US20040086903A1

US20040086903A1 - Epididymal lipocalin gene and uses thereof

Info

Publication number: US20040086903A1
Application number: US10/451,867
Authority: US
Inventors: Jean-Jacques Lareyre; Marie-Claire Orgebin-Crist; Robert Matusik
Original assignee: Individual
Current assignee: Individual
Priority date: 2000-12-29
Filing date: 2001-12-27
Publication date: 2004-05-06
Also published as: WO2002053701A2; WO2002053701A9; WO2002053701A3; US8580941B2; AU2002246758A1; US20090118210A1

Abstract

Isolated nucleic acids comprising a lipocalin gene promoter region, isolated nucleic acids comprising a human lipocalin gene, isolated nucleic acids encoding a lipocalin polypeptide, isolated lipocalin polypeptides, and uses thereof. The disclosed lipocalin nucleic acids and polypeptides can be used to generate a mouse model of male infertility, for drug discovery screens, and for therapeutic treatment of fertility-related conditions.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to U.S. Provisional Patent Application Serial No. 60/258,655 filed Dec. 29, 2000, the entire contents of which are herein incorporated by reference.[0001]

GRANT STATEMENT

[0002] This work was supported by NICHD grant HD36900. Thus, the U.S. Government has rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to epididymal function and male fertility. More particularly, the present invention provides lipocalin nucleic acid and polypeptide sequences, a lipocalin gene promoter region that directs gene expression in the epididymis, chimeric genes comprising disclosed lipocalin sequences, and uses thereof.



Table of Abbreviations

	CAT	chloramphenicol acetyl transferase
	ES	embryonic stem cell
	FCS	fluorescence correlation spectroscopy
	hEP17	human Epididymal Protein 17
	mE-RABP	mouse Epididymal Retinoic Acid Binding
		Protein
	mEP17	mouse Epididymal Protein 17
	MS	mass spectroscopy
	PCR	polymerase chain reaction
	PGK	phosphoglycerate kinase
	pLN-17	mEP17 targeting vector for homologous
		recombination
	RAR	Retinoic Acid Receptor
	RT-PCR	reverse transcription polymerase chain
		reaction
	SELDI	surface-enhanced laser desorption/
		ionization
	SPR	surface plasmon resonance
	TOF	time of flight mass spectroscopy

BACKGROUND ART

Recent studies of reproductive frequency in the United States report that 7% of married couples (greater than 2 million couples) describe difficulty in achieving a pregnancy. See Fidler and Bernstein (1999) Public Health Reports 114:494-511. Many individuals now seek medical support for conception, including infertility diagnosis and assisted reproductive treatment. The monetary costs for such services are substantial, and financial commitment must increase to include pre- and post-natal care of multiple birth pregnancies often associated with infertility treatment. A tumult of legal and ethical issues have emerged regarding the rights of parents and unborn children that are conceived by an unconventional method. The escalating magnitude of monetary, legal, and ethical concerns when considering pregnancy has established infertility as a significant public health issue.

Rational treatment approaches for many andrological disorders resulting in infertility are still lacking. See Kamischke and Nieschlag (1999) Human Reproduction 14(Suppl. 1):1-23. The cause of male infertility is often unidentifiable, referred to generally as “idiopathic infertility”, or the presumed pathology is not yet met with an unequivocal therapy. Intracytoplasmic sperm injection has been a successful method for enabling fertilization in many cases, yet a less interventive treatment is still sought. The historical use of approaches now deemed ineffective emphasizes the importance of thorough studies during early stages of therapy development. In particular, there is a need for more sophisticated diagnostic tools that detect molecular bases of male infertility and for non-surgical therapies that are supported by solid physiological data. An initial effort in this regard is the development of animal models of male infertility.

A related medical incentive is the development of new methods for contraception. See Baird and Glasier (1999) BMJ 319:969-972. The prevalence of contraceptive use is increasing worldwide, however, existing contraceptive means are limited by adverse side effects, inconvenience, and remaining instances of ineffectiveness. In particular, there are presently no safe and reversible means for male contraception. One strategy that has been explored recently is an immunological approach for disrupting endocrine or physiological events that normally promote pregnancy. Vaccines that comprise antigens of sperm plasma membrane proteins, zona pellucida proteins of the egg, or gonadotropin releasing hormone have shown success in suppressing fertility when administered to several mammalian subjects, including humans. See U.S. Pat. Nos. 6,096,318 and 6,132,270; Barber and Fayrer-Hosken (2000) J Reprod Immunol 46:103-124; Paterson et al. (2000) Cells Tissues Organs 166:228-232; Srivastav (2000) J Reprod Fertil 119:241-252; Feng et al. (1999) J Reprod Med 44:759-765; Naz (1999) Immunol Rev 171:193-202; Talwar (1999) Immunol Rev 171:173-192.

Although animal use and clinical trials of immunocontraceptive vaccines are encouraging, existing vaccines present significant complications, for example, auto-immune reactions. Thus, current research is focused on identifying new antigens that can provide safer vaccines. Animal models with implications for post-testicular human male contraceptives acting at the epididymis offer promising leads. See Nikkanen et al. (2000) Contraception 61:401-406; Cooper and Yeung (1999) Hum Reprod Update 5:141-152. To establish molecular targets for vaccine and drug development, proteins that are essential for epididymal function have been identified (Srivastav, 2000; Diekman et al. (1999) Immunol Rev 171:203-211; Costa et al. (1997) Biol Reprod 56:985-990; Sonnenberg-Riethmacher et al. (1996) Genes Dev 10:1184-1193).

During their transit through the epididymis, spermatozoa undergo biochemical and morphological changes to acquire motility and the ability to fertilize an oocyte in vivo. The maturation process occurs progressively along the epididymal duct and is believed to depend on epididymal secretory proteins. The epididymal epithelial cells secrete proteins in a highly regulated and regionalized manner such that spermatozoa encounter luminal fluid protein in a specific sequence. Indeed, each region within the epididymis is a unique microenvironment adapted with a characteristic milieu of ions, organic solutes, proteins, and steroids. See Cornwall et al. (2001) in “ The Epididymis”, Plenum Press. Spatially-restricted gene expression as well as regional differences in cellular morphology define three distinctive regions of the epididymis known as the caput, corpus, and cauda. These structural subdivisions and highly regionalized gene expression therein are observed in the epididymis of several organisms, including humans (Krull et al. (1993) Mol Reprod Dev 34:16-34).

Regionalization of the epididymis likely fulfills an essential and cumulative role in the maturation and survival of spermatozoa. In support thereof, targeted mutation of the mouse c-ros tyrosine kinase receptor confers male sterility, although sperm production is not affected. c-ros is normally expressed in the initial segment of the epididymis, and animals lacking c-ros function show specific underdevelopment and lack of cellular differentiation within the initial segment (Sonnenberg-Riethmacher et al., 1996). Sperm taken from a c-ros mutant mouse are less motile due to flagellar angulation, suggesting that failure of differentiation of epithelial cells in one segment of the epididymis can affect sperm maturation and survival (Yeung et al. (1999) Biol Reprod 61:1062-1069).

To generate regionalization within the epididymal epithelium, gene expression is precisely controlled, in part through transcriptional regulation. Transcription factors modulate transcription by binding DNA cis-regulatory sequences, most often located upstream of the gene promoter and transcription start site, and by concomitantly affecting assembly of cellular transcriptional machinery at the relevant promoter. Therefore, cis-regulatory sequences of epididymal-specific genes and the transcription factors that are operative through these sites are important elements in understanding epididymal function as it contributes to sperm maturation. Current approaches to identify mechanisms involved in region-specific gene expression in the epididymis have been limited by a lack of identified transcriptional regulatory proteins which are key to this process. See Cornwall et al. (2001).

Candidate regulators include components of retinoid signaling pathways. Most elements known to be involved in retinoid signaling are present in the epididymis, including epididymal retinoic acid binding protein (mE-RABP), cellular retinol-binding protein type I (CRBP I), cellular retinoic acid binding protein type I (CRABP I), retinoic acid receptor alpha (RARα), retinoic acid, and retinyl esters. Moreover, studies addressing the function of such elements emphasize the important role of retinoid signaling pathways in epididymal integrity. In retinoid deficient animals, there is widespread squamous metaplasia and keratinization of the epididymal epithelium (Wolbach (1925) J Exp Med 42:753-777), and abnormal synthesis and secretion of several epididymal proteins (Astraudo et al. (1995)Arch Androl35:247-259). Similarly, overexpression of a dominant negative form of RARα leads to disorganization of the epididymal epithelium and concomitant infertility (Costa et al., 1997). In a related study, RARα knockout mice display aspermatogenesis and vacuolization of the epididymal epithelium (Lufkin et al. (1993) Proc Natl Acad Sci USA 90:7225-7229), and animals lacking both RARα and RARγ function show epididymal dysplasia (Mendelsohn et al. (1994) Development 120:2749-2771).

The mE-RABP protein is of particular interest among regulators of retinoid signaling, as it appears to be expressed selectively in the mid and distal caput of the epididymis. mE-RABP is a member of a family of secreted lipocalin proteins. Structural analyses reveal that lipocalins comprise an eight-stranded β barrel that is closed at one end by an α-helical turn, thereby forming a hydrophobic binding cavity. This hydrophobic pocket is well-adapted for noncovalent binding and transport of small lipophilic ligands. mE-RABP binds active retinoids (9-cis and all-trans retinoic acid), and functions as a retinoid carrier protein in the epididymis. See Ong et al. (2000) Biochim Biophys Acta 1482(1-2):209-17.

Recent studies by the co-inventors of the present application have identified a similar gene encoding a 17 kDa lipocalin, Mouse Epididymal Protein of 17 kDa (mEP17) (Lareyre et al. (2001) Endocrinology 142:1296-1306). mEP17 and mE-RABP are significantly related by several measures that collectively suggest mEP17 also functions as a regulator of retinoid signaling in the epididymis. First, mE-RABP and mEP17 are positioned adjacent to each other on mouse chromosome 2. Exon/intron boundaries are strictly conserved between mE-RABP and mEP17, supporting that these genes arose by gene duplication. Second, mEP17 shows regionalized expression in the epididymis. mEP17 expression is limited to the initial segment of the caput epididymis, while mE-RABP is expressed in the adjacent mid and distal caput epididymis. Third, the mEP17 protein contains two motifs (G-X—W and T-D-Y) and two cysteine residues that are characteristic features shared by members of the lipocalin protein family. With the exception of these motifs, mEP17 shows low sequence similarity with other known lipocalins. However, it is well established that lipocalin family members do not show significant sequence homology (average 25% identity and 50% homology between representative members). Rather, lipocalins are more clearly related by assessing homology of secondary and tertiary structure. The tryptophan residue of the G-X—W motif is required for binding of lipophilic ligands, and the two cysteine residues form a intramolecular disulfide bond that influences ligand affinity. In addition, a putative signal sequence at the amino-terminal of the mEP17 precursor suggests that it is cleaved to generate a mature secreted protein, consistent with its identification as a lipocalin. These structural similarities between mEP17 and other lipocalins, most significantly mE-RABP, suggest that mEP17 is also a carrier for retinoid ligands.

The present invention relates to a current challenge in developing animal models of infertility, male fertility treatments, and male contraceptives. To this end, the present invention provides an isolated promoter region of the mEP17 gene, an isolated nucleic acid molecule encoding a human mEP17 gene (hEP17), an isolated promoter region of hEP17, and chimeric genes comprising the disclosed sequences. Host cells expressing a recombinant EP17 gene or an mEP17 promoter region operably linked to a reporter gene sequence are useful in screening assays for discovery of substances that modulate EP17. A chimeric gene comprising an mEP17 promoter region can also be used to direct transcription of a heterologous nucleotide sequence in the epididymis of a host organism. The present invention further provides an EP17 polypeptide that can be used for vaccine or drug development. By provision of epididymal lipocalin nucleotide and polypeptide sequences, and methods for using the same, the present invention meets a long-felt need for advancement in fertility research.

SUMMARY OF THE INVENTION

The present invention provides an isolated promoter region of an EP17 gene that reconstitutes endogenous expression in epididymis. In one preferred embodiment, a promoter region of the invention comprises a 5.3 kb fragment (GenBank Accession No. AF08222) of mouse genomic clone 10983 (Genome Systems, Inc.) between the EcoRV and SalI restriction sites, or functional portion thereof. More preferably, the functional portion of the promoter region comprises a TATA box and at least one cis-acting regulatory sequence selected from the group including but not limited to a Sp-1 binding site, an AP-1 binding site, a retinoic acid receptor binding site, an androgen receptor binding site, a C-Ets binding site, a SRY binding site, an APA binding site, a C/EBP binding site, and combinations thereof. Most preferably, an isolated promoter region of the present invention comprises the nucleotide sequence of SEQ ID NO:1, a nucleic acid molecule substantially identical to SEQ ID NO:1, or a 20 base pair nucleotide sequence identical to a contiguous 20 base pair nucleotide portion of SEQ ID NO:1.

The present invention also provides a human EP17 gene. Preferably, the human EP17 gene comprises the sequence set forth as SEQ ID:2, a nucleic acid molecule that is substantially similar to SEQ ID NO:2; or a nucleic acid molecule comprising a 20 base pair nucleotide sequence that is identical to a contiguous 20 base pair sequence of SEQ ID NOs:2. The present invention further provides an isolated promoter region derived from a human EP17 gene. In this case, a hEP17 promoter region is preferably an about 5160 base pair region immediately upstream of the human EP17 transcription start site. More preferably, an isolated promoter region of the present invention comprises a TATA box and at least one cis-acting regulatory sequence selected from the group including but not limited to Sp-1 binding site, an AP-1 binding site, a cAMP response element binding protein (CREB) binding site, a SRY-related HMG-box gene 5 (Sox5) binding site, a Sex-determining region Y gene product (SRY) binding site, a c-Ets binding site, a GATA binding site, an Octamer transcription factor 1 (Oct-1) binding site, and combinations thereof. Most preferably, an isolated promoter of the present invention comprises the nucleotide sequence of SEQ ID NO:5, a nucleic acid molecule substantially identical to SEQ ID NO:5, or a 20 base pair nucleotide sequence identical to a contiguous 20 base pair nucleotide portion of SEQ ID NO:5.

The present invention further provides a chimeric gene comprising an EP17 promoter region operably linked to a heterologous nucleotide sequence. Preferably, the EP17 promoter region comprises the nucleic acid molecule of SEQ ID NOs:1 or 5, or functional portion thereof. In a preferred embodiment, a chimeric gene of the invention is carried in a vector and expressed in a host cell including but not limited to a bacterial cell, a hamster cell, a mouse cell, or a human cell.

The present invention also provides a transgenic animal having a transgene that comprises a chimeric gene of the present invention. In a preferred embodiment, expression of the chimeric gene alters fertility of the host animal.

The present invention also provides a method for identifying a substance that regulates EP17 expression using a chimeric gene that includes an isolated EP17 promoter region operably linked to a reporter gene. According to this method, a gene expression system is established that includes the chimeric gene and components required for gene transcription and translation so that reporter gene expression is assayable. To select a substance that regulates EP17 expression, the method further provides the steps of using the gene expression system to determine a baseline level of reporter gene expression in the absence of a candidate regulator, providing a plurality of candidate regulators to the gene expression system, and assaying a level of reporter gene expression in the presence of a candidate regulator. A candidate regulator is selected whose presence results in an altered level of reporter gene expression when compared to the baseline level. Preferably, the Isolated EP17 promoter region used in this method comprises the sequence of SEQ ID NOs:1 or 5, or functional portion thereof.

In another aspect of the invention, a method is provided for producing an epididymal cell line using a chimeric gene comprising an EP17 promoter operably linked to a gene encoding a selectable marker. According to the method, a transgenic animal is generated that expresses a selectable marker gene. Preferably, the selectable marker gene is an antibiotic resistance gene. More preferably, the antibiotic resistance gene is a neomycin resistance gene. Epididymal cells are procured from the transgenic animal and stably reproduced in cell culture using selection of the marker gene. Preferably, the EP17 promoter region used to perform this method is the nucleic acid molecule of SEQ ID NO:1, or functional portion thereof.

Another aspect of the present invention pertains to a method for mutagenizing an EP17 locus by homologous recombination. The method uses a targeting vector having an isolated EP17 promoter region, a marker gene, and an isolated EP17 3′flanking region. In a vector so constructed, the marker gene is positioned between the promoter region and the 3′flanking region. In one embodiment, the targeting vector further comprises a mutant EP17 coding sequence, also positioned between the promoter region and the 3′ flanking region. The targeting vector is linearized by digestion with a restriction endonuclease at a site other than within the promoter region, marker gene, 3′ flanking region, and optional mutant EP17 coding sequence. The linearized vector is introduced into embryonic stem cells and is assayed by detecting the marker gene in the stem cells. Stem cells bearing the vector are used to create a transgenic vertebrate animal. According to the method, a homologous recombination event is mediated at the EP17 locus, thereby exchanging native mEP17 gene sequences positioned between the promoter region and the 3′ flanking region with vector nucleotide sequences positioned the same. In a more preferred embodiment, male EP17 mutant animals produced by the disclosed method are sterile.

The present invention also discloses a human EP17 polypeptide and an isolated nucleic acid sequence encoding the same. Preferably, an isolated EP17 polypeptide, or functional portion thereof, comprises a polypeptide encoded by the nucleic acid molecule of SEQ ID NO:3, a polypeptide encoded by a nucleic acid molecule that is substantially identical to SEQ ID NO:3, a polypeptide fragment encoded by a 20 nucleotide sequence that is identical to a contiguous 20 nucleotide sequence of SEQ ID NO:3; a polypeptide having an amino acid sequence of SEQ ID NO:4, a polypeptide that is a biological equivalent of the polypeptide of SEQ ID NO:4, or a polypeptide that is immunologically cross-reactive with an antibody that shows specific binding with a polypeptide comprising some or all amino acids of SEQ ID NO:4. Preferably, the polypeptide of the present invention comprises a human EP17 polypeptide.

The present invention further teaches chimeric genes having a heterologous promoter that drives expression of a nucleic acid sequence encoding an EP17 polypeptide. Preferably, the chimeric gene is carried in a vector and introduced into a host cell so that an EP17 polypeptide of the present invention is produced. Preferred host cells include but are not limited to a bacterial cell, a hamster cell, a mouse cell, or a human cell.

In another aspect of the invention, a method is provided for detecting a nucleic acid molecule that encodes an EP17 polypeptide. According to the method, a biological sample having nucleic acid material is hybridized under stringent hybridization conditions to an EP17 nucleic acid molecule of the present invention. Such hybridization enables a nucleic acid molecule of the biological sample and the EP17 nucleic acid molecule to form a detectable duplex structure. Preferably, the EP17 nucleic acid molecule includes some or all nucleotides of SEQ ID NOs:1, 2, 3, or 5. Also preferably, the biological sample comprises human nucleic acid material.

The present invention further teaches an antibody that specifically recognizes an EP17 polypeptide. Preferably, the antibody recognizes some or all amino acids of SEQ ID NO:4. A method for producing an EP17 antibody is also disclosed, and the method comprises recombinantly or synthetically producing an EP17 polypeptide, or portion thereof; formulating the EP17 polypeptide so that it is an effective immunogen; immunizing an animal with the formulated polypeptide to generate an immune response that includes production of EP17 antibodies; and collecting blood serum from the immunized animal containing antibodies that specifically recognize an EP17 polypeptide. Preferably, the EP17 polypeptide used as an immunogen includes some or all amino acid sequences of SEQ ID NO:4.

A method is also provided for detecting a level of EP17 polypeptide using an antibody that specifically recognizes an EP17 polypeptide. According to the method, a biological sample is obtained from an experimental subject and a control subject, and EP17 polypeptide is detected in the sample by immunochemical reaction with the EP17 antibody. Preferably, the antibody recognizes amino acids of SEQ ID NO:4 and is prepared according to a method of the present invention for producing such an antibody.

The present invention further discloses a method for identifying a compound that modulates EP17 function. The method comprises: exposing an isolated EP17 polypeptide to a plurality of compounds; and assaying binding of a compound to the isolated EP17 polypeptide. A compound is selected that demonstrates specific binding to the isolated EP17 polypeptide. Preferably, the EP17 polypeptide used in the binding assay of the method includes some or all amino acids of SEQ ID NO:4.

The present invention further provides a method for modulating EP17 function in a subject. According to the method, a pharmaceutical composition is prepared that includes a substance capable of modulating EP17 expression or function, and a carrier. An effective dose of the pharmaceutical composition is administered to a subject, whereby EP17 activity is altered in the subject. In a preferred embodiment, the substance used to perform this method shows specific binding to some or all amino acids of SEQ ID NO:4 and was discovered by a screening assay method of the present invention. In another embodiment, EP17 function is disrupted by immunizing a subject with an effective dose of the disclosed EP17 polypeptide. The immune system of the subject produces an antibody that specifically recognizes the—EP17 polypeptide, and preferably recognizes some or all of amino acids of SEQ ID NO:4. In a further embodiment, a gene therapy vector is used, the vector comprising a nucleotide sequence encoding an EP17 polypeptide. Alternatively, the gene therapy vector comprises a nucleotide sequence encoding a nucleic acid molecule, a peptide, or a protein that interacts with an EP17 nucleic acid or polypeptide. Preferably, the subject is a human subject.

A method is also provided for expressing a nucleotide sequence of interest in epididymis using an EP17 promoter region. According to the method, a gene therapy vector is prepared comprising an EP17 promoter region operably linked to a nucleotide sequence of interest. A gene therapy vector so-constructed is administered to a subject, whereby the nucleotide sequence of interest is expressed in epididymis. Preferably, the EP17 promoter comprises SE ID NO:5, or functional portion thereof. Also preferably, the subject is a human subject.

The invention further provides a method for diminishing the fertile capacity of a subject. According to the method, a chemical compound, peptide, or antibody that interacts with an EP17 polypeptide is identified. Preferably, the polypeptide is the sequence of SEQ ID NO:4 or 6. A pharmaceutical preparation is prepared comprising such a chemical compound, peptide, or antibody, and a carrier. An effective dose of the pharmaceutical composition is administered to a subject, whereby the fertile capacity of the subject is diminished.

The invention further provides a method for promoting the fertile capacity of a subject. In this case, a chemical compound or peptide that interacts with an EP17 polypeptide is identified. Preferably, the polypeptide is the sequence of SEQ ID NO:4 or 6. A pharmaceutical composition comprising the chemical compound or peptide and a carrier is prepared. An effective dose of the pharmaceutical composition is administered to a subject, whereby the fertile capacity of the subject is improved.

Accordingly, it is an object of the present invention to provide novel EP17 nucleic acid and polypeptide sequences, and novel methods relating thereto. This object is achieved in whole or in part by the present invention.

