separated into two pools.
- the cDNAs in each pool are cleaved with a first restriction endonuclease, called an "anchoring enzyme," having a recognition site which is likely to be present at least once in most cDNAs.
- the fragments which contain the 5' or 3' most region of the cleaved cDNA are isolated by binding to a capture medium such as streptavidin coated beads.
- a first oligonucleotide linker having a first sequence for hybridization of an amplification primer and an internal restriction site for a "tagging endonuclease" is ligated to the digested cDNAs in the first pool. Digestion with the second endonuclease produces short "tag" fragments from the cDNAs.
- a second oligonucleotide having a second sequence for hybridization of an amplification primer and an internal restriction site is ligated to the digested cDNAs in the second pool.
- the cDNA fragments in the second pool are also digested with the "tagging endonuclease" to generate short "tag” fragments derived from the cDNAs in the second pool.
- the "tags” resulting from digestion of the first and second pools with the anchoring enzyme and the tagging endonuclease are ligated to one another to produce "ditags.”
- the ditags are concatamerized to produce ligation products containing from 2 to 200 ditags.
- the tag sequences are then determined and compared to the sequences of the 5' ESTs or cDNAs to determine which 5' ESTs or cDNAs are expressed in the cell, tissue, organism, or other source of nucleic acids from which the tags were derived. In this way, the expression pattern of the 5' ESTs or cDNAs in the cell, tissue, organism, or other source of nucleic acids is obtained.
- arrays means a one dimensional, two dimensional, or multidimensional arrangement of full length cDNAs (i.e. cDNAs which include the coding sequence for the signal peptide, the coding sequence for the mature protein, and a stop codon), cDNAs, 5' ESTs or fragments of the full length cDNAs, cDNAs, or 5' ESTs of sufficient length to permit specific detection of gene expression.
- the fragments are at least 15 nucleotides in length. More preferably, the fragments are at least 100 nucleotides in length. More preferably, the fragments are more than 100 nucleotides in length. In some embodiments the fragments may be more than 500 nucleotides in length.
- cDNAs, cDNAs, 5' ESTs, or fragments thereof in a complementary DNA microarray as described by Schena ef al. (Science 270:467-470, 1995; Proc. Natl. Acad. Sci. U.S.A. 93:10614-10619, 1996).
- Full length cDNAs, cDNAs, 5' ESTs or fragments thereof are amplified by PCR and arrayed from 96-well microtiter plates onto silylated microscope slides using high-speed robotics.
- Printed arrays are incubated in a humid chamber to allow rehydration of the array elements and rinsed, once in 0.2% SDS for 1 min, twice in water for 1 min and once for 5 min in sodium borohydride solution.
- the arrays are submerged in water for 2 min at 95°C, transferred into 0.2% SDS for 1 min, rinsed twice with water, air dried and stored in the dark at 25°C.
- Probes are hybridized to 1 cm 2 microarrays under a 14 x 14 mm glass coverslip for 6-12 hours at 60°C. Arrays are washed for 5 min at 25°C in low stringency wash buffer (1 x SSC/0.2% SDS), then for 10 min at room temperature in high stringency wash buffer (0.1 x SSC/0.2% SDS). Arrays are scanned in 0.1 x SSC using a fluorescence laser scanning device fitted with a custom filter set. Accurate differential expression measurements are obtained by taking the average of the ratios of two independent hybridizations.
- Quantitative analysis of the expression of genes may also be performed with full length cDNAs, cDNAs, 5' ESTs, or fragments thereof in complementary DNA arrays as described by Pietu ef al. (Genome Research 6:492-503, 1996).
- the full length cDNAs, cDNAs, 5' ESTs or fragments thereof are PCR amplified and spotted on membranes.
- mRNAs originating from various tissues or cells are labeled with radioactive nucleotides. After hybridization and washing in controlled conditions, the hybridized mRNAs are detected by phospho-imaging or autoradiography. Duplicate experiments are performed and a quantitative analysis of differentially expressed mRNAs is then performed.
- expression analysis of the 5' ESTs or cDNAs can be done through high density nucleotide arrays as described by Lockhart ef al. (Nature Biotechnology 14: 1675-1680, 1996) and Sosnowsky ef al. (Proc. Natl. Acad. Sci. 94:1119-1123, 1997).
- Oligonucleotides of 15-50 nucleotides corresponding to sequences of the 5' ESTs or cDNAs are synthesized directly on the chip (Lockhart ef al., supra) or synthesized and then addressed to the chip (Sosnowski ef al., supra).
- the oligonucleotides are about 20 nucleotides in length.
- cDNA probes labeled with an appropriate compound such as biotin, digoxigenin or fluorescent dye, are synthesized from the appropriate mRNA population and then randomly fragmented to an average size of 50 to 100 nucleotides. The said probes are then hybridized to the chip. After washing as described in Lockhart ef al., supra and application of different electric fields (Sosnowsky ef al., Proc. Natl. Acad. Sci. 94:1119-1123)., the dyes or labeling compounds are detected and quantified. Duplicate hybridizations are performed.
- an appropriate compound such as biotin, digoxigenin or fluorescent dye
- clones which include the 5'end of their corresponding mRNA and which encode a new protein with a signal peptide, as determined in the aforementioned procedure may be subcloned into an appropriate vector such as pED6dpc2 (DiscoverEase, Genetics Institute, Cambridge, MA) before full sequencing.
- polyA tail and polyadenylation signal structural features, e.g. polyA tail and polyadenylation signal, of the sequences of cDNAs were subsequently determined using the algorithm, parameters and criteria defined in figure 1.
- a polyA tail was defined as a homopolymeric stretch of at least 11 A with at most one alternative base within it.
- the polyA tail search was restricted to the last 100 nt of the sequence and limited to stretches of 11 consecutive A's because sequencing reactions are often not readable after such a polyA stretch.
- the polyA tail was clipped from the full-length sequence.
- the 50 bp preceding the polyA tail were searched for the canonic polyadenylation AAUAAA signal allowing one mismatch to account for possible sequencing errors as well as known variation in the canonical sequence of the polyadenylation signal.
- Functional features, e.g. ORFs and signal sequences, of the sequences of cDNAs were subsequently determined as follows.
- the 3 upper strand frames of cDNAs were searched for ORFs defined as the maximum length fragments beginning with a translation initiation codon and ending with a stop codon. ORFs encoding at least 80 amino acids were preferred. Each found ORF was then scanned for the presence of a signal peptide using the matrix method described in example 10. Sequences of cDNAs were then compared, on a nucleotidic or proteic basis, to public sequences available at the time of filing.
- Sequences of cDNA clones were then compared pairwise with BLAST after masking of the repeat sequences. Sequences containing at least 90% homology over 30 nucleotides were clustered in the same class. Each cluster was then subjected to a clustal analysis that detects sequences resulting from internal priming or from alternative splicing, identical sequences or sequences with several frameshifts. This automatic analysis served as a basis for manual selection of the sequences. b) Manual sequence selection
- EXAMPLE 14 Characterization of Full-length cDNAs The procedure described above was used to obtain or full length cDNAs derived from a variety of tissues. The following list provides a few examples of thus obtained cDNAs.
- This cDNA encodes a potentially secreted protein (SEQ ID NO:4) with a signal peptide having a von Heijne score of 5.3.
- the full length cDNA of SEQ ID NO:5 (internal identification number 108-004- 5-0-B12-FLC) was obtained.
- This cDNA encodes a potentially secreted protein (SEQ ID NO:6) with a signal peptide having a von Heijne score of 7.0.
- the full length cDNA of SEQ ID NO:7 (internal identification number 108-013- 5-0-G5-FLC) was obtained.
- This cDNA encodes a potentially secreted protein (SEQ ID NO:8) with a signal peptide having a von Heijne score of 9.4.
- polypeptides encoded by the extended or full-length cDNAs may be screened for the presence of known structural or functional motifs or for the presence of signatures, small amino acid sequences which are well conserved amongst the members of a protein family.
- 108-013-5-0-H9-FLC shows homologies with a family of lysophospholipases conserved among eukaryotes (yeast, rabbit, rodents and human). In addition, some members of this family exhibit a calcium-independent phospholipase A2 activity (Portilla ef al., J. Am. Soc. Nephro., 9 :1178-1186 (1998)). All members of this family exhibit the active site consensus GXSXG motif of carboxylesterases that is also found in the protein of SEQ ID NO :10 (position 54 to 58).
- this protein may be a membrane protein with one transmembrane domain as predicted by the software TopPred II (Claras and von Heijne, CABIOS applic. Notes, 10 :685-686 (1994)).
- TopPred II Claras and von Heijne, CABIOS applic. Notes, 10 :685-686 (1994)
- this protein or part therein may be useful in diagnosing and/or treating several disorders including, but not limited to, cancer, diabetes, and neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. It may also be useful in modulating inflammatory responses to infectious agents and/or to suppress graft rejection.
- the protein of SEQ ID NO: 12 encoded by the full-length cDNA SEQ ID NO:11 shows remote homology to a subfamily of beta4-galactosyltransferases widely conserved in animals (human, rodents, cow and chicken).
- Such enzymes usually type II membrane proteins located in the endoplasmic reticulum or in the Golgi apparatus, catalyze the biosynthesis of glycoproteins, glycolipid glycans and lactose.
- Biochem., 123:1000-1009 are pretty well conserved in the protein of SEQ ID NO: 12, especially the region I containing the DVD motif (positions 163-165) thought to be involved either in UDP binding or in the catalytic process itself.
- the protein of SEQ ID NO: 12 has the typical structure of a type II protein. Indeed, it contains a short 28-amino-acid-long N-terminal tail, a transmembrane segment from positions 29 to 49 and a large 278-amino-acid-long C-terminal tail as predicted by the software TopPred II (Claras and von Heijne, CABIOS applic. Notes, 10 :685-686 (1994)).
- the protein of SEQ ID NO: 12 may play a role in the biosynthesis of polysaccharides, and of the carbohydrate moieties of glycoproteins and glycolipids and/or in cell-cell recognition.
- this protein may be useful in diagnosing and/or treating several types of disorders including, but not limited to, cancer, atherosclerosis, cardiovascular disorders, autoimmune disorders and rheumatic diseases including rheumatoid arthritis.
- the protein of SEQ ID NO: 14 encoded by the extended cDNA SEQ ID NO: 13 exhibits the typical PROSITE signature for amino acid permeases (positions 5 to 66) which are integral membrane proteins involved in the transport of amino acids into the cell.
- the protein of SEQ ID NO: 14 has a transmembrane segment from positions 9 to 29 as predicted by the software TopPred II (Claras and von Heijne, CABIOS applic. Notes, 10 :685-686 (1994)). Taken together, these data suggest that the protein of SEQ ID NO: 14 may be involved in amino acid transport.
- this protein may be useful in diagnosing and/or treating several types of disorders including, but not limited to, cancer, aminoacidurias, neurodegenerative diseases, anorexia, chronic fatigue, coronary vascular disease, diphtheria, hypoglycemia, male infertility, muscular and myopathies.
- Bacterial clones containing plasmids containing the full length cDNAs described above are presently stored in the inventor's laboratories under the internal identification numbers provided above. The inserts may be recovered from the deposited materials by growing an aliquot of the appropriate bacterial clone in the appropriate medium.
- the plasmid DNA can then be isolated using plasmid isolation procedures familiar to those skilled in the art such as alkaline lysis minipreps or large scale alkaline lysis plasmid isolation procedures. If desired the plasmid DNA may be further enriched by centrifugation on a cesium chloride gradient, size exclusion chromatography, or anion exchange chromatography. The plasmid DNA obtained using these procedures may then be manipulated using standard cloning techniques familiar to those skilled in the art. Alternatively, a PCR can be done with primers designed at both ends of the cDNA insertion. The PCR product which corresponds to the cDNA can then be manipulated using standard cloning techniques familiar to those skilled in the art.
- Table I provides the sequence identification numbers of the cDNAs of the present invention, the locations of the first and last nucleicotides of the full coding sequences in SEQ ID NOs: 24-73 (i.e.
- nucleotides encoding both the signal peptide and the mature protein listed under the heading FCS location in Table I
- the locations of the first and last nucleotides in SEQ ID NOs: 24-73 which encode the signal peptides listed under the heading SigPep Location in Table I
- the locations of the first and last nucleotides in SEQ ID NOs: 24-73 which encode the mature proteins generated by cleavage of the signal peptides listed under the heading Mature Polypeptide Location in Table I
- the locations in SEQ ID NOs: 24- 73 of stop codons listed under the heading Stop Codon Location in Table I
- the locations of the first and last nucleotides in SEQ ID NOs: 24-73 of the polyA signals listed under the heading Poly A Signal Location in Table I
- the locations of the first and last nucleotides of the polyA sites listed under the heading Poly A Site Location in Table I).
- Table II lists the sequence identification numbers of the polypeptides of SEQ ID NOs: 74-123, the locations of the first and last amino acid residues of SEQ ID NOs: 74-123 in the full length polypeptide (second column), the locations of the first and last amino acid residues of SEQ ID NOs: 74-123 in the signal peptides (third column), and the locations of the first and last amino acid residues of SEQ ID NOs: 74-123 in the mature polypeptide created by cleaving the signal peptide from the full length polypeptide (fourth column).
- nucleotide sequences of the sequences of SEQ ID NOs: 24-73 and the amino acid sequences encoded by SEQ ID NOs: 24-73 are provided in the appended sequence listing. In some instances, the sequences are preliminary and may include some incorrect or ambiguous sequences or amino acids. All instances of the symbol "n" in the nucleic acid sequences mean that the nucleotide can be adenine, guanine, cytosine or thymine. For each amino acid sequence, Applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
- polypeptide sequences in the Sequence Listing contain the symbol "Xaa.” These "Xaa” symbols indicate either (1) a residue which cannot be identified because of nucleotide sequence ambiguity or (2) a stop codon in the determined sequence where applicants believe one should not exist (if the sequence were determined more accurately). Thus, "Xaa” indicates that a residue may be any of the twenty amino acids. In some instances, several possible identities of the unknown amino acids may be suggested by the genetic code.
- the sequences of SEQ ID NOs: 24-73 can readily be screened for any errors therein and any sequence ambiguities can be resolved by resequencing a fragment containing such errors or ambiguities on both strands.
- Nucleic acid fragments for resolving sequencing errors or ambiguities may be obtained from the deposited clones or can be isolated using the techniques described herein. Resolution of any such ambiguities or errors may be facilitated by using primers which hybridize to sequences located close to the ambiguous or erroneous sequences. For example, the primers may hybridize to sequences within 50-75 bases of the ambiguity or error. Upon resolution of an error or ambiguity, the corresponding corrections can be made in the protein sequences encoded by the DNA containing the error or ambiguity. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein, and determining its sequence. EXAMPLE 15
- nucleic acid sequences of the present invention were grouped based on their homology to known sequences as follows. All sequences were compared to EMBL release 58 and daily releases available at the time of filing using BLASTN. In some instances, the cDNAs did not match any known vertebrate sequence nor any publicly available EST sequence, thus being completely new.
- Table III gives the sequence identification numbers of these cDNAs (first column) and the positions of preferred fragments within these sequences (second column entitled "Positions of preferred fragments"). Each fragment is represented by x-y where x and y are the start and end positions respectively of a given preferred fragment. Preferred fragments are separated from each other by a coma.
- polynucleotide described in Table III refers to the all of the preferred polynucleotide fragments defined in Table III in this manner.
- the present invention encompasses isolated, purified, or recombinant nucleic acids which consist of, consist essentially of, or comprise a contiguous span of one of the sequences of SEQ ID Nos. 24-73 or a sequence complementary thereto, said continguous span comprising at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 5 2000 nucleotides of the sequence of SEQ ID Nos.
- contiguous span of these lengths is consistent with the lengths of the particular sequence, wherein the contiguous span comprises at least 1, 2, 3, 5, 10, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400 or 500 of a polynucleotide described in Table III, or a sequence complementary thereto.
- the present invention also encompasses isolated, purified, or recombinant nucleic acids comprising, consisting essentially of, 0 or consisting of a contiguous span of at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 nucleotides of a polynucleotide described in Table III or a sequence complementary thereto, to the extent that a contiguous span of these lengths is consistent with the length of the particular sequence described in Table III.
- the present invention also encompasses isolated, purified, or recombinant nucleic acids which comprise, consist of or consist essentially of a polynucleotide described in 5 Table III, or a sequence complementary thereto.
- the present invention further encompasses any combination of the nucleic acids listed in this paragraph.
- Cells containing the cDNAs (SEQ ID NOs: 24-73) of the present invention in the vector pBluescriptll SK- (Stratagene) are maintained in permanent deposit by the inventors at Genset, S.A., 24 Rue Royale, 75008 Paris, France. 0
- ECACC European Collection of Cell Cultures
- Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
- An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The design of the oligonucleotide probe should preferably follow these parameters:
- the probe is designed to have a T m of approx. 80 ⁇ C (assuming 2 degrees for each A or T and 4 degrees for each G or C).
- DC may also be used provided that specificity is not lost.
- the oligonucleotide should preferably be labeled with (-[ 32 P]ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantified by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4X10 6 dpm/pmole.
- the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 Dl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 Dg/ml.
- the culture should preferably be grown to saturation at 37QC, and the saturated culture should preferably be diluted in fresh L-broth.
- Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 Dg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 370C.
- Other known methods of obtaining distinct, well-separated colonies can also be employed.
- Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
- the filter is then preferably incubated at 65DC for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaC1/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 pg/ml of yeast RNA, and 10 mM EDTA (approximately 10 ml per 150 mm filter).
- 6X SSC 20X stock is 175.3 g NaC1/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH
- the probe is then added to the hybridization mix at a concentration greater than or equal to 1X10 6 dpm/ml.
- the filter is then preferably incubated at 65QC with gentle agitation overnight.
- the filter is then preferably washed in 500 ml of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes.
- a third wash with 0.1X SSC/0.5% SDS at 65DC for 30 minutes to 1 hour is optional.
- the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film.
- Other known hybridization methods can also be employed.
- the positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
- the plasmid DNA obtained using these procedures may then be manipulated using standard cloning techniques familiar to those skilled in the art.
- a PCR can be done with primers designed at both ends of the cDNA insertion.
- the PCR product which corresponds to the cDNA can then be manipulated using standard cloning techniques familiar to those skilled in the art.
- the cDNA clone obtained by the process described in Examples 1 through 13 may not include the entire coding sequence of the protein encoded by the corresponding mRNA, although they do include sequences derived from the 5'ends of their corresponding mRNA.
- Such 5'EST can be used to isolate extended cDNAs which contain sequences adjacent to the 5' ESTs.
- Such obtained extended cDNAs may include the entire coding sequence of the protein encoded by the corresponding mRNA, including the authentic translation start site.
