MXPA02006580A - Genes encoding abc1 paralogs and the polypeptides derived therefrom. - Google Patents

Abstract

The present invention relates to three novel genes and polypeptides derived therefrom encoding PDABC proteins. The invention also describes methods for using the novel gene and polypeptides in the detection of genetic deletions of the gene, subcellular localization of the polypeptide, binding assays in connection with the chemical databases, gene therapy, and identification of chemicals which may be used in the therapeutic treatment of PDABCmediated diseases.

Description

GENES THAT CODE APPLY TO THE ABC1 PARALOGS AND THE POLYPEPTIDES DERIVED FROM THEMSELVES FIELD OF THE INVENTION The present invention relates to the identification of the PD-ATP binding cassette gene (hereinafter "PD-ABC gene") and the derived and identified polypeptides thereof and the use of PD-ABC genes for drug analysis tests and diagnostic and therapeutic methods for the treatment of inflammatory diseases and cardiovascular, mediated by the expression of a mutant form or by the aberrant levels or activity of the PD-ABC genes. The invention is based on the discovery that the sequences of the PD-ABC genes encoding the polypeptides that are paralogos for the ATP-link cassette transporter gene family, the human gene localizes to a chromosomal region involved in the disease Cardiovascular and abnormal HDL metabolism and the human gene is expressed in cells involved in inflammatory and cardiovascular diseases. In particular, the sequences of the novel human PD-ABC gene and the derived and identified polypeptides thereof encoding human PD-ABC polypeptides are described. In addition, the chromosomal location of PD-ABC for human chromosome 19p13.3 and the expression of PD-ABC in the spleen, thymus, peripheral blood leukocytes, bone marrow, lymph nodes and additional tissues are described. The invention also describes vectors and host cells comprising PD-ABC genes and methods for using PD-ABC genes, polypeptides and antibodies specifically signaling polypeptides in the detection of genetic alterations of PD-ABC genes, subcellular location of polypeptides, gene therapy applications or binding tests in connection with chemical databases. The invention also relates to the development of property analysis strategies for molecules that modify PD-ABC protein activities, diagnostics for syndromes associated with the expression of altered PD-ABC protein and methods for identification of the compounds that modulate the expression, synthesis or activity of PD-ABC proteins / genes and to use those compounds such as those identified as therapeutic agents in the treatment of mediated diseases with the PD-ABC. occluding by way of example and without limit, coronary artery disease (CAD).

BACKGROUND OF THE INVENTION ATP cassette transporters (ABCs) constitute a large family of trans-membrane proteins that transport a wide variety of substrates across cell membranes (Higgins, CF, Annu, Rev. Cell. Biol. 1992; 8: 67-113). The members of this transporter family have two hydrophobic domains, each containing six trans-membrane segments. In addition, they have two cytoplasmic ATP binding cassettes or nuclear binding folds (NBF) at the carboxyl terminus of each hydrophobic domain. ATP binding and hydrolysis in NBF provide energy for transport activity (Higgins, C.F. Annu., Rev. Cell, Biol., 1992; 8: 67-113). Although different members share significant homology, the transporters have diverse substrate specificities. Multidrug-resistant p-glycoprotein (MDR) transports organic chemicals with unrelated structures while the related transporter MRP is associated with the translocation of the phospholipid membrane (Gottesman, MM, et al., Annu., Rev. Biochem. , 1993; 62: 385-428; Smith JJ et al., Cell, 1993; 75: 451-462; Rutes, S., et al., Cell, 1994; 77: 1071-1081). The physiological importance of ABC transporters has been highlighted by the findings that genetic defects in some ABC transporters have been linked to human diseases. Mutations in the trans-membrane conductance regulator gene of cystic fibrosis are the cause of cystic fibrosis (Riordan, J.R. et al., Science, 1989; 245: 1066-1073). Genetic mutations or truncations in ABCR, an ABCA transporter ABC (Broccardo, C, et al, Biochim, Biophys, Acta, 1999; 1461: 395-404), result in Stargardt disease, a disease of the degenerative retina ( Allikmets, R., et al., Nat. Genet., 1997; 15: 236-246). ABC1, also a member of the ABCA subfamily and was isolated from mice by PCR based on sequence homology (Luciani, M-F., Et al., Genomics, 1994; 21: 150-159). ABC1 was initially found to be required for submersion of the apoptotic cells and for the anion that is transported across the membranes (Luciani, MF., Et al., EMBO J., 1996; 15: 226-235; Becq. , F., et al., J. Biol.

Chem., 1997; 272: 2695-2699). The expression of human AUC1 is regulated during the differentiation of macrophages and by cholesterol loading (Langmann T., et al., Biochem Biophys, Res. Comm., 1999; 257: 29-33). Recently, ABC1 has been identified as the defective gene in Tangier's disease (TD), a rare form of lipoprotein deficiency (Bodzich, M., et al., Nature Genetics, 1999; 22: 347-351; Broccardo, C, et al., Biochim, Byophys, Acta, 1999; 1461: 395-404, Brooks-Wilson, A., et al., Nature Genetics, 1999; 22: 336-345; Rust, S., et al. , Nature Genetics, 1999,22: 352-355). Both pharmacological and genetic evidence suggest that ABC1 transports cholesterol and phospholipid through the cell membrane in peripheral tissues (Remaley, AT, et al., Proc. Nati, Acad. Sci. USA, 1999; 96: 12685 -12690; Young, SG, et al., Nature Genetics, 1999; 22: 316-318). A phenotype similar to that in the TD patients was observed in the ABC1 of the drugged mice (McNeish, J., et al., Proc. Nati, Acad. Sci. USA, 2000; 97: 4245-4250; Orso, E, et al., Nature Genetics, 2000; 24: 192-196).

The involvement of ABC1 in the lipoprotein metabolism leads to the search for close homologues of ABC1 that must be involved in the expiration of cholesterol and the transport inverse of cholesterol. This document describes the isolation and specific tissue expression of a novel ABC transporter that is the closest homologue ABC1 to date. In addition, an alternately linked variant having a different specific tissue expression design was also identified. These findings suggest that PD-ABC must have an important physiological role.

An increased risk for CAD has been associated with a low level of high density lipoprotein (HDL) particles. In fact, almost half of all patients with CAD have low levels of HDL cholesterol. As a result, recent efforts to prevent CAD have focused on methods to increase the levels of HDL particles. HDL particles are important to undo the body by excess cholesterol by transporting cholesterol from the cells to the liver (reverse cholesterol transport), where cholesterol metabolism and eventual excretion take place. Tangier disease and familial high-density lipoprotein (FHA) deficiency are characterized by extremely low plasma levels of HDL and increased levels of cellular cholesterol, with resultant premature arteriosclerosis (Rogler, et al., Arterioscler Thromb Vasc Biol., 1995; 15 (5): 683-90; Marcil, et al., Arterioscler Thromb Vass Biol., 1999; 19 (1): 159-69). Mutations in the ATP-binding cassette transporter-1 (ABC1) gene have recently been identified in TD and FHA patients (Brooks-Wilson, A., et al., Nature Genetics, 1999; 22: 336-345; Bodzioch; , M, et al., Nature Genetics, 1999; 22: 347-351). These studies show that the ABC1 protein plays a critical role in the expiration of cholesterol from the cells within the HDL particles (Marcil, et al., Lancet, 1999; 354: 1340-1346). It has become clear that the increased activity of the ABC1 protein, through the use of small molecule compounds, may be a way to raise HDL levels and to prevent CAD.

Although the ABC1 protein / gene was recently identified as playing a role in HDL metabolism, it is clear that other genes will also be involved. These other genes have not been identified yet. It would be beneficial if those genes had been identified. By gaining an understanding of the biochemical mechanisms behind this path, new opportunities can be achieved to treat and diagnose diseases related to the abnormal production (high or low) of HDL particles. Another established path, a better understanding of the molecular mechanisms of the HDL-mediated expiration of intracellular cholesterol, will improve the design of therapeutic drugs that treat diseases related to abnormal cholesterol levels and HDL production.

Dyslipidemia, said alterations in the metabolism of HDL or CAD result from the dyslipidemia that has been associated with a number of diseases. Such diseases include diabetes (for review, Evans et al., Curr Opin Lipidol, 1999; 10 (5): 387-391), liver disease by fat (Marchesini et al., Am J Med, 1999; 107 (5) : 450-455), obesity (for review, Indulski et al., Cent Eur J Public Healt, 1999; 7 (3): 122-129), insulin resistance (for review, Bailey, et al, Brochem Pharmacol, 1999; 58 (10): 1511-1520), alcoholism (for review, Barahona et al., Recent Dev Alcohol, 1998; 14: 97-134), retinal degeneration (Gordon, et al., Am J OFthalmol, 1991; 112 (4): 385-391), hypertension (for review, Giannattasio, et al. al., Pathol Biol (Paris), 1999; 47 (7): 744-751) and vascular diseases in general.

SUMMARY OF THE INVENTION To determine if other genes exist that may also be important in the regulation of cholesterol levels, commercially available sequence databases were searched for genes related to 1 (the cassette transporter ATP binding in this document "ABC1"). The family of the ABC transporter gene is the largest known gene family and the ABC transporter genes have various substrates including sugars, amino acids, peptides and antibiotics (for review, Croop JM, Methods Enzymol, 1998; 292: 101-116). By developing searches of the homology of a proprietary database of EST sequences in clusters, generated using algorithms and tools from Compugen Systems, Ltd., novel human gene sequences were identified that are 48% identical and 64% similar to the amino acid level of ABC1. These genes (hereinafter referred to as PD-ABC Form 1 and Form 2) represent the closest human paralogs of the ABC1 gene in this identified manner. The PD-ABC genes and the polypeptides they encode are expressed in various cells and tissues and both polynucleotide sequences for the full-length genes and any binding variant and their encoded proteins thereof are identified herein. The polynucleotide sequence of PD-ABC Forma 1 is identified in SEQ ID NO 1 and the amino acid sequences of the PD-ABC Forma 1 protein were encoded by the novel gene set forth in SEQ ID NO 2. The polynucleotide sequence of PD-ABC Form 2 was identified in SEQ ID NO 3 and the amino acid sequences of the PD-ABC protein by the novel gene set forth in SEQ ID NO 4.

Additionally, the alignment of these genes to high-throughput genomic sequences in the Genbank database leads to the "in-lysic" localization of these genes to the human chromosome 19p13.3. Searches of the Mendelial Heritage database in Men's line (OMIM) leads to the discovery that this same region of human chromosome 19 has been genetically linked to susceptibility to atherosclerosis (Nishina, et al., PhÍAS, 1992; 89: 708-712; Naggert, et al. ., Clin Genet, 1997: 236-240). Affected individuals of the families that show binding for 19p13.3 have low levels of HDL particles, characteristic of mutations in the ABC1 gene. The level of identity of these novel genes for the ABC1 gene and the genetic binding for a point involved in the susceptibility to atherosclerosis, identifies these genes as a target for drugs to prevent CAD. In other words, because the PD-ABC proteins Form 1 and 2 share the amino acid homology for the ABC1 protein, it is very likely that they share some functional and structural characteristics with the ABC1.

Another aspect of the invention is to provide purified PD-ABC Form 1 and 2 proteins. The purified proteins can be obtained from either recombinant cells or naturally occurring cells. The purified PD-ABC proteins of the invention can be of mammalian origin. Including PD-ABC proteins derived from humans, are examples of the various proteins specially provided for primates. The invention also provides allelic variants and biologically active derivatives of PD-ABC proteins of natural occurrence.

Another aspect of the invention is to provide polynucleotides that encode the PD-ABC Form 1 and 2 proteins of the invention and provide polynucleotides complementary to the polynucleotide coding strand. The polynucleotides of the invention can be used to provide recombinant expression of PD-ABC proteins. The polynucleotides of the invention may also be used for gene therapy purposes as well as 1) to treat diseases that may result from alterations of PD-ABC genes or alterations of the cellular pathways involved with PD-ABC genes / proteins, 2) test for the presence of a disease or susceptibility to a disease due to alterations or omissions of genes / PD-ABC proteins, 3) analyzing or altering the subcellular location of the PD-ABC polypeptides, 4) #Dnar or isolate discrete classes of similar RNA for PD-ABC genes; 5) express discrete classes of RNA to alter levels of PD-ABC genes.

The invention also relates to oligonucleotide molecules useful as probes or primers, wherein said oligonucleotide molecules hybridize especially to any nucleotide sequence that comprises or relates to the PD-ABC genes, particularly the sequences of SEQ ID NOS 1 and 3. These oligonucleotides are useful as either primers for use in various processes such as amplification and DNA micro-sequencing or as probes for DNA recognition in hybridization assays.

A nucleic acid probe or primer according to the invention comprises at least 8 consecutive nucleotides of a polynucleotide of SEQ ID NOS 1 or 3, preferably from 8 to 200 consecutive nucleotides, more particularly of 10, 15, 20 or 30 a 100 consecutive nucleotides, more preferably 10 to 90 nucleotides and more preferably 20 to 80 consecutive nucleotides of a polynucleotide of SEQ ID NOS 1 or 2. Preferred probes or primers of the invention comprise the oligonucleotides selected from the group consisting of oligonucleotides set forth in the following examples.

The invention also comprises a method for the amplification of a region of PD-ABC genes. The method comprises the step of: contacting a test sample suspected of containing the desired PD-ABC sequences or portions thereof with amplification reaction reagents, comprising a pair of amplification primers such as those described above, initiators being located on one side of the PD-ABC nucleotide regions to be amplified. The method may further comprise the step of detecting the amplification product. For example, the amplification product can be detected using a detection probe that can hybridize to an internal region of the amplified sequences. Alternatively, the amplification product can be detected with Any of the primers used for the amplification reaction of themselves, dt) ctonally in a labeled form.

The invention also relates to diagnostic equipment for detecting the presence of at least one copy of a PD-ABC DNA Form 1 or Form 2 in a test sample, said kits containing an initiator, a pair of primers or a DNA probe. according to the invention.

In a first embodiment, the kit comprises primers such as those described above, preferably inverse and tracking primers that are used to amplify genes or PD-ABC fragments thereof.

In a second embodiment, the kit comprises a DNA probe for hybridization, which is or eventually becomes immobilized on a solid support, which is capable of hybridizing to a gene or PD-ABC fragment thereof. Techniques for immobilizing a nucleotide primer or probe on a solid support are well known to those skilled in the art.

The kits of the present invention may further comprise optional elements including appropriate amplification reagents such as DNA polymerases, when The kit comprises primers, reagents useful in hybridization reactions and useful reagents to reveal the presence of a hybridization reaction between a labeled hybridization probe and a PD-ABC gene.

Another aspect of the invention is to provide antibodies capable of binding proteins 25 PD-ABC of the invention. The antibodies can be polyclonal or monoclonal. The invention also provides methods for using the subject antibodies to detect and measure the expression of PD-ABC proteins either in vitro or in vivo or to detect proteins that interact with PD-ABC proteins or the molecules that regulate any of the activities of PD-ABC proteins.

Another aspect of the invention is to provide tests for the detection of proteins that interact with PD-ABC proteins / genes using genetic advances. A preferred embodiment involves the use of two-yeast hybrid advances for this analysis (Bartel and Fields, The Two-Hybrid Yeast System, Oxford University Press, 1997).

Another aspect of the invention is to provide tests for the detection or analysis of therapeutic compounds that interfere with or mimic in any way, the interaction between PD-ABC proteins and the ligands that bind PD-ABC proteins.

In a first embodiment, said method for the analysis of a candidate substance comprising the following steps: a) providing a polypeptide comprising the amino acid sequence of SEQ ID NO 2 or 4 or a peptide fragment or a variant thereof; b) obtain a candidate substance; c) contacting said polypeptide with said candidate substance and d) detecting the complexes formed between said polypeptide and said candidate substance. In one embodiment of the method of analysis defined above, complexes formed between the polypeptide and the candidate substance are further incubated in the presence of a polyclonal or monoclonal antibody that specifically binds to a PD-ABC protein of the invention or to the peptide fragment. or the variant thereof. The candidate substance or molecule to be analyzed for interacting with the PD-ABC polynucleotide can be of various nature, including without being limited to natural or synthetic organic compounds or molecules of biological origin such as polypeptides.

In another embodiment of the present method of analysis, the increment concentrations of a substance which is responsible for the binding of the PD-ABC protein with the candidate substance considered, simultaneously or before the addition of the candidate substance or molecule, when performed Stage c) of said method. By this technique, the elimination and optionally the quantification of the complexes formed between the PD-ABC protein or the peptide fragment or the variant thereof and the candidate substance or molecule to be analyzed allows one skilled in the art to determine the affinity value of said substance or molecule of said PD-ABC protein or the peptide fragment thereof or variant thereof.

The invention also describes equipment useful to perform in the method of analysis described above in this document. Preferably, said kits comprise a PD-ABC protein having the amino acid sequence of SEQ ID NO. 2 or 4 or a peptide fragment or a variant thereof and optionally means useful for detecting the complex formed between the PD-ABC protein or its peptide or variant fragment and the candidate substance. In a preferred embodiment, the detection means consist of monoclonal or polyclonal antibodies directed against the PD-ABC protein or a peptide fragment or a variant thereof.

The tests of the invention therefore comprise the step of measuring the effect of a compound of interest on the link between the PD-ABC proteins and the ligands that bind PD-ABC proteins. The link can be measured in a variety of ways, including the use of the labeled PD-ABC protein or labeled ligands.

