CA2483323A1 - Contraceptive targets - Google Patents

Contraceptive targets Download PDF

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CA2483323A1
CA2483323A1 CA002483323A CA2483323A CA2483323A1 CA 2483323 A1 CA2483323 A1 CA 2483323A1 CA 002483323 A CA002483323 A CA 002483323A CA 2483323 A CA2483323 A CA 2483323A CA 2483323 A1 CA2483323 A1 CA 2483323A1
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seq
expression
peptide
protein
modulator
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Martin M. Matzuk
Pei Wang
Xuemei Wu
Yuchen Bai
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Wyeth LLC
Baylor College of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates generally to ovary-specific genes (O1-180, O1- 184 and O1-236) and the proteins they encode. Also provided are methods for detecting cell proliferative or degenerative disorders in reproductive tissues. Yet further, the invention provides methods for scrounge of compoun ds that interact and/or modulate the expression or activity of the ovary-specif ic genes. These compounds are possible contraceptive agents and/or fertility agents.

Description

CONTRACEPTIVE TARGETS
[0001] This application is a continuation-in-part of International Application Number PCT/LTS02/13245, which was filed on April 26, 2002 and claims priority to U.S.
Provisional Application Number 60/442,164 filed on January 23, 2003, U.S.
Provisional Application Number 60/439,781, which was filed on January 13, 2003; U.S.
Provisional Application Number 60/434,165, which was filed on December 17, 2002 and U.S.
Provisional Application Number 60/411,262 filed September 17, 2002.
BACKGROUND OF THE INVENTION
A. Field of the Invention [0002] The present invention relates generally to ovary-specific genes and the proteins they encode.
B. Description of Related Art [0003] Reproductive development and fwction are complex processes involving both genetically-determined and physiological events. Identification of the critical protein products of genes involved in these processes is necessary to characterize how these processes are regulated. Although important molecular events occur during the early phases of mammalian oogenesis and folliculogenesis, to date, few "candidate" regulatory molecules have been identified and characterized thoroughly. Several studies have suggested that both endocrine factors, such luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary, as well as paracrine factors secreted from the oocyte influence folliculogenesis. FSH
and LH are known to bind to granulosa and thecal cells which in turn are required for oocyte growth and maturation and maintenance of oocyte meiotic competence. Likewise, oocytes may secrete factors which are necessary for normal granulosa cell and thecal cell function. Because oocyte growth is cooxdinated with the development and growth of the surrounding somatic cells (i.e., granulosa cells initially and thecal cells later), understanding the molecular events at early stages will give important clues about the paracrine factors mediating the reciprocal interactions between oocytes and somatic cells, the development of competence for trophic hormone stimulation, the process of follicular recruitment, and the regulation of the ovulation process.
[0004] Disruption of the hypothalamic-pituitary-gonadal reproductive axis by administration of steroids containing synthetic estrogens and progestins has been one of the oldest methods of hormonal contraception. However, the latest report of the Institute of Medicine emphasizes the importance of developing strategies for new contraceptives. According to the report, some of the long-term contraceptive strategies for women include inhibition of ovulation, prevention of fertilization, or blocking of implantation of a fertilized egg into the uterine lining. Furthermore, infertility affects ~15% of couples, and in ~40%
of the cases, the female is believed to be the sole cause of the infertility. Thus, it is critical to identify novel ovary-specific gene products which could be potential targets for new contraceptive agents as well as determining the etiology of specif c forms of female infertility.
[0005] One function of the ovary is to produce an oocyte that is fully capable of supplying all the necessary proteins and factors for fertilization and early embryonic development. Oocyte-derived mRNA and proteins are necessary for the removal of the sperm nuclear envelope, the decondensation of the sperm nucleus (including the removal of protamines), the assembly of histones on the sperm DNA and chromatin condensation, the completion of oocyte meiotic maturation and extrusion of the second polar body, the formation of male and female pronuclei, the fusion of male and female pronuclei, the replication of DNA, and the initiation of zygote and early embryonic cleavages [reviewed in (Perreault, 1992)].
Oocyte-derived factors are necessary since the sperm contains mainly DNA
(i.e., no cytoplasm or nucleoplasm), and many of the factors necessary for early post-fertilization events in mammals are acquired during oocyte meiotic maturation (McLay and Clarke, 1997). These oocyte proteins are predicted to be highly conserved through evolution since oocytes can efficiently remodel heterologous sperm or somatic cell nuclei into pronuclei (Perreault, 1992).
Although histones are involved in the modification of the sperm chromatin to resemble that of a somatic cell, the other non-histone proteins involved in these processes are unknown in mammals. In Xenopus laevis, a key factor in sperm decondensation is nucleoplasmin which was isolated and cloned over a decade ago (Burglin et al., 1987; Dingwall et al., 1987). Sperm chromatin decondensation occurs after a spermatotozoon enters an egg. In Xenopus laevis, although reduction of the protamine disulfide bonds by ooplasmic glutathione is important, nucleoplasmin (also called nucleoplasmin A or Xnpm2) is necessary and sufficient to initiate the decondensation of sperm nuclei (Philpott et al., 1991). Nucleoplasmin, an acidic, thermostable protein, is the most abundant protein in the nucleus of Xenopus laevis oocytes and eggs, making up 7-10% of the total nuclear protein (Krohne and Franke, 1980a; Mills et al., 1980). After germinal vesicle breakdown, nucleoplasmin [present in the egg nucleoplasm but not bound to DNA (Mills et al., 1980)], is released into the ooplasm where it functions to bind protamines tightly and strip them from the sperm nucleus within 5 minutes of sperm entry, resulting in sperm decondensation (Ohsumi and Katagiri, 199I; Philpott and Leno, 1992;
Philpott et al., 1991). This process allows egg histones to subsequently bind the sperm DNA.
Immunodepletion of nucleoplasmin from egg extracts prevents sperm decondensation (Philpott et al., 1991). Direct interaction of nucleoplasmin with protamine was observed in in vitro experiments. The data suggest that the nucleoplasmin is bound to protamine in a I :1 ratio and that the polyglutamic acid tract in nucleoplasmin plays a critical role for binding to protamine (Iwata et al., 1999).
Interestingly, injection of sperm DNA into oocyte nuclei, male or female pronuclei of fertilized eggs, or nuclei of 2 cell embryos leads to sperm decondensation (Maeda et al., 1998), suggesting that nucleoplasmin is f~mctional at all of these stages. Nucleoplasmin can also interact with histones as a pentamer (Earnshaw et al., 1980; Laskey et al., 1993).
Nucleoplasmin binds specifically to histories H2A and H2B and along with the proteins N1/N2 that bind histories H3 and H4, can promote nucleosome assembly onto DNA (Dilworth et al., 1987;
Laskey et al., 1993). These observations suggest that during oogenesis and during oogenesis and at fertilization, the oocyte-derived nucleoplasmin interacts with the female pronucleus and male pronucleus, interacts with histories, and is required in some way for chromatin assembly.
(Laskey et al., 1993; Philpott et al., 199I). Although "ubiquitous" proteins with low homology to nucleoplasmin have been cloned in mammals and Drosophila (Chan et al., 1989;
Crevel et al., 1997; Ito et al., 1996; MacArthur arid Shackleford, 1997b; Schmidt-Zachmann and Franke, 1988), an oocyte-equivalent ortholog in mammals had not yet been identified.
[0006] The basic functional unit within the ovary is the follicle, which consists of the oocyte and its surrounding somatic cells. Fertility in female mammals depends on the ability of the ovaries to produce Graafian (pre-ovulatory) (pre-ovulatory) follicles, which ovulate fertilizable oocytes at mid-cycle (Erickson and Shimasaki, 2000). This process, termed folliculogenesis, requires a precise coordinate regulation between extraovarian and intraovarian factors (Richards, et al., 1995). Compared to the knowledge of extraovarian regulatory hormones at the levels of the hypothalamus (i. e., GnRH) and anterior pituitary (i. e., FSH and LH), little is known about paracrine and autocxine factors within the ovaries, though oocyte-somatic cell communication leas been long recognized as important (Falck, 1959).
Accumulating evidence shows that factors secreted by the oocyte promote the proliferation of surrounding granulosa cells, and inhibit premature luteinization of these cells during folliculogenesis (El-Fouly et al., 1970; Charming, 1970). Oocyte factors have been implicated in controlling granulosa cell synthesis of hyaluronic acid, urokinase plasminogen activator (uPA), LH
receptor, steroidsand prostaglandins and prostaglandins (El-Fouly et al., 1970; Nelcola and Nalbandov, 1971; Salustri et al., 1985; Vanderhyden et al., 1993; Eppig et al., 1997a, b).
[0007] Several novel regulatory proteins have been recently discovered within oocytes. Growth differentiation factor 9 (GDF-9 or Gdf~), a member of transforming growth factor (3 (TGF-(3) superfamily, is one of the most important signaling factors. Oocyte expression of GDF-9 begins at the primary follicle stage, and persists through ovulation in the mouse (McGrath et al., 1995; Elvin et al., 2000). Female Gdf~ knockout mice are infertile due to a block of folliculogenesis at the type 3b (primary) follicle stage, accompanied by defects in granulosa cell growth and differentiation, theca cell formation, and oocyte meiotic competence (Dong et al., I996; Carabatsos et al., 1998, Elvin et al, 1999A). Also, recombinant GDF-9 affects the expression of the genes encoding hyaluronan synthase 2 (Has2), cyclooxygenase 2 (Cox2), steroid acute regulatory protein (SCAR), the prostaglandin E2 receptor EP2, pentraxin 3, the LH receptor and uPA (Elvin et al., 1999B, Elvin et al., 2000).
[0008] To identify key proteins in the hypothalamic-pituitary-gonadal axis, several important knockout mouse models have been generated, including four which have ovarian defects. Mice lacking the gonadal/pituitary peptide inhibin have secondary infertility due to the onset of ovarian or testicular tumors which appear as early as 4 weeks of age (Matzuk et al., 1992). Mice lacking activin receptor type II (Acv~2) survive to adulthood but display reproductive defects. Male mice show reduced testes size and demonstrate delayed fertility (Matzuk, et al. 1995). In contxast, female mice have a block in folliculogenesis at the early antral follicle stage leading to infertility. Consistent with the known role of activins in FSH
homeostasis, both pituitary and serum FSH levels are dramatically reduced in these Acv~2 knockout mice. Female mice lacking FSH, due to a mutation in the FSHbeta gene, are infertile (Kumar et al., 1997). However, these mice have an earlier block in folliculogenesis prior to antral follicle formation. Thus, FSH is not required for formation of a mufti-layer pre-antral follicle, but it is required for progression to antral follicle formation.
Finally, growth differentiation factor 9(Gdf~) knockout mice have been used to determine at which stage in follicular development GDF-9 is required (bong et al., 1996). Within the ovary, expression of Gdf~ mRNA is limited to the oocyte and is seen at the early one-layer primary follicle stage and persists through ovulation. Absence of GDF-9 results in ovaries that fail to demonstrate any normal follicles beyond the primary follicle stage. Although oocytes surrounded by a single layer of granulosa cells are present and appear normal histologically, no normal two-layered follicles are present. Follicles beyond the one-layer stage are abnormal, contain atypical granulosa cells, and display asymmetric growth of these cells. Furthermore, as determined by light and electron microscopy, a thecal cell layer does not form in these GdfJ
knockout ovaries (bong et al., 1996; Elvin et al., 1999). Thus, in contrast to kit ligand and other growth factors which are synthesized by the somatic cells and influence oocyte growth, GDF-9 functions in the reciprocal manner as an oocyte-derived growth factor which is required for somatic cell function.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides three ovary-specific and oocyte-specific polynucleotide sequences, O1-180 (also known as zygote arrest 1 (Zap°I
)) (SEQ.ID.NO.1, SEQ.ID.NO.11, SEQ.ID.NO.12, SEQ.ID.N0.13, SEQ.ID.N0.28, SEQ.ID.NO.30, SEQ.ID.NO.31, SEQ.ID.NO.33, SEQ.ID.NO.35, SEQ.ID.N0.37, SEQ.ID.N0.38, SEQ.ID.N0.40 and SEQ.ID.N0.41), O1-184 (SEQ.ID.NO.3) and O1-236 (also known as nucleoplasmin (Npf~r2)) (SEQ.ID.NO.S, SEQ.ID.N0.7, SEQ.ID.N0.8; SEQ.ID.NO.10, SEQ.117.N0.14 and SEQ.ID.NO.43), the protein products they encode, fragments, homologues, and derivatives thereof, and antibodies which are immunoreactive with these protein products.
These genes and their protein products appear to relate to various cell proliferative or degenerative disorders, especially those involving ovarian tumors, such as germ cell tumors and granulosa cell tumors, or infertility, such as premature ovarian failure.
[0010] In a specific embodiment, the present invention provides nucleic acid molecules that are specific to gonadal tissue. These specific nucleic acids may be a naturally-occurring cDNA, genomic DNA, RNA, or a fragment of one of these nucleic acids, or may be a non-naturally-occurring nucleic acid molecule. If the specific nucleic acid is genomic DNA, then it is a gonadal specific gene. In one embodiment, the nucleic acid molecule encodes a polypeptide that is specific to the gonads. In another preferred embodiment, the nucleic acid molecule encodes a polypeptide that comprises an amino acid sequence of O1-180 (SEQ.ID.N0.2, SEQ.ID.N0.16, SEQ.ID.N0.29, SEQ.ID.NO.32, SEQ.ID.N0.34, SEQ.I17.N0.36 and SEQ.ID.N0.39), O1-184 (SEQ.ID.N0.4), O1-236 (SEQ.ID.N0.6, SEQ.ID.NO.9, and SEQ.ID.N0.42). In yet another, the nucleic acid molecule comprises a nucleic acid sequence of Ol-180 (also known as zygote arrest 1 (Zarl)) (SEQ.ID.NO.1, SEQ.ID.NO.11, SEQ.ID.N0.12, SEQ.ID.NO.I3, SEQ.ID.N0.28, SEQ.ID.N0.30, SEQ.ID.N0.31, SEQ.ID.N0.33, SEQ.ID.N0.35, SEQ.ID.NO.37, SEQ.ID.N0.38, SEQ.ID.N0.40 and SEQ.ID.N0.41), OI-I84 (SEQ.ID.N0.3) and O1-236 (also known as nucleoplasmin (Npm2)) (SEQ.ID.N0.5, SEQ.ID.N0.7, SEQ.ID.NO.8; SEQ.ID.NO.10, SEQ.ID.N0.14 and SEQ.ID.N0.43). By nucleic acid molecule, it is also meant to be inclusive of sequences that selectively hybridize or exhibit substantial sequence similarity to a nucleic acid molecule encoding a gonadal specific protein, or that selectively hybridize or exhibit substantial sequence similarity to a gonadal specific nucleic acids, as well as allelic variants of a nucleic acid molecule encoding a gonadal specific protein, and allelic variants of a gonadal specific nucleic acids. Nucleic acid molecules comprising a part of a nucleic acid sequence that encodes a gonadal specific protein or that comprises a part of a nucleic acid sequence of gonadal specific nucleic acids are also provided.
[OOlI] Thus, in one embodiment, the invention provides methods for detecting cell proliferative or degenerative disorders of ovarian origin and which are associated with O1-180, O1-184 or O1-236. In another embodiment, the invention provides method of treating cell proliferative or degenerative disorders associated with abnormal levels of expression of O1-180, Ol-184 or Ol-236, by suppressing or enhancing their respective activities.
[0012] In a specific embodiment, the present invention provides a pharmaceutical composition comprising a modulator of Ol-180, Ol-184 and/or O1-236 expression dispersed in a pharmaceutically acceptable carrier. The modulator may suppress or enhance transcription of an Ol-180, O1-184 and/or O1-236 gene. The modulator may be a polypeptide sequence, a protein, a small molecule, or a polynucleotide sequence. Specifically, the polynucleotide sequence is DNA or RNA. In further embodiments, the polynucleotide sequence is comprised in an expression vector operatively linked to a promoter.
[0013] A further embodiment of the present invention is a pharmaceutical composition comprising a modulator of O1-I80, OI-184 and/or OI-236 activity dispersed in a pbannaceutically acceptable carrier. The composition may inhibit or stimulate O1-180, O1-184 and/or Ol-236 activity. The composition may be a protein, polypeptide sequence, small molecule, or polynucleotide sequence. It is envisioned that the composition may block or enhance the interaction of the nucleic acid sequences in question with the other protein partners.

[0014] Another embodiment of the present invention is a method of modulating contraception comprising administering to an animal an effective amount of a modulator of 01-180, Ol-184 and/or Ol-236 activity and/or expression dispersed in a pharmacologically acceptable carrier, wherein said amount is capable of decreasing conception.
The animal may be a male or a female.
[OOISj A further embodiment is a method of enhancing fertility comprising administering to an animal an effective amount of a modulator of O1-180, O1-184 and/or 01-236 activity andlor expression dispersed in a pharmacologically acceptable carrier, wherein said amount is capable of increasing conception.
[0016] Yet further, another embodiment is a method of screening for a modulator of O1-180, O1-184 and/or Ol-236 expression comprising the steps of: providing a cell expressing an O1-180, Ol-184 and/or O1-236 polypeptide; contacting said cell with a candidate modulator; measuring O1-180, O1-184 and/or O1-236 expression; and comparing the 01-180, O1-184 and/or Ol-236 expression in the presence of the candidate modulator with the expression of O1-180, O1-184 and/or O1-236 in the absence of the candidate modulator;
wherein a difference in the expression of Ol-180, O1-184 and/or O1-236 in the presence of the candidate modulator, as compared with the expression of O1-180, O1-184 and/or O1-236 in the absence of the candidate modulator, identifies the candidate modulator as a modulator of O1-180, Ol-184 and/or O1-236 expression.
[0017] A specific embodiment of the present invention is a method of identifying compounds that modulate the activity of O1-180, O1-I84 and/or Ol-236 comprising the steps of obtaining an isolated O1-180, O1-184 and/or O1-236 polypeptide or functional equivalent thereof; admixing the O1-180, O1-184 and/or O1-236 polypeptide or functional equivalent thereof with a candidate compound; and measuring an effect of said candidate compound on the activity of O1-I80, O1-184 and/or O1-236.
[0018] Another embodiment is method of screening for a compound which modulates the activity of O1-180, O1-184 and/or O1-236 comprising exposing Ol-180, Ol-184 and/or OI-236 or a O1-180, O1-184 and/or O1-236 binding fragment thereof to a candidate compound; and determining whether said compound binds to O1-180, O1-184 and/or O1-236 or the Ol-180, Ol-184 andlor O1-236 binding partner thereof; and fiu-ther determining whether said compound modulates O1-180 or the OI-I80 interaction with a binding partner.
[0019] Yet further, another embodiment is a method of screening for an interactive compound which binds with O1-180, Ol-184 and/or Ol-236 comprising exposing a O1-180, 01-184 and/or Ol-236 protein, or a fragment thereof to a compound; and determining whether said compound bound to the Ol-180, Ol-184 and/or O1-236.
[0020] Another embodiment is a method of identifying a compound that effects O1-180, Ol-184 and/or Ol-236 activity. The method comprises the steps of providing a group of transgenic animals having (1) a regulatable one or more 01-180, O1-184 and/or O1-236 protein/genes, (2) a knock-out of one or more O1-180, O1-184 and/or OI-236 protein/genes, or (3) a knock-in of one or more O1-180, O1-184 and/or O1-236 protein/genes;
providing a second group of control animals respectively for the group of transgeiuc animals; and exposing the transgenic animal group and control animal group to a potential O1-180, O1-I84 andlor OI-236-modulating compounds; and comparing the transgenic animal group and the control animal group and determining the effect of the compound on one or more proteins related to infertility or fertility in the transgenic animals as compared to the control animals.
[0021] In specific embodiments, the present invention provides a method of detecting a binding interaction of a first peptide and a second peptide of a peptide binding pair, comprising culturing at least one eukaryotic cell under conditions suitable to detect the selected phenotype; wherein the cell comprises; a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thexeof joined to a transcriptional activation protein DNA binding domain; a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation pxotein of a transcriptional activation domain; wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and a reporter element activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter element produces a selected phenotype; detecting the binding interaction of the peptide binding pair by determining the level of the expression of the reporter element which produces the selected phenotype; wherein said first or second peptide is an O1-180, Ol-184 andlor O1-236 peptide and the other peptide is a test peptide, preferably selected peptides/proteins present in a reproductive tissue. In specific embodiments the reproductive tissue is an ovary or testis.
[0022] A further embodiment is a rescue screen for detecting the binding interaction of a first peptide and a second peptide of a peptide binding pair.
The screen comprises the steps of culturing at least one eukaryotic cell under conditions to detect a selected phenotype or the absence of such phenotype, wherein the cell comprises; a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a DNA binding domain of a transcriptional activation protein; a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation domain of a transcriptional activation protein;
wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and a reporter element activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter element prevents exhibition of a selected phenotype; detecting the ability of the test peptide to interact with O1-180, Ol-184 and/or O1-236 by determining whether the test peptide affects the expression of the reporter element which prevents exhibition of the selected phenotype, wherein said first or second peptide is an O1-180, O1-184 and/or 01-236 peptide and the other peptide is a test peptide, preferably selected peptides/proteins present in a reproductive tissue. In specific embodiments, the reproductive tissue is an ovary, testis, epididymis, vas deferens, etc.
[0023] Yet further, another embodiment is a method of identifying binding partners for Ol-180, Ol-184 and/or O1-236 comprising the steps of: exposing the protein to a potential binding partner; and determining if the potential binding partner binds to O1-180, O1-184 and/or O1-236.
[0024] The present invention provides key in vita o and i~ vivo reagents for studying ovarian development and function. The possible applications of these reagents are far-reaching, and are expected to range from use as tools in the study of development to therapeutic reagents against cancer. The major application of these novel ovarian gene products is to use them as reagents to evaluate and/or develop potential contraceptives to modulate ovulation in women in a reversible or irreversible manner. It will also be expected that these novel ovarian gene products will be useful to screen for genetic mutations in components of those signaling pathways that are associated with some forms of human infertility or gynecological cancers. In addition, depending on the phenotypes of humans with mutations in these genes or signaling pathways, the inventors may consider using these novel ovarian gene products as reagent tools to generate a number of mutant mice for the further study of oogenesis, folliculogenesis, and/or early embryogenesis as maternal effect genes. Such knockout mouse models will provide key insights into the roles of these gene products in human female reproduction and permit the use of these gene products as practical reagents for evaluation and development of new contraceptives.
[0025] Still fiuther, another embodiment of the present invention comprises a method of treating an animal suffering from infertility by screening for a modulator that modulates the activity and/or expression of O1-180, O1-184 and/or Ol-236 comprising the steps of obtaining an isolated O1-180, Ol-184 and/or O1-236 polypeptide or functional equivalent thereof; admixing the O1-180, Ol-184 and/or O1-236 polypeptide or functional equivalent thereof with a candidate compound; measuring an effect of said candidate compound on the activity and/or expression of O1-180, O1-184 and/or O1-236, and administering to the subject an effective amount of the modulator to increase conception.
[0026] Still further, another embodiment of the present invention comprises a method of modulating conception or fertility in an animal by screening for a modulator that modulates the activity and/or expression of O1-180, O1-184 and/or O1-236 comprising the steps of obtaining an isolated O1-180, O1-184 andlor O1-236 polypeptide or functional equivalent thereof; admixing the O1-180, O1-184 and/or O1-236 polypeptide or functional equivalent thereof with a candidate compound; measuring an effect of said candidate compound on the activity and/or expression of O1-180, Ol-I84 and/or O1-236, and administering to the subject an effective amount of the modulator to decrease conception and/or increase conception. Thus, the modulator can be a contraceptive or a fertility agent.
[0027] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention.
It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that IO

such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings.
[0029] FIGURE 1. Mufti-tissue Northern blot analysis of ovary-specific genes.
Northern blot analysis was performed on total RNA using O1-180, Ol-184, and O1-236 probes.
These gene products demonstrate an ovary-specific pattern (OV, ovary; WT, wild-type; -/-, Gdfp knockout) as shoran. The migration positions of 18S and 28S ribosomal RNA are indicated. All lanes had approximately equal loading as demonstrated using an 18S rRNA cDNA
probe. (Br, brain; Lu, lung; He, heart; St, stomach; Sp, spleen; Li, liver; Si, small intestine; Ki, kidney; Te, testes, Ut, uterus).
[0030] FIGURES 2A-2F. In situ hybridization analysis of ovary-specific genes in mouse ovaries. In situ hybridization was performed using anti-sense probes to O1-180 (Figures 2A-2B), O1-184 (Figures 2C-2D) and O1-236 (Figures 2E-2F). Figures 2A, 2C, and 2E are brightfield analysis of the ovaries. Figures 2B, 2D, and 2F are darkfield analysis of the same ovary sections. All genes demonstrate specific expression in the oocyte beginning at the one layer primary follicle stage (small arrows) and continuing through the antral follicle stage (large arrows).
[0031] FIGURES 3A and 3B. In situ hybridization analysis of O1-236 in mouse ovaries. In situ hybridization was performed using probe O1-236 (partial N~m2 cDNA
fragment). Brightfield analysis (Figure 3A) and darkfield analysis (Figure 3B) of the O1-236 mRNA in the same adult ovary sections. The probe demonstrates specific expression in alI
growing oocytes. Oocyte-specif c expression is first seen in the early one layer primary follicle (type 3a), with higher expression in the one layer type 3b follicle and all subsequent stages including antral (an) follicles.
[0032] FIGURE 4. Amino acid sequence conservation among Xenopus laevis (SEQ.ID.NO.15), mouse (SEQ.ID.N0.6), rat (SEQ.ID.N0.42) and human (SEQ.ID.N0.9) NPM2 proteins. Using the NCBI blast search tools and Megalign software, comparison of mouse (m), human (h), (r) rat and Xenopus laevis NPM2 amino acid sequences reveals high identity.
Spaces between the amino acids indicate gaps to aid in the alignment. Inter-species amino acid identity is highlighted in black. The conserved bipartite nuclear localization sequence is indicated by asterisks (*); a line is drawn over the acidic histone binding region.
[0033] FIGURE 5. Chromosomal localization of the mouse Npm2 gene. (Top) Map figure from the T31 radiation hybrid database at The Jackson Laboratory showing Chromosome 14 data. The map is depicted with the centromere toward the top.
Distances between adjacent loci in centiRay3000 are shown to the left of the chromosome bar. The positions of some of the chromosome 14 MIT markers are shown on the right. The mouse Npm2 gene is positioned between Dl4Mit203 and Dl4Mit32. Missing typings were inferred from surrounding data where assignment was unambiguous. (Bottom) Haplotype figure from the T31 radiation hybrid database at The Jackson Laboratory showing part of Chromosome 14 with loci linked to Npm2. Loci are listed in the best fit order with the most proximal at the top. The black boxes represent hybrid cell lines scoring positive for the mouse fragment and the white boxes represent cell lines scoring as negative. The grey box indicates an untyped or ambiguous line.
The number of lines with each haplotype is given at the bottom of each column of boxes.
Missing typings were inferred from surrounding data where assigmnent was unambiguous.
[0034] FIGURES 6A-6H. Analysis of Npm2 mRNA and NPM2 protein in mouse ovaries and early embryos. In situ hybridization was performed using probe O1-236 (partial Npm2 cDNA fragment). Brightfield analysis (Figure 6A) and darkfield analysis (Figure 6B) of the O1-236 mRNA in the same adult ovary sections. Arrows and arrowheads denote expression of the Npm2 mRNA in oocytes from follicles at various stages of follicular development.
(Figure 6C) Immunohistochemistry of ovaries from a 5-week old mouse stained for NPM2 in the nuclei of oocytes from type 3 through to antral follicles. (Figure 6D) In preovulatory GVB
oocytes induced by luteinizing hormone (hCG), NPM2 is evenly stained in the cytoplasm (arrow). An LH (hCG) unresponsive preantral follicle (upper xight) continues to demonstrate an oocyte with NPM2 protein localized to the nucleus. (Figure 6E) After fertilization, NPM2 begins to localize in the pronuclei; the formation of one pronucleus (arrow), is in the process of fomning and some of NPM2 staining continues to be present in the cytoplasm of this early one cell embryo. (Figure 6F) The pronuclei stain strongly in an advanced one cell embryo where very little NPM2 remains in the cytoplasm. NPM2 antibodies also specifically stain the nuclei of two cell (Figure 6G) and eight cell (Figure 6H) embryos.
[0035] FIGURES 7A-7C. Gene targeting construct for a knockout of Npm2 and genotype analysis of offspring from heterozygote intercrosses. Figure 7A shows the targeting strategy used to delete exon 2, exon 3, and the junction region of exon 4. PGK-hprt and MC1-tk expression cassettes. Recombination was detected by Southern blot analysis using 5' and 3' probes. (B, BamHl; Bg, Bgl II; P, Pst I). Figure 7B shows a Southern blot analysis of genomic DNA isolated from intercrosses of NpnZ2+~ mice. The 3' probe identifies the wild-type 7.5-kb band and the mutant 10.3-kb band when DNA was digested with Bgl II. Figure 7C
shows that when DNA was digested with Fst 1, the exon 2 probe only detected the wild-type 4.5-kb fragment.
[0036] FIGURES 8A-8F. Histological analysis of ovaries from wild-type, Npna2+~
and Npm2-~~ mice. (Figure 8A-8D) Immunohistochemistry of ovaries from 6-week old mice stained for Npm2 in the nuclei of oocytes (Figure 8A and Figure 8C for Npm2+~
ovaries; Figure 8B and Figure 8D for Npm2-~ ovaries). (Figures 8E-8F) PAS (Periodic acid Schiff)/hematoxylin staining of ovaries from 12 week old mice wild-type (Figure 8E) and Npm2-~
(Figure 8F) ovaries. Arrows show large antral follicles.
[0037] FIGURES 9A-9F. In vitro culture of eggs (metaphase II) and fluorescent-labeling of DNA from fertilized eggs from Npn22 ~ and control mice. Eggs were isolated from the oviducts of immature mice after superovulation and cultured in vitro.
Pictures were taken under a microscope at 45 (Figures 9A-9B), 55 (Figures 9C-9D) and 96 (Figures 9E-9F) hours of culture. Most fertilized eggs from wild-type mice form 2-cell and 4-cell embryos by 45 and 55 hours post-hCG (white arrows), while few Npm2 Npm2-~ eggs cleave to form multicellular embryos, and even fewer form blastocysts compared to wild-type controls.
[0038] FIGURES 10 and 1 OB. The percent cleavage of in vivo fertilized embryos to various stages is shown after oviduct collection (Figure l0A) and subsequent 24 hour culture (Figure l OB). Times are given as hours post-hCG.
[0039] FIGURES 11A-11D. Wild-type (Figure 11A) and Npna2-~- (Figure lOB) fertilized oocytes are TUNEL negative, with the exception of their TUNEL
positive polar bodies.
(Figure lIC and 11D) Later, DNA within fragmenting Npm2 null embryos stain TUNEL
positive.
[0040] FIGURE 12. Transcription-requiring complex (TRC) proteins were extracted from wild-type (WT) and null (-/-) 2-cell embryos after culture in 35S-labeled methionine. As a negative control, actinomycin D (ActD) inhibited transcription and TRC
production.
[0041] FIGURES 13A-13Z. WT and mutant oocytes and embryos were analyzed.
Immunofluorescence analysis of wild-type or Npm2 null oocytes (Figures 13A-13J), 1-cell embryos (Figures 13I~-13V), or 8-cell embryos (Figures 13W-13Z) was performed using the indicated antibodies. DNA was counterstained with DAPI (Figures 13A-13L, 130-13P, and 13S-13Z) ox To-pro-3 (Figures 13Q-13R).
[0042] FIGURE I4. Analysis of ribosomal RNAs is shown in oocytes and 1-cell embryos. An RNAse protection assay was performed to quantify 18S and 28S rRNAs in wild-type (WT) and Npm2 null GV stage oocytes, metaphase II oocytes, and 1-cell embryos. Small quantities of untreated full-length probe served as indicators that the digestion went to completion (Lanes 1 and 8). Phosphorimager analysis to quantify WT and Npm2 null rRNA
signals (i. e., comparing Lane 2 and 5; 3 and 6; 4 and 7;9 and 12; 10 and 13;
and 11 and 14) result in ratios ranging from 0.69 to 1.40.
[0043] FIGURE 15. Absolute levels of protein synthesis in oocytes and 1-cell embryos are shown. In all cases, the addition of 3.0 mg/mL unlabeled methionine competed effectively with the incorporation of the 35S-labeled methionine.
[0044] FIGURES 16A-16H. In situ hybridization was used to detect Npml and Npm3 mRNAs in ovaries of wild-type mice. Npml mRNA was highly expressed in oocytes of small follicles (Figures 16A-16B), secondary follicles (Figures I6C-16D) and large antral follicles (Figures 16E-16F) (arrows). Sections are shown in brightfield (Figures 16A, 16C, and 16E) and darkfield (Figures 16B, 16D, and 16F) to demonstrate the histology and highlight the hybridization signal, respectively. Npna3 mRNA was detected in all stages of oocytes in the adult ovary (Figures 16G-16H).
[0045] FIGURES 17A and 178. Expression analysis of Zygote arrest 1 in mouse and human tissues. Figure 17A shows a Northern blot analysis with the Za~l cDNA fragment in total RNA derived from wildtype tissues and Gdf9-~ ovaries. Figure 17B shows RT-PCR
analysis of human ZARl. (Br, brain; Lu, lung; He, heart; St, stomach; Sp, spleen; Li, liver; SI, small intestine; Ki, kidney; Te, testes; Ut, uterus; Co, colon; Pr, prostate;
Pl, placenta; Pa, pancreas; Mu, muscle).
[0046] FIGURE 18. Comparison of the mouse and human ZARl amino acid sequences.
[0047] FIGURE 19A and 19B. Comparison of the Zarl gene and the Zarl psl pseudogene. Sequences of exons, exon-intron boundaries and the size of each intron are shown.
Different nucleotides between the two genes and consensus polyadenylation sequence are underlined. The translation start codon and stop COd011 are S110Wn 111 bold.
Upper case: exon sequences; lower case: intron sequences.
[0048] FIGURES 20A and 20B. Maps of mouse chromosome 5, showing the position in centiMorgan (cM) of the marker best linked to the Zarl gene (Figure 20A) and its related pseudogene (Figure 20B).
[0049] FIGURE 21. Western blot analysis of recombinant ZARl .
[0050] FIGURES 22A-22F. Expression of Za~l in PMSG-treated wild-type (Figures 22A and 22B) and Gdfp-~' (Figures 22C-22F) ovaries was analyzed by i~
situ hybf°idizatioiz with a specific antisense probe. Both brightfield (Figures 22A, 22C and 22E) and corresponding daxkfield (Figures 22B, 22D and 22F) images of the same ovary sections are presented. Areas of sections of Figures 22C and 22D are shown at higher magnification (Figures 22E and 22F). The expression of the Zaf-1 gene was detected at early primary follicle (type 3a) through to antral follicle (type 8) stage, but not in primordial follicles (type 2), in wild-type or Gdf~-~ ovaries. In Gdf~'~ ovaries, the follicle numbers increase per unit volume due to follicle arrest at the primary stage, and hence more Za~l positive signals were detected in each section.
[0051] FIGURES 23A-23D. Mouse Zar~l gene structure and targeting strategy.
Figure 23A shows a targeting vector, which was constructed by replacing Exon 1 (which contains the ATG start codon) and part of intron 1 with a PGK-Hprt expression cassette.
Targeted ES cell clones containing a wild-type (WT), a pseudogene allele (Zaf~l ~sl ), and a mutant (MUT) allele were confirmed by Southern blot analysis and injected into blastocysts to produce chimeric male mice, which were bred to produce F1 Zar~l+~ offspring.
Southern blot analysis (Figure 23B) of genomic DNA is derived from offspring of one litter from a heterozygous mating. Figure 23C shows Northern blot analysis of ovarian mRNA
from wild-type, Za~l+~, and Zap°I'~ females using the full-length Zarl cDNA. On longer exposure, a smaller transcript of unknown relevance was observed in Za~~l'~ ovaries, and the expression level of Zarl in wild-type mice is approximately twice the levels of the Zarl +~ .
GapdlZ was used as a control for equal loading on the Northern blot (Figure 23D).
[0052] FIGURES 24A-24J. Mouse ZARl protein expression. An anti-mouse ZARl polyclonal antibody was used for immunohistochemistry (Figures 24A-24D) and immunofluorescence analysis (Figures 24E-24J) to detect ZAR1 expression.
Similar to the Zarl mRNA, ZARl protein expression begins in oocytes of primary follicles and continues through all follicle stages in wild-type ovaries (Figures 24A, 24B). ZARl is also detected in Gdf~
ovaries (Figures 24C), whereas no protein was detected in Za~l'~ ovaries (Figure 24D). ZARl protein was detected predominantly in the cytoplasm of fully-grown, prophase I-arrested oocytes from Zarl+~ (Figure 24E) but not Za~l ~ mice (Figure 24F). ZAR1 is expressed in wild-type oocytes, during the progression from MI (Figure 24G) to MII (Figure 24H), and persists in zygotes at the 1-cell stage, collected 6 h post-fertilization (Figure 24I).
However, ZARl expression is dramatically reduced in 2-cell stage embryos (Figure 24J), with bright staining evident only in polar body remnants.
[0053] FIGURES 25A - 25D. Development of embryos derived from Zarl+~' and Zarl-~- mice. Adult Zarl~~' (Figure 25A) and Zarl-~' (Figure 25B) females were mated with stud males. Whereas all zygotes from Zarl+~' female mice progressed to the blastocyst stage (Figure 25A), most zygotes from Zarl'~ mice remained at the 1-cell stage, and many degenerating embryos were detected (Figure 25B). At 24 h post-fertilization, the arrested zygotes from Zarl'~' females were labeled with anti-(3 tubulin and propidium iodide to assess microtubule and chromatin configurations, respectively (Figure 2SC). Decondensed chromatin was evident in both the maternal and paternal pronucleus. Additionally, the microtubules show an interphase configuration, with no assembled spindle apparatus. In a second experiment, the fertilized zygotes were placed in medium with BrdU at 8 h post-fertilization and cultured overnight (Figure 25D). Immunofluoresence analysis shows BrdU incorporation in both pronuclei of an arrested zygote from a Zarl-~- female indicative of entry into S-phase.
[0054] FIGURES 26A-26B. Cell-free transcription/translation of Za~l, Polr2c (DNA directed RNA polymerase II polypeptide C), Gnb2 (Guanine nucleotide binding protein, beta 2), Polr2g (DNA directed RNA polymerase II polypeptide G), and Lmol (LIM
only 1) cDNAs. Autoradiogxaph of [3SS] Met-labeled proteins from cell-free i~ vitro transcriptioutranslation and co-immunoprecipitation by anti-HA polyclonal antibody (Figure 26A) or anti-MYC monoclonal antibody (Figure 26B). The position of molecular mass standards in kDa is shown at the right. The HA-tagged POLR2C, GNB2, POLR2G, and LMOl bind to the MYC-tagged ZARl.
[0055] FIGURE 27. Amino Acid sequence comparison of ZAR1 proteins from homo sapiens, Mus musculus, Xenopus laevis, Danio rerio and Fugu rubripes.
DETAILED DESCRIPTION OF THE INVENTION
[0056] It is readily apparent to one skilled in the art that various embodiments and modifications can be made to the invention disclosed in this Application without departing from the scope and spirit of the invention.
[0057] As used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."
[0058] As used herein, the term "animal" xefers to a mammal, such as human, non-human primates, horse, cow, elephant, cat, dog, rat or mouse. In specific embodiments, the animal is a human.

[0059] As used herein, the term "antibody" is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG
and/or IgM are preferred because they axe the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. Thus, one of skill in the art understands that the term "antibody" refers to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')Z, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
[0060] As used herein, the term "binding protein" refers to proteins that demonstrate binding affinity for a specific higand. Binding proteins may be produced from separate and distinct genes. For a given ligand, the binding proteins that are produced from specific genes are distinct from the ligand binding domain of the receptor or its soluble receptor.
[0061] As used herein, the term "binding partner" or "interacting proteins"
refer to a molecule capable of binding another molecule with specificity, as for example, an antigen and an antigen-specific antibody or an enzyme and its inhibitor. Binding partners may include, for example, biotin and avidin or streptavidin, IgG and protein A, receptor-Iigand couples, protein-protein interaction, and complementary pohynucheotide strands. The term "
binding partner" may also refer to polypeptides, lipids, small molecules, or nucheic acids that bind to O1-180, OI-236 and/or Ol-184 in cells. A change in the interaction between a protein and a binding partner can manifest itself as an increased or decreased probability that the interaction forms, ox an increased or decreased concentration of O1-180, O1-236 and/or O1-184 in cells -binding partner complex.
[0062] As used herein, the term "O1-I80 binding fragment", "Ol-184 binding fragment" and/or "O1-236 binding fragment" refers to the nucleic acid fragment and/or amino acid fragment of Ol-180, Ol-184 and/or Ol-236 respectively that is capable of binding to the binding partner or interacting protein, for example polypeptides, lipids, small molecules, or nucheic acids.
[0063] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terns also include their progeny, which are any and ahl subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaxyotic or eukaryotic cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located ih vitf°o or in vivo. For example, host cells may be located in a transgenic animal. Host cell can be used as a recipient for vectors and may include any transformable organisms that are capable of replicating a vector andlor expressing a heterologous nucleic acid encoded by a vector.
[0064] As used herein, the term "conception" refers to the union of the male sperm and the ovum of the female; fertilization.
[0065] As used herein, the term "contraception" refers to the prevention or blocking of conception. A contraceptive device, thus, refers to any process, device, or method that prevents conception. Well known categories of contraceptives include, steroids, chemical barrier, physical barrier; combinations of chemical and physical barriers; use of immunocontraeeptive methods by giving either antibodies to the reproductive antigen of interest or by developing a natural immune response to the administered reproductive antigen; abstinence and permanent surgical procedures. Contraceptives can be administered to either males or females.
[0066] As used herein, the term "complementary" is used to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.
[0067] As used herein, the term "DNA" is defined as deoxyribonucleic acid.
[0068] As used herein, "cDNA" refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double-stranded form using, for example, the Klenow fragment of DNA polymerase I.
[0069] As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Included within the term "DNA
segment" are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.