An object of the invention having been stated above, other objects and advantages of the present invention will become apparent to those skilled in the art after a study of the following description of the invention, Figures and non-limiting Examples. [0033]

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts genomic organization of the mEP17 gene. mEP17 is located upstream from mE-RABP within the locus[A3,B] of the [0034] mouse chromosome 2. Exon sizes are indicated in nucleotides. The major transcription initiation sites of both genes are represented with broken arrows. Primer “FwmEP17cDNA” (SEQ ID NO:7) was used for primer extension analysis. Two motifs G-X—W and T-D-Y and two cysteine residues (C) that contribute to the three dimensional structure of lipocalin proteins are also indicated.
FIG. 2A presents a Northern blot showing epididymis-specific expression of the mEP17 gene. Total RNA was extracted from individual tissues and hybridized with [[0035] ³²P]-labeled mEP17 cDNA. Two major transcripts of 1 kb and 3.1 kb in size were detected only in the epididymis.
FIG. 2B shows Northern blot analysis of total RNA extracted from the epididymis, hybridized with [[0036] ³²P]-labeled intron 1 of the mEP17 gene or with [³²P]-labeled mEP17 cDNA used as probes. The intron 1 probe only detected the 3.1 kb transcripts, suggesting that these transcripts are likely unspliced mEP17 precursor RNA.
FIG. 3 shows region-specific expression of the mEP17 gene in the initial segment of the epididymis. In situ hybridization of mEP17 transcripts is detected in the initial segment (IS) but not in the efferent duct (ED) and mid/distal caput epididymis (Cp). [0037]
FIGS. 4A and 4B show in situ hybridization of mEP17 in epididymal tissue, and also show cell-specific expression of the mEP17 gene. [0038]
FIG. 4A shows a high magnification view of the boxed region of FIG. 3 at the boundary between the initial (IS) and proximal caput epididymis (Cp). mEP17 mRNA is highly expressed only in the principal cells of the initial segment (IS). No staining is observed in the conjunctive tissue (CT) and in the epithelial cells of the proximal caput epididymis (Cp). [0039]
FIG. 4B shows hybridization of a section of the initial segment with a sense strand digoxygenin-labeled mEP17 RNA. No signal is detected. [0040]
FIG. 5 presents a comparison of the genomic structure of the mouse and human EP17 genes. The major transcription initiation sites (TIS) of both genes are indicated by broken arrows. The lipocalin-specific motifs (G-X—W, T-D-Y, and 2 cysteine residues) are also indicated. Black boxes indicate exons, and the line region between the boxes indicate introns. Numbers below the boxes and line regions indicate exon and intron sizes in base pairs. [0041]
FIG. 6 presents BLAST results using human EP17 cDNA sequence of SEQ ID NO:3 as the query sequence. The highest homologies were observed with other epididymal lipocalins (E-RABP and prostaglandin H[0042] ₂-D isomerases).
FIG. 7 presents a comparison of the amino acid sequences of mouse EP17 and human EP17 proteins. Conserved lipocalin motifs are indicated. The mouse and human EP17 proteins share 61% overall identity. [0043]
FIG. 8 shows hydropathic analysis of the murine and human EP17 proteins. [0044]
FIG. 9 depicts a restriction enzyme map of plasmids derived from BAC clone 10983 aligned with the mEP17/mE-RABP genomic region. The promoter region of mEP17, mEP17 exons, the intergenic region, and mE-RABP exons are indicated. [0045]
FIG. 10 shows primer extension analysis of the 5′ end of mEP17 mRNA. Total RNA extracted from the epididymis (Ep) or transfer (t) RNA was reverse transcribed with [[0046] ³²P]-radiolabeled mEP17PE2 primer (SEQ ID NO:7) and extended using Avian Myeloblastosis virus (AMV) reverse transcriptase. Lanes labeled “C”, “T”, “A” and “G” are [³⁵S]-radiolabeled DNA sequencing reactions carried out using the mEP17PE2 primer (SEQ ID NO:7) and the pHindIII clone (indicated in FIG. 9) as template. The localization of two major (arrows) and two minor (arrowheads) transcription initiation sites are indicated.
FIG. 11 shows the nucleotide sequence of the mEP17 5.3 kb promoter region. Putative cis-DNA regulatory elements within the 5′ flanking region are underlined, including binding sites for androgen receptor (ARSB), retinoic acid receptor (RARE), Stimulating Protein 1 (SP-1), Activator Protein 1 (AP-1), Octamer transcription factor 1 (Oct-1), and Sox-5 (SRY-related [0047] Sequence #5 Protein). A consensus TATA box is indicated.
FIG. 12 depicts constructs used in functional assays of the mEP17 promoter, each construct comprising a different fragment of the 5.3 kb mEP17 promoter region (solid lines), an open reading frame encoding an exemplary reporter gene (chloramphenicol acetyltransferase, solid bar labeled “CAT”) operably linked to a promoter fragment, and the polyA tail region of [0048] Simian virus 40 large T antigen.
FIG. 13 shows the nucleotide sequence of the human EP17 promoter region. Putative cis-DNA regulatory elements are underlined, including a Stimulatory Protein 1 (Sp-1) binding site, an Activator Protein 1 (AP-1) binding site, a cAMP response element binding protein (CREB) site, a SRY-related HMG-box gene (Sox5) binding site, a Sex-determining region Y gene product (SRY) binding site, a c-Ets binding site, a GATA binding site, and an Octamer transcription factor 1 (Oct-1) binding site. [0049]
FIG. 14 presents a comparison of the putative cis-DNA regulatory elements in the mouse and human EP17 promoter regions. [0050]
FIGS. 15A, 15B, and [0051] 15C present experiments demonstrating hormonal regulation of mEP17 transcription.
FIG. 15A shows Northern blot analysis of epididymal total RNA (10 μg/lane) extracted from intact (I) and castrated animals at 5, 10 , 20, and 30 days following castration (C5, C10, C20 and C30 respectively), hybridized with [[0052] ³²P]-labeled mEP17 cDNA.
FIG. 15B shows Northern blot analysis to detect mEP17 RNA four days following hemicastration. Levels of mEP17 RNA in the epididymis of the castrated side (HI) are reduced to 0.7% of RNA levels in the epididymis of the non-castrated side (HC). [0053]
FIG. 15C shows Northern blot analysis to detect [0054] mEP17 cDNA 5 days after castration (C5) and 5 days after castration and androgen replacement (P).
FIG. 16 depicts homologous recombination at the mEP17 locus using the pLN-17 vector. The mEP17/mE-RABP genomic region is presented at the top. mEP17 exons are indicated by hatched rectangles. mE-RABP exons are indicated by open rectangles. The mEP17 targeting plasmid pLN-17 is designed so that 1.4 kb of [0055] mEP17 5′ flanking region is positioned immediately upstream of the vector PGK neomycin sequence, and 10.9 kb of mEP17 3′ flanking region and mE-RABP gene is positioned immediately downstream of the vector PGK neomycin sequence. mEP17 sequences carried in the pLN-17 targeting vector mediate homologous recombination, depicted as an “X” between the genomic region and the targeting plasmid. The recombination event creates a genomic reorganization wherein the entire mEP17 coding sequence is replaced by the PGK neomycin sequence.
FIG. 17 shows recombinant production of mEP17 protein using the pBAD/gIII vector (Invitrogen). Protein extracted from [0056] E.coli transformed with pBAD/gIII-mEP17 is resolved by polyacrylamide gel electrophoresis.
FIG. 17A shows Coomassie blue staining that identifies two enriched protein species (boxed). [0057]
FIG. 17B shows Western blot analysis using an anti-his tag antibody to detect two recombinant proteins of approximately 21 and 23 kDa, corresponding to the processed and non-processed mEP17 isoforms.[0058]

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acids comprising a lipocalin gene promoter region (representative embodiments set forth as SEQ ID NOs:1 and 5), isolated nucleic acids comprising a human lipocalin gene (a representative embodiment set forth as SEQ ID NO:2), isolated nucleic acids encoding a lipocalin polypeptide (a representative embodiment set forth as SEQ ID NO:3), isolated lipocalin polypeptides (a representative embodiment set forth as SEQ ID NO:4), and uses thereof. The disclosed lipocalin nucleic acids and polypeptides can be used according to methods of the present invention to generate a mouse model of male infertility, for drug discovery screens, and for therapeutic treatment of fertility-related conditions, among other uses. [0059]

I. Definitions

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the invention. The entire contents of all publications mentioned herein, including the discussion of the background art presented above, are hereby fully incorporated by reference. [0060]

I.A. EP17 Nucleic Acids

The nucleic acid molecules provided by the present invention include the isolated nucleic acid molecules of SEQ ID NOs:1, 2, 3, and 5, sequences substantially similar to sequences of SEQ ID NOs:1, 2, 3, and 5, conservative variants thereof, subsequences and elongated sequences thereof, complementary DNA molecules, and corresponding RNA molecules. The present invention also encompasses genes, cDNAs, chimeric genes, and vectors comprising disclosed EP17 nucleic acid sequences. [0061]
The term “nucleic acid molecule” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar properties as the reference natural nucleic acid. Unless otherwise indicated, a particular nucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions), complementary sequences, subsequences, elongated sequences, as well as the sequence explicitly indicated. The terms “nucleic acid molecule” or “nucleotide sequence” can also be used in place of “gene”, “cDNA”, or “mRNA”. Nucleic acids can be derived from any source, including any organism. [0062]
The term “isolated”, as used in the context of a nucleic acid molecule, indicates that the nucleic acid molecule exists apart from its native environment and is not a product of nature. An isolated DNA molecule can exist in a purified form or can exist in a non-native environment such as a transgenic host cell. [0063]
The term “Purified”, when applied to a nucleic acid, denotes that the nucleic acid is essentially free of other cellular components with which it is associated in the natural state. Preferably, a purified nucleic acid molecule is a homogeneous dry or aqueous solution. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure. [0064]
The term “substantially identical”, the context of two nucleotide or amino acid sequences, can also be defined as two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90-95%, and most preferably at least 99% nucleotide or amino acid sequence identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms (described herein below under the heading [0065] Nucleotide and Amino Acid Sequence Comparisons) or by visual inspection. Preferably, the substantial identity exists in nucleotide sequences of at least 50 residues, more preferably in nucleotide sequence of at least about 100 residues, more preferably in nucleotide sequences of at least about 150 residues, and most preferably in nucleotide sequences comprising complete coding sequences. In one aspect, polymorphic sequences can be substantially identical sequences. The term “polymorphic” refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. An allelic difference can be as small as one base pair.
Another indication that two nucleotide sequences are substantially identical is that the two molecules specifically or substantially hybridize to each other under stringent conditions. In the context of nucleic acid hybridization, two nucleic acid sequences being compared can be designated a “probe” and a “target”. A “probe” is a reference nucleic acid molecule, and a “target” is a test nucleic acid molecule, often found within a heterogenous population of nucleic acid molecules. A “target sequence” is synonymous with a “test sequence”. [0066]
A preferred nucleotide sequence employed for hybridization studies or assays includes probe sequences that are complementary to or mimic at least an about 14 to 40 nucleotide sequence of a nucleic acid molecule of the present invention. Preferably, probes comprise 14 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of any of SEQ ID NOs:1, 2, 3, and 5. Such fragments can be readily prepared by, for example, directly synthesizing the fragment by chemical synthesis, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production. The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA). The phrase “binds substantially to” refers to complementary hybridization between a probe nucleic acid molecule and a target nucleic acid molecule and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired hybridization. [0067]
“Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern blot analysis are both sequence- and environment-dependent. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) “[0068] Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes” part I chapter 2, Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5 C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize specifically to its target subsequence, but to no other sequences.
The T[0069] _mis the temperature (underdefined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T_mfor a particular probe. An example of stringent hybridization conditions for Southern or Northern Blot analysis of complementary nucleic acids having more than about 100 complementary residues is overnight hybridization in 50% formamide with 1 mg of heparin at 42° C. An example of highly stringent wash conditions is 15 minutes in 0.15 M NaCl at 65° C. An example of stringent wash conditions is 15 minutes in 0.2×SSC buffer at 65° C. (See Sambrook (1989) for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of medium stringency wash conditions for a duplex of more than about 100 nucleotides, is 15 minutes in 1×SSC at 45° C. An example of low stringency wash for a duplex of more than about 100 nucleotides, is 15 minutes in 4-6×SSC at 40° C. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0-8.3, and the temperature is typically at least about 30° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2-fold (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
The following are examples of hybridization and wash conditions that can be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a probe nucleotide sequence preferably hybridizes to a target nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO[0070] ₄, 1 mM EDTA at 50° C. followed by washing in 2×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed by washing in 1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed by washing in 0.5×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at 65° C.
A further indication that two nucleic acid sequences are substantially identical is that proteins encoded by the nucleic acids are substantially identical, share an overall three-dimensional structure, are biologically functional equivalents; or are immunologically cross-reactive. These terms are defined further under the heading EP17 Polypeptides herein below. Nucleic acid molecules that do not hybridize to each other under stringent conditions are still substantially identical if the corresponding proteins are substantially identical. This can occur, for example, when two nucleotide sequences are significantly degenerate as permitted by the genetic code. [0071]
The term “conservatively substituted variants” refers to nucleic acid sequences having degenerate codon substitutions wherein the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) [0072] Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608; Rossolini et al. (1994) Mol Cell Probes 8:91-98).
The term “subsequence” refers to a sequence of nucleic acids that comprises a part of a longer nucleic acid sequence. An exemplary subsequence is a probe, described herein above, or a primer. The term “primer” as used herein refers to a contiguous sequence comprising about 8 or more deoxyribonucleotides or ribonucleotides, preferably 10-20 nucleotides, and more preferably 20-30. nucleotides of a selected nucleic acid molecule. The primers of the invention encompass oligonucleotides of sufficient length and appropriate sequence so as to provide initiation of polymerization on a nucleic acid molecule of the present invention. [0073]
The term “elongated sequence” refers to an addition of nucleotides (or other analogous molecules) incorporated into the nucleic acid. For example, a polymerase (e.g., a DNA polymerase), .g., a polymerase which adds sequences at the 3′ terminus of the nucleic acid molecule. In addition, the nucleotide sequence can be combined with other DNA sequences, such as promoters, promoter regions, enhancers, polyadenylation signals, intronic sequences, additional restriction enzyme sites, multiple cloning sites, and other coding segments. [0074]
The term “complementary sequence”, as used herein, indicates two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between base pairs. As used herein, the term “complementary sequences” means nucleotide sequences which are substantially complementary, as can be assessed by the same nucleotide comparison set forth above, or is defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein. A particular example of a complementary nucleic acid segment is an antisense oligonucleotide. [0075]
The term “gene” refers broadly to any segment of DNA associated with a biological function. A gene encompasses sequences including but not limited to a coding sequence, a promoter region, a cis-regulatory sequence, a non-expressed DNA segment is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof. A gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation of an existing sequence. [0076]
The term “promoter region” defines a nucleotide sequence within a gene that is positioned 5′ to a coding sequence of a same gene and functions to direct transcription of the coding sequence. The promoter region includes a transcriptional start site and at least one cis-regulatory element. The present invention encompasses nucleic acid sequences that comprise a promoter region of an EP17 gene, or functional portion thereof. [0077]
The term “cis-acting regulatory sequence” or “cis-regulatory motif” or “response element”, as used herein, each refer to a nucleotide sequence that enables responsiveness to a regulatory transcription factor. Responsiveness can encompass a decrease or an increase in transcriptional output and is mediated by binding of the transcription factor to the DNA molecule comprising the response element. [0078]
The term “transcription factor” generally refers to a protein that modulates gene expression by interaction with the cis-regulatory element and cellular components for transcription, including RNA Polymerase, Transcription Associated Factors (TAFs), chromatin-remodeling proteins, and any other relevant protein that impacts gene transcription. [0079]
The term “gene expression” generally refers to the cellular processes by which a biologically active polypeptide is produced from a DNA sequence. [0080]
A “functional portion” of a promoter gene fragment is a nucleotide sequence within a promoter region that is required for normal gene transcription. To determine nucleotide sequences that are functional, the expression of a reporter gene is assayed when variably placed under the direction of a promoter region fragment. [0081]
Promoter region fragments can be conveniently made by enzymatic digestion of a larger fragment using restriction endonucleases or DNAse I. Preferably, a functional promoter region fragment comprises about 5000 nucleotides, more preferably 2000 nucleotides, more preferably about1000 nucleotides, more preferably a functional promoter region fragment comprises about 500 nucleotides, even more preferably a functional promoter region fragment comprises about 100 nucleotides, and even more preferably a functional promoter region fragment comprises about 20 nucleotides. [0082]
The terms “reporter gene” or “marker gene” or “selectable marker” each refer to a heterologous gene encoding a product that is readily observed and/or quantitated. A reporter gene is heterologous in that it originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form. Non-limiting examples of detectable reporter genes that can be operably linked to a transcriptional regulatory region can be found in Alam and Cook (1990) [0083] Anal Biochem 188:245-254 and PCT International Publication No. WO 97/47763. Preferred reporter genes for transcriptional analyses include the lacZ gene (See, e.g., Rose and Botstein (1983) Meth Enzymol 101:167-180), Green Fluorescent Protein (GFP) (Cubitt et al. (1995) Trends Biochem Sci 20:448455), luciferase, or chloramphenicol acetyl transferase (CAT). Preferred reporter genes for methods to produce transgenic animals include but are not limited to antibiotic resistance genes, and more preferably the antibiotic resistance gene confers neomycin resistance. Any suitable reporter and detection method can be used, and it will be appreciated by one of skill in the art that no particular choice is essential to or a limitation of the present invention.
An amount of reporter gene can be assayed by any method for qualitatively or preferably, quantitatively determining presence or activity of the reporter gene product. The amount of reporter gene expression directed by each test promoter region fragment is compared to an amount of reporter gene expression to a control construct comprising the reporter gene in the absence of a promoter region fragment. A promoter region fragment is identified as having promoter activity when there is significant increase in an amount of reporter gene expression in a test construct as compared to a control construct. The term “significant increase”, as used herein, refers to an quantified change in a measurable quality that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater relative to a control measurement, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater. [0084]
The present invention also encompasses chimeric genes comprising the disclosed EP17 sequences. The term “chimeric gene”, as used herein, refers to an EP17 promoter region operably linked to an open reading frame, wherein the nucleotide sequence created is not naturally occurring. In this regard, the open reading frame is also described as a “heterologous sequence”. The term “chimeric gene” also encompasses a promoter region operably linked to an EP17 coding sequence, a nucleotide sequence producing an antisense RNA molecule, a RNA molecule having tertiary structure, such as a hairpin structure, or a double-stranded RNA molecule. [0085]
The term “operably linked”, as used herein, refers to a promoter region that is connected to a nucleotide sequence in such a way that the transcription of that nucleotide sequence is controlled and regulated by that promoter region. Techniques for operatively linking a promoter region to a nucleotide sequence are well known in the art. [0086]
The terms “heterologous gene”, “heterologous DNA sequence”, “heterologous nucleotide sequence”, “exogenous nucleic acid molecule”, or “exogenous DNA segment”, as used herein, each refer to a sequence that originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example by mutagenesis or by isolation from native cis-regulatory sequences. The terms also includes non-naturally occurring multiple copies of a naturally occurring nucleotide sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid wherein the element is not ordinarily found. [0087]
The present invention further includes vectors comprising the disclosed EP17 sequences, including plasmids, cosmids, and viral vectors. The term “vector”, as used herein refers to a DNA molecule having sequences that enable its replication in a compatible host cell. A vector also includes nucleotide sequences to permit ligation of nucleotide sequences within the vector, wherein such nucleotide sequences are also replicated in a compatible host cell. A vector can also mediate recombinant production of an EP17 polypeptide, as described further herein below. Preferred vectors include but are not limited to pBluescript (Stratagene), pUC18, pBLCAT3 (Luckow and Schutz (1987) [0088] Nucleic Acids Res 15:5490), pLNTK (Gorman et al. (1996) Immunity 5:241-252), and pBAD/gIII (Stratagene). A preferred host cell is a mammalian cell; more preferably the cell is a Chinese hamster ovary cell, a HeLa cell, a baby hamster kidney cell, or a mouse cell; more preferably the cell is a mouse epididymal cell; even more preferably the cell is a human cell.
Nucleic acids of the present invention can be cloned, synthesized, recombinantly altered, mutagenized, or combinations thereof. Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are well known in the art. Exemplary, non-limiting methods are described by Sambrook et al., eds. (1989) “[0089] Molecular Cloning”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; by Silhavy et al. (1984) “Experiments with Gene Fusions”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; by Ausubel et al. (1992) Current Protocols in Molecular Biology John Wylie and Sons, Inc. New York; and by Glover, ed. (1985) “DNA Cloning: A Practical Approach”, MRL Press, Ltd., Oxford, U.K. Site-specific mutagenesis to create base pair changes, deletions, or small insertions are also well known in the art as exemplified by publications, see. e.g., Adelman et al., (1983) DNA 2:183; Sambrook et al. (1989).
Sequences detected by methods of the invention can be detected, subcloned, sequenced, and further evaluated by any measure well known in the art using any method usually applied to the detection of a specific DNA sequence including but not limited to dideoxy sequencing, PCR, oligomer restriction (Saiki et al., [0090] Bio/Technology 3:1008-1012 (1985), allele-specific oligonucleotide (ASO) probe analysis (Conner et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:278), and oligonucleotide ligation assays (OLAs) (Landgren et. al. (1988) Science 241:1007). Molecular techniques for DNA analysis have been reviewed (Landgren et. al. (1988) Science 242:229-237).

I.B. EP17 Polypeptides

The polypeptides provided by the present invention include the isolated polypeptide of SEQ ID NO:4, polypeptides substantially similar to sequences of SEQ ID NO:4, EP17 polypeptide fragments, fusion proteins comprising EP17 amino acid sequences, biologically functional analogs, and polypeptides that cross-react with an antibody that specifically recognizes an EP17 polypeptide. [0091]
The term “isolated”, as used in the context of a polypeptide, indicates that the polypeptide exists apart from its native environment and is not a product of nature. An isolated polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell. [0092]
The term “purified”, when applied to a polypeptide, denotes that the polypeptide is essentially free of other cellular components with which it is associated in the natural state. Preferably, a polypeptide is a homogeneous solid or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide which is the predominant species present in a preparation is substantially purified. The term “purified” denotes that a polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the polypeptide is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure. [0093]
The term “substantially identical” in the context of two or more polypeptides sequences is measured by (a) polypeptide sequences having about 35%, or 45%, or preferably from 45-55%, or more preferably 55-66%, or most preferably 65% or greater amino acids which are identical. or functionally equivalent. Percent “identity” and methods for determining identity are defined herein below under the heading [0094] Nucleotide and Amino Acid Sequence Comparisons.
Substantially identical polypeptides also encompass two or more polypeptides sharing a conserved three-dimensional structure. Computational methods can be used to compare structural representations, and structural superpositions can be generated and easily tuned to identify similarities around important active sites or ligand binding sites. See Henikoff et al. (2000) [0095] Electrophoresis 21(9):1700-1706; Huang et al. (2000) Pac Symp Biocomput 230-241; Saqi et al. (1999) Bioinformatics 15(6):521-522; and Barton (1998) Acta Crystallogr D Biol Crystallogr 54:1139-1146.
The term “functionally equivalent” in the context of amino acid sequences is well known in the art and is based on the relative similarity of the amino acid side-chain substituents. See Henikoff and Henikoff (2000) [0096] Adv Protein Chem 54:73-97. Relevant factors for consideration include side-chain hydrophobicity, hydrophilicity, charge, and size. For example, arginine, lysine, and histidine are all positively charged residues; that alanine, glycine, and serine are all of similar size; and that phenylalanine, tryptophan, and tyrosine all have a generally similar shape. By this analysis, described further herein below, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine; are defined herein as biologically functional equivalents.
In making biologically functional equivalent amino acid substitutions, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). [0097]
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al. (1982) [0098] J Mol Biol 157:105.). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 of the original value is preferred, those which are within ±1 of the original value are particularly preferred, and those within ±0.5 of the original value are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein. [0099]
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). [0100]
In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 of the original value is preferred, those which are within ±1 of the original value are particularly preferred, and those within ±0.5 of the original value are even more particularly preferred. [0101]
The present invention also encompasses EP17 polypeptide fragments or functional portions of an EP17 polypeptide. Such functional portion need not comprise all or substantially all of the amino acid sequence of a native lipocalin gene product. The term “functional” includes any biological activity or feature of EP17, including immunogenicity. [0102]
The present invention also includes longer sequences an EP17 polypeptide, or portion thereof. For example, one or more amino acids can be added to the N-terminal or C-terminal of an EP17 polypeptide. Fusion proteins comprising EP17 polypeptide sequences are also provided within the scope of the present invention. Methods of preparing such proteins are known in the art. [0103]
The present invention also encompasses functional analogs of an EP17 polypeptide. Functional analogs share at least one biological function with an EP17 polypeptide. An exemplary function is immunogenicity. In the context of amino acid sequence, biologically functional analogs, as used herein, are peptides in which certain, but not most or all, of the amino acids can be substituted. Functional analogs can be created at the level of the corresponding nucleic acid molecule, altering such sequence to encode desired amino acid changes. In one embodiment, changes can be introduced to improve the antigenicity of the protein. In another embodiment, an EP17 polypeptide sequence is varied so as to assess the activity of a mutant EP17 polypeptide. [0104]
The present invention also encompasses recombinant production of the disclosed EP17 polypeptides. Briefly, a nucleic acid sequence encoding an EP17 polypeptide, or portion thereof, is cloned into a expression cassette, the cassette is introduced into a host organism, where it is recombinantly produced. [0105]
The term “expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest can be chimeric. The expression cassette can also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. [0106]
The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. Exemplary promoters include [0107] Simian virus 40 early promoter, a long terminal repeat promoter from retrovirus, an action promoter, a heat shock promoter, and a metallothionein protein. In the case of a multicellular organism, the promoter and promoter region can direct expression to a particular tissue or organ or stage of development. Exemplary tissue-specific promoter regions include a mE-RABP promoter and an EP17 promoter, described herein above. Suitable expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus, yeast vectors, bacteriophage vectors (e.g., lambda phage), and plasmid and cosmid DNA vectors.
The term “host cell”, as used herein, refers to a cell into which a heterologous nucleic acid molecule has been introduced. Transformed cells, tissues, or organisms are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. [0108]
A host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. For example, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. Expression in a bacterial system can be used to produce a non-glycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in animal cells can be used to ensure “native” glycosylation of a heterologous protein. [0109]
Expression constructs are transfected into a host cell by any standard method, including electroporation, calcium phosphate precipitation, DEAE-Dextran transfection, liposome-mediated transfection, and infection using a retrovirus. The EP17-encoding nucleotide sequence carried in the expression construct can be stably integrated into the genome of the host or it can be present as an extrachromosomal molecule. [0110]
Isolated polypeptides and recombinantly produced polypeptides can be purified and characterized using a variety of standard techniques that are well known to the skilled artisan. See, [0111] e.g. chapter 16 of Ausubel et al. (1992), Bodanszky, et al. (1976) “Peptide Synthesis”, John Wiley and Sons, Second Edition, New York., and Zimmer et al. (1993) “Peptides”pp. 393-394, ESCOM Science Publishers, B. V.