- Examples 16 and 17 below describe methods for obtaining extended cDNAs using 5' ESTs.
- Example 17 also describes methods to obtain cDNA, mRNA or genomic DNA homologous to cDNA, 5'ESTs, or fragment thereof.
- Examples 16 and 17 can also be used to obtain cDNAs which encode less than the entire coding sequence of proteins encoded by the genes corresponding to the 5' ESTs.
- the cDNAs isolated using these methods encode at least 5, 8, 10, 12, 15, 20, 25, 30, 35, 40,
- EXAMPLE 16 General Method for Using 5' ESTs to Clone and Seguence cDNAs which Include the Entire Coding Region and the Authentic 5'End of the Corresponding mRNA The following general method may be used to quickly and efficiently isolate cDNAs including sequence adjacent to the sequences of the 5' ESTs used to obtain them. This method, illustrated in Figure 3, may be applied to obtain cDNAs for any 5' EST.
- the method takes advantage of the known 5' sequence of the mRNA.
- a reverse transcription reaction is conducted on purified mRNA with a poly dT primer containing a nucleotide sequence at its 5' end allowing the addition of a known sequence at the end of the cDNA which corresponds to the 3' end of the mRNA.
- a primer and a commercially-available reverse transcriptase enzyme are added to a buffered mRNA sample yielding a reverse transcript anchored at the 3' polyA site of the RNAs. Nucleotide monomers are then added to complete the first strand synthesis.
- RNA hybridized to the first cDNA strand After removal of the mRNA hybridized to the first cDNA strand by alkaline hydrolysis, the products of the alkaline hydrolysis and the residual poly dT primer can be eliminated with an exclusion column. Subsequently, a pair of nested primers on each end is designed based on the known 5' sequence from the 5' EST and the known 3' end added by the poly dT primer used in the first strand synthesis. Software used to design primers is either based on GC content and melting temperatures of oligonucleotides, such as OSP (illier and Green, PCR Meth. Appl.
- OSP illier and Green, PCR Meth. Appl.
- the nested primers at the 5' end and the nested primers at the 3' end are separated from one another by four to nine bases. These primer sequences may be selected to have melting temperatures and specificities suitable for use in PCR.
- a first PCR run is performed using the outer primer from each of the nested pairs.
- a second PCR run using the inner primer from each of the nested pairs is then performed on a small aliquot of the first PCR product. Thereafter, the primers and remaining nucleotide monomers are removed. Due to the lack of position constraints on the design of 5' nested primers compatible for PCR use using the OSP software, amplicons of two types are obtained.
- the second 5' primer is located upstream of the translation initiation codon thus yielding a nested PCR product containing the entire coding sequence.
- Such a cDNA may be used in a direct cloning procedure such as the one described in example 4.
- the second 5' primer is located downstream of the translation initiation codon, thereby yielding a PCR product containing only part of the ORF.
- intermediate steps are necessary to obtain both the complete coding sequence and a PCR product containing the full coding sequence.
- the complete coding sequence can be assembled from several partial sequences determined directly from different PCR products. Once the full coding sequence has been completely determined, new primers compatible for PCR use are then designed to obtain amplicons containing the whole coding region.
- 3' primers compatible for PCR use are located inside the 3' UTR of the corresponding mRNA, thus yielding amplicons which lack part of this region, i.e. the polyA tract and sometimes the polyadenylation signal, as illustrated in Figure 3.
- Such obtained cDNAs are then cloned into an appropriate vector using a procedure essentially similar to the one described in example 4.
- Full-length PCR products are then sequenced using a procedure similar to the one described in example 11. Completion of the sequencing of a given cDNA fragment may be assessed by comparing the sequence length to the size of the corresponding nested PCR product. When Northern blot data are available, the size of the mRNA detected for a given PCR product may also be used to finally assess that the sequence is complete. Sequences which do not fulfill these criteria are discarded and will undergo a new isolation procedure.
- Full-length PCR products are then cloned in an appropriate vector.
- the cDNAs can be cloned into a vector using a procedure similar to the one described in example 4.
- Such full-length cDNA clones are then double-sequenced and submitted to computer analyses using procedure essentially similar to the ones described in Examples 11 through 13.
- full-length cDNA clones obtained from amplicons lacking part of the 3'UTR may lack polyadenylations sites and signals.
- EXAMPLE 17 Methods for Obtaining cDNAs or Nucleic Acids Homologous to cDNAs or Fragments Thereof
- traditional hybridization based methods may also be employed. These methods may also be used to obtain the genomic DNAs which encode the mRNAs from which the cDNA is derived, mRNAs corresponding to the cDNAs, or nucleic acids which are homologous to cDNAs or fragments thereof.
- cDNAs of the present invention or fragments thereof, including 5'ESTs may also be used to isolate cDNAs or nucleic acids homologous to cDNAs from a cDNA library or a genomic DNA library as follows.
- Such cDNA libraries or genomic DNA libraries may be obtained from a commercial source or made using techniques familiar to those skilled in the art such as the one described in Examples 1 through 5.
- An example of such hybridization-based methods is provided below. Techniques for identifying cDNA clones in a cDNA library which hybridize to a given probe sequence are disclosed in Sambrook ef al., Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring Harbor Laboratory Press, 1989. The same techniques may be used to isolate genomic DNAs.
- cDNA or genomic DNA clones which hybridize to the detectable probe are identified and isolated for further manipulation as follows.
- a probe comprising at least 10 consecutive nucleotides from the cDNA or fragment thereof is labeled with a detectable label such as a radioisotope or a fluorescent molecule.
- the probe comprises at least 12, 15, or 17 consecutive nucleotides from the cDNA or fragment thereof. More preferably, the probe comprises 20 to 30 consecutive nucleotides from the cDNA or fragment thereof. In some embodiments, the probe comprises more than 30 nucleotides from the cDNA or fragment thereof.
- Techniques for labeling the probe are well known and include phosphorylation with polynucleotide kinase, nick translation, in vitro transcription, and non radioactive techniques. The cDNAs or genomic DNAs in the library are transferred to a nitrocellulose or nylon filter and denatured.
- the filter After blocking of non specific sites, the filter is incubated with the labeled probe for an amount of time sufficient to allow binding of the probe to cDNAs or genomic DNAs containing a sequence capable of hybridizing thereto.
- the stringency of the hybridization conditions used to identify cDNAs or genomic DNAs which hybridize to the detectable probe By varying the stringency of the hybridization conditions used to identify cDNAs or genomic DNAs which hybridize to the detectable probe, cDNAs or genomic DNAs having different levels of homology to the probe can be identified and isolated as described below.
- the melting temperature of the probe may be calculated using the following formulas:
- Tm melting temperature
- Prehybridization may be carried out in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, 100 ⁇ g denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS, 100 ⁇ g denatured fragmented salmon sperm DNA, 50% formamide.
- 6X SSC 6X SSC
- 5X Denhardt's reagent 0.5% SDS
- 100 ⁇ g denatured fragmented salmon sperm DNA 50% formamide.
- the formulas for SSC and Denhardt's solutions are listed in Sambrook et al, supra.
- Hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured before addition to the hybridization 5 solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25°C below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 15-25°C below the Tm. Preferably, for hybridizations in 6X SSC, the hybridization is conducted at approximately 10 68°C. Preferably, for hybridizations in 50% formamide containing solutions, the hybridization is conducted at approximately 42°C.
- the above procedure may be modified to identify cDNAs or genomic DNAs having decreasing levels of homology to the probe sequence.
- less stringent conditions may be used.
- the hybridization temperature may be decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a 25 sodium concentration of approximately 1M.
- the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be "moderate” conditions above 50°C and "low” conditions below 50°C.
- the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C.
- concentration of formamide in the hybridization buffer 30 may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe.
- the filter may be washed with 6X SSC, 0.5% SDS at 50°C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide.
- cDNAs or genomic DNAs which have hybridized to the probe are identified by autoradiography or other conventional techniques. 35 3.
- the nucleotide sequences of the hybridized nucleic acid and the cDNA or fragment thereof from which the probe was derived are compared.
- the sequences of the cDNA or fragment thereof from which the probe was derived and the sequences of the cDNA or genomic DNA which hybridized to the detectable probe may be stored on a computer readable medium as described below and compared to one another using any of a variety of algorithms familiar to those skilled in the art such as those described below.
- the polypeptide sequence encoded by the hybridized nucleic acid and the polypeptide sequence encoded by the cDNA or fragment thereof from which the probe was derived are compared.
- the sequences of the polypeptide encoded by the cDNA or fragment thereof from which the probe was derived and the polypeptide sequence encoded by the cDNA or genomic DNA which hybridized to the detectable probe may be stored on a computer readable medium as described below and compared to one another using any of a variety of algorithms familiar to those skilled in the art such as those described below.
- Protein and/or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BUSTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol. 275(3):403-410; Thompson ef al., 1994, Nucleic Acids Res. 22(2j:4673-4680; Higgins ef al., 1996, Methods Enzymol. 266:383-402; Altschul et al., 1990, J. Mol. Biol. 215(3J:403-410; Altschul ef al., 1993, Nature Genetics 3:266-272).
- sequence comparison algorithms and programs include, but are by no means limited to, TBLASTN, BUSTP, FASTA,
- BLAST Basic Local Alignment Search Tool
- BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database
- BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database
- TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands).
- TBLASTX compares the six-frame translations of a nucleotide query sequence against the six- frame translations of a nucleotide sequence database.
- the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
- High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.
- the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992, Science 256:1443-1445; Henikoff and Henikoff, 1993, Proteins .7:49-61).
- the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation)
- the BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology.
- the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).
- the parameters used with the above algorithms may be adapted depending on the sequence length and degree of homology studied. In some embodiments, the parameters may be the default parameters used by the algorithms in the absence of instructions from the user.
- the homology level is manually adjusted by calculating the number of nucleotides of the cDNA or fragment thereof which are not matched or aligned with the hybridizing sequence, determining the percentage of total nucleotides of the hybridizing sequence which the non-matched or non-aligned nucleotides represent, and subtracting this percentage from the homology level.
- the homology level would be adjusted as follows.
- nucleic acids having at least 95% nucleic acid homology, at least 96% nucleic acid homology, at least 97% nucleic acid homology, at least 98% nucleic acid homology, at least 99% nucleic acid homology, or more than 99% nucleic acid homology to the cDNA or fragment thereof from which the probe was derived may be obtained and identified.
- Such nucleic acids may be allelic variants or related nucleic acids from other species.
- by using progressively less stringent hybridization conditions one can obtain and identify nucleic acids having at least 90%, at least 85%, at least 80% or at least 75% homology to the cDNA or fragment thereof from which the probe was derived.
- nucleic acids encoding proteins having at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80% or at least 75% homology to the protein encoded by the cDNA or fragment thereof from which the probe was derived.
- the homology levels can be determined using the "default" opening penalty and the "default” gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff ef al., in: Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)).
- the level of polypeptide homology may be determined using the FASTDB algorithm described by Brutlag ef al. Comp. App. Biosci. 6:237-245, 1990.
- the results may be manually corrected as follows. First, the number of amino acid residues of the amino acid sequence encoded by the cDNA or fragment thereof which are not matched or aligned with the homologous sequence is determined. Then, the percentage of the length of the sequence encoded by the cDNA or fragment thereof which the non-matched or non-aligned amino acids represent is calculated. This percentage is subtracted from the homology level.
- cDNAs may be prepared by obtaining mRNA from the tissue, cell, or organism of interest using mRNA preparation procedures utilizing polyA selection procedures or other techniques known to those skilled in the art.
- a first primer capable of hybridizing to the polyA tail of the mRNA is hybridized to the mRNA and a reverse transcription reaction is performed to generate a first cDNA strand.
- the first cDNA strand is hybridized to a second primer containing at least 10 consecutive nucleotides of the sequences of SEQ ID NOs 24-73.
- the primer comprises at least 10, 12, 15, 17, 18, 20, 23, 25, or 28 consecutive nucleotides from the sequences of SEQ ID NOs 24-73. In some embodiments, the primer comprises more than 30 nucleotides from the sequences of SEQ ID NOs 24-73. If it is desired to obtain cDNAs containing the full protein coding sequence, including the authentic translation initiation site, the second primer used contains sequences located upstream of the translation initiation site. The second primer is extended to generate a second cDNA strand complementary to the first cDNA strand. Alternatively, RT-PCR may be performed as described above using primers from both ends of the cDNA to be obtained.
- cDNAs containing 5' fragments of the mRNA may be prepared by hybridizing an mRNA comprising the sequences of SEQ ID NOs. 24-73 with a primer comprising a complementary to a fragment of the known cDNA, genomic DNA or fragment thereof hybridizing the primer to the mRNAs, and reverse transcribing the hybridized primer to make a first cDNA strand from the mRNAs.
- the primer comprises at least 10, 12, 15, 17, 18, 20, 23, 25, or 28 consecutive nucleotides of the sequences complementary to SEQ ID NOs. 24-73.
- a second cDNA strand complementary to the first cDNA strand is synthesized.
- the second cDNA strand may be made by hybridizing a primer complementary to sequences in the first cDNA strand to the first cDNA strand and extending the primer to generate the second cDNA strand.
- the double stranded cDNAs made using the methods described above are isolated and cloned.
- the cDNAs may be cloned into vectors such as plasmids or viral vectors capable of replicating in an appropriate host cell.
- the host cell may be a bacterial, mammalian, avian, or insect cell.
- cDNAs are prepared from mRNA and cloned into double stranded phagemids as follows.
- the cDNA library in the double stranded phagemids is then rendered single stranded by treatment with an endonuclease, such as the Gene II product of the phage F1 and an exonuclease (Chang ef al., Gene 127:95-8, 1993).
- an endonuclease such as the Gene II product of the phage F1 and an exonuclease (Chang ef al., Gene 127:95-8, 1993).
- a biotinylated oligonucleotide comprising the sequence of a fragment of a known cDNA, genomic DNA or fragment thereof is hybridized to the single stranded phagemids.
- the fragment comprises at least 10, 12, 15, 17, 18, 20, 23, 25, or 28 consecutive nucleotides of the sequences of SEQ ID NOs. 24-73.
- Hybrids between the biotinylated oligonucleotide and phagemids are isolated by incubating the hybrids with streptavidin coated paramagnetic beads and retrieving the beads with a magnet (Fry ef al., Biotechniques, 13: 124-131, 1992). Thereafter, the resulting phagemids are released from the beads and converted into double stranded DNA using a primer specific for the cDNA or fragment thereof used to design the biotinylated oligonucleotide. Alternatively, protocols such as the Gene Trapper kit (Gibco BRL) may be used. The resulting double stranded DNA is transformed into bacteria. Homologous cDNAs or full length cDNAs containing the cDNA or fragment thereof sequence are identified by colony PCR or colony hybridization.
- a plurality of cDNAs containing full-length protein coding sequences or fragments of the protein coding sequences may be provided as cDNA libraries for subsequent evaluation of the encoded proteins or use in diagnostic assays as described below.
- cDNAs prepared by any method described therein may be subsequently engineered to obtain nucleic acids which include desired fragments of the cDNA using conventional techniques such as subcloning, PCR, or in vitro oligonucleotide synthesis.
- nucleic acids which include only the full coding sequences i.e.
- sequences encoding the signal peptide and the mature protein remaining after the signal peptide peptide is cleaved off may be obtained using techniques known to those skilled in the art. Alternatively, conventional techniques may be applied to obtain nucleic acids which contain only the coding sequence for the mature protein remaining after the signal peptide is cleaved off or nucleic acids which contain only the coding sequences for the signal peptides.
- nucleic acids containing any other desired fragment of the coding sequences for the encoded protein may be. obtained.
- the nucleic acid may contain at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 consecutive bases of a cDNA.
- a cDNA Once a cDNA has been obtained, it can be sequenced to determine the amino acid sequence it encodes. Once the encoded amino acid sequence has been determined, one can create and identify any of the many conceivable cDNAs that will encode that protein by simply using the degeneracy of the genetic code. For example, allelic variants or other homologous nucleic acids can be identified as described below.
- nucleic acids encoding the desired amino acid sequence can be synthesized in vitro.
- the coding sequence may be selected using the known codon or codon pair preferences for the host organism in which the cDNA is to be expressed. IV. Use of cDNA or Fragments Thereof to Express Proteins and Uses of Those Expressed Proteins
- cDNAs containing the full protein coding sequences of their corresponding mRNAs or portions thereof may be used to express the secreted proteins or portions thereof which they encode as described below.
- the cDNAs may contain the sequences encoding the signal peptide to facilitate secretion of the expressed protein. It will be appreciated that a plurality of extended cDNAs containing the full protein coding sequences or portions thereof may be simultaneously cloned into expression vectors to create an expression library for analysis of the encoded proteins as described below.
- nucleic acids containing the coding sequence for the proteins or fragments thereof to be expressed are obtained as described above and cloned into a suitable expression vector.
- the nucleic acids may contain the sequences encoding the signal peptide to facilitate secretion of the expressed protein.
- the nucleic acid may comprise the sequence of one of SEQ ID NOs: 24-73 listed in Table I and in the accompanying sequence listing.
- the nucleic acid may comprise those nucleotides which make up the full coding sequence of one of the sequences of SEQ ID NOs: 24-73 as defined in Table I above.
- nucleic acid used to express the protein or fragment thereof may comprise those nucleotides which encode the mature protein (i.e. the protein created by cleaving the signal peptide off) encoded by one of the sequences of SEQ ID NOs: 24-73 as defined in Table I above.
- claims relating to nucleic acids containing the sequence encoding the mature protein encompass equivalents to the sequences listed in Table I, such as sequences encoding biologically active proteins resulting from post-translational modification, enzymatic cleavage, or other readily identifiable variations from or equivalents to the secreted proteins in addition to cleavage of the signal peptide.
- the extent of the mature polypeptides differ from those indicated in Table II as a result of any of the preceding factors, the scope of claims relating to polypeptides comprising the sequence of a mature protein included in the sequence of one of SEQ ID NOs. 74-123 is not to be construed as excluding any readily identifiable variations from or equivalents to the sequences listed in Table II.
- claims relating to polypeptides comprising the sequence of the mature protein encompass equivalents to the sequences listed in Table II, such as biologically active proteins resulting from post-translational modification, enzymatic cleavage, or other readily identifiable variations from or equivalents to the secreted proteins in addition to cleavage of the signal peptide. It will also be appreciated that should the biologically active form of the polypeptides included in the sequence of one of SEQ ID NOs.