[0070] As used herein, the term "expression construct" or "transgene" is defined as any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed can be inserted into the vector. The transcript is translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product.
In other embodiments, expression only includes transcription of the nucleic acid encoding genes of interest. In the present invention, the term "therapeutic construct" may also be used to refer to the expression construct or transgene. One skilled in the art realizes that the present invention utilizes the expression construct or transgene as a therapy to treat infertility. het further, the present invention utilizes the expression construct or transgene as a "prophylactic construct" for contraception. Thus, the "prophylactic construct" is a contraceptive.
[0071] As used herein, the terns "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described ihf °a.
[0072] As used herein, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. This functional term includes both genomic sequences, cDNA sequences and engineered segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins and mutant. Thus, one of skill in the art is aware that the term "native gene" or "endogenous gene" refers to a gene as found in nature with its own regulatory sequences and the term "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from the same source, but arranged in a manner different than that found in nature.

[0073] As used herein, the term "gonadal" or "gonadal tissue" or "gonads"
refers to tissue that is related to the male and female sex organs. Gonadal tissue is not limited to the ovaries and/or testes; it may also include the embryonic tissue that develops into the ovaries and/or testes.
[0074] As used herein, the terms "identity" or "similarity", as known in the art, are relationships between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Both identity and similarity can be readily calculated by known methods such as those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing:
Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987;
Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991. Methods commonly employed to determine identity or similarity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988). Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG
program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Atschul, S. F. et al., J Molec. Biol., 215, 403 (1990)).
[0075] As used herein, the term "homologous" refers to the degree of sequence similarity between two polymers (i.e. polypeptide molecules or nucleic acid molecules). The homology percentage figures referred to herein reflect the maximal homology possible between the two polymers, i. e., the percent homology when the two polymers are so aligned as to have the greatest number of matched (homologous) positions.
[0076] As used herein, the term "percent homology" refers to the extent of amino acid sequence identity between polypeptides. The homology between any two polypeptides is a direct function of the total number of matching amino acids at a given position in either sequence, e.g., if half of the total number of amino acids in either of the sequences are the same then the two sequences are said to exhibit 50% homology.
[0077] The term "fragment", "analog", and "derivative" when referring to the polypeptide of the present invention (e.g., O1-180 (SEQ.ID.N0.2, SEQ.ID.N0.16, SEQ.ID.N0.29, SEQ.TD.NO.32, SEQ.ID.N0.34, SEQ.ID.NO.36 and SEQ.TD.N0.39), O1-(SEQ.ID.N0.4), Ol-236 (SEQ.ID.N0.6, SEQ.ID.N0.9, and SEQ.ID.N0.42)), refers to a polypeptide which may retain essentially the same biological function or activity as such polypeptide. Thus, an analog includes a precursor protein that can be activated by cleavage of the precursor protein portion to produce an active mature polypeptide. The fragment, analog, or derivative of the polypeptide of the present invention (O1-180 (SEQ.TD.N0.2, SEQ.ID.N0.16, SEQ.ID.N0.29, SEQ.ID.N0.32, SEQ.ID.N0.34, SEQ.ID.N0.36 and SEQ.ID.N0.39), O1-(SEQ.ID.N0.4), O1-236 (SEQ.ID.N0.6, SEQ.ID.N0.9, and SEQ.ID.N0.42)), may be one in which one or more of the amino acids are substituted with a conserved or non-conserved amino acid residues and such amino acid residues may or may not be one encoded by the genetic code, or one in which one or more of the amino acid residues includes a substituent group, or one in which the polypeptide is fused with a compound such as polyethylene glycol to increase the half life of the polypeptide, or one in which additional amino acids are fused to the polypeptide such as a signal peptide or a sequence such as polyhistidine tag which is employed for the purification of the polypeptide or the precursor protein. Such fragments, analogs, or derivatives are deemed to be within the scope of the present invention.
[0078] The term "functional equivalent" as used herein is defined as a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to perform the biologic function of interest of the wild-type or reference protein. Thus, as used herein, the term functional equivalent includes truncations, deletions, insertions or substitutions of 01-I80 (SEQ.ID.NO.2, SEQ.ID.NO.16, SEQ.ID.NO.29, SEQ.ID.N0.32, SEQ.ID.NO.34, SEQ.ID.N0.36 and SEQ.ID.NO.39), O1-184 (SEQ.ID.NO.4), Ol-236 (SEQ.ID.N0.6, SEQ.ID.NO.9, and SEQ.ID.NO.42)) which retains their function to play a role in in fertility and embryonic development. This also can be accomplished to the degeneracy of the genetic code, i.e., the presence of multiple codons, which encode for the same amino acids. In one example, one of skill in the art may wish to introduce a restriction enzyme recognition sequence into a polynucleotide while not disturbing the ability of that polynucleotide to encode a protein. In another example, a polynucleotide may be (and encode) a functional equivalent with more significant changes. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigezi-binding regions of antibodies, binding sites on substrate molecules, receptors, and such like. So-called "conservative" changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges of the protein's ability to carry out its designed function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.
[0079] The term "hyperproliferative disease" is defined as a disease that results from a hyperproliferation of cells. Hyperproliferative disease is further defined as cancer. The hyperproliferation of cells results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. Exemplary hyperproliferative diseases include, but are not limited to cancer or autoimmune diseases. Other hyperproliferative diseases can include vascular occlusion, restenosis, atherosclerosis, or inflammatory bowel disease.
[0080) As used herein, the term "fertility" refers to the quality of being productive or able to conceive. Fertility relates to both male and female animals.
[0081] As used herein, the term "infertility" refers to the inability or diminished ability to conceive or produce offspring. Infertility can be present in either male or female. In the present invention, administration of a composition to enhance infertility or decrease fertility is reversible. Examples of infertility include, without limitation, azoospermia; genetic disorders associated with defective spermatogenesis (e.g., l~linefelter's syndrome and gonadal dysgenesis);
oligospermia, varicocele, and other sperm disorders relating to low sperm counts, sperm motility, and sperm morphology; and ovulatory dysfunction (e.g., polycystic ovary syndrome (PCOS) or chronic anovulation).
[0082] As used herein, the terms "O1-180", "Ool", "zygote arrest 1 (Za>~I)", "ZARI" or "ZARl" are interchangeable. Zaf°I and ZARI denote the mouse and human DNA
sequence, respectively. ZAR1 denotes the mouse and the human amino acid sequences.

[0083] As used herein, the terms "O1-236", "nucleoplasmin 2 (Npm2)", "NPM2"
or "NPM2" are interchangeable. Npm2 and NPM2 denote the mouse and human DNA
sequence, respectively. NPM2 denotes the mouse and the human amino acid sequences.
[0084] As used herein, the term "modulate" refers to the suppression, enhancement, or induction of a function. For example, "modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. "Modulate" or "regulate" also refers to methods, conditions, or agents which increase or decrease the biological activity of a protein, enzyme, inhibitor, signal transducer, receptor, transcription activator, co-factor, and the like.
This change in activity can be an increase or decrease of mRNA translation, DNA transcription, and/or mRNA or protein degradation, which may in turn correspond to an increase or decrease in biological activity. Such enhancement or inhibition may be contingent upon occurrence of a specific event, such as activation of a signal transduction pathway and/or may be manifest only in particular cell types.
[0085] As used herein, the term "modulated activity" refers to any activity, condition, disease or phenotype that is modulated by a biologically active form of a protein.
Modulation may be affected by affecting the concentration of biologically active protein, e.g., by regulating expression or degradation, or by direct agonistic or antagonistic effect as, for example, through inhibition, activation, binding, or release of substrate, modification either chemically or structurally, or by direct or indirect interaction which may involve additional factors.
[0086] As used herein, the term "modulator" refers to any composition and/or compound that alters the expression of a specific activity, such as O1-236 activity or expression, O-180 activity or expression, and/or O1-184 activity or expression. The modulator is intended to comprise any composition or compound, e.g., antibody, small molecule, peptide, oligopeptide, polypeptide, or protein.
[0087] The term "small molecule" refers to a synthetic or naturally occurring chemical compound, for instance a peptide or oligonucleotide that may optionally be derivatized, natural product or any other low molecular weight (typically less than about 5 kDalton) organic, bioinorganic or inorganic compound, of either natural or synthetic origin.
Such small molecules may be a therapeutically deliverable substance or may be further derivatized to facilitate delivery.
[0088] The term "operatively linked" refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operatively linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i. e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operatively linked to regulatory sequences in sense or antisense orientation. As used herein, the term "peptide binding pair" refers to any pair of peptides having a known binding affinity for which the DNA
sequence is known or can be deduced. The peptides of the peptide binding pair must exhibit preferential binding for each other over any other components of the modified cell.
[0089] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic and/or prophylactic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
[0090] As used herein, the terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "oligonucleotide", refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA, and mean any chain of two or more nucleotides. The polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. The antisense oligonuculeotide may comprise a modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanne, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, S- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, S'-methoxycarboxymethyluracil, S-methoxyuracil, 2-methylthio-N6-isopentenyladenine, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, S-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, S-methyluracil, uracil- S-oxyacetic acid methylester, uracil-S-oxyacetic acid, S-methyl-2- thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. A
nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double-or single-stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This includes single- and double-stranded molecules, i. e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thin-uracil, thio-guanine and fluoro-uracil, or containing carbohydrate, or lipids.
[0091] As used herein, the term "polypeptide" is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term polypeptide is interchangeable with the terms "peptides" and "proteins".
[0092] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
[0093] As used herein, the term "purified protein or peptide", is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
[0094] As used herein, the term "RNA" is defined as ribonucleic acid.
[0095] As used herein, "messenger RNA (mRNA)" refers to the RNA that is without introns and can be translated into polypeptides by the cell.
[0096] As used herein, the term "RNA interference" or "RNAi" is an RNA
molecule that is used to inhibit a particular gene of interest.

[0097] As used herein, the term "regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA
processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
[0098] As used herein, the term "sense" refers to sequences of nucleic acids that are in the same orientation as the coding mRNA nucleic acid sequence. A DNA
sequence linked to a promoter in a "sense orientation" is linked such that an RNA molecule which contains sequences identical to an mRNA is transcribed. The produced RNA molecule, however, need not be transcribed into a functional protein.
[0099] As used herein, the term an "anti-sense" copy of a particular polynucleotide refers to a complementary sequence that is capable of hydrogen bonding to the polynucleotide and can therefor be capable of modulating expression of the polynucleotide.
These are DNA, RNA or analogs thereof, including analogs having altered backbones, as described above. The polynucleotide to which the anti-sense copy binds may be in single-stranded form or in double-stranded form. A DNA sequence linked to a promoter in an "anti-sense orientation" may be linked to the promoter such that an RNA molecule complementary to the coding mRNA of the target gene is produced.
[0100] As used herein, the terms "sense" strand and an "anti-sense" strand when used in the same context refer to single-stranded polynucleotides that are complementary to each other. They may be opposing strands of a double-stranded polynucleotide, or one strand may be predicted from the other according to generally accepted base-pairing rules.
Unless otherwise specified or implied, the assignment of one or the other strand as "sense" or "antisense" is arbitrary.
[0101] The term ''effective amount" or "therapeutically effective amount" as used herein refers to an amount that results in an improvement or remediation of the symptoms of the disease or condition.
[0102] The term "treating" and "treatment" as used herein refers to administering to a subject a therapeutically effective amount of the pharmaceutical composition and/or modulator so that the subject has an improvement in the disease and/or condition. The improvement is any improvement or remediation of the symptoms. The improvement is an observable or measurable improvement. Thus, one of skill in the art realizes that a treatment may improve the disease and/or condition, but may not be a complete cure for the disease and/or condition.
[0103] As used herein, the term "under transcriptional control" or "operatively linked" is defined as the promoter that is in the 'correct location and orientation in relation to the nucleic acid to control RNA polymerase iutiation and expression of the gene.
[0104] The present invention provides three novel proteins, O1-180 (SEQ.ID.N0.2, SEQ.ID.NO.16, SEQ.ID.N0.29, SEQ.ID.N0.32, SEQ.ID.N0.34, SEQ.ID.N0.36 and SEQ.ID.N0.39), O1-184 (SEQ.ID.NO.4), O1-236 (SEQ.ID.N0.6, SEQ.ID.N0.9, and SEQ.ID.NO.42), the polynucleotide sequences that encode them, and fragments and derivatives thereof. Expression of O1-180, O1-184, O1-236 is highly tissue-specific, being expressed in cells primarily of ovarian tissue. In one embodiment, the invention provides a method for detection of a cell proliferative or degenerative disorder of the ovary, which is associated with expression of Ol-I80, O1-184 or O1-236. In another embodiment, the invention provides a method for treating a cell proliferative or degenerative disorder associated with abnormal expression of O1- O1-180, O1-184, Ol-236 by using an agent which suppresses or enhances their respective activities.
[0105] Based on the known activities of many other ovary specific proteins, it can be expected that O1-180, Ol-184 and O1-236, as well as fragments and derivatives thereof, will also possess biological activities that will make them useful as diagnostic and therapeutic reagents.
[0106] For example, GDF-9 is an oocyte-expressed gene product which has a similar pattern of expression as Ol-180, O1-184, and Ol-236. It has been shown that mice lacking GDF-9 are infertile at a very early stage of follicular development, at the one-layer primary follicle stage. These studies demonstrate that agents which block GDF-9 function would be useful as contraceptive agents in human females. Since O1-180, O1-184, and O1-236 have an expression pattern in the oocyte (Figure 2) which is nearly identical to GDF-9, this suggests that mice and humans or any other mammal lacking any of all of these gene products may also be infertile. Thus, blocking the function of any or all of these gene products may result in a contraceptive action.

[0107) Another regulatory protein that has been found to have ovary-specific expression is inhibin, a specific and potent polypeptide inhibitor of the pituitary secretion of FSH. Inhibin has been isolated from ovarian follicular fluid. Because of its suppression of FSH, inhibin has been advanced as a potential contraceptive in both males and females. Ol-180, 01-184 and O1-236, may possess similar biological activity since they are also ovarian specific peptides. Inhibin has also been shown to be useful as a marker for certain ovarian tumors (Lappohn et al., 1989). O1-180, O1-184, O1-236 may also be useful as markers for identifying primary and metastatic neoplasms of ovarian origin. Likewise, mice which lack inhibin develop granulosa cell tumors (Matzuk et al., 1992). Similarly, O1-180, Ol-184 and O1-236 may be useful as indicators of developmental or reproductive anomalies in prenatal screening procedures.
[0108] Mullerian inhibiting substance (MIS or anti-Mullerian hormone) peptide, which is produced by the testis and is responsible for the regression of the Mullerian ducts in the male embryo, has been shown to inhibit the growth of human ovarian cancer in nude mice (Donahoe et al., 1981). O1-180, O1-184 and O1-236 may function similarly and may, therefore, be targets for anti-cancer agents, such as for the treatment of ovarian cancer.
[0109] O1-180, O1-184 and O1-236, and agonists and antagonists thereof can be used to identify agents which inhibit fertility (e.g., act as a contraceptive) in a mammal (e.g., human). Additionally, O1-180, O1-184 and Ol-236 and agonists and antagonists thereof can be used to identify agents which enhance fertility (e.g., increase the success of in vivo or i~ vitro fertilization) in a mammal. Likewise, assays of these or related oocyte-expressed gene products can be used in diagnostic assays fox detecting forms of infertility (e.g., in an assay to analyze activity of these gene products) or other diseases (e.g., germ cell tumors, polycystic ovary syndrome).
T. Proteins [0110] In an effort to identify other novel ovarian-expressed genes that may play key functions in ovarian physiology, fertilization and early cleavage events, the inventors used a subtractive hybridization approach. Several novel oocyte-expressed genes have been identified by the inventors which are important in regulating oogenesis, folliculogenesis, fertilization, and/or early embryogenesis. One of these oocyte-specific gene products, nucleoplasmin 2 (01-236 or NPM2), is the mammalian ortholog of Xenopus laevis nucleoplasmin (xNPM2) (Burglin et al., 1987; Dingwall et al., 1987). The 207 amino acid open reading frame of demonstrated high homology to the family of proteins called nucleoplasmins or nucleophosmins (nomenclature designation = species). NPM2 human gene, Npm2 mouse gene, and XtZptaz2 Xenopus gene; NPM2 = protein in all species). Human nucleoplasmin gene (NPMI
also called N038; accession # M23613) maps to human chromosome Sq35, encodes a 294 amino acid protein, and has orthologs in mouse (Npml, also called B23, accession #
Q61937) and Xenopus laevis (~hlvy~al or N038 accession # X05496). Mouse nucleoplasmiunucleophosmin homolog Npm3, which has been mapped to mouse chromosome 19, encodes a protein of 175 amino acids [accession # U64450, (MacArthur and Shackleford, 1997a)], and there is an apparent human NPM3 homolog gene (accession # AF08I280). In contrast to Npm2, the genes Npml and Npm3 are ubiquitously expressed, and the structure of the mouse Npm2 gene is considerably divergent compared to the mouse Npm3 gene (MacArthur and Shackleford, 1997a).
[0111] In the present invention, Ol-180 (SEQ.ID.N0.2, SEQ.ID.N0.16, SEQ,ID.N0,29, SEQ.ID.N0.32, SEQ.ID.NO,34, SEQ.ID.N0.36, and SEQ.ID.N0.39), Ol-(SEQ.ID.N0.4) and O1-236 (SEQ.ID.N06, SEQ.ID.N0.9, and SEQ.ID.N0.42) identified these proteins using subtractive hybridization. These identified proteins or agents which act on these pathways may also function as growth stimulatory factors and, therefore, be useful for the survival of various cell populations ih vitf°o. In particular, if Ol-180, O1-184 and/or O1-236 play a role in oocyte maturation, they may be useful targets for ih vitro fertilization procedures, e.g., in enhancing the success rate.
[0112] In this patent, the terms "O1-180 gene product" "O1-184 gene product"
and "Ol-236 gene product" refer to proteins and polypeptides having amino acid sequences that are substantially identical to the native O1-180, O1-184 and/or O1-236 amino acid sequences (or RNA, if applicable) or that are biologically active, in that they are capable of performing functional activities similar to an endogenous OI-180, O1-184 and/or O1-236 andlor cross-reacting with anti-Ol-I80, O1-184 andlor O1-236 antibody raised against Ol-180, O1-184 and/or 01-236.
[0113] The terms "O1-180 gene product" "Ol-184 gene product" and "O1-236 gene product" also include analogs of the respective molecules that exhibit at least some biological activity in common with their native counterparts. Such analogs include, but are not limited to, truncated polypeptides and polypeptides having fewer amino acids than the native polypeptide.
[0114] In addition to the entire O1-180, O1-184 or O1-236 molecules, the present invention also relates to fragments of the polypeptides that may or may not retain the functions described below. Fragments, including the N-terminus of the molecule, may be generated by genetic engineering of translation stop sites within the coding region.
Alternatively, treatment of the O1-180, O1-I84 or O1-236 with proteolytic enzymes, known as proteases, can produce a variety of N-terminal, C-terminal and intenlal fragments. Fragments of proteins are seen to include any peptide that contains 6 contiguous amino acids or more that are identical to 6 contiguous amino acids of sequences of SEQ.ID.N0.2, SEQ.ID.N0.4, SEQ.ID.N0.6, SEQ.ID.N0.9, SEQ.ID.N0.16, SEQ.ID.N0.29, SEQ.ID.N0.32, SEQ.ID.N0.34, SEQ.ID.N0.36, SEQ.ID.N0.39, and SEQ.ID.N0.42. Fragments that contain 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200 or more contiguous amino acids or more that are identical to a corresponding number of amino acids of any of the sequences of SEQ.ID.N0.2, SEQ.ID.N0.4, SEQ.ID.N0.6, SEQ.ID.NO.9, SEQ.ID.NO.I6, SEQ.ID.N0.29, SEQ.ID.N0.32, SEQ.ID.N0.34, SEQ.ID.N0.36, SEQ.ID.N0.39, and SEQ.ID.N0.42 are also contemplated. Fragments may be used to generate antibodies. Particularly useful fragments will be those that make up domains of O1-180, O1-184 or O1-236. Domains are defined as portions of the proteins having a discrete tertiary structure and that is maintained in the absence of the remainder of the protein. Such structures can be found by techniques known to those skilled in the art. The protein is partially digested with a protease such as subtilisin, trypsin, chymotrypsin or the like and then subjected to polyacrylamide gel electrophoresis to separate the protein fragments. The fragments can then be transferred to a PVDF membrane and subjected to micro sequencing to determine the amino acid sequence of the N-terminal of the fragments.
[0115] The term substantially pure as used herein refers to O1-180, O1-1~4 and O1-236 which are substantially free of other proteins, lipids, carbohydrates or other materials with which they are naturally associated. One skilled in the art can purify O1-180, O1-184 and Ol-236 using standard techniques for protein purification. The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the O1-180, O1-184 and O1-236 polypeptides can also be determined by amino-terminal amino acid sequence analysis. Ol-180, O1-184 and O1-236 polypeptides include functional fragments of the polypeptides, as long as their activities remain. Smaller peptides containing the biological activities of 01-180, Ol-184 and O1-236 may also be used in the present invention.
A. Variants [0116] Amino acid sequence variants of the O1-180, O1-236 and/or Ol-184 polypeptides can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity.
Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
[0117] The polypeptides of the invention include the disclosed sequences and conservative variations thereof. The term conservative variation as used herein denotes the replacement of an amino acid residue by another, biologically similar residue.
Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
[0118] The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.

[0119] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (I~yte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structiu~e of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
[0120] Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+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).
[0121] It is klloWn in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, z. e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within ~1 are particularly preferred, and those within X0.5 are even more particularly preferred.
[0122] 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. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U.S. Patent 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 (-I.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
[0123] It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ~2 is preferred, those that are within ~1 are particularly preferred, and those witlun X0.5 are even more particularly preferred.
[0124] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine;
glutamine and asparagine; and valine, leucine and isoleucine.
B. Domain Switching [0125] An interesting series of mutants can be created by substituting homologous regions of various proteins. This is known, in certain contexts, as "domain switching."
[0126] Domain switching involves the generation of chimeric molecules using different but, in this case, related polypeptides. By comparing various O1-180, O1-236 and/or O1-184 proteins or polypeptides, one can make predictions as to the functionally significant regions of these molecules. It is possible, then, to switch related domains of these molecules in an effort to determine the criticality of these regions to Ol-180, O1-236 and/or O1-184 function.
These molecules may have additional value in that these "chimeras" can be distinguished from natural molecules, while possibly providing the same function.
C. Fusion Proteins [0127] A specialized kind of insertional variant is the fusion protein. This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide. For example, a fusion protein of the present invention can includes the addition of a protein transduction domains, for example, but not limited to Antennepedia transduction domain (ANTP), HSV1 (VP22) and HIV-1(Tat). Fusion proteins containing protein transduction domains (PTDs) can traverse biological membranes efficiently, thus delivering the protein of interest (O1-180, O1-236 and/or O1-184 or variant thereof, such as an activator or inhibitor) into the cell. (Tremblay, 2001;
Forman et al., 2003).
[0128] Yet further, inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, other cellular targeting signals or transmembrane regions.
D. Synthetic Peptides [0129] The present invention also describes smaller Ol-180, OI-236 and/or Ol-184-related peptides for use in various embodiments of the present invention.
Because of their relatively small size, the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols.
See, for example, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); and Baxany and Merrifield (1979), each incorporated herein by reference. Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides. Alternatively, recombinant DNA
technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable fox expression.
E. Antigen Compositions [0130] The present invention also provides for the use of OI-180, O1-236 and/or O1-184 proteins or polypeptides as antigens for the immunization of animals relating to the production of antibodies. Antibodies, which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibodies, are provided.
Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler et al., Nature, 256:495, 1975).
The term antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab')2, which are capable of binding an epitopic determinant on O1-I80, OI-184 or O1-236.
[0131] It is envisioned that O1-I80, O1-236 and/or O1-184 proteins, polypeptides or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i. e., pharmaceutically acceptable.
Preferred agents are the carriers are keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).
1. Antibody Production [0132] In certain embodiments, the present invention provides antibodies that bind with high specificity to the Ol-180, Ol-236 and/or Ol-184 polypeptides provided herein. Thus, antibodies that bind to the polypeptide of O1-180 (SEQ.ID.N0.2, SEQ.ID.N0.16, SEQ.ID.N0.29, SEQ.ID.N0.32, SEQ.ID.N0.34, SEQ.ID.NO.36 and SEQ.ID.N0.39), Ol-(SEQ.ID.N0.4), O1-236 (SEQ.ID.N0.6, SEQ.ID.NO.9, and SEQ.ID.N0.42) are provided. In addition to antibodies generated against the full length proteins, antibodies may also be generated in response to smaller constructs comprising epitopic core regions, including wild-type and mutant epitopes.
[0133] Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred. The invention thus provides monoclonal antibodies of the human, marine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, marine monoclonal antibodies will often be preferred. However, humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.
[0134] A polyclonal antibody is prepared by immunizing an animal with an immunogenic O1-180, O1-236 and/or O1-184 composition in accordance with the present invention and collecting antisera from that immunized animal.
[0135] A wide range of animal species can be used for the production of antisera.
Typically the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
[0136] As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
Exemplary and preferred 36 , carriers are keyhole limpet hemocyanin (I~LH) and bovine serum albumin (BSA).
Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
[0137] As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.
[0138] The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.
[0139] A second, booster injection, may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.
[0140] For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots.
The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G
chromatography.
[0141] MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified O1-180, O1-236 and/or O1-184 protein, polypeptide, peptide or domain, be it a wild-type or mutant composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.
[0142] The animals are injected with antigen, generally as described above.
The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary.
The antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster injections with the same antigen would occur at approximately two-week intervals.
[0143] Following immunization, somatic cells with the potential fox producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
[0144] Often, a panel of animals will have been immunized and the spleen of an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 10' to 2 x 1 Og lymphocytes.
[0145] The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
[0146] Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X631Ag8, X63-Ag8.653, NSlll.Ag 4 l, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and 5194/SXXO Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

[0147] One preferred marine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse marine myeloma SP2/0 non-producer cell line.
[0148] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986).
[0149] Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10-6 to 1 ae 10-8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thyrnidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
[0150] The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B
cells.

[0151] This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
[0152] The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. First, a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as sexum or ascites fluid, can then be tapped to provide MAbs in high concentration. Second, the individual cell lines could be cultured i~
vitf°o, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
[0153] MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods, which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.
[0154] It is also contemplated that a molecular cloning approach may be used to generate monoclonals. For this, 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 using cells expressing the antigen and control cells. The advantages of this approach over conventional hybridoma techniques are that approximately I04 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which fiuther increases the chance of finding appropriate antibodies.
[0155] Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coli.
2. Antibody Conjugates [0156] The present invention further provides antibodies against O1-180, O1-andlor Ol-184, generally of the monoclonal type, that are linked to one or more other agents to form an antibody conjugate. Any antibody of sufficient selectivity, specificity and affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art.
[0157] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. "Detectable labels" are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and further quantified if desired. Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, as may be termed "immunotoxins" (described in U.S. Patents 5,686,072, 5,578,706, 4,792,447, 5,045,451, 4,664,911 and 5,767,072, each incorporated herein by reference).
[0158] Antibody conjugates are thus preferred for use as diagnostic agents.
Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use i~ vivo diagnostic protocols, generally known as "antibody-directed imaging." Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, e.g., U.S. Patents 5,021,236 and 4,472,509, both incorporated herein by reference). Certain attachment methods involve the use of a metal chelate complex .employing, for example, an organic chelating agent such a DTPA
attached to the antibody (U.S. Patent 4,472,509). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.

[0159] In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention Zl~astatine, l4carbon, slchromium, 36chlorine, s~cobalt, s8cobalt, 6~copper, ls2Eu, 6~gallium, 3hydrogen, 123iodine, lasiodine, 131iodine, lxtindium, s9iron, 3aphosphorus, lg6rhenium, ls8rhenium, ~sselenium, 3ssulphur, and 99"'teclmicium. l2sl is often being preferred for use in certain embodiments, and 99"'techniciumand lindium are also often preferred due to their low energy and suitability for long range detection.
[0160] Minor modifications of the recombinant O1-I80, O1-184 and O1-236 primary amino acid sequences may result in proteins which have substantially equivalent activity as compared to the respective O1-180, Ol-184 and O1-236 polypeptides described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the biological activity of OI-180, Ol-184 or O1-236 still exists. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule which would have broader utility. For example, one could remove amino or carboxy terminal amino acids which may not be required for biological activity of O1-180, O1-184 or O1-236.
[0161] For the purpose of this invention, the term derivative shall mean any molecules which are within the skill of the ordinary practitioner to make and use, which are made by modifying the subject compound, and which do not destroy the activity of the derivatized compound. Compounds which meet the foregoing criteria which diminish, but do not destroy, the activity of the derivatized compound are considered to be within the scope of the term derivative. Thus, according to the invention, a derivative of a compound comprising amino acids in a sequence corresponding to the sequence of Ol-180, Ol-184 or O1-236, need not comprise a sequence of amino acids that corresponds exactly to the sequence of O1-180, O1-184 or O1-236, so long as it retains a measurable amount of the activity of the O1-180, Ol-184 or O1-236.
[0162] Equally, the same considerations may be employed to create a protein, polypeptide or peptide with countervailing, e.g., antagonistic properties.
This is relevant to the present invention in which O1-I80, OI-184 or Ol-236 mutants or analogues may be generated.
For example, a Ol-I80, O1-184 or Ol-236 mutant may be generated and tested for Ol-I80, 01-184 or Ol-236 activity to identify those residues important for O1-180, O1-184 or O1-236 activity. Ol-180, Ol-184 or O1-236 mutants may also be synthesized to reflect a O1-180, Ol-184 or O1-236 mutant that occurs in the human population and that is linked to the development of cancer. Also, O1-180, Ol-184 or O1-236 mutants may be used as antagonists to inhibit or enhance fertility. Thus, Ol-180, O1-184 or O1-236 mutants may be used as potential contraceptive compositions and/or fertility enhancement compositions.
II. Nucleic Acids [0163] The term "O1-180 gene" "Ol-180 polynucleotide" or "Ol-180 nucleic acid" refers to any DNA sequence that is substantially identical to a DNA
sequence encoding an O1-180 gene product as defined above. Similar terms for O1-184 and/or O1-236 are within the scope of the present invention. The term also refers to RNA or antisense sequences compatible with such DNA sequences. An "O1-180, O1-184 or O1-236 gene or O1-180, O1-184 or O1-236 polynucleotide" may also comprise any combination of associated control sequences.
[0164] Thus, nucleic acid compositions encoding O1-180, O1-184 and/or Ol-236 are herein provided and are also available to a skilled artisan at accessible databases, including the National Center for Biotechnology Information's GenBank database and/or commercially available databases, such as from Celera Genomics, Inc. (Rockville, MD). Also included are splice variants that encode different forms of the protein, if applicable. The nucleic acid sequences may be naturally occurring or synthetic.
[0165] As used herein, the terms " O1-180, O1-184 and/or O1-236 nucleic acid sequence," "OI-180, O1-184 and/or O1-236 polynucleotide," and "O1-180, Ol-184 and/or 01-236 gene" refer to nucleic acids provided herein, homologs thereof, and sequences having substantial similarity and function, respectively. A skilled artisan recognizes that the sequences are within the scope of the present invention if they encode a product which regulates at least one of the following functions oocyte maturation and furthermoxe knows how to obtain such sequences, as is standard in the art.
[0166] Specific polynucleotides of the present invention include sequences encoding the O1-180 (SEQ.ID.NO.1, SEQ.ID.NO.11, SEQ.ID.N0.13, SEQ.ID.N0.12, SEQ.ID.N0.28 (accession # AY191415), SEQ.ID.NO.30 (accession # AY191416), SEQ.ff~.N0.31, SEQ.ID.N0.33, SEQ.ID.N0.35, SEQ.ID.NO.37, SEQ.ID.N0.38, SEQ.ID.N0.40 (accession number AY193889) and SEQ.ID.N0.41 (accession #
AY193890)), Ol-184 (SEQ.ID.N0.3) or O1-236 (SEQ.ID.NO.S, SEQ.ID.N0.7, SEQ.ID.N0.8, SEQ.ID.NO.10, SEQ.ID.N0.14, and SEQ.ID.N0.43) proteins and fragments and derivatives thereof. These polynucleotides include DNA, cDNA and RNA sequences which encode Ol-180, Ol-184 or O1-236. It is understood that all polynucleotides encoding all or a portion of Ol-180,.
O1-184 and/or 01-236 are also included herein, as long as they encode a polypeptide with the activity of O1-180 (SEQ.ID.NO.1, SEQ.ID.NO.11, SEQ.ID.N0.13, SEQ.ID.N0.12, SEQ.ID.N0.28, SEQ.ID.N0.30, SEQ.ID.N0.31, SEQ.ID.N0.33, SEQ.ID.N0.35, SEQ.ID.N0.37, SEQ.ID.N0.38, SEQ.ID.N0.40 and SEQ.ID.N0.41), Ol-184 (SEQ.ID.N0.3) or O1-236 (SEQ.ID.NO.S, SEQ.ID.N0.7, SEQ.ID.N0.8, SEQ.ID.NO.10, SEQ.ID.N0.14 and' SEQ.ID.N0.43). Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For example, polynucleotides of O1-180 (SEQ.ID.N0.1, SEQ.ID.NO.11, SEQ.ID.N0.13, SEQ.ID.N0.12, SEQ.ID.N0.28, SEQ.ID.N0.30, SEQ.ID.N0.31, SEQ.ID.N0.33, SEQ.ID.N0.35, SEQ.ID.N0.37, SEQ.ID.N0.38, SEQ.ID.N0.40 and SEQ.ID.N0.41), O1-184 (SEQ.ID.N0.3) or Ol-236 (SEQ.ID.N0.5, SEQ.ID.N0.7, SEQ.ID.N0.8, SEQ.ID.NO.10, SEQ.ID.N0.14, SEQ.ID.N0,43) may be subjected to site-directed mutagenesis. The polynucleotide sequences for O1-180, O1-184 and Ol-236 also includes antisense sequences. The polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequences of Ol-180, O1-184 and O1-236 polypeptides encoded by the nucleotide sequences are functionally unchanged.
[0167] The term "substantially identical" and/or "homologous", when used to define either a O1-180, O1-184 and/or O1-236 amino acid sequence or Ol-180, O1-184 and/or O1-236 polynucleotide sequence, means that a particular subject sequence, for example, a mutant sequence, varies from the sequence of natural O1-180, O1-184 and/or O1-236, respectively, by one or more substitutions, deletions, or additions, the net effect of which is to retain at least some biological activity of the O1-180, O1-184 and/or Ol-236 protein, respectively. Alternatively, DNA analog sequences are "substantially identical" and/or "homologous" to specific DNA sequences disclosed herein if: (a) the DNA analog sequence is derived fiom coding regions of the natural O1-180, O1-184 and/or O1-236 gene, respectively; or (b) the DNA analog sequence is capable of hybridization of DNA sequences of (a) under moderately stringent conditions and which encode biologically active O1-180, O1-184 and/or O1-236, respectively; or (c) DNA sequences which are degenerative as a result of the genetic code to the DNA analog sequences defined in (a) or (b). Substantially identical analog proteins will be greater than about 40%, about 45%, about 50%, about 55%, about 60%, about 65% about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range derivable therein similar to the corresponding sequence of the native protein.
Sequences having lesser degrees of similarity but comparable biological activity are considered to be equivalents. In determining polynucleotide sequences, all subject polynucleotide sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference polynucleotide sequence, regardless of differences in codon sequence.
A. Complentary Nucleic acids [0168] The present invention also encompasses a nucleic acid that is complementary to a Ol-180, O1-184 and/or O1-236 nucleic acid. In particular embodiments the invention encompasses a nucleic acid or a nucleic acid segment complementary to the sequence set forth in SEQ ID NO: O1-180 (SEQ.ID.NO.l, SEQ.ID.NO.11, SEQ.ID.NO.13, SEQ.ID.N0.12, SEQ.ID.NO.28, SEQ.ID.N0.30, SEQ.ID.N0.31, SEQ.ID.N0.33, SEQ.ID.N0.35, SEQ.ID.N0.37, SEQ.ID.NO.38, SEQ.ID.N0.40 and SEQ.ID.N0.41), O1-(SEQ.ID.N0.3) or O1-236 (SEQ.ID.NO.S, SEQ.ID.NO.7, SEQ.ID.NO.B, SEQ.ID.NO.10, SEQ.ID.NO.14, SEQ.ID.NO,43). A nucleic acid is "complement(s)" or is "complementary" to another nucleic acid when it is capable of base-pairing with another nucleic acid according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules. As used herein "another nucleic acid" may refer to a separate molecule or a spatial separated sequence of the same molecule.
[0169] As used herein, the term "complementary" or "complement(s)" also refers to a nucleic acid comprising a sequence of consecutive nucleobases or semiconsecutive nucleobases (e.g., one or more nucleobase moieties are not present in the molecule) capable of hybridizing to another nucleic acid strand or duplex even if less than all the nucleobases do not base pair with a counterpart nucleobase. In certain embodiments, a "complementary" nucleic acid comprises a sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78°l0, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range derivable therein, of the nucleobase sequence is capable of base-pairing with a single or double stranded nucleic acid molecule during hybridization. In certain embodiments, the term "complementary" refers to a nucleic acid that may hybridize to another nucleic acid strand or duplex in stringent conditions, as would be understood by one of ordinary skill in the art.
[0170] In certain embodiments, a "partly complementary" nucleic acid comprises a sequence that may hybridize in low stringency conditions to a single or double stranded nucleic acid, or contains a sequence in which less than about 70% of the nucleobase sequence is capable of base-pairing with a single or double stranded nucleic acid molecule during hybridization.
B. Hybridization of Nucleic Acids [0171] As used herein, "hybridization", "hybridizes" or "capable of hybridizing" is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term "hybridization", "hybridize(s)" or "capable of hybridizing" encompasses the terms "stringent condition(s)" or "high stringency" and the terms "low stringency" or "low stringency condition(s)" or "moderately stringent conditions".
[0172] As used herein "stringent condition(s)" or "high stringency" axe those conditions that allow hybridization between or within one or more nucleic acid strands) containing complementary sequence(s), but precludes hybridization of random sequences.
Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand.
Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA
transcript or a nucleic acid segment thereof, and the Like.
[0173] Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCI at temperatures of about 50°C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of fornamide, tetramethylammonium chloride or other solvents) in a hybridization mixture.
[0174] It is also understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls. Depending on the application envisioned it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of a nucleic acid towards a target sequence. In a non-limiting example, identification or isolation of a related target nucleic acid that does not hybridize to a nucleic acid under stringent conditions may be achieved by hybridization at low temperature and/or high ionic strength. For example, a medium or moderate stringency condition could be provided by about 0.1 to 0.25 M NaCI at temperatures of about 37°C to about 55°C. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. In another example, a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Of course, it is within the skill of one in the art to further modify the low or high stringency conditions to suite a particular application. For example, in other embodiments, hybridization may be achieved under conditions of, 50 mM Tris-HCl (pH 8.3), 75 mM KCI, 3 mM MgCl2, 1.0 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH ~.3), 50 mM KCI, 1.5 mM MgCl2, at temperatures ranging from approximately 40°C to about 72°C.
[0175] DNA sequences of the invention can be obtained by several methods. For example, the DNA can be isolated using hybridization or amplification techniques which are well known in the art. These include, but are not limited to: 1 ) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences, 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features, or 3) use of oligonucleotides related to these sequences and the technique of the polymerase chain reaction.
[0176] Preferably, the O1-180, O1-1 ~4 and O1-236 polynucleotides of the invention are derived from a mammalian organism, and most preferably from a mouse, rat, elephant, pig, cow or human. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequence must be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate.
This includes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of cDNA
clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA done by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Wallace et al., 1981).
[0177] The development of specific DNA sequences encoding O1-180, O1-184 and O1-236 can also be obtained by: 1) isolation of double-stranded DNA
sequences from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptides of interest; and 3) in vitro synthesis of a double- stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
[0178] Of the three above-noted methods for developing specific DNA sequences for use in recombinant procedures, the isolation of genomic DNA isolates is the least common.
This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of introns.
[0179] The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known. When the entire sequence of amino acid residues of the desired polypeptides is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA
sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries, which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the polypeptide are known, the production of labeled single or double-stranded DNA
or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay et al., 1983).
[0180] A cDNA expression library, such as lambda gtl l, can be screened indirectly for O1-180, O1-184 and/or O1-236 peptides having at least one epitope, using antibodies specific for O1-180, O1-184 and/or O1-236. Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of 01-180, O1-184 and/or Ol-236 cDNA.
III. Expression Vectors [0181] DNA sequences encoding O1-180, Ol-184 or O1-236 can be expressed i~
vity°o by DNA transfer into a suitable host cell. Host cells are cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell.
It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term host cell is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
[0182] In the present invention, the O1-180, Ol-184 and/or O1-236 polynucleotide sequences may be inserted into a recombinant expression vector. The term recombinant expression vectors refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the O1-180, O1-184 or Ol-236 genetic sequences.
Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al., 1987), the pMSXND
expression vector for expression in mammalian cells (Lee and Nathans, 1988) and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein 1, or polyhedrin promoters). Polynucleotide sequences encoding O1-180, Ol-184 or O1-236 can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
A. Selectable Markers [0183] In certain embodiments of the invention, the expression cassette and/or constructs of the present invention contain nucleic acid constructs whose expression is identified zh vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) are employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable markers are well known to one of skill in the art and include reporters such as EGFP, (3ga1 or chloramphenicol acetyltransferase (CAT).
B. Control Regions 1. Promoters [0184] The particular promoter employed to control the expression of a polynucleotide sequence of interest is not believed to be important, so long as it is capable of directing the expression of the polynucleotide in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the polynucleotide sequence coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter.
[0185] In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, 13-actin, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.
[0186] Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product. For example in the case where expression of a transgene, or transgenes when a multicistronic vector is utilized, is toxic to the cells in which the vector is produced in, it is desirable to prohibit or reduce expression of one or more of the transgenes. Examples of transgenes that are toxic to the producer cell line are pro-apoptotic and cytokine genes. Several inducible promoter systems are available for production of viral vectors where the transgene product is toxic.
[0187] The ecdysone system (Invitrogen, Carlsbad, CA) is one such system. This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility. The system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene high levels of mRNA transcripts are attained. In this system, both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone-responsive promoter which drives expression of the gene of interest is on another plasmid. Engineering of this type of system into the gene transfer vector of interest would therefore be useful. Cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would then allow for the production of the gene transfer vector without expression of a potentially toxic transgene. At the appropriate time, expression of the transgene could be activated with ecdysone or muristeron A.
[0188] Another inducible system that would be useful is the Tet-OffrM or Tet-OnTM
system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, 1992; Gossen et al., 1995). This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline. In the Tet-OnTM system, gene expression is turned on in the presence of doxycycline, whereas in the Tet-OffrM system, gene expression is turned on in the absence of doxycycline.
These systems are based on two regulatory elements derived from the tetracycline resistance operon of E. coli. The tetracycline operator sequence to which the tetracycline repressor binds, and the tetracycline repressor protein. The gene of interest is cloned into a plasmid behind a promoter that has tetracycline-responsive elements present in it. A second plasmid contains a regulatory element called the tetracycline-controlled transactivator, which is composed, in the Tet-OffrM system, of the VP16 domain from the herpes simplex virus and the wild-type tertracycline repressor. Thus in the absence of doxycycline, transcription is constitutively on. In the Tet-OnTM system, the tetracycline repressor is not wild type and in the presence of doxycycline activates transcription.
For gene therapy vector production, the Tet-OffrM system would be preferable so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constitutively on.
[0189] Viral promoters with varying strengths of activity can be utilized depending on the level of expression desired. In mammalian cells, the CMV immediate early promoter is often used to provide strong transcriptional activation. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired. When expression of a transgene in cells is desired, retroviral promoters such as the LTRs from MLV or MMTV are often used. Other viral promoters that are used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as from the ElA, E2A, or MLP region, AAV LTR, HSV-TK, and avian sarcoma virus.
[0190] Similarly tissue specific promoters are used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non-targeted tissues. For example, promoters such as an oocyte-specific promoter: Zp3 promoter (Lira et al., 1990), a spermatocyte-specific promoter: PGK2 promoter (Zhang et al., 1999);
and a spermatid-specific promoter: Protamine promoter (Peschon et al., 1987).
[0191] In certain indications, it is desirable to activate transcription at specific times after administration of the vector. This is done with such promoters as those that are hormone or cytokine regulatable. Cytokine and inflammatory protein responsive promoters that can be used include I~ and T I~ininogen (Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone et al., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBP
alpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson et al., 1990), IL-8, alpha-1 acid glycoprotein (Drowse and Baumann, 1988), alpha-1 antitypsin, lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et al., 1991), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothionein (heavy metal and glucocorticoid inducible), Stromelysin (inducible by phorbol ester, interleukin-1 and EGF), alpha-2 macroglobulin and alpha-1 antichymotrypsin.
[0192] It is envisioned that any of the above promoters alone or in combination with another can be useful according to the present invention depending on the action desired. In addition, this list of promoters should not be construed to be exhaustive or limiting, those of skill in the art will know of other promoters that are used in conjunction with the promoters and methods disclosed herein.
2. Enhancers [0193] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance;
this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA
synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
[0194] Any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) can be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