I.C. Nucleotide and Amino Acid Sequence Comparisons

The terms “identical” or percent “identity” in the context of two or more nucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms disclosed herein or by visual inspection. [0112]
The term “substantially identical” in regards to a nucleotide or polypeptide sequence means that a particular sequence varies from the sequence of a naturally occurring sequence by one or more deletions, substitutions, or additions, the net effect of which is to retain at least some of biological activity of the natural gene, gene product, or sequence. Such sequences include “mutant” sequences, or sequences wherein the biological activity is altered to some degree but retains at least some of the original biological activity. The term “naturally occurring”, as used herein, is used to describe a composition that can be found in nature as distinct from being artificially produced by man. For example, a protein or nucleotide sequence present in an organism, which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring. [0113]
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer program, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are selected. The sequence comparison algorithm then calculates the percent sequence identity for the designated test sequence(s) relative to the reference sequence, based on the selected program parameters. [0114]
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) [0115] Adv Appl Math 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol Biol 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc Natl Acad Sci USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.), or by visual inspection (See generally, Ausubel et al. (1992)).
A preferred algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) [0116] J Mol Biol 215: 403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always<0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength W=11, an expectation E10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See Henikoff and Henikoff (1989) Proc Natl Aced Sci USA 89:10915.
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See. e.g., Karlin and Altschul (1993) [0117] Proc Natl Acad Sci USA 90:5873-5887. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

I.D. Antibodies

The present invention also provides an antibody immunoreactive with an EP17 polypeptide. The term “antibody” indicates an immunoglobulin protein, or functional portion thereof, including a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, Fab fragments, and an Fab expression library. “Functional portion” refers to the part of the protein that binds a molecule of interest. In a preferred embodiment, an antibody of the invention is a monoclonal antibody. Techniques for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane (1988) “[0118] Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). A monoclonal antibody of the present invention can be readily prepared through use of well-known techniques such as the hybridoma techniques exemplified in U.S. Pat. No 4,196,265 and the phage-displayed techniques disclosed in U.S. Pat. No. 5,260,203.
The phrase “specifically (or selectively) binds to an antibody”, or “specifically (or selectively) immunoreactive with”, when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biological materials. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not show significant binding to other proteins present in the sample. Specific binding to an antibody under such conditions can require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to a protein with an amino acid sequence encoded by any of the nucleic acid sequences of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with unrelated proteins. [0119]
The use of a molecular cloning approach to generate antibodies, particularly monoclonal antibodies, and more particularly single chain monoclonal antibodies, are also provided. The production of single chain antibodies has been described in the art. See, e.g., U.S. Pat. No. 5,260,203. For this approach, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning on endothelial tissue. The advantages of this approach over conventional hybridoma techniques are that approximately 10[0120] ⁴times as many antibodies can be produced and screened in a single round, and that new specificities are generated by heavy (H) and light (L) chain combinations in a single chain, which further increases the chance of finding appropriate antibodies. Thus, an antibody of the present invention, or a “derivative” of an antibody of the present invention, pertains to a single polypeptide chain binding molecule which has binding specificity and affinity substantially similar to the binding specificity and affinity of the light and heavy chain aggregate variable region of an antibody described herein.
The term “immunochemical reaction”, as used herein, refers to any of a variety of immunoassay formats used to detect antibodies specifically bound to a particular protein, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. See Harlow and Lane (1988) for a description of immunoassay formats and conditions. [0121]

I.E. Protein Binding Assays

The term “binding” refers to an affinity between two molecules, for example, a ligand and a receptor, means a preferential binding of one molecule for another in a mixture of molecules. The binding of the molecules can be considered specific if the binding affinity is about 1×10[0122] ⁴M⁻¹to about 1×10⁶M⁻¹or greater. Binding of two molecules also encompasses a quality or state of mutual action such that an activity of one protein or compound on another protein is inhibitory (in the case of an antagonist) or enhancing (in the case of an agonist).
Fluorescence Correlation Spectroscopy (FCS) theory was developed in 1972 but it is only in recent years that the technology to perform FCS became available (Madge et al. (1972) [0123] Phys Re. Lett 29:705-708; Maiti et al. (1997) Proc Natl Acad Sci USA, 94: 11753-11757). FCS measures the average diffusion rate of a fluorescent molecule within a small sample volume. The sample size can be as low as 10³fluorescent molecules and the sample volume as low as the cytoplasm of a single bacterium. The diffusion rate is a function of the mass of the molecule and decreases as the mass increases. FCS can therefore be applied to protein-ligand interaction analysis by measuring the change in mass and therefore in diffusion rate of a molecule upon binding. In a typical experiment, the target to be analyzed is expressed as a recombinant protein with a sequence tag, such as a poly-histidine sequence, inserted at the N-terminus or C-terminus. The expression takes place in E. coli, yeast or mammalian cells. The protein is purified by chromatography. For example, the poly-histidine tag can be used to bind the expressed protein to a metal chelate column such as Ni2+ chelated on iminodiacetic acid agarose. The protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODIPY™ (Molecular Probes, Eugene, Oreg.). The protein is then exposed in solution to the potential ligand, and its diffusion rate is determined by FCS using instrumentation available from Carl Zeiss, Inc. (Thornwood, N.Y.). Ligand binding is determined by changes in the diffusion rate of the protein.
Surface-Enhanced Laser Desorption/Ionization (SELDI) was invented by Hutchens and Yip (1993) [0124] Rapid Commun Mass Spectrom 7:576-580). When coupled to a time-of-flight mass spectrometer (TOF), SELDI provides means to rapidly analyze molecules retained on a chip. It can be applied to ligand-protein interaction analysis by covalently binding the target protein on the chip and analyze by MS the small molecules that bind to this protein (Worrall et al. (1998) Anal. Biochem. 70: 750-756). In a typical experiment, the target to be analyzed is expressed as described for FCS. The purified protein is then used in the assay without further preparation. It is bound to the SELDI chip either by utilizing the poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. The chip thus prepared is then exposed to the potential ligand via, for example, a delivery system able to pipet the ligands in a sequential manner (autosampler). The chip is then submitted to washes of increasing stringency, for example a series of washes with buffer solutions containing an increasing ionic strength. After each wash, the bound material is analyzed by submitting the chip to SELDI-TOF. Ligands that specifically bind the target are identified by the stringency of the wash needed to elute them.
Biacore relies on changes in the refractive index at the surface layer upon binding of a ligand to a protein immobilized on the layer. In this system, a collection of small ligands is injected sequentially in a 2-5 microliter cell, wherein the protein is immobilized within the cell. Binding is detected by surface plasmon resonance (SPR) by recording laser light refracting from the surface. In general, the refractive index change for a given change of mass concentration at the surface layer is practically the same for all proteins and peptides, allowing a single method to be applicable for any protein (Liedberg et al. (1983) [0125] Sensors Actuators 4:299-304; Malmquist (1993) Nature 361:186-187). In a typical experiment, the target to be analyzed is expressed as described for FCS. The purified protein is then used in the assay without further preparation. It is bound to the Biacore chip either by utilizing the poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. The chip thus prepared is then exposed to the potential ligand via the delivery system incorporated in the instruments sold by Biacore (Uppsala, Sweden) to pipet the ligands in a sequential manner (autosampler). The SPR signal on the chip is recorded and changes in the refractive index indicate an interaction between the immobilized target and the ligand. Analysis of the signal kinetics on rate and off rate allows the discrimination between non-specific and specific interaction.

I.F. Transgenic Animals

It is also within the scope of the present invention to prepare a transgenic animal to mutagenize the EP17 locus or to express a transgene comprising nucleic acid sequences of the present invention. The term “transgenic animal”, indicates an animal comprising a germline insertion of a heterologous nucleic acid. Transgenic animals of the present invention are understood to encompass not only the end product of a transformation method, but also transgenic progeny thereof. [0126]
The term “transgene”, as used herein indicates a heterologous nucleic acid molecule that has been transformed into a host cell. For intended use in the creation of a transgenic animal, the transgene includes genomic sequences of the host organism at a selected locus or site of transgene integration to mediate a homologous recombination event. A transgene further comprises nucleic acid sequences of interest, for example a targeted modification of the gene residing within the locus, a reporter gene, or a expression cassette, each defined herein above. [0127]
Transgene integration can be used to create gene mutations, including “knock-out”, “knock-in”, or a “knock-down” mutations. The term “knock-out” refers to a homologous recombination event that renders a gene inactive. Gene knock-out is generally accomplished by integration of the transgene at a chromosomal loci, thereby interrupting a gene residing at that loci. The term “knock-in” refers to in vivo replacement at a targeted locus. Knock-in mutations can modify a gene sequence to create a loss-of-function or gain-of-function mutation. The term “gene knock-down” refers to a homologous recombination event wherein the transgene partially eliminates gene function. A knock-down animal can be created by transgenic expression of an antisense molecule, wherein a transgene comprising the antisense sequence and a relevant promoter are integrated into the genome at a non-essential loci. Expression of the antisense or ribozyme molecule disrupts the corresponding gene function, although this disruption is generally incomplete (Luyckx et al. (1999) [0128] Proc Natl Acad Sci U S A 96(21):12174-12179).
Conditional mutation can be accomplished using transgenic methods in combination with the Cre-recombinase system in mice. Briefly, in one instance, a transgenic mouse is derived that expresses Cre-recombinase under the direction of an inducible promoter. A second transgenic mouse bears a mutation of a gene of interest as well as a lox-P-flanked endogenous gene sequence. Such transgenic mice are mated, the resulting progeny having both the Cre-recombinase and lox-P-flanked transgenes. Induction of Cre recombinase catalyzes excision of the lox-P-flanked transgene, thereby excising a portion of the endogenous gene sequence and revealing the mutated sequence. Conditional knockout can be varied according to the temporal and spatial features of Cre recombinase expression, inherent in the selection of a promoter to drive Cre recombinase. See Postic et al. (1999) [0129] J Biol Chem 275(1):305-315; and Sauer (1998) Methods 14(4):381-392.
Transgenes can also be used for heterologous expression in a host organism without generating phenotypically apparent mutations. By this method, nucleotide sequences of interest are introduced into the genome at a nonessential loci, whereby insertion alone does not disrupt an essential gene function. [0130]
Techniques for the preparation of transgenic animals are known in the art. Exemplary techniques are described in U.S. Pat. No. 5,489,742 (transgenic rats); U.S. Pat. Nos. 4,736,866, 5,550,316, 5,614,396, 5,625,125 and 5,648,061 (transgenic mice); U.S. Pat. Nos. 5,573,933 (transgenic pigs); 5,162,215 (transgenic avian species) and U.S. Pat. No. 5,741,957 (transgenic bovine species). Briefly, nucleotide sequences of interest are cloned into a vector (e.g., pLNK—Gorman et al. 1996), and the construct is transformed into a germ cell. In the germ cell, a chromosomal rearrangement event takes place wherein the nucleic acid sequences of interest are integrated into the genome of the germ cell by homologous recombination. Fertilization and propagation of the transformed germ cell results in a transgenic animal. Homozygosity of the mutation is accomplished by intercrossing. [0131]

I.G. Therapeutic Methods

The present invention further provides methods for discovering substances that can be used as pharmaceutical compositions. The term “pharmaceutical composition” or “drug” as used herein, each refer to any substance having a biological activity. Substances discovered by methods of the present invention include but are not limited to polypeptide, proteins, peptides, chemical compounds, and antibodies. [0132]
A composition of the present invention is typically formulated using acceptable vehicles, adjuvants, and carriers as desired. [0133]
Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable compositions. [0134]
Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic diluent or solvent, for example, as a solution in 1,3-butanediol. [0135]
A vector can be used as a carrier, for example an adenovirus vector, can be used for gene therapy methods. The vector is purified to sufficiently render it essentially free of undesirable contaminants, such as defective interfering adenovirus particles or endotoxins and other pyrogens such that it does not cause any untoward reactions in the individual receiving the vector construct. A preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation. [0136]
A transfected cell can also serve as a carrier. By way of example, a liver cell can be removed from an organism, transfected with a nucleic acid sequence of the present invention using methods set forth above and then the transfected cell returned to the organism (e.g. injected intra-vascularly). [0137]
Monoclonal antibodies or polypeptides of the invention can be administered parenterally by injection or by gradual infusion over time. Although the tissue to be treated can typically be accessed in the body by systemic administration and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery means are provided where there is a likelihood that the tissue targeted contains the target molecule and are known to those of skill in the art. [0138]
Representative antibodies for use in the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, single chain immunoglobulins or antibodies, those portions of an immunoglobulin molecule that contain the paratope, including antibody fragments. It is contemplated to be within the scope of the present invention that a monovalent modulator can optionally be used. [0139]
Methods of preparing “humanized” antibodies are generally well known in the art, and can readily be applied to the antibodies of the present invention. Humanized monoclonal antibodies offer particular advantages over monoclonal antibodies derived from other mammals, particularly insofar as they can be used therapeutically in humans. Specifically, humanized antibodies are not cleared from the circulation as rapidly as “foreign” antigens, and do not activate the immune system in the same manner as foreign antigens and foreign antibodies. [0140]
With respect to the therapeutic methods of the present invention, a preferred subject is a vertebrate subject. A preferred vertebrate is warm-blooded; a preferred warm-blooded vertebrate is a mammal. A preferred mammal is a mouse or, most preferably, a human. As used herein and in the claims, the term “patient” includes both human and animal patients. Thus, veterinary therapeutic uses are provided in accordance with the present invention. [0141]
Also provided is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economical importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered and/or kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans. Thus, provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses, poultry, and the like. [0142]
As used herein, the term “experimental subject” refers to any subject or sample in which the desired measurement is unknown. The term “control subject” refers to any subject or sample in which a desired measure is unknown. [0143]
As used herein, an “effective” dose refers to one a dose(s) administered to an individual patient sufficient to cause a change in EP17 activity. After review of the disclosure herein of the present invention, one of ordinary skill in the art can tailor the dosages to an individual patient, taking into account the particular formulation and method of administration to be used with the composition as well as patient height, weight, severity of symptoms, and stage of the biological condition to be treated. Such adjustments or variations, as well as evaluation of when and how to make such adjustments or variations, are well known to those of ordinary skill in the art of medicine. [0144]
A therapeutically effective amount can comprise a range of amounts. One skilled in the art can readily assess the potency and efficacy of an EP17 modulator of this invention and adjust the therapeutic regimen accordingly. A modulator of EP17 biological activity can be evaluated by a variety of means including the use of a responsive reporter gene, interaction of lipocalin polypeptides with a monoclonal antibody, and fertility assays, each technique described herein. [0145]
Additional formulation and dose techniques have been described in the art, see for example, those described in U.S. Pat. Nos. 5,326,902 and 5,234,933, and PCT Publication WO 93/25521. [0146]
For the purposes described above, the identified substances can normally be administered systemically, parenterally, or orally. The term “parenteral” as used herein includes intravenous, intra-muscular, intra-arterial injection, or infusion techniques. Other compositions for administration include liquids for external use, and endermic liniments (ointment, etc.), suppositories, and pessaries which comprise one or more of the active substance(s) and can be prepared by known methods. [0147]

II. Cloning and Expression of the Mouse EP17 Gene

The present co-inventors have identified a new lipocalin, mEP17, that is adjacent to a related lipocalin-encoding gene, mE-RABP, on mouse chromosome 2 (FIG. 1). The genomic organization of the mEP17 gene was determined by prediction of exons within a BAC genomic clone and further supported by cloning of the mEP17 cDNA (Lareyre et al., 2001). FIG. 1 depicts the genomic organization of the mEP17 locus. Exon sizes are indicated in nucleotides. The major transcription initiation sites of both genes are represented with broken arrows. Primer FwMEP17cDNA (SEQ ID NO:7) was used for primer extension analysis, as described herein below. The G-X—W and T-D-Y and motifs and two cysteine residues (C) are also indicated. [0148]
To isolate the mEP17 promoter region, two clones containing 6.3 kb EcoRV restriction fragments within the 5′ flanking region were isolated from the genomic BAC clone 10983 (FIG. 9). DNA sequencing analysis of both clones revealed that the 6.3 kb fragment contains 5.4 kb of the 5′ flanking region of the mEP17 gene. Cloning methods are provided in Example 1. [0149]
The tissue distribution of mRNA encoding the mEP17 protein was examined by Northern blot analysis of total RNA from twelve different tissues, including spleen, liver, heart, lung, brain, kidney, testis, epididymis, vas deferens, seminal vesicles, uterus, and ovary (FIG. 2A). Hybridization of Northern blots with a [[0150] ³²P]-radiolabeled mEP17 cDNA probe revealed two RNA species of about 3.1 kb and 1 kb only in the epididymis. The total length of the mEP17 gene, including exons and intron, is 3.1 kb. To determine whether the 3.1 kb RNA could be the precursor RNA, two epididymal RNA samples were run side by side and hybridized individually with the cDNA probe or with a probe encompassing intron 1 of the mEP17 gene (FIG. 2B). The first intron 1 probe hybridized with the 3.1 kb RNA but not the 1 kb RNA, indicating that the 3.1 kb RNA is an unspliced precursor RNA.
To investigate the tissue-, region-, and cell-specific profile of gene expression, in situ hybridization of mEP17 transcripts was carried out using sense and antisense digoxygenin-labeled riboprobes generated from mEP17 cDNA (FIGS. 3 and 4[0151] a), as described in Example 2 below. mEP17 mRNA was detected only in the principal cells of the initial segment of the epididymis and is localized basally. Hybridization was not detected when the sense riboprobe was used (FIG. 4B). mEP17 gene expression was high throughout the initial segment (IS). A checkerboard pattern was observed at the boundary between the initial segment and the proximal caput epididymis, wherein some cells expressed mEP17 and some cells did not. mEP17 mRNA was not detected in the efferent ducts (ED), mid and distal caput (Cp), corpus and cauda epididymis using sense or antisense probes.

III. Isolation of Human EP17

The present invention also provides a human EP17 gene. Preferably, the human EP17 gene comprises the sequence set forth as SEQ ID:2, a nucleic acid molecule that is substantially similar to SEQ ID NO:2, or a nucleic acid molecule comprising a 20 base pair nucleotide sequence that is identical to a contiguous 20 base pair sequence of SEQ ID NO:2. [0152]
The mouse EP17 sequence was used to query databases of human genomic sequence, including GenBank and proprietary databases of Celera Genomics Corp. (Rockville, Md.). Two DNA fragments derived from human chromosome 9 were identified (Accession numbers AL35598.7 and 449425.3) in GenBank, although neither sequence or the combination of the sequences predicts the hEP17 gene. A genomic region having sequences corresponding to the hEP17 gene was also identified in Celera's database (Accession number GA 65 373998). The genomic sequence derived from Celera's database was unannotated and did not predict the hEP17 gene. [0153]

The hEP17 gene disclosed herein (SEQ ID NO:2) was predicted by comparing unannotated genomic sequence and the gene structure of mouse EP17. Conserved intron/exon boundaries and conserved nucleotide sequence were recognized and used to construct the gene map depicted in FIG. 5 and Table 1. Preferably, the human EP17 gene comprises a coding region and a promoter region set forth as SEQ ID NOs:3 and 5, respectively.

	TABLE 1


	base pairs

Feature	from	to

Exon 1	5160	5255
Intron 1	5256	12495
Exon 2	12496	12626
Intron 2	12627	13072
Exon 3	13073	13143
Intron 3	13144	14148
Exon 4	14149	14253
Intron 4	14254	14331
Exon 5	14332	14421
Intron 5	14422	14504
Exon 6	14505	14530
Intron 6	14531	15155
Exon 7	15156	15279

The predicted hEP17 gene displays sequence homology with other lipocalins, most notably with m-ERABP and prostaglandin H[0155] ₂-D isomerases s (FIG. 6). The mouse and human EP17 proteins share 61% overall identity and have conserved lipocalin motifs (G-X—W, T-D-Y, and two cysteine residues) at similar positions (FIG. 7).
The amino terminal regions of both mouse and human EP17 proteins are predicted to be a signal peptide since in each case the region is highly hydrophobic, similar to the signal peptide of the mE-RABP protein, and in agreement with the sliding window/matrix scoring method and -1,-3 rule for predicting a peptide cleavage (von Heiji (1986) [0156] Nucleic Acid Res 14:4683-4690) (FIG. 8). This observation implies that the human and mouse EP17 genes encode secreted proteins.

IV. Characterization of an EP17 Promoter Region

The transcription initiation sites of the mEP17 gene were determined by primer extension using epididymal total RNA as a template and the EP17PE2 primer (SEQ ID NO:7) designed according to sequence in the first exon (FIG. 10). Primer extension methods are described in Example 4 below. Two major transcription initiation sites were localized 22 and 18 nucleotides from the putative translation initiation site, and were numbered +1 and +5, respectively. Two minor transcription initiation sites were also detected at position +2 and +4. As shown in FIG. 10, total RNA extracted from the epididymis (Ep) or transfer (t) RNA was reverse transcribed with [[0157] ³²P]-radiolabeled EP17PE2 primer (SEQ ID NO:7) and extended using Avian Myeloblastosis Virus (AMV) reverse transcriptase. Lanes labeled “C”, “T”, “A” and “G” are [³⁵S]-radiolabeled DNA sequencing reactions carried out using the EP17PE2 primer (SEQ ID NO:7) and the pHindIII clone (shown in FIG. 9) as template. The localization of two major (arrows) and two minor (arrowheads) transcription initiation sites are indicated.
The 5′ flanking sequence closest to the transcription start site was analyzed further. A 2.5-kb EcoRI restriction fragment comprising this sequence was isolated from the genomic BAC clone 10983 (shown in FIG. 9). A computer analysis to identify putative cis-regulatory sites was carried out using TFSEARCH version 1.3 (Yutaka Akiyama: “TFSEARCH: Searching Transcription Factor Binding Sites”, http://www.rwcp.or.jp/papia/). This analysis revealed the presence of several binding sites for known transcription factors, including binding sites for androgen receptor (ARSB), retinoic acid receptor (RARE), Stimulating Protein 1 (SP-1), Activator Protein 1 (AP-1), Activator Protein 4 (AP4), SRY (Sex-determining Region Y protein), C-Ets (cellular ets oncogene), C/EBP (CCMT/enhancer binding protein), and Sox-5 (SRY-related [0158] sequence #5 protein). Putative cis-regulatory sites are underlined in FIG. 11. A consensus TATA box and CMT-box are indicated. Major transcription initiation sites are marked by long arrows, and minor transcription initiation sites are marked by arrowheads. The computer analysis was carried out using TFSEARCH version 1.3 [Yukata Akiyama: “TFSEARCH: Searching Transcription Factor Binding Sites”, http://www.rwcp.or.jp/papia/].
To define functional sequences within the promoter region, several chimeric reporter genes were constructed by ligation of various portions of the 5′ flanking region of the EP17 gene and a reporter gene (FIG. 12), as described in Example 5 below. Each chimeric gene comprises a different fragment of the 5.3 kb EP17 promoter region, an open reading frame encoding a reporter gene operably linked to a promoter fragment, and the polyA tail region of [0159] Simian Virus 40 large T antigen. FIG. 12 indicates one preferred reporter, chloramphenicol acetyltransferase (CAT). These constructs are alternatively used for in vitro and in vivo assays of EP17 promoter region function.
A preferable in vitro technique for evaluating EP17 promoter function is a transient transfection assay. According to this method, each chimeric reporter gene is introduced into a relevant host cell, and the resulting level of reporter gene expression is quantitated. Preferred host cells include HeLa and PC-3 cells, or normal or immortalized epididymal cells, described herein below. In these experiments, luciferase is a preferable reporter gene in that it demonstrates increased sensitivity of detection. Transient transfection assays are performed as described in Example 6. Additional methods for making an expression system comprising a promoter region operably linked to a heterologous reporter sequence are disclosed in U.S. Pat. No. 6,087,111. [0160]
To analyze the function of an EP17 promoter region in vivo, transgenic mice bearing each chimeric gene are generated as described in Example 7 below, and a level of reporter gene expression in each mouse is determined. For these experiments, CAT is a preferred reporter gene as it displays low endogenous activity in the epididymis. Several assays are performed to characterize CAT expression in transgenic animals, including PCR using CAT-specific primers, CAT enzymatic assays, immunohistochemistry using an antiCAT antibody, and in situ hybridization using a CAT-specific probe. Methods for performing these assays can be found in Lareyre, J. J., et al. (1999) [0161] J. Biol. Chem. 274:8282-8290, in Lareyre et al. (2001) and Examples 2, 8, and 9.
A transgenic mouse bearing the entire 5.3 [0162] kb 5′ flanking region of the EP17 gene operably linked to the CAT gene shows CAT expression in the caput epididymis, demonstrating that the 5.3 kb promoter region of the EP17 gene contains sequences required for the region-specific expression of the EP17 gene. Shorter sequences of the EP17 promoter region can be used to define a minimal sequence requisite for EP17 gene expression. In determining a promoter region that reproduces endogenous EP17 expression, the expression profile of each chimeric gene can be carefully compared to the profile of EP17 gene expression as determined by in situ hybridization.
Within a candidate promoter region or response element, the presence of regulatory proteins bound to a nucleic acid sequence can be detected using a variety of methods well known to those skilled in the art (Ausubel et al., 1992). Briefly, in vivo footprinting assays demonstrate protection of DNA sequences from chemical and enzymatic modification within living or permeabilized cells. Similarly, in vitro footprinting assays show protection of DNA sequences from chemical or enzymatic modification using protein extracts. Nitrocellulose filter-binding assays and gel electrophoresis mobility shift assays (EMSAs) track the presence of radiolabeled regulatory DNA elements based on provision of candidate transcription factors. [0163]
Genomic clones derived from GenBank and proprietary databases (Celera Genomics Corp., Rockville, Md.) were used to predict an hEP17 promoter region comprising an about 5150 base pair region immediately upstream of the hEP17 transcription start site (FIG. 13). This region is similar to the promoter region of mEP17, having putative cis-DNA regulatory elements included but not limited to a Sp-1 binding site, an AP-1 binding site, a cAMP response element binding protein (CREB) binding site, a SRY-related HMG box gene 5 (Sox 5) binding site, a Sex-determining region Y gene product (SRY) binding site, a c-Ets binding site, a GATA binding site, and an Octamer trasncription factor 1 (Oct-1) binding site (FIG. 14). The hEP17 promoter is further characterized in a manner as described herein above regarding the mouse EP17 promoter region. [0164]