- nucleic acids encoding the biologically active form of the polypeptides differ from those identified as the mature polypeptide in Table II or the nucleotides encoding the mature polypeptide in Table I as a result of a sequencing error, reverse transcription or amplification error, mRNA splicing, post-translational modification of the encoded protein, enzymatic cleavage of the encoded protein, or other biological factors, one skilled in the art would be readily able to identify the amino acids in the biologically active form of the polypeptides and the nucleic acids encoding the biologically active form of the polypeptides. In such instances, the claims relating to polypetides comprising the mature protein included in one of SEQ ID NOs. 74-123 or nucleic acids comprising the nucleotides of one of SEQ ID NOs. 24-73 encoding the mature protein shall not be construed to exclude any readily identifiable variations from the sequences listed in Table I and Table II.
- the nucleic acid used to express the protein or fragment thereof may comprise those nucleotides which encode the signal peptide encoded by one of the sequences of SEQ ID NOs: 24-73 as defined in Table I above.
- the nucleic acid may encode a polypeptide comprising at least 5 consecutive amino acids of one of the sequences of SEQ ID NOs: 74-123.
- the nucleic acid may encode a polypeptide comprising at least 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 consecutive amino acids of one of the sequences of SEQ ID NOs: 74-123.
- the nucleic acids inserted into the expression vectors may also contain sequences upstream of the sequences encoding the signal peptide, such as sequences which regulate expression levels or sequences which confer tissue specific expression.
- the nucleic acid encoding the protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology.
- the expression vector may be any of the mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, MA), Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego, California). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence may be optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, ef al., U.S. Patent No. 5,082,767.
- the following is provided as one exemplary method to express the proteins encoded by the cDNAs or the nucleic acids described above.
- the methionine initiation codon for the gene and the poly A signal of the gene are identified. If the nucleic acid encoding the polypeptide to be expressed lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques.
- this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using Bgll and Sail restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene).
- pXT1 contains the LTRs and a fragment of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection.
- the vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable neomycin gene.
- the cDNA or fragment thereof encoding the polypeptide to be expressed is obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the cDNA or fragment thereof and containing restriction endonuclease sequences for Pst I incorporated into the 5'primer and Bglll at the 5' end of the corresponding cDNA 3' primer, taking care to ensure that the cDNA is positioned in frame with the poly A signal.
- the purified fragment obtained from the resulting PCR reaction is digested with Pstl, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXT1, now containing a poly A signal and digested with Bglll.
- the ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, New York) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600ug/ml G418 (Sigma, St. Louis, Missouri). Preferably the expressed protein is released into the culture medium, thereby facilitating purification.
- the cDNAs may be cloned into pED6dpc2 (DiscoverEase, Genetics Institute, Cambridge, MA).
- the resulting pED6dpc2 constructs may be transfected into a suitable host cell, such as COS 1 cells. Methotrexate resistant cells are selected and expanded.
- the protein expressed from the cDNA is released into the culture medium thereby facilitating purification. Proteins in the culture medium are separated by gel electrophoresis. If desired, the proteins may be ammonium sulfate precipitated or separated based on size or charge prior to electrophoresis.
- the expression vector lacking a cDNA insert is introduced into host cells or organisms and the proteins in the medium are harvested.
- the secreted proteins present in the medium are detected using techniques such as Coomassie or silver staining or using antibodies against the protein encoded by the cDNA. Coomassie and silver staining techniques are familiar to those skilled in the art.
- Antibodies capable of specifically recognizing the protein of interest may be generated using synthetic 15-mer peptides having a sequence encoded by the appropriate 5' EST, cDNA, or fragment thereof.
- the synthetic peptides are injected into mice to generate antibody to the polypeptide encoded by the 5' EST, cDNA, or fragment thereof.
- Secreted proteins from the host cells or organisms containing an expression vector which contains the cDNA or a fragment thereof are compared to those from the control cells or organism.
- the presence of a band in the medium from the cells containing the expression vector which is absent in the medium from the control cells indicates that the cDNA encodes a secreted protein.
- the band corresponding to the protein encoded by the cDNA will have a mobility near that expected based on the number of amino acids in the open reading frame of the cDNA.
- the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
- the proteins expressed from host ceils containing an expression vector containing an insert encoding a secreted protein or fragment thereof can be compared to the proteins expressed in host cells containing the expression vector without an insert.
- the presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the desired protein or fragment thereof is being expressed.
- the band will have the mobility expected for the secreted protein or fragment thereof.
- the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
- the protein encoded by the cDNA may be purified using standard immunochromatography techniques.
- a solution containing the secreted protein such as the culture medium or a cell extract, is applied to a column having antibodies against the secreted protein attached to the chromatography matrix.
- the secreted protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins.
- the specifically bound secreted protein is then released from the column and recovered using standard techniques.
- the cDNA sequence or fragment thereof may be incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides.
- the coding sequence of the cDNA or fragment thereof is inserted in frame with the gene encoding the other half of the chimera.
- the other half of the chimera may be D-globin or a nickel binding polypeptide encoding sequence.
- a chromatography matrix having antibody to D-globin or nickel attached thereto is then used to purify the chimeric protein.
- Protease cleavage sites may be engineered between the 0-globin gene or the nickel binding polypeptide and the cDNA or fragment thereof.
- the two polypeptides of the chimera may be separated from one another by protease digestion.
- One useful expression vector for generating D-globin chimerics is pSG5 (Stratagene), which encodes rabbit D-globin. Intron II of the rabbit D-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression.
- pSG5 which encodes rabbit D-globin. Intron II of the rabbit D-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression.
- Standard methods are published in methods texts such as Davis ef al., (Basic Methods in Molecular Biology, L.G. Davis, M.D. Dibner, and J.F. Battey, ed., Elsevier Press, NY, 1986) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega.
- Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro ExpressTM Translation Kit (Stratagene
- the purified proteins may be tested for the ability to bind to the surface of various cell types as described below. It will be appreciated that a plurality of proteins expressed from these cDNAs may be included in a panel of proteins to be simultaneously evaluated for the activities specifically described below, as well as other biological roles for which assays for determining activity are available.
- polypeptide to be expressed may also be a product of transgenic animals, i.e., as a component of the milk of transgenic cows, goats, pigs or sheeps which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein of interest.
- transgenic animals i.e., as a component of the milk of transgenic cows, goats, pigs or sheeps which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein of interest.
- the proteins encoded by the cDNAs, or fragments thereof are cloned into expression vectors such as those described in the previous example.
- the proteins are purified by size, charge, immunochromatography or other techniques familiar to those skilled in the art.
- the proteins are labeled using techniques known to those skilled in the art.
- the labeled proteins are incubated with cells or cell lines derived from a variety of organs or tissues to allow the proteins to bind to any receptor present on the cell surface. Following the incubation, the cells are washed to remove non-specifically bound protein.
- the labeled proteins are detected by autoradiography. Alternatively, unlabeled proteins may be incubated with the cells and detected with antibodies having a detectable label, such as a fluorescent molecule, attached thereto.
- Specificity of cell surface binding may be analyzed by conducting a competition analysis in which various amounts of unlabeled protein are incubated along with the labeled protein.
- the amount of labeled protein bound to the cell surface decreases as the amount of competitive unlabeled protein increases.
- various amounts of an unlabeled protein unrelated to the labeled protein is included in some binding reactions.
- the amount of labeled protein bound to the cell surface does not decrease in binding reactions containing increasing amounts of unrelated unlabeled protein, indicating that the protein encoded by the cDNA binds specifically to the cell surface.
- secreted proteins have been shown to have a number of important physiological effects and, consequently, represent a valuable therapeutic resource.
- the secreted proteins encoded by the cDNAs or fragments thereof made using any of the methods described therein may be evaluated to determine their physiological activities as described below.
- secreted proteins may act as cytokines or may affect cellular proliferation or differentiation.
- Many protein factors discovered to date including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.
- the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7c and CMK.
- the proteins encoded by the above cDNAs or fragments thereof may be evaluated for their ability to regulate T cell or thymocyte proliferation in assays such as those described above or in the following references: Current Protocols in Immunology, Ed. by J.E. Coligan ef al., Greene Publishing Associates and Wiley-lnterscience; Takai ef al. J. Immunol. 137:3494-3500, 1986. Bertagnolli ef al. J. Immunol. 145:1706-1712, 1990. Bertagnolli et al., Cellular Immunology 133:327-34 , 1991. Bertagnolli, ef al. J. Immunol.
- the proteins encoded by the cDNAs may also be assayed for the ability to regulate the proliferation and differentiation of hematopoietic or lymphopoietic cells.
- Many assays for such activity are familiar to those skilled in the art, including the assays in the following references: Bottomly, K., Davis, L.S. and Lipsky, P.E., Measurement of Human and Murine Interleukin 2 and Interleukin 4, Current Protocols in Immunology.. J.E. Coligan ef al. Eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries ef al., J. Exp. Med.
- proteins which exhibit cytokine, cell proliferation, or cell differentiation activity may then be formulated as pharmaceuticals and used to treat clinical conditions in which induction of cell proliferation or differentiation is beneficial.
- genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.
- the proteins encoded by the cDNAs may also be evaluated for their effects as immune regulators.
- the proteins may be evaluated for their activity to influence thymocyte or splenocyte cytotoxicity.
- Numerous assays for such activity are familiar to those skilled in the art including the assays described in the following references: Chapter 3 (In vitro Assays for Mouse Lymphocyte Function 3.1-3.19) and Chapter 7 (Immunologic studies in Humans) in Current Protocols in Immunology. J.E. Coligan ef al. Eds,
- the proteins encoded by the cDNAs may also be evaluated for their effects on T-cell dependent immunoglobulin responses and isotype switching. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Maliszewski, J. Immunol. 144:3028-3033, 1990; Mond, J.J. and Brunswick, M Assays for B Cell Function: In vitro Antibody Production, Vol 1 pp. 3.8.1-3.8.16 in Current Protocols in Immunology. J.E. Coligan et al Eds., John Wiley and Sons, Toronto. 1994. The proteins encoded by the cDNAs may also be evaluated for their effect on immune effector cells, including their effect on Th1 cells and cytotoxic lymphocytes.
- the proteins encoded by the cDNAs may also be evaluated for their effect on dendritic cell mediated activation of naive T-cells.
- Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Guery ef al., J. Immunol.
- the proteins encoded by the cDNAs may also be evaluated for their influence on the lifetime of lymphocytes.
- Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233- 243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca ef al., International Journal of Oncology 1 :639-648, 1992.
- Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica ef al., Blood 84:111-117, 1994; Fine ef al., Cellular immunology 155:111-122, 1994; Galy ef al., Blood 85:2770-2778, 1995; Toki ef al., Proc. Nat. Acad Sci. USA 88:7548- 7551, 1991.
- Those proteins which exhibit activity as immune system regulators activity may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of immune activity is beneficial.
- the protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytoiytic activity of NK cells and other cell populations.
- SCID severe combined immunodeficiency
- These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
- infectious diseases caused by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
- a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
- Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
- a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
- Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention.
- T- cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
- Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
- Tolerance which involves inducing non- responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
- Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus- host disease (GVHD).
- B lymphocyte antigen functions such as, for example, B7
- GVHD graft-versus- host disease
- blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
- rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
- a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7- 1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal.
- Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
- the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
- Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
- the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
- appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4lg fusion proteins in vivo as described in Lenschow ef al., Science 257:789-792 (1992) and Turka ef al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
- murine models of GVHD see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
- Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
- Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
- Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
- Administration of reagents which block costimulation of T cells by disrupting receptor ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.
- blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
- the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/pr/pr mice or NZB hybrid mice, murine autoimmuno collagen arthritis, diabetes mellitus in OD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
- Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory form of B lymphocyte antigens systemically.
- anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
- the infected cells would now be capable of delivering a costimulatory signal to T cells in vivo, thereby activating the T cells.
- up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
- Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
- a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
- tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1-like activity and/or B7-3-like activity.
- the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
- gene therapy techniques can be used to target a tumor cell for transfection in vivo.
- tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acids encoding all or a fragment of (e.g., a cytoplasmic- domain truncated fragment) of an MHC class I D chain protein and D 2 microglobulin protein or an MHC class II D chain protein and an MHC class II D chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
- nucleic acids encoding all or a fragment of (e.g., a cytoplasmic- domain truncated fragment) of an MHC class I D chain protein and D 2 microglobulin protein or an MHC class II D chain protein and an MHC class II D chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
- a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain. can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
- a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
- genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.
- the proteins encoded by the cDNAs or fragments thereof may also be evaluated for their hematopoiesis regulating activity. For example, the effect of the proteins on embryonic stem cell differentiation may be evaluated. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Johansson ef al. Cellular Biology 15:141-151, 1995; Keller ef al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan ef a/., Blood 81:2903- 2915, 1993.
- the proteins encoded by the cDNAs or fragments thereof may also be evaluated for their influence on the lifetime of stem cells and stem cell differentiation.
- Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Freshney, M.G. Methylcellulose Colony Forming Assays, in Culture of Hematopoietic Cells. R.I. Freshney, ef al. Eds. pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama ef al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; McNiece, I.K. and Briddell, R.A.
- a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
- erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with
- Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H1 and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978). Those proteins which are involved in the regulation of tissue growth may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of tissue growth is beneficial.
- a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of bums, incisions and ulcers.
- a protein of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
- Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
- a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
- a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
- tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
- a protein of the present invention which induces tendon/ligamentlike tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
- Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
- compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
- the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
- the compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
- compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
- the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e., for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.
- Further conditions which 5 may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.
- Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
- Proteins of the invention may also be useful to promote better or faster closure of non-healing 10 wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
- a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium) muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or 15 for promoting the growth of cells comprising such tissues.
- organs including, for example, pancreas, liver, intestine, kidney, skin, endothelium
- vascular including vascular endothelium tissue, or 15 for promoting the growth of cells comprising such tissues.
- Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to generate.
- a protein of the invention may also exhibit angiogenic activity.
- a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic 20 cytokine damage.
- a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
- genes encoding these proteins or nucleic acids 25 regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.
- the proteins encoded by the cDNAs or fragments thereof may also be evaluated for their ability to regulate reproductive hormones, such as follicle stimulating hormone.
- reproductive hormones such as follicle stimulating hormone.
- Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Vale ef al.,
- a protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins are characterized by their ability to stimulate the release of folic stimulating hormone (FSH).
- FSH follicle stimulating hormone
- FSH folic stimulating hormone
- a protein of the present invention alone or in heterodimers with a member of the inhibin D family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.
- the protein of the invention as a homodimer or as a heterodimer with other protein subunits of the inhibin-B group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885.
- a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
- genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.
- EXAMPLE 25 Assaying the Proteins Expressed from cDNAs or Fragments Thereof for Chemotactic/Chemokinetic Activity
- the proteins encoded by the cDNAs or fragments thereof may also be evaluated for chemotactic/chemokinetic activity.
- a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, cosinophils, epithelial and/or endothelial cells.
- Chemotactic and chmokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
- Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
- a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
- the protein or peptide has the ability to directly stimulate directed movement of cells.
- Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
- the activity of a protein of the invention may, among other means, be measured by the following methods:
- Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as 5 well as the ability of a protein to induce the adhension of one cell population to another cell population.
- Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-lnterscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub ef al. J. Clin. invest.
- EXAMPLE 26 Assaying the Proteins Expressed from cDNAs or Fragments Thereof for Regulation of Blood Clotting 15
- the proteins encoded by the cDNAs or fragments thereof may also be evaluated for their effects on blood clotting.
- Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Linet ef al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick ef al., Thrombosis Res. 45:413-419, 1987; Humphrey ef al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
- a protein of the invention may also exhibit hemostatic or thrombolytic activity.
- a protein is expected to be useful in treatment of various coagulations disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds 25 resulting from trauma, surgery or other causes.
- a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke)).
- genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the 30 proteins as desired.
- proteins encoded by the cDNAs or a fragment thereof may also be evaluated for their
- proteins of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
- receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune respones).
- Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
- a protein of the present invention may themselves be useful as inhibitors of receptor/ligand interactions.
- the proteins encoded by the cDNAs or a fragment thereof may also be evaluated for anti- inflammatory activity.
- the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
- Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
- SIRS systemic inflammatory response syndrome
- a protein of the invention may exhibit other anti-tumor activities.
- a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
- a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
- a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, eofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation
- Proteins which interact with the polypeptides encoded by cDNAs or fragments thereof, such as receptor proteins may be identified using two hybrid systems such as the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech). As described in the manual accompanying the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1 , Clontech), the cDNAs or fragments thereof, are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain of the yeast transcriptional activator GAL4.
- cDNAs in a cDNA library which encode proteins which might interact with the polypeptides encoded by the cDNAs or fragments thereof are inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4.
- the two expression plasmids are transformed into yeast and the yeast are plated on selection medium which selects for expression of selectable markers on each of the expression vectors as well as GAL4 dependent expression of the HIS3 gene.
- Transformants capable of growing on medium lacking histidine are screened for GAL4 dependent lacZ expression. Those cells which are positive in both the histidine selection and the lacZ assay contain plasmids encoding proteins which interact with the polypeptide encoded by the cDNAs or fragments thereof.
- the system described in Lustig ef al., Methods in Enzymology 283: 83-99 (1997), may be used for identifying molecules which interact with the polypeptides encoded by cDNAs.
- in vitro transcription reactions are performed on a pool of vectors containing cDNA inserts cloned downstream of a promoter which drives in vitro transcription.
- the resulting pools of mRNAs are introduced into Xenopus laevis oocytes.
- the oocytes are then assayed for a desired acitivity.
- pooled in vitro transcription products produced as described above may be translated in vitro.
- the pooled in vitro translation products can be assayed for a desired activity or for interaction with a known polypeptide.
- Proteins or other molecules interacting with polypeptides encoded by cDNAs can be found by a variety of additional techniques.
- affinity columns containing the polypeptide encoded by the cDNA or a fragment thereof can be constructed.
- the affinity column contains chimeric proteins in which the protein encoded by the cDNA or a fragment thereof is fused to glutathione S-transferase.
- Proteins interacting with the polypeptide attached to the column can then be isolated and analyzed on 2-D electrophoresis gel as described in Ramunsen ef al. Electrophoresis, 18, 588-598 (1997).
- the proteins retained on the affinity column can be purified by electrophoresis based methods and sequenced. The same method can be used to isolate antibodies, to screen phage display products, or to screen phage display human antibodies.
- Proteins interacting with polypeptides encoded by cDNAs or fragments thereof can also be screened by using an Optical Biosensor as described in Edwards & Leatherbarrow, Analytical Biochemistry, 246, 1-6 (1997).
- the main advantage of the method is that it allows the determination of the association rate between the protein and other interacting molecules. Thus, it is possible to specifically select interacting molecules with a high or low association rate.
- a target molecule is linked to the sensor surface (through a carboxymethl dextran matrix) and a sample of test molecules is placed in contact with the target molecules. The binding of a test molecule to the target molecule causes a change in the refractive index and/ or thickness.