Another aspect of the invention is to provide tests for the discovery of proteins that interact directly with PD-ABC proteins. The tests of the invention comprise a method for detecting said alterations in cells or in biochemical tests. These interactions can be detected in a variety of ways, including the use of the PD-ABC proteins encoding the cDNA or PD-ABC proteins themselves or fragments or modifications thereof. t »» l? á? r.1 .1. &? ? ... fc «8 JS, ..., gtJhx.r *. * ?? ¡¡¡¡¡¡¡¡, In a time, 'c:' ac, preferred of the present invention, the PD-ABC genes represent novel targets that can be used to develop high-throughput assays for the identification of chemicals and the interaction of proteins that increase the activity of PD-ABCs. Recently, compounds that alter the activity of PD-ABC genes can be tested for their efficiency in the prevention of CAD. The points can also be of use for basic search and pharmacogenetic studies related to HDL metabolism.

In another preferred embodiment of the present invention, the protein product of the PD-ABC genes can serve as novel therapeutic targets for the treatment of CAD and dyslipidemia.

In another preferred embodiment of the present invention, a genetic model for the study of CAD and dyslipidemia can be created by altering the PD-ABC genes in animals such as mice.

In a further preferred embodiment of the present invention, polymorphisms in PD-ABC genes can identify members of the population at risk for CAD and a genetic test should be created using the sequences of these genes to identify such people.

In a preferred embodiment of the present invention, polymorphisms in the sequence of the PD-ABC genes could be used to select the appropriate methods of therapy for CAD and dyslipidemia.

In a further preferred embodiment of the present invention, the sequence of the genes PD-ABC could be used to create anti-sense RNA or antibody probes that could be used later for the therapeutic treatment of CAD or dyslipidemia.

In a further preferred embodiment of the present invention, the sequences of the PD-ABC genes could be used to identify the interaction genes, which by themselves could serve as therapeutic targets.

In a further preferred embodiment of the present invention, the nucleotide sequences that include and surround the PD-ABC genes could be used to identify the novel regulatory factors of these genes. These factors could become therapeutic targets for dyslipidemia and CAD.

In a further preferred embodiment of the present invention, the protein product of the PD-ABC genes could be used to identify compounds that are selective for a particular member of the ABC transporter family.

Preferred sequences, polypeptides and methods for making and using the invention are described herein. However, it is understood that the invention is not limited only to the particular sequences, polypeptides and methods described. The sequences, polypeptides and methodologies may vary and the terminology used in this document is for the purpose of describing the particular modalities. The foregoing should not be considered as limiting the invention in any way given that the scope of the protection depends on the claims. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. All patents and all publications mentioned in this document are incorporated in their entirety as a reference.

BRIEF DESCRIPTION OF THE INVENTION Figure 1. Alignment of the predicted amino acid sequence of human PD-ABC for human ABC1 and ABCR. The amino acids are shown in a single letter code. The identical residues across all the sequences are highlighted in the dark area and the homologous residues they are marked in the shaded area. Dashes indicate intervals entered within the sequences to maximize alignment. 25 Í.Í.? ? HL ^ -? - «*« * ** - -? .A .... ^^ ^ jlg ^ i ..., KA » Figure 2. Structures of the union variants and the exon / intron organization. Diagram A represents the intron and exon locations in each variant as well as the alternate binding site. The trans-membrane domains (TM1 = N-terminal, TM2 = C-terminal) and the nucleotide binding folds (NBF) are indicated.

Form IBIIHUI IB ill ll flll H-HH-B Form 2 iflim ii iw imiwiiiii ipii iin Figure 3. Expression of PD-ABC in multiple tissues. A, Northern hybridization analysis of PD-ABC expression in various human tissues. A hybridization with mRNA of tissues ÉA, isí, t. ..mm. .r ,. . ,. *. & A, .aAAiAi-go .. ^^ .. A ^. indicated were hybridized with a PD-ABC probe and a GAPDH probe, respectively. The * PD-ABC and GAPDH bands were indicated with arrows. B, Northem hybridization analysis of PD-ABC expression in tissues or cells of the immune system. Hybridization was carried out as mentioned above and the two forms of PD-ABC were indicated.

CAPI) H (I.S kb) Figure 4. Tissue distribution of the PD-ABC binding variants. The Rapid Analysis Gene Expression panels were used as templates and the polymerase reaction chains for reverse transcription were run with the specific primer pairs for the two variants, respectively. The PCR products were resolved on agarose gel. Line 1, fetal liver; line 2, fetal brain; line 3, bone marrow; line 4, PBL (peripheral blood leukocytes); line 5, skin; line 6; prostate; line 7, uterus; line 8, ovaries; line 9, pancreas; line 10, adrenal; line 11, thyroid; line 12, salivary; line 13, placenta; line 14, testicles; line 15, stomach; line 16, muscle; line 17, small intestine; line 18, lungs; line 19, colon; line 20, liver; line 21, spleen; line 22, kidney; line 23, heart; line 24, brain.

Formal Form 2 DETAILED DESCRIPTION OF THE INVENTION Within this application, unless stated otherwise, the techniques used can be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego CA), "Guide for Protein Purification" in Methods in Enzymology (MP Deutshcer , ed., (1990) Academic Press, Inc); PCR Protocols: A Guide to Methods and Applications (Innis, et al., 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd. Ed. (R. Freshney, 1987. Liss, Inc. New York, NY) and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc. Clifton, N.J.) Sequence Analysis Primer (Gribskov, et al., 1994, Oxford University Press).

In one aspect, the present invention provides novel purified and isolated polynucleotides, hereinafter referred to as genes of paralog 1 of the ATP binding cassette transporter (PD-ABC), which encode PD-ABC proteins, wherein the sequences of polynucleotides are substantially similar to those shown in SEQ ID NOS 1 and 3 and the polynucleotide sequences are substantially similar to those shown in SEQ ID NOS 2 and 4. The term "PD-ABC" is widely used herein. Unless stated otherwise, the term "PD-ABC" includes any form of PD-ABC derived from OS natural mammals and the like. It is preferred that the terms PD-ABC include all mammals, including but not limited to primates and humans.

The polynucleotides provided to be able to encode PD-ABC proteins or portions thereof. The polynucleotides of the invention can be produced by a variety of methods including chemical synthesis in vitro using the well-known solid phase technique, by cloning or combinations thereof. The polynucleotide of the invention can be derived from the genomic or cDNA libraries. Those skilled in the art are familiar with the degeneracy of the genetic code and can rapidly design polynucleotides encoding PD-ABC proteins having polynucleotide sequence homology or partial for the naturally occurring polynucleotide sequences encoding the proteins PD-ABC. The polynucleotides of the invention can be single stranded or double stranded. The polynucleotide complementary to polynucleotides encoding PD-ABC proteins is also provided.

The polynucleotides encoding the PD-ABC protein can be obtained from the cDNA libraries prepared from tissue believed to possess the PD-ABC protein or the mRNA and to express it at a detectable level. For example, a cDNA library can be constructed by obtaining the polyadenylated cRNA of a known cell line to express the PD-ABC protein and using the mRNA as a template to synthesize the double-stranded cDNA.

Libraries, either the cDNA or the genomic, were analyzed with probes designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries, appropriate probes include monoclonal and polyclonal antibodies that specifically recognize and bind to a PD-ABC protein. For cDNA libraries, appropriate probes include carefully selected oligonucleotide probes (usually about 20-80 bases in length) that encode the suspected or known portions of a PD-ABC protein of the same or different species and / or the cDNAs i.fcA¿AA «** A» homologs or complementary or fragments thereof encoding the same or a similar gene and / or homologous genomic cDNAs or fragments thereof. Analysis of the cDNA or the genomic library with the selected probe can be carried out using standard procedures as described in Chapters 10-12 of Sambrook, et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989).

A preferred method for practicing this invention is to use the carefully selected oligonucleotide sequences to analyze the cDNA libraries from various tissues. The sequences of oligonucleotides selected as probes should be sufficient in length and sufficiently ambiguous so as to minimize false positives. The actual nucleotide sequence (s) is / are usually designated based on the regions of a PD-ABC gene having the last redundancy of the codon. Oligonucleotides can degenerate into one or more positions. The use of degenerate oligonucleotides is of particular importance where the library is analyzed from species in which the use of the preferential codon is not known.

The oligonucleotide should be labeled so that it can be detected in the hybridization of the DNA in the library that is being analyzed. The preferred method of dialing is to use the ATP (ie, T32P) and the polynucleotide kinase to radiolabel the 5 'end of the oligonucleotide. However, other methods can be used to label the oligonucleotide, including but not limited to biotinylation or labeling of the enzyme.

The cDNAs encoding PD-ABC proteins can also be identified and isolated by other techniques known from recombinant DNA technology, such as by direct expression cloning or by the use of the polymerase chain reaction (PCR) as described in FIG. described in U.S. Patent No. 4,683,195 in section 14 of Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, New York or in Chapter 15 of Current Protocols in Molecular Biology, Ausubel, et al., edds., Green Publishing Associates and Wiley-lnterscience 1991. This method requires the use of oligonucleotide probes that will hybridize to DNA encoding a PD-ABC protein.

As defined herein, "substantially similar" includes the identical sequences, as well as omissions, substitutions or additions for a DNA, RNA or protein sequence that maintains any biologically active portion thereof of the protein product and possesses any of the motives This includes, but is not limited to, any PD-ABC binding variant that is found to exist. Preferably, the DNA sequences according to the invention consist essentially of the DNA sequence of SED ID NOS 0 1 or 3. These novel isolated and purified DNA sequences can be used to direct the expression of PD-ABC proteins and for the mutational analysis of the function of PD-ABC proteins.

The mutated sequences of compliance can be identified in a routine manner by those skilled in the art using the teachings provided herein and techniques well known in the art.

In a preferred embodiment, the present invention comprises a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO. 1 or 3 under 0 conditions of high stringency hybridization. As used herein, the term "high stringency hybridization conditions" refers to hybridization on a filter support at 65 ° C in a low-salt hybridization stabilizer for the probe of interest of 2x108 cpm / μg for between about 8 to 24 hours followed by washing in 1% SDS, 20 mM phosphate stabilizer and 1 mM EDTA at 65 ° C for about 30 minutes for 4 5 hours. In a preferred embodiment, the low-salt hybridization stabilizer comprises between 0.5% to 10% SDS and 0.05 M and 0.5 M sodium phosphate. In a more preferred embodiment, the low salt hybridization stabilizer comprises 7% SDS and 0.125 M sodium phosphate.

Lit »ßO¡áÍr ?? - r¿ií i i 'As shown in the art, numerous equivalent conditions may be employed to comprise either high or low conditions of severity. Factors such as the length and nature of the sequence (DNA, RNA, base composition), the nature of the target (DNA, RNA, base composition, presence in the solution or immobilization, etc.) and the concentration of the salts and other components (ie, the presence or absence of formamide, dextran sulfate and / or polyethylene glycol) are considered and the hybridization solution can be varied to generate conditions of high or low severity different from, but equivalent to, the conditions listed above.

The term "severity conditions" as used herein, is the "severity" that occurs within a range of approximately Tm-5 ° C (5 ° C below the melting temperature (Tm) of the probe) to approximately 20 ° C to 25 ° C below Tm. As will be understood by one skilled in the art, the severity of hybridization can be altered to identify or detect related or identical polynucleotide sequences.

The polynucleotides of the invention have a variety of uses, some of which have been identified or will be addressed in greater detail, inflates. The particular uses for a given polynucleotide depend in part on the specific polynucleotide modality of interest. The polynucleotides of the invention can be used as hybridization probes to coat the PD-ABC nucleotide sequences of the genetic libraries. The polynucleotides of the invention can also be used as primers for the amplification of the PD-ABC gene sequences encoding the polynucleotides or a portion thereof through PCR and other similar amplification methods. The polynucleotides of the invention can also be used as probes and amplification primers to detect mutations in the PDABC protein encoding genes that have been correlated with diseases, particularly diseases related to an altered function for PD-ABC proteins. Including but not limited to those diseases established above.

The invention also provides a variety of expression vectors comprising a polynucleotide PD-ABC gene or a substantially similar thereto subcloned in an extra-chromosomal vector sequence. This aspect of the invention allows in vitro expression of the PD-ABC genes, further allowing an analysis of the PD-ABC gene regulation and the structure of the PD-ABC protein and function. As used herein, the term "extra-chromosomal vector" includes but is not limited to plasmids, bacteriophages, cosmids, retrovirus and artificial chromosomes. In a preferred embodiment, the extra-chromosomal vector comprises an expression vector which allows production of PD-ABC protein when the recombinant DNA molecule is inserted into a host cell. Such vectors are well known in the art and include but are not limited to those with the T3 and T7 polymerase promoters, the SV40 promoter, the CMV promoter or any promoter that can direct expression of the gene or that it is desired to test for the ability to direct the expression of the gene.

In a preferred embodiment, the subject expression vectors comprise a polynucleotide sequence that encodes the PD-ABC protein in functional combination with one or more promoter sequences so as to provide for the expression of the PD-ABC protein (or an anti-sense copy of the appropriate sequence for the inhibition of the expression of an endogenous gene). The vectors may comprise additional polynucleotide sequences for gene expression, regulation or convenient manipulation of the vector, such additional sequences include terminators, reporters, enhancers, selective markers, packaging sites and the like. The detailed description of the vectors of polynucleotide expression and their use can be found in, inter alia, Gene Expression Technology: Methods in Enzymology, Vol 185 Goeddel, ed, Academic Press Inc., San Diego, CA (1991), Protein Expression in Animal Cells, Roth, ed., Academic Press, San Diego, CA (1994).

The polynucleotide expression vectors of the invention have a variety of uses. Such uses include genetic engineering for host cells to express PD-ABC proteins. In a further aspect, the present invention provides host cells mammals and oocytes Xenopus. Transfection of host cells with recombinant DNA molecules is well known in the art (Sambrook, et al, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989) and as used herein, includes but is not limited to transfection of calcium phosphate, transfection of dextran sulfate, electrophoresis, lipofection and viral infection. This aspect of the invention allows in vitro and in vivo expression of PD-ABCs and their gene products, further enabling high level expression of PD-ABC proteins. In a further aspect of the invention, the RNA molecules containing PD-ABCs can be injected into the Xenopus oocytes and the transport of the substrates can be measured using standard electrophysiological techniques.

In another aspect of the invention, the transgenic animals can be constructed by injecting the nucleotide sequence for a PD-ABC cloned into the appropriate expression vectors within the germ cells.

Other uses of polynucleotide expression vectors, described in more detail, inflate, include their use for gene therapy for diseases and conditions in which use may be desirable to express PD-ABC proteins at higher levels than levels. of expression of natural occurrence. Alternatively, the use of the subject vectors for anti-sense expression to reduce the natural occurrence levels of PD-ABC proteins may be desirable.

The polynucleotide sequence of SEQ ID NOS 2 and 4 is mapped to human chromosomes using the nucleotide sequences for the cDNA from library sources to generate probes. The sequences were mapped to a particular chromosome or to a specific region of the chromosome using well-known techniques. These include on-site hybridization for chromosomal extensions and PCR-based mapping by amplifying the DNA of the standard radiation hybrid cell lines. (Verma, et al., (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, NYC).

In another aspect, the present invention provides a substantially purified recombinant protein comprising a polypeptide substantially similar to the PD-ABC polypeptide shown in SEQ ID NOS 2 or 4. In addition, this aspect of the invention enables the use of PD-ABC proteins in the in vitro tests described later. As used herein, the term "substantially similar" includes omissions, substitutions and additions to the sequence SEQ ID NOS 2 and 4 introduced by any in vitro means or any genetic alterations naturally observed in vivo. As used in this document, the term "substantially purified" means that the protein should be free of the detectable contamination protein, but the PD-ABC protein can be co-purified with an interaction protein or as an oligomer. In a more preferred embodiment, the protein sequence according to the invention comprises an amino acid sequence of SEQ ID NOS 2 or 4. The mutated sequences according to the invention can be identified in a routine manner by those skilled in the art using the teachings provided in this document and techniques well known in the art. This aspect of the invention provides a novel purified protein that can be used by in vitro tests and as a component of a pharmaceutical composition.

PD-ABC proteins can be used to discover molecules that interfere with their activities. For example, molecules that prevent the binding of PD-ABCs to ligands or to other molecules.

The PD-ABC proteins of the present invention have a putative biological activity of modulating the cellular expiration of cholesterol. The PD-ABC proteins of the invention can be isolated from a variety of species of mammalian animals. The mammal species preferred for isolation are primates and humans. The invention also contemplates allele variants of PD-ABC proteins that can be prepared from a variety of mammalian tissues. Preferably, PD-ABC proteins are obtained from recombinant host cells genetically treated to express significant amounts of PD-ABC proteins. PD-ABC proteins can be isolated from recombinant or non-recombinant cells in a variety of ways well known to a person skilled in the art.