3. Polyadenylation Signals [0195] Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence is employed such as human or bovine growth hormone and polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
4. Integration sequences [0196] In instances wherein it is beneficial that the expression vector replicate in a cell, the vector may integrate into the genome of the cell by way of integration sequences, i.e., retrovirus long terminal repeat sequences (LTRs), the adeno-associated virus ITR sequences, which are present in the vector, or alternatively, the vector may itself comprise an origin of DNA
replication and other sequence which facilitate replication of the vector in the cell while the vector maintains an episomal form. For example, the expression vector may optionally comprise an Epstein-Barr virus (EBV) origin of DNA replication and sequences which encode the EBV
EBNA-1 protein in order that episomal replication of the vector is facilitated in a cell into which the vector is introduced. For example, DNA constructs having the EBV origin and the nuclear antigen EBNA-1 coding are capable of replication to high copy number in mammalian cells and are commercially available from, for example, Invitrogen (San Diego, CA).
[0197] It is important to note that in the present invention it is not necessary for the expression vector to be integrated into the genome of the cell for proper protein expression.
Rather, the expression vector may also be present in a desired cell in the form of an episomal molecule. For example, there are certain cell types in which it is not necessary that the expression vector replicate in order to express the desired protein. These cells are those which do not normally replicate and yet are fully capable of gene expression. An expression vector is introduced into non-dividing cells and express the protein encoded thereby in the absence of replication of the expression vector.
C. Methods of Gene Transfer [0198] In order to mediate the effect of the transgene expression in a cell, it will be necessary to transfer the expression constructs of the present invention into a cell. Such transfer may employ viral or non-viral methods of gene .transfer. This section provides a discussion of methods and compositions of gene transfer. Still further, one of skill in the art is aware that isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention, may be carried out by conventional means inducing preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
1. Non-viral Transfer [0199] Several non-viral methods for the transfer of expression construct into cells are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (copal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;
Fraley et al., 1979), cell sonication (Fechheimer et al., 198?), gene bombardment using high velocity microprojectiles (Yang et al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988).
[0200] In a specific embodiment of the present invention, the expression construct is complexed to a cationic polymer. Cationic polymers, which are water-soluble complexes, are well known in the art and have been utilized as a delivery system for DNA
plasmids. This strategy employs the use of a soluble system, which will convey the DNA into the cells via a receptor-mediated endocytosis (Wu & Wu 1988). One skilled in the art realizes that the complexing nucleic acids with a cationic polymer will help neutralize the negative charge of the nucleic acid allowing increased endocytic uptake.
[0201) Tn a particular embodiment of the invention, the expression construct is entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). The addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules (Radler et al., 1997). These DNA-lipid complexes are potential non-viral vectors for use in gene therapy.
SS

[0202] Liposome-mediated nucleic acid delivery and expression of foreign DNA
ih vitro has been very successful. Using the (3-lactamase gene, Wong et al., (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et al., (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection. Also included are various commercial approaches involving "lipofection" technology.
[0203] In certain embodiments of the invention, the liposome is complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome is complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the liposome is complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid i~ vitro and in vivo, then they are applicable for the present invention.
[0204] In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al., (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a therapeutic gene also is specifically delivered into a cell type such as prostate, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes. For example, the human prostate-specific antigen (Watt et al., 1986) is used as the receptor for mediated delivery of a nucleic acid in prostate tissue.
[0205] In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct is performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is applicable particularly for transfer in vitf°o, however, it is applied for in vivo use as well. Dubensky et al., (1984) successfully injected polyomavirus DNA in the form of CaP04 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of CaPO4 precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a CAM also is transferred in a similar manner i~c vivo and express CAM.
[0206] Another embodiment of the invention for transferring a naked DNA
expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
2. Viral Vector-Mediated Transfer [0207] In certain embodiments, transgene is incorporated into a viral particle to mediate gene transfer to a cell. Typically, the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus. The present methods are advantageously employed using a variety of viral vectors, as discussed below.
a. Adenovirus [0208] Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. The roughly 36 kB viral genome is bounded by 100-200 base pair (bp) inverted terminal repeats (ITR), in wluch are contained cis-acting elements necessary for viral DNA
replication and packaging. The early (E) and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA
replication.
[0209] The E1 region (ElA and ElB) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA
replication.
These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan, 1990). The products of the late genes (L1, L2, L3, L4 and LS), including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP (located at 16.8 map units) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5' tripartite leader (TL) sequence which makes them preferred mRNAs for translation.
[0210] In order for adenovirus to be optimized for gene therapy, it is necessary to maximize the carrying capacity so that large segments of DNA can be included.
It also is very desirable to reduce the toxicity and immunologic reaction associated with certain adenoviral products. The two goals are, to an extent, coterminous in that elimination of adenoviral genes serves both ends. By practice of the present invention, it is possible achieve both these goals while retaining the ability to manipulate the therapeutic constructs with relative ease.
[0211] The large displacement of DNA is possible because the cis elements required for viral DNA replication all are localized in the inverted terminal repeats (ITR) (100-200 bp) at either end of the linear viral genome. Plasmids containing ITR's can replicate in the presence of a non-defective adenovirus (Hay et al., 1984). Therefore, inclusion of these elements in an adenoviral vector should permit replication.
[0212] In addition, the packaging signal for viral encapsidation is localized between 194-385 by (0.5-1.1 map units) at the left end of the viral genome (Hearing et al., 1987).
This signal mimics the protein recognition site in bacteriophage ~, DNA where a specific sequence close to the left end, but outside the cohesive end sequence, mediates the binding to proteins that are required for insertion of the DNA into the head structure.
El substitution vectors of Ad have demonstrated that a 450 by (0-1.25 map units) fragment at the left end of the viral genome could direct packaging in 293 cells (Levrero et al., 1991).
[0213] Previously, it has been shown that certain regions of the adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line. There also have been reports of complementation of replication deficient adenoviral vectors by "helping"
vectors, e.g., wild-type virus or conditionally defective mutants.
[0214] Replication-deficient adenoviral vectors can be complemented, in traps, by helper virus. This observation alone does not permit isolation of the replication-deficient vectors, however, since the presence of helper virus, needed to provide replicative functions, would contaminate any preparation. Thus, an. additional element was needed that would add specificity to the replication and/or packaging of the replication-deficient vector. That element, as provided for in the present invention, derives from the packaging function of adenovirus.
[0215] It has been shov~ni that a packaging signal for adenovirus exists in the left end of the conventional adenovirus map (Tibbetts, 1977). Later studies showed that a mutant with a deletion in the ElA (194-358 bp) region of the genome grew poorly even in a cell line that complemented the early (ElA) function (Hearing and Shenk, 1983). When a compensating adenoviral DNA (0-353 bp) was recombined into the right end of the mutant, the virus was packaged normally. Further mutational analysis identified a short, repeated, position-dependent element in the left end of the Ad5 genome. One copy of the repeat was found to be sufficient for efficient packaging if present at either end of the genome, but not when moved towards the interior of the Ad5 DNA molecule (Hearing et al., 1987).
[0216] By using mutated versions of the packaging signal, it is possible to create helper viruses that are packaged with varying efficiencies. Typically, the mutations are point mutations or deletions. When helper viruses with low efficiency packaging are grown in helper cells, the virus is packaged, albeit at reduced rates compared to wild-type virus, thereby permitting propagation of the helper. When these helper viruses are grown in cells along with virus that contains wild-type packaging signals, however, the wild-type packaging signals are recognized preferentially over the mutated versions. Given a limiting amount of packaging factor, the virus containing the wild-type signals are packaged selectively when compared to the helpers. If the preference is great enough, stocks approaching homogeneity should be achieved.
b. Retrovirus [0217] The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes - gag, pol and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene, termed ~I', functions as a signal for packaging of the genome into virions.
Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome.
These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990).
[0218] In order to construct a retroviral vector, a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol and env genes but without the LTR and ~ components is constructed (Mann et al., 1983). When a recombinant plasmid containing a human cDNA, together with the retroviral LTR and ~I' sequences is introduced into this cell line (by calcium phosphate precipitation for example), the ~I' sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression of many types of retroviruses require the division of host cells (Paskind et al., 1975).
[0219] An approach designed to allow specific targeting of retrovirus vectors recently was developed based on the chemical modification of a retrovirus by the chemical addition of galactose residues to the viral envelope.
[0220] A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, the infection of a variety of human cells that bore those surface antigens was demonstrated with an ecotropic virus in vitro (Roux et. al., 1989).
c. Adeno-associated Virus [0221] AAV utilizes a linear, single-stranded DNA of about 4700 base pairs.
Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription.

[0222] The three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, pl9 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced. The splice site, derived from map units 42-46, is the same for each transcript.
The four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.
[0223] AAV is not associated with any pathologic state in humans.
Interestingly, for efficient replication, AAV requires "helping" functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus. The best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAV replication. Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block.
[0224] The terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV
genome (Samulski et al., 1987), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV. The ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i. e., stable and site-specific integration. The ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration.
[0225] AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vita°o, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and ifz vivo (Carter and Flotte, 1995 ; Chatterjee et al., 1995; Ferrari et al., 1996; Fisher et al., 1996; Flotte et al., 1993; Goodman et al., 1994; Kaplitt et al., 1994; 1996, Kessler et al., 1996;
Koeberl et al., 1997;
Mizukami et al., 1996).
[0226] AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1995;
Flotte et al., 1993).

Similarly, the prospects for treatment of muscular dystrophy by AAV-mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor IX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart, appear promising since AAV-mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al., 1996; Flotte et al., 1993; I~aplitt et al., 1994;
1996; I~oeberl et al., 1997;
McCown et al., 1996; Ping et al., 1996; Xiao et al., 1996).
d. Other Viral Vectors [0227] Other viral vectors are employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) canary pox virus, and herpes viruses are employed. These viruses offer several features for use in gene transfer into various mammalian cells.
[0228] Once the construct has been delivered into the cell, the nucleic acid encoding the transgene are positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the transgene is stably integrated into the genome of the cell. This integration is in the cognate location and orientation via homologous recombination (gene replacement) or it is integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid is stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.
How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
IV. Diagnostic Uses [0229] The term cell-degenerative disorder denotes the loss of any type of cell in the ovary, either directly or indirectly. For example, in the absence of GDF-9, there is a block in the growth of the granulosa cells leading to eventual degeneration (i. e., death) of the oocytes (bong et al., 1996). This death of the oocyte appears to lead to differentiation of the granulosa cells. In addition, in the absence of GDF-9, no normal thecal cell layer is formed around the follicles. Thus, in the absence of one oocyte-specific protein, GDF-9, there are defects in three different cell lineages, oocytes, granulosa cells, and thecal cells. In a similar way, death or differentiation of these various cell lineages could be affected by absence or misexpression of O1-180, Ol-184, or O1-236. Furthermore, absence or misexpression of O1-180, O1-184, or 01-236 could result in defects in the oocytelegg leading to the inability of the egg to be fertilized by spermatozoa. Alternatively, embxyos may not develop or halt development during the eaxly stage of embryogenesis or show defects in fertilization secondary to absence of these oocyte derived factors.
[0230] Therefore, 01-180, O1-184 or Ol-236 compositions may be employed as a diagnostic or prognostic indicator of infertility in general. Moxe specifically, point mutations, deletions, insertions or regulatory perturbations can be identified. The present invention contemplates further the diagnosis of infertility detecting changes in the levels of O1-180, O1-I84 or O1-236 expression.
[0231] One embodiment of the instant invention comprises a method for detecting variation in the expression of O1-180, Ol-184 or O1-236. This may comprise determining the level of O1-180, Ol-184 or O1-236 expressed, or determining specific alterations in the expressed product. In specific embodiments, alterations are detected in the expression of 01-180, O1-184 or O1-236.
[0232] The biological sample can be tissue or fluid. Various embodiments include cells from the testes and ovaries. Other embodiments include fluid samples such as vaginal fluid or seminal fluid.
[0233] Nucleic acids used are isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al., 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA (cDNA). In one embodiment, the RNA is whole cell RNA; in another, it is poly-A RNA. Normally, the nucleic acid is amplified.
[0234] Depending on the format, the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification. Next, the identified product is detected. In certain applications, the detection may be performed by visual means (e.g., ethidium bromide staining of a gel).
Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintography of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).

[0235] Following detection, one may compare the results seen in a given patient with a statistically significant reference group of normal patients and patients that have been diagnosed with infertility.
[0236] It is contemplated that other mutations in the 01-180, O1-184 or O1-236 polynucleotide sequences may be identified in accordance with the present invention by detecting a nucleotide change in particular nucleic acids (U.S. Patent 4,988,617, incorporated herein by reference). A variety of different assays are contemplated in this regard, including but not limited to, fluorescent in situ hybridization (FISH; U.S. Patent 5,633,365 and U.S. Patent 5,665,549, each incorporated herein by reference), direct DNA sequencing, PFGE
analysis, Southern or Northern blotting, single-stranded conformation analysis (SSCA), RNAse protection assay, allele-specific oligonucleotide (ASO) (e.g., U.S. Patent 5,639,611), dot blot analysis, denaturing gradient gel electrophoresis (e.g., U.S. Patent 5,190,856 incorporated herein by reference), RFLP (e.g., U.S. Patent 5,324,631 incorporated herein by reference) and PCRTM_ SSCP. Methods for detecting and quantitating gene sequences, such as mutated genes and oncogenes, in for example biological fluids are described in U.S. Patent 5,496,699, incorporated herein by reference.
[0237] Yet further, it is contemplated by that chip-based DNA technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996) can be used for diagnosis of infertility. Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al., (1994); Fodor et al., (1991).
[0238] Antibodies can be used in characterizing the Ol-180, O1-184 or 01-236 content through techniques such as ELISAs and Western blot analysis. This may provide a prenatal screen or in counseling for those individuals seeking to have children.
[0239] The steps of various other useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al., (1987). Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of radioimmunoassays (RIA) and immunobead capture assay.

Immunohistochemical detection using tissue sections also is particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the lilce also may be used in connection with the present invention.
[0240] The antibodies of the invention can be bound to many different carriers and used to detect the presence of an antigen comprising the polypeptide of the invention. Samples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain such, using routine experimentation.
[0241] There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds.
Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain such, using routine experimentation.
[0242] Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl, puridoxal, and fluorescein, which can react with specific anti-hapten antibodies.
[0243] In using the monoclonal antibodies of the invention for the ifs vivo detection of antigen, the detectably labeled antibody is given a dose which is diagnostically effective. The term diagnostically effective means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the antigen composing a polypeptide of the invention for which the monoclonal antibodies are specific.
The concentration of detectably labeled monoclonal antibody which is administered should be sufficient such that the binding to those cells having the polypeptide is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio. As a rule, the dosage of detestably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. Such dosages may vary, for example, depending on whether multiple injections are given, antigenic burden, and other factors known to those of skill in the art.
[0244] For ih vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting a given radioisotope. The radioisotope chosen must have a type of decay which is detectable for a given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that deleterious radiation with respect to the host is minimized. Ideally, a radioisotope used for i~r vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which may readily be detested by conventional gamma cameras.
[0245] For in vivo diagnosis, radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to immunoglobulins are the bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules. Typical examples of metallic ions which can be bound to the monoclonal antibodies of the invention are lllln, 9~Ru, 6~Ga, 68Ga, ~2As, $9Zr and 2oiTi.
[0246] The monoclonal antibodies of the invention can also be labeled with a paramagnetic isotope for purposes of i~ vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include ls~Gd, ssMn, lszDy, ssCr and s6Fe.
[0247] The term cell-proliferative disorder or hyperproliferative disorder denotes malignant as well as non-malignant cell populations which often appear to differ from the surrounding tissue both morphologically and genotypically. The Ol-180, O1-184 and O1-236 polynucleotides that are antisense molecules are useful in treating malignancies of the various organ systems, pauticularly, for example, the ovaries. Essentially, any disorder which is etiologically linked to altered expression of O1-180, O1-184 or O1-236 could be considered susceptible to treatment with a O1-180, O1-184 or Ol-236 suppressing reagent, respectively.
[0248] The invention provides a method for detecting a cell proliferative disorder of the ovary which comprises contacting an anti-O1-180, O1-184 or Ol-236 antibody with a cell suspected of having an O1-180, O1-184 or O1-236 associated disorder and detecting binding to the antibody. The antibody reactive with O1-180, Ol-184 or Ol-236 is labeled with a compound which allows detection of binding to Ol-180, O1-184 or O1-236, respectively.
For purposes of the invention, an antibody specific for an O1-180, O1-184 or O1-236 polypeptide may be used to detect the level of O1-180, O1-184 or O1-236, respectively, in biological fluids and tissues. Any specimen containing a detectable amount of antigen can be used. A preferred sample of this invention is tissue of ovarian origin, specifically tissue containing oocytes or ovarian follicular fluid. The level of Ol-180, O1-184 or O1-236 in the suspect cell can be compared with the level in a normal cell to determine whether the subject has an Ol-180, O1-184 or O1-236-associated cell proliferative disorder. Preferably the subject is human. The antibodies of the invention can be used in any subject in which, it is desirable to administer ivr vitro or in vivo immunodiagnosis or immunotherapy. The antibodies of the invention are suited for use, for example, in immuno assays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (ELISA) assay. Detection of the antigens using the antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
V. Therapeutic Uses [0249] Due to the expression of Ol-180, Ol-184 and O1-236 in the reproductive tract, there are a variety of applications using the polypeptides, polynucleotides and antibodies of the invention, related to contraception, fertility and pregnancy. O1-180, Ol-184 and O1-236 could play a role in regulation of the menstrual cycle and, therefore, could be useful in various contraceptive regimens.

[0250] It is also contemplated that O1-180, O1-184, or O1-236 polynucleotide sequences, polypeptide sequences, antibodies, fragments thereof or mutants thereof may be used to inhibit or enhance early embryogenesis by distrubing the maternal genome.
One of skill in the art is aware that disruptions of the maternal genome that cause phenotypes in embryonic development are termed maternal effect mutations. Two such examples have been characterized in mice using knockout technology. In each example, the gene product is normally accumulated in growing oocytes and persists in the early developing embryo and the phenotype affects offspring of knockout females, regardless of their genotype or gender. The first identified gene encodes MATER (maternal antigen that embryos require), which is necessary for development beyond the two-cell stage and has been implicated in establishing embryonic genome transcription patterns (Tong et al., 2000). The second identified gene encodes DNMTlo, an oocyte-specific DNA methyltransferase critical for maintaining imprinting patterns established in the embryonic genome and the viability of the developing mouse during the last third of gestation (Howell et al., 2001). Presumably many other oocyte-derived factors mediate the complexities of early embryogenesis, thus, it is contemplated that the O1-180 and O1-236 are maternal effect genes since they function in processes of early embryogenesis.
[0251] In further embodiments, it is contemplated that O1-236 may play a role in in chromatin remodeling during early embryoonic development. For example, studies have predicted the presence of a mammalian nuclear protein that is necessary for oocyte remodeling of sperm DNA, and is released into the ooplasm at germinal vesicle breakdown (Maeda et al., 1998). Yet further, it is known that oocytes can efficiently remodel not only sperm nuclei during fertilization, but also somatic cell nuclei. Thus, the inventors have contemplated the role of NPM2 in nuclear transfer cloning (Zuccotti et al., 2000). It envisioned that NPM2 (encoded by O1-236) is a critical factor in mammalian oocytes for chromatin remodeling during early embryonic development. Thus, supplementing enucleated oocytes with NPM2 may facilitate cloning by nuclear transfer technologies.
[0252] The monoclonal antibodies of the invention can be used ih vitro and in vivo to monitor the course of amelioration of an O1-180, Ol-184 or Ol-236-associated disease in a subject. Thus, for example, by measuring the increase or decrease in the number of cells expressing antigen comprising a polypeptide of the invention or changes in the concentration of such antigen present in various body fluids, it would be possible to determine whether a particular therapeutic regimen aimed at ameliorating the O1-180, O1-184 or O1-236-associated disease is effective. The term ameliorate denotes a lessening of the detrimental effect of the O1-180, O1-184 or O1-236-associated disease in the subject receiving therapy.
[0253] The present invention identifies nucleotide sequences that can be expressed in an altered manner as compared to expression in a normal cell, therefore, it is possible to design appropriate therapeutic or diagnostic techniques directed to this sequence. Thus, where a cell-proliferative disorder is associated with the expression of O1-180, O1-184 or O1-236, nucleic acid sequences that interfere with the expression of Ol-180, Ol-184 or O1-236, respectively, at the translational level can be used. Tlus approach utilizes, for example, antisense nucleic acids or ribozymes to block translation of a specific O1-180, O1-184 or O1-236 mRNA, either by masking that mRNA with an antisense nucleic acid or by cleaving it with a ribozyme.
[0254) Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target O1-180, O1-184 or O1-236-producing cell. The use of antisense methods to inhibit the ih vitf°o translation of genes is well knows in the art (Marcus-Sakura, 1988).
[0255) Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
[0256] There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, 1988) and "hammerhead"-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while "hammerhead"-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA
species and 18-based recognition sequences are preferable to shorter recognition sequences.
[0257] It is also contemplated in the present invention that double-stranded RNA is used as an interference molecule, e.g., RNA interference (RNAi). RNA
interference is used to "knock down" or inhibit a particular gene of interest by simply injecting, bathing or feeding to the organism of interest the double-stranded RNA molecule. This technique selectively "knock downs" gene function without requiring transfection or recombinant techniques (Diet, 2001;
Hammond, 2001; Stein P, et al., 2002; Svoboda P, et al., 2001; Svoboda P, et al., 2000). Thus, in certain embodiments, double-stranded O1-180, O1-184 or O1-236 RNA is synthesized or produced using standard molecular techniques described herein.
[0258] The present invention also provides gene therapy for the treatment of cell proliferative or degenerative disorders which are mediated by O1-180, O1-184 or O1-236 proteins. Such therapy would achieve its therapeutic effect by introduction of the respective 01-180, O1-184 or 01-236 cDNAs or O1-180, O1-184, or O1-236 antisense polynucleotide into cells having the proliferative or degenerative disorder. Delivery of O1-180, O1-184, or Ol-236 cDNAs or antisense O1-180, Ol-184 or Ol-236 polynucleotides can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
Especially preferred for therapeutic delivery of cDNAs or antisense sequences is the use of targeted liposomes.
[0259] Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus.
Preferably, the retroviral vector is a derivative of a marine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to:
Moloney marine leukemia virus (MoMuLV), Harvey marine sarcoma virus (HaMuSV), marine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting an O1-180, Ol-184 or O1-236 sequence of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein.
Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing an O1-180, O1-184 or O1-236 cDNA or O1-180, Ol-184, or 01-236 antisense polynucleotides.
[0260] Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packing mechanism to recognize an RNA
transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include, but are not limited to yr2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
[0261] Alternatively NIH 3'T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
[0262] Another targeted delivery system for O1-180, O1-184 or O1-236 cDNAs or O1-180, O1-184, or O1-236 antisense polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome.
Liposomes are artificial membrane vesicles which are useful as delivery vehicles ih vitro and ih vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 ~,m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al., 1981).
In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high exigency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Manning et al., 1988).
[0263] The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent canons.
[0264] Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
[0265] The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
[0266] The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand.
VI. Screening for Modulators [0267] As used herein, the term "candidate substance" refers to any molecule that may potentially modulate O1-180, O1-184 or Ol-236 activity, expression or function. Candidate compounds may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. The candidate substance can be a polynucleotide, a polypeptide, a small molecule, etc. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds.
[0268] One basic approach to search for a candidate substance is screening of compound libraries. One may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to "brute force"
the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries, is a rapid and efficient way to screen a large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds. It will be understood that an undesirable compound includes compounds that are typically toxic, but have been modified to reduce the toxicity or compounds that typically have little effect with minimal toxicity and are used in combination with another compound to produce the desired effect.
[0269] In specific embodiments, a small molecule library that is created by chemical genetics may be screened to identify a candidate substance that may be a modulator of the present invention (Schreiber et al., 2001a; Schreiber et al., 2001b).
Chemical genetics is the technology that uses small molecules to modulate the functions of proteins rapidly and conditionally. The basic approach requires identification of compounds that regulate pathways and bind to proteins with high specificity. Small molecules are prepared using diversity-oriented synthesis, and the split-pool strategy to allow spatial segregation on individual polymer beads.

Each bead contains compounds to generate a stock solution that can be used for many biological assays.
[0270] The goal of rational drug design is to produce structural analogs of biologically active target compounds. By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a molecule like Ol-180, O1-184 or O1-236 polypeptide, and then design a molecule for its ability to interact with O1-180, O1-184 or 01-236 polypeptide. This could be accomplished by X-ray crystallography, computer modeling or by a combination of both approaches. The same approach may be applied to identifying interacting molecules of O1-180, O1-184 or O1-236 polypeptides and/or polynucleotides.
[0271] It also is possible to use antibodies to ascertain the structure of a target compound or activator. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
[0272] It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.
[0273] Thus, the present invention contemplates the use of O1-180, O1-184 or 236 and active fragments, and nucleic acids coding therefore, in the screening of compounds for activity in either stimulating O1-180, O1-184 or O1-236, overcoming the lack of O1-180, 01-184 or O1-236 or blocking or inhibiting the effect of an O1-180, O1-184 or O1-236 molecule.
These assays may make use of a variety of different formats and may depend on the kind of "activity" for which the screen is being conducted.

[0274] In one embodiment, the invention is to be applied for the screening of compounds that bind to the Ol-180, O1-184 or O1-236 polypeptide or fragment thereof. The polypeptide or fragment may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the polypeptide or the compound may be labeled, thereby permitting the determination of binding.
[0275] In another embodiment, the assay may measure the inhibition of binding of O1-180, O1-184 or O1-236 to a natural or artificial substrate or binding partner. Competitive binding assays can be performed in which one of the agents (O1-180, O1-184 or Ol-236, binding partner or compound) is labeled. Usually, the polypeptide will be the labeled species.
One may measure the amount of free label versus bound label to determine binding or inhibition of binding.
[0276] Another tecluzique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with O1-180, O1-184 or O1-236 and washed. Bound polypeptide is detected by various methods.
[0277] Purified O1-180, O1-184 or O1-236 can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase.
Also, fusion proteins containing a reactive region (preferably a terminal region) may be used to link the Ol-180, Ol-184 or O1-236 active region to a solid phase.
[0278] Various cell lines containing wild-type or natural or engineered mutations in Ol-180, O1-184 or O1-236 gene can be used to study various functional attributes of O1-180, O1-184 or O1-236 and how a candidate compound affects these attributes.
Methods for engineering mutations are described elsewhere in this document, as are naturally-occurring mutations in O1-180, O1-184 or O1-236 that lead to, contribute to and/or otherwise cause infertility. In such assays, the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell. Depending on the assay, cell culture may be required. The cell may then be examined by virtue of a number of different physiologic assays. Alternatively, molecular analysis may be performed in which the function of O1-180, O1-184 or O1-236, or related pathways, may be explored.
[0279] In a specific embodiment, yeast two-hybrid analysis is performed by standard means in the art with the polypeptides of the present invention, i.e., O1-180, O1-184 or O1-236. Two hybrid screen is used to elucidate or characterize the function of a protein by identifying other proteins with which it interacts. The protein of unknown function, herein referred to as the "bait" is produced as a chimeric protein additionally containing the DNA
binding domain of GAL4. Plasmids containing nucleotide sequences which express this chimeric protein are transformed into yeast cells, which also contain a representative plasmid from a library containing the GAL4 activation domain fused to different nucleotide sequences encoding different potential target proteins. If the bait protein physically interacts with a target protein, the GAL4 activation domain and GAL4 DNA binding domain are tethered and are thereby able to act conjunctively to promote transcription of a reporter gene.
If no interaction occurs between the bait protein and the potential target protein in a particular cell, the GAL4 components remain separate and unable to promote reporter gene transcription on their own.
One skilled in the art is aware that different reporter genes can be utilized, including [3-galactosidase, HIS3, ADE2, or URA3. Furthermore, multiple reporter sequences, each under the control of a different inducible promoter, can be utilized within the same cell to indicate interaction of the GAL4 components (and thus a specific bait and target protein). A skilled artisan is aware that use of multiple reporter sequences decreases the chances of obtaining false positive candidates. Also, alternative DNA-binding domain/activation domain components may be used, such as LexA. One skilled in the art is aware that any activation domain may be paired with any DNA binding domain so long as they are able to generate transactivation of a reporter gene. Furthermore, a skilled artisan is aware that either of the two components may be of prokaryotic origin, as long as the other component is present and they jointly allow transactivation of the reporter gene, as with the LexA system.
[0280] Two hybrid experimental reagents and design are well known to those skilled in the art (see The Yeast Two-Hybrid System by P. L. Bartel and S.
Fields (eds.) (Oxford University Press, 1997), including the most updated improvements of the system (Fashena et al., 2000). A skilled artisan is aware of commercially available vectors, such as the MatchmakerTM
Systems from Clontech (Palo Alto, CA) or the HybriZAP~ 2.1 Two Hybrid System (Stratagene;

La Jolla, CA), or vectors available through the research community (Yang et al., 1995; James et al., 1996). In alternative embodiments, organisms other than yeast are used for two hybrid analysis, such as mammals (Mammalian Two Hybrid Assay I~it from Stratagene (La Jolla, CA)) or E. coli (Hu et al., 2000).
[0281] In an alternative embodiment, a two hybrid system is utilized wherein protein-protein interactions are detected in a cytoplasmic-based assay. In this embodiment, proteins are expressed in the cytoplasm, which allows posttranslational modifications to occur and permits transcriptional activators and inhibitors to be used as bait in the screen. An example of such a system is the CytoTrap~ Two-Hybrid System from Stratagene (La Jolla, CA), in which a target protein becomes anchored to a cell membrane of a yeast which contains a temperature sensitive mutation in the cdc25 gene, the yeast homologue for hSos (a guanyl nucleotide exchange factor). Upon binding of a bait protein to the target, hSos is localized to the membrane, which allows activation of RAS by promoting GDP/GTP exchange. RAS
then activates a signaling cascade which allows growth at 37°G of a mutant yeast cdc25H. Vectors (such as pMyr and pSos) and other experimental details are available for this system to a skilled artisan through Stratagene (La Jolla, CA). (See also, for example, U.S. Patent No. 5,776,689, herein incorporated by reference).
[0282] Thus, in accordance with an embodiment of the present invention, there is a method of screening for a peptide which interacts with O1-180, O1-184 or O1-236 comprising introducing into a cell a first nucleic acid comprising a DNA segment encoding a test peptide, wherein the test peptide is fused to a DNA binding domain, and a second nucleic acid comprising a DNA segment encoding at least part of O1-180, Ol-184 or O1-236, respectively, wherein at least part of Ol-180, O1-184 or O1-236 respectively, is fused to a DNA activation domain. Subsequently, there is an assay for interaction between the test peptide and the Ol-180, Ol-184 or O1-236 polypeptide or fragment thereof by assaying for interaction between the DNA
binding domain and the DNA activation domain. For example, the assay for interaction between the DNA binding and activation domains may be activation of expression of (3-galactosidase.
[0283] An alternative method is screening of lambda.gtll, lambda.LZAP
(Stratagene) or equivalent cDNA expression libraries with recombinant O1-180, Ol-184 or 01-236. Recombinant O1-180, O1-184 or O1-236 or fragments thereof are fused to small peptide tags such as FLAG, HSV or GST. The peptide tags can possess convenient phosphorylation sites for a kinase such as heart muscle creatine kinase or they can be biotinylated. Recombinant O1-180, O1-184 or Ol-236 can be phosphorylated with 32[P] or used unlabeled and detected with streptavidin or antibodies against the tags lambdagtllcDNA expression libraries are made from cells of interest and are incubated with the recombinant O1-180, O1-184 or O1-236, washed and cDNA clones which interact with O1-180, 01-184 or O1-236 isolated.
Such methods are routinely used by skilled artisans. See, e.g., Sambrook (supra).
[0284] Another method is the screening of a mammalian expression library in which the cDNAs are cloned into a vector between a mammalian promoter and polyadenylation site and transiently transfected in cells. Forty-eight hours later the binding protein is detected by incubation of fixed and washed cells with a labeled O1-180, O1-184 or Ol-236.
In this manner, pools of cDNAs containing the cDNA encoding the binding protein of interest can be selected and the cDNA of interest can be isolated by further subdivision of each pool followed by cycles of transient transfection, binding and autoradiography. Alternatively, the cDNA of interest can be isolated by transfecting the entire cDNA library into mammalian cells and panning the cells on a dish containing the O1-180, O1-184 or O1-236 bound to the plate. Cells which attach after washing are lysed and the plasmid DNA isolated, amplified in bacteria, and the cycle of transfection and panning repeated until a single cDNA clone is obtained. See Seed et al., 1987 and Aruffo et al., 1987 which are herein incorporated by reference. If the binding protein is secreted, its cDNA can be obtained by a similar pooling strategy once a binding or neutralizing assay has been established for assaying supernatants from transiently transfected cells. General methods for screening supernatants are disclosed in Wong et al., (1985).
[0285] Another alternative method is the isolation of proteins interacting with the O1-180, Ol-184 or O1-236 directly from cells. Fusion proteins of Ol-180, O1-184 or Ol-236 with GST or small peptide tags are made and immobilized on beads.
Biosynthetically labeled or unlabeled protein extracts from the cells of interest are prepared, incubated with the beads and washed with buffer. Proteins interacting with the Ol-180, O1-184 or Ol-236 are eluted specifically from the beads and analyzed by SDS-PAGE. Binding partner primary amino acid sequence data are obtained by microsequencing. Optionally, the cells can be treated with agents that induce a functional response such as tyrosine phosphorylation of cellular proteins. An example of such an agent would be a growth factor or cytokine such as interleukin-2.

[0286] Another alternative method is immunoaffmity purification. Recombinant O1-180, O1-184 or O1-236 is incubated with labeled or wlabeled cell extracts and immunoprecipitated with anti- O1-180, O1-184 or O1-236 antibodies. The immunoprecipitate is recovered with protein A-Sepharose and analyzed by SDS-PAGE. Unlabelled proteins are labeled by biotinylation and detected on SDS gels with streptavidin. Binding partner proteins are analyzed by microsequencing. Further, standard biochemical purification steps known to those skilled in the ant may be used prior to microsequencing.
[0287] Yet another alternative method is screening of peptide libraries for binding partners. Recombinant tagged or labeled O1-180, O1-184 or O1-236 is used to select peptides from a peptide or phosphopeptide library which interact with the O1-180, O1-184 or O1-236.
Sequencing of the peptides leads to identification of consensus peptide sequences which might be found in interacting proteins.
[0288] The present invention also encompasses the use of various animal models.
Thus, any identity seen between human and other animal Ol-180, O1-184 or O1-236 provides an excellent opportunity to examine the function of O1-180, O1-184 or O1-236 in a whole animal system where it is normally expressed. By developing or isolating mutant cells lines that fail to express normal Ol-180, O1-184 or Ol-236, one can generate models in mice that enable one to study the mechanism of O1-180, O1-184 or O1-236 and its role in oogenesis and embryonic development.
[0289] Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route that could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Specifically contemplated are systemic intravenous injection, regional administration via blood or lymph supply and intratumoral injection.
[0290] Determining the effectiveness of a compound i~ vivo may involve a variety of different criteria. Such criteria include, but are not limited to, increased fertility, decreased fertility or contraception.

[0291] In one embodiment of the invention, transgenic animals are produced which contain a functional transgene encoding a functional Ol-180, Ol-184 or O1-236 polypeptide or variants thereof. Transgenic animals expressing O1-180, O1-184 or O1-236 transgenes, recombinant cell lines derived from such animals and transgenic embryos may be useful in methods for screening for and identifying agents that induce or repress function of O1-180, 01-184 or O1-236. Transgenic animals of the present invention also can be used as models for studying disease states.
[0292] In one embodiment of the invention, an Ol-180, O1-184 or Ol-236 transgene is introduced into a non-human host to produce a transgenic animal expressing an 01-180, O1-184 or O1-236. The transgenic animal is produced by the integration of the transgene into the genome in a manner that permits the expression of the transgene.
Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S.
Patent 4,873,191; which is incorporated herein by reference), Brinster et al., 1985;
which is incorporated herein by reference in its entirety) and in "Manipulating the Mouse Embryo; A
Laboratory Manual" 2nd edition (eds., Hogan, Beddington, Costantimi and Long, Cold Spring Harbor Laboratory Press, 1994; which is incorporated herein by reference in its entirety).
Expression of the transgene may be regulatable by incorporating sequences such as cytokine or hormone response elements. This is done with such promoters as those that are hormone or cytokine regulatable. Cytokine and inflammatory protein responsive promoters that can be used include K and T Kininogen (I~ageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone et al., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson et al., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988), alpha-1 antitypsin, lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et al., 1991), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothionein (heavy metal and glucocorticoid inducible), Stromelysin (inducible by phorbol ester, interleukin-1 and EGF), alpha-2 macroglobulin and alpha-1 antichymotrypsin.
[0293] It may be desirable to replace the endogenous O1-180, Ol-184 or O1-236 by homologous recombination between the transgene and the endogenous gene; or the endogenous gene may be eliminated by deletion as in the preparation of "knock-out" animals.