- the target molecule can be one of the polypeptides encoded by cDNAs or a fragment thereof and the test sample can be a collection of proteins extracted from tissues or cells, a pool of expressed proteins, combinatorial peptide and/ or chemical libraries.or phage displayed peptides.
- the tissues or cells from which the test proteins are extracted can originate from any species.
- a target protein is immobilized and the test population is a collection of unique polypeptides encoded by the cDNAs or fragments thereof.
- proteins expressed from the cDNAs or fragments may be assayed for numerous activities in addition to those specifically enumerated above.
- the expressed proteins may be evaluated for applications involving control and regulation of inflammation, tumor proliferation or metastasis, infection, or other clinical conditions.
- the proteins expressed from the cDNAs or fragments thereof may be useful as nutritional agents or cosmetic agents.
- the proteins expressed from the cDNAs or fragments thereof may be used to generate antibodies capable of specifically binding to the expressed protein or fragments thereof as described below.
- the antibodies may capable of binding a full length protein encoded by one of the sequences of SEQ ID NOs. 24-73, a mature protein encoded by one of the sequences of SEQ ID NOs. 24-73, or a signal peptide encoded by one of the sequences of SEQ ID Nos. 24-73.
- the antibodies may be capable of binding fragments of the proteins expressed from the cDNAs which comprise at least 10 amino acids of the sequences of SEQ ID NOs: 74-123.
- the antibodies may be capable of binding fragments of the proteins expressed from the cDNAs which comprise at least 15 amino acids of the sequences of SEQ ID NOs: 74-123. In other embodiments, the antibodies may be capable of binding fragments of the proteins expressed from the cDNAs which comprise at least 25 amino acids of the sequences of SEQ ID NOs: 74-123. In further embodiments, the antibodies may be capable of binding fragments of the proteins expressed from the cDNAs which comprise at least 40 amino acids of the sequences of SEQ ID NOs: 74-123.
- Substantially pure protein or polypeptide is isolated from the transfected or transformed cells as described in example 18.
- concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml.
- Monoclonal or polyclonal antibody to the protein can then be prepared as follows: A. Monoclonal Antibody Production by Hybridoma Fusion
- Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, C, Nature 256:495 (1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).
- HAT media aminopterin
- the successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
- Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as Elisa, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. ef al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2. B. Polyclonal Antibody Production by Immunization
- Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogenicity.
- Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant.
- host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable.
- An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).
- Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, 0. ef al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 DM).. Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980).
- Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.
- the antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.
- the cDNAs of the present invention may be used as reagents in isolation procedures, diagnostic assays, and forensic procedures.
- sequences from the cDNAs may be detectably labeled and used as probes to isolate other sequences capable of hybridizing to them.
- sequences from the cDNAs may be used to design PCR primers to be used in isolation, diagnostic, or forensic procedures.
- the cDNAs may be used to prepare PCR primers for a variety of applications, including isolation procedures for cloning nucleic acids capable of hybridizing to such sequences, diagnostic techniques and forensic techniques.
- the PCR primers are at least 10 bases, and preferably at least 12, 15, or 17 bases in length. More preferably, the PCR primers are at least 20-30 bases in length. In some embodiments, the PCR primers may be more than 30 bases in length. It is preferred that the primer pairs have approximately the same G/C ratio, so that melting temperatures are approximately the same.
- a variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B.A. Ed.
- PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.
- a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.
- the nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample.
- the hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites.
- Probes derived from cDNAs or fragments thereof (or genomic DNAs obtainable therefrom) may be labeled with detectable labels familiar to those skilled in the art, including radioisotopes and non-radioactive labels, to provide a detectable probe.
- the detectable probe may be single stranded or double stranded and may be made using techniques known in the art, including in vitro transcription, nick translation, or kinase reactions.
- a nucleic acid sample containing a sequence capable of hybridizing to the labeled probe is contacted with the labeled probe. If the nucleic acid in the sample is double stranded, it may be denatured prior to contacting the probe.
- the nucleic acid sample may be immobilized on a surface such as a nitrocellulose or nylon membrane.
- the nucleic acid sample may comprise nucleic acids obtained from a variety of sources, including genomic DNA, cDNA libraries, RNA, or tissue samples.
- Procedures used to detect the presence of nucleic acids capable of hybridizing to the detectable probe include well known techniques such as Southern blotting, Northern blotting, dot blotting, colony hybridization, and plaque hybridization.
- the nucleic acid capable of hybridizing to the labeled probe may be cloned into vectors such as expression vectors, sequencing vectors, or in vitro transcription vectors to facilitate the characterization and expression of the hybridizing nucleic acids in the sample.
- vectors such as expression vectors, sequencing vectors, or in vitro transcription vectors to facilitate the characterization and expression of the hybridizing nucleic acids in the sample.
- such techniques may be used to isolate and clone sequences in a genomic library or cDNA library which are capable of hybridizing to the detectable probe as described in example 17 above.
- PCR primers made as described in example 32 above may be used in forensic analyses, such as the DNA fingerprinting techniques described in Examples 34-38 below. Such analyses may utilize detectable probes or primers based on the sequences of the cDNAs or fragments thereof (or genomic DNAs obtainable therefrom).
- DNA samples are isolated from forensic specimens of, for example, hair, semen, blood or skin cells by conventional methods.
- Corresponding sequences are obtained from a test subject.
- Each of these identification DNAs is then sequenced using standard techniques, and a simple database comparison determines the differences, if any, between the sequences from the subject and those from the sample.
- Statistically significant differences between the suspect's DNA sequences and those from the sample conclusively prove a lack of identity. This lack of identity can be proven, for example, with only one sequence. Identity, on the other hand, should be demonstrated with a large number of sequences, all matching.
- a minimum of 50 statistically identical sequences of 100 bases in length are used to prove identity between the suspect and the sample.
- primers are prepared from a large number of sequences from Table I and the appended sequence listing. Preferably, 20 to 50 different primers are used. These primers are used to obtain a corresponding number of PCR-generated DNA segments from the individual in question in accordance with example 32. Each of these DNA segments is sequenced, using the methods set forth in example 34. The database of sequences generated through this procedure uniquely identifies the individual from whom the sequences were obtained. The same panel of primers may then be used at any later time to absolutely correlate tissue or other biological specimen with that individual. EXAMPLE 36
- the procedure of example 35 is repeated to obtain a panel of at least 10 amplified sequences from an individual and a specimen.
- the panel contains at least 50 amplified sequences. More preferably, the panel contains 100 amplified sequences. In some embodiments, the panel contains 200 amplified sequences.
- This PCR-generated DNA is then digested with one or a combination of, preferably, four base specific restriction enzymes. Such enzymes are commercially available and known to those of skill in the art. After digestion, the resultant gene fragments are size separated in multiple duplicate wells on an agarose gel and transferred to nitrocellulose using Southern blotting techniques well known to those with skill in the art. For a review of Southern blotting see Davis ef al. (Basic Methods in Molecular Biology, 1986, Elsevier Press, pp 62-65).
- a panel of probes based on the sequences of the cDNAs (or genomic DNAs obtainable therefrom), or fragments thereof of at least 10 bases, are radioactiveiy or colorimetrically labeled using methods known in the art, such as nick translation or end labeling, and hybridized to the Southern blot using techniques known in the art (Davis ef al., supra).
- the probe comprises at least 12, 15, or 17 consecutive nucleotides from the cDNA (or genomic DNAs obtainable therefrom). More preferably, the probe comprises at least 20-30 consecutive nucleotides from the cDNA (or genomic DNAs obtainable therefrom). In some embodiments, the probe comprises more than 30 nucleotides from the cDNA (or genomic DNAs obtainable therefrom). In other embodiments, the probe comprises at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the cDNA (or genomic DNAs obtainable therefrom).
- At least 5 to 10 of these labeled probes are used, and more preferably at least about 20 or 30 are used to provide a unique pattern.
- the resultant bands appearing from the hybridization of a large sample of cDNAs (or genomic DNAs obtainable therefrom) will be a unique identifier. Since the restriction enzyme cleavage will be different for every individual, the band pattern on the Southern blot will also be unique. Increasing the number of cDNA probes will provide a statistically higher level of confidence in the identification since there will be an increased number of sets of bands used for identification.
- Another technique for identifying individuals using the cDNA sequences disclosed herein utilizes a dot blot hybridization technique.
- Genomic DNA is isolated from nuclei of subject to be identified. Oligonucleotide probes of approximately 30 bp in length are synthesized that correspond to at least 10, preferably 50 sequences from the cDNAs or genomic DNAs obtainable therefrom. The probes are used to hybridize to the genomic DNA through conditions known to those in the art. The oligonucleotides are end labeled with P 32 using polynucleotide kinase (Pharmacia). Dot Blots are created by spotting the genomic DNA onto nitrocellulose or the like using a vacuum dot blot manifold (BioRad, Richmond California).
- the nitrocellulose filter containing the genomic sequences is baked or UV linked to the filter, prehybridized and hybridized with labeled probe using techniques known in the art (Davis ef al. supra).
- the 32 P labeled DNA fragments are sequentially hybridized with successively stringent conditions to detect minimal differences between the 30 bp sequence and the DNA.
- Tetramethylammonium chloride is useful for identifying clones containing small numbers of nucleotide mismatches (Wood ef al., Proc. Natl. Acad. Sci. USA 82(6):1585-1588 (1985)).
- a unique pattern of dots distinguishes one individual from another individual.
- cDNAs or oligonucleotides containing at least 10 consecutive bases from these sequences can be used as probes in the following alternative fingerprinting technique.
- the probe comprises at least
- the probe comprises at least 20-30 consecutive nucleotides from the cDNA (or genomic DNAs obtainable therefrom). In some embodiments, the probe comprises more than 30 nucleotides from the cDNA (or genomic DNAs obtainable therefrom). In other embodiments, the probe comprises at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the cDNA (or genomic DNAs obtainable therefrom).
- a plurality of probes having sequences from different genes are used in the alternative fingerprinting technique.
- Example 38 below provides a representative alternative fingerprinting procedure in which the probes are derived from cDNAs.
- EXAMPLE 38 Alternative "Fingerprint" Identification Technigue 20-mer oligonucleotides are prepared from a large number, e.g. 50, 100, or 200, of cDNA sequences (or genomic DNAs obtainable therefrom) using commercially available oligonucleotide services such as Genset, Paris, France. Cell samples from the test subject are processed for DNA using techniques well known to those with skill in the art. The nucleic acid is digested with restriction enzymes such as EcoRI and Xbal. Following digestion, samples are applied to wells for electrophoresis.
- restriction enzymes such as EcoRI and Xbal
- the procedure may be modified to accommodate polyacrylamide electrophoresis, however in this example, samples containing 5 ug of DNA are loaded into wells and separated on 0.8% agarose gels. The gels are transferred onto nitrocellulose using standard Southern blotting techniques.
- the number of probe sequences used can be varied for additional accuracy or clarity.
- the antibodies generated in Examples 18 and 31 above may be used to identify the tissue type or cell species from which a sample is derived as described above.
- EXAMPLE 39 Identification of Tissue Types or Cell Species by Means of Labeled Tissue Specific Antibodies Identification of specific tissues is accomplished by the visualization of tissue specific antigens by means of antibody preparations according to Examples 18 and 31 which are conjugated, directly or indirectly to a detectable marker. Selected labeled antibody species bind to their specific antigen binding partner in tissue sections, cell suspensions, or in extracts of soluble proteins from a tissue sample to provide a pattern for qualitative or semi-qualitative interpretation.
- Antisera for these procedures must have a potency exceeding that of the native preparation, and for that reason, antibodies are concentrated to a mg/ml level by isolation of the gamma globulin fraction, for example, by ion-exchange chromatography or by ammonium sulfate fractionation. Also, to provide the most specific antisera, unwanted antibodies, for example to common proteins, must be removed from the gamma globulin fraction, for example by means of insoluble immunoabsorbents, before the antibodies are labeled with the marker. Either monoclonal or heterologous antisera is suitable for either procedure.
- Purified, high-titer antibodies prepared as described above, are conjugated to a detectable marker, as described, for example, by Fudenberg, H., Chap. 26 in: Basic 503 Clinical Immunology, 3rd Ed. Lange, Los Altos, California (1980) or Rose, N. ef al., Chap. 12 in: Methods in Immunodiagnosis, 2d Ed. John Wiley 503 Sons, New York (1980).
- a fluorescent marker either fluorescein or rhodamine, is preferred, but antibodies can also be labeled with an enzyme that supports a color producing reaction with a substrate, such as horseradish peroxidase. Markers can be added to tissue-bound antibody in a second step, as described below.
- the specific antitissue antibodies can be labeled with ferritin or other electron dense particles, and localization of the ferritin coupled antigen-antibody complexes achieved by means of an electron microscope.
- the antibodies are radiolabeled, with, for example 125 l, and detected by overlaying the antibody treated preparation with photographic emulsion.
- Preparations to carry out the procedures can comprise monoclonal or polyclonal antibodies to a single protein or peptide identified as specific to a tissue type, for example, brain tissue, or antibody preparations to several antigenically distinct tissue specific antigens can be used in panels, independently or in mixtures, as required.
- Tissue sections and cell suspensions are prepared for immunohistochemical examination according to common histological techniques. Multiple cryostat sections (about 4 Dm, unfixed) of the unknown tissue and known control, are mounted and each slide covered with different dilutions of the antibody preparation. Sections of known and unknown tissues should also be treated with preparations to provide a positive control, a negative control, for example, pre-immune sera, and a control for non-specific staining, for example, buffer. Treated sections are incubated in a humid chamber for 30 min at room temperature, rinsed, then washed in buffer for 30-45 min. Excess fluid is blotted away, and the marker developed.
- tissue specific antibody was not labeled in the first incubation, it can be labeled at this time in a second antibody-antibody reaction, for example, by adding fluorescein- or enzyme-conjugated antibody against the immunoglobulin class of the antiserum-producing species, for example, fluorescein labeled antibody to mouse IgG.
- fluorescein- or enzyme-conjugated antibody against the immunoglobulin class of the antiserum-producing species for example, fluorescein labeled antibody to mouse IgG.
- fluorescein labeled antibody to mouse IgG.
- the antigen found in the tissues by the above procedure can be quantified by measuring the intensity of color or fluorescence on the tissue section, and calibrating that signal using appropriate standards.
- B. Identification of Tissue Specific Soluble Proteins The visualization of tissue specific proteins and identification of unknown tissues from that procedure is carried out using the labeled antibody reagents and detection strategy as described for immunohistochemistry; however the sample is prepared according to an electrophoretic technique to distribute the proteins extracted from the tissue in an orderly array on the basis of molecular weight for detection. A tissue sample is homogenized using a Virtis apparatus; cell suspensions are disrupted by
- SDS polyacrylamide electrophoresis as described, for example, by Davis, L. ef al., Section 19-2 in: Basic Methods in Molecular Biology (P. Leder, ed), Elsevier, New York (1986), using a range of amounts of polyacrylamide in a set of gels to resolve the entire molecular weight range of proteins to be detected in the sample.
- a size marker is run in parallel for purposes of estimating molecular weights of the constituent proteins.
- Sample size for analysis is a convenient volume of from 5 to55 Dl, and containing from about 1 to 100 Dg protein.
- An aliquot of each of the resolved proteins is transferred by blotting to a nitrocellulose filter paper, a process that maintains the pattern of resolution. Multiple copies are prepared.
- a detectable label can be attached to the primary tissue antigen-primary antibody complex according to various strategies and permutations thereof.
- the primary specific antibody can be labeled; alternatively, the unlabeled complex can be bound by a labeled secondary anti-lgG antibody.
- either the primary or secondary antibody is conjugated to a biotin molecule, which can, in a subsequent step, bind an avidin conjugated marker.
- enzyme labeled or radioactive protein A which has the property of binding to any IgG, is bound in a final step to either the primary or secondary antibody.
- tissue specific antigen binding at levels above those seen in control tissues to one or more tissue specific antibodies, prepared from the gene sequences identified from cDNA sequences, can identify tissues of unknown origin, for example, forensic samples, or differentiated tumor tissue that has metastasized to foreign bodily sites.
- cDNAs may be mapped to their chromosomal locations
- example 40 below describes radiation hybrid (RH) mapping of human chromosomal regions using cDNAs.
- example 41 below describes a representative procedure for mapping a cDNA (or a genomic DNA obtainable therefrom) to its location on a human chromosome,
- example 42 below describes mapping of cDNAs (or genomic DNAs obtainable therefrom) on metaphase chromosomes by Fluorescence In Situ Hybridization (FISH).
- FISH Fluorescence In Situ Hybridization
- RH mapping is a somatic cell genetic approach that can be used for high resolution mapping of the human genome.
- cell lines containing one or more human chromosomes are lethally irradiated, breaking each chromosome into fragments whose size depends on the radiation dose. These fragments are rescued by fusion with cultured rodent cells, yielding subclones containing different fragments of the human genome.
- This technique is described by Benham ef al. (Genomics 4:509-517, 1989) and Cox ef al., (Science 250:245-250, 1990). The random and independent nature of the subclones permits efficient mapping of any human genome marker.
- Human DNA isolated from a panel of 80-100 cell lines provides a mapping reagent for ordering cDNAs (or genomic DNAs obtainable therefrom), in this approach, the frequency of breakage between markers is used to measure distance, allowing construction of fine resolution maps as has been done using conventional ESTs (Schuler et al., Science 274:540-546, 1996).
- 5 RH mapping has been used to generate a high-resolution whole genome radiation hybrid map of human chromosome 17q22-q25.3 across the genes for growth hormone (GH) and thymidine kinase (TK) (Foster ef al., Genomics 33:185-192, 1996), the region surrounding the Gorlin syndrome gene (Obermayr ef al., Eur. J.
- EXAMPLE 41 Mapping of cDNAs to Human Chromosomes using PCR technigues cDNAs (or genomic DNAs obtainable therefrom) may be assigned to human chromosomes using
- oligonucleotide primer pairs are designed from the cDNA sequence (or the sequence of a genomic DNA obtainable therefrom) to minimize the chance of amplifying through an intron.
- the oligonucleotide primers are 18-23 bp in length and are designed for PCR amplification.
- the creation of PCR primers from known sequences is well known to those with skill in the art. For a review of PCR technology see Erlich, H.A., PCR Technology; Principles and Applications for DNA
- the primers are used in polymerase chain reactions (PCR) to amplify templates from total human genomic DNA.
- PCR conditions are as follows: 60 ng of genomic DNA is used as a template for PCR with 80 ng of each oligonucleotide primer, 0.6 unit of Taq polymerase, and 1 DCu of a 32 P-labeled deoxycytidine triphosphate.