The term "(S) PD-ABC protein" as used herein refers not only to proteins that have the sequence of amino acid residues of PD-ABC proteins of natural occurrence but also refers to functional derivatives and the natural occurrence variants of the PD-ABC protein. A "functional derivative" of a native polypeptide is a compound having a qualitative biological activity in common with the native PD-ABC proteins. In addition, a functional derivative of the native PD-ABC protein is a compound that has a qualitative biological activity in common with a native PD-ABC protein, i.e., the transport substrates through the biological membranes. "Functional derivatives" include, but are not limited to, fragments of native polypeptides of any animal species (including humans) and derivatives of any animal species (including humans) and derivatives of native polypeptides (human and non-human) and their fragments, provide that they have a biological activity in common with a respective native polypeptide. The "fragments" comprise the regions within the sequence of a mature native polypeptide. The term "derivative" is used to define the amino acid sequence and the glycosylation variants and the covalent modifications of a native polypeptide, wherein the term "variant" refers to the amino acid sequence and the glycosylation variants within this definition . Preferably, the functional derivatives are polypeptides having at least about 70% amino acid sequence similarity, more preferably about 80% similarity of the amino acid sequence, even more preferably at least 90% of the similarity of the sequence of amino acids, more preferably at least 99% of the similarity of the amino acid sequence to the sequence of a corresponding native polypeptide. More preferably, the functional derivatives of a PD-ABC protein or similar to the region or regions within the native polypeptide sequence that directly participates in the binding of the ligand. The phrase "functional derivative" specifically includes peptides and small organic molecules that have a qualitative biological activity in composing with a native PD-ABC protein.

"Identity" or "homology" with respect to a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are similar to the residues of a corresponding native polypeptide, after aligning the sequences and enter the intervals, if necessary, to achieve maximum percentage homology. Either the terminal extensions C or N and not in the inserts, nor in the alternatively linked variants, should be constructed as reducing identity or homology. Methods and computer programs for alignment are well known in the art.

The amino acid sequence variants of the native PD-ABC proteins or the PD-ABC protein fragments are prepared by methods known in the art by introducing the appropriate nucleotide changes into a native or PD-ABC proteins that encode the DNA or by in vitro synthesis of the desired polypeptides. There are two main variants in the construction of variants of amino acid sequences: the location of the mutation site and the nature of the mutation. With the exception of naturally occurring alleles, which do not require manipulation of the DNA sequence encoding the PD-ABC proteins, the amino acid sequence variants of the PD-ABC proteins are preferably constructed by mutating the DNA, to arrive in an allele or in a variant of the amino acid sequence that does not occur naturally.

Alternatively or in addition, the amino acid alterations may be at sites that differ in PD-ABC proteins from several species or in highly conserved regions, depending on the purpose to be achieved.

Sites in these locations will typically be modified in series, that is, through (1) replace first with the conservative selections and later with more radical selections depending on the results achieved, (2) eliminate the white residue or residues or (3) insert residues of the same or different class adjacent to the localized site or combinations of options 1 to 3.

One useful technique is called "Alanine analysis" by Cunningham and Wells, Science, 1989; 244: 1081-1085. A residue or group of white residues is identified and replaced by alanine or polyalanine. These domains that demonstrate functional sensitivity to alanine substitutions are subsequently refined by additional introduction or other substituents at or for the alanine substitution sites.

After identification of the desired mutation (s), the gene encoding a PD-ABC protein variant can, for example, be obtained by chemical synthesis.

More preferably, the DNA encoding an amino acid sequence variant of the PD-ABC protein is prepared by site-directed mutagenesis of the DNA encoding an early prepared variant or a non-variant version of the PD-ABC protein. Site-directed mutagenesis (site-specific) allows the production of PD-ABC protein variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides to providing a starter sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the junction junction being cross-linked. Typically, an initiator of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, site-specific mutagenesis techniques are well known in the art, as exemplified by publications such as, Edelman, et al., DNA, 1983; 2: 183. As will be appreciated, the site-specific mutagenesis technique typically employs a phage vector that exists in both double-stranded and single-stranded forms. Typical vectors useful in site-directed mutagenesis include vectors such as M13 phage. This and other phage vectors are commercially available and their use is well known to those skilled in the art. An efficient and versatile method for the construction of site-directed mutations of the oligodeoxyribonucleotide in the DNA fragments using the M13 derived vectors was published by Zoller, M.J. and Smith, M., Nucleic Acids Res. 1982; 10; 6487-6500. Also, plasmid vectors containing an origin of replication of one strand phage, Veira, et al., Meth. Enzymol. 1987; 153.3 can be used to obtain the DNA of a strand. Alternatively, nucleotide substitutions are introduced by synthesis of the appropriate DNA fragment in vitro and amplification thereof by PCR procedures known in the art.

In general, site-specific mutagenesis can be performed by obtaining a single-stranded or double-stranded vector that includes within its sequence a DNA sequence encoding the relevant protein. An oligonucleotide primer is prepared by supporting the desired mutated sequence, generally, synthetically, for example, by the method of Crea, et al., Proc. Nati Acad. Sci. USA, 1978; 75: 5765. This initiator is subsequently harvested with the vector containing the protein sequence of a strand and subjected to the DNA polymerization enzymes such as the Klenow fragment of E. Coli polymerase I to complete the synthesis of the strand that supports the mutation . In addition, a heteroduplex is formed where one strand encodes the original non-mutated sequence and the second strand supports the desired mutation. This heteroduplex vector is subsequently used to transform the appropriate host cells such as HB101 cells and the clones are selected which include the recombinant vectors that support the arrangement of the mutated sequence. Then, the mutated region can be removed and placed in an appropriate expression vector for protein production.

The PCR technique can also be used in the creation of amino acid sequence variants of the PD-ABC protein. When small amounts of template DNA are t t Bu. ^ f tt < ? u? ^ »^^^. iJto ^^ J > fc ^ || l, i 'irirtifrH ifréiMi fi fi as the initial material in a PCR, the primers that differ slightly in the sequence of the corresponding region in a template DNA can be used to generate relatively large amounts of a specific DNA fragment which differs from the template sequence only in the positions where the primers differ from the template. For the introduction of a mutation within a plasmid DNA, one of the primers is designed to cover the position of the mutation and to contain the mutation; the sequence of the other primers should be identified for an elongation of the sequence in the opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer be located within 500 to 5000 nucleotides of that first, so that in the latter the total amplified region of the DNA circumscribed by the primers can be easily sequenced. PCR amplification using a pair of primers as the one describes the results in a population of the DNA fragments that differ in the position of the specific mutation by the primer and possibly in other positions, since the copy of the template is prone to something to error.

Additional details of the prior mutagenesis techniques and the like are found in general textbooks, such as, for example, Sambrook, et al., Molecular Cloning: H Laboratory Manual 2nd. edition, Cold Spring Harbor Press, Cold Spring Harbor Press, Cold Spring Harbor (1989) and Current Protocols In Molecular Biology, Ausubel, et al., eds., John Wiley and Sons (1995).

The naturally occurring amino acids are divided into groups based on the common side chain properties: (1) hydrophobic, norleucine, met, ala, val, leu, ile; (2) neutral hydrophobic: cys, ser.tier; (3) Acids: asp. glu; (4) basic: asn, gin, his, lys, erg; (5) residues that influence the orientation of the chain: gly, pro and t i itiilü ilililinmiiit f-tffifitMlilT- * má? * «~ -. ^^« ^. «« ^^ ^ rm. ^. m. M. (6) aromatics: trp, tyr, pine.

Conservative substitutions involve the exchange of a member within a group by another member within the same group, where non-conservative substitutions 5 involve the exchange of a member in one of these classes for the other. Variants obtained by non-conservative substitutions are expected to result in significant changes in the properties / biological function of the variant obtained and may result in PD-ABC protein variants with the biological activities of the PD-ABC protein, ie , the modulation of cholesterol expiration. The amino acid positions that are conserved among the various 10 species are generally substituted in a relatively conservative manner if the objective is to retain the biological function.

The omissions of the amino acid sequence generally range from about 1 to 30 residues, more preferably about 1 to 10 residues and typically are contiguous. The omissions can be introduced within the regions not directly involved in the link of the ligand.

The amino acid insertions include the carboxyl and / or amino terminal fusions oscillating at the length of a residue for polypeptides containing a hundred or more 20 residues, as well as intra-sequence insertions of single or multiple amino acid residues. Intra-sequence insertions (ie, insertions within the amino acid sequence of the PD-ABC protein) can generally range from about 1 to 10 residues, more preferably 1 to 5 residues, more preferably 1 to 3 residues. Examples of terminal inserts include PD-ABC proteins with an N-terminal methionyl residue, a N terminal sequence of natural occurrence, a direct expression artifact in the recombinant cell culture and the fusion of a heterologous N-terminal sequence for the N-terminus of PD-ABC proteins to facilitate the secretion of PD proteins -ABC mature from the recombinant host cells. Said signal sequences will generally be obtained from and in addition homologous to, the host cell species attempted. Appropriate sequences include STII or Ipp for E. coli, alpha factor for yeast and viral signals such as herpes gD for mammalian cells. Other insertion variants of the native PD-ABC protein molecules include fusion of the C or N term of a PD-ABC protein for immunogenic polypeptides, i.e., bacterial polypeptides such as beta-lactamase or an enzyme encoded by E. Cold of the trp point or the yeast protein and the C-terminal fusions with the proteins having a long half-life such as the immunoglobulin regions (preferably the immunoglobulin constant region), albumin or ferritin as described in FIG. Published PCT application WO 89/02922.

Since it is commonly difficult to predict in advance the characteristics of a variant PD-ABC protein, it will be appreciated that the analysis will be necessary to select the optimal variant. For this purpose, biochemical analysis tests, such as those described in this document, will subsequently be available quickly.

In a further aspect, the present invention provides antibodies and methods for detecting antibodies that selectively bind to polypeptides with an amino acid sequence substantially similar to the amino acid sequence of the sequence of SEQ ID NOS 2 or 4. As described in more detail, it inflates, the antibody of the present invention can be a polyclonal or monoclonal antibody, prepared by using all or a portion of the sequence of SEQ ID NOS 2 or 4 or the modified portions thereof to cause a immune response in a host animal in accordance with standard techniques (Harlow and Lane (1988), eds Antibody: A Laboratory Manual, Cold Spring Harbor Press). In a preferred embodiment, the complete polypeptide sequence of SEQ ID NOS 2 or 4 is used to elicit the production of polyclonal antibodies in a host animal.

The method for detecting PD-ABC antibodies comprises contacting the cells with an antibody that recognizes a PD-ABC protein and incubating the cells in a manner that allows Detection of the PD-ABC antibody-protein complex. Standard conditions for the detection of antigen antibodies can be used to fulfill this aspect of the invention (Harlow and Lane, 1988). This aspect of the invention allows the detection of PD-ABC proteins both in vitro and in vivo.

The subject invention provides methods for the treatment of a variety of diseases characterized by undesirably abnormal cell levels of PD-ABCs. Diseases can be treated through gene therapy in vitro or in vivo. Protocols for gene therapy through the use of viral vectors can be found among other places in Viral Vector Gene Therapy and Neuroscience Applications, Kaplit and Lowry, Academic Press, San Diego (1995). The applications of gene therapy typically involve the labeling of host cells or tissues in need of therapy, designating the vector constructs capable of expressing a desired gene product in the identified cells and sending the constructs to the cells in a way that results in efficient transduction of host cells. The cells or tissues marked by gene therapy are those typically that are affected by the disease in which the construction of the vector is designated for treatment.

The gene therapy methods of the present invention comprise the step of introducing a vector for the expression of the PD-ABC protein (or anti-sense inhibitor RNA) within a patient's cell. The patient's cell can be in the patient, that is, in gene therapy in vivo, or external to the patient and subsequently reintroduced into the patient, that is, in in vitro gene therapy. Diseases that can be treated by subject gene therapy methods include, but are not limited to, those associated with dyslipidemia. Dyslipidemia, such as alterations in HDL metabolism or CAD resulting from dyslipidemia, have been associated with a number of diseases. These diseases include diabetes, liver disease due to fats, obesity, insulin resistance, alcoholism, retinal degeneration, hypertension and vascular diseases in general.

In a preferred aspect of the invention, there is provided a method for protecting mammalian cells from abnormal levels of PD-ABC in cells, which comprises introducing into mammalian cells an expression vector comprising a sequence of DNA substantially similar to the DNA sequence shown in SEQ ID NOS 1 5 or 3, which is operably linked to a DNA sequence that promotes expression of the DNA sequence and incubation of the cells under conditions wherein the sequence of DNA of SEQ ID NOS 1 or 3 will be expressed at high levels in mammalian cells. The appropriate expression vectors are as described above. In a preferred embodiment, the coding region of a human PD-ABC gene is subcloned into an expression vector under the transcriptional control of the cytomegalovirus (CMV) promoter to allow expression of the constitutive PD-ABC gene.

In another preferred aspect of the present invention, there is provided a method for treating or preventing abnormal levels of PD-ABC, comprising the introduction of a vector of Expression of mammalian cells comprising a DNA that is anti-sense for a sequence substantially similar to the DNA sequence shown in SEQ ID NOS 1 or 3 that is operationally linked to a DNA sequence that promotes expression of the sequence of anti-sense DNA. The cells are then cultured under conditions wherein the anti-sense DNA sequence of SEQ ID NOS 1 or 3 will be expressed at high levels in the cells of 20 mammals In a more preferred embodiment, the DNA sequence consists essentially of SEQ ID NOS 1 or 3. In a further preferred embodiment, the expression vector comprises an adenoviral vector in which cDNA PD-ABC is operatively linked in an anti-sense orientation for a CMV promoter for the constitutive expression of the anti-sense cDNA PD-ABC in a host cell. In a preferred embodiment, the adenoviral expression vector PD-ABC is introduced into the cells by injection into a mammal.

Another aspect of the invention is to provide useful tests to determine if a compound of interest can bind to PD-ABC proteins. This link may interfere with or resemble, the binding of ligands for ABC1 or this link may affect the function of PD-ABC in transport substrates through the membranes or the modulation of cholesterol expiration. The test comprises the steps of measuring the binding of a compound of interest to a PD-ABC protein. Neither the PD-ABC protein nor the compound of interest to be tested can be labeled with a detectable marker, i.e., a fluorescent or radioactive label, so as to provide detection of complex formation between the compound of interest and the PD- protein. ABC. In another modality of the object tests, the tests will involve the measurement of the interference, that is, the competitive binding of a compound of interest with the binding interaction between PD-ABC proteins and a ligand already known to bind the ABC1 protein. . For example, the effect of the amounts of increase of a compound of interest in the formation of complexes between the labeled ligand of radioactivity and a PD-ABC protein can be measured by quantifying the formation of the PD-ABC protein complex formation of the labeled ligand. In another modality of the subject tests, the tests involve the measurement of the alteration, that is to say the noncompetitive inhibition of a compound of interest with the activity of the PD-ABC proteins.

Polyclonal antibodies to PD-ABC proteins are generally raised in animals by intraperitoneal (ip) or subcutaneous (se) injections of a PDABC protein and an adjuvant. It may be useful to conjugate the PD-ABC protein or a fragment containing the target amino acid sequence for a protein that is immunogenic in the species to be immunized, i.e. keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or inhibitor of soybean trypsin using a bi-functional or derivatizing agent, for example maleicidobenzoyl sulfosuccinamide ester (conjugation through cistern residues), N-hydroxysuccinamide (via lysine residues), glutaraldehyde, succinic anhydride, SOCI2 or Ri- NsCsNR, wherein R and Ri are different alkyl groups.

The animals were immunized against the immunogenic conjugates or derivatives by combining 1 mg or 1 fig of the conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally into the sites multiple One month later the animals were augmented with 1/5 to 1/10 of the original amount of the conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals were bled and the serum was tested for the solution concentration of the anti-PD-ABC protein antibody. The animals were increased until reaching the concentration in solution. Preferably, the animal is augmented with the conjugate of the same PD-ABC protein, but it can also be conjugated to a different protein and / or through a different cross-linking reagent. The conjugates can also be made in the recombinant cell culture as protein fusions. Also the aggregation agents such as alum that are used to improve the immune response.

The monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population that are identical except for possible naturally occurring mutations that may occur in minor amounts. In addition, the "monoclonal" modifier indicates the character of the antibody as not being a mixture of discrete antibodies. For example, the monoclonal antibodies of the anti-PD-ABC protein of the invention can be made using the hybridoma method first described by Kohier & Milstein, Nature, 1975; 256: 495 or can be made by recombinant DNA methods (Cabilly et al., US Patent No. 4,816,567).

The antibodies can also be generated using the phage display. In this advancement, libraries of random sequence peptides are generated in the antibody genes cloned within the phage. These phage libraries are analyzed for the antibodies by analysis against the immobilized protein. (Hoogenboom-HR, Trends-Biotechnol., 1997; 15 (2): 62-70).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, hybridizes as described above to cause the lymphocytes to be produced or capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, the lymphocytes can be immunized in vitro. The lymphocytes subsequently focus with the myeloma cells using an appropriate fusion agent, such as polyethylene glycol to form a hybridoma cell (CODIG, Monoclonal Antibodies: Principles and Practice, pp. 59-103 [Academic Press, 1986]).

The antibodies specific for the PD-ABC protein of the invention have a number of uses. The antibodies can be used to purify PD-ABC proteins from recombinant and non-recombinant cells. The subject antibodies can be used to detect and / or quantitate the presence of PD-ABC proteins in tissue samples, ie blood, skin and the like. The quantification of PD-ABC proteins can be used in diagnostic form for those diseases and genetic or physiological conditions that have been correlated with the particular levels of expression levels of the PD-ABC protein.

In a future aspect, the present invention provides a diagnostic test for detecting cells containing the PD-ABC omissions, comprising the isolation of the total genomic DNA from the cell and subjecting the genomic DNA for PCR amplification using primers derived from the DNA sequence of SEQ ID NOS 1 or 3.