Typcially, targeting vectors that contain a portion of the gene of interest and a selection marker are generated and transfected into embryonic stem (ES) cells. These targeting vectors are electroporated into the hprt-negative ES cell line a.nd selected in HAT and FIAU. ES cells with the correct mutation are injected into blastocysts to generate chimeras and eventually heterozygotes and homozygotes for the mutant O1-180, O1-184 and O1-236 genes.
Thus, the absence of O1-180, O1-184 or O1-236 in "knock-out" mice permits the study of the effects that loss of O1-180, O1-184 or O1-236 protein has on a cell in vivo.
[0294] As noted above, transgenic animals and cell lines derived from such animals may find use in certain testing experiments. In this regard, transgenic animals and cell lines capable of expressing wild-type or mutant Ol-180, O1-184 or O1-236 may be exposed to test substances. These test substances can be screened for the ability to enhance wild-type O1-180, O1-184 or Ol-236 expression and or function or impair the expression or function of mutant O1-180, O1-184 or Ol-236.
VII. Formulations and Routes for Administration to Patients [0295] Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions - expression vectors, virus stocks, proteins, antibodies and drugs -in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
[0296] One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
[0297] The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route.
This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradennal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
Such compositions would nornlally be administered as pharmaceutically acceptable compositions, described supra.
[0298] The active compounds also may be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0299] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0300] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0301] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
Supplementary active ingredients can also be incorporated into the compositions.
[0302] For oral administration the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient also may be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
[0303] The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0304] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCI
solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FICA Office of Biologics standards.
VIII. Examples [0305] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1 Creation of a cDNA subtractive hybridization library [0306] Ovaries from Gdf~ knockout mice are histologically very different from wild-type ovaries due to the early block in folliculogenesis. In particular, one layer primary follicles are relatively enriched in Gdf~ knockout ovaries and abnormal follicular nests are formed after oocyte loss. The inventors took advantage of these differences in ovary composition and related them to alterations in gene expression patterns to clone novel ovary-expressed transcripts which are upregulated in the Gdf~ knockout ovaries.
[0307] Ovaries from either Gdf9 knockout mice (C57BL/6/129SvEv hybrid) or wild-type mice were collected and polyA+ mRNA was made from each pool. Using a modified version of the CLONTECH PCR-Select Subtraction kit, the inventors generated a pBluescript SK+plasmid-based cDNA library which was expected to be enriched for sequences upregulated in the Gdf~ knockout ovaries.
[0308] Ligations into the NotI site of pBluescript SK+ were performed with a low molar ratio of EagI-digested cDNA fragment inserts to vector to prevent multiple inserts into the vector. Transformations were performed, and > 1000 independent bacterial clones were picked and stored in glycerol at -80°C. The remainder of the ligation mix was stored at -80°C for future transformations.
Example 2 Initial sequence analysis of pOvaryl (p01) Library inserts [0309] The inventors performed sequence analysis of 331 inserts from the pOl subtractive hybridization of cDNA library. An Applied Biosystems 373 DNA
Sequencer was used to sequence these clones. BLAST searches were performed using the National Center for Biotechnology Information databases. Novel sequences were analyzed for open reading frames and compared to previously identified novel sequences using DNASTAR analysis programs. A
summary of the data is presented in Table 1. As shown, the majority of the clones were known genes or matched mouse or human ESTs. 9.4% of the clones failed to match any known sequence in the database.
Table 1. Summary of database searches of p01 cDNA clones O1 cDNA Matches Number identified Percents a Known Genes 180 54.4%

Mouse /Human EST 120 36.2%

RARE ESTs (1 EST match)(8) (2.4%) ESTs from 2-cell library(3) (0.9%) No match 31 9.4%

Total 331 100%

Example 3 Northern blot analysis [0310] Northern blot analysis was performed using standard techniques well known and used in the art. Briefly, total RNA from tissues was obtained by the method (Leedo Medical Laboratories, Inc.). RNA was isolated from the following tissues:
ovaries, brain, lung, heart, stomach, spleen, liver, small intestine, kidney, testes, uterus, colon, prostate, placenta, pancreas, and muscle. Agarose gel electrophoresis of RNA, transfer to nylon membranes, and subsequent hybridization were performed by standard methods (Sambrook et al., 1989).
Example 4 In situ hybridization ~0311J In situ hybridization of ovaries was performed as described previously (Albrecht et al., 1997; Elvin et al., 1999) using partial Npml, Npm2, or Npm3, Zarl, or O1-184 cDNA fragments. The "sense"probe revealed no hybridization (data not shown).
Briefly, the cDNA fragments in pBluescript SK+or T-vector (Promega, Madison,WI) served as templates for generating sense and antisense strands with [35S ]-dUTP using the Riboprobe combination system (Promega). Sections were exposed to photographic emulsion for 4-7 days at 4 0 C. After the slides were developed and fixed, they were counterstained with hematoxylin.
Example 5 Oocyte Collection and Embryo Culture [0312] To collect non-SN (surrounded nucleolus) configuration oocytes, ovaries of day old mice were digested with collagenase as described (Eppig, 1978).
[0313] For GV-stage oocytes, adult females were injected intraperitoneally with 5 IU PMSG (pregnant mare serum gonadotropin) and oocytes were recovered 46 hours later by large follicle puncture.
[0314] For metaphase II oocytes or ih vivo fertilized embryos, females were treated with 5 IU PMSG followed by 5 IU hCG (human chorionic gonadotropin) to induce superovulation as described (Hogan et al., 1994). For fertilization, females were mated overnight with stud males and vaginal plugs checked the following morning. Mature metaphase II oocytes and 1-cell embryos were recovered from oviducts 18-24 hours post-hCG treatment in M2 media (Sigma, St.Louis, MO) with hyaluronidase. Collections of embryos 45, 55, and 72 hours post-hCG were accomplished by flushing oviducts with M2 media. For 24 hour culture experiments, eggs and embryos were kept under 5%C02 in M16 media (Sigma) until the transition to blastocyst stage when they were transferred to M15. For one experiment, colcemid was added to the media to arrest cells in mitosis (Sigma #D1925;160 ng/mL).
[0315] For in vita°o fertilization, sexually mature mice were injected with 5 IU of PMSG, cumulus-enclosed oocyte complexes were isolated 46 hours later, and cultured for 17 hours in minimal essential medium with 5% serum. Mature MII-stage eggs were mixed with capacitated sperm from wild-type (C57BL/6J x SJL/J) F1 mice as described (Eppig, 1999). The development of zygotes and two-cell-stage embryos were assessed at 6 and 24 h after fertilization, respectively.
Example 6 Expression analysis and cDNA screening of ovarian-expressed genes [0316] Northern blot analysis was performed on all cDNAs which failed to match sequences in any database. Additionally, sequences matching ESTs derived predominantly from mouse 2-cell embryo cDNA libraries (e.g., Za~l, O1-184, and Npm2) were analyzed. The rationale for analyzing this last group of ESTs was that mRNAs expressed at high levels in oocytes may persist until the 2-cell stage and may play a role in early embryonic development including fertilization of the egg or fusion of the male and female pronuclei.
[0317] The results of the initial screen of novel ovarian genes is presented in Table 2. Northern blot analysis of 23 clones demonstrated that 8 of these clones were upregulated in the Gdf9 knockout ovary indicating that the subtractive hybridization protocol used was adequate. Northern blot analysis using total RNA isolated from either adult C57BL/6/129SvEv hybrid mice (the ovarian RNA) or Swiss WEBSTER mice (all other tissues) also demonstrated that four of these clones including 2 clones which matched ESTs sequenced from 2-cell libraries were only expressed in the ovary (Figure 1). The O1-236 fragment probe (749 bp) detected a transcript of approximately 1.0 kb (Figure 1). Several clones have so far been analyzed for their ovarian localization by irr situ hybridization analysis (Figure 2). Clones O1-180 (herein after referred to as Zarl), Ol-184, and O1-236 (herein after referred to as Npm~) were oocyte-specific and expressed in oocytes of primary (one-layer) preantral follicles through ovulation (Figure 2).

Table 2. Analysis of ovarian cDNAs with no known function PO1 Adult Upregulated Database Further studies cDNA mRNA in match (in sitrc hybridization;
ExpressionGdf9 knockout chromosomal mapping) ovary 24 Multiple No - No 27 Multi le Yes - Oocyte-specific by in situ 37 Multiple Yes - No 70 Multi le No - No 91 1 EST (2-cell) 97 Multi le No ? No 101 Multi le Nol - No 114 Multiple No - No 110 Multi le Yes - No 126 Multiple Yes - No 180 Ovary- Yes - Oocyte-specific by iu (Zarl specific situ ) 184 Ovary- Yes >1 EST (All Oocyte-specific by ih s ecific 2- situ cell) 186 Ovary- Yes - Granulosa cell-specific specific by in situ 223 Multiple No - No 224 Multiple No - No 236 Ovary- Yes 6 EST (2 Oocyte-specific by iu (Npm2) specific c-cell situ and others) 255 Multiple No "zinc-forger"
domains 279 Multiple No - No 317 Multi le No - No 330 Multiple No - No 331 Multiple No - No 332 Multi le No - No 334 Multiple No - No 371 Multiple No - No [0318] The O1-236 gene product was oocyte-specific (Figure 3). O1-236 was not expressed in oocytes of primordial (type 2) or small type 3a follicles (Pedersen et al., 1968), but was first detected in oocytes of intermediate-size type 3a follicles and all type 3b follicles (i.e., follicles with >20 granulosa cells surrounding the oocyte in largest cross-section). Expression of the O1-236 mRNA persisted through the antral follicle stage. Interestingly, the oocyte-specific expression pattern of the O1-236 gene product paralleled the expression of other oocyte-specific genes which the inventors have studied including Gdfp (McGrath et al., 1995) and bone morphogenetic protein (Dube et al., 1998).
Example 7 Cloning of mouse Npm2 [0319] Wild-type ovary and Gdf~ knockout ZAP Express ovary cDNA libraries were synthesized and were screened to isolate full-length cDNAs for the above-mentioned three clones. Each full-length cDNA was again subjected to database searches and analyzed for an open reading frame, initiation ATG, and protein homology. The full-length cDNAs approximate the mRNA sizes determined from Northern blot analysis. Database searches using the predicted amino acid sequence permitted the identification of important domains (e.g., signal peptide sequences, transmembrane domains, zinc fingers, etc.) which were useful to define the possible function and cellular localization of the novel protein.
[0320] The O1-236 partial cDNA fragment identified in Example 1 was used to screen Matzuk laboratory ZAP Express (Stratagene) ovarian cDNA libraries generated from either wild-type or Gdf~ deficient ovaries (Dube et al., 1998). In brief, approximately 300,000 clones of either wild-type or Gdfp knockout mouse ovary cDNA libraries were hybridized to [alpha-32P] dCTP random-primed probes in Church's solution at 63°C.
Filters were washed with O.1X Church's solution and exposed overnight at -80°C.
[0321] Upon primary screening of the mouse ovarian cDNA libraries, the Ol-236 cDNA fragment detected 22 positive phage clones out of 300,000 screened. Two of these clones (236-1 and 236-3), which approximated the mRNA size and which were derived from the two independent libraries, were analyzed further by restriction endonuclease digestion and DNA
sequence analysis. These independent clones formed a 984 by overlapping contig (excluding the polyA sequences) and encoded a 207 amino acid open reading frame. Including the polyA tail, this sequence approximated the 1.0 kb mRNA seen by Northern blot analysis, which suggested that nearly all of the 5' UTR sequence had been isolated. When the nucleotide sequence was subjected to public database search, no significant matches were derived.
However, a database search with the 207 amino acid open reading frame demonstrated high homology with several nucleoplasmin homologs from several species. Interestingly, O1-236 showed highest homology with Xenopus laevis nucleoplasmin. At the amino acid level, O1-236 was 48%
identical to Xenopus laevis nucleoplasmin (Figure 4). Based on this homology and the expression patterns of both gene products in oocytes, the inventors termed the gene Npm~ since it was the mammalian ortholog of Xenopus laevis nucleoplasmin [called Xnpna2 in (MacArthur et al., 1997)]. Thus, herein after O1-236 is referred to as Npm2.
Example 8 Cloning of human NPM2 [0322] Using the Nprn2 cDNA sequence to search the EST database, two human cDNA clones containing sequences homologous to the mouse Npm2 were found.
Sequence analysis of these two ESTs was performed. The two independent clones formed a 923 by overlapping contig which encoded a 214 amino acid open reading frame. At the amino acid level, human NPM2 was 48% and 67% identical to Xnpm2 and mouse NPM2, respectably (Figure 4).
[0323] Still further, Figure 4 shows that the 207 amino acid of the mouse NPM2 shares 39.5% identity with Xenopus NPM2. Subsequently, human and rat NPM2 proteins were 61.4% and 81.6% identical with mouse NPM2.
[0324] When the frog and mammalian NPM2 sequences were compared, several interesting features were realized. Nucleoplasmin had a bipartite nuclear localization signal consisting of KR-(X)10- KKKK (Dingwall et al., 1987). Deletion of either of these basic amino acid clusters in nucleoplasmin prevented translocation to the nucleus (Robbins et al., 1991).
When the mouse and human NPM2 sequences were analyzed, this bipartite sequence was 100%
conserved between the two proteins (Figure 4). Thus, mammalian NPM2 was predicated to translocate to the nucleus where it would primarily function.
[0325] Also, conserved between NPM2 and nucleoplasmin was a long stretch of negatively charged residues. Amino acids 125-144 of NPM2 and amino acids 128-146 of nucleoplasmin are mostly glutamic acid and aspartic acid residues, with 19 out of the 20 residues for NPM2 and 16 out of the 19 residues for nucleoplasmin either Asp or Glu.
This region of Xenopus laevis nucleoplasmin has been implicated to bind the positively charged protamines and histones. Thus, a similar function for this acidic region of NPM2 was predicted.
[0326] The last obvious feature of the NPM2 and nucleoplasmin sequences was the high number of serine and threonine residues. The NPM2 sequence contained 19 serine and 17 threonines (i.e., 17.2% of the residues) and nucleoplasmin had 12 serine and 11 threonine residues (i.e., 11.5°/~ of the residues). Several putative phosphorylation sequences that were conserved between the two proteins are shown in Figure 4. Phosphorylation of nucleoplasmin is believed to increase its translocation to the nucleus and also its activity (Sealy et al., 1986, Cotten et al., 1986, Vancurova et al., 1995, Leno et al., 1996). Similarly, phosphorylation may also alter NPM2 activity. It is envisioned that phosphorylation may act to regulate when NPM2 acts, making it inactive until the critical time (i. e., histone addition to male and female pronuclei or during transcriptional arrest).
[0327] A specific putative phosphorylation site is, for example, casein kinase II.
Casein kinase II specifically interacts with nucleoplasmin and phosphorylates it, and an inhibitor of casein kinase II blocks nuclear transport of Xenopus laevis nucleoplasmin (Vancurova et al., 1995). Interestingly, two of the predicted casein kinase II phosphorylation sites are conserved between frog nucleoplasmin 2 (Ser125 and Ser177), mouse NPM2 (Thr123 and Ser184), and human NPM2 (Thr127 and Ser191). Although other phosphorylation sites are likely important, a casein kinase II-NPM2 interaction i~ vivo may be predicted in mammals.
[0328] Since both mouse and human NPM2 and Xenopus laevis nucleoplasm are oocyte (and egg)-specific at the mRNA level and share highest identity, it was concluded that mammalian NPM2 and frog nucleoplasmin were orthologs.
Example 9 Structure of the Npm2 gene [0329] One of the full length Npm2 cDNAs (clone 236-1) was used to screen a mouse 129/SvEv genomic library (Stratagene) to identify the mouse Npf~a~ gene.
500,000 phage were screened and 12 positive were identified. Two of these overlapping phage clones, 236-13 and 236-14 (~37 kb of total genomic sequence), were used to determine the structure of the mouse Npm2 gene. The mouse Npm2 was encoded by 9 exons and spanned ~6.6 kb (SEQ ID
NO: 7). Two moderate size introns (introns 4 and 5) contributed the majority of the gene size.
The initiation ATG codon resided in exon 2 and the termination codon in exon 9. The splice donor and acceptor sites (SEQ ID NO: 7) matched well with the consensus sequences found in rodents, and all of the intron-exon boundaries conformed to the "GT-AG" rule (Senapathy et al., 1990). A consensus polyadenylation signal sequence was found upstream of the polyA tracts which were present in the two isolated cDNAs (SEQ ID NO: 5).

Example 10 Chromosomal mapping of the mouse Npm2 gene [0330] Chromosomal mapping of genes in the mouse identifies candidate genes associated with spontaneous or induced mouse mutations. To further aid in the functional analysis of the isolated novel ovary-specific cDNAs, these mouse genes were mapped using the Research Genetics Radiation Hybrid Panel. Table 3 shows the genes that were mapped using this technique. Also, identification of the syntenic region on the human chromosome may identify one or more of these novel ovarian genes as candidate genes for known human diseases which map to these regions.
Table 3. Analysis of partial or full-length cDNAs PO1 cDNA ORF Database Homolog Ol-180 361 as No O 1-184 426 No Ol-236 207 Yes; Xenopus laevis nucleoplasmin homolog (~1 % similar) [0331] To map the mouse Npm2 gene, the inventors used the Research Genetics radiation hybrid panel, The Jackson Laboratory Backcross DNA Panel Mapping Resource, and The Jackson Laboratory Mouse Radiation Hybrid Database. Forward (SEQ.ID.N0.17:
GCAAAGAAGCCAGTGACCAAGAAATGA) and reverse (SEQ.ID.N0.18: CCTGATCATG
CAAATTTTATTGTGGCC) primers within the last exon were used to PCR amplify a 229 by fragment from mouse, but not hamster. Using these primers, the mouse Npm~ gene was mapped to the middle of chromosome 14 (Figure 5). Npm2 showed linkage to Dl4Mit32 with a LOD of 11.2 and also had a LOD of 7.8 to D l4Mit203. This region was syntenic with human chromosome 8p21.
Example 11 Ovarian-specific expression of mouse Npm2 ~0332J Ih situ hybridization was performed as described previously (Albrecht et al., 1997; Elvin et al., 1999) and Example 4.
[0333] Briefly, ovaries were dissected from C57B16/129SvEv mice and fixed overnight in 4% paraformaldehyde in PBS before processing, embedded in paraffin and sectioned at 5 um. The fragment Npm2 was used as the template for generating sense and antisense strands with [a,32P]-dUTP using the Riboprobe T7/SP6 combination system (Promega).
Hybridization was carried out at 50-55°C with 5x106 cpm for each riboprobe per slide for 16 hours in 50% deionized formamide/0.3 M NaCI/20 mM Tris-HCl (pH 8.0)/5 mM
EDTA/10 mM
NaP04 (pH8.0)/10% dextran sulphate/lxDenhardts/0.5 mg/ml yeast RNA. High stringency washes were carried out in 2xSSC/50% formamide and O.1X SSC at 65°C.
Dehydrated sections were dipped in NTB-2 emulsion (Eastman Kodak, Rochester, NY) and exposed for 4-7 days at 40°C. After the slides were developed and fixed, they were stained with hematoxylin and mounted for photography.
[0334] The Npyrr2 gene product was oocyte-specific (Figures 6A and 6B). The probe demonstrated specific expression in all growing oocytes. Oocyte-specific expression was first seen in the early one layer primary follicle (type 3a), with higher expression in the one layer type 3b follicle and all subsequent stages including antral (an) follicles.
The "sense" probe did not detect a signal for this oocyte-specific gene.
Example 12 Subcellular localization of NPM2 [0335] The subcellular localization of NPM2 protein was determined by immunohistostaining of mouse ovaries with anti-NPM2 antibodies.
[0336] The cDNA encoding the full-length mouse NPM2 protein was amplified by PCR to introduce a BamHl site before the start codon and a XhoI site before the stop codon.
This PCR fragment was cloned into pET-23b(+)(Novagen) to produce a His-tagged protein and sequenced to confirm the absence of mutations. The recombinant NPM2 protein was purified as described in the pET System Manual (Novagen). Two goats were immunized with the purified His-tagged NPM2 to produce specific and high affinity antibodies.
[0337] Ovaries were fixed in 4% paraformaldehyde in PBS for 2 h, processed, embedded in paraffin, and sectioned at 5 um thickness. Goat anti-NPM2 polyclonal antiserum was diluted 1:2000 in Common Antibody Dilute (BioGenex). The pre-immune goat serum from the same goat was used as a control. All sections were blocked for 10 min in Universal Blocking Reagent (BioGenex), and incubated with the primary antibody for 1 h at room temperature.
NPM2 detection was accomplished using anti-goat biotinylated secondary antibody, streptavidin-conjugated alkaline phosphatase label and New Fuschin substrate (BioGenex Laboratories, Inc., San Racoon, CA).
[0338] One to eight-cell embryos and blastocysts were fixed in 4%
paraformaldehyde in PBS for 2 h in 96-well round bottom plate, washed with 0.85% saline, and embedded in a few drops of 1.5% agaxose. The agarose-containing embryos were dehydrated, embedded in paraffin, and analyzed as described above.
[0339] Consistent with the expression pattern of Npm2 mRNA, NPM2 protein was expressed in oocytes from type 3 to antral follicle stages. In randomly cycling mice, the anti-NPM2 antibody strongly and specifically stained the nucleus (Figure 6C). The oocyte nucleus is also called the germinal vesicle (GV). The preovulatory surge of luteinizing hormone (LH) accelerates the maturation of GV oocytes and promotes GV breakdown (GVB). When mice were injected with PMSG and hCG to induce superovulation, the NPM2 protein redistributed in the oocytes of antral follicles after germinal vesicle breakdown. In preovulatory GVB oocytes, the NPM2 was evenly distributed in the cytoplasm of the oocyte (Figure 6D). Since xNPM2 has been implied to play a role in sperm DNA decondensation and pronuclei formation after fertilization, this redistribution suggested that the cytoplasmic NPM2 was now properly positioned to interact with the sperm nucleus at the time of fertilization. To examine the NPM2 expression after fertilization, early embryos were fixed, sectioned and stained with anti-NPM2 antibodies. In zygotes, NPM2 began to translocate back to the nucleus. Figure 6E shows an intermediate stage in which one pronucleus was formed but other was not yet complete and some NPM2 was still present in the cytoplasm. At a later point (Figure 6F), all of the NPM2 was present in the pronuclei. In two-cell (Figure 6G) and eight-cell (Figure 6H) embryos, the antibody continued to detect the NPM2 protein exclusively in the nucleus. NPM2 continued to be detected at significantly reduced levels in blastocysts (embryonic day 3.5), but in embryonic day 6.5 embryos, NPM2 expression was undetectable.
Example 13 Generation of Npm2 knockout mice [0340] To study the role of NPM2 in mammalian oocyte development and early embryo development, the inventors disrupted the mouse Npm2 locus using ES cell technology.
[0341] The targeting vector was constructed to delete exon 2 which contains the translation initiation codon and also exon 3 and the exon 4 splice junction (Figure 7A). Outside sectioned at 5 um. The fragment Np of exon 2, only one other ATG was present in the remaining sequence (exon 6), and this ATG
was positioned downstream of the acidic domain and between the bipartite nuclear localization consensus sequence. The deletion targeting vector contains from left to right, 2.2 kb of 5' Npm2 homology, a PGI~-hprt expression cassette, 4.6 lcb of 3'Nprn2 homology and an MC1-tk (thymidine kinase) expression cassette. The linearized Nprn~ targeting vector was electroporated into AB2.1 ES cells. ES cell clones were selected in M15 medium containing HAT
(hypoxanthine, aminopterine and thymidine and FIAU [1-(2'-deoxy-2'-fluoro-B-D-arabinofuranosyl)-5'-iodouracil]. Culturing of ES cells and collection and injection of blastocysts (Matzuk et al., 1992).
[0342] For genomic Southern blot analysis, BgIII-digested DNA was transferred to GeneScreen Plus nylon membrane and probed with an external 190 by PCR
synthesized fragment corresponding to exon 9 sequence (3' probe). An internal 200 by PCR
synthesized fragment (49 by exon 1 plus 150 by 5' upstream sequence) was also used to distinguish the wild-type and Npm2 null (Npm2t'mzuk~ herein called Npm2-~ ) alleles when DNA was digested with BamHl. Genotype analysis of 230 F2 offspring from these intercrosses (Figure 7B; Table 4) was consistent with a normal Mendelian ratio of 1:2:1, and a similar number of male and female homozygotes (Npm2-~ ) were produced. Therefore, Npm2 homozygous mutant male and female mice were viable and appeared to have normal sexual differentiation demonstrating that Npm2 was not required prior to birth.
Table 4. Heterozygous mating -/- +/- Wild type Total Male 27 71 19 117 Female 27 53 33 113 Total 54 124 52 230 [0343] To confirm that the mice genotyped as Npna2 homozygotes lacked Npm2, a cDNA probe that hybridized to exon 2 of the wild-type Nprrr2 gene was used for Southern blot analysis. As shown (Figure 7C), this probe failed to detect any signal in DNA
derived from homozygous (Npm2-~ ) mice in which exon 2 had been deleted. Furthermore, Nprn2 immunohistochemical analysis was performed on Nprn2 homozygotes and controls.
Whereas the expression of NPM2 protein was noted in the ovaries from the heterozygous controls (Figure 8A
and 8C), no protein was detected in oocytes in the homozygote ovaries (Figure 8B and 8D).
[0344] This confirmed that the Npna2tmizuk mutation was a null allele and that Nprn2 homozygotes were completely laclcing NPM2 protein.
Example 14 Loss of NPM2 results in female infertility and subfertility [0345] To study the function of NPM2 in reproductive function, adult homozygous hybrid (C57B1/6/129SvEv) male or female mice were intercrossed with control hybrid mice (C57B1/6/129SvEv) mice. Consistent with the female-specific expression of Npm2 mRNA and protein, Npm2-~ male mice were fertile and had no gross or histological defects in the testes.
Similarly, intercrosses of 9 female and male Npm2+~' males over 6 months resulted in a normal number of litters (n=54; 1.00 ~ 0.06 litters/month), with 8.98 ~ 0.31 offspring/litter. In contrast, only 11 of 14 Npm2-~- females became pregnant over this period, yielding 40 litters (0.48 ~ 0.11 litters/month) with 2.65 ~ 0.24 offspring/litter. Thus, deficiency of NPM2 leads to subfertility and infertility in females, but not males.
Example 15 Early cleavage defect in Npm~null fertilized eggs [0346] To determine the causes of the fertility defects in the Npm2 ~ female mice, ovaries were first examined morphologically and histologically. There was no significant difference between Npm2-~ and control ovaries at the gross or histological levels (Figure 8E and 8F). Normal folliculogenesis including the formation of corpora lutea were observed in the Npm2-~ ovaries suggesting that ovulation occurred in these mice.
[0347] To confirm that ovulation was occurring and to further study the cause of the infertility and subfertility of the Npm2-~ mice, pharmacological superovulation of wild-type, heterozygous, and homozygous mice was performed and the eggs were collected from the oviducts and cultured in vitro as described in Example 5.
~0348J In vitro maturation and fertilization of Npm2 null eggs were apparently normal, but there was reduced cleavage to the 2-cell stage (Table 5). In vivo fertilized Npm2 null eggs were recovered 24 hours after hCG treatment. However, mostly asynchronously fragmenting and dying embryos were found 45-55 hours post-hCG (Figure 10A, Figures 9A-9D), and few Npm2 null embryos were cultured to the blastocyst stage (Figure l OB, Figures 9G-9H). Thus, the defect in Npm2 null mice appeared to result in a reduced viability of embryos.
Table 5: Early developmental potential of eggs matured and fertilized in vitro Genotype Number Oocytes Eggs Eggs 2-cell stage of recovered matured fertilizedembryos (%)*

females Wild type4 93 69 48 46 (96%) Npm2-~- 4 75 62 49 9 (18%)**

*Percentage of eggs fertilized proceeding to 2-cell stage embryos.
* * P<0.0005 (x2 test) Example 16 DNA Damage [0349] Next, TUNEL (TdT-mediated dUTP nick end labeling) assays were performed to determine DNA damage. TUNEL assays rely on a terminal deoxynucleotidyl transferase (TdT) to label free ends of DNA with fluorescent dUTP conjugates.
[0350] Briefly, oocytes and early embryos were collected from oviducts, fixed, permeabilized, and incubated with TdT and labeled nucleotides. These were then washed and imaged by deconvolution microscopy. For BrDU incorporation assays, fully-grown oocytes were ivy vitro matured and fertilized as described above. Approximately 8 h after fertilization, zygotes that had formed pronuclei were transferred to medium supplemented with 50 ~M
BrDU for overnight culture (Ferreira et al., 1997). Incorporation was assessed by immunofluorescence using a mouse monoclonal antibody against BrDU (Roche, #1170376).
[0351] Nuclei of these embryos were TUNEL positive, although there was no evidence that DNA damage caused embryo loss, and 1-cell embryos collected from null females 20 hrs post-hCG exhibited TUNEL staining only within polar bodies (Figures 11A-11D). All developmental defects occurred when eggs were fertilized with wild-type spermatozoa, indicating that maternal NPM2 was crucial in early embryogenesis.
Example 17 Transcription-Requiring Complex (TRC) Quantification [0352] TRC proteins were extracted as described by Conover et al., 1991.
Briefly, two-cell embryos estimated to be in early S-phase were collected from oviducts and cultured for two hours in M16 media supplemented with amino acids, including 35S-methionine. The addition of 1 ~g/mL actinomycin D (Sigma #A1410) served as a negative control.
Insoluble proteins remained in the zona after extraction with 2%Triton X-100, 0.3 M ICI, and 50 mM Tris-HCl pH
7.4. These proteins were electrophoresed, and the gel was then fixed in isopropanol and glacial acetic acid, soaked in Amplify (Amersham Pharmacia Biotech), and exposed to X-GMAT film or phosphorimaged oveniight.
[0353] Two-cell embryos lacking NPM2 synthesized the Transcription-Requiring Complex (TRC) of proteins, which indicated some zygotic gene transcription and translation (Latham et al., 1992), albeit at reduced levels (30%) as compared to wild-type 2-cell embryos (Figure 12). It has been suggested that NPM2 may function in the translational activation of specific maternal maternal RNAs in early embryos as has been proposed for xNPM2 (Meric et al., 1997). Because a few Nprn2 null embryos developed to birth, potential compensatory mechanisms exist. Transcriptional activation of paternal Npm2 was not involved as Npm2-~-males sire pups when mated with Npm~-~- females.
Example 18 Analysis of WT and mutant oocytes and embryos [0354] Immunofluorescence of formaldehyde-fixed unfertilized eggs and early embryos was undertaken as described previously (Yan et al., 2002) to analyze WT and mutant oocytes and embryos.
[0355] Briefly, oocytes were collected and fixed in 2-4% formaldehyde or 70%
ethanol, blocked in PBS with 10% fetal calf serum, permeabilized with Triton X-100, and treated with primary and secondary antibodies. After washing, DNA was counterstained with DAPI or To-pro-3 and images were taken using confocal or deconvolution microscopy. The following primary antibodies were used: goat NPM2 antisera (1:500); rabbit anti- acetyl-Histone H3 (Upstate Biotechnology 06-599;1:200); goat anti-fibrillarin (Santa Cruz Biotechnology sc -11335;1:100); mouse monoclonal anti-tubulin antibody (Sigma T-6793; 1:300);
goat anti-lamin B (Santa Cruz sc-6217;1:300); mouse anti-hypoacetylated histone H3 (Upstate Biotechnology 06-755;1:500); rabbit anti-histone H3 phosphorylated at SerlO (Upstate Biotechnology 06-570;1:500); and mouse monoclonal anti-histone H1 (Santa Cruz sc-8030; 1/200).
The following secondary antibodies were used: AlexaFluor594 rabbit anti-goat (Molecular probes A-11080;

1:500); AlexaFluor568 goat anti-rabbit (Molecular Probes A-11011; 1:500); and AlexaFluor488 goat anti-mouse (Molecular Probes A-11001; 1:500).
[0356] Oocytes from PMSG-treated wild-type females exhibited an organization of heterochromatin surrounding the prominent nucleolus, termed the SN (surrounded nucleolus) configuration (Figure 13C). The SN configuration was characteristic of advanced oocyte development, as SN oocytes were larger and were found in gonadotropin-dependent follicles.
The condensation of chromatin correlated with transcriptional silencing, competence to reswne meiosis, the appearance of M-phase characteristics, and post-fertilization embryo developmental potential (Bouniol-Baly et al., 1999; Mattson et al., 1990; Wickramasinghe et al., 1991; Zuccotti et al., 1998). In contrast to wild-type oocytes, the DNA in Npm2 null oocytes was amorphous and diffused with no condensation around the nucleolus (Figure 13D). The loss of nucleolar clearing was also illustrated by immunofluorescence to detect acetylated histone H3 in these oocytes (Figures 13E-13F), as well as the less mature non-SN oocytes isolated from 10 day old untreated mice (Figures 13A-13B). Immunofluorescence to localize the nucleolar protein fibrillarin demonstrated dispersed nucleolar-like bodies in Npm2 null oocytes compared to the single organized nucleolus observed in controls (Figures 13G-13H). These anomalies were observed in hundreds of oocytes examined from more than 30 Npm2'~' females.
Thus, NPM2 was essential for organization of oocyte nuclear and nucleolar domains and the compaction of oocyte chromatin during the final stages of oocyte development.
[0357] Meiosis progresses essentially normally in the absence of NPM2, with no obvious defects in metaphase II arrest, spindle formation, chromosomal segregation, or extrusion of polar bodies (Figures 13I-13J). Sperm DNA decondensation occurs normally without NPM2;
fertilization was followed by the formation of both maternal and paternal pronuclei surrounded by nuclear envelopes (Figures 13K-13L), and there was no persistent protamine 2B detectable in male pronuclei (data not shown). DNA replication in the first S phase also proceeded without NPM2 (Figures 13M-13N). However, as in Npm2'~' oocytes, normal nucleoli were absent in Npm2 null 1-cell embryos, and immunofluorescence to detect acetylated histone H3 in zygotes showed no nucleolar clearing compared to controls (Figures 130-13P).
Hypoacetylated histone H3, which was normally associated with compact chromatin rimming the pronuclei nucleoli, was undetectable (Figures 13Q-13R). Treatment with colcemid to inhibit spindle formation arrested both wild-type (n=34) and Npm2 null (n=28) 1-cell embryos in metaphase with condensed chromosomes staining for phosphorylated histone H3; this indicated that the first mitosis initiated in essentially all cases (Figures 13S-13T). Without colcemid treatment, spindle forms in wild-type zygotes 13-15 hours after pronuclear formation (Figure 13U), and all zygotes complete mitosis by 19 hours. In contrast, N~anz2 null zygotes were observed with metaphase spindle from 13-19 hours following pronuclear formation (Figure 13V) and immediately preceding fragmentation, suggesting abnormal exit from the first mitosis. A few multi-cellular embryos lacking NPM2 were recovered, and their nuclei contain somatic linker histone H1 (Figures 13W-13X); however, their nuclei remained relatively amorphous until the blastocyst stage (Figures 13Y-13Z). Thus, mammalian NPM2 was crucial for histone deacetylation and heterochromatin formation surrounding nucleoli in oocytes and early zygotes.
Example 19 RNAse Protection Assay [0358] An RNAse protection assay was performed to quantify 18S and 28S rRNAs in wild-type (WT) and Npm~ null GV stage oocytes, metaphase II oocytes, and 1-cell embryos.
3aP-labeled antisense probes for 18S and 28S rRNAs were prepared using the Ambion MAXIscript kit templates (Austin, TX). Total RNA from 30 oocytes or embryos was prepared for probe hybridization using the Ambion Direct Protect Lysate lcit and then incubated with probe, treated with nuclease cocktail, and electrophoresed as recommended by the manufacturer and as described in (Tong et al., 1995). Protected fragments were detected by autoradiography and quantified by phosphorimaging (Johnston et al., 1990).
[0359] As shown in Figure 14, there are no major differences apparent in ribosomal RNA content in the absence of NPM2.
Example 20 Total Protein Synthesis [0360] Absolute rates of protein synthesis were quantified as described by Schultz et al., 1978. Briefly, GV-stage oocytes, metaphase II oocytes, and 1-cell embryos were collected and incubated for 2 hours in M16 media supplemented with amino acids, including 250 ~Ci of ass-methionine/mL, and either 0.3 mg/mL or 3.0 mg/mL of non-radioactive methionine. After the incubation, twenty oocytes or embryos were removed from each group, extensively washed in fresh M16 media, and lysed by freezing and thawing. Total protein was precipitated by the addition of 20 ~,L of lug/uL BSA and 20 ~.L of 10% trichloroacetic acid (TCA).
Pellets were washed with 5% TCA, dissolved in 1M NaOH for 1 hour at 37°C, acidified with HCI, and assayed by scintillation counting.
[0361] As shown in Figure 15, WT and Npm~ null oocytes and embryos displayed comparable counts or levels of protein synthesis.
Example 21 In situ hybridization to detect Npml and Npm3 [0362] Despite the low homology in the primary amino acid sequences of NPM2 and other "ubiquitous" nucleoplasmins (Chan et al., 1989; MacArthur et al.
1997: Schmidt-Zachmann et al., 1988), these more widely expressed nucleoplasmins are karyophilic, negatively charged proteins that may share functional redundancy with NPM2 in oocytes and developing embryos.
~0363J In situ hybridization to measure Npml and Nmp3 mRNA in mouse oocytes was performed as described in Example 4. Npml mRNA was detected with an 35 S-UTP labeled antisense riboprobe corresponding to nucleotides 131-551 of NM 008722. Figures shows that Npml mIRNA was highly expressed in oocytes of small follicles (A-B), secondary follicles (C-D) and large antral follicles (E-F) (arrows). Sections are shown in brightfield (A, C, and E) and darkfield (B, D, and F) to demonstrate the histology and highlight the hybridization signal, respectively. Npm3 mRNA was studied using a probe corresponding to 41-657 of NM 008723. Figure 16G-Figure 16H show that Npm3 mRNA was detected in all stages of oocytes in the adult ovary, although at levels more comparable to the expression observed in the surrounding somatic cells (G-H). Thus, in situ hybridization revealed that both nucleophosmin 1 (Npml ; B23) and nucleoplasmin 3 (Npm3) mRNAs were expressed in mouse oocytes.
Example 22 Cloning of Zarl [0364] Partial cDNAs from the library of Example 1 were subcloned and sequenced and all sequences were grouped into contigs and analyzed by BLAST
searches.
Novel sequences were analyzed further by Northern blot analysis. A partial 325 nucleotide cDNA designated ovary 1-clone 180 [O1-180, herein after referred to as zygote arrest 1 (Za~l)]
identified a 1.4kb transcript only in the ovary (Figure 17A).

[0365] Next, a ZAP-express mouse ovary cDNA library was screened to isolate the full-length Zarl cDNA. Excluding the polyA tail, the full-length Zanl cDNA was about l.4kb, and encoded an open reading frame from nucleotides 28 to 1110. The Zarl cDNA
was homologous to several ESTs in the database, including ESTs in a mouse sixteen-cell embryo cDNA library (AU044294) and a mouse unfertilized egg cDNA library (AU023153).
The polypeptide predicted from the Zarl cDNA ORF consisted of 361 amino acids (Figure 11), with a molecular mass of 40 kDa. Searching the public protein database failed to identify any known protein homologues. A bipartite nuclear localization signal was found at positions 333 to 350 (SEQ.ID.N0.19: Lys-Arg-Pro-His-Arg-Gln-Asp-Leu-Cys-Gly-Arg-Cys-Lys-Asp-Lys-Arg-Leu-Ser), which strongly suggested that Zap°I migrates to the oocyte or embryo nucleus.
[0366] To clone the mouse Zarl gene, both mouse 129/S6SvEv genomic ~, Fix II
phage .and 129X1/SvJ BAC libraries were screened with the full-length Zarl cDNA or PCR
primers (SEQ.ID.N0.20 5'-GTAGAAAAGGGGACTGTAGTCACT-3', and SEQ ID NO: 21 5'-TGCATCTCCCACACAAGTCTTGCC-3) and the recovered clones were characterized by Southern blot analysis and sequencing. The mouse Za~l gene (SEQ ID NO:11) spanned 4.Okb, and exon 1 encoded the majority of the protein. Both the Zarl gene and a related pseudogene (Za~l psl) contained four exons. The related Zarl psl (SEQ ID NO:12) gene contained a 13-nt gap in exon 1 (Figure 19), which was predicted to result in a frameshift and early protein termination in exon 2. Whereas RT-PCR with Zay~l-specific primers confirmed that it was ovary-specific, the related gene-specific primers failed to detect a transcript in all tissues examined. This established the related gene as a pseudogene (Za~l psl ).
Example 23 Chromosomal mapping of the tar-1 [0367] The whole genome-radiation hybrid panel T31 (McCarthy et al., 1997) were purchased from Research Genetics (Huntsville, AL) and used according to the manufacturer's instruction. The panel was constructed by fusing irradiated mouse embryo primary cells (129aa) with hamster cells. Because the sequence of the hamster homologues for Zap-1 is unknown, the inventors designed the reverse primers from the 3'-untranslated region of the marine sequence to minimize the risk of coamplification of the hamster homologues (Makalowski and Boguski, 1998). Zar-1 gene specific primers were (SEQ.ID.N0.20) 5'-CTAGAAAAGGGGACTGTAGTCACT-3' and (SEQ.ID.NO.21) 5'-TGCATCTCCCACA
CAAGTCTTGCC-3'; Zar-1 ps-1 gene specific primers were (SEQ.ID.NO.22) 5'-CTAGAAAAGGGGACTATAGGCACC-3' and (SEQ.ID.N0.23) 5'-TGCATCTCTCA
CACAAGTGTTGCT-3'. Specificity of the two sets of primers was tested with A23 hamster DNA and 129 mouse DNA. The PCR reactions were performed in 15,1 final volume, containing 1 ~,l of each panel DNA, 1.25u of Taq platinum DNA polymerase (Gibco, Rockville, MD), companion reagents (0.25mM dNTPs, l.SmM MgCl2, lxPCR buffer), and 0.4~M of each primer. An initial denaturation step of 4 min at 94°G was followed by amplification for 30 cycles (40s at 94°C, 30s at 60°C, and 30s at 72°C) and final elongation at 72°C for 7min.
[0368] The data for each gene were submitted for analysis at the Jackson Laboratory Mouse Radiation Hybrid Mapper Server. Both genes were placed in the same region on mouse chromosome 5. The Zar-1 locus was at 40cM, between two markers DSBuc48 and Txk, while the Zap-1 psl gene lies at 4lcM, between Tec and DSMit356, just distal to the coding locus (Figure 20).
Example 24 Isolation of human ZARl [0369] To identify the human ortholog of the mouse Za~l gene, a full-length mouse ovary cDNA was used for BLAST searches and to screen a human genomic library.
A human genomic sequence was identified from both the non-redundant database and a human genomic library. The entire human gene spanned 4.1 kb and also contained four exons;
its four exons shared 50%, 86%, 84%, and 78% nucleotide homology with mouse Za~l exons 1 to 4, respectively. The ZARI gene was located on human chromosome 4p12, which is syntenic to the Zarl locus on mouse chromosome 5. No pseudogene was found in the human genome.
Example 25 Expression of human ZARI
[0370] RT-PCR analysis of human ZARI was performed using standard techniques well known and used in the art. The following primers were used to amplify cDNA derived from multiple human tissues (SEQ.ID.N0.24) 5'-GGAGGTGTGGACGAAGAAGG-3' and (SEQ.ID.N0.25) 5'-AAGCTGAAGGTGCTGTCGCAGG-3'. GAPDH was used as a control using the primers (SEQ.ID.NO.26) 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3', and (SEQ.ID.N0.27) 5'-CATGTGGGCCATGAGGTCCACCAC-3'.
[0371] As shown in Figure 17B, Human ZARI exons 1 to 4 were transcribed exclusively in the ovary and testis based on multiple-tissue RT-PCR analysis.
The human ZARI

mRNA is predicted to be at least 1.3 kb and encode a larger protein of 424 amino acids. Human and mouse ZARl proteins share 58% amino acid identity (Figure 18) although the carboxyl-tenninus of the ZAR1 proteins, encoded by exons 2-4, were highly conserved and showed 91%
similarity between mouse and human. This suggests that the ZAR1 carboxyl-terminus region may be functionally more important.
Example 26 Protein expression of ZARl [0372] Western blot amalysis was performed using standard techniques well known and used in the art. Briefly, ovarian protein was isolated from wildtype and Gdf9-~ mice.
Antibodies to ZARl were used to compare the size of the recombinant ZAR1 protein to a native ZARl protein. Figure 21 revealed that the recombinant ZAR1 protein is similar in size to the native ZARl protein from isolated ovaries from Gdf~ ~ mice.
Example 27 Localization of Zar1 in mouse ovaries ~0373J In situ hybridization was performed with the Zarl specific probe. [a-ssS]UTP-labeled antisense and sense probes were generated by the Riboprobe combination system (Promega, Madison, WI). Hybridization was carried out according to methods described by Albrecht et al., 1997 and Elvin et al., 1999A.
~0374J In situ hybridization showed high level expression of Za~l localized to the oocytes within these ovaries. The expression of Zarl within oocytes was evident at the one-layer (primary) follicle stage through the antral follicle stage, but no expression was observed at the primordial follicle stage. Because the number of follicles was increased in Gdf~ knockout ovaries due to the arrest of follicle development at the primary follicle stage, more Za~l positive oocytes were detected in each section (Figure 22).
Example 28 Generation of Zar1 knockout mice [0375] Mouse Za~l genomic sequences were used to generate a targeting vector to mutate the Zarl gene in ES cells. The targeting vector contained 1.5 kb of genomic DNA
upstream of Zay~l exon l, a selectable marker (the PGKhprt expression cassette), 5.5 kb of Zarl sequence downstream of exon 1, and a negative selectable marker (the MCltk expression cassette) (Figure 23A). The linearized vector was electroporated into the hprt-negative AB2.2 ES

cell line, clones were selected in HAT (hypoxantine, aminopteridine and thimidine) and FIAU
[1-(2'-deoxy-2'fluoro-(3-D-arabinofuranosyl)-5-iodouracil], and DNA from the clones analyzed by Southern blot. Targeted ES cell clones were injected into blastocysts (Matzulc et al., 1992).
[0376] Two of these cell lines were used to produce chimeric male mice that were fertile and transmitted the Zarl mutant allele (Zarl t'nlZuk~ herein called Zarl-) to F 1 progeny.
Intercrossing of the Fl heterozygotes (Figure 23B) yielded 232 F2 progeny [52 wild-type (22.5%), 119 heterozygous (Za~l +~ ) (51.5%), and 60 homozygous mutant (Zarl -~ ) mice (26.0%]
from 32 litters analyzed. Thus, the mutated allele was transmitted with the expected Mendelian frequency of 1:2:1. The male (117): female (114) ratio was approximately 1:1.
[0377] Northern blot analysis with the full length Zarl cDNA showed a significant reduction of the Zarl mRNA in Zarl +~ ovaries and failed to detect the 1.4kb Za~l mRNA in the ovaries of Zar ~ mice (Figure 23 C), confirming that the Za~l t"'rzuk allele was a null allele.
Example 29 Fertility of Zarl~~ and Zarl ~ mice ~0378J Zarl+~ and Zarl-~ male and female mice showed no gross or histological abnormalities from birth through adult stages. The fertility of Zaf°l +~ and Zarl ~ mice was tested by mating over a 6 month period. Zarl ~ males showed normal fertility (7.4 ~
0.4 pups/litter).
Since Za~l was also expressed in the testis, this indicated that Zaf°I
was not essential for male fertility at a gross level.
[0379] Mating of 14 female Zarl +~ mice with male Za~l +~ mice resulted in 80 litters (0.95 litters/month/mouse) with an average litter size of 7.9 ~ 0.3 pups, which did not differ significantly from previous litter sizes of wild-type mice (8.4 ~ 0.2 pups/litter)(I~umar et al., 2000). Hence, Zaf°I+~ females displayed normal fertility. In contrast, breeding of 20 Zarl-female mice with control males failed to yield any offspring over 6 months.
Thus, ZARl plays an essential role in female fertility.
Example 30 Subcellular localization of ZARl [0380] To further define the function of ZAR1, immunostaining and indirect immunofluorescent labeling with ZAR1 antisera were used to evaluate protein expression and subcellular localization in oocytes and zygotes.