- the PCR is performed in a microplate thermocycler (Techne) under the following conditions:
- NIGMS Human-Rodent Somatic Cell Hybrid Mapping Panel Number 1 (NIGMS, Camden, NJ).
- PCR is used to screen a series of somatic cell hybrid cell lines containing defined sets of human chromosomes for the presence of a given cDNA (or genomic DNA obtainable therefrom). DNA is isolated from the somatic hybrids and used as starting templates for PCR reactions using the primer pairs from the cDNAs (or genomic DNAs obtainable therefrom). Only those somatic cell hybrids with chromosomes
- the cDNAs (or genomic DNAs obtainable therefrom) are assigned to a chromosome by analysis of the segregation pattern of PCR products from the somatic hybrid DNA templates.
- the single human chromosome present in all cell hybrids that give rise to an amplified fragment is the chromosome containing that cDNA (or genomic DNA obtainable therefrom).
- the cDNAs may be mapped to individual chromosomes using FISH as described in example 42 below.
- EXAMPLE 42 Mapping of cDNAs to Chromosomes Using Fluorescence in situ Hybridization Fluorescence in situ hybridization allows the cDNA (or genomic DNA obtainable therefrom) to be mapped to a particular location on a given chromosome.
- the chromosomes to be used for fluorescence in situ hybridization techniques may be obtained from a variety of sources including cell cultures, tissues, or whole blood.
- chromosomal localization of a cDNA is obtained by FISH as described by Cherif ef al. (Proc. Natl. Acad. Sci. U.S.A., 87:6639-6643, 1990).
- Metaphase chromosomes are prepared from phytohemagglutinin (PHA)-stimulated blood cell donors. PHA-stimulated lymphocytes from healthy males are cultured for 72 h in RPMI-1640 medium. For synchronization, methotrexate (10 DM) is added for 17 h, followed by addition of 5-bromodeoxyuridine (5- BudR, 0.1 mM) for 6 h.
- PHA phytohemagglutinin
- Colcemid (1 Dg/ml) is added for the last 15 min before harvesting the cells.
- Cells are collected, washed in RPMI, incubated with a hypotonic solution of KCI (75 mM) at 370C for 15 min and fixed in three changes of methanol:acetic acid (3:1).
- the cell suspension is dropped onto a glass slide and air dried.
- the cDNA (or genomic DNA obtainable therefrom) is labeled with biotin-16 dUTP by nick translation according to the manufacturer's instructions (Bethesda Research Laboratories, Bethesda, MD), purified using a Sephadex G-50 column (Pharmacia, Upssaia, Sweden) and precipitated.
- hybridization buffer 50% formamide, 2 X SSC, 10% dextran sulfate, 1 mg/ml sonicated salmon sperm DNA, pH 7) and the probe is denatured at 7O0C for 5-10 min. Slides kept at -20DC are treated for 1 h at 37DC with RNase A (100 Dg/ml), rinsed three times in 2 X
- Chromosome preparations are denatured in 70% formamide, 2 X SSC for 2 min at 7O0C, then dehydrated at 4DC.
- the slides are treated with proteinase K (10 Dg/100 ml in 20 mM Tris-HCl, 2 mM CaCI 2 ) at 37DC for 8 min and dehydrated.
- the hybridization mixture containing the probe is placed on the slide, covered with a coverslip, sealed with rubber cement and incubated overnight in a humid chamber at 37DC. After hybridization and post-hybridization washes, the biotinylated probe is detected by avidin-FITC and amplified with additional layers of biotinylated goat anti-avidin and avidin-FITC.
- fluorescent R-bands are obtained as previously described (Cherif ef al., supra.). The slides are observed under a LEICA fluorescence microscope (DMRXA). Chromosomes are counterstained with propidium iodide and the fluorescent signal of the probe appears as two symmetrical yellow-green spots on both chromatids of the fluorescent R-band chromosome (red). Thus, a particular cDNA (or genomic DNA obtainable therefrom) may be localized to a particular cytogenetic R-band on a given chromosome.
- DMRXA LEICA fluorescence microscope
- cDNAs to Construct or Expand Chromosome Maps
- Chromosome mapping involves assigning a given unique sequence to a particular chromosome as described above. Once the unique sequence has been mapped to a given chromosome, it is ordered relative to other unique sequences located on the same chromosome.
- One approach to chromosome mapping utilizes a series of yeast artificial chromosomes (YACs) bearing several thousand long inserts derived from the chromosomes of the organism from which the cDNAs (or genomic DNAs obtainable therefrom) are obtained. This approach is described in Ramaiah Nagaraja ef al. Genome Research 7:210- 222, March 1997. Briefly, in this approach each chromosome is broken into overlapping pieces which are inserted into the YAC vector.
- the YAC inserts are screened using PCR or other methods to determine whether they include the cDNA (or genomic DNA obtainable therefrom) whose position is to be determined. Once an insert has been found which includes the cDNA (or genomic DNA obtainable therefrom), the insert can be analyzed by PCR or other methods to determine whether the insert also contains other sequences known to be on the chromosome or in the region from which the cDNA (or genomic DNA obtainable therefrom) was derived. This process can be repeated for each insert in the YAC library to determine the location of each of the cDNAs (or genomic DNAs obtainable therefrom) relative to one another and to other known chromosomal markers. In this way, a high resolution map of the distribution of numerous unique markers along each of the organisms chromosomes may be obtained.
- cDNAs may also be used to identify genes associated with a particular phenotype, such as hereditary disease or drug response.
- This example illustrates an approach useful for the association of cDNAs (or genomic DNAs obtainable therefrom) with particular phenotypic characteristics.
- a particular cDNA or genomic DNA obtainable therefrom is used as a test probe to associate that cDNA (or genomic DNA obtainable therefrom) with a particular phenotypic characteristic.
- CDNAs are mapped to a particular location on a human chromosome using techniques such as those described in Examples 40 and 41 or other techniques known in the art.
- a search of Mendelian Inheritance in Man (V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) reveals the region of the human chromosome which contains the cDNA (or genomic DNA obtainable therefrom) to be a very gene rich region containing several known genes and several diseases or phenotypes for which genes have not been identified. The gene corresponding to this cDNA (or genomic DNA obtainable therefrom) thus becomes an immediate candidate for each of these genetic diseases.
- PCR primers from the cDNA are used to screen genomic DNA, mRNA or cDNA obtained from the patients.
- CDNAs (or genomic DNAs obtainable therefrom) that are not amplified in the patients can be positively associated with a particular disease by further analysis.
- the PCR analysis may yield fragments of different lengths when the samples are derived from an individual having the phenotype associated with the disease than when the sample is derived from a healthy individual, indicating that the gene containing the cDNA may be responsible for the genetic disease.
- the present cDNAs may also be used to construct secretion vectors capable of directing the secretion of the proteins encoded by genes inserted in the vectors.
- Such secretion vectors may facilitate the purification or enrichment of the proteins encoded by genes inserted therein by reducing the number of background proteins from which the desired protein must be purified or enriched. Exemplary secretion vectors are described below.
- the secretion vectors of the present invention include a promoter capable of directing gene expression in the host cell, tissue, or organism of interest.
- promoters include the Rous Sarcoma Virus promoter, the SV40 promoter, the human cytomegalovirus promoter, and other promoters familiar to those skilled in the art.
- the host cell, tissue, or organism may be any cell, tissue, or organism which recognizes the signal peptide encoded by the signal sequence in the cDNA (or genomic DNA obtainable therefrom). Suitable hosts include mammalian cells, tissues or organisms, avian cells, tissues, or organisms, insect cells, tissues or organisms, or yeast.
- the secretion vector contains cloning sites for inserting genes encoding the proteins which are to be secreted.
- the cloning sites facilitate the cloning of the insert gene in frame with the signal sequence such that a fusion protein in which the signal peptide is fused to the protein encoded by the inserted gene is expressed from the mRNA transcribed from the promoter.
- the signal peptide directs the extracellular secretion of the fusion protein.
- the secretion vector may be DNA or RNA and may integrate into the chromosome of the host, be stably maintained as an extrachromosomal replicon in the host, be an artificial chromosome, or be transiently present in the host.
- the secretion vector is maintained in multiple copies in each host cell.
- multiple copies means at least 2,5, 10, 20, 25, 50 or more than 50 copies per cell.
- the multiple copies are maintained extrachromosomally.
- the multiple copies result from amplification of a chromosomal sequence.
- nucleic acid backbones suitable for use as secretion vectors are known to those skilled in the art, including retroviral vectors, SV40 vectors, Bovine Papilloma Virus vectors, yeast integrating plasmids, yeast episomal plasmids, yeast artificial chromosomes, human artificial chromosomes, P element vectors, baculovirus vectors, or bacterial plasmids capable of being transiently introduced into the host.
- the secretion vector may also contain a polyA signal such that the polyA signal is located downstream of the gene inserted into the secretion vector.
- the secretion vector is introduced into the host cell, tissue, or organism using calcium phosphate precipitation, DEAE-Dextran, electroporation, liposome-mediated transfection, viral particles or as naked DNA.
- the protein encoded by the inserted gene is then purified or enriched from the supernatant using conventional techniques such as ammonium sulfate precipitation, immunoprecipitation, immunochromatography, size exclusion chromatography, ion exchange chromatography, and hplc.
- the secreted protein may be in a sufficiently enriched or pure state in the supernatant or growth media of the host to permit it to be used for its intended purpose without further enrichment.
- the signal sequences may also be inserted into vectors designed for gene therapy.
- the signal sequence is operably linked to a promoter such that mRNA transcribed from the promoter encodes the signal peptide.
- a cloning site is located downstream of the signal sequence such that a gene encoding a protein whose secretion is desired may readily be inserted into the vector and fused to the signal sequence.
- the vector is introduced into an appropriate host cell. The protein expressed from the promoter is secreted extracellularly, thereby producing a therapeutic effect.
- the cDNAs or 5' ESTs may also be used to clone sequences located upstream of the cDNAs or 5' ESTs which are capable of regulating gene expression, including promoter sequences, enhancer sequences, and other upstream sequences which influence transcription or translation levels. Once identified and cloned, these upstream regulatory sequences may be used in expression vectors designed to direct the expression of an inserted gene in a desired spatial, temporal, developmental, or quantitative fashion.
- the next example describes a method for cloning sequences upstream of the cDNAs or 5' ESTs.
- Sequences derived from cDNAs or 5' ESTs may be used to isolate the promoters of the corresponding genes using chromosome walking techniques.
- chromosome walking technique which utilizes the GenomeWalker- kit available from Clontech, five complete genomic DNA samples are each digested with a different restriction enzyme which has a 6 base recognition site and leaves a blunt end. Following digestion, oligonucleotide adapters are ligated to each end of the resulting genomic DNA fragments.
- a first PCR reaction is performed according to the manufacturer's instructions using an outer adaptor primer provided in the kit and an outer gene specific primer.
- the gene specific primer should be selected to be specific for the cDNA or 5' EST of interest and should have a melting temperature, length, and location in the cDNA or 5' EST which is consistent with its use in PCR reactions.
- Each first PCR reaction contains 5ng of genomic DNA, 5 ⁇ l of 10X Tth reaction buffer, 0.2 mM of each dNTP, 0.2 ⁇ M each of outer adaptor primer and outer gene specific primer, 1.1 mM of Mg(OAc) 2 , and 1 ⁇ l of the Tth polymerase 50X mix in a total volume of 50 ⁇ l.
- the reaction cycle for the first PCR reaction is as follows: 1 min at 940C / 2 sec at 94DC, 3 min at 720C (7 cycles) / 2 sec at 940C, 3 min at 67DC (32 cycles) / 5 min at 670C.
- the product of the first PCR reaction is diluted and used as a template for a second PCR reaction according to the manufacturer's instructions using a pair of nested primers which are located internally on the amplicon resulting from the first PCR reaction.
- 5 ⁇ l of the reaction product of the first PCR reaction mixture may be diluted 180 times. Reactions are made in a 50 ⁇ l volume having a composition identical to that of the first PCR reaction except the nested primers are used.
- the first nested primer is specific for the adaptor, and is provided with the GenomeWalkerO kit.
- the second nested primer is specific for the particular cDNA or 5' EST for which the promoter is to be cloned and should have a melting temperature, length, and location in the cDNA or 5' EST which is consistent with its use in PCR reactions.
- the reaction parameters of the second PCR reaction are as follows: 1 min at 940C / 2 sec at 94DC, 3 min at 72DC (6 cycles) / 2 sec at 940C, 3 min at 67DC (25 cycles) / 5 min at 67DC
- the product of the second PCR reaction is purified, cloned, and sequenced using standard techniques.
- two or more human genomic DNA libraries can be constructed by using two or more restriction enzymes.
- the digested genomic DNA is cloned into vectors which can be converted into single stranded, circular, or linear DNA.
- a biotinylated oligonucleotide comprising at least 15 nucleotides from the cDNA or 5' EST sequence is hybridized to the single stranded DNA. Hybrids between the biotinylated oligonucleotide and the single stranded DNA containing the cDNA or EST sequence are isolated as described in example 17 above.
- the single stranded DNA containing the cDNA or EST sequence is released from the beads and converted into double stranded DNA using a primer specific for the cDNA or 5' EST sequence or a primer corresponding to a sequence included in the cloning vector.
- the resulting double stranded DNA is transformed into bacteria.
- DNAs containing the 5' EST or cDNA sequences are identified by colony PCR or colony hybridization.
- promoters and transcription start sites within the upstream sequences may be identified by comparing the sequences upstream of the cDNAs or 5' ESTs with databases containing known transcription start sites, transcription factor binding sites, or promoter sequences.
- promoters in the upstream sequences may be identified using promoter reporter vectors as described below.
- each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, D galactosidase, or green fluorescent protein.
- the sequences upstream of the cDNAs or 5' ESTs are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell.
- the level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site.
- the presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert.
- the upstream sequences can be cloned into vectors which contain an enhancer for augmenting transcription levels from weak promoter sequences. A significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present in the inserted upstream sequence.
- Appropriate host cells for the promoter reporter vectors may be chosen based on the results of the above described determination of expression patterns of the cDNAs and ESTs. For example, if the expression pattern analysis indicates that the mRNA corresponding to a particular cDNA or fragment thereof is expressed in fibroblasts, the promoter reporter vector may be introduced into a human fibroblast cell line.
- Promoter sequences within the upstream genomic DNA may be further defined by constructing nested deletions in the upstream DNA using conventional techniques such as Exonuclease III digestion.
- the resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity. In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into the cloning sites in the promoter reporter vectors.
- EXAMPLE 48 Cloning and Identification of Promoters Using the method described in example 47 above with 5' ESTs, sequences upstream of several genes were obtained. Using the primer pairs GGG AAG ATG GAG ATA GTA TTG CCT G (SEQ ID NO:15) and CTG CCA TGT ACA TGA TAG AGA GAT TC (SEQ ID NO-16), the promoter having the internal designation P13H2 (SEQ ID NO:17) was obtained.
- the promoter having the internal designation P15B4 (SEQ ID NO:20) was obtained.
- the promoter having the internal designation P29B6 (SEQ ID NO:23) was obtained.
- Figure 4 provides a schematic description of the promoters isolated and the way they are assembled with the corresponding 5' tags.
- the upstream sequences were screened for the presence of motifs resembling transcription factor binding sites or known transcription start sites using the computer program Matlnspector release 2.0, August 1996.
- Figure 5 describes the transcription factor binding sites present in each of these promoters.
- the columns labeled matrice provides the name of the Matlnspector matrix used.
- the column labeled position provides the 5' postion of the promoter site. Numeration of the sequence starts from the transcription site as determined by matching the genomic sequence with the 5' EST sequence.
- the column labeled "orientation” indicates the DNA strand on which the site is found, with the + strand being the coding strand as determined by matching the genomic sequence with the sequence of the 5' EST.
- the column labeled “score” provides the Matlnspector score found for this site.
- the column labeled "length” provides the length of the site in nucleotides.
- the column labeled "sequence” provides the sequence of the site found.
- the promoters and other regulatory sequences located upstream of the cDNAs or 5' ESTs may be used to design expression vectors capable of directing the expression of an inserted gene in a desired spatial, temporal, developmental, or quantitative manner.
- a promoter capable of directing the desired spatial, temporal, developmental, and quantitative patterns may be selected using the results of trie expression analysis described in example 10 above. For example, if a promoter which confers a high level of expression in muscle is desired, the promoter sequence upstream of a cDNA or 5' EST derived from an mRNA which is expressed at a high level in muscle, as determined by the method of example 10, may be used in the expression vector.
- the desired promoter is placed near multiple restriction sites to facilitate the cloning of the desired insert downstream of the promoter, such that the promoter is able to drive expression of the inserted gene.
- the promoter may be inserted in conventional nucleic acid backbones designed for extrachromosomal replication, integration into the host chromosomes or transient expression.
- Suitable backbones for the present expression vectors include retroviral backbones, backbones from eukaryotic episomes such as SV40 or Bovine Papilloma Virus, backbones from bacterial episomes, or artificial chromosomes.
- the expression vectors also include a polyA signal downstream of the multiple restriction sites for directing the polyadenylation of mRNA transcribed from the gene inserted into the expression vector.
- proteins which interact with the promoter may be identified as described in example 49 below.
- EXAMPLE 49 Identification of Proteins Which Interact with Promoter Sequences, Upstream Regulatory Seguences, or mRNA Sequences within the promoter region which are likely to bind transcription factors may be identified by homology to known transcription factor binding sites or through conventional mutagenesis or deletion analyses of reporter plasmids containing the promoter sequence. For example, deletions may be made in a reporter plasmid containing the promoter sequence of interest operably linked to an assayable reporter gene. The reporter plasmids carrying various deletions within the promoter region are transfected into an appropriate host cell and the effects of the deletions on expression levels is assessed.
- Transcription factor binding sites within the regions in which deletions reduce expression levels may be further localized using site directed mutagenesis, linker scanning analysis, or other techniques familiar to those skilled in the art.
- Nucleic acids encoding proteins which interact with sequences in the promoter may be identified using one- hybrid systems such as those described in the manual accompanying the Matchmaker One-Hybrid System kit avalilabe from Clontech (Catalog No. K1603-1). Briefly, the Matchmaker One-hybrid system is used as follows.
- the target sequence for which it is desired to identify binding proteins is cloned upstream of a selectable reporter gene and integrated into the yeast genome. Preferably, multiple copies of the target sequences are inserted into the reporter plasmid in tandem.
- the yeast are plated on selective media to select cells expressing the selectable marker linked to the promoter sequence.
- the colonies which grow on the selective media contain genes encoding proteins which bind the target sequence.
- the inserts in the genes encoding the fusion proteins are further characterized by sequencing.
- the inserts may be inserted into expression vectors or in vitro transcription vectors. Binding of the polypeptides encoded by the inserts to the promoter DNA may be confirmed by techniques familiar to those skilled in the art, such as gel shift analysis or DNAse protection analysis.