This aspect of the invention enables the detection of omissions of PD-ABC in any cell type and can be used in the genetic examination or as a laboratory tool. The PCR primers can be selected in a manner that allows the amplification of a sufficiently long PD-AABC gene fragment to be detected by gel electrophoresis.

Detection can be done by any method, including, but not limited to, dialing ethidium bromide with polyacrylamide or agarose gels, autoradiographic detection of the radiolabelled PD-ABC gene fragments, Southern-label hybridization and sequence analysis. DNA In a preferred embodiment, the detection is carried out by electrophoresis in polyacrylamide gel, followed by DNA sequence analysis to verify the identity of the omissions. PCR conditions are routinely determined based on the length and base content of the primers selected in accordance with techniques well known in the art (Sambrook, et al., 1989).

A further aspect of the present invention provides a diagnostic test for detecting cells that contain omissions of PD-ABC, comprising the isolation of total cellular RNA and subjecting the RNA to PCR amplification of reverse transcription using primers derivatives of the DNA sequence of SEQ ID NOS 1 or 3. This aspect of the invention enables the detection of omissions of PD-ABC in any type of cell and can 15 used in genetic testing or as a laboratory tool.

Reverse transcription is carried out routinely via standard techniques (Ausubel, et al., In Current Protocols in Molecular Viology, ed., John Wiley and Sons, Inc. 1994) and PCR is carried out as described above. The present invention can be understood with reference to the following examples which are intended for purposes of illustration only and should not be construed to limit the scope of the invention, as defined by the appended claims. 25 MATERIALS AND METHODS Sequence Analysis TBLASTN investigations using the ABCR and ABC1 protein sequences as questions were conducted against a branched EST database generated using the -rrrriwftei iiiMltf j? i r - • a ^^ aá .. .. ..

Compugen LEADS ™ platform. A single branching EST consisting of four ESTs was identified (access numbers AI733552, H21585). The 4 ESTs represent two different isoforms of PD-ABC. The BLASTN investigations of the high-throughput genomic sequence (HTG) division of the Genbank ™ database lead to the identification of an HTG sequence (accession number AC011558) containing the entire coding region of PD-ABC.

Isolation of the cDNAs for PD-ABC Two ESTs (I.M.A.G.E. # 160038 and I.M.A.G.E. # 182933) in the database containing partial open reading frames that share significant homology with human ABC1 after translation. The ESTs were obtained from ATCC and completely sequenced. The ESTs have insertion sizes of 1.2 kb (I.MA.G.E. # 160038) and 1.1 kb (I.MA.G.E. # 182933), respectively. The two clones are identical in their overlap region of 1 kb. Using a region that is common for both ESTs as a probe, the cDNA clones were isolated from a mixture of three human cDNA libraries: adult brain, skeletal muscle and mammary glands (EdgeBiosystems, Gaithersburg, MD). DNA sequencing was carried out with synthetic and universal primers. PCR amplification of the 5 'cDNA ends.

The largest clone was sequenced and found to be missing at the 5 'end. We obtained the database investigations that generated the ramifications of the high-throughput genomic sequence that share identity with the cDNA sequence. Three pairs of primers were synthesized based on the high-throughput genomic sequences. These areas of primer pairs are as follows: 1. forward primer, 5'-TCTCACCATGGCCTTCTGGACACAG-3 'reverse primer, 5'-CACGTAGCGCAGGTCGGTCAGGG-3' 2. forward primer, 5'-GCTGATTGGAGCCCTGGACAGCCA-3 'reverse primer, 5' - GTCCACATAGCACGGATAGGGCAT-3 '3. forward primer, 5' - TCGTGTACCTGCAAGACCTGGTG-3 tmm H? .d.? j. tAl? - 1. ... M. ^ í ...,. ,., -, ^ «fa. - ^ «- ^ - ^^ -»? M. «* -« MfcH * ^ »rt 111 j '| | tjA || l || [¡| i i í reverse primer, 5 '- CAGAGCCAGGCTCTCGCAGCC-3' PCR reactions were carried out with the human pituitary gland or cDNAs from Marathon-Ready of the thymus (Clontech). The reaction was started with an initial denaturation of 5 minutes at 94 ° C, followed by 28 cycles of 30 seconds at 94 ° C, 2 minutes at 60 ° C, 2 minutes at 72 ° C with a final extension of 10 minutes at 72 ° C. ° C. The PCR reactions with three pairs of primers generated three bands with the following sizes, respectively: 1.8 kb, 800 bp and 600 bp. The PCR products were ligated into the pCRII-TOPO vector (invitrogen) and sequenced with the synthetic and universal primers.

Northern Hybridization Analysis A labeled digoxigenin (DIG) probe for Northern hybridization was generated using a PCR labeling kit (Boehringer Mannheim) with primers based on the sequence of a partial PD-ABC cDNA fragment. The forward primer was 5'-CAGCTTCACTCTTGTCCTCATTGAG-3 'and the reverse primer was 5'- TTTATGCAGGTGAGCACCACATAG-3'. The 262 bp of the PCR product was the PD-ABC partial cDNA fragment or the human spleen cDNA (Clontech). The main tissue spot 12 (Origine, Rockville, MD) and a main spot of tissue 6 (Clontech) were hybridized with the probe and developed in accordance with the manufacturer's instruction (Boehringer Mannheim). The same spots were cut and hydrated with a DIG-labeled β-actin or GAODH probe for the purpose of control.

Tissue Distribution by RT-PCR Tissue-specific expression of the two PD-ABC variants was carried out by the reverse transcription polymerase chain reaction (hereinafter "RT-PCR"). The Rapid Analysis Gene Expression Panels (Origene) were used as PCR templates. The specific primers for Form 1 are 5'- CCCCTCTTCCTTCTCTTCACACTAC-3 '(front starter) and 5'- ^ mmmm * tSmjAft *. my tf, .mtm £? '"i .m * ?? ~? É *.? T,.

AGCAGCCCAAAACACTCACCAC-3 '(reverse primer); Specific initiators for Form 2 are 5'-TGGGAGAGGACGAGGATGTAG-3 '(forward primer) and 5'- AGGTGTTCAGTAAAGGATGATGGG-3' (reverse primer). The PCR reaction was carried out with 35 cycles as follows: 95 ° C, 1 minute, 62 ° C, 1 minute, 72 ° C 1 minute. PCR products were separated on 1% NuSieve gels (FMC).

RESULTS AND DISCUSSION Isolation and primary structure of PD-ABC ABC1 is a member of the ABCA subfamily (Broccardo, C. et al., Biochim, Biophys.

Acta, 1999; 1461: 395-404) and associated with TD. Recent pharmacological studies show that ABC1 is responsible for cholesterol and transport of phospholipids (Lawn, R.M., et al., J Clin Invest. 104, R25-31, 1999). Two members of the additional ABCA subfamily, ABC2 and ABCR, have been described (Luciani, MF., Et al., EMBO J., 1996; 15: 226-235; Allikmets, R., et al., Nat. Genet. , 1997; 15: 236-246). The functions of ABCR and ABC2 are unknown although ABCR has been proposed as a flipase for N-retinylidene-phosphatidylethanolamine (Weng, J., et al., Cell, 1999; 98: 13-23). To look for other ABC1 homologs, especially those that are also involved in cholesterol metabolism, the databases of the novel sequences that share the homology with ABC1 and ABCR were investigated. The two overlaps of the ESTs were identified that contained a partial open reading frame. 80% of the 5 'end of the open reading frame is similar with ABC1. While 20% of the 3 'end of the open reading frame does not share any homology with ABC1. In addition, this open reading frame does not contain the corresponding NBF as predicted in ABC1 (Bodzioch, M., et al., Nature Genetics, 1999; 22: 347-351; Brooks-Wilson, A., et al., Nature Genetics, 1999; 22: 336-345; Rust, S., et al., Nature Genetics, 1999; 22: 352-355).

Using a probe based on the homologous sequence of ABC1, the cDNA clones were obtained from the cDNA libraries. The largest clone was sequenced and is identical to the EST sequence TO ? . * m *. t- .1 *., .. «itaj.t ^^ tuAUL ^ A. ^, .. t» ^. tf3it ^ ... M ... Ui. ^. ^ < ..- m., m ?? tMm ^ ?. -M & S at the 5 'end. Interestingly, the 3 'end of this clone is different from that of the EST. In addition, this clone contains the second NBF after translation and the complete amino acid sequence shows homology with ABC1. It was predicted that there are two forms of PDABC that originate the alternating union. The cDNA clone represents Form 1, which contains the second NBF; the EST represents Form 2, which lacks the second NBF. The alternative binding site shown in Figure 2 was identified and the sizes of the introns and exons as well as the intron-exon binding areas are shown in Tables 1 and 2.

N > n o I heard Oí Table 1 Exon number Exon size Intron size Binding site 5"Binding site 3 'Position, bp 1 67 gacagccggt AACGCCCCAG 68 2 95 80 CTCCCCGCAG ggtccagctc accatgaatg GTGAGCCAGA 161 3 142 227 TGGCTTGTTT gccacttccc cgactccctg TGAGCCAGAG 303 4 112 91 CTGGGGACAG ggtctcccgg cgcagcacgg GTGAGGAGGC 415 5 84 138 TCCTCCCCCA gcccagcctc ctgcgcacgg TAGGGTGTCG 499 6 81 347 TTTCTCCCCA ggaatccctg ggcccaggag GTACGAGGCC 579 7213213 ACCTCTCTCT agctcctggc gcagcctgag TGAGTGACTG 791 8142 82 CCCATCCCAG gccccgcctg ggcccaggtg GGGGCAGCCT 933 9119246 GTGCTGTCCA caggtgaacc catgctgcag CCCCGCACAC 1047 10170733 CTGCGCCCCC cagcggctcc agtgacggag AGGCCCTCAC GTGGGATGCG 1215 11 233 256 1447 • agtgcctgtc agggacaggt CAGGCGAGGG í- 12181994 GCCCCTCTCG caggttttgg gacgacgtgt GCCAGAGCTC 1624 13227394 CCCACCCCAG gttcctgcgt tcaagctggg CGCGCCTCGG 1850 14223126 TGCCTCTCAC agctggggga cgtggccgcg GTGAGAGCCG 2067 15203 74 CTTTTCCGCA gagcctgctg tgtgcccagg TGGGCCGATG 2270 16113 1237 CCCCTC CCCG caggccagta gacccaaagg GTGAGGCACT 2380 17 174 260 CATCTCTGCA gtgctggtag accaccctgt GAGCCCCCAA 2554 18 132 undet. CTATCCACAG gtccatcttg gtttgacatg TGCGTCTCGG 2685 19 142 102 CTGGCCGCAG gctgaccgtg ctctctggtg AGCCCATCCC 2827 20 140 151 ATCTCTACCA ggtgggatgc ccgagaaggt AAGAGCTGGG 2964 21 187 352 CCCCGTGCCT aggtcgcacg aatgagaagg TGGGGACCGG 3148 22 75 85 CTCTGCCCGC aggctgacac gcagagtcgg TGAGGGCCGG 3221 23203 undet. TGTCCACACA ggcactcctc cctcgaggag GTGTGAGGCC 3423 NJ C? O Ul Ül Table 1 (continued) Exon No. Exon size Intron size Binding site 5 'Binding site 3' Position, bp 24 50 255 GTCTCCCCAG atcttcctga gatatggagg GTGCGGCCAC 3472 25 105 169 TGCAGGGCCA gatggcagct ggggaaccag GTAAGTCCTT 3577 26 151 80 TCATCCCTCA cagctgggtc gttcgcccag GTGAGGAGGG 3726 27 129 224 CTGCACCCAC cccagatcgt ccttcttcag GTGGGTGCAG 3851 28 103 83 CCCTCACACA cagtgaggac tcccacaggt GAGGCGTCTT 3952 29257214 CCATTGTCTG caggttctcg tgcgccaggg GTGAGCCATG 4205 30 35555 CTCTCCCACA gcctgaagac gaggtcaggt GAGGAGGGGT 4240 31176126 GCCCCACAGA tacggaggct cagtctcaag GTGGGAACTG 4416 32172 85 ACCCCCACCC agatctggtt ggctgcactg GAGGGACTCA 4587 33 177 407 TCACCTCCAG gatggcctcc ctgggacatg TGCGGGGGCG 4764 34 116 229 CTCCAATGCA gtgtaactac tactgtatgg GTGAGGCCCC 4880 *. 35145485 GCCTGTGCCC gctggtcgat ctctgatcag ül GTGGGGCACC 5025 36 127 84 CACCCTTGAG cagaagctgc agcgcttggg GAGGCGGAGG 5150 37131347 TGTGGATATA ggagacaggc cctgccacag TTAGTGAGGT 5280 38123 72 CTGTCCCCAG gcccagggtg gaccaaggta GGTGTGGTCA 5403 39 64 78 GCACTCTCCC aggtataccg ccctggtgag GTGAGTCCAG 5463 40108 2695 GCATCCCTGT agtgttttgg caggccacag GTGAGGGGTG 5570 41 144281 TGTCCCTTAT cagcgtggcc ggttgcccag GTGAGCCCAC 5712 42138 1228 5848 CCCCACCCCA cagaccgctg gtgtttctgg TGCGTGGGAG 43104 80 GTGCTCCCCA ggacgagccg cctcccatag GTGGGCCGGG 5951 44 94 296 6044 GGGTCCCGAC agcatggagg tcaagggcag GTGAGCCGGC 45246674 CCCACTCACT gcagattcgc tggaggaggt GATCACGGCG 6287 46232 95 6516 GCCCACCGCT aggtattctt tangagccct GGACTCAGGC NJ NJ ül O l or ül - • Table 2 Exon No. Exor size i Intron size Joint site 5 'Joint site 3' Position, Bp January 67 gacagccggt AACGCCCCAG 68 2 95 80 CTCCCCGCAG ggtccagctc accatgaatg GTGAGCCAGA 161 3142227 TGGCTTGTTT gccacttccc cgactccctg TGAGCCAGAG 303 4112 91 CTGGGGACAG ggtctcccgg cgcagcacgg GTGAGGAGGC 415 5 84 138 TCCTCCCCCA gcccagcctc ctgcgcacgg TAGGGTGTCG 499 6 81 347 TTTCTCCCCA ggaatccctg ggcccaggag GTACGAGGCC 579 7213213 ACCTCTCTCT agctcctggc gcagcctgag TGAGTGACTG 791 8142 82 933 CCCATCCCAG gccccgcctg ggcccaggtg GGGGCAGCCT 9,119,246 GTGCTGTCCA caggtgaacc catgctgcag CCCCGCACAC 1047 10170733 CTGCGCCCCC cagcggctcc agtgacggag AGGCCCTCAC 1215 11233256 GTGGGATGCG agtgcctgtc agggacaggt CAGGCGAGGG 1447 12181994 1624 GCCCCTCTCG caggttttgg gacgacgtgt GCCAGAGCTC O) 13227394 CCCACCCCAG gttcctgcgt tcaagctggg CGCGCCTCGG 1850 14223126 TGCCTCTCAC agctggggga cgtggccgcg GTGAGAGCCG 2067 74 15 203 C1 1 January 1CCGCA gagcctgctg tgtgcccagg TGGGCCGATG 2270 1237 16113 2380 CCCCTCCCCG caggccagta gacccaaagg GTGAGGCACT 17174260 CATCTCTGCA gtgctggtag accaccctgt GAGCCCCCAA 2554 18132 undet. CTATCCACAG gtccatcttg gtttgacatg TGCGTCTCGG 2685 19 142 102 CTGGCCGCAG gctgaccgtg ctctctggtg AGCCCATCCC 2827 20 140 151 ATCTCTACCA ggtgggatgc ccgagaaggt AAGAGCTGGG 2964 21 187 352 CCCCGTGCCT aggtcgcacg aatgagaagg TGGGGACCGG 3148 22 75 85 CTCTGCCCGC aggctgacac gcagagtcgg TGAGGGCCGG 3221 23203 undet. TGTCCACACA ggcactcctc cctegaggag GTGTGAGGCC 3423 24 50 255 GTCTCCCCAG atcttcctga gatatggagg GTGCGGCCAC 3472 25 105 169 TGCAGGGCCA gatggcagct GTAAGTCCTT 3577 NJ NJ ül O ül üi Table 2 (continued) Exon No. Exon size Intron size Binding site 5 'Binding site 3' Position, bp 26 151 80 TCATCCCTCA cagctgggtc gttcgcccag GTGAGGAGGG 3726 27 129 224 CTGCACCCAC cccagatcgt ccttcttcag GTGGGTGCAG 3851 28 103 83 CCCTCACACA cagtgaggac tcccacaggt GAGGCGTCTT 3952 29 257 214 CCATTGTCTG caggttctcg tgcgccaggg GTGAGCCATG 4205 30 35555 CTCTCCCACA gcctgaagac gaggtcaggt GAGGAGGGGT 4240 31176126 GCCCCACAGA tacggaggct cagtctcaag GTGGGAACTG 4416 32172 85 ACCCCCACCC agatctggtt ggctgcactg GAGGGACTCA 4587 33177407 TCACCTCCAG gatggcctcc ctgggacatg TGCGGGGGCG 4764 34116229 CTCCAATGCA gtgtaactac tactgtatgg GTGAGGCCCC 4880 35 145 485 GCCTGTGCCC gctggtcgat ctctgatcag GTGGGGCACC 5025 -? 36 127 84 CACCCTTGAG cagaagctgc agcgcttggg GAGGCGGAGG 5150 37 131 347 TGTGGATATA ggagacaggc cctgccacag TTAGTGAGGT 5280 38a 123 72 CTGTCCCCAG gcccagggtg 48 Using a variety of techniques (Materials and Methods), the full-length PD-ABC coding region was obtained. The full-length PD-ABC contains an open reading frame of 2146 amino acids and is a typical ABC transporter. The PD-ABC is currently the closest ortholog to ABC1 in the public database. The sequence was aligned with ABC1 and ABCR (Figure 1). The homology between PD-ABC and ABC1 is 66%. The regions Most conserved PD-ABC alignment corresponds to the nucleotide and trans-membrane binding domains.