[0381] Briefly, immunohistochemistry was performed using ZAR1 antibodies. To prepare the ZAR1 antibodies, a partial mouse Zar~l cDNA [nucleotides 151-1056]
was subcloned into pET23b vector, and fused recombinant ZAR1 protein (T7-tag at N-terminal and His-tag at C-terminal) was injected into goats to produce polyclonal antibodies (CoCalico Biologicals, Inc., Reamstown, PA). Next, immunostaining was performed using the primary antibody (diluted at 1:1,000) (Yan et al., 2002). Incubation with secondary antibody and visualization of positive cells were performed using the New Fuchsin kit (BioGenex, San Ramon, CA).
Preimmune serum was used in control sections.
[0382] The ZAR1 protein localized predominantly to the cytoplasm of oocytes in both wild-type (Figures 24A-24B) and Gdf~ ~ ovaries (Figure 17C). Consistent with the ifz situ hyb~idizatio~c analysis, ZAR1 protein was present from the primary through antral follicle stages (Figure 24B).
[0383] Immunofluorescence analysis of oocytes and embryos was performed (Yan et al., 2002) to evaluate protein expression. Reaction with the ZAR1 goat antisera (diluted 1/1000 in block solution) was carried out for 1 h, followed by exposure to 3 ~g/ml of FITG-conjugated anti-rabbit IgG (Jackson Immuno Research Laboratories, West Grove PA) for 45 min. DNA was labeled with propidium iodide (10 ~,g/ml, for 10 min). Negative control samples were evaluated with pre-immune serum.
[0384] Moreover, ZAR1 protein was distributed diffusely throughout the cytoplasm of fully-grown oocytes isolated from Zarl +~ mice (Figure 24E), and consistent with the above Northern blot, ovaries (Figure 24D) and oocytes (Figure 24F) from Zaf°I-~ females exhibited no protein. ZAR1 was also detected, after the resumption of meiosis and progression to metaphase-I (Figure 24G) and metaphase-II (Figure 24H).
[0385] Next, fertilized zygotes, which failed to undergo the first mitotic division by 24 h post-fertilization, were evaluated to determine chromatin and microtubule configurations.
The zygotes were fixed, permeabilized, and blocked as indicated. All subsequent steps, including rinses, were carried out at 37°C in block solution. Microtubules were labeled with anti-[3-tubulin (3.8 ~ghnl, for 1 h) and a FITC-conjugated anti-mouse secondary antibody (1.3 ~,g/ml, for 45 min), while DNA was labeled with propidium iodide (10 ~,g/ml, for 10 min).
Fluorescence was detected using a TCS-NT laser scanning confocal microscope equipped with an air-cooled argon ion laser system (L,eica Microsystems).
[0386] ZARl persisted in the cytoplasm of early 1-cell zygotes post-fertilization (Figure 19I) but was dramatically reduced in 2-cell embryos (Figure 24J).
Thus, ZARl functions at any stage of oogenesis from the primary follicle stage through the formation of 2-cell embryos. The rapid disappearance of ZARl at the 2-cell stage, however, suggested a critical role in the oocyte-to-embryo transition.
Example 31 In vitro oocyte maturation and fertilization [0387] Sexually mature, heterozygous control and Zar 1-~ female mice were injected with 5 IU of PMSG to stimulate preovulatory follicle development.
Cumulus-enclosed oocyte complexes were isolated 48 h later and cultured for 17 h in Minimal Essential Medium with 5% senun. The surrounding somatic cells were subsequently removed, and the oocytes were examined to determine the progression of meiosis. Mature MII-stage eggs were fertilized ih vitro with sperm from wild type (C57BL/6J x SJL/J) F1 mice 19. Development of zygotes and 2-cell stage embryos was assessed at 6 and 24 h post-fertilization, respectively.
Blastocyst formation was evaluated on day 5.
Example 32 Zarl embryonic development [0388] To confirm that ovulation was occurring and to further study the cause of the infertility and subfertility of the Zarl -~ and Zar~l +~ mice, pharmacological superovulation of Zarl -~ , and Za~l +~ mice was performed and the eggs were collected from the oviducts and cultured ih vitro.
[0389] Briefly, Twenty-five-day-old Za~l +~ and Za~l -~ female mice were inj ected with PMSG (i.p., 5.0 IU/mouse), and given hCG (i.p., 5 IU/mouse) 48 h later.
Mice were then caged overnight with (C57BL/6J x 12956/SvEv)F1 stud males. The following morning, eggs and/or embryos were recovered in M2 medium, counted, and cultured is vitro up to 4 days in M16 medium (Sigma, St. Louis, MO). Alternatively, adult mutant females were mated to stud males, uteri and oviducts flushed on day 3.5, and embryos collected and cultured in M16 medium.

[0390] Superovulation with exogenous gonadotropins demonstrated similar numbers of oocytes from Za~l-~ (34.31 ~ 4.12; n=14) and Zarl+l (31.63 ~ 4.78;
n=8) females.
Yet further, the majority of Zaf~l~~ and Zarl-~ oocytes resumed meiosis and progressed to metaphase-II during a 17-h culture.
[0391] Next, metaphase-II oocytes were fertilized i~ vitf°o or after mating with stud males, embryos were recovered from the reproductive tract and cultured for up to 4 days or from adult females on day 3.5 (Table 7). Most oocytes from Zarl ~ females formed two distinct pronuclei within 8 h post-insemination similar to the controls. However, while the first cleavage occurs in 89.3% of i~ vivo fertilized embryos from Za~l +~ females and 86.5%
of i~ vitro fertilized embryos from Zarl +~ females, "apparent" 2-cell embryos (some of which appeared fragmented) were observed in 20.8% of in vivo fertilized embryos and 19.1% of in vitro fertilized embryos from Za~l ~ females. Most 2-cell embryos from Za~l +~ mice progressed to the morula and blastocyst stages by the fourth day of culture (Table 7);
however, embryos from Za~l-~ mice either remained at the one- or 2-cell stage or degenerated.
Whereas 100% of the embryos isolated from the uteri of adult Zarl ~~ females developed to blastocysts by day 3.5, only fragmented, 1-cell,;and a single 2-cell embryo could be observed in the Zaf°I-~ females (Figures 25A-25B and Table 6). Therefore, an arrest of early embryo cleavage at the zygote stage accounts for the infertility of Zaf°I-~ females.
Table 6: Evaluation of ih vitro and in vivo oocyte maturation and embryo development.
Age Genotype N Total Qocytes/ % MII-stage % 2 pronuclei % 2-Cell % Blastocyst embryos 3-4wk +/- 6 156 74.45.5 92.92.1 86.51.4 82.11.5 3-4wk -/- 5 137 62.94.3 82.47.5 19.19.1 0.0 Adult +/- 6 41 - - 0.0 100 Adult -/- 9 19 - - 5.3 0.0 Example 33 DNA synthesis [0392] In further determining the cause of the infertility of the Zarl-~ mice, DNA
synthesis was performed. Briefly, fully-grown oocytes from Za~l+~ and Zap°I-~ mice were ih vitro matured and fertilized, as previously described. Approximately 8 h post-fertilization, the zygotes that had formed a male and female pronucleus (PN) were transferred to media supplemented with 50 ~M bromo-deoxyuridine (BrdU) for overnight culture. At 24 h post-fertilization, embryos were fixed and processed to assess BrdU incorporation (Ferreira et al., 1997). Fluorescence was detected using a confocal microscope.
[0393] Analysis of the timing of the embryonic block showed that the arrested zygotes from Zarl-~ females progressed through Gl and successfully entered S-phase. The chromatin of both maternal and paternal pronuclei was completely decondensed (Figure 25C).
BrdU was readily incorporated into both pronuclei of embryos from Zarl-~
females (Figure 25D), indicating active DNA synthesis in S-phase. The microtubule network showed an interphase configuration with no assembled spindle apparatus. Ih vitf°o fertilized oocytes were treated with colcemid, a reagent that depolymerized microtubules to arrest cells at M-phase. As expected, all of the zygotes derived from Zaf°I +~ females arrested at M-phase after colcemid treatment. Only a few zygotes from Zarl-~ females were similarly arrested;
this corresponded to the number of 2-cell embryos normally observed in this group. Hence, the small percent of fertilized oocytes from Zarl-~ females that progressed to the 2-cell stage entered M-phase, yet the majority arrested earlier, presiunably at the S/G2 transition or the G2 stage of the first meiotic division. Thus, the maternal and paternal genomes remain separated in discrete pronuclei, and the two haploid genomes failed to unite, indicating that the completion of fertilization has not occurred.
Example 34 Yeast Two-hybrid screening [0394] Yeast Two hybrid screen was used to elucidate or characterize the function of a protein by identifying other proteins with which it interacts.
[0395] The full-length open reading frame of mouse Zarl was subcloned into the pGBI~T7 vector for expression as a GAL4 DNA binding fusion protein. Ovarian and oocyte cDNA libraries were subcloned into the pGADT7 vector to be expressed as transactivation domain fusion proteins. In this yeast two-hybrid system, interactions between ZARl and proteins encoded by library cDNAs are expected to reconstitute transactivating complexes, which bind to GAL4 DNA and promote transcription of selectable markers. To identify ZAR1-interacting proteins, ovary cDNA transformants were screened by mating. Colonies grew on Leu-/Trp-/Ade-/His-/X-alpha-Gal selection plates and certain isolated plasmids with inserts were sequenced. Four of these sequences corresponded to Polr2c (DNA directed RNA
polymerase II
polypeptide C), Gnb2 (Guanine nucleotide binding protein, beta 2), Polr2g (DNA
directed RNA
polymerase II polypeptide G), and Lmol (LIM only 1).
Example 35 Cell-free transcriptionltranslation of Zarl [0396] Cell-free ih vitr°o transcription/translation of Zap°I
was performed to confirm in vitro protein interaction. Briefly, the pGBKT7 (MYC-Tagged) and pGADT7 (HA-Tagged) vectors were used as templates for in vitf°o transcription/translation using [35S] Met and the TNT
T7 Coupled Reticulocyte Lysate System (Promega, Madison, WI). In vitro translated proteins were combined at room temperature for 1 h, and reciprocal co-immunoprecipitation experiments were performed using mouse anti-MYC monoclonal or rabbit anti-HA polyclonal antibodies (Clontech).
[0397] Cell-free i~r vitf°o transcription/translation of Za~l, Polr2c (DNA directed RNA polymerase II polypeptide C), Gnb2 (Guanine nucleotide binding protein, beta 2), Polr2g (DNA directed RNA polymerase II polypeptide G), and Lmol (LIM only 1) cDNAs was performed. The cDNAs for Polr2c, Gnb2 and Lmoll were inserted into the pGADT7 (HA-Tagged) vector and Za~l cDNA was inserted into the pGBKT7 (MYC-Tagged) vector.
The i~
vitro translated proteins were then co-immunoprecipitated and analyzed on a SDS-PAGE.
[0398] Figures 26A and 27B demonstrates that POLR2C, GNB2, POLR2G, and LMO1 bind to the ZAR1.
Example 36 ZARl interactions in CHO cells [0399] To confirm that ZARl binds to POLR2C, GNB2, POLR2G, and LMO1, co-immunoprecipitation studies are performed using extracts of transiently transfected Chinese hamster ovary (CHO) cells.
[0400] Briefly, CHO-Kl cells (American Type Culture Collection, Manassas, VA) are cultured in Dulbecco's modified Eagle's medium l Ham's F-12 (DMEM/F-12) containing 10% fetal bovine serum (FBS) and are grown to 90-95% confluence in 6 cm dishes. To express tagged proteins, mouse cDNAs encoding the open reading frames of ZAR1, POLR2C, GNB2, POLR2G, and LMOl are inserted into pCMV-Tag4A/FLAG-C and pCMV-TagSA/MYC-C
vectors (Stratagene, La Jolla, CA) and are transiently transfected using LipofectAMINE 2000 (Invitrogen Life Technologies). Twenty-four hours after transfection, cells are harvested, lysed in lysis buffer [50 mM TrisHCl, pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 % Triton X-100 and protease inhibitor cocktail (Sigma, Saint Louis, MO)] and are analyzed by immunoprecipitation and SDS-PAGE.
[0401] The MYC-tagged constructs are detected with anti-MYG antibodies and FLAG-tagged constructs are detected with anti-FLAG antibodies.
Example 37 Conformation of binding of the binding partners [0402] To confirm that ZARl binds to POLR2C, GNB2, POLR2G, and LMO1, ovarian protein is isolated from mice. Next, immunoprecipitation experiments are performed using ZAR1 antibodies. Western blot analysis is performed using antibodies to POLR2C, GNB2, POLR2G, and LMOl.
Example 38 Generation of knockout mice lacking novel ovary-expressed genes [0403] Using the gene sequence obtained above, the inventors generate a targeting vector to mutate the O1-184 gene in embryonic stem (ES) cells. This targeting vector is electroporated into the hprt-negative AB2.1 ES cell line and selected in HAT
and FIAU. Clones are processed for Southern blot analysis and screened using 5' and 3' external probes. ES cells with the correct mutation are injected into blastocysts to generate chimeras and eventually heterozygotes and homozygotes for the mutant O1-184 gene.
[0404] Since expression of Ol-184 was limited to the ovary, the inventors anticipate that these O1-184-knockout mice are viable, but that females lacking this gene product can have fertility alterations (i. e., be infertile, subfertile, or superfertile). Mutant mice are analyzed for morphological, histological and biochemical information relating to intraovarian proteins required for folliculogenesis, oogenesis, or fertilization using techniques well within the ability of the person of ordinary skill in the art. It is envisioned that the absence of this protein can result in female mice having increased or decreased fertility. These studies will lead a search for human reproductive conditions with similar idiopathic phenotypes.

Example 39 Generation of O1-184 Transgenic Animals [0405] The O1-184 gene is flanked by genomic sequences and is transferred by microinjection into a fertilized egg. The microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene. Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish. These animals are generated to overexpress O1-184 or express a mutant form of the polypeptide.
[0406] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention.
Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.
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SEQUENCE LISTING
<110> Matzuk, Martin M.
Wu, Xuemei Wang, Pei Bai, Yuchen <120> Contraceptive Targets <130> P01925W03 <140> Not Assigned <141> 2003-04-24 <150> US 60/434,165 <151> 2002-12-17 <150> US 60/439,781 <151> 2003-01-Z3 <150> US 60/442,164 <151> 2003-01-23 <150> PCT/US 02/13245 <151> 2002-04-26 <150> US 60/411,262 <151> 2002-09-17 <160> 43 <170> PatentIn version 3.1 <210> 1 <211> 1258 <212> DNA
<213> Mus musculus <400>

ggcgggcgacgcgcgggacgcacccatgttcccggcgagcacgttccacccctgcccgca 60 tccttatccgcaggccaccaaagccggggatggctggaggttcggagecaggggctgccg 120 aCCCgCgCCCCCCtCCttCCtccccggctacagacagctcatggccgcggagtacgtcga 180 ecgccaccagcgggcacagctcatggccctgctgtcgcggatgggtccccggtcggtcag 240 cagccgtgacgctgcggtgcaggtgaacccgcgccgcgacgcctcggtgcagtgttcact 300 cgggcgccgcacgctgcagcctgcagggtgCCgagCCagCCCCgaCgCCCgatcgggttc 360 ctgtcaaccccgtggccacgccggcgccgggagatccccgcgatcctggcagaccgtagc 420 cccgttctcgtccgtgaccttctgtggcctctectcctcactggaggttgcgggaggcag 480 gcagacacccacgaagggagaggggagcccggcatcctcggggacccgggaaccggagcc 540 gagagaggtggccgcgaggaaagcggtcccccagccgcgaagcgaggagggcgatgttca 600 ggctgcagggcaggccgggtgggagcagcagccaccaccggaggaccggaacagtgtggc 660 ggcgatgcagtctgagcctgggagcgaggagccatgtcctgccgcagagatggctcagga 720 CCCCggtgattCggatgCCCCtCgagaCCaggCCtCCCCgcaaagcacggagaaggacaa 780 ggagcgcctgcgtttccagttcttagagcagaagtacggctactatcactgcaaggactg 840 caaaatccggtgggagagcgcctatgtgtggtgtgtgcagggcaccagtaaggtgtactt 900 caaacagttctgccgagtgtgtgagaaatcctacaacccttacagagtggaggacatcac 960 ctgtcaaagttgtaaaagaactagatgtgcctgcccagtcagacttcgccacgtggaccc 1020 taaacgcccccatcggcaagacttgtgtgggagatgcaaggacaaacgcctgtcctgcga 1080 cagcaccttcagcttcaaatacatcatttagtgagagtcgaaaacgtttctgctagatgg 1140 ggctaatggaatggacaagtgacgtttctcccctcttcacctcttccctttccaaattct 1200 tcatgacagacagtattacttgagtataaagcctgtgaataaaaggtattgcaaacaa 1258 <210> 2 <211> 361 <212> PRT
<213> Mus musculus <400> 2 Met Phe Pro Ala Ser Thr Phe His Pro Cys Pro His Pro Tyr Pro Gln Ala Thr Lys Ala Gly Asp Gly Trp Arg Phe Gly Ala Arg Gly Cys Arg Pro Ala Pro Pro Ser Phe Leu Pro Gly Tyr Arg Gln Leu Met Ala Ala Glu Tyr Val Asp Ser His Gln Arg Ala Gln Leu Met Ala Leu Leu Ser Arg Met Gly Pro Arg Ser Val Sex Ser Arg Asp Ala Ala Val Gln Val Asn Pro Arg Arg Asp Ala Ser Val Gln Cys Ser Leu Gly Arg Arg Thr Leu Gln Pro Ala Gly Cys Arg Ala Ser Pro Asp Ala Arg Ser Gly Ser Cys Gln Pro Arg Gly His Ala Gly Ala Gly Arg Ser Pro Arg Ser Trp Gln Thr Val Ala Pro Phe Ser Ser Val Thr Phe Cys Gly Leu Ser Ser Ser Leu Glu Val Ala Gly Gly Arg Gln Thr Pro Thr Lys Gly Glu Gly Ser Pro A1a Ser Ser Gly Thr Arg Glu Pro Glu Pro Arg Glu VaI Ala Ala Arg Lys Ala Val Pro Gln Pro Arg Ser Glu GIu Gly Asp Val Gln l80 185 190 Ala Ala Gly GIn Ala Gly Trp Glu Gln Gln Pro Pro Pro Glu Asp Arg Asn Ser Val Ala Ala Met Gln Ser Glu Pro Gly Ser Glu Glu Pro Cys .

Pro Ala Ala Glu Met Ala Gln Asp Pro Gly Asp Ser Asp Ala Pro Arg Asp Gln Ala Ser Pro Gln Ser Thr Glu Gln Asp Lys Glu Arg Leu Arg Phe Gln Phe Leu Glu Gln Lys Tyr Gly Tyr Tyr His Cys Lys Asp Cys Lys Ile Arg Trp Glu Ser AIa Tyr Val Trp Cys Val Gln Gly Thr Ser Lys Val Tyr Phe Lys Gln Phe Cys Arg Val Cys Glu Lys Ser Tyr Asn Pro Tyr Arg Val Glu Asp Ile Thr Cys Gln Ser Cys Lys Arg Thr Arg Cys Ala Cys Pro Val Arg Phe Arg His Val Asp Pro Lys Arg Pro His Arg Gln Asp Leu Cys Gly Arg Cys Lys Asp Lys Arg Leu Ser Cys Asp Ser Thr Phe Ser Phe Lys Tyr Ile Ile <210> 3 <211> 1817 <212> DNA
<213> Mus musculus <400> 3 gtCaCagCtt tCCCCtgCCC gaatatggtg atctgtctcc attgtccaga tcaggatgat 60 tctttagaag aagtcacaga ggaatgctat tccccaccca ccctccagaa cctggcaatt 120 cagagtctac tgagggatga ggccttggcc atttctgctc tcacggacct gccccagagt 180 ctgttcccagtaatttttgaggaggccttcactgatggatatatagggatcttgaaggcc240 atgatacctgtgtggcccttcccatacctttctttaggaaagcagataaataattgcaac300 ctggagactttgaaggctatgcttgagggactagatatactgcttgcacaaaaggttcaa360 accagtaggtgcaaactcagagtaattaattggagagaagatgacttgaagatatgggct420 ggatcccatgaaggtgaaggcttaccagatttcaggacagagaagcagccaattgagaac480 agtgctggctgtgaggtgaagaaagaattgaaggtgacgactgaagtccttcgcatgaag540 ggcagacttgatgaatctaccacatacttgttgcagtgggcccagcagagaaaagattct600 attcatctattctgtagaaagctactaattgaaggcttaaccaaagcctcagtgatagaa660 atcttcaaaactgtacacgcagactgtatacaggagcttatcctaagatgtatctgcata720 gaagagttggcttttcttaatccctacctgaaactgatgaaaagtcttttcacactcaca780 ctagatcacatcataggtaccttcagtttgggtgattctgaaaagcttgatgaggagaca840 atattcagcttgatttctcaacttcccacactccactgtctccagaaactctatgtaaat900 gatgtcccttttataaaaggcaacctgaaagaatacctcaggtgcctgaaaaagcccttg960 gagacactttgcatcagtaactgtgacctctcacagtcagacttggattgcctgccctat1020 tgcctgaatatttgtgaactcaaacatctgcatattagtgatatatatttatgtgattta1080 CCCCttgagCCtCttggttttCtCCttgagagagttggagataccctgaaaaccctggaa1140 ttggattcatgttgtatagtggactttcagttcagtgccttgctgcctgccctaagccaa1200 tgttctcacctcagagaggtcactttctatgataatgatgtttctctgcctttcttgaaa1260 acaacttetacaccacacagccctgctgagtcagetgatctatgagtgttaCCCtgCCCC1320 tctagagtgctatgatgacagtggtgtaatactaacacacagattagaaagtttttgtcc1380 tgagcttctggatatactgagagccaaaagacagctccatagtgtctcctttcaaacaac1440 caaatgctctaaatgtggtgggtgctacatttatgatcggcatacccaatgttgccgttt1500 tgtggaactactataagcttgattgtgaaactgagaaatagaaacttagtattggggact1560 gatgaaatcctaagtgaatgtccactgctaaatggagcatgaaaatgtcaatcacctaaa1620 agtctgagat acacaggaaa gtcaataact tcctctgagc tggtgaatgg atgttgcatc 1680 tgtagaaagt atcaagcact tgtagtttga atgtgttaca atagaagcac cattttatga 1740 gactggccca atctgttgac tgcatacaat aaatctgttg acttattaaa tttttaaaaa 1800 aaaaaaaaaa aaaaaaa 1817 <210> 4 <211> 426 <212> PRT
<213> Mus musculus <400> 4 Met Val Ile Cys Leu His Cys Pro Asp Gln Asp Asp Ser Leu Glu Glu Val Thr Glu Glu Cys Tyr Ser Pro Pro Thr Leu Gln Asn Leu Ala Ile Gln Ser Leu Leu Arg Asp Glu Ala Leu Ala Ile Ser Ala Leu Thr Asp Leu Pro Gln Ser Leu Phe Pro Val Ile Phe Glu Glu Ala Phe Thr Asp Gly Tyr Ile Gly Ile Leu Lys Ala Met Ile Pro Val Trp Pro Phe Pro Tyr Leu Ser Leu Gly Lys Gln Ile Asn Asn Cys Asn Leu Glu Thr Leu Lys Ala Met Leu Glu Gly Leu Asp Ile Leu Leu Ala Gln Lys Val Gln 100 1.05 110 Thr Ser Arg Cys Lys Leu Arg Val Tle Asn Trp Arg Glu Asp Asp Leu Lys Ile Trp Ala Gly Ser His Glu Gly Glu Gly Leu Pro Asp Phe Arg Thr Glu Lys Gln Pro Ile Glu Asn Ser Ala Gly Cys Glu Val Lys Lys Glu Leu Lys Val Thr Thr Glu Val Leu Arg Met Lys Gly Arg Leu Asp Glu Ser Thr Thr Tyr Leu Leu Gln Trp Ala Gln Gln Arg Lys Asp Ser Ile His Leu Phe Cys Arg Lys Leu Leu Ile Glu Gly Leu Thr Lys Ala Ser Val Ile Glu Ile Phe Lys Thr Val His Ala Asp Cys Ile Gln Glu Leu Ile Leu Arg Cys Ile Cys Ile Glu Glu Leu Ala Phe Leu Asn Pro Tyr Leu Lys Leu Met Lys Ser Leu Phe Thr Leu Thr Leu Asp His Ile Ile Gly Thr Phe Ser Leu Gly Asp Ser Glu Lys Leu Asp Glu Glu Thr I1e Phe Ser Leu Ile Ser Gln Leu Pro Thr Leu His Cys Leu Gln Lys Leu Tyr Val Asn Asp Val Pro Phe Ile Lys Gly Asn Leu Lys Glu Tyr Leu Arg Cys Leu Lys Lys Pro Leu Glu Thr Leu Cys Ile Ser Asn Cys Asp Leu Ser Gln Ser Asp Leu Asp Cys Leu Pro Tyr Cys Leu Asn I1e Cys Glu Leu Lys His Leu His Ile Ser Asp Ile Tyr Leu Cys Asp Leu Leu Leu Glu Pro Leu Gly Phe Leu Leu Glu Arg Val Gly Asp Thr Leu Lys Thr Leu Glu Leu Asp Ser Cys Cys Ile Val Asp Phe Gln Phe Ser 370 375 ~ 380 Ala Leu Leu Pro Ala Leu Ser Gln Cys Ser His Leu Arg Glu Val Thr Phe Tyr Asp Asn Asp Val Ser Leu Pro Phe Leu Lys Thr Thr Ser Thr 405 . 410 415 Pro His Ser Pro Ala Glu Ser Ala Asp Leu <210> 5 <211> 1018 <212> DNA
<213> Mus musculus <400>

gccatattgaggacctgcagtagaggtggaacccatgactggcagcgcaaacacagtgat60 aacagctgagctccaagcaaggacccaggaccttgcctcaccacagacataatctttccc120 cacaacacctccaccaagccgccctgtaaatcgacatgagtcgccacagcaccagcagcg180 tgaccgaaaccacagcaaaaaacatgctctggggtagtgaactcaatcaggaaaagcaga240 cttgcacctttagaggccaaggcgagaagaaggacagctgtaaactcttgctcagcacga300 tCtgcctgggggagaaagccaaagaggaggtgaaccgtgtggaagtcctctcccaggaag360 gcagaaaaccaCCaatcactattgctacgctgaaggcatcagtcctgcccatggtcactg420 tgtcaggtatagagctttctcctccagtaacttttcggctcaggactggctcaggacctg480 tgttcctcagtggcctggaatgttatgagacttcggacctgacctgggaagatgacgagg540 aagaggaggaagaggaggaggaagaggatgaagatgaggatgcagatatatcgctagagg600 agatacctgtcaaacaagtcaaaagggtggctccccagaagcagatgagcatagcaaaga660 aaaagaaggtggaaaaagaagaggatgaaacagtagtgaggcccagccctcaggacaaga720 gtccctggaagaaggagaaatctacacccagagcaaagaagccagtgaccaagaaatgac780 ctcatcttagCatCttCtgCgtccaaggcaggatgtccagcagctgtgttttggtgcagg840 tgtccagccccaccaccctagtctgaatgtaataaggtggtgtggctgtaaccctgtaac900 ccagccctccagtttccggaggtttttggtgaagagcccccagcaagttcgcctagggcc960 acaataaaatttgcatgatcaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1018 <210> 6 <211> 207 <212> PRT
<213> Mus musculus <400> 6 Met Ser Arg Hzs Ser Thr Ser Ser Val Thr Glu Thr Thr Ala Lys Asn Met Leu Trp Gly Ser Glu Leu Asn Gln Glu Lys Gln Thr Cys Thr Phe Arg Gly Gln Gly Glu Lys Lys Asp Ser Cys Lys Leu Leu Leu Ser Thr Ile Cys Leu Gly Glu Lys Ala Lys Glu Glu Val Asn Arg Val Glu Val Leu Ser Gln Glu Gly Arg Lys Pro Pro Ile Thr Ile Ala Thr Leu Lys 65 70 75 g0 A1a Ser Val Leu Pro Met Val Thr Val Ser Gly Ile Glu Leu Ser Pro Pro Val Thr Phe Arg Leu Arg Thr Gly Ser Gly Pro Val Phe Leu Ser Gly Leu Glu Cys Tyr Glu Thr Ser Asp Leu Thr Trp Glu Asp Asp Glu G1u Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Asp Glu Asp Ala Asp Ile Ser Leu Glu Glu Ile Pro Val Lys Gln Val Lys Arg Val Ala Pro Gln Lys Gln Met Ser Ile Ala Lys Lys Lys Lys Val Glu Lys Glu Glu Asp Glu Thr Val Val Arg Pro Ser Pro Gln Asp Lys Ser Pro Trp Lys Lys Glu Lys Ser Thr Pro Arg Ala Lys Lys Pro Val Thr Lys Lys <210> 7 <211> 6970 <212> DNA
<213> Mus musculus <220>
<221> misc_feature <222> (1) . (6970) <223> n equals unknown <400>

acagcagaggtgatgctcagaaatcaagttttaacagagggccaggtgcttctagagtag60 gaggggattgCaCaCCtCCCCdCCCCCtCCtctttcccaggcttcttaacagcctgctgt120 gggaagctgacccttagatggagccctgaagccatattgaggacctgcagtagaggtgga180 acccatgactggcagcgcagtaagcttgagcaggnnnnnnnnnnnnnnnnnnnnnnnnnn240 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn420 nnnnnnnrinnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn480 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn540 nnnnnnnnnnnnnnnnnctttgcattactcagaacacagtgataacagctgagctccaag600 caaggacccaggaccttgcctcaccacagacataatctttccccacaacacctccaccaa660 gccgccctgtaaatcgacatgagtcgccacagcaccagcagcgtgaccgaaaccacagca720 aaaaacatgctctggggtaagggctaaggctnnnnnnnnnnnnnnnnnnnnnnnnnnnnn780 nrinnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn840 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnngtcttcgctgtgcag900 gtagtgaactcaatcaggaaaagcagacttgcacctttagaggccaatgcgagaagaagg960 acagctgtaaactcttgctcagcacggtgggtgtctcccaannnnnnnnnnnnnnrinnnn1020 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1080 nnnnnnnnnnnnncatcacctttctcagatctgcctgggggagaaagccaaagaggaggt1140 gaaccgtgtggaagtcctctcccaggaaggcagaaaaccaccaatcactattgctacgct1200 gaaggcatcagtcctgcccatggtgagtcttctctccnnnnrinnnnnrinnnnnnnnnnnn1260 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnrinnnnnnnnnnnnnnnnnnnnnnnnnnnn1320 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnrinnnnnnnnnnnnnnnnnn1380 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1440 nrinnnnrinnnnnnnnnnrinnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1500 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1560 nrinnnnnnnnnnnnnnnrinnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1620 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1680 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1740 nnnnnnnrinnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnrinnnnnnnnnnnn1800 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1860 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnrinnnnnnnnnnnn1920 nnnnnnnrinnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1980 nnnnnnnnnnnnnnnnnrinnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2040 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2100 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnrinnnnnnnnnnnn2160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2280 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2340 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2400 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2460 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2520 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2580 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2640 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2700 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2760 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2820 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3000 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3060 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3120 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3180 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3240 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3300 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3360 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3420 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3480 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3540 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3600 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3660 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3720 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3780 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3840 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3900 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3960 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn4020 nnnnnnnnnnnnnnnnnagaagggggacacaggtcactgtgtcaggtatagagctttctc4080 ctccagtaacttttcggctcaggactggetcaggacctgtgttcctcagtggcctggaat4140 gttatggtaagttgtagcctannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn4200 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn4260 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn4320 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn4380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4860 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn4920 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn4980 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5040 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5100 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5160 ririnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5220 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5280 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5340 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5400 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5460 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnggctaccca5520 ttccagagacttcggacctgacctgggaagatgacgaggaagaggaggaagaggaggagg5580 aagaggatgaagatgaggatgcagatatatcgctagaggagatacctgtcaaacaagtca5640 aaagggtggctccccagaagcagatgagcatagcaaaggtggggggaaaagaannnnnnn5700 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5760 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn5820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt 5880 ggtttttgtt ccagaaaaag aaggtggaaa aagaagagga tgaaacagta gtgaggtaat 5940 tcatgcagtt nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6360 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn6420 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnctattccctttccaggcccagccctcagga6480 caagagtccctggaagaaggtgagcaataagaagnnnnnnnnnnnnnnnnnnnnnnnnnn6540 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn6600 nnnnnnctcttatctgcacaggagaaatctacacccagagcaaagaagccagtgaccaag6660 aaatgacctcatcttagcatcttctgcgtccaaggcaggatgtccagcagctgtgttctg6720 gtgcaggtgtccagccccaccaccctagtctgaatgtaataaggtggtgtggctgtaacc6780 ctgtaacccagccctccagtttccggaggtttttggtgaagagcccccagcaagttcgcc6840 tagggccacaataaaatttgcatgatcaggaCCtCCCt gCCtCCCCCtccctggatgg6900 Ct gtctcctcgctgctgcgatagctcatgtgcccagcagagggcaaccacgagcaagaaacc6960 agccccatgt 6970 <210> 8 <211> 1207 <212> ANA
<213> Human <400>

agggggcgccaggaggcctcggcgggtccgcaattggccgggacagcttctcacgaaagg60 tcctgggccggcatcatcagcctcacctgggaactggttagaactacaaattccctcggc120 cccacccagaccgacgccaagggcagctgtggagtggggcgcggcaatgcgccccttaac180 agccctccaggcttcttagcccgggcttggacagccgccttccggccagaggggatgagg240 ttgcgctgcgctccgggagcgccgatggcgtgactggccccgcgcggagcagcgacactg300 cccggccagcccgcttctctgcccggagccatgaatctcagtagcgccagtagcacgagg360 aaaaggcagt gacgaccgtg ctctggggct gcgagctcag tcaggagagg cggacttgga 420 ccttcagaccccagctggaggggaagcagagctgcaggctgttgcttcatacgatttgct480 tgggggagaaagccaaagaggagatgcatcgcgtggagatCCtgCCCCCagcaaaccagg540 aggacaagaagatgcagccggtcaccattgcctcactccaggcctcagtcctccccatgg600 tctccatggtaggagtgcagctttctcccccagttactttccagctccgggctggctcag660 gacccgtgttcctcagtggccaggaacgttatgaagcatcagacctaacctgggaggagg720 aggaggaagaagaaggggaggaggaggaagaggaagaggaagatgatgaggatgaggatg780 cagatatatctctggaggagcaaagccctgtcaaacaagtcaaaaggctggtgccccaga840 agcaggcgagcgtggctaagaaaaaaaagctggaaaaagaagaagaggaaataagagcca900 gcgttagagacaagagccctgtgaaaaaggccaaagccacagccagagccaagaagccag960 gattcaagaaatgaggagccacgccttggggggcacggtgcaaagtgggccttccctggg1020 ctgtgctgcaggcacagggtgcccctgtccagcccctccacctgtgtctgaatgcaacag1080 gggtgttgcgggggcaacatgagagcccctcacccccaactctccactttcaggaggccc1140 ccagtgaagagccccacctcggggtcacaataaagttgcctggtcaggaaaaaaaaaaaa1200 aaaaaaa 1207 <210>

<211>

<212>
PRT

<213>
Human <400> 9 Met Asn Leu Ser Ser Ala Ser Ser Thr Glu Glu Lys Ala Val Thr Thr Val Leu Trp Gly Cys Glu Leu Ser Gln Glu Arg Arg Thr Trp Thr Phe Arg Pro Gln Leu GIu Gly Lys Gln Ser Cys Arg Leu Leu Leu His Thr Ile Cys Leu Gly Glu Lys Ala Lys Glu Glu Met His Arg Val Glu Ile Leu Pro Pro Ala Asn Gln Glu Asp Lys Lys Met Gln Pro Val Thr Ile Ala Ser Leu Gln Ala Ser Val Leu Pro Met Val Ser Met Val Gly Val Gln Leu Ser Pro Pro Val Thr Phe Gln Leu Arg Ala Gly Ser Gly Pro Val Phe Leu Sex Gly Gln Glu Arg Tyr Glu Ala Ser Asp Leu Thr Trp Glu Glu Glu Glu Glu Glu Glu Gly Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Glu Asp Glu Asp Ala Asp Ile Ser Leu Glu Glu Gln Ser Pro Val Lys Gln Val Lys Arg Leu Val Pro Gln Lys Gln Ala Ser Val Ala 7.65 170 175 Lys Lys Lys Lys Leu Glu Lys Glu Glu Glu Glu Ile Arg Ala Ser Val Arg Asp Lys Ser Pro Val Lys Lys Ala Lys Ala Thr Ala Arg Ala Lys Lys Pro Gly Phe Lys Lys <210> ZO
<212> 19952 <212> DNA
<213> Human <400>

ggcatccccatatgatggttactagcggctgggaagtgggggtggggggaggatgaacag60 aggttgattaatgggtacaaacatactgtttgatggaaataagataagttgaaataagaa120 ataagttgatagtacagtagggtgactatagttaacaataatttattgtatatttcaaaa180 tagctagaagagaagatttgaaatgtttccaacacaaagaaatgataaatgtttagccgg240 gcccagtggctcatgcctgtaatcccagcactttgggaggcctaggcaggaggatcactg300 aggtcaggagttcgagaccaacctggccaacatggtgaaaccccatctctagtaaaaata360 tgaaaattagctgggcatggtggtaagcacctataaacccagctacttgggaggctgagg420 caggagaatcgcttgaacatgggaggcagaggttatagtgagctgagatggcaccaccgc480 actccagcctgggtgatgagagtgaaacgccatctccaaaaaaaaaaaaaaaaagaaaag540 aaataatgttgaaggtaccccagttaccctgatttgctcattacacattgtgtgcaagta600 taaaaatatcatatgtaccccataaatatgtacaattaatatgtatcagtttaaaaagtt660 aatgacgatggtaccaatattatagcctcatttagcagatgaggaaactgaggcactgag720 ctatgaatta acacacctga aatcacagag cacagtccag acttgaaccc agactgtcca 780 gttccagtgt cccagctcta ggtcatgacc tcagggtcat CCCCCtCCCC tgCC'tCCatt 840 tagccttcac tgtgaccccc agctgcagcc tgacatcagt gtgattattc acggggtggg 900 tagcctgggg ccactgaagg ccggtttgct ttgagcactg ctctccaatg aggctggaag 960 ccctttgagg ggctgtcgta ttCaCCgCgg gatgCCCagg tCCCgCCCaa ttggcggaat 1020 caccgtttgt tgagtgaatt cttgaacgtc tgtgcatggc atgcatgtgc ctgccatttg 1080 ctcatcttta ccacacactt ggctgcaatg gctgtcacct tcaataggcg ccttgccagg 1140 gacagagagc tcagggacag ccaaggagac tggaactgct cagctgggga tagggagcct 1200 agggggccct ggcaggcccc caagcctctc ctgggttgtc ctggccaatt cacagggagg 1260 ctgaccccag cttccaggaa taaaagattc tgacctctcc gtggcaatga gcccctgtcc 1320 caggggtgtg cgggagatcc ctggttatct gaggtgtctc cagggtatat ccgctgaaac 2380 cccacttcct cttcactgcc cactgagcct gggaccagct gctggtcacg tgcttggccc 1440 tgtagcgtcg ctagccgtgc tcctcagttg tgctccccgc eccctgccgc ggcgcctcgc 1500 CtCCCggCtC aCCtCCCCa.C CCCaCCtgCC CgCtgCggCt ctccggcggg agatctcacc 1560 gttctggaga cagggctcgc tcgctctcac ggtaggctgg aagaacgggc tgtctgggcc 1620 ttaggaaagg cccatgctgt ataaggcatg gggaaaggaa aggaagaaag gcaacgaaca 1680 agaaggaggg cttccaactg cagcttcctg ccggctgcag gcctcccttc ctaagctgag 1740 ctgaggcttc ctctccatgg gctggggagg gggcgccagg aggectcggc gggtccgcaa 1800 ttggccggga cagcttctca cgaaaggtcc tgggccggca tcatcagcct cacctgggaa 1860 ctggttagaa ctacaaattc cctcggcccc acccagaccg acgccaaggg cagctgtgga 1920 gtggggcgcg gcaatgcgcc ccttaacagc cctccaggtg attctgccgc gcagaggagg 1980 aaagaatggg agaagggaag gggagaggga ggcggcttct ttgcgactaa ttggacacct 2040 gcccttcccc ttcccaggct tcttagcccg ggcttggaca gccgccttcc ggccagaggg 2100 gatgaggttg cgctgcgctc cgggagcgcc gatggcgtga ctggccccgc gcggagcagc 2160 gacagtaagg ctgtgtgggg ggagctggga cctaagccgc gcgcacaccc ctttctctgc 2220 gtctggtgga ggtgcacaga ggcttttgag tcaggcccaa gcgcagccag gtgacctccc 2280 cgcggccttt caagcctgag CtCggtggaC agCtCCCtCt cccgtgagtc ccgctgtcct 2340 gtacgcgccc ggtcgagccc cgggctgcgc aCCCCgctag gaggtgggta ctcgtcctec 2400 aggagttgcc ggtgagccct tgaccgtggc aggtcccctc cagccgcgag cgacccctca 2460 gtacctgccg atgcctgctg gtctctggca tcctccagtc gagggteagg gtcagggagc 2520 aaggcctcac gcgggcgccc tccttgcagc tgeccggcca gcccgcttct ctgcccggag 2580 ccatgaatctcagtagcgccagtagcacggaggaaaaggcagtgacgaccgtgctctggg2640 gtgagtggggactcaggctccttcccagagacacgccccacctccggtgcgcggcagctt2700 ggggcgcaggtgagcccctcctttgggaacgaatggagggccccacttccctccctttct2760 cctccgcaggctgcgagctcagtcaggagaggcggacttggaccttcagaccccagctgg2820 aggggaagcagagctgcaggctgttgcttcatacggtaggtgttcccaaaagaggggagg2880 aagatggtgtccgggaactttctggtcccaacggagggctatggatttctcccgtcggcc2940 ctcagggtgatgaggggcctctattttcaaccccgctcagatttgcttgggggagaaagc3000 caaagaggagatgcatcgcgtggagatcctgcccccagcaaaccaggaggacaagaagat3060 gcagccggtcaccattgcctCSCtCCaggCCtCagtCCtCCCCatggtgCgcatttccct3120 gctggctggaagactgctgtcagcctcaccctcacccttgggtggggatggacacacacg3180 agggtgcattcaccctacagaaatgagccatgctagggaggtagcacagcccatgcagaa3240 agccggggtccagcccagctCaCCCCttCCtagagctgtgtgaccttgggccagttaagc3300 tgtctgcaaaaattacactttaaaccaggggtccecaactgccgggtcaaggaccagaac3360.