- the present invention also comprises the use of cDNAs (or genomic DNAs obtainable therefrom) in gene therapy strategies, including antisense and triple helix strategies as described in Examples 50 and 51 below.
- cDNAs or genomic DNAs obtainable therefrom
- antisense approaches nucleic acid sequences complementary to an mRNA are hybridized to the mRNA intracellulariy, thereby blocking the expression of the protein encoded by the mRNA.
- the antisense sequences may prevent gene expression through a variety of mechanisms.
- the antisense sequences may inhibit the ability of ribosomes to translate the mRNA.
- the antisense sequences may block transport of the mRNA from the nucleus to the cytoplasm, thereby limiting the amount of mRNA available for translation.
- antisense sequences may inhibit gene expression is by interfering with mRNA splicing.
- the antisense nucleic acid may be inco ⁇ orated in a ribozyme capable of specifically cleaving the target mRNA.
- the antisense nucleic acid molecules to be used in gene therapy may be either DNA or RNA sequences. They may comprise a sequence complementary to the sequence of the cDNA (or genomic DNA obtainable therefrom).
- the antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression of the mRNA in the duplex.
- Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green ef al., Ann. Rev. Biochem., 55:569-597 (1986) and Izant and Weintraub, Cell, 36:1007-1015 (1984).
- antisense molecules are obtained from a nucleotide sequence encoding a protein by reversing the orientation of the coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed in the cell.
- the antisense molecules may be transcribed using in vitro transcription systems such as those which employ T7 or SP6 polymerase to generate the transcript.
- Another approach involves transcription of the antisense nucleic acids in vivo by operably linking DNA containing the antisense sequence to a promoter in an expression vector.
- oligonucleotides which are complementary to the strand normally transcribed in the cell may be synthesized in vitro.
- the antisense nucleic acids are complementary to the corresponding mRNA and are capable of hybridizing to the mRNA to create a duplex.
- the antisense sequences may contain modified sugar phosphate backbones to increase stability and make them less sensitive to RNase activity. Examples of modifications suitable for use in antisense strategies include 2' O- methyl RNA oligonucleotides and Protein-nucleic acid (PNA) oligonucleotides. Further examples are described by Rossi ef al., Pharmacol. Then, 50(2):245-254, (1991).
- antisense oligonucleotides complementary to the sequence of the cDNA may be used.
- stable and semi-stable antisense oligonucleotides described in International Application No. PCT WO94/23026 are used. In these moleucles, the 3' end or both the 3' and 5' ends are engaged in intramolecular hydrogen bonding between complementary base pairs. These molecules are better able to withstand exonuclease attacks and exhibit increased stability compared to conventional antisense oligonucleotides.
- the covalently cross-linked antisense oligonucleotides described in International Application No. WO 96/31523 are used.
- oligonucleotides comprise one or more, respectively, inter- or intra-oligonucleotide covalent cross-linkages, wherein the linkage consists of an amide bond between a primary amine group of one strand and a carboxyl group of the other strand or of the same strand, respectively, the primary amine group being directly substituted in the 2' position of the strand nucleotide monosaccharide ring, and the carboxyl group being carried by an aliphatic spacer group substituted on a nucleotide or nucleotide analog of the other strand or the same strand, respectively.
- WO 92/18522 may also be used. These molecules are stable to degradation and contain at least one transcription control recognition sequence which binds to control proteins and are effective as decoys therefor. These molecules may contain "hairpin” structures, “dumbbell” structures, “modified dumbbell” structures, "cross-linked” decoy structures and “loop” structures.
- the cyclic double-stranded oligonucleotides described in European Patent Application No. 0 572 287 A2 are used. These ligated oligonucleotide "dumbbells" contain the binding site for a transcription factor and inhibit expression of the gene under control of the transcription factor by sequestering the factor. Use of the closed antisense oligonucleotides disclosed in International Application No. WO
- 92/19732 is also contemplated. Because these molecules have no free ends, they are more resistant to degradation by exonucleases than are conventional oligonucleotides. These oligonucleotides may be multifunctional, interacting with several regions which are not adjacent to the target mRNA.
- the appropriate level of antisense nucleic acids required to inhibit gene expression may be determined using in vitro expression analysis.
- the antisense molecule may be introduced into the cells by diffusion, injection, infection or transfection using procedures known in the art.
- the antisense nucleic acids can be introduced into the body as a bare or naked oligonucleotide, oligonucleotide encapsulated in lipid, oligonucleotide sequence encapsidated by viral protein, or as an oligonucleotide operably linked to a promoter contained in an expression vector.
- the expression vector may be any of a variety of expression vectors known in the art, including retroviral or viral vectors, vectors capable of extrachromosomal replication, or integrating vectors.
- the vectors may be DNA or RNA.
- the antisense molecules are introduced onto cell samples at a number of different concentrations preferably between 1x10 10 M to 1x10- 4 M. Once the minimum concentration that can adequately control gene expression is identified, the optimized dose is translated into a dosage suitable for use in vivo. For example, an inhibiting concentration in culture of 1x10 7 translates into a dose of approximately 0.6 mg/kg bodyweight. Levels of oligonucleotide approaching 100 mg/kg bodyweight or higher may be possible after testing the toxicity of the oligonucleotide in laboratory animals. It is additionally contemplated that cells from the vertebrate are removed, treated with the antisense oligonucleotide, and reintroduced into the vertebrate.
- the antisense oligonucleotide sequence is incorporated into a ribozyme sequence to enable the antisense to specifically bind and cleave its target mRNA.
- ribozyme and antisense oligonucleotides see Rossi ef al., supra.
- the polypeptide encoded by the gene is first identified, so that the effectiveness of antisense inhibition on translation can be monitored using techniques that include but are not limited to antibody-mediated tests such as RIAs and ELISA, functional assays, or radiolabeling.
- the cDNAs of the present invention may also be used in gene therapy approaches based on intracellular triple helix formation. Triple helix oligonucleotides are used to inhibit transcription from a genome. They are particularly useful for studying alterations in cell activity as it is associated with a particular gene.
- the cDNAs (or genomic DNAs obtainable therefrom) of the present invention or, more preferably, a fragment of those sequences, can be used to inhibit gene expression in individuals having diseases associated with expression of a particular gene.
- a fragment of the cDNA (or genomic DNA obtainable therefrom) can be used to study the effect of inhibiting transcription of a particular gene within a cell.
- homopurine sequences were considered the most useful for triple helix strategies.
- homopyrimidine sequences can also inhibit gene expression. Such homopyrimidine oligonucleotides bind to the major groove at homopurine:homopyrimidine sequences.
- both types of sequences from the cDNA or from the gene corresponding to the cDNA are contemplated within the scope of this invention.
- RNA sequences of the cDNAs are scanned to identify 10- mer to 20-mer homopyrimidine or homopurine stretches which could be used in triple-helix based strategies for inhibiting gene expression.
- their efficiency in inhibiting gene expression is assessed by introducing varying amounts of oligonucleotides containing the candidate sequences into tissue culture cells which normally express the target gene.
- the oligonucleotides may be prepared on an oligonucleotide synthesizer or they may be purchased commercially from a company specializing in custom oligonucleotide synthesis, such as GENSET, Paris, France.
- oligonucleotides may be introduced into the cells using a variety of methods known to those skilled in the art, including but not limited to calcium phosphate precipitation, DEAE-Dextran, electroporation, liposome-mediated transfection or native uptake.
- Treated cells are monitored for altered cell function or reduced gene expression using techniques such as Northern blotting, RNase protection assays, or PCR based strategies to monitor the transcription levels of the target gene in cells which have been treated with the oligonucleotide .
- the cell functions to be monitored are predicted based upon the homologies of the target gene corresponding to the cDNA from which the oligonucleotide was derived with known gene sequences that have been associated with a particular function.
- the cell functions can also be predicted based on the presence of abnormal physiologies within cells derived from individuals with a particular inherited disease, particularly when the cDNA is associated with the disease using techniques described in example 44.
- oligonucleotides which are effective in inhibiting gene expression in tissue culture cells may then be introduced in vivo using the techniques described above and in example 50 at a dosage calculated based on the in vitro results, as described in example 50.
- the natural (beta) anomers of the oligonucleotide units can be replaced with alpha anomers to render the oligonucleotide more resistant to nucleases.
- an intercalating agent such as ethidium bromide, or the like, can be attached to the 3' end of the alpha oligonucleotide to stabilize the triple helix.
- the cDNAs of the present invention may also be used to express an encoded protein in a host organism to produce a beneficial effect.
- the encoded protein may be transiently expressed in the host organism or stably expressed in the host organism.
- the encoded protein may have any of the activities described above.
- the encoded protein may be a protein which the host organism lacks or, alternatively, the encoded protein may augment the existing levels of the protein in the host organism. A full length cDNA encoding the signal peptide and the mature protein, or a cDNA encoding only the mature protein is introduced into the host organism.
- the cDNA may be introduced into the host organism using a variety of techniques known to those of skill in the art.
- the cDNA may be injected into the host organism as naked DNA such that the encoded protein is expressed in the host organism, thereby producing a beneficial effect.
- the cDNA may be cloned into an expression vector downstream of a promoter which is active in the host organism.
- the expression vector may be any of the expression vectors designed for use in gene therapy, including viral or retroviral vectors.
- the expression vector may be directly introduced into the host organism such that the encoded protein is expressed in the host organism to produce a beneficial effect.
- the expression vector may be introduced into cells in vitro. Cells containing the expression vector are thereafter selected and introduced into the host organism, where they express the encoded protein to produce a beneficial effect.
- EXAMPLE 53 Use Of Signal Peptides To Import Proteins Into Cells
- the short core hydrophobic region (h) of signal peptides encoded by the cDNAs of the present invention or fragment thereof may also be used as a carrier to import a peptide or a protein of interest, so- called cargo, into tissue culture cells (Lin ef al., J. Biol. Chem., 270: 14225-14258 (1995); Du ef al., J. Peptide Res., 51: 235-243 (1998); Rojas et al., Nature Biotech., 16: 370-375 (1998)).
- nucleic acids can be genetically engineered, using techniques familiar to those skilled in the art, in order to link the cDNA sequence or fragment thereof encoding the h region to the 5' or the 3' end of a DNA sequence coding for a cargo polypeptide.
- Such genetically engineered nucleic acids are then translated either in vitro or in vivo after transfection into appropriate cells, using conventional techniques to produce the resulting cell permeable polypeptide. Suitable hosts cells are then simply incubated with the cell permeable polypeptide which is then translocated across the membrane.
- This method may be applied to study diverse intracellular functions and cellular processes. For instance, it has been used to probe functionally relevant domains of intracellular proteins and to examine protein-protein interactions involved in signal transduction pathways (Lin ef al., supra; Lin ef al., J. Biol. Chem., 271: 5305-5308 (1996); Rojas et al., J. Biol.
- Such techniques may be used in cellular therapy to import proteins producing therapeutic effects. For instance, cells isolated from a patient may be treated with imported therapeutic proteins and then re- introduced into the host organism.
- the h region of signal peptides of the present invention could be used in combination with a nuclear localization signal to deliver nucleic acids into cell nucleus.
- Such oligonucleotides may be antisense oligonucleotides or oligonucleotides designed to form triple helixes, as described in examples 50 and 51 respectively, in order to inhibit processing and maturation of a target cellular RNA.
- cDNA codes of SEQ ID NOs. 24-73 encompasses the nucleotide sequences of SEQ ID NOs. 24-73, fragments of SEQ ID NOs. 24-73, nucleotide sequences homologous to SEQ ID NOs. 24-73 or homologous to fragments of SEQ ID NOs. 24-73, and sequences complementary to all of the preceding sequences.
- the fragments include fragments of SEQ ID NOs. 24-73 comprising at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 consecutive nucleotides of SEQ ID NOs. 24-73.
- the fragments are novel fragments.
- the fragments include polynucleotides described in Table III or fragments thereof comprising at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 consecutive nucleotides of the polynucleotides described in Table III.
- Homologous sequences and fragments of SEQ ID NOs. 24-73 refer to a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% homology to these sequences. Homology may be determined using any of the computer programs and parameters described in example 17, including BLAST2N with the default parameters or with any modified parameters.
- Homologous sequences also include RNA sequences in which uridines replace the thymines in the cDNA codes of SEQ ID NOs. 24-73.
- the homologous sequences may be obtained using any of the procedures described herein or may result from the correction of a sequencing error as described above.
- the homologous sequences and fragments of SEQ ID NOs. 24-73 include polynucleotides described in Table III or fragments comprising at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 consecutive nucleotides of the polynucleotides described in Table III. It will be appreciated that the cDNA codes of SEQ ID NOs.
- polypeptide codes of SEQ ID NOS. 74-123 encompasses the polypeptide sequences of SEQ ID NOs. 74-123 which are encoded by the cDNAs of SEQ ID NOs. 24-73, polypeptide sequences homologous to the polypeptides of SEQ ID NOS. 74-123, or fragments of any of the preceding sequences.
- Homologous polypeptide sequences refer to a polypeptide sequence having at least 99%, 98%, 5 97%, 96%, 95%, 90%, 85%, 80%, 75% homology to one of the polypeptide sequences of SEQ ID NOS. 74- 123. Homology may be determined using any of the computer programs and parameters described herein, including FASTA with the default parameters or with any modified parameters. The homologous sequences may be obtained using any of the procedures described herein or may result from the correction of a sequencing error as described above.
- the polypeptide fragments comprise at least 5, 8, 10, 12, 15, 20, 25,
- the fragments are novel fragments.
- the fragments include polypeptides encoded by the polynucleotides described in Table III, or fragments thereof comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids of the polypeptides encoded by the polynucleotides described in Table III. It will be appreciated that the polypeptide codes of the SEQ ID NOS. 74-123 can be
- the words "recorded” and “stored” refer to a process for storing information on a computer medium.
- a skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the cDNA codes of SEQ ID NOs. 24-73, one or more of the polypeptide codes of SEQ ID NOS. 74-123.
- Another aspect of the present invention is a computer readable
- Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 polypeptide codes of SEQ ID NOS. 74-123.
- Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
- the computer readable media may
- ROM 30 be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
- Embodiments of the present invention include systems, particularly computer systems which store and manipulate the sequence information described herein.
- One example of a computer system 100 is
- a computer system refers to the hardware components, software components, and data storage components used to analyze the nucleotide sequences of the cDNA codes of SEQ ID NOs. 24-73, or the amino acid sequences of the polypeptide codes of SEQ ID NOS. 74-123.
- the computer system 100 is a Sun Enterprise 1000 server (Sun Microsystems, Palo Alto, CA).
- the computer system 100 preferably includes a processor for processing, accessing and manipulating the sequence data.
- the processor 105 can be any well-known type of central processing unit, such as the Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq or International Business Machines.
- the computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components.
- the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/or other computer readable media having data recorded thereon.
- the computer system 100 further includes one or more data retrieving device 118 for reading the data stored on the internal data storage devices 110.
- the data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, etc.
- the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon.
- the computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.
- the computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125a-c in a network or wide area network to provide centralized access to the computer system 100.
- Software for accessing and processing the nucleotide sequences of the cDNA codes of SEQ ID NOs. 24-73, or the amino acid sequences of the polypeptide codes of SEQ ID NOS. 74-123 may reside in main memory 115 during execution.
- the computer system 100 may further comprise a sequence comparer for comparing the above-described cDNA codes of SEQ ID NOs. 24-73 or polypeptide codes of SEQ ID NOS. 74-123 stored on a computer readable medium to reference nucleotide or polypeptide sequences stored on a computer readable medium.
- a "sequence comparer" refers to one or more programs which are implemented on the computer system 100 to compare a nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences and/or compounds including but not limited to peptides, peptidomimetics, and chemicals stored within the data storage means.
- the sequence comparer may compare the nucleotide sequences of the cDNA codes of SEQ ID NOs. 24-73, or the amino acid sequences of the polypeptide codes of SEQ ID NOS. 74-123 stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies, motifs implicated in biological function, or structural motifs.
- sequence comparer programs identified elsewhere in this patent specification are particularly contemplated for use in this aspect of the invention.
- Figure 7 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
- the database of sequences can be a private database stored within the computer system 100, or a public database such as GENBANK, PIR or
- the process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100.
- the memory could be any type of memory, including RAM or an internal storage device.
- the process 200 then moves to a state 204 wherein a database of sequences is opened for analysis and comparison.
- the process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer.
- a comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database.
- Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user of the computer system.
- the term "same” is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as “same” in the process 200. If a determination is made that the two sequences are the same, the process 200 moves to a state
- the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220. However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database.
- one aspect of the present invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid code of SEQ ID NOs. 24-73 or a polypeptide code of SEQ ID NOS. 74-123, a data storage device having retrievably stored thereon reference nucleotide sequences or polypeptide sequences to be compared to the nucleic acid code of SEQ ID NOs. 24-73 or polypeptide code of SEQ ID NOS.
- the sequence comparer may indicate a homology level between the sequences compared or identify structural motifs in the above described nucleic acid code of SEQ ID NOs. 24-73 and polypeptide codes of SEQ ID NOS. 74-123 or it may identify structural motifs in sequences which are compared to these cDNA codes and polypeptide codes.
- the data storage device may have stored thereon the sequences of at least 2, 5, 10, 15, 20, 25, 30, or 50 of the cDNA codes of SEQ ID NOs. 24-73 or polypeptide codes of SEQ ID NOS. 74-123.
- Another aspect of the present invention is a method for determining the level of homology between a nucleic acid code of SEQ ID NOs. 24-73 and a reference nucleotide sequence, comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through the use of a computer program which determines homology levels and determining homology between the nucleic acid code and the reference nucleotide sequence with the computer program.
- the computer program may be any of a number of computer programs for determining homology levels, including those specifically enumerated herein, including BLAST2N with the default parameters or with any modified parameters.
- the method may be implemented using the computer systems described above.
- the method may also be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 of the above described cDNA codes of SEQ ID NOs. 24-73 through use of the computer program and determining homology between the cDNA codes and reference nucleotide sequences .
- Figure 8 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous.
- the process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory.
- the second sequence to be compared is then stored to a memory at a state 256.
- the process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherein the first character of the second sequence is read.
- the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U.
- the sequence is a protein sequence, then it should be in the single letter amino acid code so that the first and sequence sequences can be easily compared.
- the level of homology is determined by calculating the profragment of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with a every character in a second sequence, the homology level would be 100%.
- the computer program may be a computer program which compares the nucleotide sequences of the cDNA codes of the present invention, to reference nucleotide sequences in order to determine whether the nucleic acid code of SEQ ID NOs. 24-73 differs from a reference nucleic acid sequence at one or more positions.