The existence of the two PD-ABC variants is interesting, especially in that Form 2 does not contain the second NBF (Figure 2). In ABC transporters, NBFs are required for ATP binding and hydrolysis, which provides energy to transport the substrates. The lack of the second NBF in other ABC transporters usually res in a dysfunctional transporter. In certain TD patients, nonsense mutations or omissions in the C-terminus of ABC1 are responsible for the loss of cholesterol expiration, suggesting that the second 15 NBF is required for transport activity. The loss of the second NBF in Form 2, the PDABC can resin a transporter that has no activity. This conveyor, however, may still be capable of bonding substrates and therefore serves as a transport regulator by competing substrates with the Form 1 conveyor. Alternatively, the first NBF must be essential to provide energy for transport 20 as in the ABC half-life transporters such as ABC8 (Klucken, J., et al., Proc. Nati, Acad. Sci. USA., 2000; 97: 817-822). pseudogen The genomic sequences for PD-ABC were divided from the human chromosome 19p13.3.

By aligning the cDNA sequence of PD-ABC for the genomic sequences, they were able to determine the limits of the exon-intron of the gene (Figure 2B). The coding region of PD-ABC Form 1 was contained in 47 exons and covered 20 kb of the genomic sequence (Figure 2). Form 2 of PD-ABC uses an alternative polyadenylation signal function found in intron number 38. This res in a termination of the PD-ABC transcript. The intron / exon boundaries of PD-ABC. Interestingly, the intron / exon structure of PD-ABC is highly similar to that of ABC1 and ABCR (data not shown).

PD-ABC tissue distribution The tissue distribution of PD-ABC is examined by Northern hybridization analysis using a common probe for both Form 1 and Form 2. A band with a size between 8 and 9 kb was observed (Figure 3A ). Transcription was only detected in the spleen, suggesting that PD-ABC is expressed specifically in the spleen. No expression was observed in other tissues examined, including brain, heart, lung, liver and muscles. The same spot was further hybridized with a GAPDH probe to show that the specific expression of the spleen is not a result of uneven loading of mRNA samples.

The specific expression of the spleen forced to examine the expression of PD-ABC in the cells or tissues of the immune system. Indeed, it was found that PD-ABC is widely expressed in the tissues of the immune system tested, including the lymph node, the thymus, the peripheral blood leukocytes, the bone marrow and the fetal liver (Figure 3B). Interestingly, there are two bands in the leukocytes of peripheral blood and fetal liver. The two transcripts are expressed almost equally in both tissues, while the minor message is only expressed slightly in the bone marrow. The two transcripts can represent the two identified variants (Figure 3A). To further assess PD-ABC expression in various tissues in a In a broader scope, point hybridization analyzes were carried out with human tissues (Table 3).

TABLE 3 Expression of PD-ABC in various human tissues evaluated by dot hybridization analysis Expression mRNA of mRNA Expression of Relative PD-ABC1 tissue Relative PD-ABC 1 Complete brain Transverse colon Brain cut Descending colon Frontal lobe Rectum Paper lobe Kidney Papetal lobe Skeletal muscle Temporal lobe Spleen P g * of the cerebral cortex Thymus Brain bridge Leukocytes of the blood pepfépca Left cerebellum Lymph node Right cerebellum Bone marrow Callous cofus Trachea Tonsils Lung Nucleus caudate Placenta Hippocampus Bladder Medulla oblongata Uterus Prostate Bone Black substance Testicles Nucleus accumbens Ovaries Thalamus Liver Pituitary gland Pancreas Spine Adrenal gland Heart Mammary gland Aorta Leukemia HL-60 Right atrium Hela S3 Left atrium Leukemia, k-562 Left ventricle Leukemia, MOLT- < * Right ventricle Burkitt lymphoma Interventicular septum Colorectal adenocarcinoma Heart apex Lung carcinoma Esophagus Fetal brain Stomach Fetal heart Duodenum Fetal kidney Jejunum Fetal fetus Fetal fetus Fetal thymus Fetal thymus Fetal lung Fetal colon Colonization by dot was densitomatically quantified and the values expressed as numbers of spots on a linear scale Consistent with Figure 3B, PD-ABC is expressed primarily in the immune system. In addition, it is also highly expressed in the pituitary gland.

The expression template of PD-ABC in the immune system suggests that PD-ABC may have a physiological role in those tissues or organs. The binding of ABC transporters to the immune system has been previously documented.

ABC1 is required to take over apoptotic cells by macrophages. In addition, there is a close correlation between interleukin-1β secretion and ABC1 activity as demonstrated in studies with ABC1 inhibitors (Harmon, Y., et al., Blood, 1997; 90: 2911-2915). These findings suggest that ABC1 must be involved in the secretion of interleukin-1β and play roles in inflammatory reactions. Given the great homology between PD-ABC1 and ABC1, the two transporters can have similar biological functions. In contrast to the ubiquitous expression of ABC1, the expression PD-ABC is almost specific in the immune system. This is a strong indication that PD-ABC is involved in certain immunological pathways.

Expression of two isoforms of PD-ABC The expression of the two variants was examined in tissues with RT-PCR. Although RT-PCR is not absolutely quantitative, the appearance of the PCR product in the templates of the different tissues may provide a general tendency of abundance in transcription. The expression of two PD-ABC variants in 24 human tissues was examined and the expression templates were found to be different. Most of the 24 tissues express both Forms 1 and 2 (Figure 4). However, the prostate and ovary express Form 1 in preference. Whereas tissues including fetal brain, skin, uterus, pancreas, adrenal gland, salivary gland and colon preferably express Form 2 (Figure 4). Interestingly, a long band was found in the bone marrow and leukocytes of the bone marrow with the specific primers APRA Form 2 (Figure 4). This suggests that there must be another form of PD-ABC without the second NBF in these tissues. The lack of the second NBF in Form 2 most likely affects the transport activity and the tissue specific expression in this way can serve as a special physiological purpose.

Conclusion A new ABC transporter, which is the closest homologue ABC1, has been identified and isolated. In addition, an alternately linked variant has been identified. The transporter is expressed mainly in the immune system and can play a role in the immune response.

In addition, the expression of the smaller alternatively linked transcript of PD-ABC is more restrictive than the original form. The tissue-specific expression template and the alternative binding of PD-ABC suggests that PD-ABC should have a similar function as ABC1, but in a more restrictive and regulated manner.

It is understood that the invention is not limited to exact details of operation or that the exact compounds, compositions, methods, procedures or modalities shown and described, such as modifications and obvious equivalents are shown and described as obvious modifications and equivalents that will be apparent to those The person skilled in the art is therefore limited only by the full scope of the following claims.

LIST OF SEQUENCES < 110 > Johns, Margaret A Tafuri, Sherrie R Wang, Minghan < 120 > GENES THAT CODIFY THE ABC1 PARALOGS AND THE POLYPEPTIDES DERIVED FROM THEMSELVES < 130 > Paralogs ABC1 that encode the Gene < 140 > < 141 > < 150 > 60 / 215,405 < 151 > 2000-06-30 < 160 > 4 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 6522 < 212 > DNA < 213 > Homo sapxens < 400 > 1 atggccttct ggacacagct gatgctgctg atttcatgta ctctggaaga tcgccggaga 60 cagccggtcc agctcctggt cgaattgctg tggcctctct tcctcttctt catcctggtg X20 gctgttcgcc actcccaccc gcccctggag caccatgaat gccacttccc aaacaagcca 180 ctgccatcgg cgggcaccgt gccctggctc cagggtctca tctgtaatgt gaacaacacc 240 tgctttccgc agctgacacc gggcgaggag cccgggcgcc tgagcaactt caacgactcc 300 ctggtctcce ggctgctagc cgatgcccgc actgtgctgg gaggggccag tgcccacagg 360 acgctggctg gcctagggaa gctgatcgcc acgctgaggg ctgcacgcag cacggcccag 420 cctcaaccaa ccaagcagtc tccactggaa ccacccatgc tggatgtcgc ggagctgctg 480 acgtcactgc tgcgcacgga atccctgggg ttggcactgg gccaagccca ggagcccttg 540 cacagcttgt tggaggccgc tgaggacctg gcccaggagc tcctggcgct gcgeagcctg 600 gtggagcttc gggcactgct gcagagaccc cgagggacca gcggccccct ggagttgctg 660 tcagaggccc tctgcagtgt caggggacct agcagcacag tgggcccctc cctcaactgg 720 tacgaggcta gtgacctgat ggagctggtg gggcaggagc cagaatccgc cctgccagac 780 agcagcctga gccccgcctg ctcggagctg attggagccc tggacagcca cccgctgtcc 840 cgcc tgctct ggagacgcct gaagcctctg atcctcggga agctactctt tgcaccagat 900 acacctttta cccggaagct catggcccag gtgaaccgga ccttcgagga gctcaccctg 960 ctgagggatg tccgggaggt gtgggagatg ctgggacccc ggatcttcac cttcatgaac 1020 gacagttcca atgtggccat gctgcagcgg ctcctgcaga tgcaggatga aggaagaagg 1080 cagcccagac ctggaggccg ggaccacatg gaggccctgc gatcctttct ggaccctggg 1140 agcggtggct acagctggca ggacgcacac gctgatgtgg ggcacctggt gggcacgctg 1200 ggccgagtga cggagtgcct gtccttggac aagctggagg cggcaccctc agaggcagcc 1260 n..jam,? a, -a .1. « ofcggtgtcgc gggccctgca actgctcgcg gaacatcgat tctgggccgg cgtcgtcttc 1320 ttgggacctg aggactcttc agaccccaca gagcacccaa ccccagacct gggccccggc 1380 cacgtgcgca tcaaaatccg catggacatt gacgtggtca cgaggaccaa taagatcagg 1440 gacaggtttt gggaccctgg cccagccgcg gaccccctga ccgacctgcg ctacgtgtgg 1500 ggcggcttcg tgtacctgca agacctggtg gagcgtgcag ccgtccgcgt gctcagcggc 1560 gccaaccccc gggccggcct ctacctgcag cagatgccct atccgtgcta tgtggacgac 1620 gtgttcctgc gtgtgctgag ccggtcgctg ccgctcttcc tgacgctggc ctggatctac 1680 tccgtgacac tgacagtgaa ggccgtggtg cgggagaagg agacgcggct gcgggacacc 1740 atgcgcgccg tggggctcag ccgcgcggtg ctctggctag gctggttcct cagctgcctc 1800 gggcccttcc tgctcagcgc cgcgctgctg gttctggtgc tcaagctggg ggacatcctc 1860 ccctgcagcc acccgggcgt ggtcttcctg ttcttggcag ccttcgcggt ggccacggtg 1920 acccagagct tcctgctcag cgccttcttc tcccgcgcca acctggctgc ggcctgcggc 1980 ggcctggcct acttctccct ctacctgccc tacgtgctgt gtgtggcttg gcgggaccgg 2040 ctgcccgcgg gtggccgcgt ggccgcgagc ctgctgtcgc ccgtggcctt cggcttcggc 2100 tgcgagagcc tggctctgct ggaggagcag ggcgagggcg cgcagtggca caacgtgggc 2160 acccggccta cggcagacgt cttcagcctg gcccaggtct ctggccttct gctgctggac 2220 gcggcgctct acggcctcgc cacctggtac ctggaagctg tgtgcccagg ccagtacggg 2280 atccctgaac catggaattt tccttttcgg aggagctact ggtgcggacc tcggcccccc 2340 aagagtccag ccccttgccc caccccgctg gacccaaagg tgctggtaga agaggcaccg 2400 cccggcctga gtcctggcgt ctccgttcgc agcctggaga agcgctttcc tggaagcccg 2460 cagccagccc tgcgggggct cagcctggac ttctaccagg gccacatcac cgccttcctg 2S20 ggccacaacg gggccggcaa gaccaccacc ctgtccatct tgagtggcct cttcccaccc 2580 agtggtggct ctgccttcat cctgggccac gacgtccgct ccagcatggc cgccatccgg 2640 ccccacctgg gcgtctgtcc tcagtacaac gtgctgtttg acatgctgac cgtggacgag 2700 cacgtctggt tctatgggcg gctgaagggt ctgagtgccg ctgtagtggg ccccgagcag 2760 gaccgtctgc tgcaggatgt ggggctggtc tccaagcaga gtgtgcagac tcgccacctc 2820 tgcaacggaa tctggtggga gctgtccgtg gccattgcct ttgtgggcgg ctcccaagtt 2880 gttatcctgg acgagcctac ggctggcgtg gatcctgctt cccgccgcgg tatttgggag 2940 c tgctgctca aataccgaga aggtcgcacg ctgatcctct ccacccacca cctggatgag 3000 gcagagctgc tgggagaccg tgtggctgtg gtggcaggtg gccgcttgtg ctgctgtggc 3060 tccccactct tcctgcgccg tcacctgggc tccggctact acctgacgct ggtgaaggcc 3120 cgcctgcccc tgaccaccaa tgagaaggct gacactgaca tggagggcag tgtggacacc 3180 aggcaggaaa agaagaatgg cagccagggc agcagagtcg gcactcctca gctgctggcc 3240 ctggtacagc actgggtgcc cggggcacgg ctggtggagg agctgccaca cgagctggtg 3300 ctggtgctgc cctacacggg tgcccatgac ggcagcttcg ccacactctt ccgagagcta 3360 gacacgcggc tggcggagct gaggctcact ggctacggga tctccgacac cagcctcgag 3420 gagatcttcc tgaaggtggt ggaggagtgt gctgcggaca cagatatgga ggatggcagc 3480 tgcgggcagc acctatgcac aggcattgct ggcctagacg taaccctgcg gctcaagatg 3S40 ccgccacagg agacagcgct ggagaacggg gaaccagctg ggtcagcccc agagactgac 3600 cagggctctg ggccagacgc cgtgggccgg gtacagggct gggcactgac ccgccagcag 3660 ctccaggccc tgcttctcaa gcgctttctg cttgcccgcc gcagccgccg cggcctgttc 3720 gcccagatcg tgctgcctgc cctctttgtg ggcctggccc tcgtgttcag cctcatcgtg 3780 cctcctt tcg ggcactaccc ggctctgcgg ctcagtccca ccatgtacgg TGCT aggtg 3840 tccttcttca gtgaggacgc cccaggggac cctggacgtg cccggctgct cgaggcgctg 3900 caggactgga ctgcaggagg gtgcagcata ggagccccca gttctcggca gctcccacag 3960 ccagaagttc ctgctgaagt ggccaaggtc ttggccagtg gcaactggac cccagagtct 4020 ccatccccag cctgccagtg tagccagccc ggtgcccggc gcctgctgcc cgactgcccg 4080 gctgcagctg gtggtccccc tccgccccag gcagtgaccg gctctgggga agtggttcag 4140 aacctgacag gccggaacct gtctgacttc ctggtcaaga cctacccgcg cctggtgcgc 4200 agactaagaa cagggcctga gtgggtgaat gaggtcaggt acggaggctt ctcgctgggg 4260 ggccgagacc caggcctgcc ctcgggccaa gagttgggcc gctcagtgga ggagttgtgg 4320 gcgctgctga gtcccctgcc tggcggggcc ctcgaccgtg tcctgaaaaa cctcacagcc 4380 tgggctcaca gcctggatgc tcaggacagt ctcaagatct ggttcaacaa caaaggctgg 4440 cactccatgg tggcctttgt caaccgagcc agcaacgcaa tcctccgtgc tcacctgccc 4500 ccaggcccgg cccgccacgc ccacagcatc accacactca accacccctt gaacctcacc 4560 aaggagcagc tgtctgaggc tgcactgatg gcctcctcgg tggacgtcct cgtetccatc 4620 tgtgtggtct ttgccatgtc ctttgtcccg gccagcttca ctcttgtcct cattgaggag 4680 cgagtcaccc gagccaagca cctgcagctc atggggggcc tgtcccccac cctctactgg 4740 cttggcaact ttctctggga catgtgtaac tacttggtgc cagcatgcat cgtggtgctc 4800 atctttctgg ccttccagca gagggcatat gtggcccctg ccaacctgcc tgctctcctg 4860 ctgttgctac tactgtatgg ctggtcgatc acaccgctca tgtacccagc ctccttcttc 4920 ttctccgtgc ccagcacagc ctatgtggtg ctcacctgca taaacctctt tattggcat c 4980 aatggaagca tggccacctt tgtgcttgag ctcttctctg atcagaagct gcaggaggtg 5040 agccggatct tgaaacaggt cttccttatc ttcccccact tctgcttggg ccgggggctc 5100 attgacatgg tgcggaacca ggccatggct gatgcctttg agcgcttggg agacaggcag 5160 ttccagtcac ccctgcgctg ggaggtggtc ggcaagaacc tcttggccat ggtgatacag 5220 gggcccctct tccttctctt cacactactg ctgcagcacc gaagccaact cctgccacag 5280 cccagggtga ggtctctgcc actcctggga gaggaggacg aggatgtagc ccgtgaacgg 5340 gagcgggtgg tccaaggagc cacccagggg gatgtgttgg tgctgaggaa cttgaccaag 5400 gtataccgtg ggcagaggat gccagctgtt gaccgcttgt gcctggggat tccccctggt 5460 gagtgttttg ggctgctggg tgtgaatgga gcagggaaga cgtccacgtt tcgcatggtg 5520 acgggggaca cattggccag caggggcgag gctgtgctgg caggccacag cgtggcccgg 5580 gaacccagtg ctgcgcacct cagcatggga tactgccctc aatccgatgc catctttgag 5640 ctgctgacgg gccgcgagca cctggagctg cttgcgcgcc tgcgcggtgt cccggaggcc 5700 caggttgccc agaccgctgg ctcgggcctg gcgcgtctgg gactctcatg gtacgcagac 5760 cggcctgcag gcacctacag cggagggaac aaacgcaagc tggcgacggc c ctggcgctg 5820 gttggggacc cagccgtggt gtttctggac gagccgacca caggcatgga ccccagcgcg 5880 cggcgcttcc tttggaacag ccttttggcc gtggtgcggg agggccgttc agtgatgctc 5940 gcatggagga acctcccata gtgtgaagcg ctctgctcgc gcctagccat catggtgaat 6000 gggcggttcc gctgcctggg cagcccgcaa catctcaagg gcagattcgc ggcgggtcac 6060 acactgaccc tgcgggtgcc cgccgcaagg tcccagccgg cagcggcctt cgtggcggcc 6120 gagttccctg ggtcggagct gcgcgaggca catggaggcc gcctgcgctt ccagctgccg 6180 ccgggagggc gctgcgccct ggcgcgcgtc tttggagagc tggcggtgca cggcgcagag 6240 cacggcgtgg aggacttttc cgtgagccag acgatgctgg aggaggtatt cttgtacttc 6300 tccaaggacc aggggaagga cgaggacacc gaagagcaga aggaggcagg agtgggagtg 6360 caggcctgca gaccccgcgc gcaccccaaa cgcgtcagcc agttcctcga tgaccctagc 6420 actgccgaaa ctgtgctctg agcctccctc ccttgcgggg cccgcggggg aggccctggg 6480 gaatggcaag ggcaaggtaa aatgcctang agcccttgaa tt 6522 < 210 > 2 < 211 > 2146 < 212 > PRT < 213 > Homo sapiens < 400 > 2 j, it..í, * .í.i ¿. garH-. * i, .fefc Met Wing Phe Trp Thr Gln Leu Met Leu Leu Leu Trp Lys Asn Phe Met 1 5 10 15 Tyr Arg Arg Arg Gln Pro Val Gln Leu Leu Val Glu Leu Leu Trp Pro 20 25 30 Leu Phe Leu Phe Phe He Leu Val Wing Val Arg His Ser üxs Pro Pro 35 40 45 Leu Glu His His Glu Cys Has Phe Pro Asn Lys Pro Leu Pro Be Wing 50 55 60 Gly Thr Val Pro Trp Leu Gln Gly Leu He Cys Asn Val Asn Asn Thr 65 70 75 80 Cys Phe Pro Gln Leu Thr Pro Gly Glu Glu Pro Gly Arg Leu Ser Asn 85 90 95 Phe Asn Asp Ser Leu Val Ser Arg Leu Leu Ala Asp Ala Arg Thr Val 100 105 110 Leu Gly Gly Wing Being Wing His Arg Thr Leu Wing Gly Leu Gly Lys Leu 115 120 125 He Wing Thr Leu Arg Wing Wing Arg Being Thr Wing Gln Pro Gln Pro Thr 130 135 140 Lys Gln Pro Pro Leu Glu Pro Pro Met Leu Asp Val Wing Glu Leu Leu 145 150 155 160 Thr Ser Leu Leu Arg Thr Glu Ser Leu Gly Leu Ala Leu Gly Gln Wing 165 170 175 Gln Glu Pro Leu His Ser Leu Leu Glu Wing Wing Glu Asp Leu Wing Gln 180 185 190 Glu Leu Leu Ala Leu Arg Ser Leu Val Glu Leu Arg Ala Leu Leu Gln 195 200 205 Arg Pro Arg Gly Thr Ser Gly Pro Leu Glu Leu Leu Ser Glu Ala Leu 210 215 220 Cys Ser Val Arg Gly Pro Ser Ser Thr Val Gly Pro Ser Leu Asn Trp 225 230 235 240 Tyr Glu Wing Being Asp Leu Met Glu Leu Val Gly Gln Glu Pro Glu Ser 245 250 255 I * * * 4 - * -. 4 * Í * Í..l. *,. l M * ^? . - > »A- < - m «- ^ J» ^^ .. ^ .. .r. ^^ ....