V. Methods for Identifying Regulators of Gene Expression

The nucleic acid sequences of the present invention can be used to identify regulators of EP17 gene expression. Several molecular cloning strategies can be used to identify substances that specifically bind EP17 cis-regulatory elements. A preferred promoter region to be used in such assays is an EP17 promoter region from mouse or human, more preferably the promoter region includes some or all amino-acids of SEQ ID NOs:1 or 5. FIGS. [0165] 15A-15C presents data mEP17 expression is not regulated by hormones. However, studies in which spermatogenesis was disrupted suggest that an EP17 lipocalin can be regulated by germ cell-associated factors.
In one embodiment, a cDNA library in an express,ion vector, such as the lambda-gt11 vector, can be screened for cDNA clones that encode an EP17 regulatory element DNA-binding activity by probing the library with a labeled EP17 DNA fragment, or synthetic oligonucleotide (Singh et al. (1989) [0166] Biotechniques 7:252-261). Preferably the nucleotide sequence selected as a probe has already been demonstrated as a protein binding site using a protein-DNA binding assay described above.
In another embodiment, transcriptional regulatory proteins are identified using the yeast one-hybrid system (Luo et al. (1996) [0167] Biotechniques 20(4):564-568; Vidal et al. (1996) Proc Natl Acad Sci USA 93(19):10315-10320; Li and Herskowitz (1993) Science 262:1870-1874). In this case, a cis-regulatory element of an EP17 gene is operably fused as an upstream activating sequence (UAS) to one, or typically more, yeast reporter genes such as the lacZ gene, the URA3 gene, the LEU2 gene, the HIS3 gene; or the LYS2 gene, and the reporter gene fusion construct(s) is inserted into an appropriate yeast host strain. It is expected that the reporter genes are not transcriptionally active in the engineered yeast host strain, for lack of a transcriptional activator protein to bind the UAS derived from the EP17 promoter region. The engineered yeast host strain is transformed with a library of cDNAs inserted in a yeast activation domain fusion protein expression vector, e.g. pGAD, where the coding regions of the cDNA inserts are fused to a functional yeast activation domain coding segment, such as those derived from the GAL4 or VP16 activators. Transformed yeast cells that acquire a cDNA encoding a protein that binds a cis-regulatory element of an EP17 gene can be identified based on the concerted activation of the reporter genes, either by genetic selection for prototrophy (e.g. LEU2, HIS3, or LYS2 reporters) or by screening with chromogenic substrates (lacZ reporter) by methods known in the art.
In another embodiment, an in situ filter detection method is used to clone a cDNA encoding the sequence-specific DNA-binding protein as described in Example 10. [0168]
In a more preferred embodiment, one-hybrid analysis and in situ filter detection methods are used sequentially. For example, an initial collection of candidate transcription factors is identified by one-hybrid analysis, and this initial collection is secondarily screened using in situ filter detection. This combination of techniques provides a smaller but more confident pool of candidate regulators than selected by either technique alone. [0169]
A candidate regulator to be tested by these methods can be a purified molecule, a homogenous sample, or a mixture of molecules or compounds. More than one modulatable transcriptional regulatory sequence can be screened simultaneously. [0170]
In accordance with the present invention there is also provided a rapid and high throughput screening method that relies on the methods described above. This screening method comprises separately contacting each compound with a plurality of substantially identical samples. In such a screening method the plurality of samples preferably comprises more than about 10[0171] ⁴samples, or more preferably comprises more than about 5×10⁴samples. In an alternative high-throughput strategy, each sample can be contacted with a plurality of candidate compounds.
The present invention also provides an in vivo assay for discovery of modulators of EP17 expression. In this case, a transgenic mouse is made such that a transgene comprising an EP17 promoter and a reporter gene is expressed and a level of reporter gene expression is assayable. Such transgenic animals can be used for the identification of drugs, pharmaceuticals, therapies, and interventions that are effective in modulating EP17 expression. [0172]

VI. Method for Heterologous Gene Expression in Epididymis

The present invention enables epididymal expression of a heterologous nucleic acid sequence. In this case, a transgenic animal is generated which bears a transgene that includes an EP17 promoter region and a nucleotide sequence of interest. A preferred EP17 promoter is the nucleotide sequence of SEQ ID NO:1 or 5, more preferably a minimal functional portion of SEQ ID NO:1 or 5 that drives appropriate epididymal expression, as determined by methods described herein above. [0173]
In one embodiment, this method enables assay of the function of a gene of interest in epididymis to the exclusion of other sites of gene function. For example, the heterologous sequence can encode an antisense or ribozyme nucleic acid molecule. When expressed in epididymis under the direction of an EP17 promoter region, the function of a gene corresponding to the antisense or ribozyme nucleic acid molecule is disrupted in epididymis but not other tissues. [0174]
In another embodiment, an EP17 promoter drives expression of a toxin, for example, thymidine kinase plus ganciclovir. Expression of the chimeric gene targets degeneration of the initial segment of the epididymis. In this case, the transgenic animal can be used as animal model of infertility, described further herein below. [0175]
In another embodiment, an EP17 promoter region drives expression of a therapeutic gene or nucleotide sequence, as described herein below. [0176]

VII. Method for Producing an Epididymal Cell Line

Another aspect of the invention is a method for producing an epididymal cell line. According to this method, a chimeric gene is constructed to express a gene encoding a selectable marker under the control of an EP17 promoter region, and the chimeric gene is used to create a transgenic animal expressing the selectable marker in epithelial cells of the initial segment of the epididymis. Preferably, the selectable marker confers antibiotic resistance, and more preferably, the selectable marker confers neomycin resistance, which can be used even in selection of epididymal cells from non-epididymal cells in culture. Also preferably, the EP17 promoter region used to perform this method is the sequence of SEQ ID NO:1, or functional portion thereof. Similarly, using the mE-RABP promoter, a neomycin-resistant immortalized cell line from the distal caput can be generated by this method. [0177]
Also provided is a method for generating an immortalized epididymal cell line. In this case, a transgenic animal is obtained, having a transgene that encodes an oncogenic virus directed by a constitutive promoter. A preferred oncogenic virus comprises a temperature-sensitive (ts) [0178] Simian virus 40 large T antigen (Tegtmeyer (1975) J Virol 15(3):613-618). The ts-Simian virus 40 large T-antigen is completely inactive at non-permissive temperature (39° C.), partially inactive at body temperature, and substantially active at a permissive temperature (33° C.). Immortalized epithelial cells are procured from ts-Simian virus 40 large T-antigen mouse are reproduced in culture. In one embodiment, epididymal cells may be selected using the EP17 promoter operably linked to the neomycin resistant gene. Since the EP17 promoter is expressed in the initial segment, neo-selection will provide a pure population of epithelial cells from that segment. A neomycin-resistant immortalized cell line from the distal caput has been generated by this method using the E-RABP promoter and maintained in culture for 12 months.

VIII. Method for Homologous Recombination at the EP17 Locus

Another aspect of the invention is a method for mutagenizing the EP17 locus by homologous recombination. The method uses a targeting vector having an isolated EP17 promoter region, a marker gene, and an [0179] isolated EP17 3′ flanking region. In a vector so constructed, the marker gene is positioned between the promoter region and the 3′ flanking region. In another embodiment, the targeting vector further comprises a mutant EP17 coding sequence, also positioned between the promoter region and the 3′ flanking region. The targeting vector is linearized by digestion with a restriction endonuclease at a site other than within the promoter region, marker gene, 3′ flanking region, and optional mutant EP17 coding sequence.
In a preferred embodiment, the linearized vector is electroporated into embryonic stem cells, and successful electroporation is assayed by detecting the marker gene in the stem cells. Stem cells bearing the vector are used to create a transgenic animal. According to the method, a homologous recombination event is mediated at the EP17 locus, thereby exchanging native EP17 gene sequences positioned between the promoter region and the 3′ flanking region with vector nucleotide sequences positioned the same. [0180]
The nucleic acids and methods of the present invention enable knockout, knock-in, and knock-down mutations of the EP17 gene. The phenotype of EP17 mutant animals can be characterized to reveal EP17 function. The expression of EP17 in epididymis, and the known functional importance of regulated epididymal gene expression for male fertility, suggest that EP17 will have a determinative role in sperm maturation. Methods for generating MEP17 mutant mice are provided in Example 11. [0181]
A preferred knock-out mutation removes part of the EP17 coding region (exon1) and can be generated using a targeting vector as depicted in FIG. 16. Preferred knock-in mutations include mutation of any one of amino acids within the conserved lipocalin motifs to any amino acid that is non-conservative substitution. Other preferred knock-in mutations are targeted replacement of one or both of the conserved cysteine residues with an amino acid(s) that is a non-conservative substitution. [0182]

IX. Method for Detecting a EP17 Nucleic Acid Molecule

In another aspect of the invention, a method is provided for detecting a nucleic acid molecule that encodes an EP17 polypeptide. According to the method, a biological sample having nucleic acid material is procured and hybridized under stringent hybridization conditions to an EP17 nucleic acid molecule of the present invention. Such hybridization enables a nucleic acid molecule of the biological sample and the EP17 nucleic acid molecule to form a detectable duplex structure. Preferably, the EP17 nucleic acid molecule includes some or all nucleotides of SEQ ID NO:1, 2, 3, or 5. also preferably, the biological sample comprises human nucleic acid material. [0183]
In another embodiment, genetic assays based on nucleic acid molecules of the present invention can be used to screen for genetic variants by a number of PCR-based techniques, including single-strand conformation polymorphism (SSCP) analysis (Orita, M., et al. (1989) [0184] Proc Natl Acad Sci USA 86(8):2766-2770), SSCP/heteroduplex analysis, enzyme mismatch cleavage, and direct sequence analysis of amplified exons (Kestila et al. (1998) Mol Cell 1 (4):575-582; Yuan et al. (1999) Hum Mutat 14(5):440-446). Automated methods can also be applied to large-scale characterization of single nucleotide. polymorphisms (Brookes (1999) Gene 234(2):177-186; Wang et al. (1998) Science 280(5366):1077-82). The present invention further provides assays to detect a mutation of a variant EP17 locus by methods such as allele-specific hybridization (Stoneking et al. (1991) Am J Hum Genet 48(2):370-82), or restriction analysis of amplified genomic DNA containing the specific mutation.

X. Recombinant Production of an EP17 Polypeptide

The present invention also provides a method for recombinant production of a EP17 polypeptide, as described in Example 12. Preferably, the recombinant polypeptide comprises some or all of the amino acid sequences of SEQ ID NO:4 or 6. [0185]
Mouse EP17 protein was recombinantly produced using the pBAD/gIII vector (Invitrogen of Carlsbad, Calif.). To confirm the production of EP17 protein, total protein derived from transformed [0186] E.coli was resolved on a polyacrylamide gel, and Coomassie blue staining revealed two enriched bands of approximately 21 kDa and 23 kDa. Western blot analysis using an anti-his tag antibody revealed the same two proteins, which correspond to the processed and unprocessed EP17 isoforms, respectively (FIG. 17)
Recombinantly produced proteins are useful for a variety of purposes, including structural determination of an EP17 polypeptide, generation of an antibody that recognizes an EP17 polypeptide, and screening assays to identify a chemical compound or peptide that interacts with an EP17 polypeptide, described further herein below. [0187]

XI. Production of EP17 Antibodies

In another aspect, the present invention provides a method of producing an antibody immunoreactive with a lipocalin polypeptide, the method comprising recombinantly or synthetically producing an EP17 polypeptide, or portion thereof, to be used as an antigen. The EP17 polypeptide is formulated so that it is used as an effective immunogen. An animal is immunized with the formulated EP17 polypeptide, generating an immune response in the animal. The immune response is characterized by the production of antibodies that can be collected from the blood serum of the animal. Preferred embodiments of the method use a polypeptide as of SEQ ID NO:4 or 6. [0188]
The present invention also encompasses antibodies produced by this method. [0189]
The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the EP17 polypeptide sequences of the invention, e.g., for cloning of EP17 nucleic acids, immunopurification of EP17 polypeptides, imaging EP17 polypeptides in a biological sample, measuring levels thereof in appropriate biological samples, and in diagnostic methods. [0190]

XII. Method for Detecting an EP17 Polypeptide

In another aspect of the invention, a method is provided for detecting a level of EP17 polypeptide using an antibody that specifically recognizes an EP17 polypeptide, or portion thereof. In a preferred embodiment, biological samples from an experimental subject and a control subject are obtained, and EP17 polypeptide is detected in each sample by immunochemical reaction with the EP17 antibody. More preferably, the antibody recognizes amino acids of SEQ ID NO:4 or 6 and is prepared according to a method of the present invention for producing such an antibody. [0191]
In one embodiment, an EP17 antibody is used to screen a biological sample for the presence of a lipocalin polypeptide. A biological sample to be screened can be a biological fluid such as extracellular or intracellular fluid, or a cell or tissue extract or homogenate. A biological sample can also be an isolated cell (e.g., in culture) or a collection of cells such as in a tissue sample or histology sample. A tissue sample can be suspended in a liquid medium or fixed onto a solid support such as a microscope slide. In accordance with a screening assay method, a biological sample is exposed to an antibody immunoreactive with an EP17 polypeptide whose presence is being assayed, and the formation of antibody-polypeptide complexes is detected. Techniques for detecting such antibody-antigen conjugates or complexes are well known in the art and include but are not limited to centrifugation, affinity chromatography and the like, and binding of a labeled secondary antibody to the antibody-candidate receptor complex. [0192]

XIII. Screening for Small Molecule Ligands that Interact with EP17

The present invention further discloses a method for identifying a compound that modulates EP17 function. According to the method, an EP17 polypeptide is exposed to a plurality of compounds, and binding of a compound to the isolated EP17 polypeptide is assayed. A compound is selected that demonstrates specific binding to the isolated EP17 polypeptide. Preferably, the EP17 polypeptide used in the binding assay of the method includes some or all amino acids of SEQ ID NO:4 or 6. [0193]
Several techniques can be used to detect interactions between a protein and a chemical ligand without employing an in vivo ligand. Representative methods include, but are not limited to, fluorescence correlation spectroscopy, surface-enhanced laser desorption/ionization, and biacore technology, as described in Example 13. These methods are amenable to automated; high-throughput screening. [0194]
Candidate regulators include but are not limited to proteins, peptides, and chemical compounds. Structural analysis of these selectants can provide information about ligand-target molecule interactions that enable the development of pharmaceuticals based on these lead structures. [0195]
Similarly, the knowledge of the structure a native EP17 polypeptide provides an approach for rational drug design. The structure of an EP17 polypeptide can be determined by X-ray crystallography or by computational algorithms that generate three-dimensional representations. See Huang et al. (2000) [0196] Pac Symp Biocomput 230-41; Saqi et al. (1999) Bioinformatics 15:521-522. Computer models can further predict binding of a protein structure to various substrate molecules, that can be synthesized and tested. Additional drug design techniques are described in U.S. Pat. Nos. 5,834,228 and 5,872,011.

XIV. Animal Model of Male Infertility

The present invention further pertains to an animal model of male infertility. Such a model is prepared by several methods. [0197]
Using a transgenic approach, knock-out, knock-in, or knock-down mutation of the EP17 gene can suppress fertility. In another embodiment, expression of a toxin, for example, thymidine kinase plus ganciclovir, under the direction of an EP17 promoter targets degeneration of the initial segment of the epididymis and thereby compromises fertility. [0198]
The present invention also teaches that an animal model of fertility is prepared by immunizing an animal with an EP17 polypeptide. The resulting immune response in the animal comprises a production of antibodies that specifically bind an EP17 polypeptide, thereby disrupting its biological activity. [0199]
A method is also provided for generating an animal model of infertility by administering to an animal a compound that disrupts EP17 expression or function. Such a compound is discovered by methods disclosed herein. [0200]
Animal models of male infertility can be characterized according to several measures, including in vivo and in vitro assays of fertility, as described in Examples 14 and 15 below, and morphological inspection of the epididymis. [0201]

XV. Therapeutic Applications

Another aspect of the present invention is a therapeutic method comprising administering to a subject a substance that modulates lipocalin biological activity. Therapeutic substances include but are not limited to chemical compounds, antibodies, and gene therapy vectors. [0202]
Compounds that are discovered by the methods disclosed herein is useful for therapeutic applications related to male fertility. For example, a compound that mimics EP17 function, when administered to an infertile male subject, can regulate fertility by promoting spermatozoa maturation in the epididymis. Conversely, a compound that interferes with EP17 function can act to suppress spermatozoa maturation when administered to a fertile subject. [0203]
The present invention also provides a method for disrupting EP17 function by immunizing a subject with an effective dose of the disclosed EP17 polypeptide. The immune system of the subject produces an antibody that specifically recognizes the EP17 polypeptide, and binding of the antibody to the EP17 polypeptide abolishes EP17 function. In a preferred embodiment, the antibody recognizes some or all of the amino acids of SEQ ID NO:4 or 6 and is prepared according to a method of the present invention for producing such an antibody. [0204]
Several studies have demonstrated the utility of immunotherapeutic approaches to contraception and teach methods for preparing and administering such vaccines, including U.S. Pat. Nos. 6,132,720 and 6,096,318, Feng et al. (1999), and Naz (1999). U.S. Pat. No. 6,096,318 additionally discloses methods for chemical modification of immunogenic proteins, and fragments thereof, which elicit an amplified immune response in a subject receiving an injection of the modified polypeptide. Briefly, the antigen modification is accomplished by attaching the protein to a carrier such as a bacterial toxin or by polymerization of protein fragments. This method has been used to modify human chorionic gonadotropin, an antigen that is effective for immunological contraception in mammals. [0205]
The present invention further provides lipocalin nucleic acid sequences and gene therapy methods for modulating lipocalin activity in a target cell. The gene therapy vector can encode an EP17 lipocalin, preferably comprising the amino acid sequences of SEQ ID NO:4 or 6. Alternatively, a gene therapy vector can include sequences encoding a nucleic acid molecule, peptide, or protein that interacts with an EP17 lipocalin. This modulation can affect spermatozoa maturation in the vicinity of a lipocalin-secreting cell. Additionally, a gene therapy vector can include an EP17 promoter sequence of the present invention to provide tissue specific expression of a gene of interest in a subject. Preferably, the EP17 promoter regions used to perform this method is the nucleotide sequence of SEQ ID NO:1 or 5, or functional portion thereof. [0206]
Vehicles for delivery of a gene therapy vector include but are not limited to a liposome, a cell, and a virus. Preferably, a cell is transformed or transfected with the DNA molecule or is derived from such a transformed or transfected cell. An exemplary and preferred transformed or transfected cell is a epididymal cell. Alternatively, the vehicle is a virus, including a retroviral vector, adenoviral vector or vaccinia virus whose genome has been manipulated in alternative ways so as to render the virus non-pathogenic. Methods for creating such a viral mutation are detailed in U.S. Pat. No. 4,769,331. Exemplary gene therapy methods are described in U.S. Pat. Nos. 5,279,833; 5,286,634; 5,399,346; 5,646,008; 5,651,964; 5,641,484; and 5,643,567. [0207]
The ability for adenovirus gene therapy vectors to infect male germ cells to the exclusion of embryos fertilized by infected sperm was demonstrated by Hall et al. (2000) [0208] Hum Gene Ther 11(12):1705-1712. High titers of the vector were injected directly into mouse testis and epididymis, or alternatively, sperm were exposed to the virus immediately prior to or during in vitro fertilization. The vector carried the bacterial lacZ gene under the direction of the Rous sarcoma virus promoter, and infection was assayed by enzymatic or immunologic detection of β-galactosidase. lacZ expression was assayed during the several weeks following injection, and in preimplantation embryos produced by in vitro fertilization with sperm exposed to gene therapy vector. lacZ expression was observed in sperm but not in embryos, supporting a conclusion that adenovirus vectors pose minimal risk for germ line integration when exposed to male reproductive cells. These studies teach methods for construction of a gene therapy vector and effective administration of a vector in the male reproductive system, as proposed for administration of nucleic acids of the present invention.
The invention further provides a method for diminishing the fertile capacity of a subject. According to the method, a chemical compound, peptide, or antibody that interacts with an EP17 polypeptide, preferably the polypeptide of SEQ ID NO:4 or 6, is identified. A pharmaceutical preparation is prepared comprising such a chemical compound, peptide, or antibody, and a carrier. An effective dose of the pharmaceutical composition is administered to a subject, whereby the fertile capacity of the subject is diminished. [0209]
The invention further provides a method for promoting the fertile capacity of a subject. In this case, a chemical compound or peptide that interacts with an EP17 polypeptide, preferably the polypeptide of SEQ ID NO:4 or 6, is identified. A pharmaceutical composition comprising the chemical compound or peptide and a carrier is prepared. An effective dose of the pharmaceutical composition is administered to a subject, whereby the fertile capacity of the subject is improved. [0210]

XVI. Summary

Summarily, the provisions of an EP17 promoter region, a 3′ flanking genomic region of mEP17, a coding sequence of hEP17, and a hEP17 polypeptide are a significant advance in fertility-related research. The disclosed EP17 nucleic acids and polypeptides can be used according to methods of the present invention to generate a mouse model of male infertility, for drug discovery screens, and for therapeutic treatment of fertility-related conditions. [0211]

EXAMPLES

The following Examples have been included to illustrate modes of the invention. Certain aspects of the following Examples are described in terms of techniques and procedures found or contemplated by the present co-inventors to work well in the practice of the invention. These Examples illustrate standard laboratory practices of the co-inventors. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modification and alteration can be employed without departing from the scope of the invention. [0212]

Example 1

Cloning of Mouse EP17 Promoter Region

Two clones containing 6.3 kb EcoRV restriction fragments were isolated from the genomic BAC clone 10983 (FIG. 9). The DNA fragments were subcloned in pBjuescript SK+ (Promega, Madison, Wis.) using appropriate enzymes. DNA templates for sequencing were purified using the “Plasmid Midi kit” (Qiagen Inc., Santa Clarita, Calif.). Sequencing reactions were performed as described in the Thermo Sequenase™ fluorescent labeled primer cycle sequencing kit (Perkin-Elmer, Foster City, Calif.). DNA fragments were separated in a denaturing PAGE (6% acrylamide gel) and analyzed using an ABI 373A automated sequencer (PE, Applied Biosystems, Foster City, Calif.). Nucleotide sequences were analyzed using the GeneJockey™ software available from Biosoft of Ferguson, Mo. DNA sequencing analysis of both clones revealed that the 6.3 kb fragment contains 5.4 [0213] kb 5′ flanking region of the mEP17 gene.

Example 2

In Situ Hybridization

Nonisotopic in situ hybridization was performed on 4-6 μm thick cryosections of fresh-frozen mouse epididymis. Sections were fixed in 4% formaldehyde in 0.1 M sodium phosphate buffer pH 7.2 and then incubated for 10 minutes in PBS containing 5 μg/ml proteinase K. See Sambrook et al. (1992) for a description of PBS. After two rinses in PBS, sections were incubated in 0.25% acetic anhydride in 0.1 M triethanolamine pH 8.0 for 15 minutes. Sense and antisense riboprobes were prepared in 20 μl transcription reactions containing SP6 (Promega, Madison, Wis.) or T7 (New England Biolabs, Beverly, Mass.) polymerase, 1× transcription buffer, 1 mM each of ATP, CTP, and GTP, 0.65 mM UTP, 0.35 mM digoxygenin-UTP (Roche Diagnostics Corp, Indianapolis, Ind.), and 1 μg linearized F3 plasmid carrying the mEP17 cDNA. Unincorporated nucleotides were removed on a Chroma Spin-100 STE column (Clontech, Palo Alto, Calif.). [0214]
Labeled riboprobes were denatured for 5 minutes at 80° C., diluted in hybridization buffer composed of 50% (vol/vol) formamide, 10% (wt/vol)dextran sulfate, 4×SSC, 1× Denhardt's reagent, 0.5 mg/ml yeast tRNA, and incubated with the sections overnight at 55° C. See Sambrook et al., 1992 for a description of SSC buffer. The slides were washed at room temperature for 5 minutes in 2×SSC, rinsed in STE buffer (500 mM NaCl, 20 mM Tris-HCl pH 7.5, 1 mM EDTA), and then incubated for 30 minutes in STE containing 40 μg/ml RNase A. The sections were washed sequentially for 5 minutes each in 2×SSC, 50% formamide at 50° C., then at room temperature with 1×SSC, and finally with 0.5×SSC. [0215]
To detect hybridized probes, slides were rinsed in TN buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl), blocked for 1 hour in blocking solution (TN buffer containing 2% horse serum and 0.1% Triton X-100), and incubated for 1 hour in 1:500 diluted alkaline phosphatase conjugated antidigoxygenin (Roche Diagnostics Corp.) in blocking solution. Slides were rinsed three times in blocking solution and then in a substrate buffer of 100 mM This-HCl pH 9.5, 100 mM NaCl, 50 mM MgCl[0216] ₂. Color development was in substrate buffer containing 0.17 mM 5-bromo4-chloro-3-indolyl phosphate, 10 mM N-ethyl-maleimide, and 1 mM levamisole as an inhibitor of endogenous alkaline phosphatase. Color development was stopped with 10 mM This-HCl pH 8.0 and 1 mM EDTA. Sections were examined and photographed with a Zeiss Axiophot using both bright field and phase contrast optics.

Example 3

Cloning Human EP17

To recover a full-length human EP17 gene, a hybridization screen is performed using a human epididymal genomic library probed with the nucleotide sequence of SEQ ID NO:2, 3 or 5, or portion thereof. Positive colonies are selected, a subset sequenced, and a clone corresponding to the full-length cDNA is recovered. Alternatively, primers from the predicted 5′ and 3′ ends of SEQ ID NO:2 are used in polymerase chain reaction with a human epididymal genomic DNA as template to amplify a fragment representing the full-length clone. [0217]
To recover a full-length human EP17 cDNA, a hybridization screen is performed using a human epididymal cDNA library probed with the nucleotide sequence of SEQ ID NO:2 or 3, or portion thereof. Positive colonies are selected, a subset sequenced, and a clone corresponding to the full-length cDNA is recovered. Alternatively, primers from the predicted 5′ and 3′ ends of SEQ ID NO:3 are used in polymerase chain reaction with a human epididymal cDNA as template to amplify a fragment representing the full-length clone. [0218]

Example 4

Primer Extension Analysis to Identify Transcription Initiation Sites

Total RNA was extracted from the mouse epididymis using a method described previously (Chomczynski and Sacchi (1987) [0219] Anal Biochem 162:156-159). The EP17PE2 primer (SEQ ID NO:7) specific for mEP17 mRNA was radiolabeled using T4 nucleic acid sequence kinase in the presence of 100 μCi [γ-³²P]-ATP (3000 Ci/mmol) (Amersham) according to the manufacturer's instructions (New England Biolabs). For each reaction, 10 μg of epididymal total RNA or transfer RNA was hybridized to 1 pmol (10⁵dpm) of EP17PE2 primer for 12 hours at 35° C. in 10 μl of a solution containing 0.04 M [1,4]-piperazine diethanesulfonic acid (PIPES), pH 6.4, 1 μM EDTA, and 80% (vol/vol) formamide.
Reverse transcription was performed in 20 μl containing 50 μM Tris-HCl pH 8.3, 30 μM KCl, 8 μM MgCl[0220] ₂, 6 μM DTT, 0.5 mM of each dNTP, and 50 units Avian Myeloblastosis Virus (AMV) reverse transcriptase (Promega, Madison, Wis.). Samples were incubated for 30 minutes at 42° C., and then, 50 units of AMV reverse transcriptase were added again and incubated for 1 hour more. Elongated radiolabeled fragments were loaded on a denaturing PAGE (7% polyacrylamide gel) nextto sequencing reactions carried out using the Sequenase sequencing kit (Amersham, USB). The clone pHindIII (shown in FIG. 9) and the EP17PE2 primer (SEQ ID NO:7) were used as template and primer, respectively.