- a program records the length and identity of inserted, deleted or substituted nucleotides with respect to the sequence of either the reference polynucleotide or the nucleic acid code of SEQ ID NOs. 24-73.
- the computer program may be a program which determines whether the nucleotide sequences of the cDNA codes of SEQ ID NOs.
- 24-73 contain a biallelic marker or single nucleotide polymorphism (SNP) with respect to a reference nucleotide sequence.
- This single nucleotide polymorphism may comprise a single base substitution, insertion, or deletion, while this biallelic marker may comprise about one to ten consecutive bases substituted, inserted or deleted.
- Another aspect of the present invention is a method for determining the level of homology between a polypeptide code of SEQ ID NOS. 74-123 and a reference polypeptide sequence, comprising the steps of reading the polypeptide code of SEQ ID NOS. 74-123 and the reference polypeptide sequence through use of a computer program which determines homology levels and determining homology between the polypeptide code and the reference polypeptide sequence using the computer program.
- another aspect of the present invention is a method for determining whether a nucleic acid code of SEQ ID NOs. 24-73 differs at one or more nucleotides from a reference nucleotide sequence comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through use of a computer program which identifies differences between nucleic acid sequences and identifying differences between the nucleic acid code and the reference nucleotide sequence with the computer program.
- the computer program is a program which identifies single nucleotide polymorphisms. The method may be implemented by the computer systems described above and the method illustrated in Figure 8.
- the method may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 50 of the cD A codes of SEQ ID NOs. 24-73 and the reference nucleotide sequences through the use of the computer program and identifying differences between the cDNA codes and the reference nucleotide sequences with the computer program.
- the computer based system may further comprise an identifier for identifying features within the nucleotide sequences of the cDNA codes of SEQ ID NOs. 24-73 or the amino acid sequences of the polypeptide codes of SEQ ID NOS. 74-123.
- An "identifier" refers to one or more programs which identifies certain features within the above- described nucleotide sequences of the cDNA codes of SEQ ID NOs. 24-73 or the amino acid sequences of the polypeptide codes of SEQ ID NOS. 74-123.
- the identifier may comprise a program which identifies an open reading frame in the cDNAs codes of SEQ ID NOs. 24-73.
- Figure 9 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence.
- the process 300 begins at a start state 302 and then moves to a state 5 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100.
- the process 300 then moves to a state 306 wherein a database of sequence features is opened.
- a database would include a list of each feature's attributes along with the name of the feature. For example, a feature name could be "Initiation Codon" and the attribute would be "ATG". Another example would be the feature name "TAATAA Box" and the feature attribute would be "TAATAA”.
- An example of 0 such a database is produced by the University of Wisconsin Genetics Computer Group (www.gcg.com).
- the process 300 moves to a state 308 wherein the first feature is read from the database.
- a comparison of the attribute of the first feature with the first sequence is then made at a state 310.
- a determination is then made at a decision state 316 whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process 300 5 moves to a state 318 wherein the name of the found feature is displayed to the user.
- the process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324. However, if more features do exist in the database, then the process 300 reads the next sequence feature at a state 326 and loops back to the state 310 wherein the attribute of the next feature is 0 compared against the first sequence.
- the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database.
- the identifier may comprise a molecular modeling program which 5 determines the 3-dimensional structure of the polypeptides codes of SEQ ID NOS. 74-123.
- the molecular modeling program identifies target sequences that are most compatible with profiles representing the structural environments of the residues in known three-dimensional protein structures. (See, e.g., Eisenberg ef al., U.S. Patent No. 5,436,850 issued July 25, 1995).
- the known three-dimensional structures of proteins in a given family are superimposed to define 0 the structurally conserved regions in that family.
- This protein modeling technique also uses the known three- dimensional structure of a homologous protein to approximate the structure of the polypeptide codes of SEQ ID NOS. 74-123.
- Hybrid methods in which fold recognition is performed using Multiple Sequence Threading (MST), structural equivalencies are deduced from the threading output using a distance geometry program DRAGON to construct a low resolution model, and a full-atom representation is constructed using a molecular modeling package such as QUANTA.
- MST Multiple Sequence Threading
- candidate templates are first identified by using the novel fold recognition algorithm MST, which is capable of performing simultaneous threading of multiple aligned sequences onto one or more 3-D structures.
- MST novel fold recognition algorithm
- the structural equivalencies obtained from the MST output are converted into inter-residue distance restraints and fed into the distance geometry program DRAGON, together with auxiliary information obtained from secondary structure predictions.
- the program combines the restraints in an unbiased manner and rapidly generates a large number of low resolution model confirmations.
- these low resolution model confirmations are converted into full-atom models and subjected to energy minimization using the molecular modeling package QUANTA. (See e.g., Asz ⁇ di et al., Proteins:Structure, Function, and Genetics, Supplement 1 :38-42 (1997)).
- Another aspect of the present invention is a method of identifying a feature within the cDNA codes of SEQ ID NOs. 24-73 or the polypeptide codes of SEQ ID NOS. 74-123 comprising reading the nucleic acid code(s) or the polypeptide code(s) through the use of a computer program which identifies features therein and identifying features within the nucleic acid code(s) or polypeptide code(s) with the computer program.
- computer program comprises a computer program which identifies open reading frames.
- the computer program identifies structural motifs in a polypeptide sequence.
- the computer program comprises a molecular modeling program. The method may be performed by reading a single sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 of the cDNA codes of SEQ ID NOs. 24-73 or the polypeptide codes of SEQ ID NOS. 74-123 through the use of the computer program and identifying features within the cDNA codes or polypeptide codes with the computer program.
- the cDNA codes of SEQ ID NOs. 24-73 or the polypeptide codes of SEQ ID NOS. 74-123 may be stored and manipulated in a variety of data processor programs in a variety of formats.
- the cDNA codes of SEQ ID NOs. 24-73 or the polypeptide codes of SEQ ID NOS. 74-123 may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE.
- sequence comparers, identifiers, or sources of reference nucleotide or polypeptide sequences to be compared to the cDNA codes of SEQ ID NOs. 24-73 or the polypeptide codes of SEQ ID NOS. 74-123.
- the following list is intended not to limit the invention but to provide guidance to programs and databases which are useful with the cDNA codes of SEQ ID NOs. 24-73 or the polypeptide codes of SEQ ID NOS. 74-123.
- the programs and databases which may be used include, but are not limited to: MacPattem (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403 (1990)), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988)), FASTDB (Brutlag ef al. Comp. App. Biosci.
- Motifs which may be detected using the above programs include sequences encoding Ieucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
- the present invention also comprises methods of making the cDNA of SEQ ID Nos.24-73, genomic DNA obtainable therefrom, or fragment thereof.
- the methods comprise sequentially linking together nucleotides to produce the nucleic acids having the preceding sequences.
- a variety of methods of synthesizing nucleic acids are known to those skilled in the art.
- synthesis is conducted on a solid support.
- 3' phosphoramidite methods in which the 3' terminal base of the desired oligonucleotide is immobilized on an insoluble carrier.
- the nucleotide base to be added is blocked at the 5' hydroxyl and activated at the 3' hydroxyl so as to cause coupling with the immobilized nucleotide base. Deblocking of the new immobilized nucleotide compound and repetition of the cycle will produce the desired polynucleotide.
- polynucleotides may be prepared as described in U.S. Patent No. 5,049,656. In some embodiments, several polynucleotides prepared as described above are ligated together to generate longer polynucleotides having a desired sequence.
- the present invention also comprises methods of making the polynucleotides encoded by the cDNA of SEQ ID Nos.24-73, genomic DNA obtainable therefrom, or fragments thereof and methods of making the polypeptides of SEQ ID Nos.74-123 or fragments thereof.
- the methods comprise sequentially linking together amino acids to produce the nucleic polypeptides having the preceding sequences.
- the polypeptides made by these methods are 150 amino acids or less in length. In other embodiments, the polypeptides made by these methods are 120 amino acids or less in length.
- polypeptides A variety of methods of making polypeptides are known to those skilled in the art, including methods in which the carboxy! terminal amino acid is bound to polyvinyl benzene or another suitable resin.
- the amino acid to be added possesses blocking groups on its amino moiety and any side chain reactive groups so that only its carboxyl moiety can react.
- the carboxyl group is activated with carbodiimide or another activating agent and allowed to couple to the immobilized amino acid. After removal of the blocking group, the cycle is repeated to generate a polypeptide having the desired sequence.
- the methods described in U.S. Patent No. 5,049,656 may be used.
- EXAMPLE 57 Functional Analysis of Predicted Protein Seguences Following double-sequencing, contigs were assembled for each of the cDNAs of the present invention and each was compared to known sequences available at the time of filing. These sequences originate from the following databases : Genbank (release 108), EMBL (release 58 and daily releases), Genseq (release 35.3) Swissprot (release 37), Genbank (release 108 and daily releases up to October, 15, 1998), Genseq (release 32) PIR (release 53) and Swissprot (release 35). In some cases, based on homology with other proteins, new open reading frames than the one previously selected were chosen. For example, the new open reading frame of SEQ ID NO: 27 does not contain a signal peptide anymore.
- the first category contains proteins of the present invention exhibiting at least 80% identical amino acid residues on the whole length of the matched protein. They are cleariy close homologues, which most probably have the same function or a very similar function as the matched protein.
- the second category contains proteins of the present invention exhibiting more remote homologies (35 to 80% over the whole protein) indicating that the protein of the present invention is likely to have functions similar to those of the matched protein.
- the third category contains proteins exhibiting homology to a domain of a known protein indicating that the matched protein and the protein of the invention may share similar features such as functional domains. It should be noted that, in the numbering of amino acids in the protein sequences discussed below, in figures 10 to 13 and in Table V, the first methionine encountered is designated as amino acid number 1.
- the first amino acid of the mature protein resulting from cleavage of the signal peptide is designated as amino acid number 1 and the first amino acid of the signal peptide is designated with the appropriate negative number, in accordance with the regulations governing sequence listings.
- Protein of SEQ ID NO: 76 (internal designation 105-095-1 -0-D 10-FLC )
- the protein of SEQ ID NO: 76 encoded by the cDNA of SEQ ID NO:26 exhibits identity to the human parotid secretory protein HPSP (Genseq accession number W60682 and SEQ ID NO : 124) as shown by the alignment of figure 10. Antagonists of this protein may be used to treat cancer and autoimmune diseases particularly of secretory or gastrointestinal tissue.
- Protein of SEQ ID NO: 76 or part thereof may play a role in cell differentiation and/or proliferation. Thus, this protein or part therein, may be useful in diagnosing and/or treating several disorders including, but not limited to cancer and autoimmune diseases.
- Protein of SEQ ID NO: 93 (internal designation 117-007-2-0-C4-FLC)
- the protein of SEQ ID NO: 93 encoded by the cDNA of SEQ ID NO:43 exhibits identity to a human protein thought to be transmembraneous (Genseq accession number W88491 and SEQ ID NO : 125) as shown by the alignment of figure 11.
- This protein displays homology to alpha-2-HS glycoprotein precursors (fetuins) of human and pigs, which belong to the cystatin superfamily.
- the 382-amino-acid-long protein of SEQ ID NO: 93 which is similar in size to fetuins, displays a cystatin-like domain with 12 conserved cysteines (positions 36, 93, 104, 117, 137, 151 , 154, 216, 224, 237, 254 and 368, in bold in figure 11) and a conserved region around the second cysteine (positions 89 to 96, underlined in figure 11) although the 5 typical PROSITE signatures for fetuins is not present.
- the potential active site QxVxG is also present in the protein of the invention (positions 198 to 202, in italics in figure 11).
- the cystatin superfamily contain evolutionarily related proteins with diverse functions such as cysteine protease inhibitors, stefins, fetuins and kininogens (see review by Brown and Dziegielewska, Prof. Science, 6:5-12 (1997)).
- this protein or part therein may be useful in diagnosing and/or treating several disorders including, but not limited to, cancer, and especially tumor progression and metastasis, chronic inflammation, neurodegenerative diseases such as Alzheimer disease, diabetes, hypertension and immune disorders. It may also be useful in treating patients with cardiovascular disorders by modulating their blood coagulation properties.
- the protein of SEQ ID NO: 75 encoded by the cDNA of SEQ ID NO:25 exhibits homology to a murine putative sialyltransferase protein (TREMBL accession number 088725 and SEQ ID NO : 126) as shown by the alignment of figure 12.
- Sialyltransferases are type II transmembrane proteins involved in the biosynthesis of sialosides which are important in a large variety of biological processes such as cell-cell
- the protein of SEQ ID NO: 75 displays the two conserved motifs of the sialyltransferase protein family, namely the centrally located sialylmotifL (positions 73 to 120, in bold in figure 12) thought to be involved in the recognition of the sugar nucleotide donor common to all sialyltransferases and the sialylmotifS (positions 211 to 233, in italics
- the 302-amino-acid long protein of SEQ ID NO: 75 has a size similar to the one of the members of the sialyltransferase family.
- the protein of the invention has a predicted transmembrane structure. Indeed, it contains 2 potential transmembrane segment (positions 7 to 27 and 206 to 226, underlined in figure 12) as predicted by the software TopPred II (Claras and von Heijne, CABIOS applic. Notes, 10 :685-
- Proteins of SEQ ID NOs: 75 may play a role in the biosynthesis of sialyl-glycoconjugates, probably as a sialyltransferase.
- this protein or part therein may be useful in diagnosing and/or treating several disorders including, but not limited to, cancer, cystic fibrosis and hypothyroidism.
- Proteins of SEQ ID NOs: 104 (internal designation 108-008-5-O-C5-FL)
- the protein of SEQ ID NO: 104 encoded by the cDNA of SEQ ID NO: 54 exhibits extensive homology over the whole length of the murine recombination activating gene 1 inducing protein (Genbank accession number X96618 and SEQ ID NO : 177).
- the amino acid residues are identical except for the positions 6, 7, 10-13, 17, 25, 34-35, 42, 51, 56, 62, 68, 71, 74, 78, 91, 93, 95-96, 106, 121-122, 151-152, 159, 162-163, 170-171, 176-177, 188, 190, 192, 196, 199, 202-203, 206, 210, 215 and 217 of the 221 amino acid long matched protein.
- This protein with 4 potential transmembrane segments is involved in the induction of the recombination of V(D)J segments in T cells (Muraguchi ef al, Leuk Lymphoma, 30 :73-85 (1998)).
- Proteins of SEQ ID NO: 104 may play a role in lymphocyte repertoire formation.
- this protein or part therein may be useful in diagnosing and/or treating several disorders including, but not limited to, cancer, immunological disorders and inflammatory disorders. It may also be useful to modulate the inflammatory or immune response to infectious agents, such as HIV.
- Proteins of SEQ ID NO: 87 (internal designation 116-073-4-0-C8-FLC1
- this protein displays the characteristic alpha-lactalbumin/lysozyme C PROSITE signature of this family of glysosyl hydrolases, family 22 (positions 162 to 180, see Table V).
- Lysozymes C are bacteriolytic defensive enzymes and alpha-lactalbumin is the regulatory subunit of lactose synthetase. Lysozymes C and alpha-lactalbumin appear to be evolutionary related (Qasba and Kumar, Crit. Rev. Biochem. Mol. Biol. 32:255-306 (1997)).
- the protein of SEQ ID NO: 87 or part thereof may play a role in glycoprotein and/or peptidoglycan metabolism, probably as a glycosyl hydrolase.
- this protein or part thereof may be useful in diagnosing and/or treating several disorders including, but not limited to, cancer and amyloidosis. It may also be useful in modulating defensive responses to infectious agents such as bacteria.
- Proteins of SEQ ID NO: 86 (internal designation 116-054-3-0-G12-FLC)
- the protein of SEQ ID NO: 86 encoded by the cDNA of SEQ ID NO:36 found in liver shows homology to the MLRQ subunit of NADH-uniquinone oxidoreductase (complex I) of bovine, murine and human species (Genbank accession numbers X64897, U59509 and EMBL accession number U94586 respectively).
- the 83-amino-acid-long protein of SEQ ID NO: 86 has a size similar to those of known MLRQ subunits.
- Complex I is part of the mitochondrial electron transport chain and is involved in the dehydrogenation of NADH and the transportation of electrons to coenzyme Q. It is also thought to play a role in the regulation of apoptosis and necrosis. Mitochondriocytopathies due to complex I deficiency are frequently encountered and affect tissues with a high-energy demand such as brain (mental retardation, convulsions, movement disorders), heart (cardiomyopathy, conduction disorders), kidney (Fanconi syndrome), skeletal muscle (exercise intolerance, muscle weakness, hypotonia) and/or eye (opthmaloplegia, ptosis, cataract and retinopathy).
- brain mental retardation, convulsions, movement disorders
- heart cardiomyopathy, conduction disorders
- kidney Kidni syndrome
- skeletal muscle exercise intolerance, muscle weakness, hypotonia
- eye opthmaloplegia, ptosis, cataract and retinopathy.
- the protein of SEQ ID NO: 86 may be a NADH-ubiquinone oxidoreductase MLRQ-like protein.
- this protein or part thereof may be useful in diagnosing and/or treating several disorders including, but not limited to, brain disorders (mental retardation, convulsions, movement disorders), heart disorders (cardiomyopathy, conduction disorders), kidney disorders (Fanconi syndrome), skeletal muscle disorders (exercise intolerance, muscle weakness, hypotonia) and/or eye disorders (opthmalmoplegia, ptosis, cataract and retinopathy).
- Protein of SEQ ID NO: 91 (internal designation 117-005-4-0-E5-FLC)
- the protein of SEQ ID NO :91 encoded by the cDNA of SEQ ID NO:41 found in liver shows homology over domains of a family of mitochondrial substrate carrier proteins found in the inner mitochondrial membrane. These carrier proteins are evolutionary related and consist of three tandem repeats of a domain of approximately one hundred residues with each of these domains containing two transmembrane regions.
- the 308-amino-acid-long protein of SEQ ID NO:91 has a size similar to the one of mitochondrial carrier proteins and displays the characteristic PROSITE signature of this protein family three times (positions 19 to 28, 115 to 124 and 237 to 246, see Table V).
- the protein of SEQ ID NO: 91 has 6 potential transmembrane segments of 20 amino acids, 4 being predicted with a high level of confidence (positions 1-21, 54-74, 135-155 and 217-237) and 2 with a lower level of confidence (positions 96-116 and 191-211), using the TopPred II software (Claras and von Heijne, CABIOS applic. Notes, 10 :685- 686 (1994)).
- this protein or part thereof may play a role in energy transfer, probably as a mitochondrial substrate carrier protein.
- this protein or part thereof may be useful in diagnosing and/or treating several disorders including, but not limited to, mitochondriocytopathies and obesity.