Met Wing Phe Trp Thr Gln Leu Met Leu Leu Leu Trp Lys Asn Phe Met 1 5 10 15 Tyr Arg Arg Gln Pro Val Gln Leu Leu Val Glu Leu Leu Trp Pro 20 25 30 Leu Phe Leu Phe Phe He Leu Val Wing Val Arg His Ser His Pro Pro 35 40 45 Leu Glu His His Glu Cys His Phe Pro Asn Lys Pro Leu Pro Be Ala 50 55 60 Gly Thr Val Pro Trp Leu Gln Gly Leu He Cys Asn Val Asn Asn Thr 65 70 75 80 Cys Phe Pro Gln Leu Thr Pro Gly Glu Glu Pro Gly Arg Leu Ser Asn 85 90 95 Phe Asn Asp Ser Leu Val Ser Arg Leu Leu Ala Asp Ala Arg Thr Val 100 105 110 Leu Gly Gly Wing Being Wing His Arg Thr Leu Wing Gly Leu Gly Lys Leu 115 120 125 He Wing Thr Leu Arg Wing Wing Arg Ser Thr Wing Gln Pro Gln Pro Thr 130 135 140 Lys Gln Ser Pro Leu Glu Pro Pro Met Leu Asp Val Wing Glu Leu Leu 145 150 155 160 Thr Ser Leu Leu Arg Thr Glu Ser Leu Gly Leu Ala Leu Gly Gln Ala 165 170 175 Gln Glu Pro Leu His Ser Leu Leu Glu Wing Wing Glu Asp Leu Wing Gln 180 185 190 Glu Leu Leu Ala Leu Arg Ser Leu Val Glu Leu Arg Ala Leu Leu Gln 195 200 205 Arg Pro Arg Gly Thr Ser Gly Pro Leu Glu Leu Leu Ser Glu Ala Leu 210 215 220 Cys Ser Val Arg Gly Pro Ser Ser Thr Val Gly Pro Ser Leu Asn Trp 225 230 235 240 Tyr Glu Wing Being Asp Leu Met Glu Leu Val Gly Gln Glu Pro Glu Ser 245 250 255 Ala Ala Val Arg Val Leu Ser Giy Ala Asn Pro Arg Ala Gly Leu Tyr 515 520 525 Leu Gln Gln Met Pro Pro Tyr Pro Cys Tyr Val Asp Asp Val Phe Leu Arg 530 535 540 Val Leu Ser Arg Ser Leu Pro Leu Phe Leu Thr Leu Wing Trp He Tyr 545 550 555 560 Ser Val Thr Leu Thr Val Lys Val Val Arg Glu Lys Glu Thr Arg 565 570 575 Leu Arg Asp Thr Met Arg Ala Val Gly Leu Ser Arg Ala Val Leu Trp 580 585 590 Leu Gly Trp Phe Leu Ser cys Leu Gly Pro Phe Leu Leu Ser Ala Ala 595 600 605 Leu Leu Val Leu Val Leu Lys Leu Gly Asp He Leu Pro Cys Ser His 610 615 620 Pro Gly Val Val Phe Leu Phe Leu Ala Ala Phe Ala Val Ala Thr Val 625 630 635 640 Thr Gln Ser Phe Leu Leu be Wing Phe Phe Ser Arg Wing Asn Leu Wing 645 650 655 Ala Ala Cys Gly Gly Leu Ala Tyr Phe Ser Leu Tyr Leu Pro Tyr Val 660 665 670 Leu Cys Val Wing Trp Arg Asp Arg Leu Pro Wing Gly Gly Arg Val Wing 675 680 685 Wing Ser Leu Leu Ser Pro Val Wing Phe Gly Phe Gly Cys Glu Ser Leu 690 695 700 Wing Leu Leu Glu Glu Gln Gly Glu Gly Wing Gln Trp His Asn Val Gly 705 710 715 720 Thr Arg Pro Thr Wing Asp Val Phe Ser Leu Wing Gln Val Ser Gly Leu 725 730 735 Leu Leu Leu Asp Ala Ala Leu Tyr Gly Leu Wing Thr Trp Tyr Leu Glu 740 745 750 Wing Val Cys Pro Gly Gln Tyr Gly He Pro Glu Pro Trp Asn Phe Pro 755 760 765 Phe Arg Arg Ser Tyr Trp Cys Gly Pro Arg Pro Pro Lys Ser Pro Wing 770 775 780 Pro Cys Pro Thr Pro Leu Asp Pro Lys Val Leu Val Glu Pro Wing 785 790 795 800 Pro Gly Leu Ser Pro Gly Val Ser Val Arg Ser Leu Glu Lys Arg Phe 805 810 815 Pro Gly Pro Pro Gln Pro Ala Leu Arg Gly Leu Ser Leu Asp Phe Tyr 820 825 830 Gln Gly His He Thr Wing Phe Leu Gly His Asn Gly Wing Gly Lys Thr 835 840 845 Thr Thr Leu Ser He Leu Ser Gly Leu Phe Pro Pro Ser Gly Gly Ser 850 855 860 Wing Phe He Leu Gly His Asp Val Arg Ser Ser Met Wing Wing He Arg 865 870 875 880 Pro Hxs Leu Gly Val Cys Pro Gln Tyr Asn Val Leu Phe Asp Met Leu 885 890 895 Thr Val Asp Glu Hxs Val Trp Phe Tyr Gly Arg Leu Lys Gly Leu Ser 900 905 910 Wing Wing Val Val Gly Pro Glu Gln Asp Arg Leu Leu Gln Asp Val Gly 915 920 925 Leu Val Ser Lys Gln Ser Val Gln Thr Arg His Leu Ser Gly Gly Met 930 935 940 Gln Arg Lys Leu Ser Val Wing He Wing Phe Val Gly Gly Ser Gln Val 945 950 955 960 Val He Leu Asp Glu Pro Thr Wing Gly Val Asp Pro Wing Ser Arg Arg 965 970 975 Gly He Trp Glu Leu Leu Leu Lys Tyr Arg Glu Gly Arg Thr Leu He 980 985 990 Leu Ser Thr His His Leu Asp Glu Wing Glu Leu Leu Gly Asp Arg Val 995 1000 1005 Wing Val Val Wing Gly Gly Arg Leu Cys Cys Cys Gly Ser Pro Leu Phe 1010 1015 1020 ? ád? M Leu Arg Arg His Leu Gly Ser, Éfty Tyr Tyr Leu Thr Leu Val Lys Wing 1025 1030 1035 1040 Arg Leu Pro Leu Thr Thr Asn Glu Lys Wing Asp Thr Asp Met Glu Gly 1045 1050 1055 Ser Val Asp Thr Arg Gln Glu Lys Lys Asn Gly Ser Gln Gly Ser Arg 1060 1065 1070 Val Gly Thr Pro Gln Leu Leu Ala Leu Val Gln Hxs Trp Val Pro Gly 1075 1080 1085 Wing Arg Leu Val Glu Glu Leu Pro Hxs Glu Leu Val Leu Val Leu Pro 1090 1095 1100 Tyr Thr Gly Wing His Asp Gly Ser Phe Wing Thr Leu Phe Arg slu Leu 1105 1110 1115 1120 Asp Thr Arg Leu Wing Glu Leu Arg Leu Thr Gly Tyr Gly He Ser Asp 1125 1130 1135 Thr Ser Leu Glu Glu He Phe Leu Lys Val Val Glu Glu Cys Ala Ala 1140 1145 1150 Asp Thr Asp Met Glu Asp Gly Ser Cys Gly Gln His Leu Cys Thr Gly 1155 1160 1165 He Wing Gly Leu Asp Val Thr Leu Arg Leu Lys Met Pro Pro Gln Glu 1170 1175 1180 Thr Ala Leu Glu Asn Gly Glu Pro Wing Gly Be Wing Pro Glu Thr Asp 1185 1190 1195 1200 Gln Gly Ser Gly Pro Asp Wing Val Gly Arg Val Gln Gly Trp Wing Leu 1205 1210 1215 Thr Arg Gln Gln Leu Gln Wing Leu Leu Leu Lys Arg Phe Leu Leu Wing 1220 1225 1230 Arg Arg Being Arg Arg Gly Leu Phe Wing Gln He Val Leu Pro Wing Leu 1235 1240 1245 Phe Val Gly Leu Ala Leu Val Phe Ser Leu He Val Pro Pro Phe .Gly 1250 1255 1260 His Tyr Pro Ala Leu Arg Leu Ser Pro Thr Met Tyr Gly Ala Gln Val 1265 1270 1275 1280 »M-? ? t. ? ~ * »Ato * ~ *. - «» **** »* J Be Phe Phe Ser Glu Asp Wing Pro Gly Asp Pro Gly Arg Wing Arg Leu 1285 1290 1295 Leu Glu Ala Leu Leu Gln Glu Ala Gly Leu Glu Glu Pro Pro Val Gln 1300 1305 1310 His Ser Ser His Arg Phe Ser Ala Pro Glu Val Pro Ala Glu Val Ala 1315 1320 1325 Lys Val Leu Ala Ser Gly Asn Trp Thr Pro Glu Ser Pro Ser Pro Ala 1330 1335 1340 Cys Gln Cys Ser Gln Pro Gly Wing Arg Arg Leu Pro Leu Asp Cys Pro 1345 1350 1355 1360 Wing Wing Wing Gly Gly Pro Pro Pro Gln Wing Val Thr Gly Ser Gly 1365 1370 1375 Glu Val Val Gln Asn Leu Thr Gly Arg Asn Leu Ser Asp Phe Leu Val 1380 1385 1390 Lys Thr Tyr Pro Arg Leu Val Arg Gln Gly Leu Lys Thr Lys Lys Trp 1395 1400 1405 Val Asn Glu Val Arg Tyr Gly Gly Phe Ser Leu Gly Gly Arg Asp Pro 1410 1415 1420 Gly Leu Pro Ser Gly Gln Glu Leu Gly Arg Ser Val Glu Glu Leu Trp 1425 1430 1435 1440 Ala Leu Leu Ser Pro Leu Pro Gly Gly Ala Leu Asp Arg Val Leu Lys 1445 1450 1455 Asn Leu Thr Ala Trp Ala His Ser Leu Asp Ala Gln Asp Ser Leu Lys 1460 1465 1470 He Trp Phe Asn Asn Lys Gly Trp His Ser Val Val Ala Phe Val Asn 1475 1480 1485 Arg Ala Ser Asn Ala He Leu Arg Ala His Leu Pro Pro Gly Pro Wing 1490 1495 1500 Arg Hxs Wing His Ser He Thr Thu Leu Asn His Pro Leu Asn Leu .Thr 1505 1510 1515 1520 Lys Glu Gln Leu Ser Glu Wing Wing Leu Met Wing Being Val Val 1525 1530 1535 Leu Val Ser He Cys Val Val Phe Wing Met Ser Phe Val Pro Wing Ser 1540 1545 1550 Phe Thr Leu Val Leu He Glu Glu Arg Val Thr Arg Wing Lys His Leu 1555 1560 1565 Gln Leu Met Gly Gly Leu Ser Pro Thr Leu Tyr Trp Leu Gly Asn Phe 1570 1575 1580 Leu Trp Asp Met Cys Asn Tyr Leu Val Pro Wing Cys He Val Val Leu 1585 1590 1595 1600 He Phe Leu Wing Phe Gln Gln Arg Wing Tyr Val Wing Pro Wing Asn Leu 1605 1610 1615 Pro Wing Leu Leu Leu Leu Leu Leu Tyr Gly Trp Ser He Thr Pro 1620 1625 1630 Leu Met Tyr Pro Wing Being Phe Phe Phe Ser Val Pro Being Thr Wing Tyr 1635 1640 1645 Val Val Leu Thr Cys He Asn Leu Phe He Gly He Asn Gly Ser Met 1650 1655 1660 Wing Thr Phe Val Leu Glu Leu Phe Ser Asp Gln Lys Leu Gln Glu Val 1665 1670 1675 1680 Ser Arg He Leu Lys Gln Val Phe Leu He Phe Pro His Phe Cys Leu 1685 1690 1695 Gly Arg Gly Leu He Asp Met Val Arg Asn Gln Wing Met Wing Asp Wing 1700 1705 1710 Phe Glu Arg Leu Gly Asp Arg Gln Phe Gln Ser Pro Leu Arg Trp Glu 1715 1720 1725 Val Val Gly Lys Asn Leu Leu Wing Met Val He Gln Gly Pro Leu Phe 1730 1735 1740 Leu Leu Phe Thr Leu Leu Leu Gln Hxs Arg Ser Gln Leu Leu Pro Gln 1745 1750 1755 1760 Pro Arg Val Arg Ser Leu Pro Leu Leu Gly Glu Glu Asp Glu Asp Val 1765 1770 1775 Wing Arg Glu Arg Glu Arg Val Val Gln Gly Wing Thr Gln Gly Asp Val 1780 1785 1790 Leu Val Leu Arg Asn Leu Thr Lys Val Tyr Arg Gly Gln Arg Met Pro 1795 1800 1805 Wing Val Asp Arg Leu Cys Leu Gly Pro Pro Gly Glu Cys Phe Gly 1810 1815 1820 Leu Leu Gly Val Asn Gly Wing Gly Lys Thr Ser Thr Phe Arg Met Val 1825 1830 1835 1840 Thr Gly Asp Thr Leu Wing Ser Arg Gly Glu Wing Val Leu Wing Gly Hxs 1845 1850 1855 Ser Val Wing Arg Glu Pro Wing Wing Hxs Leu Ser Met Gly Tyr Cys 1860 1865 1870 Pro Gln Ser Asp Wing He Phe Glu Leu Leu Thr Gly Arg Glu His Leu 1875 1880 1885 Glu Leu Leu Wing Arg Leu Arg Gly Val Pro Glu Wing Gln Val Wing Gln 1890 1895 1900 Thr Ala Gly Ser Gly Leu Ala Arg Leu Gly Leu Ser Trp Tyr Wing Asp 1905 1910 1915 1920 Arg Pro Wing Gly Thr Tyr Ser Gly Gly Asn Lys Arg Lys Leu Wing Thr 1925 1930 1935 Wing Leu Wing Leu Val Gly Asp Pro Wing Val Val Phe Leu Asp Glu Pro 1940 1945 1950 Thr Thr Gly Met Asp Pro Be Wing Arg Arg Phe Leu Trp Asn Ser Leu 1955 1960 1965 Leu Ala Val Val Arg Glu Gly Arg Ser Val Met Leu Thr Ser His Ser 1970 1975 1980 Met Glu Glu Cys Glu Ala Leu Cys Ser Arg Leu Ala He Met Val Asn 1985 1990 1995 2000 Gly Arg Phe Arg Cys Leu Gly Ser Pro Gln His Leu Lys Gly Arg Phe 2005 2010 2015 Ala Ala Gly His Thr Leu Thr Leu Arg Val Pro Ala Ala Arg Ser Gln 2020 2025 2030 Pro Ala Ala Ala Phe Ala Ala Ala Glu Phe Pro Gly Ser Glu Leu Arg 2035 2040 2045 Glu Wing Hxs Gly Gly Arg Leu Arg Phe Gln Leu Pro Pro Gly Gly Arg 2050 2055 2060 cys Ala Leu Ala Arg Val Phe Gly Glu Leu Ala Val Hxs Gly Ala Glu 2065 2070 2075 2080 Hxs Gly Val Glu Asp Phe Ser Val Ser Gln Thr Met Leu Glu Glu Val 2085 2090 2095 Phe Leu Tyr Phe Ser Lys Asp Gln Gly Lys Asp Glu Asp Thr Glu Glu 2100 2105 2110 Gln Lys Glu Wing Gly Val Gly Val Asp Pro Wing Pro Gly Leu Gln Hxs 2115 2120 2125 Pro Lys Arg Val Ser Gln Phe Leu Asp Asp Pro Ser Thr Ala Glu Thr 2130 2135 2140 Val Leu 2145 < 210 > 3 < 211 > 5669 < 212 > DNA < 213 > Homo sapxens < 400 > 3 atggccttct ggacacagct gatgctgctg atttcatgta ctctggaaga tcgccggaga 60 cagccggtcc agctcctggt cgaattgctg tggcctctct tcctcttctt catcctggtg 120 gctgttcgcc actcccaccc gcccctggag caccatgaat gccacttccc aaacaagcca 180 ctgccatcgg cgggcaccgt gccctggctc cagggtctca tctgtaatgt gaacaacacc 240 tgctttccgc agctgacacc gggcgaggag cccgggcgcc tgagcaactt caacgactcc 300 ctggtctccc ggctgctagc cgatgcccgc actgtgctgg gaggggccag tgcccacagg 360 acgctggctg gcctagggaa gctgatcgcc acgctgaggg ctgcacgcag cacggcccag 420 cctcaaccaa ccaagcagtc tccactggaa ccacccatgc tggatgtcgc ggagctgctg 480 acgtcactgc tgcgcacgga atccctgggg ttggcactgg gccaagccca ggagcccttg 540 cacagcttgt tggaggccgc tgaggacctg gcccaggagc tcctggcgct gcgcagcctg 600 gtggagcttc gggcactgct gcagagaccc cgagggacca gcggccccct ggagttgctg 660 tcagaggccc tctgcagtgt caggggacct agcagcacag tgggcccctc cctcaactgg 720 tacgaggcta gtgacctgat ggagctggtg gggcaggagc cagaatccgc cctgccagac 780 agcagcctga gccccgcctg ctcggagctg attggagccc tggacagcca cccgctgtcc 840 cgcctgctct ggagacg cct gaagcctctg atcctcggga agctactctt tgcaccagat 900 acacctttta cccggaagct catggcccag gtgaaccgga ccttcgagga gctcaccctg 960 ctgagggatg tccgggaggt gtgggagatg ctgggacccc ggatcttcac cttcatgaac 1020 gacagttcca atgtggccat gctgcagcgg ctcctgcaga tgcaggatga aggaagaagg 1080 cagcccagac ctggaggccg ggaccacatg gaggccctgc gatcctttct ggaccctggg 1140 agcggtggct acagctggca ggacgcacac gctgatgtgg ggcacctggt gggcacgctg 1200 ggccgagtga cggagtgcct gtccttggac aagctggagg cggcaccctc agaggcagcc 1260 ctggtgtcgc gggccctgca actgotn ^? g gaacatcgat tctgggccgg cgtcgtcttc 1320 ttgggacctg aggactcttc agaccccaca gagcacccaa ccccagacct gggccccggc 1380 tcaaaatccg cacgtgcgca gacgtggtca catggacattí cgaggaccaa taagatcagg 1440 gacaggtttt gggaccctgg cccagccgcg gaccccctga ccgacctgcg ctacgtgtgg 1500 ggcggcttcg tgtacctgca agacctggtg gagcgtgcag ccgtccgcgt gctcagcggc 1560 gccaaccccc gggccggcct ctacctgcag cagatgccct atccgtgcta tgtggacgac 1620 gtgttcctgc gtgtgctgag ccggtcgctg ccgctcttcc tgacgcfeggc ctggatctac 1680 tccgtgacac tgacagtgaa ggccgtggtg cgggagaagg agacgcggct gcgggacacc 1740 atgcgcgccg tggggctcag ccgcgcggtg ctctggctag gctggttcct cagctgcctc 1800 gggcccttcc tgctcagcgc cgcgctgctg gttctggtgc tcaagctggg ggacatcctc 1860 ccctgcagcc acccgggcgt ggtcttcctg ttcttggcag ccttcgcggt ggccacggtg 1920 acccagagct tcctgctcag cgccttcttc tcccgcgcca acctggctgc ggcctgcggc 198p ggcctggcct acttctccct ctacctgccc tacgtgctgt gtgtggcttg gcggga ccgg 2040 ctgcccgcgg gtggccgcgt ggccgcgagc ctgctgtcgc ccgtggcctt cggcttcggc 2100 tgcgagagcc tggctctgct ggaggagcag ggcgagggcg cgcagtggca caacgtgggc 2160 acccggccta cggcagacgt cttcagcctg gcccaggtct ctggccttct gctgctggac 2220 gcggcgctct acggcctcgc cacctggtac ctggaagctg tgtgcccagg ccagtacggg 2280 atccctgaac catggaattt tccttttcgg aggagctact ggtgcggacc tcggcccccc 2340 aagagtccag ccccttgccc caccccgctg gacccaaagg tgctggtaga agaggcaccg 2400 cccggcctga gtcctggcgt ctccgttcgc agcctggaga agcgctttcc tggaagcccg 2460 cagccagccc tgcgggggct cagcctggac ttctaccagg gccacatcac cgccttcctg 2520 ggccacaacg gggccggcaa gaccaccacc ctgtccatct tgagtggcct cttcccaccc 2580 agtggtggct ctgccttcat cctgggccac gacgtccgct ccagcatggc cgccatccgg 2640 ccccacctgg gcgtctgtcc tcagtacaac gtgctgtttg acatgctgac cgtggacgag 2700 cacgtctggt tctatgggcg gctgaagggt ctgagtgccg ctgtagtggg ccccgagcag 2760 gaccgtctgc tgcaggatgt ggggctggtc