Example 5

Chimeric Gene Expression Constructs

DNA fragments derived from the BAC clone 10983 were generated using appropriate restriction enzymes. DNA fragments were resolved on an agarose gel, purified from the agarose, and ligated into the promoterless pBLCAT3 plasmid (Luckow, 1987) by standard methods. This construction enabled expression of the CAT gene by a mEP17 promoter region fragment. [0221]

Example 6

Transient Transfection Assays

Unless otherwise indicated, all media, cell culture and transfection reagents were obtained from GIBCO BRL (Life Technologies, Rockville, Md.). PC-3 and HeLa cells were cultured in F12K Nutrient Mixture (Kaighn's Modification) or Dulbecco's Modified Eagle Medium (DMEM) supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin and 10% (v/v) charcoal/dextran treated fetal bovine serum (FBS, Hyclone, Logan, Utah). Both cultures were maintained at 37° C. in humidified air with 5% CO[0222] ₂. Plasmids were prepared with the QIAGEN™ plasmid kit. Lipofectin reagent and PLUS reagent (Gibco BRL) were used according to the manufacturer's protocol. Briefly, cells were plated at 2×10⁵cells/well in 6-well plates the day before transfection. After 24 hours, 5 μl of PLUS reagent, 0.5 μg of chimeric construct, 0.5 μg of androgen or glucocorticoid expression vector and 0.05 μg of pRL-CMV, were diluted in 100 μl of DMEM and incubated for 15 minutes at room temperature. The two solutions were combined, gently mixed, and incubated for 15 minutes at room temperature. Four or eight μl of Lipofectin reagent was diluted in DMEM and incubated for 15 minutes at room temperature. The two solutions were combined, gently mixed, and incubated for 15 minutes at room temperature. While complexes were forming, medium was replaced with 800 μl of fresh DMEM. Following incubation, the transfection mixtures were added to the wells. Cells were incubated for 4 hours at 37° C. at 5% CO₂. After incubation, medium was replaced with 2 ml of DMEM containing 10% FBS and appropriate hormones. After 24 hours, cells were washed once with phosphate buffered saline, 500 μl of passive lysis buffer (Promega) were added and cells were incubated for 15 minutes at room temperature in a shaker. The cell lysates were transferred to fresh tubes, centrifuged at 12,000 rpm for 30 seconds to remove debris and stored at −80° C. For efficiency control, Renilla luciferase activity (pRL-CMV) was monitored.

Example 7

Transgenic Mice

The chimeric gene comprising the 5.3 kb EP17 promoter region fragment and the CAT reporter gene was excised from the pUC18 vector by restriction enzyme digest. DNA fragments were purified on a 0.8% (w/v) agarose gel using the AgarACE™ enzyme (Promega). Transgenic mice (strain B6D2; Harlan Sprague-Dawley) were generated by microinjection of the DNA into the male pronucleus of a fertilized oocyte using standard techniques (Palmiter and Brinster(1985) [0223] Cell 141:343-345). Seven independent transgenic lines carrying the CAT reporter gene were obtained. Caput epididymis-specific CAT activity was detected in three transgenic mouse lines. CAT expression was restricted to the initial segment of the caput epididymis as observed for the mEP17 gene. Thus, the 5.3 kb fragment of the mEP17 5′ flanking region is sufficient of region-specific expression and can be used for heterologous expression in the initial segment of the caput epididymis.

Example 8

PCR Assay of Transgenic Mice

Transgenic animals were identified by PCR-based screening using DNA isolated from the tail of each animal. Approximately 1 cm of the tail was digested overnight at 55° C. in a Proteinase K digestion mix (10 mM Tris-Cl, pH 7.5,75 mM NaCl, 25 mM EDTA, 1% SDS, 0.5 mg/ml Proteinase K). DNA was extracted with one volume of phenol/chloroform/isoamyl alcohol (25/24/1) and precipitated at room temperature with two volumes of absolute ethanol. Samples were centrifuged at 10,000×g at 4° C. for 15 minutes, washed with 70% ethanol, centrifuged at 10,000×g at 4° C. for 15 minutes, and dried for 2 hours at room temperature. [0224]
500 ng of genomic DNA were mixed with 1×PCR buffer II (Perkin Elmer), 2 units of Taq DNA polymerase (Promega), 1.5 mM MgCl[0225] ₂, 1 μM concentration of each primer (primer 1, SEQ ID NO:8; primer 2, SEQ ID NO:9; casein forward primer, SEQ ID NO:10; casein reverse primer, SEQ ID NO:11), and 0.2 mM dNTP. DNA fragments were amplified for 30 cycles (95° C., 1 minute; 50° C. 45 seconds, 72° C., 45 seconds) and 1 cycle (95° C., 1 minute; 50° C. 45 seconds; 72° C., 10 minutes). PCR products were analyzed on a 2% (w/v) agarose gel.

Example 9

CAT Enzymatic Assay in Transgenic Mice

To monitor CAT activity, organs were dissected from a transgenic animal and homogenized by 20 strokes with a B pestle in a glass Dounce homogenizer in 200 μl of0.1M Tris-HCl, 0.1% Triton X-100, pH 7.8. Insoluble material was removed by centrifugation (14,500×g at 4° C. for 5 minutes). CAT assays were performed by the two-phase flour diffusion method as described previously (Nachtigal et al. (1989) [0226] Nuc Acid Res 17:4327-4337). Briefly, cell lysate (50 to 200 μg) is added to a scintillation vial with a lysis buffer to give a total volume of 200 μl. The solution is heated to 65° C. for 10 mintues, cooled to room temperature, and a reaction mix (75 μl), containing 2 μl ³H-acetyl CoA (Amersham Pharmacia Biotech), 50 μl of 5 mM chloramphenicol (in water), 7.5 μl of 1M Tris-HCl (pH 7.8) and 15.5 μl of water, was added. The reaction mixture was carefully overlaid with 3 ml of organic phase scintillation cocktail. After 30 minutes, the samples were counted for at least 5 minutes. Quantitative values for CAT activity were determined by regression analysis to give counts per minute, per mg (cpm/min/mg) of protein cell lysate.

Example 10

In Situ Filter Detection

About 10[0227] ⁷λgt11 clones of a cDNA expression library are prepared from RNA containing poly(A)⁺ RNA of the mouse distal caput epididymis. Clones are plated and replicated on nitrocellulose filters. After denaturaion and renaturation, the filter-bound proteins are screened with a concatenated oligonucleotide probe containing the nucleotide sequence of the cis-DNA regulatory element containing the nucleotide sequence of the cis-DNA regulatory element. The probe is prepared by nick translation with a specific activity of >10⁸μg. Duplicate screening using a probe carrying a mutated cis-DNA regulatory element is carried out to eliminate false positive clones.

Example 11

Mouse EP17 Knock-Out

To recover genomic DNA sequence necessary for homologous recombination, a 129/SvEv mouse genomic DNA library was screened using mE-RABP cDNA as a probe. BAC clone 170K23 was isolated, having 5.3 kb flanking region and all exons of the mEP17 gene. The targeting vector comprises a 5.3 kb EcoRV-SalI fragment of the 5′ promoter region and a 1.9 [0228] kb 3′ flanking region (FIG. 9). The entire mEP17 coding region is replaced with a PGKneomycin cassette from the pLNTK vector (Gorman et al., 1996), so that the PGKneomycin cassette is positioned between the 5′ promoter region and the 3′ flanking region. The targeting vector is linearized using an appropriate restriction enzyme, and the linearized vector is electroporated into TL1 embryonic stem (ES) cells. ES cells are selected based on demonstrated resistance to geneticin after 24 hours. Resistant cells are further screened by Southern blot analysis using a probe designed according to sequence of the targeting vector.
Clones bearing the transgene are injected into blastocysts according to standard procedures (Joyner (1993) “[0229] Gene Targeting—A Practical Approach” IRL Press, Oxford). Chimeric mice bearing the transgene are crossed with C57BLU6 females and agouti offspring are analyzed by PCR and Southern blot analysis for presence of the targeted allele. mEP17 homozygous mutant mice are obtained by crossing heterozygous mice having one native allele and a knock-out allele. mEP17 homozygous mutant mice are confirmed as such by demonstrating a loss of mEP17 expression by standard methods, including Northern blot analysis, RNAse protection assays, Western blot analysis, and immunohistochemistry.

Example 12

Recombinant Production of EP17 Protein

The mature protein coding sequence was cloned into the prokaryotic expression vector pBAD/gIII (Invitrogen). To simplify purification, the pBAD/gIII vector encodes a leader peptide which directs the recombinant protein into bacterial periplasmic space, thereby minimizing any potential toxic effect. The pBAD/gIII vector also encodes a C-terminal polyhistidine tag for detection with an anti-His antibody and for purification with ProBond resin (Invitrogen). The pBAD/gIII vector carrying the mEP17 coding sequence was transformed into [0230] E.coli according to the manufacturer's conditions. Transformed E.coli were cultured and recombinant protein was extracted. To confirm the production of mEP17 protein, protein derived from transformed E.coli was resolved on a polyacrylamide gel and Western blot analysis was performed according to standard techniques.

Example 13

In Vitro Binding Assays

Recombinant protein is obtained, for example, according to the approach described in Example 12 herein above. The protein is immobilized on chips appropriate for ligand binding assays. The protein immobilized on the chip is exposed to sample compound in solution according to methods well known in the art. While the sample compound is in contact with the immobilized protein, measurements capable of detecting protein-ligand interactions are conducted. Measurement techniques include, but are not limited to, SEDLI, biacore, and FCS, as described above. Compounds found to bind the protein are readily discovered in this approach and are subjected to further characterization. [0231]

Example 14

In Vivo Fertility Assay

Five wild type heterozygous transgenic and five mutant homozygous transgenic male mice are individually mated with five wild type C57BL/6 females during an overnight interval. Females exhibiting a vaginal plug the next morning are isolated. If pregnancy occurs, the genotype of the offspring are analyzed by PCR amplification of tail DNA using primers to detect the transgene. A lower percentage of pregnant females resulting from mating with homozygous mutant males suggests male subfertility. [0232]

Example 15

In Vitro Fertility Assay

The method used is essentially that of Wolf and Inoue (1976). In brief, male mice are killed, and each cauda epididymis is rapidly excised and minced in 1 ml of Toyoda's medium pre-equilibrated at 37° C. under 5% (vol/vol) carbon dioxide in air. The minced tissue is left at 37° C. for 30 minutes before the tissues pieces are removed. An aliquot is taken for sperm counting, and the incubation is continued for a further 30 minutes. Female mice are induced to superovulate by injections of PMSG and hCG. The female mice are killed, and their oviducts are removed and placed into Biggers, Whitten, and Whittingham medium (BWW). Under a microscope, the oviduct is pricked, and the cumulus mass is removed and treated with hyaluronidase. The denuded eggs are washed through three changes of medium before being allotted to 100 μl droplets of medium under silicon oil. [0233]
Approximately 10[0234] ⁵spermatozoa are added to each drop (i.e. 10⁶sperm/ml), and the dishes are incubated at 37° C. under 5% (vol/vol) carbon dioxide in air for 5 hours. At this time, some eggs are removed and washed by repeated micropipetting before the number of attached spermatozoa is scored. The rest of the eggs are transferred to fresh medium and incubated for a further 24 hours, at which time eggs are scored for evidence of fertilization and development. Experiments are conducted with spermatozoa from a mutant and a wild type male and eggs from a common pool of females.

Example 16

Regulation of EP17 Protein

The regulation of mEP17 protein expression was investigated using Western blotting and immunohistochemistry in castrated mice, castrated testosterone supplemented mice, unilateral castrated mice, unilateral cryptorchid mice, and busulphan-treated mice. As previously observed at the mRNA level (FIGS. [0235] 15A-15C), mEP17 protein disappeared from the initial segment two days after bilateral castration and was not restored by testosterone treatment. Similarly, after unilateral castration, mEP17 protein disappeared from the castrated side, but not from the non-castrated side. These data suggest that mEP17 is not regulated by circulating androgens, but can be regulated by testicular factors provided via the efferent ducts.
To determine whether germ cell-associated testicular factors can regulate mEP17, spermatogenesis was disrupted using cryptorchidism or busulphan treatment. One month following cryptorchidism, mEP17 protein was not detected in the initial segment of the cryptorchid epididymis but was detected at normal levels in the scrotal epididymis. In cryptorchidism, the testis and the epididymis are exposed to abdominal temperature. To distinguish the effects of abdominal temperature on the testis or epididymis, spermatogenesis was also disrupted by busulphan treatment. Following a 35-day treatment, the level of mEP17 protein was drastically reduced when compared to untreated controls. Collectively, these observations suggest that mEP17 is regulated by germ cell-associated factors. [0236]