- the protein of SEQ ID NO: 91 encoded by the cDNA of SEQ ID NO: 41 exhibits homology to apolipoprotein A-IV related protein.
- Lipoproteins such as HDL and LDL contain characteristic apolipoproteins that are responsible for targeting them to certain tissues and for activating enzymes required for the trafficking of the lipid fraction of the lipoprotein (including cholesterol).
- Apolipoprotein A-IV-related protein (AA4RP) is a member of the apolipoprotein family; it is 52% similar (29% identical) to Apolipoprotein A-IV ( ApoA-IV) and therefore is likely to have a similar function. ApoA-IV is found associated with the chylomicron and HDL fraction of blood.
- AA4RP circulates in the blood, and is therefore easily amenable to therapeutic intervention, by direct administration into the blood of synthetic peptide analogs that mimic its activity or function as competitive antagonists (dominant negatives). Since this protein is involved in fat transport and in cholesterol trafficking within the body and mediates the changes in blood cholesterol in response to dietary changes, interventions targeted at this protein will be useful for cholesterol lowering and anti-atherosclerosis therapeutics, and in the control of diabetes and obesity.
- Proteins of SEQ ID NO: 74 (internal designation 105-016-3-0-E3-FLC)
- the 325-amino-acid-long protein of SEQ ID NO: 74 encoded by the cDNA of SEQ ID NO: 24 shows homology over the whole length of the 332-amino-acid-long murine neural proliferation differentiation and control 1 protein or NPDC-1 (Genbank accession number X67209) which is thought to play an important role in the control of neural cell proliferation and differentiation as well as in cell survival probably by interacting directly or not with cell cycle regulators such as E2F-1 (Galiana ef al., Proc. Natl. Acad. Sci. USA 92:1560-1564 (1995); Dupont ef al., J. Neurosci. Res. 51:257-267 (1998)).
- Protein of SEQ ID NO: 74 or part thereof may play a role in cell proliferation and differentiation. Thus, this protein or part thereof, may be useful in diagnosing and/or treating several disorders including, but not limited to cancer and neurodegenerative disorders.
- Protein of SEQ ID NO: 111 (internal designation 108-013-5-Q-H9-FU
- the protein of SEQ ID NO : 111 encoded by the extended cDNA SEQ ID NO: 61 shows homologies with a family of lysophospholipases conserved among eukaryotes (yeast, rabbit, rodents and human).
- Protein of SEQ ID NO:111 may play a role in fatty acid metabolism, probably as a phospholipase.
- this protein or part therein may be useful in diagnosing and/or treating several disorders including, but not limited to, cancer, neurodegenerative disorders such as Parkinson's and Alzheimer's diseases, diabetes. It may also be useful in modulating inflammatory responses to infectious agents and/or to suppress graft rejection.
- Protein of SEQ ID NOs:101 (internal designation 108-005-5-O-F9-FL1
- the protein of SEQ ID NO:71 encoded by the extended cDNA SEQ ID NO: 51 shows homology with the Drosophila rhythmically expressed gene 2 protein (Genbank accession number U65492). Expression of the mRNA coding for the matched protein is dependent on the interplay between light-dark cycle, feeding conditions and expression of the per gene which is essential to the function of the endogenous circadian pacemaker (Van Gelder ef al., Curr. Biol., 5 :1424-1436 (1995)).
- Protein of SEQ ID NO: 101 may play a role in circadian control.
- this protein or part therein may be useful in diagnosing and/or treating several disorders including, but not limited to, insomnia, depression, stress and other disorders of the circadian rhythm.
- such a protein may be useful in modulating the physiological response to night work or to jet lag.
- Gangliosides are believed to be involved in neural cell development, differentiation, survival and pathology, maybe as modulators of membrane properties (Brigande and Seyfried, Ann. N Y. Acad. Sci. 845:215-218 (1998); Schengrund and Mummert, Ann. N. Y. Acad. Sci. 845:278-284 (1998)).
- Protein of SEQ ID NO: 94 may play a role in central nervous system development and differentiation. Thus, this protein or part thereof, may be useful in diagnosing and treating several disorders including, but not limited to, cancer and neuronal disorders.
- Protein of SEQ ID NO: 89 (internal designation 117-005-2-0-E10-FLC1
- the protein of SEQ ID NO: 89 encoded by the cDNA of SEQ ID NO:39 shows remote homology to domains of apolipoprotein A-IV of human, murine and chicken species (Genbank accession numbers M13654, M13966, and EMBL accession number 093601 respectively).
- 366-amino-acid-long protein of SEQ ID NO: 89 has a size similar to those of above-mentioned apolipoprotein A-IV.
- the protein of SEQ ID NO: 89 encoded by the cDNA of SEQ ID NO: 39 exhibits homology to the camitine carrier related protein.
- the camitine carrier-related protein (CCRP) is 45% similar (30% identical) to the acyl-carnitine/camitine carrier and is therefore likely to have a similar function.
- the acyl- carnitine/carnitine carrier is a mitochondrial carrier protein that is responsible for transporting fatty acids into the mitochondrion where they may be oxidized to produce energy.
- CCRP also shares underlying structural similarities with the uncoupling protein (UCP-1), another mitochondrial transporter protein which is involved in weight regulation and temperature homeostasis.
- UCP-1 uncoupling protein
- UCP protein activity is regulated by nucleotides via a 9 amino acid protein domain that is relatively well conserved in the predicted CCR protein (6 of 9 identical, 9 of 9 similar), compared to only 4 of 9 for the acyl-carnitine/camitine carrier itself. Therefore the function of the CCRP may be amenable to direct activation or inhibition via small molecule nucleotide analogs.
- Acyl-carnitine/camitine carrier is required for transport of fatty acids into mitochondria before they can be oxidized for energy, however genetic mutations of this gene do not result in disturbances of weight. This indicates that another protein must also be available for fatty acid transport, and CCRP is likely to be this transporter.
- the rate of lipid burning by the mitochondrion is dependent upon the rate of delivery of fatty acids into the mitochondrion by these transporters.
- Regulation of the activity of CCRP via its nucleotide binding domain or by other interventions to increase its availability or activity in the mitochondria, would increase the fat burning capacity of tissues. Since elevated plasma free fatty acids have been implicated in the causation of type II diabetes (NIDDM) such interventions could be designed to increase net clearance of lipids from the blood.
- NIDDM type II diabetes
- Other effects of therapeutics targeted at CCRP could be to increase fat burning by liver and muscle at the expense of fat storage by adipose tissue, with the result of decreasing weight.
- this protein or part thereof may be useful in diagnosing and treating several disorders including, but not limited to, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disorders such as coronary heart disease, neurodegenerative disorders such as Alzheimer's disease or dementia, and obesity.
- Protein of SEQ ID NO: 95 (internal designation 122-005-2-0-F11-FLC)
- the protein of SEQ ID NO: 95 encoded by the cDNA of SEQ ID NO:45 exhibits homology with domains of a family of reductases, and especially with the NADH-cytochrome b5 reductase of rat, bovine and human species (Genbank accession numbers J03867, M83104 and Y09501, respectively).
- the homology include the flavin-adenine dinucleotide-binding domain of NADH-cytochrome b5 reductase proteins which belong to a flavoenzyme family whose members are involved in photosynthesis, in the assimilation of nitrogen and sulfur, in fatty-acid oxidation, in the reduction of methemoglobin and in the metabolism of many pesticides, drugs and carcinogens.
- Protein of SEQ ID NO: 95 may play a role in cellular oxidoreduction reactions, maybe as a flavoenzyme reductase.
- this protein or part thereof may be useful in diagnosing and treating several disorders including, but not limited to, cancer, methemoglobinemia, hyperlipidemia, obesity and cardiovascular disorders. It may also be useful in regulating the metabolism of pesticides, drugs and carcinogens.
- Protein of SEQ ID NO: 106 (internal designation 108-011-5-Q-B12-FL)
- the protein of SEQ ID NO: 106 encoded by the extended cDNA SEQ ID NO: 56 shows homology to the predicted extracellular domain and part of transmembrane domain of interieukin-17 receptor of both human and murine species (Genbank accession numbers W04185 and W04184). These IL-17R proteins are thought to belong to a new family of receptors for cytokines which induce T cell proliferation, l-CAM expression and preferential maturation of haematopoietic precursors into neutrophils (Yao ef al., Cytokine., 9:794-8001 (1997)). It is also thought to play a proinflammatory role and to induce nitric oxide.
- the protein of the invention has a 21 amino acid transmembrane domain (positions 172 to 192) as predicted by the software TopPred II (Claras and von Heijne, CABIOS applic. Notes, 10 :685-686 (1994)) matching the 21 amino acid putative transmembrane domain of human interleukin-17 receptor.
- this protein or part therein may be useful in diagnosing and treating several disorders including, but not limited to, cancer, immunological disorders, septic shock and impotence.
- this protein may also be useful to modulate immune and/or inflammatory responses to infectious responses and/or to suppress graft rejection.
- Protein of SEQ ID NO: 114 (internal designation 108-014-5-O-D12-FL)
- the protein of SEQ ID NO: 114 encoded by the extended cDNA SEQ ID NO: 64 possess a cysteine-rich C3H2C3 region also found in G1 protein of Drosophila melanogaster (Swissprot accession number Q06003). This cysteine-rich region is similar to a RING type zinc finger, a domain that binds two atoms of zinc and is probably involved in mediating protein-protein interaction.
- the nucleic acid sequences of SEQ ID NOs: 24-73 or fragments thereof may also be used to construct fusion proteins in which the polypeptide sequences of SEQ ID NOs: 74-123 or fragments thereof are fused to heterologous polypeptides.
- the fragments of the polypeptides of SEQ ID NOs. 74- 123 which are included in the fusion proteins may comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids of the polypeptides of SEQ ID NOs. 74-123 or may be of any length suitable for the intended purpose of the fusion protein.
- Nucleic acids encoding the desired fusion protein are produced by cloning a nucleic acid of SEQ ID NOs.
- nucleic acid encoding the heterologous polypeptide is operably linked to a promoter in an appropriate vector, such as any of the vectors described above, and introduced into a host capable of expressing the fusion protein.
- Antibodies against the polypeptides of SEQ ID NOs. 74-123 or fragments thereof may be used in immunoaffinity chromatography to isolate the polypeptides of SEQ ID NOs. 74-123 or fragments thereof or to isolate fusion proteins containing the polypeptides of SEQ ID NOs. 74-123 or fragments thereof.
- EXAMPLE 58 Immunoaffinity Chromatography Antibodies prepared as described above are coupled to a support.
- the antibodies are monoclonal antibodies, but polyclonal antibodies may also be used.
- the support may be any of those typically employed in immunoaffinity chromatography, including Sepharose CL-4B (Pharmacia, Piscataway, NJ), Sepharose CL-2B (Pharmacia, Piscataway, NJ), Affi-gel 10 (Biorad, Richmond, CA), or glass beads.
- the antibodies may be coupled to the support using any of the coupling reagents typically used in immunoaffinity chromatography, including cyanogen bromide.
- the support is contacted with a sample which contains a target polypeptide whose isolation, purification or enrichment is desired.
- the target polypeptide may be a polypeptide of SEQ ID NOs. 74-123, a fragment thereof, or a fusion protein comprising a polypeptide of SEQ ID NOs.74-123 or a fragment thereof.
- the sample is placed in contact with the support for a sufficient amount of time and under appropriate conditions to allow at least 50% of the target polypeptide to specifically bind to the antibody coupled to the support.
- the wash solution may be any of those typically employed in immunoaffinity chromatography, including PBS, Tris-lithium chloride buffer (0.1M lysine base and 0.5M lithium chloride, pH 8.0), Tris-hydrochloride buffer (0.05M Tris-hydrochloride, pH 8.0), or Tris/Triton/NaCI buffer (50mM
- Tris.cl pH 8.0 or 9.0, 0.1% Triton X-100, and O. ⁇ MNaCI).
- the specifically bound target polypeptide is eluted from the support using the high pH or low pH elution solutions typically employed in immunoaffinity chromatography.
- the elution solutions may contain an eluant such as triethanolamine, diethylamine, calcium chloride, sodium thiocyanate, potasssium bromide, acetic acid, or glycine.
- the elution solution may also contain a detergent such as Triton X-100 or octyl- ⁇ -D-glucoside.
- the cDNAs of the present invention or fragments thereof can be used for various purposes.
- the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination for expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.
- the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
- the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
- the proteins or polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
- the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
- the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
- Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
- the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
- the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11368698P | 1998-12-22 | 1998-12-22 | |
US113686P | 1998-12-22 | ||
US14103299P | 1999-06-25 | 1999-06-25 | |
US141032P | 1999-06-25 | ||
PCT/IB1999/002058 WO2000037491A2 (fr) | 1998-12-22 | 1999-12-20 | Adn complementaires codant pour des proteines secretees avec des peptides signaux |
Publications (2)
Publication Number | Publication Date |
---|---|
EP1144444A2 true EP1144444A2 (fr) | 2001-10-17 |
EP1144444A3 EP1144444A3 (fr) | 2002-01-02 |
Family
ID=26811346
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP99959633A Withdrawn EP1144444A3 (fr) | 1998-12-22 | 1999-12-20 | Adn complementaires codant pour des proteines secretees avec des peptides signaux |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1144444A3 (fr) |
JP (1) | JP2002539767A (fr) |
AU (1) | AU1675900A (fr) |
CA (1) | CA2354369A1 (fr) |
WO (1) | WO2000037491A2 (fr) |
Families Citing this family (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6787647B1 (en) | 1998-12-22 | 2004-09-07 | Genset S.A. | Carnitine carrier related protein-1 |
WO2001000803A2 (fr) * | 1999-06-25 | 2001-01-04 | Genset | Proteine associee a l'apolipoproteine a-iv (aa4rp): sequences polypeptidiques, polynucleotidiques et marqueurs bialleliques de ces dernieres |
CN1244584A (zh) * | 1999-05-14 | 2000-02-16 | 北京医科大学 | 具有免疫细胞趋化和造血刺激活性的趋化因子 |
CN1426420A (zh) * | 2000-02-21 | 2003-06-25 | 吴羽化学工业株式会社 | 新蛋白质以及编码该蛋白质的新基因 |
US7094566B2 (en) | 2000-03-16 | 2006-08-22 | Amgen Inc., | IL-17 receptor like molecules and uses thereof |
WO2001079446A2 (fr) * | 2000-04-14 | 2001-10-25 | Hyseq, Inc. | Matieres et procedes relatifs au metabolisme lipidique |
WO2002040666A2 (fr) * | 2000-11-17 | 2002-05-23 | Xenon Genetics Inc. | Genes a regulation lipidique, utilisations de ces genes et composes permettant leur modulation |
WO2002102985A2 (fr) * | 2001-06-15 | 2002-12-27 | The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services | Pate, gene exprime dans le cancer de la prostate, la prostate et les testicules, et ses utilisations |
WO2003002737A1 (fr) * | 2001-06-27 | 2003-01-09 | Riken | Nouvelle proteine inhibitrice de la topoisomerase humaine 2$g(a) et utilisation associee |
FR2843395A1 (fr) * | 2002-08-12 | 2004-02-13 | Genfit S A | Composition et methodes pour le dosage de l'aa4rp |
FR2829581A1 (fr) * | 2001-09-07 | 2003-03-14 | Genfit S A | Methodes de criblage de molecules utiles pour la prevention ou le traitement du syndrome metabolique, des maladies cardiovasculaires et de l'atherosclerose |
EP1423708B1 (fr) * | 2001-09-07 | 2006-11-29 | Genfit | Compositions et methodes pour le dosage de l'aa4rp |
DE10344799A1 (de) * | 2003-09-26 | 2005-04-14 | Ganymed Pharmaceuticals Ag | Identifizierung von Oberflächen-assoziierten Antigenen für die Tumordiagnose und -therapie |
US7892733B1 (en) | 2004-04-22 | 2011-02-22 | Amgen Inc. | Response element regions |
DE102005013846A1 (de) * | 2005-03-24 | 2006-10-05 | Ganymed Pharmaceuticals Ag | Identifizierung von Oberflächen-assoziierten Antigenen für die Tumordiagnose und -therapie |
CA2900087C (fr) | 2005-10-17 | 2024-02-13 | Sloan Kettering Institute For Cancer Research | Peptides de liaison wt1 hla de classe ii, compositions et methodes associees comprenant ces peptides |
US9265816B2 (en) | 2006-04-10 | 2016-02-23 | Sloan Kettering Institute For Cancer Research | Immunogenic WT-1 peptides and methods of use thereof |
WO2013106834A2 (fr) | 2012-01-13 | 2013-07-18 | Memorial Sloan Kettering Cancer Center | Peptides wt-1 immunogènes et leurs procédés d'utilisation |
LT2945647T (lt) | 2013-01-15 | 2021-02-25 | Memorial Sloan Kettering Cancer Center | Imunogeniniai wt-1 peptidai ir jų panaudojimo būdai |
US10815273B2 (en) | 2013-01-15 | 2020-10-27 | Memorial Sloan Kettering Cancer Center | Immunogenic WT-1 peptides and methods of use thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0988385A2 (fr) * | 1997-01-21 | 2000-03-29 | Human Genome Sciences | Polypeptides semblables a tace ou a la matrilysime |
JP2001518793A (ja) * | 1997-04-10 | 2001-10-16 | ジェネティックス・インスチチュート・インコーポレーテッド | 分泌発現配列タグ(sESTs) |
DE19816395A1 (de) * | 1998-04-03 | 1999-10-07 | Metagen Gesellschaft Fuer Genomforschung Mbh | Menschliche Nukleinsäuresequenzen aus Ovar-Normalgewebe |
EP0976824A1 (fr) * | 1998-07-10 | 2000-02-02 | Amsterdam Molecular Therapeutics | Gène et protéine impliqué dans la régénération du foie |
-
1999
- 1999-12-20 CA CA002354369A patent/CA2354369A1/fr not_active Abandoned
- 1999-12-20 JP JP2000589560A patent/JP2002539767A/ja not_active Withdrawn
- 1999-12-20 EP EP99959633A patent/EP1144444A3/fr not_active Withdrawn
- 1999-12-20 AU AU16759/00A patent/AU1675900A/en not_active Abandoned
- 1999-12-20 WO PCT/IB1999/002058 patent/WO2000037491A2/fr active Search and Examination
Non-Patent Citations (1)
Title |
---|
See references of WO0037491A3 * |
Also Published As
Publication number | Publication date |
---|---|
EP1144444A3 (fr) | 2002-01-02 |
WO2000037491A2 (fr) | 2000-06-29 |
CA2354369A1 (fr) | 2000-06-29 |
JP2002539767A (ja) | 2002-11-26 |
AU1675900A (en) | 2000-07-12 |
WO2000037491A3 (fr) | 2001-09-20 |
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