tccaagcaga gtgtgcagac tcgccacctc 2820 tgcaacggaa tctggtggga gctgtccgtg gccattgcct ttgtgggcgg ctcccaagtt 2880 gttatcctgg acgagcctac ggctggcgtg gatcctgctt cccgccgcgg tatttgggag ctg 2940 ctgctca aataccgaga aggtcgcacg ctgatcctct ccacccacca cctggatgag 3000 gcagagctgc tgggagaccg tgtggctgtg gtggcaggtg gccgcttgtg ctgctgtggc 3060 tccccactct tcctgcgccg tcacctgggc tccggctact acctgacgct ggtgaaggcc 3120 cgcctgcccc tgaccaccaa tgagaaggct gacactgaca tggagggcag tgtggacacc 3180 aggcaggaaa agaagaatgg cagccagggc agcagagtcg gcactcctca gctgctggcc 3240 ctggtacagc actgggtgcc cggggcacgg ctggtggagg agctgccaca cgagctggtg 3300 ctggtgctgc cctacacggg tgcccatgac ggcagcttcg ccacactctt ccgagagcta 3360 gacacgcggc tggcggagct gaggctcact ggctacggga tctccgacac cagcctcgag 3420 gagatcttcc tgaaggtggt ggaggagtgt gctgcggaca cagatatgga ggatggcagc 3480 tgcgggcagc acctatgcac aggcattgct ggcctagacg taaccctgcg gctcaagatg 3540 ccgccacagg agacagcgct ggagaacggg gaaccagctg ggtcagcccc agagactgac 3600 cagggctctg ggccagacgc cgtgggccgg gtacagggct gggcactgac ccgccagcag 3660 ctccaggccc tgcttctcaa gcgctttctg cttgcccgcc gcagccgccg cggcctgttc 3720 gcccagatcg tgctgcctgc cctctttgtg ggcctggccc tcgtgttcag cctcatcgtg 3 780 cctcctttcg ggcactaccc ggctctgcgg ctcagtccca ccatgtacgg tgctcaggtg 3840 tccttcttca gtgaggacgc cccaggggac cctggacgtg cccggctgct cgaggcgctg 3900 caggactgga ctgcaggagg gtgcagcata ggagccccca gttctcggca gctcccacag 3960 ccagaagttc ctgctgaagt ggccaaggtc ttggccagtg gcaactggac cccagagtct 4020 ccatccccag cctgccagtg tagccagccc ggtgcccggc gcctgctgcc cgactgcccg 4080 gctgcagctg gtggtccccc tccgccccag gcagtgaccg gctctgggga agtggttcag 4140 aacctgacag gccggaacct gtctgacttc ctggtcaaga cctacccgcg cctggtgcgc 4200 cagggcctga agactaagaa gtgggtgaat gaggtcaggt acggaggctt ctcgctgggg 4260 ggccgagacc caggcctgcc ctcgggccaa gagttgggcc gctcagtgga ggagttgtgg 4320 gcgctgctga gtcccctgcc tggcggggcc ctcgaccgtg tcctgaaaaa cctcacagcc 4380 tgggctcaca gcctggatgc tcaggacagt ctcaagatct ggttcaacaa caaaggctgg 4440 Cactccatgg tggcctttgt caaccgagcc agcaacgcaa tcctccgtgc tcacctgccc 4500 ccaggcccgg cccgccacgc ccacagcatc accacactca accacccctt gaacctcacc 4560 aaggagcagc tgtctgaggc tgcactgatg gcctcctcgg tggacgtcct cgtctccatc 46 20 tgtgtggtct ttgccatgtc ctttgtcccg gccagcttca ctcttgtcct cattgaggag 4680 cgagtcaccc gagccaagca cctgcagctc atggggggcc tgtcccccac cctctactgg 4740 cttggcaact ttctctggga catgtgtaac tacttggtgc cagcatgcat cgtggtgctc 4800 atctttctgg ccttccagca gagggcatat gtggcccctg ccaacctgcc tgctctcctg 4860 ctgttgctac tactgtatgg ctggtcgatc acaccgctca tgtacccagc ctccttcttc 4920 ttctccgtgc ccagcacagc ctatgtggtg ctcacctgca taaacctctt tattggcatc 4980 aatggaagca tggccacctt tgtgcttgag ctcttctctg atcagaagct gcaggaggtg 5040 agccggatct tgaaacaggt cttccttatc ttcccccact tctgcttggg ccgggggctc 5100 attgacatgg tgcggaacca ggccatggct gatgcctttg agcgcttggg agacaggcag 5160 ttccagtcac ccctgcgctg ggaggtggtc ggcaagaacc tcttggccat ggtgatacag 5220 gggcccctct tccttctctt cacactactg ctgcagcacc gaagccaact cctgccacag 5280 cccagggtga ggtctctgcc actcctggga gaggaggacg aggatgtagc ccgtgaacgg 5340 gagcgggtgg tccaaggagc cacccagggg gatgtgttgg tgctgaggaa cttgaccaag 5400 gtataccgtg ggcagaggat gccagctgtt gaccgcttgt gcctggggat tccccctggt 5460 gag gtgagtc caggggtgga ggccaggtgc agggacagtg agtggctgcc ctactgcatg 5520 ccctgcccat catcctttac tgaacaccta ctgtgtatcc accacctttt attgggcacc 5580 tactgtatgc caatatttgt gctcctattt ttattttatt aaattattat ttatttaaaa 5640 aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 5669 < 210 > 4 < 211 > 1873 < 212 > PRT < 213 > Homo sapiens < 400 > 4 Met Wing Phe Trp Thr Gln Leu Met Leu Leu Leu Trp Lys Asn Phe Met 1 5 10 15 Tyr Arg Arg Gln Pro Val Gln Leu Leu Val Glu Leu Leu Trp Pro 20 25 30 Leu Phe Leu Phe Phe He Leu Val Wing Val Arg His Ser Hxs Pro Pro 35 40 45 Leu Glu His His Glu Cys His Phe Pro Asn Lys Pro Leu Pro Be Ala 50 55 60 Gly Thr Val Pro Trp Leu Gln Gly Leu He Cys Asn Val Asn Asn Thr 65 70 75 80 Cys Phe Pro Gln Leu Thr Pro Gly Glu Pro Gly Arg Leu Ser Asn 85 s 90 95 Phe Asn Asp Ser Leu Val Ser Arg Leu Leu Ala Asp Ala Arg Thr Val 100 105 110 Leu Gly Gly Ala Ser Ala His Arg Thr Leu Wing Gly Leu Gly Lys Leu 115 120 125 He Wing Thr Leu Arg Wing Wing Arg Being Thr Wing Gln Pro Gln Pro Thr 130 135 140 Lys Gln Pro Pro Leu Glu Pro Pro Met Leu Asp Val Wing Glu Leu Leu 145 150 155 160 Thr Ser Leu Leu Arg Thr Glu Ser Leu Gly Leu Ala Leu Gly Gln Wing 165 170 175 G n Glu Pro Leu Hxs Ser Leu Leu Glu Wing Ala Glu Asp Leu Ala Gln 180 '185 190 Glu Leu Leu Ala Leu Arg Ser Leu Val Glu Leu Arg Ala Leu Leu Gln 195 200 205 Arg Pro Arg Gly Thr Ser Gly Pro Leu Glu Leu Leu Ser Glu Ala Leu 210 215 220 Cys Ser Val Arg Gly Pro Ser Ser Thr Val Gly Pro Ser Leu Asn Trp 225 230 235 240 Tyr Glu Wing Ser Asp Leu Met Glu Leu Val Gly Gln Glu Pro Glu Ser 245 250 255 Wing Leu Pro Asp Being Ser Leu Ser Pro Wing Cys Ser Glu Leu He Gly 260 265 270 Ala Leu Asp Ser His Pro Leu Ser Arg Leu Leu Trp Arg Arg Leu Lys 275 280 285 Pro Leu He Leu Gly Lys Leu Leu Phe Pro Wing Pro Asp Thr Pro Phe Thr 290 295 300 Arg Lys Leu Met Wing Gln Val Asn Arg Thr Phe Glu Glu Leu Thr Leu 305 310 315 320 Leu Arg Asp Val Arg Glu Val Trp Glu Met Leu Gly Pro Arg He Phe 325 330 335 Thr Phe Met Asn Asp Ser Ser Asa Val Wing Met Leu Gln Arg Leu Leu 340 3T45 350 Gln Met Gln Asp Glu Gly Arg Arg Gln Pro Arg Pro Gly Gly Arg Asp 355 360 365 His Met Glu Ala Leu Arg Ser Phe Leu Asp Pro Gly Ser Gly Gly Tyr 370 375 380 Ser Trp Gln Asp Ala His Wing Asp Val Gly His Leu Val Gly Thr Leu 385 390 395 400 Gly Arg Val Thr Glu Cys Leu Ser Leu Asp Lys Leu Glu Ala Wing Pro 405 410 415 Ser Glu Ala Ala Leu Val Ser Arg Ala Leu Gln Leu Leu Ala Glu His 420 425 430 Arg Phe Trp Wing Gly Val Val Phe Leu Gly Pro Glu Asp Ser As Asp 435 440 445 Pro Thr Glu Hxs Pro Thr Pro Asp Leu Gly Pro Gly His Val Arg He 450 455 460 Lys He Arg Met Asp He Asp Val Val Thr Arg Thr Asn Lys He Arg 465 470 475 480 Asp Arg Phe Trp Asp Pro Gly Pro Wing Wing Asp Pro Leu Thr Asp Leu 485 490 495 Arg Tyr Val Trp Gly Gly Phe Val Tyr Leu Gln Asp Leu Val Glu Arg 500 505 510 Ala Ala Val Arg Val Leu Ser Gly Ala Asn Pro Arg Ala Gly Leu Tyr 515 520 525 Leu Gln Gln Met Pro Pro Tyr Pro Cys Tyr Val Asp Asp Val Phe Leu Arg 530 535 540 Val Leu Ser Arg Ser Leu Pro Leu Phe Leu Thr Leu Wing Trp He Tyr 545 550 555 560 Being Val Thr Leu Thr Val Lys Wing Val Val Arg Glu Lys Glu Thr Arg 565 570 575 Leu Arg Asp Thr Mss Arg Wing Val Gly Leu Ser Arg Wing Val Leu Trp 580 585 590 Leu Gly Trp Phe Leu Ser Cys Leu Gly Pro Phe Leu Leu Ser Wing Wing 595 600 605 Leu Leu Val Leu Val Leu Lys Leu Gly Asp He Leu Pro Cys Ser His 610 615 620 Pro Gly Val Val Phe Leu Phe Leu Ala Ala Phe Ala Ala Ala Thr Val 625 630 635 640 Thr Gln Ser Phe Leu Leu Ser Ala Phe Phe Ser Arg Ala Asn Leu Ala 645 650 655 Ala Ala Cys Gly Gly Leu Ala Tyr Phe Ser Leu Tyr Leu Pro Tyr Val 660 665 670 Leu Cys Val Wing Trp Arg Asp Arg Leu Pro Wing Gly Gly Arg Val Wing 675 680 685 Wing Ser Leu Leu Ser Pro Val Wing Phe Gly Phe Gly Cys Glu Ser Leu 690 695 700 Wing Leu Leu Glu Glu Gln Gly Glu Gly Wing Gln Trp His Asn Val Gly 705 710 715 720 Thr Arg Pro Thr Wing Asp Val Phe Ser Leu Wing Gln Val Ser Gly Leu 725 730 735 Leu Leu Leu Asp Wing Wing Leu Tyr Gly Leu Wing Thr Trp Tyr Leu Glu 740 745 750 Wing Val Cys Pro Gly Gln Tyr Gly He Pro Glu Pro Trp Asn Phe Pro 755 760 765 Phe Arg Arg Ser Tyr Trp Cys Gly Pro Arg Pro Pro Lys Pro Pro Wing 770 775 780 Pro Cys Pro Thr Pro Leu Asp Pro Lys Val Leu Val Glu Glu Pro Wing 785 790 795 800 Pro Gly Leu Ser Pro Gly Val Ser Val Arg Ser Leu Glu Lys Arg Phe 805 810 815 Pro Gly Ser Pro Gln Pro Wing Leu Arg Gly Leu Ser Leu Asp Phe Tyr 820 825 830 Gln Gly His He Thr Wing Phe Leu Gly His Asn Gly Wing Gly Lys Thr 835 840 845 Thr Thr Leu Ser He Leu Ser Gly Leu Phe Pro Pro Ser Gly Gly Ser 850 855 860 Wing Phe He Leu Gly His Asp Val Arg Ser Ser Met Wing Wing He Arg 865 870 875 880 Pro Hxs Leu Gly Val Cys Pro Gln Tyr Asn Val Leu Phe Asp Met Leu 885 890 895 Thr Val Asp Glu His Val Trp Phe Tyr Gly Arg Leu Lys Gly Leu Ser 900 905 910 Wing Wing Val Gly Pro Glu Gln Asp Arg Leu Leu Gln Asp Val Gly 915 920 925 Leu Val Ser Lys Gln Ser Val Gln Thr Arg Hxs Leu Ser Gly Gly Met 930 935 940 Gln Arg Lys Leu Ser Val Wing He Wing Phe Val Gly Gly Ser Gln Val 945 950 955 960 Val He Leu Asp Glu Pro Thr Wing Gly Val Asp Pro Wing Ser Arg Arg 965 970 975 Gly He Trp Glu Leu Leu Leu Lys Tyr Arg Glu Gly Arg Thr Leu He 980 985 990 Leu Ser Thr His Hxs Leu Asp Glu Wing Glu Leu Leu Gly Asp Arg Val 995 1000 1005 Wing Val Val Wing Gly Gly Arg Leu Cys Cys Cys Gly Ser Pro Leu Phe 1010 1015 1020 Leu Arg Arg Hxs Leu Gly Ser Gly Tyr Tyr Leu Thr Leu Val Lys Wing 1025 1030 1035 1040 Arg Leu Pro Leu Thr Thr Asn Glu Lys Wing Asp Thr Asp Met Glu Gly 1045 1050 1055 Ser Val Asp Thr Arg Gln Glu Lys Lys Asn Gly Ser Gln Gly Ser Arg 1060 1065 1070 Val Gly Thr Pro Gln Leu Leu Ala Leu Val Gln His Trp Val Pro Gly 1075 1080 1085 Wing Arg Leu Val Glu Glu Leu Pro Hxs Glu Leu Val Leu Val Leu Pro i .Ji ......? ^? Mki? - ?. 1090 1095 1100 Tyr Thr Gly Wing Hxs Asp Gly Ser Phe Wing Thr Leu Phe Arg Glu Leu 1105 1110 1115 1120 Asp Thr Arg Leu Wing Glu Leu Arg Leu Thr Gly Tyr Gly He Ser Asp 1125 1130 1135 Thr Ser Leu Glu Glu He Phe Leu Lys Val Val Glu Glu Cys Ala Wing 1140 1145 1150 Asp Thr Asp Met Glu Asp Gly Ser Cys Gly Gln Hxs Leu Cys Thr Gly 1155 1160 1165 He Wing Gly Leu Asp Val Thr Leu Arg Leu Lys Met Pro Pro Gln Glu 1170 1175 1180 Thr Wing Leu Glu Asn Gly Glu Pro Wing Gly Wing Pro Glu Thr Asp 1185 1190 1195 1200 Gln Gly Ser Gl ^ ro Asp Wing Val Gly Arg Val Gln Gly Trp Wing Leu '15 1210 1215 Thr Arg Gln Gln Leu Gln Ala Leu Leu Leu Lys Arg Phe Leu Leu Wing 1220 1225 1230 Arg Arg Arg Arg Gly Leu Phe Wing Gln He Val Leu Pro Wing Leu 1235 1240 1245 Phe Val Gly Leu Ala Leu Val Phe Ser Leu He Val Pro Pro Phe Gly 1250 1255 1260 Hxs Tyr Pro Ala Leu Arg Leu Ser Pro Thr Met Tyr Gly Ala Gln Val 1265 1270 1275 1280 Ser Phe Phe Ser Glu Asp Wing Pro Gly Asp Pro Gly Arg Wing Arg Leu 1285 1290 1295 Leu Glu Ala Leu Leu Gln Glu Ala Gly Leu Glu Glu Pro Pro Val Gln 1300 1305 1310 Hxs Ser Ser Hxs Arg Phe Ser Ala Pro Glu Val Pro Ala Glu Val Ala 1315 1320 1325 Lys Val Leu Wing Ser Gly Asn Trp Thr Pro Glu Ser Pro Ser Pro Wing 1330 1335 1340 Cys Gln Cys Ser Gln Pro Gly Wing Arg Arg Leu Leu Pro Asp Cys Pro 1345 1350 1355 1360 Wing Wing Wing Gly Gly Pro Pro Pro Gln Wing Wing Thr Gly Ser Gly 1365 1370 1375 Glu Val Vai Gln Asn Leu Thr Gly Arg Asn Leu Ser Asp Phe Leu Val 1380 1385 1390 Lys Thr Tyr Pro Arg Leu Val Arg Gln Gly Leu Lys Thr Lys Lys Trp 1395 1400 1405 Val Asn Glu Val Arg Tyr Gly Gly Phe Ser Leu Gly Gly Arg Asp Pro 1410 1415 1420 Gly Leu Pro Ser Gly Gln Glu Leu Gly Arg Ser Val Glu Glu Leu Trp 1425 1430 1435 1440 Ala Leu Leu Ser Pro Leu Pro Gly Gly Ala Leu Asp Arg Val Leu Lys 1445 1450 1455 Asn Leu Thr Ala Trp Ala Hxs Ser Leu Asp Ala Gln Asp Ser Leu Lys 1460 1465 1470 He Trp Phe Asn Asn Lys Gly Trp His ser Met Val Wing Phe Val Asn 1475 1480 1485 Arg Wing Ser Asn Wing He Leu Arg Wing His Leu Pro Pro Gly Pro Wing 1490 1495 1500 Arg Hxs Wing Hxs Ser He Thr Thu Leu Asn His Pro Leu Asn Leu Thr 1505 1510 1515 1520 Lys Glu Gln Leu Ser Glu Ala Ala Leu Met Ala Ser Ser Val Val 1525 1530 1535 Leu Val Ser He Cys Val Val Phe Ala Met Ser Phe Val Pro Wing Ser 1540 1545 1550 Phe Thr Leu Val Leu He Glu Glu Arg Val Thr Arg Ala Lys His Leu 1555 1560 1565 Gln Leu Met Gly Gly Leu Ser Pro Thr Leu Tyr Trp Leu Gly Asn Phe 1570 1575 1580 Leu Trp Asp Met Cys Asn Tyr Leu Val Pro Wing Cys He Val Val Leu 1585 1590 1595 1600 He Phe Leu Wing Phe Gln Gln Arg Wing Tyr Val Wing Pro Wing Asn Leu 1605 1610 1615 Pro Ala Leu Leu Leu Leu Leu Leu% am »Tyr Gly Trp Ser He Thr Pro 1620 1625 1630 Leu Met Tyr Pro Wing Being Phe Phe Phe Ser Val Pro Ser Thr Wing Tyr 1635 1640 1645 Val Val Leu Thr Cys He Asn Leu Phe He Gly He Asn Gly Ser Met 1650 1655 1660 Wing Thr Phe Val Leu Glu Leu Phe Ser Asp Gln Lys Leu Gln Glu Val 1665 1670 1675 1680 Ser Arg He Leu Lys Gln Val Phe Leu He Phe Pro Hxs Phe Cys Leu 1685 1690 1695 Gly Arg Gly Leu He Asp Met Val Arg Asn Gln Wing Met Wing Asp Wing 1700 1705 1710 Phe Glu Arg Leu Gly Asp Arg Gln Phe Gln Ser Pro Leu Arg Trp Glu 1715 1720 1725 Val Val Gly Lys Asn Leu Leu Ala Met Val He Gln Gly Pro Leu Phe 1730 1735 1740 Leu Leu Phe Thr Leu Leu Leu Gln Hxs Arg Ser Gln Leu Pro Gln 1745 1750 1755 1760 Pro Arg Val Arg Ser Leu Pro Leu Leu Gly Glu Glu Asp Glu Asp Val 1765 1770 1775 Wing Arg Glu Arg Glu Arg Val Val Gln Gly Ala Thr Gln Gly Asp Val 1780 1785 1790 Leu Val Leu Arg Asn Leu Thr Lys Val Tyr Arg Gly Gln Arg Met Pro 1795 1800 1805 Wing Val Asp Arg Leu Cys Leu Gly He Pro Pro Gly Glu Val Ser Pro 1810 181S 1820 Gly Val Glu Wing Arg Cys Arg Asp Ser Glu Trp Leu Pro Tyr Cys Met 1825 1830 1835 1840 Pro Cys Pro Ser Ser Phe Thr Glu Hxs Leu Leu Cys He Hxs Hxs Leu 1845 1850 1855 Leu Leu Gly Thr Tyr Cys Met Pro He Phe Val Leu Leu Phe Leu Phe 1860 1865 1870 Tyr ^ ^ ^ jáak