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1 11 1 5315 DNA Mus musculus 5′UTR (1)..(5315) 1 gatatctagc atgtttccag tttctggata ttttgaataa agctactatg aacacagttg 60 agccagtgtc cttgtgggat ggtggagtat cttttgggta tatgcccagg agtgatatag 120 cagggtcttg gggtagaact atttccagtt tattgagaaa ctgccacatt gatttttcag 180 agtggttgta ccagttttta ctcccaccag caatggagga gtgttccctt gtgttccata 240 ccctgtcact tgaggtgttt ttttgttttt gttttaagat tttttttttc gagacagggt 300 ttctctgtat agtcctgtct gtcctggaac tcactttgta gactaggctg gcctagaact 360 cagaaatccg cctgcctctg cctcccaagt gctgggatta aaggcgtgcg ccaccacacc 420 cggctgtttt aagatttttt taaaaaaaga tttatttata ataataaata tctaagtaca 480 ctgtagctgt cttcagacac accagaagag ggcatcagat ctcattacag atggttgtga 540 gccaccatgt ggttgctggg atttgaactc aggatctttg gaagagcagt cagtgctctt 600 aaccactgag ccatctctac agcccatcac ttgagttttt gaatcttatc cattctgaca 660 gttgtaagat ggaatctcag agtccttttg atttgcatct ccctgataac taaggacgtt 720 gaacatttct ttaagggctt ctcagccatt cgagattcct gtttaactag gaaactcttt 780 actaactcct ttcttgcctg tcacagccac tgtgaagggt tcacacataa tcgcttctct 840 gcatcgcgta ggtcctaaga ccccagtccc caggccctca aggccttttc agacttctac 900 ccaacaatgg gtcttctggg taacatgatg gtaacaccac ccaaatcagt tactagttga 960 gctagcccac cagagcacct gcctaggtca tcccttcagt tctggagcat gtaggtctac 1020 attgcctaga cccccttcta tctccagcac cggtgaggag cccctgatgg ggataattct 1080 ctaaccagct ctgtggggat tgtaggggtg agcaggaccc aagcacctag tgggtgtgtc 1140 tgagagaaag gaggaaaatc aatgatcttc ccatgtgcgt ttctctgggt aaggaggtac 1200 tcaggtgtcc tgagtgacca gtgccaacat tctcgggcta tgtgcagggt gagttttctt 1260 ctgagcttgg gtcagcaaga tcctatccct gaaatctgtg atccctacta cctgtcgagt 1320 ctcatgctta tggttttagc ttagacatgt ctggtagaag atgccaccct ggtgtcttgg 1380 ggccatcata ataagccaag gcaaagagac agaaagaatt ctcttttagt ccaagagacc 1440 gaagtcagga atccatgtgt gtggaggcta gaggagggct cttgccaatc tgcaccccca 1500 acccccagct tctggtcttt cttctgttct gggcagtctt aatcttgact gcatcactaa 1560 tatctgcctc tttcttctgc ccatctgtgt ctgaattctt ccattcataa ggatgctgtc 1620 tttgggttag ggccatctga acctgtctgc agaaaccttg tcttcaagta agatattgtt 1680 tggaggttct gggtggggga tactattcaa tccagtgtac cccatgtttt gctaaccttt 1740 gggcccccct tctcagggct tgtaccatat ctttaattgt atcatcctgt tcccaagaac 1800 cctgaaaggg cagaactatt tttcatctac tgctccacta cagggttagg gtagaagtcc 1860 ttggcatctt gatacaggca ggttcatggg gaggcctacc caaccaaacc aatgtagaaa 1920 gctgcccaat tcctacccaa gctcattgct aacaattttg gcactcacaa agccacagta 1980 gtagatgaca tgtgagtgta taaaagtcag tggatagagg gggtgggtgt aataccgtat 2040 ggcttatcct gggatggaga ctttgtggta tttcttgttc ttttcccagc ctcctagttc 2100 tctgcatgct ggtactttgc aggtcatgat aggcctctta ttatcctttc ctcttgcctg 2160 cttaccctga atgcctgctg ttttgacagt ccatggatct aaacacctgt gctgagctta 2220 catgttctcc actatcaggt agttttgaaa acgtaagaag ttagactcaa tgtcaaatca 2280 ccctgtcatt tttcctgtta ctatggtctt ggtctctctt ctcagggcct ggcaaggctt 2340 aggggttact agtcctgtaa gaattaggga tccagaagta tggcaggtct ctatggtctc 2400 tgggaaggtg ccttggagag gcatctctag tcgggtcagt ggcagtgtgg gaagttttta 2460 ctggtttata gatgctttgg tgacaggatt atctttcctt gcctttgtgg gttggcagcc 2520 ttggggggca gtgcctagct tgtggggagt atggaggatg tttgacaaag gactggtgat 2580 ggagtgattg tgccctttta gggtaggatg aacctggagt tgccaacctc aagagtgtca 2640 ctctcactgt gatctttagg gactgagaga tcctcttggg tcccacctcc ttccgtcctg 2700 tgttccttct cttgctcttc tcctttcacc caataaacag attcagcatc cgcatggctc 2760 tttgaccctt tggagatgcc cttgggcaga tcatagtgaa ctctttcttc atcccagccc 2820 tctggccact tagggttctc ctcgagagtg ggcaggatgt ttcattcaaa tttggtgaag 2880 ggaggttgct gcatccacag ggaagactga gcacagcata tgtggtatcc acgaggtcac 2940 agtgtctttg aaaccaaggt gcctactgag gatgggttgt aggctctagg ctcccttggg 3000 tctcagaaat tgtatgtatg gatatatgaa ctggaaagca ggagggccat aatgctaggt 3060 tttgattccc atctcctcct ttgatcttgg acaaatggtt tcgacctttg ggagcctaag 3120 aaataggtca gggcagtggt cactgtgctt gaagaccttg ctaggctagc tgtcatgtga 3180 tttccatgac agacctcctg gatacacata gctagctgca tccattcttt tttctcagat 3240 tgagacactg aggcacagaa gctggacagg ttactcaagg tcacacgatg gacaaattca 3300 ggggtggcat gatgtagtct ctgtgctgct cttgaacaag atgtggggta cgggggttcc 3360 tttccaggta ttatcactgc tctcgtcttc atgtcacaga attcccattc tccagtggat 3420 ctagttctac ttcaaagtcc aggcatggaa acaggaaggg gctggttctg ctccattgct 3480 tatctgtctg tggtcctcag acaacaagaa ctggaagcaa ttagccctca tagctgctgc 3540 cctcatcctg tgccagtcac ccagagaacc aggtgccatc cacccacatc tctttccctc 3600 actccagcca aacccctctc tgtatcccca agggccctgg cttgtcagtc tgttgttggg 3660 accagcaggc cctcagcttc cctcccgatg ccccaaagtc tttggatggt tccttctagt 3720 gctcactgtg cattctctcc taactcatct cttatgccgt cctcacaggc caccctaatg 3780 tcttctgtgc ctggttccct ctggagggtg ggcttcagtg gatttcctgt gagctgtcca 3840 ggtcttgagc agctccaggt gggtccaaaa gattagactg ttcctacagg atgaggggtg 3900 ctgacctggg tagagctgtt ttggggtgcc ctgggacact caagacctat ttctctgcta 3960 cacaagttgg aggttagaga tttgggtcac aaggtgacta agtcattttg ggggtgtcag 4020 tcagggaggg ctttgcattc agggtgcttc atggtccctg tttatcctgt ttctttcctt 4080 tttatttgga ggaaatagac aattgtgaga aaaactaata tttttagttt ttttattttc 4140 ctagcgaatg ggagactttg ttttagtatc aatcacaaat aaagttgatt ttaaagataa 4200 aagttgtctg ccttaaaaaa attctctgtg gtctgagagg tgtttgtttg tttctgagag 4260 gtgtgaaaaa ccttgtcgtt ggggctggga agatagctca gctggtaaag tactttcttc 4320 agaacccata taaaagccag gcatggtggt gtgtatttga aatcacatgg tgagtgctgg 4380 acagacagag atagtgggat ctctggggct cactggctag gcagcctaga ctactcagca 4440 cgcttcaggt tagtaagaaa ctctgtctca aaaaataagt aaccagccaa ttgaccaacc 4500 aatcaataaa ctaactaact agctagctag ctagctagct aactaactaa ctaactaact 4560 aactaactaa tgaagcaaaa caaatgaaaa cacaaagtga cttgtactaa aggaacaaca 4620 cctgaggttg aactctggct tccacatcca tgtgactatt tgtatgcttt ccccaaccaa 4680 gaacaaagac atgaacacta tatacacaga gacacacaga cacacaaata ccacaaacat 4740 acacaccaca cacaaacacc acatacaaac atacatacca cacacaaata ccacaaacat 4800 atataacaca cacatacaca gataccacat acaaacatat ataacacacc acacagatag 4860 agatactata taaaaaaata tacacacaca gaatggaggg aatgcagaat gctgcaaggt 4920 ataaggtata aggttcctgg ttggagactc aggcctcctt gttcagttca atattttgtt 4980 attgttttaa gcttattgtt acaaatggag aaactgagat aaatagcttg tccagttcat 5040 agctcagagc gtggggctgg acttggttgg tgcagtctta ctgcatagtt catcactgtc 5100 cacatgctag gatggaggca gcttaactgt catcttagct tggtcctaca cctctctgga 5160 tgggggttga tagcatttga gcagaagctg agtctctgag cagctgacag ccagctttgt 5220 ccaatgacat tctctaagtg gttgcacatg cttgcacact ctccaaatat aagctcccac 5280 cttgcataaa cagaagccac aagccaggcc ctgag 5315 2 30865 DNA Homo sapiens 2 tgtttatttt tattattttt tttttttaaa tttatttaac ataattttta ttaataataa 60 taataaatat agtttgtaaa ttttaaaaat aaaagagaag agataaagaa tagattaaat 120 gaattatata aatgaattaa ggtaatactc aatcctttgg atatgtggga ttatacacca 180 tataattttg ttatatttaa tattagtata tatggtaggg tgtatgtgag aaaaggtaaa 240 acttaaaaaa gcggtgaatt acaagatcgg taacgaaggt cgaaaagaaa ggaaatagcg 300 ggtgtctggt gatgtcttgg atttgagggt cgggggtttc gaactctttt taaggtccat 360 tatttaggcg cggcggggag agaggtatgt aaaaccgcaa gatggggtcc ttatcaggat 420 aaggaccacg agtgactgtt agcagaaagt aatatcatga ggaaacagta aaaaaaatac 480 ttatgagttg ggttgggagg ggactgagct ctgagaaagg accccagttc actatgtcaa 540 aaacgaaaaa aaggcaaggc gcagtggctc acgcctgtaa tcccggcact ttgggaggcc 600 aaggagggca gatcacctga ggtcaggagt ttgagaccag cctgaccaac atggtgaaat 660 cctgtctcta ctaaaaatgc aaaaaaaatt agacgggcat ggtggctact gttatcccag 720 ctacatggga ggctgaggca ggagaatcgc ttgaacccgg gaggtggaga ttgcagtgag 780 ctgagattgc accactgcac tccagcctga gtgacagagt gagaccccgt cttaaaaaac 840 aaaacaaggc caggcacggt ggctcatgcc tgcaatccca gcactctggg aggctgaggc 900 aggtggatca caaggtcagg agatcgagac catcctggct aacatggtga aaccctgtct 960 ctactaaaaa tacaaagaat tatcccagca tggtggtgga cgcctgtggt cccagctact 1020 ccggaggctg aggtgggaga atggcgtgaa cccaggaggt ggagcttgca gtgagctgag 1080 attgcgccac agcactccag cctgggcgac agagcgagac tccatctcaa aaaaagaaaa 1140 aaaaaaaaaa aaaaaaaaga aaaaaaagaa aaaagaaaaa aagaaaagaa aacgaagaaa 1200 aaccacaaat aaataaaagt tgaggtatga tgtatgcaca gctgcattcg cccctttgag 1260 ggcacagttg tgtgagttta gacaaatgca aatgttcatg gaaccactgc aagtgcgaca 1320 cagcagctct ggtgcccaca aagcttcctc gaggtccctt gcggtcaatc actgcccgca 1380 cctcagccct cagagccact gggaagcccc ctgccctgtg ggctcacctg tcctagaacg 1440 tcgtataaaa ggagtcgcag atcgcagcct tttccacctg gcttctgtcc ctctgcaggc 1500 tgcacctggg attcagccac cttcgttcct tttactgctg agtgtccatc acgtgctgct 1560 catgcagaag tgctcaagtt cgctgtaaac gtagttttca tttctcccgg gtggagacct 1620 ccataagtag gtaggttggt ggaatgtttc actgcatgag agtctgccag gaagttttgc 1680 agtgtggtcc cgtcttgttc ttcgcgcaga gggtgatagc tctgctgact cccagccctc 1740 acaggctcta gggattgagc ctgtttctgt ggaacgcctt ccatggtctg cgaggtgcac 1800 agaggggtct caaaaagctt ccaggttgca tcttcccatg gactgatgct gagcgtgttc 1860 tcatctccca tccggatgtt ttctttggtg gagtatcttt tcagatctct tgccttttta 1920 tttttatttg tttatatata tattttgaga cggaattttg ctctttcact taggttggag 1980 tgcaacggcg cgatcttggc tcactgcaac ctccccccac ccacctcact ttgggttcaa 2040 gcgattctcc tgcctcagcc tcccgagtag ctgggactac aggcgcccgc caccatgccc 2100 ggctaatttt tgtattttga gaagagatag ggtttcacca tgttggccag gctggtctcg 2160 aactcctgac ctcaggtgat ccacccgcct cggcctccca aaatgcgagg atcacaggcg 2220 tgagatgcca cacctggcct ctttttattt ttaatggagt ggcttgtttt cttattactc 2280 agtcttggga gttcctcatg gatgctggat ttcagtctcg tctcaggtgt atgcgttttg 2340 tagatgtttt ctgtagggac cagccccaca gggttggtga gtttctccct gtgtgctgag 2400 atgagagggc atagaaataa ggacacaaga aaaagacata aaagaaaaga cagctgggcc 2460 tggggaccac taccaccaag acgtggagac cggtagtggc cccgaatgtc tggctgtgct 2520 gatatttatt ggatacaaag caaaagggac agggtaaaga gtgtgagtca tctccaatga 2580 taggtaaggt gacgtgagtc acgtgtccac cggatggggg gcccttccct gtttggcagc 2640 caaggcggag agagagagag agagacagct tacatcatta tttctgcata tcagagactt 2700 ttagtacttt cactaattga ctactgctat ctagaaggca gagccaggtg tacaggatgg 2760 aacatgaagg cggactacga gcgtgaccac tgaagcacag catcacaggg agacggttag 2820 gcctctggat aactgcgggc aggtctgact gatgtcaggc cctccacagg aggtggagga 2880 gtagagtcct ctctaagctc ccccggggga aagggagact ccctttccct gtctgctaag 2940 tagcaggtgt tttcccttga cactgacgct actgctagat cacggtccgc ttggcaaccg 3000 gtgtcttccc agacgctggc gtcaccacta gaccaaggag ccctctggtg gccctgtccg 3060 gcgtaacaga aggctcgcac tcttgtcttc tggtcacttc tcactatgtc ccctcagctc 3120 ctatctctgt atggcctggt ttttcctagg ttataattgt agagcaagga ttattataat 3180 attggaataa agagtaattg ctacaagcta atgattaata atattcatat ataatcatgt 3240 ctatgatcta gatctagtat aactcttgtt attttatata ttttattaaa ctggaacagc 3300 tcgtgccctc ggtctcttgc ttcggcatct gggtggcttg ctgaccacag ttttcctgct 3360 gtatctaact gctggcttag gtgaacctgt caggggcgtg gctgggaact ggaacctcct 3420 gaagaccagt ggaggccaca agcagaagcc ctcggctcct tcccttcccc gatgctgggg 3480 gccaagggtc tttctctgac cctgtccccc cctcacgtta ccagtggagg gtgtccaggt 3540 tcttggcatt ttgaacaaag aattggacaa aacacacaaa caaagcaagg aaagaatgaa 3600 gcaacaaaag cagaaattta ttgaaaatga aagcactctt tacagggtgg gagcgggctg 3660 agcaagcagc tcaagggccc cggttacaga attttctggt gtttcaatat cctctagcgg 3720 tttaccattg gttacttggt gtatgcccta tgtaaatgaa gaggatgaag tcaaagtcat 3780 tttctcggct gagcatactg tcacgcctgt aatcccagta ctttgggagg ctgaggtggg 3840 tgtatcacct gaggtcggga gttcaagacc agcctgatga acatggagaa accccgtctc 3900 tactaaaaat acaaaattag ctgggtgtgg tggcgggtgc ctgtaatccc agatagtcag 3960 gaggctgagg caggaaaatc gcttaaaccc gggaggcaga ggctgcagag agccaaggag 4020 tgcaccactg cactccagcc tgggcaacaa gagcaaaact ccatctcaaa aaacaaacaa 4080 acaaaacgaa gtcatgtact cggtatgagc cctacgtaaa cggagaggat ggagtgaagg 4140 tacaaagcca ttcacattcc cgtcatcgtt agtgtttcca gttgatttgg ttctaggatg 4200 cccttgggct ccctgcgtcc aggccctctt ctcctgcctc actctcactc cagaggactg 4260 gcagtgctgg cctccctggc aggactcccg cccctgacca gaggggtctg ggcgtgctcc 4320 gaggccacct gctgctcctt gcccagtgtc cctgggtgct ctgtggggac acagagaggt 4380 cacttgggag gttcctgcct ctgccagact gcggcgcctt cctggcgtca ttgccccctg 4440 cacgcttata caaccagtga taaggcggta tccaatgccg taatcctccc cagggccagc 4500 ccagccctgt ccggtcccgt ccggtccctg gcctgcttgc tgggagccca gctgggggct 4560 gcatcggggg tggtgggggt ctggagtgag gggtgcccag gcctctctca cagtgtgggg 4620 gcacaagctc tgccctggac actctgccag tgtgacccta ccccccatgt tgggggtgag 4680 agaagggggc tttctgggtg gccagatccc aggagagggg gcctagccag accaacccca 4740 gcccagctct ctgcagctgt accccaaagg ccctggccca gcccagccag acagagtgtg 4800 gacagagggg cttgggtgcg agtccaggca aggcaggaca agtctctgct gtggcttggc 4860 agtggccccg tgactggtga catagtctgg caggcctagg ggacaaatcc tcagcccggg 4920 tgcatcaggc cagagcctgg ctgtctggag ccctccagga aatgaccccc cgggctgggg 4980 gacatccgag tgattgtgcc aaacaatgga caagggggcc ggagtctcag agccgaagtg 5040 ccttccgctt ccatctgctc cgcccaggag caggtgcacc caggtggtgg cctgggctat 5100 aaagctggcc ccctggggct tggggactca gcaccagggg ctggagggca ggggagggga 5160 tgatgtcatt cctgctcggc gcaatcctga ccctgctctg ggcgcccacg gctcaggctg 5220 aggttctgct gcagcctgac ttcaatgctg aaaaggtacc aggggcctct gctgtcctgt 5280 ggtgggtggg agctgggccc ctgccagaga caacgtgata attgtgacaa ctgcctttcc 5340 tggggcgctt gctctgtgcc cggccaggca cgtgctatag acccactctg cgcttaatcc 5400 taaaccaacc tgtgaatgga tcattactgc acccacttca cagctgggaa ctgaggctca 5460 cagttgcact gtgacctacc caagatgagt tcctgtccac ctgggctcag ctcacaccca 5520 agagatggcc actccgagac cccttgctgt gtgacttctg agttgtcccc tgggtccctg 5580 ggcaaggagg gccctgctgc tggctgtcct ggcctgcggg tgactaagcc cccggcagtt 5640 ctcaggcctc tggtacgtgg tctccatggc atctgactgc agggtcttcc tgggcaagaa 5700 ggaccacctg tccatgtcca ccagggccat caggcccaca gaggagggcg gcctccacgt 5760 ccacatggag ttcccggggt gagttacttg ggctgggctg cctgggctgg gctggggagc 5820 tgaggtagct gggtgccatg agtgagccgg ctgtgcaccc ccagggcgga cggctgtaac 5880 caggtggatg ccgagtacct gaaggtgggc tccgagggac acttcagagt cccgggtagg 5940 tcctgcctcc tctgccttca ggggtgctgg atgccgcttc tctgggggtg acgtggggct 6000 gactttccac ccgggcccaa gctgtccatc ccagtctcca gccccctgac cgccctcccc 6060 gcccctcctc tgtcttcggg gaacaggctc acagcccagc agcaggggca ggcatgggtg 6120 gggtcggtgc ccgggcccat ccacttctcc agcctgaaaa ggccgcctgc ctctccgcag 6180 ccttgggcta cctggacgtg cgcatcgtgg acacagacta cagctccttc gccgtccttt 6240 acatctacaa ggagctggag ggggcgctca gcaccatggt gcagctctac agtgagtgcc 6300 tgtcccacct ccccgcactg gcccctcccc aacttcccac aggccccgcc ccccgttctg 6360 cctacccccg ccccgcccgc ctgccgcccc gcgcctcctc acaggccccg ccctcacccc 6420 tccgcccccc gccttttagg ccgagtttag gctccgcctc ctcatccccg ggcccgcccc 6480 ttctggtgct gccccatgcc ctctcctgct gtgacccttc gcacacactc ctggcctggc 6540 caggaccggg tggggtggga gctgggcccg ccgtgctttg gatgtggagg aggttggctt 6600 gggggagggt cccgcggtgc atgctcctct ctggcctcct gagagtggtg ccccgccctt 6660 ccctgcctgg cctcgctgcc tgagttctgg ggctggggct gaggttcttt tttttttttt 6720 tttttttaat atggagtctc actctgtcgc ccaggctgga gtgcagtggc gcgatctcgg 6780 ctcactgcaa cctccgactc cccagttcaa gcaattctcc tgcctcagcc ccccgataag 6840 ctgggactac aggcgcccat caccatgccc gtatttgtat ttgtaaaaat ataaataatt 6900 ttttgtattt ttagtagaga cggggtttca ccgtgttagc caggatggtc tcgatctcct 6960 gacctcgtga tccgtccgcc tcggcctccc caaagtgctg ggattccagg cgtgagccac 7020 cgcgccggct ggggctcagg ttctttaact cctggggact ccccgattgg agccgacaga 7080 gtgaggctgg aggcttccgg gctgcaggga aagaggctca aggggtagtg gggcccagga 7140 ggacagccag accaccccaa cctcccagac atcttggtgt gaattgggga agggggcccc 7200 aagtttcctt cagccaggca ggagctttat gccgcgcaca acctgcccaa cccagggtgg 7260 cggccttgct gtcagccctc aggctggtgg agtgaggcca ggagaccccg tgctgcactc 7320 ctgtgagctg ctgagttcac agtgacctct gctctgccca ggccggaccc aggatgtgag 7380 tccccaggct ctgaaggcct tccaggactt ctacccgacc ctggggctcc ccgaggacat 7440 gatggtcatg ctgccccagt caggtaccct ggcaagcccc gcccgcccca tgccccccag 7500 cctctccctc ccttcccatg agcccaccct tccttcccct cacagcacag gcagtgtgcg 7560 ggcagcaggt ggtcttctct tccaggggtg ggctggcagc ccctgcagcc cctaccccac 7620 ctcccccaga acagactacg ggagaggctt tgggcagcgc tgggagggaa gctctgggct 7680 ccactctctg gggatggtgg agctgtccag tggcccttgg cacccagggg ttgggggctg 7740 cgggaagcga gggcggtcct ggccgtcagc atctcacgga tgtcttcctc cccacagatg 7800 catgcaaccc tgagagcaag gaggcgccct gacacctccg gagccccacc cccgcccttc 7860 ccaggtgggt tctccaggcc ctgcagggga tgccctggtt gcctctcccc tccctatcgc 7920 agcttgacat ctggttctgt ctgccccatg cctgccccgc tgttctgcct ggcgacccca 7980 tgcttcaggc ctcagcttag acattacctc ttccaggaag cctcccttga tttcctaagg 8040 tacccatcac agagtaccac aaatggggcg acttaaaaca gcagaagttt ggccgggtgc 8100 agtggctcat gcctatcatc ccagcacttt gggaggccga ggcaggcggg tcacctgagg 8160 ttgggagttc gagaccagcc tgaccaacat ggagaaaccc cgtctctact aaaaatacaa 8220 aacttagctg ggcgtggtgg tgcacacctg taatctcagc tacttgtgag gctgaggcag 8280 gagaatcgcc tgaacccagg aggcggaggt tgcggtgagc cgagattgca ccattacagt 8340 ccagcctggg tgacaagaga gaaactccat ctcaaaacaa acagacaaaa ccagcagacg 8400 tttatctctc ccagttctgg aggccagaag tccaaaatca agacgtccac agggctggtt 8460 cctctggaag ctctgaagga gactccctcc cacaccgccc ccccagctcc cacggctgcc 8520 gcttgtcctg ggcatttctt ggcctgtgga agcatcagcc ctgtctccgc ctccgtcctc 8580 ccacagccac cttccttctg tgcatctgtg tccaaactgc cccctagtgt ctgtgctgcc 8640 gaagcgagtg ccccacggcc ctcttcttag gagcatgcct gtgcttggat tcagggccca 8700 ctgtaaatgc aggatgatct aatctccaga ttctaaacta gtggtatctg caaagaccgt 8760 ttccaaataa ggccacgttc ggaggttcca ggtgggcctg aagttttggg gacacgattc 8820 aatccacagc tctgtgtccc cgctgtatcc acacagcccc tggctccctc cccagggcag 8880 ggtcctgcct tggaattgtg ggagccttgt cttctccccc agggcccagg agggcagacc 8940 acagccttcg cgcgcagtgc ccctcacagg agccacgtgc gggcggggtc ttgcggagat 9000 ccccccctaa accagacgcc gggagaccgc tgggtccctc gccggggctc accgccaaga 9060 atttgggcac agccacacgt cacgtgtctg acgtgacagt caccggcgtc tggagggaca 9120 ctggcccttc ctggcatggc gggaggaggt gggcggctct gaggcggggc tgtttctcct 9180 gcgtttctgc cgtgctgctg ttgcgtttcc tgcatctctg ctcctctcca tgagcttcgc 9240 ctccactccc aggaccctct ccctggagac tcgccgtcct gcctggggac actggggctg 9300 ttcagctttg cacaagtctg caccagcgtc tccctgcaca accccgctgg gttgggaaac 9360 atggggggca acaccaaatc gcccttgtcc agaaggttct gtgggcagaa catgtagccc 9420 catcccgcca tgatcttgaa gaagcagctt tcggggtaga gtgccccgcc tgggccacca 9480 cgcgtgaccg tggaggctgt gggttctgtg gtggcttcat ggacattccg gggcttttct 9540 tgccatgcag cccccttcac cgaggaccag gcagaggcca tggctcactg ccactcagcc 9600 agggactggg gcatggcact gggtcctccc gcgccgggga aggtggggaa gtgctggagc 9660 cagggatggc ctctggggca gcctctcgcc tggggcctgt ggggcagcag tggtagctgg 9720 tgtgtgtttc caggtttgca ggagttttgg gagcagaatg agctctccct ggcctgtcag 9780 tggtggcttt ggaagagtga tgcccagctt gtggggagca ggaagggcgt ttgtcgcggg 9840 agtgggtgat ggagtaggca tgcgttttct acgcaggtgg agccaaagca gcaggcgcct 9900 ttgcccctgg agtcaagacc cacagccctc ggggaccacc tggagtctct ccatcctcca 9960 ccccccgcct gtgggatgcc ttgtgggacg tctctttcta ttcaataaac agatgctgca 10020 gcctcatggc cctcacctct ttggacatgc cctgggggag ggcaatgcgg gcccctgact 10080 gaccccagca ctgctgggga gactgaggca gggaggcaag tgtgagaagg ccctggccgg 10140 gtgagcaggc cctggggtga gagggcaggg ggcaggggag aggggccctg ggaaggggca 10200 gggggcaggg gtgaagggcc ctgggtgagg gggcagggga caagggtgag gggccctggg 10260 tgaagaggca ggggacaggt gtgaggggcc ctggggaggg gcaggaggca gcagccactc 10320 ttggcctgtg tgggggctgc acccacagga agaatggagc atcctggccc agtccctatg 10380 ggtctgcagt gctgtgggga actgggcacc ctgtgaagac ggcccggcca ctggggctgt 10440 gctgggcaca caggccccgg tgctggggcg gggagctggg ggagctgcag gaggccgggg 10500 agttaggccc acttgggttt tgcccctcac cactgatatg atcgtgggca cctggtttac 10560 acccctggaa atgggtcacc gttgtggcca ggggactgcc agccagcctg agcgctccac 10620 agcagaccca cagacatagg tagctacccc gcgtactcct cgtttcaaag atcaggaaac 10680 tggcctggcg cggtggctca cacctgtaat cccagcactt tgggaggccg aggcgggtgg 10740 atcacttgag gccaggagtt tgagaccagc ctggccaaca tggcaaaacc ccgtctctac 10800 taaaaataca agacttaacc gggcgtggtg gcgcacacct gtagtcccag ctactcagga 10860 ggctgaggca ggagaatcgc ttgaacccag gaggcagagg ctgcagtgag ctgagattgt 10920 gccactgcac tccagcccgg gcgatggagc aagactctgt ctcagaaaaa aaaaaaaaaa 10980 aagaaaaaaa aatcaagaaa ccgaggcaca gaagggttgg gtaacttatc caaggctgca 11040 cagccaggaa gaggccaggc aggctgcaag cctgggtgat caggctccag ggccgggctc 11100 cacactgccc ccgggtggtg atgcctggtg cagcagaggc cctccccact gtcactgtca 11160 ctctggtcgc tggcgctgtg cctccggcct ccggagctct gactgcccac tgcgtctggc 11220 acacatttca aagggccagg cgtggggtgg cacaggaaag tgctggaggc tctggtccgt 11280 tgcccatcag tgcccacagc cctcgcagcg caggccagaa cgagcccaaa aaaacaagtg 11340 tcatgtgggg tgggggtcac cgagccccag gtggtgtggg tggagctcct cttcctctca 11400 ggctccgaga cggccccagc agcgtccacc gctgtccata cgccaggagg gtggctgggc 11460 aggctgctgt ctaggccagg ctaacccctg cggtgggcgt gggtgtcacc agggccgatg 11520 gcgcttgtgc agaaacccac gtctctgagc tgccagcagc caagctgtcc tgatgacatt 11580 cccgggtggg cgcacaagcc tgcactgtcc gtatagaatc ggcccaggct gtgcagcagg 11640 ggaacccgga gcccggaccc cgccacggag gccaggctgc cgtgcaccat cctgggtgtc 11700 ctcgtggtgc tccgggcgca ggtggcagca gccatggagg agctggaccg gcagaaggtg 11760 gcttctcctt cttagttcgt ggggctcctc ctacaccccc aacccctcag gctcagggaa 11820 ggaggcctct cccggtgtgg ggagctcgtg gggacgctgg tgcccggcta gacacttcct 11880 gttagcggca ttttcttctc cgctgagtct gtgccggctg ctgggccaga ggcacattag 11940 caggcccaga gaaggtagat gccggagacg aagattcttt cctcccgaaa atggtagggt 12000 ttttaaaagt ctcagcggaa gtcccggctc tgggccggtt gctgagggca ggaggcccat 12060 cccctggcgt ggttggcagg ctggcgagct gcgctacccg agccacctgt tctctggcgt 12120 ttctcactcc gccgcgccct gcgggctttc ttttccaggc ccctgctcct gggtcctgcc 12180 tccgaggtca ggcagggcct gtggttcctc ccgacatgtc gcagaagccc cagggactgt 12240 tccgcagctc tagatggccc agtggggagg ggctgccctg tgggcattgc tgtctgatgg 12300 cctgaaggca ccgcttggag ggacacatgc ctggggacag tgggctcaca gatgtcttgc 12360 tgctttgcca ccgagcctca cagccatctg ctgacctctc agagcccagc aggcccctgc 12420 ccgggggttc gtggaatgcc cctgggggtc tcagacccac tgctcagctc ttggccaggc 12480 tccgtatctc tctagattgg aggattctgg agggaagtcg gtgtggcctc cgatcaaagc 12540 ctggtgctga cggccccgaa gcgggtggag ggcttgttcc tcaccttgag cgggagtaac 12600 ctgaccgtga aggttgcata taacaggtaa gaggcctggg ggctgctgga gaggaagagg 12660 catgcatggc cggggactgg gcgtgggttg ctgcacccgc tgcagcgtgg ggtctgggct 12720 gagtgccgtc ccgctggtgg gccagcactc ccggtgcccg tctgcctgag ccctcgaccc 12780 ccaggctcct ggatctgcat tttatcccaa atataagcat agtgttttaa tgaagccccg 12840 tgaacttaga aggaaggaaa aggggtgtgg gacaggtgac tggctggagg gaacagaatc 12900 cccaggggaa ggccccacct ggcacattcc ccgcctcatc ctgctccagg ccctgggata 12960 ctccacagtg ctgcctgtgc ccttcctaaa gacacagccc cactccggga aggagcccag 13020 caggcgggac acaggctagc attttaccga tgggcacttt gctgcctttc agctcaggaa 13080 gctgtgagat agagaagatc gtgggctcag aaatagacag tacgggaaaa ttcgcttttc 13140 ctggtaagtg cagttgccct gtgatggcag gtggaacccg gctgtgcaca cagctaggcc 13200 ttattgttcc ccatgctgtt ccctgcactg ttccccatgc tgttccctgc actgttctct 13260 gtgctgttcc ctgcactgtt ccccatgctg ttccctgaac tattccctgt gctgttcccc 13320 atgttgttcc ctgcactgct ccctgcactg ttccacatgc tgtttcctgc actattcccc 13380 atgctgttcc ctgcactttt ctctgcgcca ttccccatgc gttccctgca ctgttccctg 13440 cactgttccc catgctgttc cctgcgctgt tccccatgct gttccctgca ctgttcccca 13500 tgctgttccc tgcgctgttc cccatgctgt tccctgcgct gttccccatg ctgttccctg 13560 cactgttccc catgctgttc cctgcaatgc tccctgcact gttccccgca ctgctccctg 13620 cactgttccc catgctgttt cctgcactat tccccatgct gttccctgca cttttctctg 13680 tgccgttccc catgcattcc ctgcactgtt ccctgcactg ttccccatgc tgttccctgc 13740 aatgctccct gcactgttcc ccgcactgct ccctgcacta ttccccatgc tgttccctgc 13800 acttttctct gtgccgttcc ccatgcattc cctgcactgt tccctgcact gttccccatg 13860 ctgttccctg cactgttccc catgctgttt cctgcaccgt tccccatgct gttccctgca 13920 gggttccctg cactgttccc catgctgttc cctgcacatt tcatgcccca gaccttccca 13980 ttctcccacc aacacactgg atcatccttc aaaagcttct gtagtgtctc caaccactca 14040 agtgctggga ctgggtgggg gcaggatgga gttagaccct gcagaccctg gccttcgagg 14100 tccgtccccc tcagacgtct cccccaacgc catggccggc tcttgaaggc cacagagaga 14160 tccacgtgct ggacaccgac tacgagggct acgccatcct gcgggtgtcc ctgatgtggc 14220 ggggcaggaa ctttcgcgtc ctcaagtact ttagtaagct tggccctggg gggctctgcc 14280 cagctgctgc tctcccaggg actgcccgcc cagcccccct gtgccccaca gctcggagcc 14340 ttgaggacaa ggaccggctg gggttctgga agtttcggga gctgacagca gacactggtc 14400 tctacctggc ggcccggcct ggtgagccca ggggccttgg ggtggaggct gggctgggcc 14460 ctgtgggctg actctgcagc tcctcatgct ggcctatcct gcagggcggt gtgccgagct 14520 cctgaaggag gtgagcttga cccccgaccc tggcctgtgc tgaagttccc gggcccctgg 14580 cccagtccct ggccctgtca ggagcccccg tggctccgcc tcccggccct gggctgggcc 14640 ttctcacccc ttcctgtgaa caggacacca aacaccactg gtgggcagct ccagagatga 14700 gtctgtctcc tggtttggaa agagctggaa cctccagggt ggtgacccta ggctgccagg 14760 cagggaccct gggaggctgg ggtcacgggg tgcagagctg ggtggggcag gggagcagaa 14820 atggcgcctt ttcttcggtg ttccgtgcag gactgccggc tgcttctgcc cccgaaggtc 14880 ccgtcggcgg cggggcacag atcctgcggg cgctgcctca gggctcccat gttgggcact 14940 gcgagaaccc agtgtctccc tcacctcgct ttgtcttggc cctagaggct gggcctgtta 15000 ccccattttg cagattgaga aggcgctcag ggagctgggt gctttgcgca aaaccaggca 15060 gcgaggacag aagtcccgcc gtgtggccct catcgaagcc ccgtggggcc tccagagacc 15120 acacgggcct gagcccctgc acttctgtgt cgcaggagct gatttaatgg agttcctgcc 15180 tcagaccaca aggttcggag cgcccgccca cccctgcccc tcctgggcac cctgcccacc 15240 aggtcacctg cacctgctct gaataaactg tgaagtcaag ccactgcctg gtgtgtcctt 15300 ccggagggcc gatgggtgac aggtgtgggg ggactcaccc gccccaggtc tggcaacagg 15360 aggtgtgttt tccgtggtta ctggacaaac agtgcggctc acgtgcaagg cagcaacccc 15420 tgccctcccg ccctgctagg gtcgtggtca ggctgcccca gggtatagac cagagacaga 15480 aacagggctc ctggtggagt ctcagcaggt ctgtcaaacg catggaggcc acacccctcc 15540 tgcctgcatc ctccatggct ccccactgcc cagggctcca ggctcacggc ccgtgctgaa 15600 ggccctaccc gatgggcccc gcttccggcc tccctggacc cccagctgcc cagccctgct 15660 cagtgtccgc ttccacagtg ggctgtcacc ccgtggcctc ggctgccacc agccgactgc 15720 atcactttct cctcccgcct ggcctgggac acgggaaggg caaggaaagt gacagtgtca 15780 gcccagaggt tctgcgccca ggcccctggg gaatcagccg gtccccacat atgcaggtga 15840 gacaacccag gcttatgagg gtggctgcct cgtcggggcg gcggaaaatg atacctgagg 15900 tcgagtggtg ccgggagctc tgggggaaca gcccgagtag ggggacctcg ccagccccca 15960 gcacggccgg gacactggaa aggccctgct aggaacaagg gctgcaccct agatgcgcct 16020 caaatccaga actgtgcccc cgagacccac aggagggaca tgtagaggct gcattctgac 16080 aagttagggg ctcctgggaa acagccgtga ctttgcgcag ctgtgcctgg ccgaagccca 16140 cgggttcaag gagcttcgcc tggacttgga tttcctgctg gaggggctgt cactctcgct 16200 gtcagctcag gctcccgtga caaaataccc cagactgggc accttaaaca acagaactgt 16260 atttcctcac cgttctggag ggtggaagtc caagattaag gtgccggaaa gtgcagtttc 16320 tggtgagggc ttcctgccca ttcgcagaag gcagcccctc gctgtgtcct cacctggcag 16380 ggagcgctct ctgggtgcct ctttttctcc taataaggat atcagcctgt taaattaggg 16440 ccactccctt tgacctcact taatctcgat tgtctcctaa gagtcccatc tcctgaaaca 16500 gtgaccatgg gatctggggc ttcaacgcag gaattttggg agggcagaat tcatcccaga 16560 acaaaagtca gtgctgttta gagggtgaca gtagatgcag ggctgccgtc atcaacaacg 16620 tcctgcccaa aagtcactgg acgtccaaag acacagaaaa gtatgaccta tgttaaaaaa 16680 aaaaagaaaa aaaaagttac taaaaactga caccgagaag acccaaagtt gtattcagaa 16740 gataaaggtc taaaacagcc attataagta gttaaaagaa aagcaagata tactctaagg 16800 aaagaaatag tgacacaata gcattaaagg actaactaat tgataaagaa ttaaggagtg 16860 aacgtaggag atcagatcag gaaatcaaga gagaaatgga aactataaaa aagaaaaaat 16920 tatggaactg aaaagtacaa