tcgtcctgttaggaaccgggcagcacagcaggaggtgagcagccagctagccagcattac3420 CCCtgagCCCtgcctcccgtcagatcagagcggcattagattctcagaggagcatgaacc3480 ctatggtaagctgtgcgtgcgaggggtctaggttgcatgcttcttatgagaatcgaatgc3540 ctgatctgaggtggagcagtttcatcccaaaaccaccccccactcccaccccatccatgc3600 aaaaattgtcttccaggaaaccattccctggttccacaatgattggagactgctgcttta3660 aaccattcactgctagtgacaggaacatggtcgatctacatattggttgtgcatccagaa3720 attctctagtttctaataacttaagttttctatgcatataataattgtgaataatggcag3780 ttcttgccttttttttttttttttttgagatggagtttcactctgtcacccaggctggag3840 tgcagtggtgcaatctcagctcactgcaatttctgcctcccaggttcaagcaattctagt3900 gectcagccttcggagtagctgggattacaggtgtgcaccaccatgcctggctaattttt3960 gtatttttagtagagacagggtttcacaatgttgcccaggctagtctcgaactcctgacc4020 tcaggtgattcgcccgccttggcctcccgaagtgctgggattacaggcgtgagtcaccac4080 gccaggtcttttttCtttCtttCtttCttttaatCCtaCtgcactggctaggccctgcag4140 tggaatattgggtgaacatgagatcaggtctgacatccttatttccttcttgttttttaa4200 aaaagaagcatttggtaagtacattttgtcaggttaaggaagtccccatctaaaaccctc4260 tgcttttaaaaaattgctttgctttgaaatcactagagggggttaaattttaccaaatgc4320 ttttctattcatatgattctaggttctttagtccacgtggtaaattgtatgaagagaata4380 atatctgagttcctttgccatcctgggataaatactactggtcatgttacaaattctaga4440 tttgttttactcatatattgagcccttttttttacctgtatacatgtgaggttggcctat4500 aagtgatagtatttaggtgttcaggacatgctagcctcatgcactgagggtggagggttc4560 tctcctattccctggaacagtgaatggaagactaggatcagctgtccttggaggtttggg4620 agacccctctgtcaaactgccctcagactttcctgttctatttacacatactttgcaggc4680 gatctcatcctttcctgtggttttcaagaccatctaaacaaatgaagactcaggaactta4740 tttctgtaaactcaacctctcttgagctccaagtcctatatccaactaaatggctttgat4800 agatatctaataaatatcccaaatttaacatgtctaaatccacatattcaatttttatct4860 ctaatccacccgcctccccttcaacctgattttctctcagaaaacagctgtttttccagt4920 tagccaagacaaagctctttttttttttttttcccctgagatggagtttcactcttatca4980 cccaggctggagtgcaatggtgcgctctcggctcactgcaacctccgcatcccatgttcg5040 agcgattctcccatctcagcctcccaagtagctgggattacaggcatgttccactacgcc5100 aggctaatttttgtaattttagtagagatgggtttttgccatgttggtcaggctggtctc5160 gaactcctgacctcaggtgatatacccgccttggcctcccaaagtgctggatttacaggc5220 atgagccaccacacccagccaaatcctcttttttcccacacccatatctgatctaccagc5280 agtcctgttgtctctgcccccatcttataccccaatggaccacatctcatcatcttccct5340 gctaccectggtacaggtgacagttgcctgtggctccattttaattgcacagccttccac5400 CtggtCtaCCtaccatcacatggtcccctgtagtctattccagggtaggcaaactagagg5460 gcttgaatctaggctgctgcctggttttgtaagtagttttactgggaacacagccacact5520 cattcgtttgtaccctgtccatggctgcttttcctccctaacagcagatttgagtagtct5580 ccatggagaccatatggtttgcaaacctaaaatattaccttctggctcttaacagaaagt5640 ttcctggtctgtgctccacacagctgccaaaaagattttttttttttttttttttttttt5700 tgagacagaatcttgctttgataccagggctggaatgcagtggcttaatctcggctcact5760 gcaacctccacctccttagtagccgggacaacaggcgctccccaccatgcccagctgatt5820 ttttttttttttttgtaatttttagtagagacggggtttcaccatgttggccaggetggt5880 cttgaacttctgacttcaagtgatccacctgcctcagcctcccaaagtgctgagattata5940 ggcgtgagccactatgcccagccaaaaagatccttttaaacacaggttagatcatgtggc6000 ttctctgctagaatagttaggtcatggctctctctcatttggaataagagccgagagtgt6060 attatggcctgcttcgaagcctttgtgttctggcctcagcaacctctctgtttcaggtgt6120 gttttatgtcttatgttccaggtatgtatcttttacacagtatgtagctagattttgttc6180 tatctggccagtatagtctgtattcattgtgattgctgatgtaattggatttgtggcatg6240 taCtttgtaC CCCtaCtttC CttgCttttt tattttCttC tccttttcct atattttatt 6300 agattaatta aagttccttt tccccttctc tactggtttg gaagttatag aatctcattc 6360 taattttttt actgtttatc tttaattttt ttaacaatca tacattactc aaagtttaga 6420 attaatgttt tatgtcctcc cagacaatcc aaggagcttt tctgattctc ctttttttat 6480 ttttttattt tcgaaatgga gtctcacttt gtttcccagg ctagagtgca gtggtgcaat 6540 cttgactcac tgcaacctct gcttcccagg tgattcaagt gattctcctg cctcagcctc 6600 ccaagtagct gggattacag gtatgcacca tcatgcccag ctaatttttg tatttttagt 6660 agagacaggg attcaccacg ttggccaggc tggtcttgaa ctcctgagct caggtgatcc 6720 tCCtgCCttg gCCtCCCaaa gtgctggaat tataggtgtg agctaccgca cctggcctga 6780 ttctcatctt tttaacttca atattattat caagtgttct agctccaact tgtcagccta 6840 ctcaacacta ctcattacta gtactattgt attttccagt taattcttat gtaggtttac 6900 tttgtttacc aattagtttg gttaccactg cttctagcac ccacttcttc cttcttgatc 6960 taatttctta tcctttttag caacatactt tagtaagcct gagaatagta aacttattca 7020 ggctttgtct ggaaatatca tgattttgcc ctcattcctt catgatggtt tggctgtgta 7080 tataattcta gatagagagt ttccttcagc tttgaagctg ttctattctg gttcccaatg 7140 ttgctgttga agtcctcaat ctgtctgatt attccctttt tggagatgtc tttcctctct 7200 ggcttatttt aagataatgt cttttgtttt tattttctat agttttacca tgatgtgttc 7260 aggtgtagat ttattttttt tgtctgttca ggacttaggt tttcagacat gaggatctat 7320 gtctttgatc aattctggaa aattattggt tgtttttcct ttgaatattg actgtcttcc 7380 agtctcccta gtcccttttc caattagata tatgttgggc cttctcactc tgtcccccgt 7440 gttagctccc ccaatgtttc tttaaatctc tcectctctg ccttatactg ggtaattcct 7500 tcacagcatt attgatcata ctaattctgc ttctgctggt tttttctgct ttttaatcat 7560 ttgatcaggt tttcttttat ttttgtttta aagacaaggt cttgctctgt tgcctaggtt 7620 ggagtgcagt ggcctgatca tggctcactg cagcctcaaa ctcctgggct caagtgatcc 7680 tcccactcag cctcctaagt agctgggact acaggcatga gccaccatgc ccagctaatg 7740 tttattattt ttttgtagag ctggagtctc actatgttat ccaggctggt ttctaatttc 7800 tgacttcaag Ca.CtCCtCCC aCCtCagCCt ctcaaagtgc tgggattata ggtgtgagcc 7860 accatgccca tcctcagctt tgggtgtttt atatctagaa agctgcattt ggttcttttt 7920 cttttcataa tgtcttgttc ttatgattat gattcctact tttatattta cttggtttta 7980 aacatataca tatatatata tatgtacata tttctgtata tacatatttt agagacaaga 8040 tcttgctctgtcgcccagtctggagtgcattgttgcaatcatagctcactgcagccttga8100 actcctgagttcaagcgatcttccggcctcagcccccccgagtagcctgggctacaggcg8160 tgcaccaacacacccagctttatctgatatttttttagatcagtgttctaagttcttggt8220 cgggctagtctgcagtcacttgatttctttctgttgactctagctggattgtttcttgta8280 tgttttgtaattttatacggtgagctcatctttagtttattttgtttttttccaaaagaa8340 ttcgatgtggcctgagtttggggagtgttccaacagagtggtttcgtgtttgcttctgcc8400 atttacctcaggaatatcataagcttgggattttctgtacatttcttggcttggcagtac8460 tcactgagtaaattcagaccccataagtgagaggtacagctatggggtatgggctctcac8520 tggagacttcttttatttccattcatgctttgttgttagcttcctttaatggtggctagg8580 gttttgatttttgtttttcatttttgaggagaagagggctggtttgggcttttgagtctc8640 taatcctcaacacttaccttgggcctctcactaagggtatagcccttgagggtcctaccc8700 tccccatggtggtctcagctacaactctcctCCttgCCtgagcccaaggccttgtctcct8760 gactgtgaagattttgttgttgttctgttgtgttttttaaagacagtcttgttctgtcgc8820 ccaggctggcacgcagtggcgcaatctcggctcactgcagcctccgcttcctgggttcaa8880 gcagttctcctgcctcagccecctgagtagctgggattacaggtgtgcaccatcacacct8940 ggctaatttttgtatttttagtagaaacagggtttcaccatgttgcccaggctggtctca9000 aactcctgacctcaagttatccgcctgccttggcctcccaaagtgcttggattacaggca9060 tgagccaccgtgcgtgctcggcctgtgtgtgagttttgaagcaaaaagtcctggctgttt9120 tCaggtCCatttCCCCtCggttgCagC3CaCCagCtCCtgcacctgcctgtcttcatttc9180 ttttttctttttttcattcatcactaatcagaaggcatcctctcttcattttttatgtac9240 gaggattcttctgtcttactgttcagccatgcagtagaaacactgaattacatcctctct9300 ggaatttctaagtgtctctggctgcagagtttgttttcacttattacctcctgtctgaac9360 ttagagtttagaagctgtaagttattcactctaagtctcagtttcctcctctgtaaagta9420 tcagtacttacatcatgggtttcttgtgaggatttaatgagataaagcagataaaatgct9480 tagcagggtgcctgacacgtggcagaagctcaaaacaataagctatcattgtcattcgag9540 aaaatttgggaagtttggaaaagtataaatacaataaataccttactatcgactgacaat9600 tatggttagcactttaatatattttaaacctttattcttatgtatatccatacattatac9660 ataagacaaa agtagtactg tagaggctct tgtcatttat aagtgtgatg attggggttt 9720 cacgctcgtg tgtaaggtgt gcctcccaca aacctggtta cgagttggca catcacctgt 9780 ctgatgtgaa gaaagcaagc agcactgtac ataaaatcat gcatctgctt tctcgtttga 9840 tcggtgtctc agtctgccca agttgctata acaaaatacc acagactgga gggctttagc 9900 aacagacgtt ttctcacact tctggaggct ttgaggtctg agatcagggg gccggcatgg 9960 ttgggttctg gtaagggtcc tctccctgag ttgcagacgg cagccttctt gctatgtgtt 10020 cacgtgtggg gcaggagagt ggagagaaag agagtgagtt ctctagtgtc tctttttgta 10080 aaggtactag ttctatcatg agggtcctgc cctcatgacc ccaaacctaa tgacctccca 20140 aggcctgcat ttctaaatac tatcacactg gaggataggg cttcaacata ggaatttgga 10200 ggatgggggg aatggceata atttagccca tactaatcag attcctctat ctgacggctg 10260 cccttcagct tggaagceca tctgtgagct tagcaagaat gggattacca ggttcaaatt 10320 cttggacaga atccacctca gcaggggctg agtagtgtgt agggtttggg gagggaaatc 10380 aactcagtat ttctgcctga ggccagtccc cgagaggtgc caggcctaag tggccctgtc 10440 ttctctgctt cctccccctg caggtctcca tggtaggagt gcagctttct cccccagtta 10500 ctttccagct ccgggctggc tcaggacccg tgttcctcag tggccaggaa cgttatggta 10560 agtcagagcc tgcgatcagg aaggtccgtg agtaccgtgc taggcagggg ctcgggacat 10620 actagctact caaacactgg agggattctt gaatgttgga agaaaatccc caaaggcaac 10680 atgacagcca gcagcctgga ctggaaggca agggcgctgg tccctgttct tcttctttac 10740 ctgccatgac ctctgtaggc tgcagcccct cacttgtaaa cttcaaagag cagttgtgaa 10800 gaataaatgg gatattcagg aaaagcactc agcgtaatac ccagcactag ggaaccactg 10860 ttcaggatgt ggctgctgca gtgatgcaga ccatagcaac gcagaccata gcaacgcaga 10920 ccatagcaac gcggcatgat gctgactcct tcaaggtccc ttcaactggc cctcttttct 10980 gtatgattat gcctcattca tcagggtact ctcctgctaa aaagccttgg caggtcccac 11040 ttctcttagg ataaggtttc aattctttag ctatttgttt tagattcttt CCCCCtCtCC 11100 tcctcttcct cccctactct gcctaccttt agccttggcc ccagcccttg ccaatatgaa 11160 tCCCCCtCCt aCCCagCCag agCCdCttCC CCtgCCCtCt ttCtaCCa.CC CCagCCCrCt 11220 gcagggcttt cctagacccc ctaccctacc etggcctcca ctgttgggag ggccagaaag 11280 ggtgccgccc tgtacaggtg gcaggcaggt aaccactgtc aactccaggc taggattcct 11340 ccagggcagt gcttggagca acacggatca cagaatggga ggtgggcatt gattctgtag 11400 ctctgaagct gtgcccctgc atcctttccc atgctattac agaagcatca gacctaacct 11460 gggaggagga ggaggaagag aaggggagga ggaggaagag gaagaggaag atgatgagga 11520 tgaggatgca gatatatctc tggaggagca aagccctgtc aaacaagtca aaaggctggt 11580 gccccagaag caggcgagcg tggctaaggt gggggaagga gcgtggctgt ttggaaggaa 11640 gtggtacccc tacagaagca cttaagaggg gtgggCCacc gggagcctgg gccagcctcc 11700 cagaatgagt gtacaggatg ggccaaggcc acctcagcta gttctggcca ggagctcagc 11760 agggaccttg tggactttgg gaatetgttg tggctctgga ctttgtctga actctcataa 11820 tacactgttt tttggttccc agaaaaaaaa gctggaaaaa gaagaagagg aaataaggta 11880 actctttcta cctattaaat tagccaaagt ctccagctga gatatacagt gttagaaaga 11940 atactgtgct gttgggatgt aCgtgtacaa atgtacacac ggtgtgtcta cctgcactcg 12000 caggcacatg ggtatggaag tgctgaaggg tggcatcacc tttctggaag agcattacaa 12060 cgttcttatc ttgggatcta attccagtga aggcaattcc ttccacagaa ttccatccaa 12120 atttcagggg aaattacctg cactaagatg cttctcacgg ccaggcttgg tggcccacgc 12180 ctgtaatccc agcactttgg gaggctaagg caggcagatc acttgaggtc aggagctcga 12240 gactagcctg gtcaacatga tgaaaccctg tctctactaa aaacacaaaa attaaccgag 12300 cctggtggca ttcttgtaat cccagctact caggaggctg aggcatgaga attgcttgag 12360 cccggggaaa agttgcagtg agccgagatc gtgccactgc gctccagcet ggatgacaga 12420 gcgagactca gtctgaaaaa acaaaatttt aaaaagacgc ttatggcatt attcgaatag 12480 tgaaaaaatg gaagcattct aaatgtctac caatataaca attcactaag ctacatccct 12540 cttctcaatg gaatattaca taatgcttat gaagaatata gcaacctgga aagtatgtgt 12600 atagttttgg tttgtttgtt taatgagaca gggttttgct ctgccaccca ggctggagtg 12660 tgatggcaca atcatggctc actgcagcct tagcttcctg ggctcaagca atcctcccac 12720 ctcagctttc caagtagcta ggactatagg cacgtgccac tatgcatggc taacttttaa 12780 gttttgtgta gagacagggt ctttctatgt tgcccaggct gatctcaaac tcctgacctc 12840 aagcaatcct cctgcctcag cctcccaaag cactggaatt acaagtgtga gcctctgcac 12900 ctgataagaa tattgatagt tcatacagca ggacataaag tcatttttat tttatttcac 12960 atatttttaa aaagagtttg accaggccgg gtacggtggc tcacgcctgt aatcccagca 13020 ctttgggaag ccaaggtggg aggatcactt gaggtcagga gttcaagacc agccttgcca 13080 acatagcaaa accctgtctc tactaaaata caaaattcag ctgggcgtgg tggcatgtgc 13140 ctgtaatccc agctactcgg gaggctgagg caggagaatc acttagaccc ggaatgcgaa 13200 ggatgcagtg aaccaagatc acaccactgc acaccagcct gggcaacaga gcaagactcc 13260 atctcaaaaa aaaaaaaaaa agtttgacca agaaaaaaat aataaccctg aaagaaaata 13320 caccaaaatg tttagtgtgg gcagtaaaga aaactataag taatgtattt tcttgtctat 13380 ttgctatatt ttgtacaaaa tggttaatat tttataatga aaaagacatt tgtgggccag 13440 gtgtggtggt gcacacctgc agtcccagct tctcaggagg ctgaggcagg aggatcactc 13500 gagcccagga ggtcgaggct gcagtgagct gtgatagtgt cactgcactc cagcctgggc 13560 aacagaacca gactccatct caaaaacaaa acaaaacaaa agacatttgt gataactaaa 13620 tgaagatgga agcctaagga aaacacatat gcgtatgcat gcacacgcac acacatccct 13680 ttgtttaaag agtccgagtg gtccccagga ggagcagcca ggcttgcttt ccagggtggg 13740 cactgggagg gccacgccgc tggtctggag etgagctctc tccctgaccc caatcccact 13800 cctgctccgc tccaccctgt tgcagagcca gcgttagaga caagagccct gtgaaaaagg 13860 tgagtaggac cagagggctt tggcccttgg gacaggcgag tattctctgg agggggctgc 13920 ctggtatgga gaagggaacg ggaccctgga gccctgcctt ccctccacag gccaaagcca 13980 cagccagagc caagaagcca ggattcaaga aatgaggagc cacgccttgg ggggcacggt 14040 gcaaagtggg ccttccctgg gctgtgctgc aggcacaggg tgcccctgtc cagcccctcc 14100 acctgtgtct gaatgcaaca ggggtgttgc gggggcaaca tgagagcccc tcacccccaa 14160 ctctccactt tcaggaggcc cccagtgaag agccccacct cggggtcaca ataaagttgc 14220 ctggtcagga ctttccttct cttecctgga gccagcctcc ttgtccgctg caccagcccc 142$0 agtgcccggc agagggcagc cttgaaccgg tgcacccggg ccctgaggtc atacctgcct 14340 ccctgcaccc agcccccgcg gcctgagcct gctgtgtccc tcgtcctcgg cacccccaat 14400 tctcccccag tggcgaggga agagcctaga gtctgccttc tgctgagctg tgtgtcaggt 14460 ggatttccag cctgcaccct ccctctgggc agagctaggt ttataggcac ccaagggcta 14520 cggctgctca agctaccaga aggggcctcg ccctaggggg ccagccccca gggtcttctc 14580 ctgaccttat tcctgtcagg cagctactgt gtgcagagca tctataggga actcagggac 14640 atccgctctc cctgcttgct tccgttaggg gccagctcat cttataggga ccteccacat 14700 gtgaagatct gtgtcaggca ggagccagag gcccgcacct tcaaaaaaac ctttgaggtg 14760 gagtagacag ggtgagcttt acagaggcgc caagccccac atgctatcga gtaggcctca 14820 gtcaagcata ggggcgaggc caagagagga ctggaaaatg gggtggggga cccccaccct 14880 ctccctggtg tgcagagggg actctggagg gctgttcacc tgtgggtgac cctggcgcag 14940 ttcctagaac aggcggacac acagatgagc cctacactct gggttatcca tgcagcgcct 15000 ctgctggctt CtCCCtgCCC CtCCCCagCa CCCtCtgggg tcaggcccga agtgaaccag 15060 tggggagctg gtcctgctgt cctgcttagt acccccaggt atggggccca ggaggtcgga 15120 gctctttgaa cacctgccta ggaaagtcaa caaccaggct ggggcctcct gtccagctat 15180 agcttctttt gagaccagag acagaggtag cagagggcag ggttgtattc attttttttt 15240 taatttttat tttttttaga gacagggtct cactctgtca ccgaggctgg agggcagtgg 15300 cacagtctta gctcacggca gCCtCgaCCt cctggactca agcaatcctC ccacctcaac 15360 ctcccaaagt gctggaacta caggcacgag ccaccacacc aaaacaaatt ttaaaatttt 15420 ttgtagagat ggggggtctt gttaagttgc ccaggccagt ctccaactcc tgggctcaag 15480 agatcctcct gcctcagcct cccaaaatgc tgggattaca gacgtcagcc actgcaccca 15540 cccagggtgg CgttCtCCCt gCatgtttCC tacacccatg agacagatgt gggtgctgtc 15600 ctgccctcca acagacaagc cactaacttt aggtcaccca gagtcccacc ctccaacaaa 15660 gggacaaacc actaacttca ggtcacccag agagtgacaa gggggactgc tccatgtgag 15720 cagctggtgc tttttgaact tggtttcatc tacagtgacc cggggtaacc caattcctca 15780 cCttcaagtc acttacagtc tagtggaaac aaaacccaac acaatttcag ttactgtctg 15840 gtgctttgaa gggagtggaa caggtgaact tgaggggcag gaggaagcag tttggtgaga 15900 aggtctcctg gaggaggcga cggacaagaa gggctcaatg ggcttcatta ggagctggca 15960 gaggacgttc ctgggaacag gaacagagca tgcaaagggt ccgaggcaga gccccacttg 16020 aagggggatc ggctgcagtg acagcttcta atacccacga cccactcctt caaccctcat 16080 aaccgtgcct taaggggatt gtgactggct CCatttcttt ctctttttaa tttttttttt 16140 agatactggg tcacactctg tCagccaggt tggagtgtag tggcacgatc atggcttact 16200 gaagcctcga actctgggct ccgcctatcc tcctgcttca gccccctcaa gtagctggga 16260 ctacaggcat gcaccactat acccagctca ttttttttta acatttttgt agagatgggg 16320 atctcactat gtggcccagg ctagtctcaa actcctgacc tcacactatc cttctatcgg 16380 cctcccaaag tgctgggatt acaggtgtga gccaccacac ctggcccagc cccatttccc 16440 agatgaggaa agtgtggcac agagaggtta gacaagttgc cccaaggtga cacggctggc 16500 agaggagcca gggagtccca ccccagagcc ctggattttg accactctgc tgatgggagg 16560 gaggcatgag ccggtgcaca gtttatgaag tcgtgtaaac tgagagcagg agttagaagt 16620 cagtcaacca tgtaatggga gtcctcaagg gacagctagg cgtttctaca gccaagcgca 16680 tatttggccc caagcacaag aaggcgcgca acagataaac cagtgatcac ttgattttga 16740 tttgcaagca ggcagtagga aataaattgc aaaggtggag gccggatgca gtgtctcatg 16800 cctataatcc cagcactttg ggaggccaag gtgggcagat cacttgaggt cgggagtttg 16860 agaccagcct gaccaacacg gagaaacccc gtctctacta aaaatacaaa attagctggg 16920 tgtggtggcg ggcacctgta atcccagcta cttgggaggc taaggcatga gaatcatttg 16980 aacccaggaa gcagaggttg cggtgagctg agatcgcgcc attgcactcc agectgaaca 17040 acaagaatga aactccgtct caaaaaagaa aaatttgcaa aggtgagggt ccatcctcat 17100 tgctagggtg CCCtttgCCC tCtgCCCttt gCCCttCCCC tgCCCCaaCt Cttctgtttt 17160 tcagcaggaa gagggtgggc tgggctcaag caggtggtgg caagtggctg acctgcaggt 17220 gggctctgtg tttgcaccag ctgggctgtt aggagaggca ggcgtgagac gaccccagct 27280 ggggggtgtt gaacttggca ctatggggtg aggattaacc acagcagccc aggctacttc 17340 tcagttccct tatcacctcc tgaaccccac ccccccagca atgaatgtta ataaacccca 17400 CCCttCttCC CtCCCCCttt ccccgagctc actccagtca agggagagag tctgacagtt 17460 taggtcaact gaggctaagc cacaaaaagg gcccctgccc ccattcttgt ggcacttgat 17520 gcgtttctgt gagtccttta tctcagctga cgtggatggc ggtggttttg acagtatccc 7.7580 tgctggtagc catttccttt ttataaactg ggaccctgaa accagagaag tgaagggact 17640 tgccacaggt cacacagcgt ataaggacca gggcaaaggg gcggggataa aatcaagggc 17700 tCCatgCtgC CtCCCCdCtt ggggCCCa,CC aCtggCttCC CCatgggCgt aaaggagcaa 17760 accaagttag aaggccaggc ctgcaggtgc ccaacaggaa gggacaagga gccacagctg 17820 tcttgcctgt gacacaggcc atccagccat gcccagagct aaccccctgg ctaagccccg 27880 aggcccagct tgactgctgg catctgttac catggagacc caggctggcc tgagggctgg 17940 gccagtgatg gcaggccctg tccccatgga tagaaacagg tgcttgggct cagaggcctt 18000 gagtggctcc cactgtcccc atggccagtg agtcccgaca gcataaattg gaaccgttac 18060 CCICCtttCg CCCCCCagCt gaCaCC'tCCa CCaaCCCaag gCCtgagCtg tCCCCtCCaC 18120 gtgtctgtgc tctctttaat gccctgcctg ggggctggga gtggtgagga tgtggatgtg 18180 aggttgaagg tttctcaggg aatgagccag agctgccaga agaggcagag tgtaacccag 18240 actgcagatg atgggaagaa cgcggaacag aagtgacctg aaggatccgc agggggaaag 18300 cagagaggtg ggcacgcggg cacctggtac cttgtcccag ccatgccacc agctagctgt 18360 gaggctttgg gcgagtccct tgccctctct ggctctcatc caaggaatga ggaagttgga 18420 acaaatgatg aatccetaag accccttcta ggtttgacat tctttgagtt gcattccaaa 18480 accctggact cccccaggta agcaaggcca gggCttgCCC CatCCtCCCC aC2.CaagCtC 18540 aggcagcacc cactcctggg ctgggttccc gaggaagagc ctgcggagag gagaccccgg 18600 agctgcctgc actggtcagt gcatgggggc aggggtggca gaccactttg tggattgatg 18660 gagctcagga aggtgagaag ggacccacag gtgagagttt cgctcccctg gtcatctctt 18720 taggtaaata aatccacatc cgccacttcc ccttcccttc ccaccctggg ggcgctgaga 18780 actccaggga gcccagagct gaggcctgag ctctgcttgc tcacactggg tcttccctca 18840 gagaccccca agccctccta tcttctgcag tcaccgtcat ccacttttct gtagggaggg 18900 aacagcatgg agctctctgt tcaccggtct ccaggacctc ggattccacc tttaatcctg 18960 aaaacccagg aaggcttctg tatccctaca atgaagcagg tttggggctg gatctgcagg 19020 gtggcaactc aatccatgca gaacagaaga agcatggact tttccattct ggctattcca 19080 ttcactagct gggccattct gagcaaacta cctcccaatg tgcctcagtt tCCtcatctg 19140 caaaatgggc tagtcgctgg cattgtaccg agcagtatga gaccggaggt catgggaaga 19200 ggctggtagg cacttcatcc caggcagctg ctcagggaca tgggacacag ggaggggact 19260 ccgagctgct cctagctcag agaggctcta gggacaggca ctggaaggaa ggggatgcag 19320 aatggtgagt ggagctgggt ctcagaacac agacatcttg aagtctgcta tgtgctgatt 19380 tcacacttga ccccccaaca ccctgaggca ttactgtcct catttctcag atggaactac 19440 agaggcccaa ggaaaagggg cttaggccag gacacccagc tcttctccaa gtgtctgtct 19500 caggctagct tttgcaactc tccattggga ttcttcctag atctccatct gtacctgcca 19560 aCCCdCCtCt gaCCCCCaaC CtgttgCgCC CagtatttgC tgccgatcag gacagcctta 19620 acccctcctt cccgggcaat cagctgctcc aagccaagcc cacccctgec ccctggaggg 19680 aggcggctcc ttttaaggct gcttctggga atttccactc cagagccaga accagagacc 19740 ccagccccac ttgacacctg cggctcaccc ttggggaggt ggggcgccag acctgagtgc 19800 aggagacgca gacctggaag ggctccccct ccctgacctg ccacacatcg agtttgtctg 19860 cgtegagttt ggccagtctg tgagggtcag gaatagagca ggagacagca gggccacctc 19920 cttcagaagg cecccaccgc tccatccctg c 19951 <210>

<211>

<212>
DNA

<213> musculus mus <400>

ggatccctgttgcagtcataccctatgggaaaagagcaacttacctatcttaaggagatt60 gggggaatttagatatttgtgcatcctctttcacttgaatgagaacaatgtaccagatcg120 tcaacagtgcacatttgacccggccagtagcaacatacctaaaactacctcaccattgta180 aaacaccctaagaatcacaagaaacttacagtttttcagagacaaatcaaggagcagagt240 tacagaattaaaaacaattcatcataccttagaattttctctaatcgaatgagttgataa300 ttgtttccatactcttagccatctttggagctatactttgaaggttaaaaatatcctaga360 ctaagttagttttcattgtaagtgctagcagcctttgctttctggtgtgaaataagaact420 aatttcaagtagaaagcacagagttcaggagagatgaagactatgttgcccaggctagtc480 ctcagcccataactgggttcttgggctaaattctagctgctccattggaaataagcaaga540 cagtgaattaagcacacacgaagagtactaaacgttgcgtggccaaggccgagaggctga600 gggtctgcctagtaacggtaacctttccctgatctctggtttatcgttttgaagaccttt660 tcagaaggagtagtctgtctgtatgtccttccatcccaagcaagtgaagagcccagagga720 gccactggatatcaagctaggtctccaagcagttatgtctaaggagcttcaggctttgtt780 tgcgaggttagaatggatgtagcttgtccagatgcctcctcagtgatgttgttgttgata840 ggttgcccatgtgtttcttttcttaccaggtttccctgtggtctctgcttgtgaccctgg900 tggttcttttcataatgactggcaccatgctgggacctgaactgctggcaagcattccca960 caactgtttacgtggtcgccatttttatgcctctggcggctacgcctcgggttatggctt1020 agctaccctcttcacctccgcccaactgcaagaggactgtgtgtctggaaacaggaagtc1080 agaacgtgcagcttctgcactgcgattcttaaacttcccgcctcgctttataggtagcat1140 gtacatgttcccttctgctctacgccttcttccagtctgccgaggcaggggtcttcgtgt1200 tgatctacaaaatgtacggaagtgagatactgcacaagcgagaggccctagacgaagacg1260 aagacaccgatatttcttataagaaactgaaagaagaggaaatggcagacacctcctatg1320 gcaccgtggggacgcatgacttagtgatgatggagaccacccagaccgccctctgactga1380 ggagatacacgggagctgaaacatcacttcctatttgtgaccattggtagcgagtatggt1440 tcgcatccgggataaagatgggttgacatttcctgtaacagatttgctcttcccactgta1500 atgtagtatctcagtattacccatgtgtttttctaactcaacagagtgtcccaatattgc1560 ttacacctggatcagctaaagtgccgcgtcctctgcttaagtagtgtgctgtttgtttgt1620 ttgttttttgttttgttttgtttttttccatttccaccagcattgetacagataggaaat1680 ggggttggaaatgtttgtaaaacagaaccatgggtttgttcaacttacaaacaaccgatt1740 ctgttcagggcgagcctgtattgagaaaagtccaaaacgggtcaaaaagggttgaaacga1800 caggatagcattgcatcgtcaagccagagaaaaccgtattaatgtgtgtgactacttgat1860 ctagtatctattgttaatggccatcaacattgtgcaggggtgaaaggcatttttccccat1920 atgtttcctgtatgtgtataaacgcatctcagctccatttatcgtctgaaggaatgattt1980 acttaggaaaatgcgtagacctcacctcagggagagaaaatgggccactttgttcatccg2040 tgggaaagggctgtggctacaggctttccttccggaaaggcctgtggctggacactgtcc2100 cactgctctggtagactggagctgtgatctgagacaacctaagaggttcagagcagtctc2160 ctaaccttggtattttgctccctaatcagacacactggcctcccttgtcttcttcatgac2220 agacatctggagctacagacatgggggcccacctggctcggctaatctcggtgatgattc2280 tggggttgaattctcatctcatctagttcccctacaaatccttgctgtggctagcaagga2340 aagctctttttctgcatccacgagggagtgggggtgggggtcgcctcttaaccagtgtgg2400 ggaaggttttgctcctcatggcaacagcaggtggtagggctttttctaccagtgcgcggc2460 cgcctatttaacgcagcgtggagggcagctgggctgcgctgatggctgcctgggcgggcg2520 aggcgcgggacgcacccatgttcccggcgagcacgttccaCCCCtgCCCgCatCCttatC2580 cgcaggccaccaaagccggggatggctggaggttcggagccaggggctgccgacccgcgc2640 CCCCCtCCttCCtCCCCggCtaCag3CagCtcatggccgcggagtacgtcgaCagCCB.CC2700 agcgggcacagctcatggccctgctgtcgcggatgggtccccggtcggtcagcagccgtg2760 acgctgcggtgcaggtgaacccgcgccgcgacgcctcggtgcagtgttcactcgggcgcc2820 gcacgctgcagcctgcagggtgccgagccagCCCCgaCgCCCgatCgggttCCtgtCaaC2880 CCCgtggCCaCgCCggCgCCgggagatccccgcgatcctggcagaccgtagccccgttct2940 cgtccgtgaccttctgtggcctctcctcctcactggaggttgcgggaggcaggcagacac3000 ccacgaagggagaggggagcccggcatcctcggggacccgggaaccggagccgagagagg3060 tggccgcgaggaaagcggtcccccagccgcgaagcgaggagggcgatgttcaggctgcag3120 ggcaggccgggtgggagcagcagccaccaccggaggaccggaacagtgtggcggcgatgc3180 agtctgagcctgggagcgaggagccatgtcctgccgcagagatggctcaggaccccggtg3240 attcggatgcccctcgagaccaggcctccccgcaaagcacggagcaggacaaggagcgcc3300 tgcgtttccaggtgaggccagcctgatggcctggacgcctccagaattgtagggctcctt3360 cagggctaagctggtggctctgggtgatgcagaacatagaattcttccatgccatccgtc3420 tggttttgtttgtttgtttgtttgtaacatgtttggtgttttgattgcatgttgtatctg3480 tacacttcgttgtagtggagagatgggagcagaagagggtgtcggatccggatcccctgg3540 gactggcgttttacagatggttgtgagtcaccatgtgagttttaggatcggaattacggt3600 cctctagaagaacagggtgttgtttcacagctgagccatctctccagctctttggcatat3660 aggattttgcagccgctgcctgttaatacaatgggaggcgtttacacaataaaaaccaac3720 ccatatgtgtcctgacccactggcagcctctgctcctggggaatgccagttgtaattatt3780 ctgatcacataaacgctacacatgaggtctccgcggagaatgcgcacagtctgggtttgg3840 accaaacttcagatggctgaaggaagataagtgcacacatggcagaaacataatcttttg3900 aacttcgttgcggggagagtCggtttCCCaaggCtCCtttttttatttCCCCtCtagatg3960 atctgtcttggttaacttgccggcttgttctataccagccccttCCCttCgtttCtgaag4020 ctgtcaactgaagcttctctctcccaaacttgcctggcttaaaaaacaaacaaacaaaaa4080 caaaacaccccccccaaaaaaaaaaacaacaaaaaaaaaaaagaaaagaaaaagaaaaag4140 aaataaaagaaaaaaaaaaccactctccccattcatcgaggccagccactgctaagctgt4200 tggatggtcttgagttgctgcctgtgctagcaaacaaggaggcacaaagagtgctgtagg4260 tcgtatacccccaccaaagaaatggagagccctgagctccaggagaggactctgagacat4320 tccttgtttttcagtcatttcaaggctggtgtgtttgaggttggggtggcagtggaatgg4380 ggtgtcagaaaaaatagaaaagtgcttggcggttgctgttcacagctgggtgtgatctct4440 taggcagaaatcccaagttttcgggcctctgtggtggtcgttcacctataaaaaattgca4500 ttaagagttcttccaagccctgccactcctaaagacttagttataaaaacttgtttccaa4560 cttgtttgtcactaagtgggaagcttgggaagtttaagaaccaggtgctaacactatgta4620 gttcataccaaatgagctagacttgggtaggtagcgggactcttttggaaacttacctag4680 catcaaggaaaatttagtattggttgaagactttcaaaggttttagaagagcctttctct4740 ttcggcataacaactttcccatgtgtgagtgtcctaatgcatcgcccacataaaatgcca4800 cgggaagaatcccaaactctaaaccgcacgatttggcttctcccttgtctgaggggggaa4860 aaaccacttatcggtctgctgctatatgaactatcttgtttggcctccgtttacatattt4920 gtttgattgagctattagttcacctggttaacttagaggttgacccaagtctaaccttac4980 taccacggtaatcttaaagtatcaagtggaatgtggtcccaggttctgaaaattagggtc5040 actcgggcatcacttgcttaaagtctggtaccctgctgttcagttcttagagcagaagta5100 cggctactatcactgcaaggactgcaaaatccggtgggagagcgcctatgtgtggtgtgt5160 gcagggcaccagtaaggtaagagacaccgtgcagccctcctgctctgctgtgttgccgag5220 tgtctgctccatgccgatgtctttctcctcgcaggtgtacttcaaacagttctgccgagt5280 gtgtgagaaatcctacaacccttacagagtggaggacatcacctgtcaagtaaaccaaac5340 gtttgcattttggaagaggggtttggtgcacgactttgagtatatttcctgaaggaggtg5400 gtttccagtagctttaggctctaccttttccctcctccttccttttcatttttgactagg5460 ttggtggtagaaagtcccctccactgtaaatggggtgtttactcccttctgctgttgtaa5520 aacttgattgcatgccctctettgcatctggttaccttgttagcagtagaaagggcttgc5580 ttaCCtggCttCttCCCaCtcggacctaagggaaaacatattgcaaaacagagtgccttt5640 ctgctagcttgagatggtacacattaccccaatgctacataggaaacacattcccaagtt5700 agcatatgaaacacaagaaattgagctctggcttttcttgagagtttacaaagggagttt5760 cctgtaagaccatcctacactgtctagctctatgcagtttacccataactgtggctaaga5820 gtttgcttgcttagtattaatttagcactgtgccaagggacttagataaccttgaaaaca5880 tttacctgttaaaattaatgacagagataaaggaattcgaattccacatctgagagccca5940 gtgcacttaaagttggtaattggagaattaattaccttagggtgggccctgtgaaaccga6000 gaatggaaagccactaaagactccatctagaaaaggggactgtagtcacttttctacaat6060 aaggggccttaaacttccctaagcttccctgcacttggttctcagtgcccagcacacagg6120 ccacttgttctgtaatctgttttgaagctccaagaatcgagtggagacagggctcaccct6180 ttgtactttt cactccgatt tttcagaagt tgtaaaagaa ctaagatgtg cctgcccagt 6240 cagacttcgc cacgtggacc ctaaacgccc ccatcggcaa gacttgtgtg ggagatgcaa 6300 ggacaaacgc ctgtcctgcg acagcacctt cagcttcaaa tacatcattt agtgagagtc 6360 gaaaacgttt ctgctagatg gggctaatgg aatggacaag tgagctttct cccctcttca 6420 cctcttccctttccaaattcttcatgacagacagtgtacttggatataaagcctgtgaat6480 aaaaggtattgcaaacaagtttgaggctttatccaattcatgtgtcagtttgaggggtgc6540 atgtgcggagagtcaataactttcttaacatttgttgatgagagtgagtcaggctgactt6600 aaggaagttaaaggcacctcattcaacaattaagatttttctttctttttgtttagtttt6660 attttatttataaatatatgagtacactgtagctgtcttcagacacaccaaaagaaggca6720 tcagatccca ttacagatag ttgtgagcca ccatgtggtt gctgggactt gaactccgga 6780 cctctggaag agcagttggt aaaccccttt cttaactgct gaaccatctc tccagcccaa 6840 atcttaaggt tttacagaca agaatattac agg 6873 <210> 12 <211> 4090 <212> DNA
<213> MOUSE PSOOL
<220>
<221> misc_~eature <222> (1) . (4090) <223> n equals unknown <400>