Claims (23)

1. CLAIMS 1. A purified and isolated DNA sequence substantially similar to the DNA sequence shown in SEQ ID NOS 1 or 3.
2. 2. A purified and isolated DNA sequence that hybridizes to the DNA sequence shown in SEQ ID NOS 1 or 3 under highly stringent hybridization conditions.
3. 3. A purified and isolated DNA sequence consisting essentially of the DNA sequence shown in SEQ ID NOS 1 or 34. A purified and isolated DNA sequence having at least 70% identity for a polynucleotide encoding the polypeptide expressed by SEQ ID NOS 2 or
4. 4.
5. 5. A purified and isolated DNA sequence that is totally complementary to the DNA sequence shown in SEQ ID NOS 1 or 3.
6. 6. A recombinant DNA molecule comprising the DNA sequence purified and isolated according to claim 2 or 3 within an extra-chromosomal vector.
7. 7. A recombinant host cell comprising a host cell transfected with the recombinant DNA molecule according to claim 6.
8. 8. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially similar to the amino acid sequence shown in SEQ ID NOS 2 or 4. lúJka.? A.L.mtámim L,. ^ .. - * »-« - > • «" «--- - * ~ -« e ***
9. 9. A recombinant polypeptide substantially purified according to claim 8, wherein the polypeptide has at least 70% similarity of the amino acid sequence to the amino acid sequence shown in SEQ ID NOS 2 or 4.
10. 10. A substantially purified polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide consists essentially of the amino acid sequence shown in SEQ ID NOS 2 64.
11. 11. An antibody that selectively binds the polypeptides with an amino acid sequence substantially similar to the amino acid sequence according to claim 8.
12. 12. A method for detecting the PD-ABC protein in cells, which comprises contacting the cells with the antibody of claim 11 and incubating the cells in a manner that allows detection of the antibody complex of the PD-ABC protein.
13. 13. A diagnostic test for detecting cells containing PD-ABC mutations, comprising the isolation of the total genomic DNA from the cell and subjecting the genomic DNA to the PCR amplification using the primers derived from the purified DNA sequence and isolated in accordance with the claims 1, 2 or 3 or by analyzing the genomic DNA directly by a hybridization method and determining whether the resulting PCR product contains a mutation.
14. 14. A diagnostic test to detect cells containing the PD-ABC mutations, which comprises isolating the total cellular RNA, subjecting the RNA to reverse transcription, PCR amplification using the primers derived from the isolated DNA sequence and purified in accordance with Claim 1, 2 or 3 and determine if the resulting PCR product contains a mutation.
15. 15. A method for the amplification of a region for the DNA sequence according to claims 1, 2 or 3, the method comprising the step of: contacting a test sample suspected of containing the desired sequence according to the claim 1, 2 or 3 or the portion thereof with the amplification reaction reagents.
16. 16. A diagnostic device for detecting the presence of at least one copy of the DNA sequence according to claim 1, 2 or 3 in a test sample, said equipment containing an initiator, a pair of primers or a probe and optionally reactive of amplification.
17. 17. A test for the detection or analysis of the therapeutic compounds that interfere with or mimic the interaction between the polypeptide according to claim 8, 9 or 10 and the ligands that bind to the polypeptide according to claim 8, 9 or 10.
18. 18. The test according to claim 17, wherein the test comprises the steps of: a) providing a polypeptide according to claim 8, 9 or 10; b) obtain a candidate substance; c) contacting said polypeptide with said candidate substance and d) detecting the complexes formed between said polypeptide and said candidate substance.
19. 19. A method for detecting mammalian cells from abnormal calcium flux, comprising introducing into mammalian cells an expression vector comprising the isolated and purified DNA sequence according to claim 1, 2 or 3, which is operatively linked to a DNA sequence that promotes high-level expression of the isolated and purified DNA sequence in mammalian cells. h A.?.A.??.-J-Í~* - «~ m. * mmmm ... mj * --- ~ - - * ~ - - * - ~» - - *. »» - - * - mm m .. *** = £. *OR? mm .. *
20. 20. A method for treating or preventing epilepsy, comprising introducing into an mammal an expression vector comprising the DNA sequence purified and isolated according to claim 1, 2 or 3, which is operably linked to a DNA sequence that promotes high level expression of the anti-sense strand of purified DNA sequence and isolated in mammalian cells.
21. 21. A method for purifying PD-ABC protein cells, comprising: transfecting a host cell with a vector comprising the isolated and purified DNA sequence according to claims 1, 2 or 3 operably linked to a promoter capable of directing the expression of the gene in a host cell; induce the expression of the purified and isolated DNA sequence in the cells; lyse the cells; Isolate the PD-ABC protein from the cells and purify the PD-ABC protein from the isolate.
22. 22. A method for treating or preventing a disease selected from the group comprising syndromes related to dyslipidemia wherein the method comprises the step of administering the polynucleotide according to claims 1, 2 or 3 to a mammal in need thereof a therapeutically amount effective
23. 23. A method for treating or preventing a disease related to the group comprising the syndromes related to dyslipidemia wherein the method comprises the step of administering the polypeptide according to claim 8, 9 or 10 to a mammal in need thereof an amount Therapeutically effective. U..i ^ ^, »J > ¿M. ^ ^ ^ ^ ^, A ^ ^ ^ ^ ^ ^ ^ ^ ^. ^ ". Jt. «M ^ > , ^^ -,. . "Mm.?r*, - ^ mmb, r -, T - A.rfA». «.A, I '.