taaatgaaaa atttactgga aagactaata ggagttggcc 16980 atggcaaaaa taatgtccat gaatttgagc acaaatcaat gtaaattatc cagtctgagg 17040 aacagagaag aaaatggttg atagcacctc aaagatatgt ggggcaagat aaagaatgta 17100 aactagcctg agaaagagaa gagagtgaga atacatgaag aaacagtggc caaaataacc 17160 caaacttgat ggaaacatca acttagccca gaggctcaga gaaacccagg caggatgaaa 17220 aagaaagcct cacaaaaatc acacctgggg acattatagt caaagcactg ccaaccaatg 17280 atttggataa aaatttaaag ggatctgtgg attaaagaca gggcacatcc aggtttggtg 17340 gctcacgtct gtaatcccag cactttggga ggctgaggca ggaagatcac ttgaggtcag 17400 aagtttgaga ccagcttagg caacatagtg agactttgtc tctctttaag aacaaacaaa 17460 aaagacaggg catgatatag tttggctctg tgtccccacc caaatctcat gtggcattat 17520 tgcaatgccc aatgttggag gtagggcttg gttggaggtg attggatcat ggaggcagat 17580 ttccccttgc tggtcttgtg acagtgagtg agttctcagg agatctggtt gtttaaaagt 17640 gtgtagcact tgccctttca ttctctctcc tgctggccat gtaaagatgt acctgcttcc 17700 ccttcacctt cagctatgat tgaaagtttc ctgaggcctc cacagctgtg attcctgtaa 17760 agcctgtgga accatgagtc aagtaaactt cttttcttca taaattaccc agtctcaggt 17820 agttcttcat agcaatgtga gaatggacta atacagggca taaacagggg gacacaatgg 17880 ctgtcttctc atgataaatg gaaaccagga gatgacaata ggccatcttt acattgatga 17940 aaaaaaaaat caacgcagag ctctttatct aggacaaata tccttcaaaa ctgagggtca 18000 aataaacata cttttcaaca aatggaagtt gagggaattt atctctagca cagcttcagt 18060 acaataaata aatgccaagg atgttcctta ggcagaagga aatgactctg gatggccact 18120 caggtctgca ggaagaagat ccgtgtgaca agtggggacc aagtgagccc actggaacct 18180 gcattccttg ccctccattt ggttacttaa ttatatcagc atggactcag cagttcctgt 18240 tttcttccat aggttacaat ttatgacaat catttctacc caatgggagt gccttcaaag 18300 tggcttcaga gtgttgttgg tacattccct gattccttga gcacattttt gctttttggc 18360 acaaaaaaca ttccaagttc ctcttgcatt ttctgtacac aggctttgaa taattcacca 18420 gagtcctggt tccttttcat atagaatggt gtgtggagaa accaagctct gagtgttggg 18480 tgtgctttta cttttactgg gtccgattgt atctaaacct tctcaacaca cagagcctgg 18540 actttccttc ctcctcttcc tccctccttc ttcttcccct ctcctgcctc cctctctctc 18600 tctccttctt cccttcccct cccatttctc cctccctctc tccctccttc ctctcctccc 18660 tgccctccct ccctcccttc cctccttcct cccctcctcc ctctctcctt ccctccctgc 18720 ctctccccat gtgtctacca aaattggagt tcacatggat atttccaatt ctaatcagac 18780 gccacagggt tcactttgat ctctcccctt tctatatgtc tatatttata attctctttt 18840 ccaacacaga gaaacccatt atccttgata tatttactca tttggtccct tctagagcac 18900 acggaaagta gtatcagaat tgctcaccca aaccactgtt agtcatgaac ctgcccatga 18960 gagcttagga tgtgtttaca gctctttctg ctgttagctt gagaatacag agaaaaacta 19020 ctgtgttcag aagttaccag gggcagtcct ttccccttca gtgtggctgt catttatcta 19080 taatgcaact atatcccatg tttggaattt gtttatttta cttttgagta taacagtata 19140 acataccaaa ttactttgat tccaaagtca aaactataca aaaacgttta ctcaaaaaag 19200 tctaacttca tccccccacc ctgttccagc tccccacccc agcagggaac tagtctcatt 19260 agactctgag gtttatcctt tgtttttgtt tgtttgtttg tttgtgtgtt tgtttgtttt 19320 tttgagacgg agtcttactc actctgtcac ccaggctgga gtgcagtggc gcaatctcgg 19380 ctcactgcaa cctctgcctc ccgggttcaa gcgattctcc tgcctccgcc tcccgagtag 19440 ctgggattac aggcttgcgc caccaccacg cccggctaat ttttgtactt ttactagaga 19500 tggggtttca ctaagttggc caggctggtc tcgaactcct gacctcaggt gatctgcccg 19560 cctcggcctc ccaaagtgct gggattatag gcatgagcca ccacacccgg ctgatgtagc 19620 gtgctgtttt gtggctgcct cgagaaccag gctccccgca gcccttaggg acactagtgg 19680 ggctggcagg tgtttccttc tcagaggcct ggcccagctg ctcggcttcc tcctttttgt 19740 atttttattt tattttagag atggggtctt gctccatcac ccaggctgga gaggagtggc 19800 acgatcacag ctcactgcag cctcaacctc ctgggctcaa gcaatcctcc tacctcagct 19860 tcctgagtag ctgggactac aggagcacgc caccatgtcc agctaatttt taaacttgct 19920 tgtagagacg aagtcttgct atgtcgccca ggctggtctc aaactcctgg actcaagtga 19980 tcctcccgcc tcagcctcct gaagtgctgg gattaccgcg agcagccacg gctccctcct 20040 gaaggctcca ggcctaagtg tcccgctccc ttgttctgtg gctcagcccc tgggcaactc 20100 ctgcttgcca tggctgtggc tggcacctcc gtggccccac tgcccggtca gtcttcgcag 20160 gcccagcccc cagtagctgt gtggtttctg ccttcctcct gcactctggt ggtcacaggc 20220 cctgctgcgt gggtacaggg caggtggcca gggctgcagc ataagctctg tgagtgcagg 20280 cggccctagg cactggagcc ttgttgcatg gacgtgctcc ttgcaccctt gttgatgaat 20340 gagcgaaggt gtgtgggagt gaatcccaga gggtgcaggc cacggcactg ggggaggacc 20400 ctcccggctg cctcccagtg ggcttcaggg gctcagatag accctgccga gcacctctgt 20460 cctctgctcc cggactgggg ctgcagagag ctctcaaggg gccttgggag gcacaagcag 20520 gccagggtcc cagggagggg aggcaggagg cagcagggcc ggatggtggg ctgcagaggt 20580 ggaaggaaca ggacagtctg ttctgggggt gctggctgtg ggggacagag gccaggccac 20640 aaagcagaat ggtgggtggg ccaggaggag gtgggagcct ttttcaaaag gctttcaggg 20700 actttttata tttaatgatt gaaacaaaag atatccaaaa ccgcagttcc tgagaaccac 20760 ttgttctcgc cctggttcct ctccttgggc ttgaccttgg cctcctgccg ggcctggcag 20820 cggacagcag tctccctgct tcccaggctg ggcgagggag agcgtgggtc cccaggaacg 20880 ggggaggtgg tgcggctggc tttaggatgc ccattctgga accttctggt ggaagaaagc 20940 aggggatcag tatagtatgg ttgatccaca cactccagac cctctcctgg ctgtgcagct 21000 ggtctgatct gggctcagag gctgccactg ctcagtgctc ccctgggtaa ttttgcagga 21060 ggccaggagg gttcctggca gttagtgcca cccacacgga gaaattcaga gccatataaa 21120 cgggtctcca gggcctggag ggactgcaca tcctgggctt gcggcgcagt gtagacctgg 21180 gaggatgggc ggcctgctgc tggctgcttt tctggctttg gtctcggtgc ccagggccca 21240 ggccgtgtgg ttgggaagac tggaccctga gcaggtacag tcctcctggg ggtggggaga 21300 gctggtcctc gggggccagc ccctccttta aggccacaca gcttctgggc cccccagggc 21360 tagcccagac cagcatgagc agtggggaat tagttgggcc agcccttggg gagtcccaca 21420 ggcaggagcc tcagggcagg aggggtggat gctggagggt tggaggctgg agggctggag 21480 aattggaggc tggagactgg aggttgcagg gctggagggt gtagggttgg agaactggag 21540 gctggagggc tggaggctgt agggctggag aactggaggc tggaggctgc tgggctggag 21600 gctggagggc tgtagaactg gaggctggag ggctggaggc tggagggctg cagaactgga 21660 ggctggaggg ctggagagct ggagactaga ggctgcaggg ctgcaggctg gaggttggaa 21720 ggctggaggg ctggagccta gaggctgtag ggctggaagg ctggaggctg gagggttgga 21780 ggtctggggg gttggaagct ggaggctgga gggctggaga actgaaggct agaggctgca 21840 aggctggaag gttggaggct ggagggcggg agggttggag actggaggct ggagggctgg 21900 atggttggag ggtggaggct ggaggctgca gggctggaag gctggaggct ggagggttgg 21960 aggctggagg ctggagggct ggatggttgg agggtggagg ctgaggctgg aggtttggag 22020 gctggaggct gaggctggag ggttggaggc tggaggctgg aggctggagg gctggcggcc 22080 ctaacgggag ccgcctcaat gcagcttctt gggccctggt acgtgcttgc ggtggcctcc 22140 cgggaaaagg gctttgccat ggagaaggac atgaagaacg tcgtgggggt ggtggtgacc 22200 ctcactccag aaaacaacct gcggacgctg tcctctcagc acgggtgagt gggcgggtcc 22260 tgccaggcct tcccgcaggc aggactgtgg ctcagccaca tcatgtactt tgcacatctg 22320 ctcccgggca ccgcggcctg ggggcttccc acggcccctc tgcacccccg tcacttttcc 22380 cactgggctc tgtgcagagc caccagccct ctgcccagct actcacatct gtgtccctgg 22440 ggcctccagg tggcactccc accttcaata ccgcggcggg cactgctctg actatgctcc 22500 ttcatgcatc cctgtcctgg gtcatctggg ctgcaggctg tggtcagagc tggggaagcc 22560 tgtcctctca gcaggccttt ttcgggaatt ctgtttaaag gaggataatg cattcggacc 22620 taagaaccac tggttctttc ataacataaa tccccaaaca atatggtcat ggacgctgtt 22680 ggccagactg gccttgtggc ctcaggcagg cgcacccgac cacatgcatg gctggtggcg 22740 gcgtgcaggg gtcgggtggg ccaggctgtg gggcggccgt gctacacagg ggacacctac 22800 agggcagccg ggctgtgggg gtagccgtgc tggggcagga gtcggtgccc gcctcccttc 22860 acagaagagt ggcctgctgg gtggcgtggt gggtgtctac cccctggcag gctggcgaca 22920 gagcccaggc ctgaaccact gagccaggta cggggcccag agagaagaac ctgccctcct 22980 caccagcgtg ccttccccct gcacctgcgc ctgcagggta tgggcagggg ccccactgca 23040 ccccccgggc caggcctggc ctcagggcga gctgggacca aggggctcca ggctgaggtg 23100 gcagctccca cgtggctttc ctagttcttc ttgctgttcc tcccgcatgg actcagcctc 23160 agtttccttc cttggcaaac accattgcac ctttgctggg ttttgctcac tctggggact 23220 gaccacttag ggagcgccgg ccccttcccc cgagtcgcag gcaaaccccc accccgcccc 23280 atccttcccc tgagtcgcag gcgaaccccc accccacccc atccttcccc tgagtcacgg 23340 gcgaaccccc accccgcccc atccttcccc tgagttgcgg gcgaaccccc accccgcccc 23400 atccttcccc tgagtcacgg gcgaaccccc accccacccc atctttcccc cgagtcgcgg 23460 gcgaacctcc cgtctgtcga gtctcctgtt gtgtcctcag cagcattcag cgttcctggt 23520 ggagatctgg cactgaggat ctctcaggaa gtgaatggga gtgtccgccc ctctcaggac 23580 tcccacgccg tccactctcc gagaacaggt ttccgggcat cggggcatct ggcagaagat 23640 ggaagctctg gcctcactgc tggctggcct ggcctggact cctgagctcc gtctcctctc 23700 accgggcccc ggggtcttga ccctgagtgg gtgacaggcc ccttcttttc caggctggga 23760 gggtgtgacc agagtgtcat ggacctgata aagcgaaact ccggatgggt gtttgagaat 23820 ccctgtgagt ctgacggcca cggcctcacc caggccgttc ttccctgcag catccccagg 23880 ccccctgcgg gcagggacgg agggctgact ccgctcccga gagccaggag gctacaggtt 23940 ctggtcttgg gattgctgcc catcctgagg gtgaagagaa gcccctggac agaggattgg 24000 aaccttgttc ccagcaggct cagacaccag tgaggtccag ccaaggtccc cataggtgca 24060 gagggcgagg ggcctgttta gtggctgtct acaggtacag ggctgggctg gtgtgaccca 24120 gccgctcctc gttcacacct gggacaggct ggggcagtgc tcgtctgtct gtccgtcagt 24180 gtctgaaagc cccttgatgc tggcaccagg tggggtcggg ggtgggggac aaagcttggg 24240 ggctctgggt gtgcttggag gggtctcctg gggagggggg ctctcacttg aattggcccc 24300 tctgtccctg tggccagcat cagctcagcg gcccctggat ggatacagca gctgccctcc 24360 cctgaggcgg ggggttttgt ttcctagcaa taggcgtgct ggagctctgg gtgctggcca 24420 ccaacttcag agactatgcc atcatcttca ctcagctgga gttcggggac gagcccttca 24480 acaccgtgga gctgtacagt aagtgtgctg tgcggggccc acgtagggag ctgtgtggtg 24540 cgtgggccgc tcggggccca cgttgggagc tgtgtggtgc gtgggccgct tgggggccgc 24600 gtggggagct gtgtggtgag tgggccgctc gggggccatg tggggagctg tgtggtgagt 24660 gggccgcttg gggccacatg gggagctgtg tggtgagtgg gccgctcggg ggccatgtgg 24720 ggagctgtgt ggtgggtggg ccgcacagga ccacatgcag gccaagttcc cccaccttga 24780 ggctcgctgc actggaggtg gctttggaag gaccggggcc agcatcccag ggcaggtggc 24840 ctccttcctg acccttccca gcccctcccc ggcccctccc cagctctcag aggttgcttc 24900 cccctgcact gccctggtgc ccccaggtct gacggagaca gccagccagg aggccatggg 24960 gctcttcacc aagtggagca ggagcctggg cttcctgtca cagtagcagg cccagctgca 25020 gaaggaccgt gagtgtccac cggagcagcc tcgggggagg cttggggcaa gttctggagc 25080 ccacagggcg tgatggggtg acagagaccc tggggttgat tctggcttct gcctgactca 25140 gtcgccctga aggagtcagg caggctgagg gggtcttcac ctgcccctgc ccaaagtcag 25200 gcctgggggt cttgtcccca cggcaggcga gtcttgtggg tggggccctg gggtgctggg 25260 gggctcagtc ctttttcaca gccagtcttg cctccccttc tagtcacctg tgctcacaag 25320 atccttctgg taagccgcca tcctgagcct caccctgggc tctcttgggg gaagggggtt 25380 ggggaggcca ccctacgcac agtgggtggg aggaccctct gcccaagccc acggggttca 25440 tggctccact gtctctaagg cagctgaggg tgtggaggag cctccatggt gggctgggcc 25500 ccctcacact cctcttggtt ttcagtgagt gctgcgtccc cagtagggat ggcgcccaca 25560 gggtcctgtg acctcggcca gtgtccaccc acctcgctca gcggctcccg gggcccagca 25620 ccagctcaga ataaagcgat tccacagcaa accaaggatg cttttgactg ggggccagcc 25680 ggggaattgc ggggaggatg gcgggggtcg tcaccaaggg ccaagccaca gaaccataga 25740 gccagctgca aagaagacgc gtgcccaacc cagtcccttg ccagagcccc tctgggtctt 25800 cagacaccca aactcaggcc cgggctcaga ggcgggggct ggtcagagga cacagcctgg 25860 gaggaagcca gcagtgagct ccaccaggac cgcagctggg gtgccagagg ctccaaggac 25920 tgcccaggac aagagagagg tggctggtgg ggctcagagc agcccctgac tccccaggct 25980 caggcttctc tgggctgggg ttgtgtctct tgcctgggca ggggcaggga ccccagacac 26040 ctgagcctgc aggtcctcct gtgttctgat gggtgtgggg ggctgcgctg cctgccgtcc 26100 tgggagaagc acaggccatg ggggtctcgg ggctggatgg ggggctgggg actgtgtgag 26160 gctcatgctg gagtgaggcg aggtgcacac agatgccttg acctgcaagg ctgggcatgc 26220 acacgcatgc acatgtgagc acacacccac acatgcacac acacacattc atgggcacgg 26280 gcacacagcc ccggcccgcc cttcggcacc tgctcaggtg cttggtgcca ggccctcaag 26340 aaggtggtgg gggctgtgtg tgtggctgcc gcgcagagct caccatgtcc acatctgccc 26400 cagaagggcc acgaccagcc ctttgcctgt cacctgggag gtctaggatg agggccaggg 26460 cttaatgagt ctcaggggca cacacagtga ccccaggccg ggcactgagg gcaggagggt 26520 cctggggcct cagaggggcc tctgtctgcc agggcagccc aggccagcag tgaccaccag 26580 gtggggcagt gccggcttcc ctggagtgtc tgggggtggt ccctgggcgg ggagagggta 26640 gcagagccca aggccacacc tgttgcctcc ctccgctccc tcccctccct cctctcccga 26700 cacaggctgg cggctccaga gaggtttaaa cactggcctt ggcggctgag ggaggagggt 26760 gaagatgagg caggggctgc tggtgctggc gctggtgctg gtgctggtgc tagtgctggc 26820 tgcagggtcc caggtgcagg agtggtaccc cagggagtcc cacgccctca actggaacaa 26880 ggtgagccat gtgccactgg tccctgagcc gggcgccggc tctggagctg gagcaggctc 26940 ctgcacagtc tcccggcagc cgcccaggag cagggctacc agcagttcca ggcacgggcg 27000 ctgcccacct ggcctctccc actgcagcct ccagcacagg ggccccgcaa gttgcagggc 27060 aaaggcaggg agcctcgagg gggttcgggg gcccaggggg gtcagggagg gccagggtgg 27120 aggggcccag agagctgtgg ccgaggaggt cgggagacgc ccagagtgca gagtgggtgt 27180 ggagcaagcg gggcatattc tgtagcggga gggcccgggc acctgctgag tgagccgtgg 27240 gagcagcact cattggcacg gcggtgccca gggcgctagg gttggcagca ggcagagttg 27300 ggggccgggg gccggcagga agggaagtgg ccgagcagta cccagccccc aggaggccat 27360 gtgggaagga gccggggcct ctgaggtagg gggcctccca cctgctggcg cccacagccc 27420 agccctttgc tgcctggggt ccctgaccag ccgggcacct gcttctctgc agccctgctc 27480 cagtggccag ggcttgagtg gccgggaccg aggcggcacc tgggcggagc tgggactccg 27540 gctcacagaa gggtcatttg tctcctgggt tctctagctg ggaacctcgg gggtggggac 27600 agcccccggt ggcagctcct gtccccgccc acacatccgc tgcgcagttt tcagggttct 27660 ggtacattct ggccactgcc actgatgccc agggattctt gccggccagg gacaagagga 27720 agctgggggc gtccgtggta aaggtgaaca aagtgggcca gctccgcgtg ctcctcgcct 27780 tcagacggtg agtgggcagc gccccccagg caagggtctc cccttccagg aggagggagc 27840 tgcctcgggg ctggaaagga gtctgggagg agcctgtctg tctggaagtt ccacaagaac 27900 cgtcccccca cttcctcccc ctaaactcag aggaagccac ctgcagcccc tgggacccca 27960 catgggtggg tgttgggagg aagctgcctg cagaccttgg gaccccacat gggtaggtgt 28020 tgggaggaag ccgcttgcag ctcctgggcc ccacacgggt gggtgttggg agtttctctc 28080 gtgccgtggg acaggcccca gtccgaattg gaaaatcctc aaaccacaga gcctcagatt 28140 gacacgagtt ggatagtgaa gcaaaaaact ggggacctgg ggccccaacc ttcccgggtc 28200 ccacgggagc aggggcggga ccgagacgtg tgggtggggc tcggtggctg gcaggcggga 28260 ggaggggttc cctggtctgc agcctgagcc cccgccttgg ccccaggttg aaggggtgcc 28320 agtcccagga ggtgatcctg aggaaagacg ggaagaagcc ggtgtttggg aacgcctgtg 28380 catacgcggc gggcccgagg gaaggacagg agggaggtgt gcttgggctc tgggccctgg 28440 aggagctgcg tgatatgcct ctgacccccc tgtgccttga gcaggcccct tgtccccgag 28500 agcacgtggc aggcaggcgt caggctggct gtgttcccag agcccacacg tcatgtgctg 28560 ggcggcctgg cccgggaggg gaggccccgg gagggcactc agtccctctg gacacctgca 28620 cctgggtggg ggctctgagg cctcgggcac caggggtcag ggctgtgggc gggcacagcc 28680 acgccatccg ggaaccagcg ggcatctctg ggcatctctt tcagtgaaag gggtgaaggc 28740 cttccacgtg ctgtccactg actacagcta cggcttggtc tacctccgcc tggggcgtgc 28800 aacccaaaac tacaagaacc tgctgctctt ccgtgagcag gcacagcgga tggcagagga 28860 tggtggaggg ggtgcagagg gaggcataga ggggcgctgg gcacaggggg ccgggcagga 28920 ggtgaggcgg gcagggagag tgggggttgg ggcggtggtc agggcagggg caatggaagc 28980 cgagaccaca gcagtcaggt cctggaggtc gctgaagcaa ggatagggga cgtcaggaag 29040 aggctgcaga gaaaggtggg ccatccaggg ggagcggagg ccaccaggat ggtggcgggt 29100 gcaggtgcaa cgttctgggt gagggacaca gtggggatga ggaggcgtgg gaggggtccc 29160 aggtaagggt ctgcccggga tggaggggtg tggaagtgac ccagtgtggg ttaagggcca 29220 caaacccaat tctagacacc aaccacttgg ggtacacgga aaggagtggc cctcccggtt 29280 ggaacgggta gaggggaggt ggggtcctgg ggtctcagtc gaggcagggc acgctcaggg 29340 tgcagcaagc tggggcacag ctgaggggct ggctggaggg gactctgcag gcagagaggg 29400 ttccccaggt gacagcgtgg tgaccctggc ttcccgagcc tggctgcctc gtaagtgggg 29460 aggggcccag cggccgctgt cctcccctgg ccaacccctg cttcacgcct aagctctgag 29520 gacatttcag cagctcctgg ggtagggatg ggggtcaggg tgcagaccct aaaatggcca 29580 gagctggagt ctctgatgtg atcctgatct caacctcaga taggcagaat gtttcgagct 29640 tccagagtct gaaggaattc atggacgctt gtgacattct ggggctctcc aaggccgccg 29700 tcatcctccc gaaagacggt aagctgtgcc ctcagccctt tgccctcctg gccctacctg 29760 cccgtcccat tggggagctg attcattggg ggaaggagag atgaaaggat ccatttgaaa 29820 ggtgccattt gaaagatccg cccaaacgtg cctcccacct cctgccgccc ctgagaatcg 29880 gggcttggcc tggcaggcct ggcaccgaca gcgagaggcg tccggagcag atgctgcatc 29940 cgaccaggcg atgccgtggc tgtggctgca ggatcggggg ctgcatggct gtgttctcgt 30000 ggtgcccaag acgagcacgc atggaaccag ggagcggccg ggggagacgt gttcacggct 30060 catgtgtctc ctccaagcag ccagcccaca tgtggctgtt ttcctctccc acagcgtccc 30120 gtacacacac catcctgcca tgagagccag cgtcctctgc tcttcccttt cgacgcgtgg 30180 tcctcccaga cccgaggagc gttgttccag atgtcaagtg gagaccacgg cttgttgaga 30240 ggtgtttggt gactctaggt cattgacagt gtgaaaatca agtggctcaa gggatccttt 30300 atgatcacag aaagatgggg gaaggaggat acaggctgac cgggtggcga gatgtcagcc 30360 aggccccttc cccactgtct tctggagcac actgcaggct gctgctattc cctgtctgct 30420 gtgaaacaga ccacaggccg gcccaacgca gtcctcgccg tgtgccttgc ctctgccctc 30480 cacgccgccg ccaccctccg cgttccttta catcctgcac tttctcagga agccgttcgc 30540 atatccaccc aacatggagg agcccccatg acgtgccagg cagcgtgctg gcccctgcgg 30600 ctgcgttagg gaacagaatc ggcaaacgca gtcccagagt tgatgcctga tcaggagaag 30660 ccaacttcag cagtgtggtc acctggatac tcgagtatag accctagatc acctggatac 30720 tcgagtatag accctagaag tgtatctagg cgcggccagg gccccacaac agcaggtctg 30780 actcattcca tccagggcag catcagggag agcttcctgg gggaagtggc atttgggctg 30840 acctgggaag ggtggtgggt gttaa 30865 3 531 DNA Homo sapiens CDS (1)..(531) 3 atg atg tca ttc ctg ctc ggc gca atc ctg acc ctg ctc tgg gcg ccc 48 Met Met Ser Phe Leu Leu Gly Ala Ile Leu Thr Leu Leu Trp Ala Pro 1 5 10 15 acg gct cag gct gag gtt ctg ctg cag cct gac ttc aat gct gaa aag 96 Thr Ala Gln Ala Glu Val Leu Leu Gln Pro Asp Phe Asn Ala Glu Lys 20 25 30 att gga gga ttc tgg agg gaa gtc ggt gtg gcc tcc gat caa agc ctg 144 Ile Gly Gly Phe Trp Arg Glu Val Gly Val Ala Ser Asp Gln Ser Leu 35 40 45 gtg ctg acg gcc ccg aag cgg gtg gag ggc ttg ttc ctc acc ttg agc 192 Val Leu Thr Ala Pro Lys Arg Val Glu Gly Leu Phe Leu Thr Leu Ser 50 55 60 ggg agt aac ctg acc gtg aag gtt gca tat aac agc tca gga agc tgt 240 Gly Ser Asn Leu Thr Val Lys Val Ala Tyr Asn Ser Ser Gly Ser Cys 65 70 75 80 gag ata gag aag atc gtg ggc tca gaa ata gac agt acg gga aaa ttc 288 Glu Ile Glu Lys Ile Val Gly Ser Glu Ile Asp Ser Thr Gly Lys Phe 85 90 95 gct ttt cct ggc cac aga gag atc cac gtg ctg gac acc gac tac gag 336 Ala Phe Pro Gly His Arg Glu Ile His Val Leu Asp Thr Asp Tyr Glu 100 105 110 ggc tac gcc atc ctg cgg gtg tcc ctg atg tgg cgg ggc agg aac ttt 384 Gly Tyr Ala Ile Leu Arg Val Ser Leu Met Trp Arg Gly Arg Asn Phe 115 120 125 cgc gtc ctc aag tac ttt act cgg agc ctt gag gac aag gac cgg ctg 432 Arg Val Leu Lys Tyr Phe Thr Arg Ser Leu Glu Asp Lys Asp Arg Leu 130 135 140 ggg ttc tgg aag ttt cgg gag ctg aca gca gac act ggt ctc tac ctg 480 Gly Phe Trp Lys Phe Arg Glu Leu Thr Ala Asp Thr Gly Leu Tyr Leu 145 150 155 160 gcg gcc cgg cct ggg cgg tgt gcc gag ctc ctg aag gag gag ctg att 528 Ala Ala Arg Pro Gly Arg Cys Ala Glu Leu Leu Lys Glu Glu Leu Ile 165 170 175 taa 531 4 176 PRT Homo sapiens 4 Met Met Ser Phe Leu Leu Gly Ala Ile Leu Thr Leu Leu Trp Ala Pro 1 5 10 15 Thr Ala Gln Ala Glu Val Leu Leu Gln Pro Asp Phe Asn Ala Glu Lys 20 25 30 Ile Gly Gly Phe Trp Arg Glu Val Gly Val Ala Ser Asp Gln Ser Leu 35 40 45 Val Leu Thr Ala Pro Lys Arg Val Glu Gly Leu Phe Leu Thr Leu Ser 50 55 60 Gly Ser Asn Leu Thr Val Lys Val Ala Tyr Asn Ser Ser Gly Ser Cys 65 70 75 80 Glu Ile Glu Lys Ile Val Gly Ser Glu Ile Asp Ser Thr Gly Lys Phe 85 90 95 Ala Phe Pro Gly His Arg Glu Ile His Val Leu Asp Thr Asp Tyr Glu 100 105 110 Gly Tyr Ala Ile Leu Arg Val Ser Leu Met Trp Arg Gly Arg Asn Phe 115 120 125 Arg Val Leu Lys Tyr Phe Thr Arg Ser Leu Glu Asp Lys Asp Arg Leu 130 135 140 Gly Phe Trp Lys Phe Arg Glu Leu Thr Ala Asp Thr Gly Leu Tyr Leu 145 150 155 160 Ala Ala Arg Pro Gly Arg Cys Ala Glu Leu Leu Lys Glu Glu Leu Ile 165 170 175 5 5159 DNA Homo sapiens 5′UTR (1)..(5159) 5 tgtttatttt tattattttt tttttttaaa tttatttaac ataattttta ttaataataa 60 taataaatat agtttgtaaa ttttaaaaat aaaagagaag agataaagaa tagattaaat 120 gaattatata aatgaattaa ggtaatactc aatcctttgg atatgtggga ttatacacca 180 tataattttg ttatatttaa tattagtata tatggtaggg tgtatgtgag aaaaggtaaa 240 acttaaaaaa gcggtgaatt acaagatcgg taacgaaggt cgaaaagaaa ggaaatagcg 300 ggtgtctggt gatgtcttgg atttgagggt cgggggtttc gaactctttt taaggtccat 360 tatttaggcg cggcggggag agaggtatgt aaaaccgcaa gatggggtcc ttatcaggat 420 aaggaccacg agtgactgtt agcagaaagt aatatcatga ggaaacagta aaaaaaatac 480 ttatgagttg ggttgggagg ggactgagct ctgagaaagg accccagttc actatgtcaa 540 aaacgaaaaa aaggcaaggc gcagtggctc acgcctgtaa tcccggcact ttgggaggcc 600 aaggagggca gatcacctga ggtcaggagt ttgagaccag cctgaccaac atggtgaaat 660 cctgtctcta ctaaaaatgc aaaaaaaatt agacgggcat ggtggctact gttatcccag 720 ctacatggga ggctgaggca ggagaatcgc ttgaacccgg gaggtggaga ttgcagtgag 780 ctgagattgc accactgcac tccagcctga gtgacagagt gagaccccgt cttaaaaaac 840 aaaacaaggc caggcacggt ggctcatgcc tgcaatccca gcactctggg aggctgaggc 900 aggtggatca caaggtcagg agatcgagac catcctggct aacatggtga aaccctgtct 960 ctactaaaaa tacaaagaat tatcccagca tggtggtgga cgcctgtggt cccagctact 1020 ccggaggctg aggtgggaga atggcgtgaa cccaggaggt ggagcttgca gtgagctgag 1080 attgcgccac agcactccag cctgggcgac agagcgagac tccatctcaa aaaaagaaaa 1140 aaaaaaaaaa aaaaaaaaga aaaaaaagaa aaaagaaaaa aagaaaagaa aacgaagaaa 1200 aaccacaaat aaataaaagt tgaggtatga tgtatgcaca gctgcattcg cccctttgag 1260 ggcacagttg tgtgagttta gacaaatgca aatgttcatg gaaccactgc aagtgcgaca 1320 cagcagctct ggtgcccaca aagcttcctc gaggtccctt gcggtcaatc actgcccgca 1380 cctcagccct cagagccact gggaagcccc ctgccctgtg ggctcacctg tcctagaacg 1440 tcgtataaaa ggagtcgcag atcgcagcct tttccacctg gcttctgtcc ctctgcaggc 1500 tgcacctggg attcagccac cttcgttcct tttactgctg agtgtccatc acgtgctgct 1560 catgcagaag tgctcaagtt cgctgtaaac gtagttttca tttctcccgg gtggagacct 1620 ccataagtag gtaggttggt ggaatgtttc actgcatgag agtctgccag gaagttttgc 1680 agtgtggtcc cgtcttgttc ttcgcgcaga gggtgatagc tctgctgact cccagccctc 1740 acaggctcta gggattgagc ctgtttctgt ggaacgcctt ccatggtctg cgaggtgcac 1800 agaggggtct caaaaagctt ccaggttgca tcttcccatg gactgatgct gagcgtgttc 1860 tcatctccca tccggatgtt ttctttggtg gagtatcttt tcagatctct tgccttttta 1920 tttttatttg tttatatata tattttgaga cggaattttg ctctttcact taggttggag 1980 tgcaacggcg cgatcttggc tcactgcaac ctccccccac ccacctcact ttgggttcaa 2040 gcgattctcc tgcctcagcc tcccgagtag ctgggactac aggcgcccgc caccatgccc 2100 ggctaatttt tgtattttga gaagagatag ggtttcacca tgttggccag gctggtctcg 2160 aactcctgac ctcaggtgat ccacccgcct cggcctccca aaatgcgagg atcacaggcg 2220 tgagatgcca cacctggcct ctttttattt ttaatggagt ggcttgtttt cttattactc 2280 agtcttggga gttcctcatg gatgctggat ttcagtctcg tctcaggtgt atgcgttttg 2340 tagatgtttt ctgtagggac cagccccaca gggttggtga gtttctccct gtgtgctgag 2400 atgagagggc atagaaataa ggacacaaga aaaagacata aaagaaaaga cagctgggcc 2460 tggggaccac taccaccaag acgtggagac cggtagtggc cccgaatgtc tggctgtgct 2520 gatatttatt ggatacaaag caaaagggac agggtaaaga gtgtgagtca tctccaatga 2580 taggtaaggt gacgtgagtc acgtgtccac cggatggggg gcccttccct gtttggcagc 2640 caaggcggag agagagagag agagacagct tacatcatta tttctgcata tcagagactt 2700 ttagtacttt cactaattga ctactgctat ctagaaggca gagccaggtg tacaggatgg 2760 aacatgaagg cggactacga gcgtgaccac tgaagcacag catcacaggg agacggttag 2820 gcctctggat aactgcgggc aggtctgact gatgtcaggc cctccacagg aggtggagga 2880 gtagagtcct ctctaagctc ccccggggga aagggagact ccctttccct gtctgctaag 2940 tagcaggtgt tttcccttga cactgacgct actgctagat cacggtccgc ttggcaaccg 3000 gtgtcttccc agacgctggc gtcaccacta gaccaaggag ccctctggtg gccctgtccg 3060 gcgtaacaga aggctcgcac tcttgtcttc tggtcacttc tcactatgtc ccctcagctc 3120 ctatctctgt atggcctggt ttttcctagg ttataattgt agagcaagga ttattataat 3180 attggaataa agagtaattg ctacaagcta atgattaata atattcatat ataatcatgt 3240 ctatgatcta gatctagtat aactcttgtt attttatata ttttattaaa ctggaacagc 3300 tcgtgccctc ggtctcttgc ttcggcatct gggtggcttg ctgaccacag ttttcctgct 3360 gtatctaact gctggcttag gtgaacctgt caggggcgtg gctgggaact ggaacctcct 3420 gaagaccagt ggaggccaca agcagaagcc ctcggctcct tcccttcccc gatgctgggg 3480 gccaagggtc tttctctgac cctgtccccc cctcacgtta ccagtggagg gtgtccaggt 3540 tcttggcatt ttgaacaaag aattggacaa aacacacaaa caaagcaagg aaagaatgaa 3600 gcaacaaaag cagaaattta ttgaaaatga aagcactctt tacagggtgg gagcgggctg 3660 agcaagcagc tcaagggccc cggttacaga attttctggt gtttcaatat cctctagcgg 3720 tttaccattg gttacttggt gtatgcccta tgtaaatgaa gaggatgaag tcaaagtcat 3780 tttctcggct gagcatactg tcacgcctgt aatcccagta ctttgggagg ctgaggtggg 3840 tgtatcacct gaggtcggga gttcaagacc agcctgatga acatggagaa accccgtctc 3900 tactaaaaat acaaaattag ctgggtgtgg tggcgggtgc ctgtaatccc agatagtcag 3960 gaggctgagg caggaaaatc gcttaaaccc gggaggcaga ggctgcagag agccaaggag 4020 tgcaccactg cactccagcc tgggcaacaa gagcaaaact ccatctcaaa aaacaaacaa 4080 acaaaacgaa gtcatgtact cggtatgagc cctacgtaaa cggagaggat ggagtgaagg 4140 tacaaagcca ttcacattcc cgtcatcgtt agtgtttcca gttgatttgg ttctaggatg 4200 cccttgggct ccctgcgtcc aggccctctt ctcctgcctc actctcactc cagaggactg 4260 gcagtgctgg cctccctggc aggactcccg cccctgacca gaggggtctg ggcgtgctcc 4320 gaggccacct gctgctcctt gcccagtgtc cctgggtgct ctgtggggac acagagaggt 4380 cacttgggag gttcctgcct ctgccagact gcggcgcctt cctggcgtca ttgccccctg 4440 cacgcttata caaccagtga taaggcggta tccaatgccg taatcctccc cagggccagc 4500 ccagccctgt ccggtcccgt ccggtccctg gcctgcttgc tgggagccca gctgggggct 4560 gcatcggggg tggtgggggt ctggagtgag gggtgcccag gcctctctca cagtgtgggg 4620 gcacaagctc tgccctggac actctgccag tgtgacccta ccccccatgt tgggggtgag 4680 agaagggggc tttctgggtg gccagatccc aggagagggg gcctagccag accaacccca 4740 gcccagctct ctgcagctgt accccaaagg ccctggccca gcccagccag acagagtgtg 4800 gacagagggg cttgggtgcg agtccaggca aggcaggaca agtctctgct gtggcttggc 4860 agtggccccg tgactggtga catagtctgg caggcctagg ggacaaatcc tcagcccggg 4920 tgcatcaggc cagagcctgg ctgtctggag ccctccagga aatgaccccc cgggctgggg 4980 gacatccgag tgattgtgcc aaacaatgga caagggggcc ggagtctcag agccgaagtg 5040 ccttccgctt ccatctgctc cgcccaggag caggtgcacc caggtggtgg cctgggctat 5100 aaagctggcc ccctggggct tggggactca gcaccagggg ctggagggca ggggagggg 5159 6 175 PRT Mus musculus mat_peptide (1)..() 6 Met Glu Ala Arg Leu Leu Ser Asn Val Cys Gly Phe Phe Leu Val Phe 1 5 10 15 Leu Leu Gln Ala Glu Ser Thr Arg Val Glu Leu Val Pro Glu Lys Ile 20 25 30 Ala Gly Phe Trp Lys Glu Val Ala Val Ala Ser Asp Gln Lys Leu Val 35 40 45 Leu Lys Ala Gln Arg Arg Val Glu Gly Leu Phe Leu Thr Phe Ser Gly 50 55 60 Gly Asn Val Thr Val Lys Ala Val Tyr Asn Ser Ser Gly Ser Cys Val 65 70 75 80 Thr Glu Ser Ser Leu Gly Ser Glu Arg Asp Thr Val Gly Glu Phe Ala 85 90 95 Phe Pro Gly Asn Arg Glu Ile His Val Leu Asp Thr Asp Tyr Glu Arg 100 105 110 Tyr Thr Ile Leu Lys Leu Thr Leu Leu Trp Gln Gly Arg Asn Phe His 115 120 125 Val Leu Lys Tyr Phe Thr Arg Ser Leu Glu Asn Glu Asp Glu Pro Gly 130 135 140 Phe Trp Leu Phe Arg Glu Met Thr Ala Asp Gln Gly Leu Tyr Met Leu 145 150 155 160 Ala Arg His Gly Arg Cys Ala Glu Leu Leu Lys Glu Gly Leu Val 165 170 175 7 27 DNA Mus musculus primer_bind (1)..(27) 7 cttctctggt acaagctcca ccctggt 27 8 24 DNA Mus musculus primer_bind (1)..(24) 8 tggatggata gatgcataca tgag 24 9 20 DNA Mus musculus primer (1)..(20) 9 caacggtggt atatccagtg 20 10 21 DNA Mus musculus primer (1)..(21) 10 gatgtgctcc aggctaaagt t 21 11 21 DNA Mus musculus primer (1)..(21) 11 agaaacggaa tgttgtggag t 21