ggcgggcgaggcgcgggacgcacccatgttCCCggCgagCaCgttCCaCCCCtgCCCgCa60 tccttatccgcaggccaccaaagccggggatggctggaggttcggagccaggggctgccg120 acccgcgccccCCtccttcctccccggctacagacagctcatggccgcggagtacgtcga180 cagccaccagcgggcacagctcatggccctgctgtcgcggatgggtccccggtcggtcag240 cagccgtgacgctgcggtgcaggtgaacccgcgccgcgacgcctcggtgcagtgttcact300 cgggcgccgcacgctgcagcctgcagggtgCCgagCCagCCCCgaCgCCCggtcgggttc360 ctgtcaaccccgtggccacgccggcgccgggagatccccgcgatcctggcagaccgtagc420 CCCgttCtCgtCCgtgaCCttCtgtggCCtctcctcctcactggaggttgcgggaggcag480 gcagacacccacgaagggagaggggagcccggcatcctcggggacccgggaaccggagcc540 gagagaggtggccgtgaggaaagcggtcccccagccgcgaagcgaggagggcgacgttca600 ggctgcagggcaggccgggtgggagcagcagccaccaccggaggaccggaacagtgtggc660 ggcgatgcagtctgagcctgggagcgaggagccatgtcctgccgcagagatggctcagga720 ccccggtgattcggatgcccctccccgcaaagcaccaagcaggacaagga gctcctgcgt780 ttccaggtgaggccagcctggnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnn840 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnn900 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnn960 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1020 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1080 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1140 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1200 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1260 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1320 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1380 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1440 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn nnnnnnnnriri nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1680 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1740 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1800 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1860 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn.nnnnnnnnnnnnnnnnnn1920 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn1980 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2040 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2100 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2160 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2220 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2280 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2340 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2400 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2460 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2520 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2580 nnnnnnnnnnnnnnnnnnnnntaccctgctgttcagttcttagagcagaagtacggctac2640 tatcactgcaaggactgcaaaatccggtgggagagcgcctatgtgtggtgtgtgcagggc2700 accagtaaggtaagagacaccgtgnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn2760 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnntctttctcctcgtaggtg2820 tacttcaaacagttctgccgagtgtgtgagaaatcctacaacccttacagagtggaggac2880 gtcacctgtcaagtaaaccaaacgtttnnnnnnnnnnnnn.nnnnnnnnnnnnnnnnnnnn2940 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn_n_ nnnnnnnnnn nnnnnnnnnn 3240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3360 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3420 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3480 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3540 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3600 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3660 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3720 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn3780 nnnnngctctgagttttcagagttgtaaaggaactagatgtgcctgcccagtcagacctc3840 gccacgtgtaccttagacgcccccatcagcaagacttgtgtgagagatgcaaggacaaac3900 gcctgtcctgcgacagcaccgtcagcttcaaatacatgatttagtgagagtcgaaaacgt3960 ttctgctagatggggctaatggaatggacaagtgagctttctcccctcttcacctcttcc4020 ctttccaaattcttcatgacagacagtgttacttggatataaagcctgtgaataaaaggt4080 attgcaaaca 4090 <210> 13 <211> 7405 <212> DNA
<213> Human <400> 13 tagatgaagatgaagatacagatatttcttataaaaaactaaaagaagaggaaatggcag 60 acacttcctatggcacagtgaaagcagaaaatataataatgatggaaaccgctcagactt 120 ctctctaaatgtggagatacacaggagcttctatcttgctgaaatattgcttcatattta 180 tagcctgtggtagtgcacatggttaacataaaagataacactggttcacatcatacatgt 240 aacaattctgatctttttaaggttcactggtgtattaaccaaacgttgtcacaaattaca 300 aatcaatgctgtaatataatttgcacctggaatggctaacgtgaagcctgaattaaatgt 360 ggtttttagtttttaccatcaccaatttctatgactgttgcaaatacagaatctattaga 420 aaacagggtcttggaaatgtagaattttggcgcactatgaggaaaaacaagctatctttg 480 taaagcataattgagtttaatgtaattgttgtaaaaaaaaaagtgtgcttgctctactta 540 aaattcctcacaatgttgaattttgacctgtattcagaagaattccaaaacaggtcagtt 600 aaataaggaaatatagtatttgtcaaaccagtatcagagaaaagttacattaatgtattt 660 gattacttgatctggtatctacttattaatgaataatcaacatttttctagtggacaagc 720 catgttcttttcgactggtcttctgcatttatgtaaacacaccccaaatcaatttatctt 780 ttgtaggaatgatttggttgggaaatttttcagaaccgctctggagcagaacaaaacggt 840 acctcccggtcaccactgggactgggggaagggacacgctccaggggagaaaacaattcg 900 cctagatgaagatccctgggtggttctccatgcactcgccgagggggctcagtgggtagc 960 ctcttagagcagctctgagaataaacttatcatctgagttaacgggtaat~agacccagaa 1020 cagttcccaaaccttggcactttcgctcacttagccagaggcacccggcctggcctcggc 1080 ttcccggtgagggagcgggggtgggggggatgcgcaggcacctggaacaatcagggcacc :1140 gggagaagccggctcagctgatgccggtgatgagtttctctcattgaaatectcctcacc 1200 tcgggcgccttggttcccttacggatcagccctttcatcacaaagaaagccctctttcca 1260 gatcatctaagggtcattgtgccaacatccgggcgtggagagtttcygtagggagaagga 1320 cgaagaggggCCCCCtCggCggggacgcgggccggtggcwggaagggcgtggagggcggt 1380 gcagcgtgcgagCCCCCgCCgagggccatccccgcctccgctcggccgcccgggcaagtc 1440 gcctatttagggtgcggcggcgggcgggagCagtgCgCCCatggCggCCCtgggggacga 1500 ggtgctggacggttacgtgttcccggcgtgCCCCCCCtgCtcgtaccggtacccataccc 1560 cgcggccaccaagggcaagggcgcggcgggcggcagctggcagcascgcggcaggggctg 1620 CCttCCCgCCtCCtCCCCCtgCtCggCgggCgCggCCtCgttgtccttcccgggctgcgg 1680 gcggctgacggccgccgagtacttcgacagctaccagcgggagcggctcatggctctcct 1740 ggcgcaggtggggccgggtctcgggccgcgcgcccgcagggccggcagctgcgacgtggc 1800 ggtgcaggtgagcccgcgcatcgacgccgcggtacagtgctcgctggggaggcgcacgct 1860 gcagcgccgggcccgcgaccccgagtccccggccggccccggggccgagggcaccacggg1920 tggcggctctttctcccagcagccatcccgtcgaggcctggagcagggcagcceccagaa1980 CggCgCCCCgCggCCCatgCgCttCCCgCgCaCCgtCgCCgtgtactcgcccctggcctt2040 gCgCCgtCtCaCCgCCttCCtggaggggcccgggcecgcggcgggcgagcagaggtccgg2100 ggcgtcggacggagagagggggccgccgcccgcgcggcttcaaggcccagaggaggggga2160 ggtgtggacgaagaaggcgccccggcggccgcagtccgacgacgacggcgaggcccaggc2220 cgcagtccgagcgagctgggagcagccggccgacggtcccgagctgccgccgcgagaggc2280 ccaggagggcgaggcggctccgcggtcggcgctaaggagcccggggcaacctccgtcggc2340 ggggagggcccgagacggcggcgacggacgggaggcggccgtcgcgggagaggggccgtc2400 gccacggagcccggagctgggcaaggagcggctgcgcttccaggtaaagcctagggcggt2460 cagggcacaggggagcccgggggtgcgggtgtcttccttgggcctggccctgtgactgct2520 tcgggcactcggaggtgcggcgcttccctaagcgtgggcyacttccgtatttccgagaca2580 gccaatgaccgcgataggtgtcttccttgacagcacagtctcatgtccccgacatccaga2640 cttactcgtggcggctgctccacgggctggccagggcgacgcccttgggacgttcttata2700 acccacatatttgcactgtaaacctcgcgcagtgggcgcataggccagccctgaccgcac2760 ggttggattacctatcagtaggcacaactgaacttcggagcacttgccggctggagagtc2820 gattcccaaggatccctctctcccatttccgcactggatgtggcaaaaacccttcaactg2880 ctgggattctggtcagcaattctgatttctcctttacgagcttccaggctattggaaagc2940 tggagctcctaaaatgccccttcctaggaatttgctttgcttttaagaagcacccccaac3000 tcagaaatccaatactgcgaaagcatttggaCtgCtCagtgttgCtgCCCgagggcagca3060 ggctgaaactttaaagggctggggcacgcagagggcagttgtgacctagcagaagtggaa3120 aggcacaagaggtggtaagaagcccgagggagtccctcgcggtCtCttCCaCggCCaCCa3180 caggctggtattccttttgaggggcggggttggtggcggtaggctgattgtgcgaggagt3240 gaatcgagaggccagggcttcCCagcgtggctgtgcaggagctgtgtgtgatcttaggcc3300 agtcataaccttectggacttagcgcagtctcacaggtgagcagactgaattaagtgctc3360 cccagagttccttccaattctgaaaggctaactctaaaaacgtgtgcataactgcttgct3420 tactgggagggaagaagggaagtttaagtaacactacttttgttcatattgaatatgaat3480 tatggcttacgtacgatttaggttcctggcaccactgtttgggagttaactagcagcatg3540 aagaatgtgatcttgggtgaacctttaaagttccttagatgtggagtcttatttttcttc3600 , agcttaatacatgtgtgcatagtctaagatcaggctttatcttaaaaggccttcctacag3660 aatcccaaactttagagaactgtttattatgtccctacttattcgtttattagccagcct3720 tatgaactgagttaatctggtatatgaactctaaggcccatgcttgcttaattgtttgac3780 taggctattaagctcacttaattactgtattgaagaggcgtacccaaacctgacctgctt3840 tctttcatgatctgaagttgccaatttcaaatatcaagtagatccttcccaacgtctgaa3900 atgaaggataattcaggtgttttgttggttaaattgacatatcctgttgttcagttctta3960 gagcagaaatatggctattaccactgcaaggactgcaacatccgctgggagagtgcttat4020 gtgtggtgtgtacagggaactaacaaggtaagaaataccaggtaactggcatcttcttgc4080 tgaaagtgtcaaggcgattttaagtttatcctctttgtcatcacaggtttacttcaaaca4140 gttttgcagaacttgtcagaagtcttataacccttaccgagtggaggatatcacctgtca4200 agtaaatcagatgttttgcattttgtctgacctgggcagtcgtcgagggtttttagtata4260 gtttgagtatacttccaaaaagaggccaggcccccagaccttaggtttcaactggctttt4320 gttaggagtggtagaaacaatactcagctgggaaacggggccttggtgttagcttctttc4380 tggccttgcaaatcttgctgttgttaacctcttctaaaactgttaacctcacttgcaata4440 tggaagaatacttgtcttacttgctacttagtctaatgtataagaaaatcaacaaaaaca4500 tgcttgtcagctaacatgaggtagtcaaggttgactgttttaccgaaacgcttcttatga4560 agcacaaccttaaagtacttaagcacagggggttagtttgtcttgcctgaaagctcacaa4620 agggacagtttaagataaatctaagttgtctagctttatggggagttgactataatggta4680 agcaagcaatatgttaactaagcattgcttaagcgcttgcttgctattaactgtgctaag4740 gggcttagctaatctttaagaggaaagaagtgactacattcgcctcttgtcacacagcta4800 atggagtctgaattgccagttgagacagcctaatcaatacacttgacccacgttggatat4860 ttaaaagcattaacaccctggggtggtggagagaaactaagtatggaaagccacttagaa4920 tcacttagatcagagctgggcatgtttctaaaagaggatgccttaaccactctgctcttg4980 gtgttcattgtcaaattcatccctgacttgttctctaccctttctcttaaacagttgttg5040 taaaagaaatttcacaattcataattggatctgatgcaatatagcagcagtacagcatgg5100 ttaaacacccactattcctagccctgtcattgctacgtaggtagggatgtagagggaaaa5160 caagattactatgggaccttgcttagagcacattcattaagtacttgaatggactagaaa5220 aatgttgaagtcctaggaaatcactaagggtttatcttctgcatgcccttctgtattttt5280 ttcccccagagttgtaaacaaacgagatgttcctgcecagtaaaacttcgccacgtggac5340 cctaaacggccccaccgtcaagatttgtgcggtagatgcaaaggcaaacgcctgtcctgt5400 gacagcactttcagcttcaaatacatcatttaggtgaaagtcagtgttgctgtgcatgcg5460 ctgatggagtagacgagtgagCttttCCgtgCCtCtCCtCCa.CCtCtCCCttctcaaaat5520 acttcatgaaaggcagtgtattctgaaaaagccttcaaataaaggtattgcaacacgatt5580 tatacattgcataaaatctgtctttgaaaataaagtttcaagagcgcttgtcttgtgcta5640 acagtctgggcctgtcacttcacctttatgaatgcttgctgatggcatagagtgggccag5700 gctctgagttaggctgcagccacttggaaaacaatttaggggggtgcttgtagacgaggt5760 ctacttatttaggcaggtctggaggactgaagcttagaaggaagttaactgaataaaaag5820 ccgcctagcgatcgcgccactgcactccagcctgggtgacagagtgagactccatctcaa5880 aaaaaaaaagctgcctagctgtaacattaaggcattcttttgggagaggtggaggcagag5940 ccatttattggttgcatgagaccgttggaggttaacgttgagtaagaatgctgagtggcg6000 gtgatggggtaggtaaaggctttagtgtccaggtgaccttaggaggtaagctactaggtg6060 gagggaggctgggaaaactaacctggctcactagtcagtttcacaaatgtggcaaaagtg6120 gggcttggaacagggtggctgtgggcagctagctgcttttaagacactgaataacctatc6180 aagtagactttgtgtttcttcaaagccttttttttttcttctttttttaaagtaggcctc6240 ctaaaatgcacttaaagatgtcaagttagaggtgtaggccttagcttttgtcttcactga6300 cttagtgctagtcgggatgccagactctaactgcgtctagtagcttctcatgacaacact6360 gaggccccactgccagaatgttcctagttgaggatgggactgagtttagagcctcaggtg6420 catctgatgaaattaaagttgtagtattggtttaattacagaaaccatataattggaacc6480 atgtgctaattatgcctccaattctctaacaaacatcccacttaagtgaaccccttacta6540 ccttaggccataggactaagtcttaacatcttggacacttgttaaaagggaccaaagtga6600 gtttgaggcctccatagaatccgtatctcaaggggaaaggcccacctgcatcaatgtgga6660 cagagatggtgcgtgtaaaatgcagattactgggcacctgtcctacctacatttaatagg6720 cgccccagctgttgctaatagaattgaagcatgagcacctgtgctcggtagagaagaggt6780 ggcatctcatggagcttccagtagagtgggcagatggccactagtttttatataaatgta6840 aaactgaggttctgacctgtgatctgcttatgtgcctctgcagtaagggacctgacttag6900 ggagatcagggaagccctgcttaaaagaacgatacctcagctaagctccagttcaaatga6960 gtcgaatgaggcaaaaatggagaggggaaaatttccagattaggttaacaacaatctgca7020 aaagccctgtggtagaaaacaaccagggggaggccacagtgtgagaatactttggaggac7080 atgacatgccaggtgaatctagcagcgccagactggaagggtgttttaggccttcattag7140 gaggatggtctctactggagtgcaggggctactcacaggcattatcagggaacaccatgg7200 CCtCaagC'tCCtgggC'tCaagtgatCCatCCtCCtggCtCagCCtCtCaagtggctggga7260 ctacaggcactttgccaggctgcactaaaaggttttaaaggtatggtggggatagtgggg7320 catggtggca tatagattaa aaaatcactc cagctgtaca gtggatacct gtagataagg 7380 agactcacag ggacctgata aagtg 7405 <210> 14 <211> 1118 <212> DNA
<213> human <400>

ggggggagctgggacctaagCCgCgCgCdCaCCCCtttCtctgcgtctggtggaggtgca 60 cagaggcttttgagtcaggcccaagcgcagccaggtgacctccccgcggcctttcaagcc 120 tgagCtCggtggaCagCtCCCtCtCCCgtgagtcccgctgtcctgtacgcgcccggtcga 180 gCCCCgggCtgCgCaCCCCgctaggagctgcccggccagcccgcttctctgcccggagcc 240 atgaatctcagtagegccagtagcacggaggaaaaggcagtgacgaccgtgctctggggc 300 tgcgagctcagtcaggagaggcggacttggaccttcagaccccagctggaggggaagcag 360 agctgcaggctgttgcttcatacgatttgcttgggggagaaagccaaagaggagatgcat 420 cgcgtggagatcctgcccccagcaaaccaggaggacaagaagatgcagccggtcaccatt 480 gcctcactccaggcctcagtcctccccatggtctccatggtaggagtgcagctttctccc 540 ccagttactttccagctccgggctggctcaggacccgtgttcctcagtggccaggaacgt 600 tatgaagcatcagacctaacctgggaggaggaggaggaagaagaaggggaggaggaggaa 660 gaggaagaggaagatgatgaggatgaggatgcagatatatctctggaggagcaaagccct 720 gtcaaacaagtcaaaaggctggtgccccagaagcaggcgagcgtggctaagaaaaaaaag 780 ctggaaaaagaagaagaggaaataagagccagcgttagagacaagagccctgtgaaaaag 840 gccaaagccacagccagagccaagaagccaggattcaagaaatgaggagccacgccttgg 900 ggggcacggtgcaaagtgggccttccctgggctgtgctgcaggcacagggtgcccctgtc 960 cagcccctccacctgtgtctgaatgcaacaggggtgttgcgggggcaacatgagagcccc 1020 trraL'L'L'L'CaaCtCtCCa.CtttCaggaggCCCCCagtgaagagccccaccteggggtcaca 1080 ataaagttgcctggtcaggaaaaaaaaaaaaaaaaaaa 1118 <210> 15 <211> 200 <212> PRT
<213> xeniopus laevis <400> 1S
Met Ala Ser Thr Val Ser Asn Thr Ser Lys Leu Glu Lys Pro Val Ser Leu Ile Trp Gly Cys Glu Leu Asn Glu Gln Asp Lys Thr Phe Glu Phe Lys Val Glu Asp Asp Glu Glu Lys Cys Glu His Gln Leu Ala Leu Arg Thr Val Cys Leu Gly Asp Lys A1a Lys Asp Glu Phe Asn Tle Val Glu Ile Val Thr Gln Glu Glu Gly Ala Glu Lys Ser Val Pro Ile Ala Thr Leu Lys Pro Ser Ile Leu Pro Met Ala Thr Met Val Gly Ile Glu Leu Thr Pro Pro Val Thr Phe Arg Leu Lys Ala Gly Ser Gly Pro Leu Tyr Ile Ser Gly Gln His Val Ala Met Glu Glu Asp Tyr Ser Trp Ala Glu Glu Glu Asp Glu Gly Glu Ala Glu Gly Glu GIu Glu GIu Glu Glu Glu Glu Asp Gln Glu Ser Pro Pro Lys Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Leu Asp Lys Glu Asp Glu Ser Ser Glu Glu Asp Ser Pro Thr Lys Lys Gly Lys Gly Ala Gly Arg Gly Arg Lys Pro Ala Ala Lys Lys <210> 16 <221> 424 <212> PRT
<223> Human <400> 16 Met Ala Ala Leu Gly Asp Glu Val Leu Asp Gly Tyr Val Phe Pro Ala Cys Pxo Pro Cys Sex Tyr Axg Tyr Pro Tyr Pro Ala Ala Thr Lys GIy Lys Gly Ala Ala Gly Gly Ser Trp Gln Gln Arg Gly Arg Gly Cys Leu Pro Ala Ser Ser Pro Cys Ser Ala Gly Ala Ala Ser Leu Ser Phe Pro Gly Cys Gly Arg Leu Thr Ala A1a Glu Tyr Phe Asp Ser Tyr Gln Arg Glu Arg Leu Met Ala Leu Leu Ala Gln Val Gly Pro Gly Leu Gly Pro Arg Ala Arg Arg Ala Gly Ser Cys Asp Val Ala Val Gln Val Ser Pro 100 l05 110 Arg Ile Asp Ala Ala Val Gln Cys Ser Leu Gly Arg Arg Thr Leu Gln Arg Arg Ala Arg Asp Pro Glu Ser Pro Ala Gly Pro Gly Ala Glu Gly Thr Thr Gly Gly Gly Ser Phe Ser Gln Gln Pro Ser Arg Arg Gly Leu 145 7.5 0 15 5 16 0 Glu Gln Gly Ser Pro Gln Asn Gly Ala Pro Arg Pro Met Arg Phe Pro Arg Thr Val Ala Val Tyr Ser Pro Leu Ala Leu Arg Arg Leu Thr Ala Phe Leu Glu Gly Pro Gly Pro Ala Ala Gly Glu Gln Arg Ser G1y Ala Ser Asp Gly Glu Arg Gly Pro Pro Pro Ala Arg Leu Gln Gly Pro Glu Glu Gly Glu Val Trp Thr Lys Lys Ala Pro Arg Arg Pro Gln Ser Asp Asp Asp Gly Glu Ala Gln Ala Ala Val Arg Ala Ser Trp Glu Gln Pro Ala Asp Gly Pro Glu Leu Pro Pro Arg Glu Ala Gln Glu Gly Glu Ala Ala Pro Arg Ser Ala Leu Arg Ser Pro Gly Gln Pro Pro Ser Ala Gly Arg Ala Arg Asp Gly Gly Asp Gly Arg Glu Ala Ala Val Ala Gly Glu Gly Pro Ser Pro Arg Ser Pro Glu Leu Gly Lys Glu Arg Leu Arg Phe Gln Phe Leu Glu Gln Lys Tyr Gly Tyr Tyr His Cys Lys Asp Cys Asn Ile Arg Trp Glu Ser Ala Tyr Val Trp Cys Val Gln Gly Thr Asn Lys Val Tyr Phe Lys Gln Phe Cys Arg Thr Cys Gln Lys Ser Tyr Asn Pro Tyr Arg Val Glu Asp Zle Thr Cys Gln Ser Cys Lys Gln Thr Arg Cys Ser Cys Pro Val Lys Leu Arg His Val Asp Pro Lys Arg Pro His Arg Gln Asp Leu Cys Gly Arg Cys Lys Gly Lys Arg Leu Ser Cys Asp Ser Thr Phe Ser Phe Lys Tyr Ile Ile <210> 17 <211> 27 <212> DNA
<213> Mouse <400> 17 gCaaagaagc cagtgaccaa gaaatga 27 <210> 18 <211> 27 <212> DNA
<213> mouse <400> 18 cctgatcatg caaattttat tgtggcc 27 <210> 19 <211> 18 <212> PRT
<213> Mouse <400> 19 Lys Arg Pro His Arg Gln Asp Leu Cys Gly Arg Cys Lys Asp Lys Arg Leu Ser <210> 20 <211> 24 <212> DNA
<213> Mouse <400> 20 ctagaaaagg ggactgtagt tact 24 <210> 21 <2l1> 24 <212> DNA
<213> Mouse <400> 21 tgcatctccc acacaagtct tgcc 24 <210> 22 <211> 24 <212> DNA
<213> Mouse <400> 22 ctagaaaagg ggactatagg tact 24 <2I0> 23 <211> 24 <212> DNA
<213> Mouse <400> 23 tgcatctctc acacaagtgt tgct 24 <210> 24 <211> 20 <212> DNA
<213> Human <400> 24 ggaggtgtgg acgaagaagg 20 <210> 25 <211> 22 <212> DNA
<213> Human <400> 25 aagctgaagg tgctgtcgca gg 22 <210> 26 <211> 26 <212> DNA
<213> Human <400> 26 tgaaggtcgg agtcaacgga tttggt 26 <210> 27 <211> 24 <212> DNA
<213> Human <400> 27 catgtgggcc atgaggtcca ccac 24 <210> 28 <211> 1260 <212> DNA
<213> Mouse <400>

tgggcgggcgaggcgcgggacgcacccatgttcccggcgagCaCgttCCaCCCCtgCCCg 60 catccttatccgcaggccaccaaagccggggatggctggaggttcggagccaggggctgc 120 CgaCCCgCgCCCCCCtCCttCCtCCCCggCtacagacagctcatggccgcggagtacgtc 180 gacagccaccagcgggcacagctcatggccctgctgtcgcggatgggtccccggtcggtc 240 agcagccgtgacgctgcggtgcaggtgaacecgcgccgcgacgcctcggtgcagtgttca 300 ctcgggcgccgcacgctgcagcctgcagggtgccgagccagccccgacgcccgatcgggt 360 tcctgtcaaccccgtggccacgccggcgccgggagatccccgcgatcctggcagaccgta 420 gccccgttctcgtccgtgaccttctgtggcctctcctcctcactggaggttgcgggaggc 480 aggcagacac ccacgaaggg agaggggagc ccggcatcct cggggacccg ggaaccggag 540 ccgagagagg tggccgcgag gaaagcggtc ccccagccgc gaagcgagga gggcgatgtt 600 caggctgcagggcaggccgggtgggagcagcagccaccaccggaggaccggaacagtgtg 660 gcggcgatgcagtctgagcctgggagcgaggagccatgtcctgccgcagagatggctcag 720 gaccccggtgattcggatgcccctcgagaccaggcctccccgcaaagcacggagcaggac 780 aaggagcgcctgcgtttccagttcttagagcagaagtacggctactatcactgcaaggac 840 tgcaaaatccggtgggagagcgcctatgtgtggtgtgtgcagggcaccagtaaggtgtac 900 ttcaaacagttctgccgagtgtgtgagaaatcctacaacccttacagagtggaggacatc 960 acctgtcaaagttgtaaaagaactagatgtgcctgcccagtcagacttcgccacgtggac7.020 cctaaacgcccccatcggcaagacttgtgtgggagatgcaaggacaaacgcctgtcctgcx080 gacagcaccttcagcttCaaatacatcatttagtgagagtcgaaaaagtttctgctagat1140 ggggctaatggaatggaCaagtgagctttctCCCCtCttCaCCtCttCCCtttccaaatt1200 cttcatgacagacagtgttacttggatataaagcctgtgaataaaaggtattgcaaacaa1260 <210> 29 <211> 361 <212> PRT
<213> Mouse <400> 29 Met Phe Pro Ala Ser Thr Phe His Pro Cys Pro His Pro Tyr Pro Gln Ala Thr Lys Ala Gly Asp Gly Trp Arg Phe Gly Ala Arg Gly Cys Arg Pro Ala Pro Pro Ser Phe Leu Pro Gly Tyr Arg Gln Leu Met Ala Ala Glu Tyr Val Asp Ser His Gln Arg Ala Gln Leu Met Ala Leu Leu Ser Arg Met Gly Pro Arg Ser Val Ser Ser Arg Asp Ala Ala Val Gln Val Asn Pro Arg Arg Asp Ala Ser Val Gln Cys Ser Leu Gly Arg Arg Thr Leu Gln Pro Ala Gly Cys Arg Ala Ser Pro Asp Ala Arg Ser Gly Ser Cys Gln Pro Arg Gly His Ala Gly Ala Gly Arg Ser Pro Arg Ser Trp Gln Thr Val Ala Pro Phe Ser Ser Val Thr Phe Cys Gly Leu Ser Ser Ser Leu Glu Val Ala Gly Gly Arg Gln Thr Pro Thr Lys Gly Glu Gly Ser Pro Ala Ser Ser Gly Thr Arg Glu Pro Glu Pro Arg Glu Val Ala A1a Arg Lys Ala Val Pro Gln Pro Arg Ser Glu Glu Gly Asp Val GIn Ala Ala Gly Gln Ala Gly Trp Glu Gln GIn Pro Pro Pro Glu Asp Arg Asn Ser Val Ala Ala Met G1n Ser Glu Pro Gly Ser Glu Glu Pro Cys Pro Ala Ala Glu Met Ala Gln Asp Pro Gly Asp Ser Asp Ala Pro Arg Asp Gln Ala Ser Pro Gln Ser Thr Glu Gln Asp Lys Glu Arg Leu Arg Phe Gln Phe Leu Glu Gln Lys Tyr Gly Tyr Tyr His Cys Lys Asp Cys Lys Ile Arg Trp Glu Ser Ala Tyr Val Trp Cys Val Gln Gly Thr Ser Lys Val Tyr Phe Lys Gln Phe Cys Arg Va1 Cys Glu Lys Ser Tyr Asn Pro Tyr Arg Val Glu Asp Ile Thr Cys Gln Ser Cys Lys Arg Thr Arg Cys Ala Cys Pro Val Arg Leu Arg His Val Asp Pro Lys Arg Pro His Arg Gln Asp Leu Cys Gly Arg Cys Lys Asp Lys Arg Leu Ser Cys Asp Ser Thr Phe Ser Phe Lys Tyr Ile Ile <210> 30 <211> 1275 <212> DNA
<213> Human <400> 30 atggcggccc tgggggacga ggtgctggac ggttacgtgt tcccggcgtg ccccccctgc 60 tcgtaccggt acccataccc cgcggccacc aagggcaagg gcgcggcggg cggcagctgg 120 cagcagcgcg gcaggggctg ccttcccgcc tcctccccct gctcggcggg cgcggcctcg l80 ttgtccttcccgggctgcgggcggctgacggccgccgagtacttcgacagctaccagcgg240 gagcggctcatggctctcctggcgcaggtggggccgggtctcgggccgcgcgcccgcagg300 gccggcagctgcgacgtggcggtgcaggtgagcccgcgcatCgaCgCCgCggtacagtgc360 tcgctggggaggcgcacgctgcagcgccgggcccgcgaccccgagtccccggecggcccc420 ggggccgagggcaccacgggtggcggctctttctcccagcagccatcccgtcgaggcctg480 gagcagggcagcccccagaacggcgccccgcggcccatgcgcttcccgcgcaccgtcgcc540 gtgtaCtCgCCCCtggCCttgCgCCgtC'tCaCCgCCttCCtggaggggcccgggcccgcg600 gcgggcgagcagaggtccggggcgtcggacggagagagggggccgccgcccgcgcggctt660 caaggcccagaggagggggaggtgtggacgaagaaggcgccccggcggccgcagtccgac720 gacgacggcgaggcccaggccgcagtccgagcgagctgggagcagccggccgacggtccc780 gagctgccgccgcgagaggcccaggagggcgaggcggctccgcggtcggcgctaaggagc840 ccggggcaacctccgtcggcggggagggcccgagacggcggcgacggacgggaggcggcc900 gtcgcgggagaggggccgtcgccacggagcccggagctgggcaaggagcggctgcgcttc960 cagttcttagagcagaaatatggctattaccactgcaaggactgcaacatccgctgggag1020 agtgcttatgtgtggtgtgtacagggaactaacaaggtttacttcaaacagttttgcaga1080 acttgtcagaagtcttataacccttaccgagtggaggatatcacctgtcaaagttgtaaa1140 caaacgagatgttcctgcccagtaaaacttcgccacgtggaccctaaacggccccaccgt1200 caagatttgtgcggtagatgcaaaggcaaacgcctgtcctgtgacagcactttcagcttc1260 aaatacatcatttag 1275 <210>

<211>

<212>
ANA

<213>
Xenopus Laevis <400>

ttcggcacgaggtggatcgctacatgtacccagcttacaatccttattcgtataggtacc60 tgaaccctaggaataaagggatgagctggagacagaagaactatttggccagttatggag120 acactggggactactgtgataattatcagagggctcagctgaaggccatcttgtctcaag180 tgaaccctaacctcacgccaaggctctgcagagcaaacacaagggatgtgggggtgcagg240 taaaccccaggcaagatgcatcagtccagtgctccttggggcccagaactttactcagaa300 ggagacctgg ggccctacgg aagcctccac cagagcaagg gagtcctgcc tcccccacaa 360 agactgtgag attccccagg actattgcgg tatattcacc tgtggctgca ggaaggttgg 420 ctccatttca ggatgaaggg gtcaatctgg aagagaaggg tgaggcagtg agaagtgaag 480 gctctgaaggagggaggcaggaaggaaagcaaggggatggagagatcaaggaacagatga540 agatggacaagacagatgaagaggaagcagcccctgctcagacaaggccaaagttccagt600 tcctggagcagaagtacggatattatcactgtaaggactgcaacatccgctgggagagcg660 cctacgtgtggtgtgtgcaggaaaccaataaggtgtacttcaagcagttctgcaggacat720 gtcagaaatcctataatccctaccgtgtggaagacatcatgtgtcagagctgcaagcaga780 cgagatgcgcgtgtcctgtcaaactgcgtcacgttgaccccaagaggccccaccgccagg840 atctgtgtgggagatgcaaaggcaaacggctctcgtgtgacagcacttttagcttcaagt900 atatcatttgattgtgtgtaattcatacatttctagctacattaattaagccaggaaagg960 gcttcatttatgtttttgttttggaggagacgactgggaaggaactgtctgtgcaatgcg1020 ctcgctttcttgtactgaataaacagaataga 1052 <210> 32 <211> 295 <212> PRT
<213> Xenopus Laevis <400> 32 Met Tyr Pro Ala Tyr Asn Pro Tyr Ser Tyr Arg Tyr Leu Asn Pro Arg Asn Lys Gly Met Ser Trp Arg Gln Lys Asn Tyr Leu Ala Ser Tyr Gly ' Asp Thr Gly Asp Tyr Cys Asp Asn Tyr Gln Arg Ala Gln Leu Lys Ala Ile Leu Ser Gln Val Asn Pro Asn Leu Thr Pro Arg Leu Cys Arg Ala Asn Thr Arg Asp Val Gly Val Gln Val Asn Pro Arg Gln Asp Ala Ser Val Gln Cys Ser Leu Gly Pro Arg Thr Leu Leu Arg Arg Arg Pro Gly A1a Leu Arg Lys Pro Pro Pro Glu Gln Gly Ser Pro Ala Ser Pro Thr Lys Thr Val Arg Phe Pro Arg Thr Ile Ala Val Tyr Ser Pro Val Ala Ala Gly Arg Leu Ala Pro Phe Gln Asp Glu Gly Val Asn Leu Glu Glu Lys Gly Glu Ala Val Arg Ser Glu Gly Ser Glu Gly Gly Arg Gln Glu Gly Lys Gln Gly Asp Gly Glu Ile Lys Glu Gln Met Lys Met Asp Lys Thr Asp Glu Glu Glu Ala Ala Pro Ala Gln Thr Arg Pro Lys Phe Gln Phe Leu Glu Gln Lys Tyr Gly Tyr Tyr His Cys Lys Asp Cys Asn Ile Arg Trp Glu Ser Ala Tyr Val Trp Cys Val Gln Glu Thr Asn Lys Val Tyr Phe Lys Gln Phe Cys Arg Thr Cys Gln Lys Ser Tyr Asn Pro Tyr Arg Val Glu Asp Ile Met Cys Gln Ser Cys Lys Gln Thr Arg Cys Ala Cys Pro Val Lys Leu Arg His Val Asp Pro Lys Arg Pro His Arg Gln Asp Leu Cys Gly Arg Cys Lys,Gly Lys Arg Leu Ser Cys Asp Ser Thr Phe Ser Phe Lys Tyr Ile Ile <210> 33 <211> 1192 <212> DNA
<213> Danio rerio <220>
<221> misc_feature <222> (1). (1192) <223> n equals unknown <400> 33 tcagctatag ggcaaggcag tggtattcat cgcagagttt cgcggggggg ctaagcgaaa 60 aatggctaca tatggaaacg agacagtcga taactatctt tactcctctt acaaccctta 120 ttactacaaa taccccaaat tcaagggctg gagacagaaa gcttacttca ccaactacgg 180 tgagggcgag acctactttg ataatcacca cagggcgcag ctgaagtcca tcttgtctca 240 gatcaaccca aatctcaccc cgcgtctgag gaaggccaac accaaagacg tcggggttca 300 ggtcaacccg aagaccgacg cgtctatcca gtgctctctg gggccgcgga cgcttctggc 360 acgcaaacgc gatgccctgc gccggcggcg gcaggaggtc cagacccctg ggagtcccgt 420 cagcagcggt ggtgtccggt tcccgcgcac ccaggccgtt tactccccag tagaatcccg 480 gagactagtgtccctcttcagagaggagggtgaagaagaggaggacacggatctcgaggt540 cacagagacggttgacagcgcagagaagctggaaagcgccgagaaaaacgtgcgcaaaca600 gggtaagaaaagcgcgaagcaaccgcttagtacagagaaaaatataaacaagcagactga660 aacaaatgaggagaacacaaacgagccagtgaaaaccgaacaagacgatctgaagtccaa720 ggctcgtgtgagatttCagtetttggagcagaagtatggattctatcattgcaaagactg780 caacctacggtgggaaagtgcttatgtgtggtgtgtccaaggaacaaacaaggtttattt840 caagcagttctgcagaacatgccagaaatcattcaacccataccgggttgaggacatagc900 atgtcagacttgcaagaaagctcgctgcacatgttctgtcaagtcgcgtcacgtggaccc960 caaaagaccccatcggcaggatctgtgcggccgctgtaaaggcaagcgtctgtcctgcga1020 cagcacgttcagcttcaagtacatcatctagcagctctgtccattgttgttcatcagctg1080 gatttgtCCaCCtCagtCtCtgCanCCtCtgCtgCtCCttcgaccgcagttggggggagg1140 gCaggCCCattCggCCCaCttaCagtCtCattttgttttttgttttgccttt 1192 <210> 34 <211> 329 <212> PRT
<213> Dario rerio <220>
<221> MISC_FEATURE
<222> (1). (329) <223> x equals unknown <400> 34 Met Ala Xaa Tyr Gly Asn Glu Thr Val Asp Asn Tyr Leu Tyr Ser Ser Tyr Asn Pro Tyr Tyr Tyr Lys Tyr Pro Lys Phe Lys Gly Trp Arg Gln Lys Ala Tyr Phe Thr Asn Tyr Gly Glu Gly Glu Thr Tyr Phe Asp Asn His His Arg Ala Gln Leu Lys Ser Ile Leu Ser Gln Ile Asn Pro Asn Leu Thr Pro Arg Leu Arg Lys Ala Asn Thr Lys Asp Val Gly Val Gln Val Asn Pro Lys Thr Asp Ala Ser Ile Gln Cys Ser Leu Gly Pro Arg Thr Leu Leu Ala Arg Lys Arg Asp Ala Leu Arg Arg Arg Arg Gln Glu Val Gln Thr Pro G1y Ser Pro Val Ser Ser Gly Gly Val Arg Phe Pro Arg Thr Gln Ala Val Tyr Ser Pro Val Glu Ser Arg Arg Leu Val Ser Leu Phe Arg Glu Glu Gly Glu Glu Glu Glu Asp Thr Asp Leu Glu Val Thr Glu Thr Val Asp Ser Ala Glu Lys Leu Glu Ser Ala Glu Lys Asn Val Arg Lys Gln Gly Lys Lys Ser Ala Lys Gln Pro Leu Ser Pro Glu Lys Asn Ile Asn Lys Gln Thr Glu Thr Asn Glu Glu Asn Thr Asn Glu Pro Val Lys Thr Glu Gln Asp Asp Leu Lys Ser Lys Ala Arg VaI Arg Phe Gln Ser Leu Glu Gln Lys Tyr Gly Phe Tyr His Cys Lys Asp Cys Asn Leu Arg Trp Glu Ser Ala Tyr Val Trp Cys Val Gln Gly Thr Asn Lys Val Tyr Phe Lys Gln Phe Cys Arg Thr Cys Gln Lys Ser Phe Asn Pro Tyr Arg Val Glu Asp Ile Ala Cys Gln Thr Cys Lys Lys Ala Arg Cys Thr Cys Ser Val Lys Ser Arg His Val Asp Pro Lys Arg Pro His Arg Gln Asp Leu Cys Gly Arg Cys Lys Gly Zys Arg Leu Ser Cys Asp Ser Thr Phe Ser Phe Lys Tyr Ile Ile <210>

<211>

<212>
DNA

<213>
Fugu rubripes <400>

atggcaacgtattgtgacgagccagtcgacagctacttctattcatcttacaacccgtat 60 atgggccggtacccccggcacagagacgccgggtggaaatataaaagttaectctcccac 120 tatggggacacttcagaagccttcagtaaccagcagcgggcccagctcaagtccatctta 180 tcccaaatcaatcccaaactcaccccgaggctcaggaaggccaacaccaaagacgtggcg 240 gtgcaggtgaacccgaagcgggacgcctcggtgcagtgctccatcggcccgcggaccctc 300 ctggtggtgaaacgagaactceggcgcaggagaaaactgaacccgggcccgcccggaact 360 ccccagaagacggagggcgaggtgcgctacccgcggaccctcgccgtgtattcgcccatt 420 gccttcaggagcgtcacctccttcctggtggagaccgggaaggaccgtcccgccgccgag 480 gcccaggccgaggagctgcccggtgagcagccggagcaaaagggcggcgagaaccaggcc 540 ggcgaggaaacgaacgcaaatctacccgaacagcgcaaaccgcaaagtgaagatgcgcaa 600 accgccgcagacgctgaggggtcaaagggcaaagcgcgtgtccgcttccagtttctggaa 6'60 cagaagtacggctactatcactgcagagaatgcaacctgcgatgggagagcgcgtacgtt 720 tggtgcgttcagggcactaacaaggtttacttcaagcagttctgtaggaaatgccaaaaa 780 gactttaacccgtaccgcgtagaggacatcacatgtcacgtatgcaacaaggcccgctgt.840 gcctgcgcagaaacgcagcgccacgttgacccaaagaggccccacaggcaggacctgtgc 900 ggcaggtgcaagggcaagcggctgtcctgcgacagcaccttcagcttcaaatacatcgtc 960 taa g63 <210>