Claims

What is claimed is:

1. An isolated promoter region, comprising an about 5.3 kb fragment (Accession No. AF082221) of mouse genomic clone 10983 (Genomesystem Inc., St. Louis, Ky.) between the EcoRV and SalI restriction sites, or functional portion thereof.

2. An isolated promoter region of claim 1, the functional portion comprising a TATA box and at least one cis-acting regulatory sequence selected from the group consisting of a Sp1-binding site, an AP-1 binding site, a retinoic acid receptor binding site, an androgen receptor binding site, a C-Ets binding site, a SRY binding site, an AP-4 binding site, a C/EBP binding site, and combinations thereof.

3. The isolated promoter region of claim 1, comprising:

(a) the nucleotide sequence of SEQ ID NO:1; or

(b) a nucleic acid molecule substantially identical to SEQ ID NO:1.

4. The isolated promoter region of claim 1, comprising a 20 base pair nucleotide sequence identical to a contiguous 20 base pair nucleotide portion of SEQ ID NO:1.

5. An isolated promoter region, or functional portion thereof, comprising an about 5150 base pair region immediately upstream of the human hEP17 transcription start site.

6. The isolated promoter region of claim 5, the functional portion comprising a TATA box and at least one cis-acting regulatory sequence selected from the group consisting of a Sp-1 binding site, an AP-1 binding site, a cAMP response element binding protein (CREB) binding site, a SRY-related HMG-box gene 5 (Sox5) binding site, a Sex-determining region Y gene product (SRY) binding site, a c-Ets binding site, a GATA binding site, an Octamer transcription factor 1 (Oct-1) binding site, and combinations thereof.

7. The isolated promoter region of claim 5, comprising:

(a) the nucleotide sequence of SEQ ID NO:5; or

(b) a nucleic acid molecule substantially identical to SEQ ID NO:5.

8. The isolated promoter region of claim 5, comprising a 20 base pair nucleotide sequence identical to a contiguous 20 base pair nucleotide portion of SEQ ID NO:5.

9. A chimeric gene comprising the isolated promoter region of claim 1 or 5 operably linked to a heterologous nucleotide sequence.

10. A vector comprising the chimeric gene of claim 9.

11. A host cell comprising the chimeric gene of claim 9.

12. The host cell of claim 11, wherein the cell is selected from the group consisting of a bacterial cell, a hamster cell, a mouse cell, and a human cell.

13. A mouse comprising a mouse cell of claim 12.

14. The mouse of claim 13, wherein expression of the chimeric gene confers male infertility in an otherwise fertile mouse.

15. A method for identifying a substance that regulates EP17 expression, the method comprising:

(a) establishing a gene expression system comprising the chimeric gene of claim 12, wherein the heterologous nucleotide sequence is a reporter gene, and components required for gene transcription and translation, whereby the reporter gene is expressed, and a level of reporter gene expression is assayable;

(b) assaying a baseline level of reporter gene expression using the gene expression system of (a) in the absence of a candidate substance;

(c) exposing the gene expression system of (a) to a plurality of candidate substances;

(d) assaying a level of reporter gene expression using the gene expression system of (a) in the presence of a candidate substance of (c); and

(e) selecting a candidate substance whose presence results in an altered level of reporter gene expression when compared to the baseline level.

16. The method of claim 15, wherein the substance is a protein, a chemical compound, or a peptide.

17. A method for identifying a substance that regulates EP17 expression, the method comprising:

(a) creating a transgenic mouse bearing a chimeric transgene comprising the promoter region of claim 1 or 5 operably linked to a reporter gene, wherein the reporter gene is expressed, and wherein a level of reporter gene expression is assayable;

(b) assaying a baseline level of reporter gene expression in the transgenic mouse in the absence of a candidate substance;

(c) administering the candidate substance to the transgenic mouse;

(d) assaying a level of reporter gene expression in the transgenic mouse following administration of the candidate substance to the mouse; and

(e) selecting a substance wherein the level of reporter gene expression in the transgenic mouse is altered following administration of the substance compared to the baseline level.

18. A method for producing an epididymal cell line, the method comprising:

(a) creating a transgenic animal bearing a chimeric transgene gene comprising the promoter region of claim 1 operably linked to a selectable marker gene that permits cell growth in the presence of a selective agent;

(b) procuring epididymal cells from the transgenic animal of (a); and

(c) reproducing the cells in vitro in the presence of the selective agent.

19. A method for mutagenizing an EP17 locus in a vertebrate animal, the method comprising:

(a) constructing a targeting vector having the isolated promoter region of claim 1, a marker gene, and an isolated 3′ flanking region of an EP17 gene, wherein the marker gene is positioned between the promoter region and the 3′ flanking region;

(b) linearizing the targeting vector by digestion with a restriction endonuclease, wherein the promoter region, marker gene, and 3′ flanking region are undigested;

(c) introducing the linearized vector into embryonic stem cells;

(d) detecting the marker gene in the embryonic stem cells;

(e) selecting embryonic stem cells having the marker gene; and

(f) generating a transgenic vertebrate animal derived from the selected embryonic stem cells, wherein the EP17 locus of the animal is altered as a result of a homologous recombination event mediated by the targeting vector.

20. An isolated EP17 polypeptide, or functional portion thereof, comprising:

(a) a polypeptide encoded by the nucleotide sequence of SEQ ID NO:3;

(b) a polypeptide encoded by a nucleic acid molecule that is substantially identical to SEQ ID NO:3;

(c) a polypeptide having the amino acid sequence of SEQ ID NO:4;

(d) a polypeptide that is a biological equivalent of the polypeptide of SEQ ID NO:4; or

(e) a polypeptide which is immunologically cross-reactive with an antibody that shows specific binding with a polypeptide of SEQ ID NO:4

21. An isolated nucleic acid molecule encoding a human EP17 polypeptide.

22. The isolated nucleic acid molecule of claim 21, comprising:

(a) the nucleotide sequence of SEQ ID NO:3; or

(b) a nucleic acid molecule substantially identical to SEQ ID NO:3.

23. The isolated nucleic acid molecule of claim 21, comprising a 20 nucleotide sequence that is identical to a contiguous 20 nucleotide sequence of SEQ ID NO:3.

24. A chimeric gene, comprising the nucleic acid molecule of claim 21 operably linked to a heterologous promoter.

25. A vector comprising the chimeric gene of claim 24.

26. A host cell comprising the chimeric gene of claim 24.

27. The host cell of claim 26, wherein the cell is selected from the group consisting of a bacterial cell, a hamster cell, a mouse cell, and a human cell.

28. A method of detecting a nucleic acid molecule that encodes an EP17 polypeptide, the method comprising:

(a) procuring a biological sample having nucleic acid material;

(b) hybridizing the nucleic acid molecule of SEQ ID NO:1, 2, 3, or 5 under stringent hybridization conditions to the biological sample of (a), thereby forming a duplex structure between the nucleic acid of SEQ ID NO:1, 2, 3, or 5 and a nucleic acid within the biological sample; and

(c) detecting the duplex structure of (b), whereby an EP17 nucleic acid molecule is detected.

29. An antibody that specifically recognizes an EP17 polypeptide encoded by the nucleotide sequence of SEQ ID NO:3; a polypeptide encoded by a nucleic acid molecule that is substantially identical to SEQ ID NO:3; a polypeptide having the amino acid sequence of SEQ ID NO:4; a polypeptide that is a biological equivalent of the polypeptide of SEQ ID NO:4; or a polypeptide which is immunologically cross-reactive with an antibody that shows specific binding with a polypeptide of SEQ ID NOs:4.

30. A method for producing an antibody that specifically recognizes an EP17 polypeptide, the method comprising:

(a) recombinantly or synthetically producing an EP17 polypeptide of claim 20, or portion thereof;

(b) formulating the polypeptide of (a) whereby it is an effective immunogen;

(c) administering to an animal the formulation of (b) to generate an immune response in the animal comprising production of antibodies, wherein antibodies are present in the blood serum of the animal; and

(d) collecting the blood serum from the animal of (c) comprising antibodies that specifically recognize an EP17 polypeptide.

31. A method for detecting a level of EP17 polypeptide, the method comprising

(a) obtaining a biological sample having peptidic material;

(b) detecting an EP17 polypeptide in the biological sample of (a) by immunochemical reaction with the antibody of claim 29, whereby an amount of EP17 polypeptide in a sample is determined.

32. A method for identifying a substance that modulates EP17 function, the method comprising:

(a) isolating an EP17 polypeptide encoded by the nucleotide sequence of SEQ ID NO:3; a polypeptide encoded by a nucleic acid molecule that is substantially identical to SEQ ID NO:3; a polypeptide having the amino acid sequence of SEQ ID NO:4; a polypeptide that is a biological equivalent of the polypeptide of SEQ ID NO:4; or a polypeptide which is immunologically cross-reactive with an antibody that shows specific binding with a polypeptide of SEQ ID NO:4;

(b) exposing the isolated EP17 polypeptide to a plurality of substances;

(c) assaying binding of a substance to the isolated EP17 polypeptide; and

(d) selecting a substance that demonstrates specific binding to the isolated EP17 polypeptide.

33. A method for modulating EP17 function in a subject, the method comprising:

(a) preparing a pharmaceutical composition, comprising a substance identified according to the method of claim 15, 17, 30, or 32, and a carrier;

(b) administering an effective dose of the pharmaceutical composition to a subject, whereby EP17 activity is altered in the subject.

34. A method for modulating EP17 function in a subject, the method comprising:

(a) preparing a gene therapy vector having a nucleotide sequence encoding an EP17 polypeptide or a nucleotide sequence encoding a nucleic acid molecule, peptide, or protein that interacts with an EP17 nucleic acid or polypeptide; and

(b) administering the gene therapy vector to a subject, whereby the function of EP17 in the subject is modulated.

35. The method of claim 34 further comprising the EP17 promoter region of claim 1 or 5.

36. A method for diminishing the fertile capacity of a subject, the method comprising:

(a) identifying a chemical compound, peptide, or antibody that interacts with the polypeptide of SEQ ID NO:4 or 6;

(b) preparing a pharmaceutical composition comprising the chemical compound, peptide, or antibody of (a) and a carrier; and

(c) administering an effective dose of the pharmaceutical composition to a subject, whereby the fertile capacity of the subject is diminished.

37. A method for promoting fertility in a subject, the method comprising:

(a) identifying a chemical compound or peptide that interacts with the polypeptide of SEQ ID NO:4 or 6;

(b) preparing a pharmaceutical composition comprising the chemical compound or peptide of (a) and a carrier; and

(c) administering the pharmaceutical composition to a subject, whereby the fertility of the subject is improved.