<211>

<212>
PRT

<213> rubripes Fugu <400> 36 Met Ala Thr Tyr Cys Asp Glu Pro Val Asp Ser Tyr Phe Tyr Ser Ser Tyr Asn Pro Tyr Met Gly Arg Tyr Pro Arg His Arg Asp Ala Gly Trp Lys Tyr Lys Ser Tyr Leu Ser His Tyr Gly Asp Thr Sex Glu Ala Phe Ser Asn Gln Gln Arg Ala Gln Leu Lys Ser Ile Leu Ser Gln Ile Asn Pro Lys Leu Thr Pro Arg Leu Arg Lys Ala Asn Thr Lys Asp Val Ala Val Gln Val Asn Pro Lys Arg Asp Ala Ser Val Gln Cys Ser Ile Gly Pro Arg Thr Leu Leu Val Val Lys Arg Glu Leu Arg Arg Arg Arg Lys Leu Asn Pro Gly Pro Pro Gly Thr Pro Gln Lys Thr Glu Gly Glu Val Arg Tyr Pro Arg Thr Leu Ala Val Tyr Ser Pro Tle Ala Phe Arg Ser Val Thr Ser Phe Leu Val Glu Thr Gly Lys Asp Arg Pro Ala Ala Glu Ala Gln Ala Glu Glu Leu Pro Gly Glu Gln Pro Glu Gln Lys Gly Gly Glu Asn Gln Ala Gly Glu Glu Thr Asn Ala Asn Leu Pro Glu Gln Arg Lys Pro Gln Ser Glu Asp Ala Gln Thr Ala Ala Asp Ala Glu Gly Ser Lys Gly Lys Ala Arg Val Arg Phe Gln Phe Leu Glu Gln Lys Tyr Gly Tyr Tyr His Cys Arg Glu Cys Asn Leu Arg Trp Glu Ser Ala Tyr Val Trp Cys Val Gln Gly Thr Asn Lys Val Tyr Phe Lys Gln Phe Cys Arg Lys Cys GIn Lys Asp Phe Asn Pro Tyr Arg Val Glu Asp Ile Thr Cys His Val Cys Asn Lys Ala Arg Cys Ala Cys Ala Glu Thr Gln Arg His Val Asp Pro Lys Arg Pro His Arg Gln Asp Leu Cys Gly Arg Cys Lys Gly Lys Arg Leu Ser Cys Asp Ser Thr Phe Ser Phe Lys Tyr Ile Val <210> 37 <221> 1280 <212> DNA
<213> RATTUS NORVEGICUS
<400>

tgggccggcgaggcgcgggacgcacccatgttcccggcgagCa.CgCCCCaCCCatgCCCg60 catccttacccgcccacggcagccaaagccggggatggctggaggtttggagccaggggc120 tgcaggcccgagCCCCCCtCCttCCtCCCCggCtaCagaCagctcatggccgcggagtac180 tttgacagctatcagcgagcgcagctcatggccttgctgtcgcgaatgggtccccggccg240 gtcagcagccgcgacgctgcggtgcaggtgaacccgcgccgcgatgcctcggtgcagtgt300 tcgctcgggcgccgcacactgcagcctggacggcgccgagccagccccgacgcccggcct360 ggttcctgccaaccccgcagccccgccagggccgggagaccccagcgatcctggcgcacc420 gtcgccctgtactcgcccgtgaccttcggtggcctctcctcctcgctggaggttgcgggg480 gacaggcaga cgcccacgaa gggagagggg agaccggcac ccacggggac ccgggaaccc 540 gagccgggagaggtggcagtgatgaaagcagtCCCCCagCCgCagagCgaggagggcgac600 gtccaggctgaagggcaggatgggcaggagcagccaccgcgggaggacccggacagtgtg660 gcggcgatgcagtctgagcccgggagtgaggagccacctcctgctgtcgagatggctcag720 gaccccagtgacgtggctgcctctagagaccgggcctccccacagagcactgagcaggac780 aaggagcgcctgcgtttccagttcttagagcagaagtacggctactatcactgcaaagac840 tgcaacatccggtgggagagcgcctatgtgtggtgtgtgcagggcaccagcaaggtgtac900 ttcaaacagttctgccgcgtgtgtgagaagtcctacaacccataccgagtggaggatatc960 acctgccaaagttgtaaaaggactagatgtgcctgcccggtcagacttcgccacgtggac1020 cctaaacgeccccatcgtcaagacttgtgtgggagatgcaaggacaaacgcctgtcctgt1080 gacagcaccttcagcttcaaatatatcatttagagagagttaaaaatggttctgctaaat1140 ggatcggaca agtgagectt CtCCCa.CgCC CCt CtCCCCt CtCC3CCtCt CCCttCtCaa 1200 aatacttcat gaaaggcagt gtactttaat ataaagcctg cgaataaaag gggttgcaaa 1260 cagtttgggg ctttatccaa 1280 <210> 38 <211> 3959 <212> DNA
<213> Rattus norvegicus <400> 38 ctgggcggcg ctgatggcag cetgggccgg cgaggcgcgg gacgcaccca tgttcccggc 60 gagcacgccc cacccatgcc cgcatectta cccgcccacg gcagccaaag ecggggatgg 120 ctggaggttt ggagccaggg gctgcaggcc cgagccccec tCCttCC'tCC CCggCtaCag 180 acagctcatg gccgcggagt actttgacag ctatcagcga gcgcagctca tggccttgct 240 gtcgcgaatgggtceccggccggtcagcagccgcgacgctgeggtgcaggtgaacccgeg300 ecgcgatgcctcggtgcagtgttcgctcgggcgccgcacactgcagcctggacggcgccg360 agccagccccgacgcccggcctggttcctgccaaccccgcagccccgecagggccgggag420.

aCCCCCgCgatcctggcgcaccgtegccetgtactegcccgtgaccttcggtggectctc480 etcctcgctggaggttgcgggggacaggcagacgcecacgaagggagaggggagaceggc540 acccacggggacccgggaacccgagcegggagaggtggcagtgatgaaagcagtecccca600 gccgcagagcgaggagggcgacgtccaggctgaagggcaggatgggcaggagcagccacc660 gcgggaggacceggacagtgtggeggcgatgcagtctgagcecgggagtgaggagccacc720 tcetgctgtcgagatggctcaggaccccagtgacgtggctgcetetagagaccgggectc780 eccacagagcactgagcaggacaaggagcgectgcgtttccaggtgaggccagcctggca840 acctggacgcttccagaattgtaggactccttcggggctaagctagtggttgtggctgat900 gcagggcatagaattcttcaatgccctcagtctgtatttaaaaaaaaaccccaaccgcgt960 atgggtgttttgattgcatgtagtgtctgtgcatttcgttggagtggagagatgggagcg1020 gaagagggegtcagatccccggaactggcgatttacaggtggttgtgagccactatgtgg1080 gtttgaggaaetgaattagggtcetetagaagaactgtgttctttaacagctgaaccatc1140 tCtCCagCtCttcagcttgtaggagcttgcagccgctgccagttaatacaatgggaggtg1200 tttacacaataaaaccaacccctacgtggcctgacccactggcagtctctgttcttggga1260 aatgactgtgtagttgattctggtcacatcaacactacaaatgaagcctccgcggcgaac1320 gcacaccagtccagtttggaecgtacttcagatggctgaaggaagataagaaaacacaca1380 gcagaatatcatcatgtgaacttggtcgcaegagagagtgggtttcccaatgctcctttt1440 gcg ctcatttccc ctctagatat ggggaaattt gcctcatttg actggtgggg ttttgttctc 1500 aactaagccc taccctccat ttttgaagct ctcaactgaa gcttctccct cccaaacttg 1560 cctggctttt aaaaaaacaa aaaaacaaaa aaacctcagc attcatccag aaggccagca 1620 ctcctaagec ttggatggtc ttgagttgca gcctaggcta accaaacaag gaactggggg 1680 gcgggggtgggggcacccaaggagcgtggccattactccctccccccaaagagatgcaga1740 gctctgagctctgggaaaggactcagaaatgccttattttctaatcatttcaaggttgtt1800 gtgtttggggttggagtggcagtggaatggggtgtcagaaaagcacttggttgccgttca1860 cagctgggggtgatcttaggcagaaaccttaagctttctggcctcctgtgtctgctcact1920 ataaaaattgcattaagagtccttccaggcccccctgccagtcctaaaggcttaattata1980 agagcttgtttgtcacttaagtgggaagcggggaagtttagaactcgggtgctaacgtta2040 catagtcataccaaatgagctatacttacataggtggcgggactttttggaaacttaact2100 agcatcatggactatttaacattgattgaagactttcgaaggtttagaagagcctttctc2160 tttcagtgtaataactctctcatctgtggaagttctaggatcactcatcaccccacataa2220 aaggcccaagggaaggatcctgagctctagacagcactatttggcttttcccttgtccga2280 ggggaaaaacacttatccatctgctggtataggaactttcttggttggcctcagtttaca2340 tatttgtttgactgaactattatttcacctgattaatttagaggttgacccaaatctaac2400 cttacttactaccatggtaaagtttcaagtatcaggttgaatctggtcccagattctgaa2460 atgagggtaactcagccatcacttgcttaaagtctgtatcctgctgttcagttcttagag2520 cagaagtaeggetactateaetgcaaagactgcaacatecggtgggagagcgcctatgtg2580 tggtgtgtgcagggcaccagcaaggtaagagactctgcggctgtcccattcctgcctggt2640 gccgagtggggagtgtcagtgtcatggggaccctctttctcatcgcaggtgtacttcaaa2700 cagttctgccgcgtgtgtgagaagtcctacaacccataccgagtggaggatatcacctgc2760 caagtaaatcaaatgtttgcattttggaaaaggggtttcgtgtgctatttcgaatatatt2820 tcttaaaagaggtgtaggtttccaggagccttaggccctactttttctttcctttttgtt2880 tttcgataggggtggtagaaagtcccctctgctgtgaaacggggtgtttgtccccttctg2940 ctcttgcaaaatttgactgcatggcctcttttgaatctgttacctcattagcagtgtaaa3000 agacttgcttacctgccacttgacttggcataagggaaaaccctgcaaaagcagagtacc3060 tttctatagcctgaggtggtacactattaccccggtgcatctaagaagcacatttccaag3120 ttagagtatgagacccaagacatggagcctcagcttttcttgagagtttgcatagggagt3180 tagctccctatgagaccttctgagaccttgtctgtccagctctgtgcagttcacccgtaa3240 tgaaggctgctaagtgcttgcttaccattaattagttcagcactgcgtcaagggacttag3300 ataaccttcaaaaagaaagaagtgacaacagttacctgctaaggttaaccacagagctaa3360 aggacttccacatctacctgagagtccagcgcacttaatgttggtaattggagaattaac3420 taccctaggatgggccctgagaaccctagaatggaaagccacgaatgctccattcagaaa3480 aggggactgcaggcaccgttctacaaacttaaagccttgagcctttctgctcttggctct3540 cggtgcccaattcacgtgccacctgttctgtagtctgctttgaagccccaagaattgagg3600 gtgggggaggggggttcatcctttgtacttttcactctgatttttcagagttgtaaaagg3660 actagatgtgcctgcccggtcagacttcgccacgtggaccctaaacgcccccatcgtcaa3720 gacttgtgtgggagatgcaaggacaaacgcctgtcctgtgacagcaccttcagcttcaaa3780 tatatcatttagagagagttaaaaatggttctgctaaatggatcggacaagtgagccttc3840 tcccacgcccetctcccctctccacctctcccttctcaaaatacttcatgaaaggcagtg3900 tactttaatataaagcctgcgaataaaaggggttgcaaacagtttggggctttatccaa 3959 <210> 39 <211> 361 <212> PRT
<213> Rattus norvegicus <400> 39 Met Phe Pro Ala Ser Thr Pro His Pro Cys Pro His Pro Tyr Pro Pro Thr Ala Ala Lys Ala Gly Asp Gly Trp Arg Phe Gly Ala Arg Gly Cys Arg Pro Glu Pro Pro Ser Phe Leu Pro Gly Tyr Arg Gln Leu Met Ala Ala Glu Tyr Phe Asp Ser Tyr Gln Arg Ala Gln Leu Met Ala Leu Leu Ser Arg Met Gly Pro Arg Pro Val Ser Ser Arg Asp Ala Ala Val Gln Val Asn Pro Arg Arg Asp Ala Ser Val Gln Cys Ser Leu Gly Arg Arg Thr Leu Gln Pro Gly Arg Arg Arg Ala Ser Pro Asp Ala Arg Pro Gly Ser Cys Gln Pro Arg Ser Pro A1a Arg Ala Gly Arg Pro Pro Arg Ser Trp Arg Thr Val Ala Leu Tyr Ser Pro Val Thr Phe Gly Gly Leu Sex Ser Ser Leu Glu Val Ala Gly Asp Arg Gln Thr Pro Thr Lys Gly Glu Gly Arg Pro AIa Pro Thr Gly Thr Arg Glu Pro Glu Pro Gly Glu Val Ala Val Met Lys Ala Val Pro Gln Pro Gln Ser Glu Glu Gly Asp Val Gln Ala Glu Gly Gln Asp Gly G1n Glu Gln Pro Pro Arg Glu Asp Pro Asp Ser Val Ala Ala Met Gln Ser Glu Pro Gly Ser Glu Glu Pro Pro Pro Ala Val Glu Met Ala Gln Asp Pro Ser Asp Val Ala Ala Ser Arg Asp Arg Ala Ser Pro Gln Ser Thr Glu Gln Asp Lys Glu Arg Leu Arg Phe Gln Phe Leu Glu Gln Lys Tyr Gly Tyr Tyr His Cys Lys Asp Cys Asn Ile Arg Trp Glu Ser Ala Tyr Val Trp Cys Val Gln Gly Thr Ser Lys Val Tyr Phe Lys Gln Phe Cys Arg Val Cys Glu Lys Ser Tyr Asn Pro Tyr Arg Val Glu Asp Ile Thr Cys Gln Ser Cys Lys Arg Thr Arg Cys Ala Cys Pro Val Arg Leu Arg His Val Asp Pro Lys Arg Pro His Arg Gln Asp Leu Cys Gly Arg Cys Lys Asp Lys Arg Leu Ser Cys Asp Ser Thr Phe Ser Phe Lys Tyr I1e Ile <210> 40 <211> 6873 <212> DNA
<213> Mouse <400>

ggatccctgttgcagtcataccctatgggaaaagagcaacttacctatcttaaggagatt60 gggggaatttagatatttgtgcatcctctttcacttgaatgagaacaatgtaccagatcg120 tcaacagtgcacatttgacccggccagtagcaacatacctaaaactacctcaccattgta180 aaacaccctaagaatcacaagaaacttacagtttttcagagacaaatcaaggagcagagt240 tacagaattaaaaacaattcatcataccttagaattttctctaatcgaatgagttgataa300 ttgtttccatactcttagccatctttggagctatactttgaaggttaaaaatatcctaga360 ctaagttagttttcattgtaagtgctagcagcctttgctttctggtgtgaaataagaact420 aatttcaagtagaaagcacagagttcaggagagatgaagactatgttgcccaggctagtc480 ctcagcccataactgggttcttgggctaaattctagctgctccattggaaataagcaaga540 cagtgaattaagcacacacgaagagtactaaacgttgcgtggccaaggccgagaggctga600 gggtctgcctagtaacggtaacctttccctgatctctggtttatcgttttgaagacottt660 tcagaaggagtagtctgtctgtatgtccttccatcccaagcaagtgaagagcccagagga720 gccactggatatcaagctaggtctccaagcagttatgtctaaggagcttcaggctttgtt780 tgcgaggttagaatggatgtagcttgtccagatgcctcctcagtgatgttgttgttgata840 ggttgcccatgtgtttcttttcttaccaggtttccctgtggtctctgcttgtgaccctgg900 tggttcttttcataatgactggcaccatgctgggacctgaactgctggcaagcattccca960 caactgtttacgtggtcgccatttttatgcctctggeggctacgcctcgggttatggctt1020 agctaccctcttcacctccgcccaactgcaagaggactgtgtgtctggaaacaggaagtc1080 agaacgtgcagcttctgcactgcgattcttaaacttcccgcctcgctttataggtagcat1140 gtacatgttccCttctgctctacgccttcttccagtctgccgaggcaggggtcttcgtgt1200 tgatctacaaaatgtacggaagtgagatactgcacaagcgagaggccctagacgaagacg1260 aagacaccgatatttcttataagaaactgaaagaagaggaaatggcagacacctcctatg1320 gcaccgtggggacgcatgacttagtgatgatggagaccacccagaccgccctctgactga1380 ggagatacacgggagctgaaacatcacttcctatttgtgaccattggtagcgagtatggt1440 tcgcatccgggataaagatgggttgacatttcctgtaacagatttgctcttcccactgta1500 atgtagtatctcagtattacccatgtgtttttctaactcaacagagtgtcccaatattgc1560 ttacacctggatcagctaaagtgccgcgtcctctgcttaagtagtgtgctgtttgtttgt1620 ttgttttttgttttgttttgtttttttccatttccaccagcattgctacagataggaaat1680 ggggttggaaatgtttgtaaaacagaaccatgggtttgttcaacttacaaacaaccgatt1740 ctgttcagggcgagcctgtattgagaaaagtccaaaacgggtcaaaaagggttgaaacga1800 caggatagcattgcatcgtcaagccagagaaaaccgtattaatgtgtgtgactacttgat1860 ctagtatctattgttaatggccatcaacattgtgcaggggtgaaaggcatttttccccat1920 atgtttcctgtatgtgtataaacgcatctcagctccatttatcgtctgaaggaatgattt1980 acttaggaaaatgcgtagacctcacctcagggagagaaaatgggccactttgttcatccg2040 tgggaaagggctgtggctacaggctttccttccggaaaggcctgtggctggacactgtcc2100 cactgctctggtagactggagctgtgatctgagacaacctaagaggttcagagcagtctc2260 ctaaccttggtattttgctccctaatcagacacactggcctcccttgtcttcttcatgac2220 agacatctggagctacagacatgggggcccacctggctcggctaatctcggtgatgattc2280 tggggttgaattctcatctcatctagttcccctacaaatccttgctgtggctagcaagga2340 aagctctttttctgcatccacgagggagtgggggtgggggtcgcctcttaaccagtgtgg2400 ggaaggttttgctcctcatggcaacagcaggtggtagggctttttCtaCCagtgCgCggC2460 cgcctatttaacgcagcgtggagggcagctgggctgcgctgatggctgcctgtgggcggg2520 cgaggcgcgggacgcacccatgttcccggcgagcacgttccacccctgcccgcatcctta2580 tccgcaggccaccaaagccggggatggctggaggttcggagccaggggctgccgacccgc2640 gCCCCCCtCCttCCtCCCCggCtaCagacagCtCatggCCgcggagtacgtcgacagcca2700 ccagcgggcacagctcatggCCCtgCtgtCgcggatgggtccecggtcggtcagcagccg2760 tgacgctgcggtgcaggtgaacccgcgccgcgacgcctcggtgcagtgttcactcgggcg2820 ccgcacgctgcagcctgcagggtgccgagccagccccgacgCCCgatCgggttCCtgtCa2880 accccgtggccacgccggcgccgggagatccccgcgatcctggcagaccgtagccccgtt2940 ctcgtccgtgaccttctgtggcctctcctcctcactggaggttgcgggaggcaggcagac3000 acccacgaagggagaggggagcccggcatcctcggggacccgggaaccggagccgagaga3060 ggtggccgcgaggaaagcggtcccccagccgcgaagcgaggagggcgatgttcaggctgc3120 agggcaggccgggtgggagcagcagccaccaccggaggaccggaacagtgtggcggcgat3180 gcagtctgagcctgggagcgaggagccatgtcctgccgcagagatggctcaggaccccgg3240 tgattcggatgcccctcgagaccaggcctccccgcaaagcacggagcaggacaaggagcg3300 cctgcgtttccaggtgaggccagcctgatggcctggacgcctccagaattgtagggctcc3360 ttcagggctaagctggtggctctgggtgatgcagaacatagaattcttccatgccatccg3420 tctggttttgtttgtttgtttgtttgtaacatgtttggtgttttgattgcatgttgtatc3480 tgtacacttcgttgtagtggagagatgggagcagaagagggtgtcggatccggatcccct3540 gggactggcgttttacagatggttgtgagtcaccatgtgagttttaggatcggaattacg3600 gtcctctagaagaacagggtgttgtttcacagctgagccatctctccagctctttggcat3660 ataggattttgcagccgctgcctgttaatacaatgggaggcgtttacacaataaaaacca3720 acccatatgtgtcctgacccactggcagcctctgctcctggggaatgccagttgtaatta3780 ttctgatcacataaacgctacacatgaggtctccgcggagaatgcgcacagtctgggttt3840 ggaccaaacttcagatggctgaaggaagataagtgcacacatggcagaaacataatcttt3900 tgaacttcgttgcggggagagtcggtttcccaaggctcctttttttatttcccctctaga3960 tgatctgtcttggttaacttgccggcttgttctataccagCCCCttCCCttCgtttCtga4020 agctgtcaactgaagcttctctctcccaaacttgcctggcttaaaaaacaaacaaacaaa4080 aacaaaacaccccccccaaaaaaaaaaacaacaaaaaaaaaaaagaaaagaaaaagaaaa4140 agaaataaaagaaaaaaaaaaccactctccccattcatcgaggecagccactgctaagct4200 gttggatggtcttgagttgctgcctgtgctagcaaacaaggaggcacaaagagtgctgta4260 ggtcgtatacccccaccaaagaaatggagagccctgagctccaggagaggactctgagac4320 attccttgtttttcagtcatttcaaggctggtgtgtttgaggttggggtggcagtggaat4380 ggggtgtcagaaaaaatagaaaagtgcttggcggttgctgttcacagctgggtgtgatct4440 cttaggcagaaatcccaagttttcgggcctctgtggtggtcgttcacctataaaaaattg4500 cattaagagttcttccaagccctgccactcctaaagacttagttataaaaacttgtttcc4560 aacttgtttgtcactaagtgggaagcttgggaagtttaagaaccaggtgctaacactatg4620 tagttcataccaaatgagctagacttgggtaggtagcgggactcttttggaaacttacct4680 agcatcaaggaaaatttagtattggttgaagactttcaaaggttttagaagagcctttct4740 ctttcggcataacaactttcccatgtgtgagtgtcctaatgcatcgcccacataaaatgc4800 cacgggaagaatcccaaactctaaaccgcacgatttggcttctcccttgtctgagggggg4860 aaaaaccacttatcggtctgctgctatatgaactatcttgtttggcctccgtttacatat4920 ttgtttgattgagctattagttcacctggttaacttagaggttgacccaagtctaacctt4980 actaccacggtaatcttaaagtatcaagtggaatgtggtcccaggttctgaaaattaggg5040 tcactcgggcatcacttgcttaaagtctggtaccctgctgttcagttcttagagcagaag5100 tacggctactatcactgcaaggactgcaaaatccggtgggagagcgcctatgtgtggtgt5160 gtgcagggcaccagtaaggtaagagacaccgtgcagccctcctgctctgctgtgttgccg5220 agtgtctgctCCatgCCgatgtCtttCtCCtcgcaggtgtacttcaaacagttctgccga5280 gtgtgtgagaaatcctacaacccttacagagtggaggacatcacctgtcaagtaaaccaa5340 acgtttgcattttggaagaggggtttggtgcacgactttgagtatatttcctgaaggagg5400 tggtttccagtagctttaggCtCtaCCttttCCC'tCCtCCttccttttcatttttgacta5460 ggttggtggtagaaagtcecctccactgtaaatggggtgtttactcccttctgctgttgt5520 aaaacttgattgcatgccctctcttgcatctggttaccttgttagcagtagaaagggctt5580 gcttacctggettcttcccactcggacctaagggaaaacatattgcaaaacagagtgcct5640 ttctgctagcttgagatggtacacattaccccaatgctacataggaaacacattcccaag5700 ttagcatatgaaacacaagaaattgagctctggcttttcttgagagtttacaaagggagt5760 ttcctgtaagaccatcctacactgtctagctctatgcagtttacccataactgtggctaa5820 gagtttgcttgcttagtattaatttagcactgtgccaagggacttagataaccttgaaaa5880 catttacctgttaaaattaatgacagagataaaggaattcgaattccacatctgagagcc5940 cagtgcacttaaagttggtaattggagaattaattaccttagggtgggccctgtgaaacc6000 gagaatggaaagccactaaagactccatctagaaaaggggactgtagtcacttttctaca6060 ataaggggccttaaacttccctaagcttccctgcacttggttctcagtgcccagcacaca6120 ggccacttgttctgtaatetgttttgaagctccaagaatcgagtggagacagggctcacc6180 ctttgtacttttcactccgatttttcagagttgtaaaagaactagatgtgcctgcccagt6240 cagacttcgccacgtggaccctaaacgcccccatcggcaagacttgtgtgggagatgcaa6300 ggacaaacgcctgtcctgcgacagcaccttcagcttcaaatacatcatttagtgagagtc6360 gaaaacgtttctgctagatggggctaatggaatggacaagtgagctttctcccctcttca6420 cctcttccctttccaaattcttcatgacagacagtgtacttggatataaagcctgtgaat6480 aaaaggtattgcaaacaagtttgaggctttatccaattcatgtgtcagtttgaggggtgc6540 atgtgcggagagtcaataactttcttaacatttgttgatgagagtgagtcaggctgactt6600 aaggaagttaaaggcacctcattcaacaattaagatttttctttctttttgtttagtttt6660 attttatttataaatatatgagtacactgtagctgtcttcagacacaccaaaagaaggca6720 tcagatcccattacagatagttgtgagccaccatgtggttgctgggacttgaactccgga6780 cctctggaagagcagttggtaaacccctttcttaactgctgaaccatctctccagcccaa6840 atcttaaggttttacagacaagaatattacagg 6873 <210> 41 <211> 6002 <212> DNA
< 213 > HITMAN
<400> 41 tagatgaagatgaagatacagatatttcttataaaaaactaaaagaagaggaaatggcag60 acacttcctatggcacagtgaaagcagaaaatataataatgatggaaaccgctcagactt120 ctctctaaatgtggagatacacaggagcttctatcttgctgaaatattgcttcatattta180 tagcctgtggtagtgcacatggttaacataaaagataacactggttcacatcatacatgt240 aacaattctgatctttttaaggttcactggtgtattaaccaaacgttgtcacaaattaca300 aatcaatgctgtaatataatttgcacctggaatggctaacgtgaagcctgaattaaatgt360 ggtttttagtttttaccatcaccaatttctatgactgttgcaaatacagaatctattaga420 aaacagggtcttggaaatgtagaattttggcgcactatgaggaaaaacaagctatctttg480 taaagcataattgagtttaatgtaattgttgtaaaaaaaaaagtgtgcttgetctactta540 aaattcctcacaatgttgaattttgacctgtattcagaagaattccaaaacaggtcagtt600 aaataaggaaatatagtatttgtcaaaccagtatcagagaaaagttacattaatgtattt660 gattacttgatctggtatctacttattaatgaataatcaacatttttctagtggacaagc720 catgttcttttcgactggtcttctgcatttatgtaaacacaccccaaatcaatttatctt780 ttgtaggaatgatttggttgggaaatttttcagaaccgctctggagcagaacaaaacggt840 acctcccggtcaccactgggactgggggaagggacacgctccaggggagaaaacaattcg900 cctagatgaagatccctgggtggttctccatgcactcgccgagggggctcagtgggtagc960 ctcttagagcagctctgagaataaacttatcatctgagttaacgggtaatagacccagaa1020 cagttcccaaaccttggcactttcgctcacttagccagaggcacccggcctggcctcggc1080 ttcccggtgagggagcgggggtgggggggatgcgcaggcacctggaacaatcagggcacc1140 gggagaagccggctcagctgatgccggtgatgagtttctctcattgaaatcctcctcacc1200 tcgggcgccttggttcccttacggatcagccctttcatcacaaagaaagccctctttcca1260 gatcatctaagggtcattgtgccaacatccgggcgtggagagtttcygtagggagaagga1320 cgaagaggggCCCCCtCggCggggacgcgggccggtggcaggaagggcgtggagggcggt1380 gcagcgtgcgagCCCCCgCCgagggccatccccgcctccgctcggccgcccgggcaagtc1440 gcctatttagggtgcggcggcgggcgggagcagtgcgcccatggcggccctgggggacga1500 ggtgctggacggttacgtgttcccggcgtgccccccctgctcgtaccggtacccataccc1560 cgcggccaccaagggcaagggcgcggcgggcggcagctggcagcagcgcggcaggggctg1620 CCttCCCgCCtCCtCCCCCtgCtCggCgggcgcggcctcgttgtccttcccgggctgcgg1680 gcggctgacggccgccgagtacttcgacagctaccagcgggagcggctcatggctctcct1740 ggcgcaggtggggccgggtctcgggccgcgcgcccgcagggccggcagctgcgacgtggc1800 ggtgcaggtgagcccgcgcatcgacgccgcggtacagtgctcgctggggaggcgcacgct1860 gCagCgCCgggCCCgCgaCCCCgagtCCCCggCCggCCCCggggccgagggcaccacggg1920 tggcggctctttctcccagcagccatcccgtcgaggcctggagcagggcagcccccagaa1980 cggcgccccgcggcccatgcgcttcccgcgcaccgtcgccgtgtactcgcccctggcctt2040 gcgccgtctcaccgccttcctggaggggcccgggcccgcggcgggcgagcagaggtccgg2100 ggcgtcggacggagagagggggccgccgcccgcgcggcttcaaggcccagaggaggggga2160 ggtgtggacgaagaaggcgccccggcggccgcagtccgacgacgacggcgaggcccaggc2220 cgcagtccgagcgagctgggagcagccggccgacggtcccgagctgccgccgcgagaggc2280 ccaggagggcgaggcggctccgcggtcggcgctaaggagcccggggcaacctccgtcggc2340 ggggagggcccgagacggcggegacggacgggaggcggccgtcgcgggagaggggccgtc2400 gccacggagcccggagctgggcaaggagcggctgcgcttccaggtaaagcctagggcggt2460 cagggcacaggggagcccgggggtgcgggtgtcttccttgggcctggccctgtgactgct2520 tcgggcactcggaggtgcggcgcttecctaagcgtgggcyacttccgtatttccgagaca2580 gccaatgaccgcgataggtgtcttccttgacagcacagtctcatgtccccgacatccaga2640 cttactcgtggcggctgctccacgggctggccagggcgacgcccttgggacgttcttata2700 acccacatatttgcactgtaaacctcgcgcagtgggcgcataggCCagCCCtgaCCgCaC2760 ggttggattacctatcagtaggcacaactgaacttcggagcacttgccggctggagagtc2820 gattcccaaggatccctctctcccatttccgcactggatgtggcaaaaacccttcaactg2880 ctgggattctggtcagcaattctgatttctcctttacgagcttccaggctattggaaagc2940 tggagctcctaaaatgecccttcctaggaatttgctttgcttttaagaagcacccccaac3000 tcagaaatccaatactgcgaaagcatttggactgctcagtgttgctgcccgagggcagca3060 ggctgaaactttaaagggctggggcacgcagagggcagttgtgacctagcagaagtggaa3120 aggcacaagaggtggtaagaagcccgagggagtccctcgcggtCtCttCCaCggCCa.CCa3180 caggctggtattccttttgaggggcggggttggtggcggtaggctgattgtgcgaggagt3240 gaatcgagaggccagggcttcccagcgtggctgtgcaggagctgtgtgtgatcttaggcc3300 agtcataaccttcctggacttagcgcagtctcacaggtgagcagactgaattaagtgctc3360 cccagagttccttccaattctgaaaggctaactctaaaaacgtgtgcataactgcttgct3420 tactgggagggaagaagggaagtttaagtaacactacttttgttcatattgaatatgaat3480 tatggcttacgtacgatttaggttcctggcaccactgtttgggagttaactagcagcatg3540 aagaatgtgatcttgggtgaacctttaaagttccttagatgtggagtcttatttttcttc3600 agcttaatacatgtgtgcatagtctaagatcaggctttatcttaaaaggccttcctacag3660 ggcgcaggtggggccgggtctcg aatcccaaactttagagaactgtttattatgtccctacttattcgtttattagccagcct3720 tatgaactgagttaatctggtatatgaactctaaggcccatgcttgcttaattgtttgac3780 taggctattaagctcacttaattactgtattgaagaggcgtacccaaacctgacctgctt3840 tctttcatgatctgaagttgccaatttcaaatatcaagtagatccttcccaacgtctgaa3900 atgaaggataattcaggtgttttgttggttaaattgacatatcctgttgttcagttctta3960 gagcagaaatatggctattaccactgcaaggactgcaacatccgctgggagagtgcttat4020 gtgtggtgtgtacagggaactaacaaggtaagaaataccaggtaactggcatcttcttgc4080 tgaaagtgtcaaggcgattttaagtttatcctctttgtcatcacaggtttacttcaaaca4140 gttttgcagaacttgtcagaagtcttataacccttaccgagtggaggatatcacctgtca4200 agtaaatcagatgttttgcattttgtctgacctgggcagtcgtcgagggtttttagtata4260 gtttgagtatacttccaaaaagaggccaggcccccagaccttaggtttcaactggctttt4320 gttaggagtggtagaaacaatactcagctgggaaacggggccttggtgttagcttctttc4380 tggccttgcaaatcttgctgttgttaacctcttctaaaactgttaacctcacttgcaata4440 tggaagaatacttgtcttacttgctacttagtctaatgtataagaaaatcaacaaaaaca4500 tgcttgtcagctaacatgaggtagtcaaggttgactgttttaccgaaacgcttcttatga4560 agcacaaccttaaagtacttaagcacagggggttagtttgtcttgcctgaaagctcacaa4620 agggacagtttaagataaatctaagttgtctagctttatggggagttgactataatggta4680 agcaagcaatatgttaactaagcattgcttaagcgcttgcttgctattaactgtgctaag4740 gggcttagctaatctttaagaggaaagaagtgactacattcgcctcctgtcacacagcta4800 atggagtctgaattgccagttgagacagcctaatcaatacacttgacccacgttggatat4860 ttaaaagcattaacaccctggggtggtggagagaaactaagtatggaaagccacttagaa4920 tcacttagatcagagctgggcatgtttctaaaagaggatgccttaaccactctgctcttg4980 gtgttcattgtcaaattcatccctgacttgttctctaccctttctcttaaacagttgttg5040 taaaagaaatttcacaattcataattggatctgatgcaatatagcagcagtacagcatgg5100 ttaaacacccactattcctagccctgtcattgctacgtaggtagggatgtagagggaaaa5160 caagattactatgggaccttgcttagagcacattcattaagtacttgaatggactagaaa5220 aatgttgaagtcctaggaaatcactaagggtttatcttctgcatgcccttctgtattttt5280 ttcccccagagttgtaaacaaacgagatgttcctgcccagtaaaacttcgccacgtggac5340 cctaaacggccccaccgtcaagatttgtgcggtagatgcaaaggcaaacgcctgtcctgt5400 gacagcactttcagcttcaaatacatcatttaggtgaaagtcagtgttgctgtgcatgcg5460 ctgatggagtagacgagtgagCttttCCgtgCCtCtCCtCCaCCtCtCCCttctcaaaat5520 acttcatgaaaggcagtgtattctgaaaaagccttcaaataaaggtattgcaacacgatt5580 tatacattgcataaaatctgtctttgaaaataaagtttcaagagcgcttgtcttgtgcta5640 acagtctgggcctgtcacttcacctttatgaatgettgctgatggcatagagtgggccag5700 gctctgagttaggctgcagccacttggaaaacaatttaggggggtgcttgtagacgaggt5760 ctacttatttaggcaggtctggaggactgaagcttagaaggaagttaactgaataaaaag5820 ccgcctagcgatcgcgccactgcactccagcctgggtgacagagtgagactccatctcaa5880 aaaaaaaagctgcctagctgtaacattaaggcattcttttgggagaggtggaggcagagc5940 catttattggttgcatgagaccgttggaggttaacgttgagtaagaatgctgagtggcgg6000 tg 6002 <210> 42 <211> 207 <212> PRT
<213> Rat <400> 42 Met Ser Arg His Ser Thr Ser Ser Met Thr Glu Thr Thr Ala Lys Asn Met Leu Trp Gly Ser Glu Leu Thr Gln Glu Lys Gln Thr Cys Thr Phe Arg Ala Gln Gly Glu Arg Lys Asp Ser Cys Lys Leu Leu Leu Ser Thr Ile Cys Leu Gly Glu Lys Ala Lys Glu Glu Val Asn Arg Val Glu Ile Leu Pro Gln Glu Asp Arg Lys Ser Pro Ile Thr Val Ala Thr Leu Lys Ala Ser Val Leu Pro Met Val Thr Val Thr Gly Ile Glu Leu Ser Pro Pro Val Thr Phe Arg Leu Arg Ala Gly Ser Gly Pro Val Phe Leu Ser 7.00 105 110 Gly Gln Glu Cys Tyr Glu Thr Ser Asp Met Ala Trp Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Asp Glu Asp Glu Asp Ala Asp Leu Ser Leu Glu Glu Ile Pro Ile Lys Gln Val Lys Arg Ala Ala Pro Gln Arg Pro Thr Ser Ile Ala Lys Arg Lys Lys Val Asp Lys Glu Glu Glu Ala Ala Val Arg Pro Ser Pro Gln Asp Lys Ser Pro Trp Lys Lys Gly Lys Ser Thr Pro Lys Pro Lys Arg Ser Thr Ser Lys Lys <210> 43 <211> 624 <212> DNA
<213> Rat <400>

atgagtcgccacagcaccagcagcatgactgaaaccacggcaaagaacatgctctggggc 60 agtgagctcactcaggaaaaacagacttgcacctttagagcccaaggcgagaggaaggac 120 agctgtaagcttttgctcagcacgatttgcctgggggagaaggccaaagaggaggtgaat l80 cgtgtggaaatccttccccaggaagacaggaaatcaccaatcactgtcgccacgctgaag 240 gcctctgtcctgcctatggtcacagtgacaggtatagagctttctcctccagtaactttt 300 cgtctcagggctggctcaggacctgtgttcctcagtggccaggaatgttatgagacttca 360 gacatggcctgggaagatgatgaggaagaggaagaggaagaggaagaggatgaagatgag 420 gatgaagatgcagatctatcgctagaagagatccctatcaaacaagtcaaaagggcagct 480 ccccagaggccgacgagcatagctaagagaaaaaaggtggacaaagaggaggaggcggca 540 gtgaggcccagccctcaggacaagagcccctggaagaagggaaaatctacacccaaacct 600 aagaggtcgacgtccaagaaatga 624

Claims (61)

Claims What is claimed is:
1. An isolated polynucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ.ID.NO.11, SEQ.ID.NO.13, SEQ.ID.NO.12, SEQ.ID.NO.28, SEQ.ID.NO.30, SEQ.ID.NO.31, SEQ.ID.NO.33, SEQ.ID.NO.35, SEQ.ID.NO.37, SEQ.ID.NO.38, SEQ.ID.NO.40, SEQ.ID.NO.41, and SEQ.ID.NO.43.
2. An isolated polynucleotide sequence encoding a protein, wherein said protein is selected from the group consisting of:
(a) a polynucleotide sequence encoding SEQ.ID.NO.16, SEQ.ID.NO.29, SEQ.ID.NO.32, SEQ.ID.NO.34, SEQ.ID.NO.36, SEQ.ID.NO.39, or SEQ.ID.NO.42;
(b) a polynucleotide sequence encoding an amino acid sequence having at least 40%
identity with SEQ.ID.NO.16, SEQ.ID.NO.29, SEQ.ID.NO.32, SEQ.ID.NO.34, SEQ.ID.NO.36, SEQ.ID.NO.39, or SEQ.ID.NO.42, (c) an isolated nucleic acid molecule that hybridizes with the polynucleotide sequence of (a) order hybridization conditions of 0.02 M to about 0.15 M NaCI
at temperatures of about 50°C to about 70°C; and (d) an isolated polynucleotide sequence that is complementary to (a), (b) or (c).
3. An expression cassette comprising the polynucleotide sequence of claim 1 or operatively linked to a promoter sequence.
4. A vector comprising the expression cassette of claim 3.
5. An isolated polypeptide sequence comprising an amino acid sequence of SEQ.ID.NO.16, SEQ.ID.NO.29, SEQ.ID.NO.32, SEQ.ID.NO.34, SEQ.ID.NO.36, SEQ.ID.NO.39 or SEQ.ID.NO.42.
6. An isolated polypeptide encoded by the polynucleotide sequence of claim 1 or 2.
7. A monoclonal antibody that specifically binds immunologically the polypeptide of claim 5.
8. A monoclonal antibody that specifically binds immunologically the polypeptide of claim 6.
9. A polyclonal antiserum, antibodies which binds immunologically to the polypeptide of claim 5.
10. A polyclonal antiserum, antibodies which binds immunologically to the polypeptide of claim 6.
11. A hybridoma cell that produces a monoclonal antibody that binds immunologically to the polypeptide of claim 5.
12. A hybridoma cell that produces a monoclonal antibody that binds immunologically to the polypeptide of claim 6.
13. A composition comprising the antibody of claim 7, 9, 9 or 10.
14. A host cell comprising the expression cassette of claim 3.
15. The host cell of claim 14, wherein the cell is a eukaryotic cell or a prokaryotic cell.
16. A transgenic animal comprising the polynucleotide sequence of claim 1 or 2.
17. The transgenic animal, wherein the animal is a rodent, a mouse or a rat.
18. A transgenic animal comprising a polynucleotide sequence selected from the group consisting of SEQ.ID.NO.11, SEQ.ID.NO.13, SEQ.ID.NO.12, SEQ.ID.NO.28, SEQ.ID.NO.30, SEQ.ID.NO.31, SEQ.ID.NO.33, SEQ.ID.NO.35, SEQ.ID.NO.37, SEQ.ID.NO.38, SEQ.ID.NO.40, SEQ.ID.NO.41, and SEQ.ID.NO43.
19. A pharmaceutical composition comprising a modulator of O1-180 expression dispersed in a pharmaceutically acceptable carrier.
20. The composition of claim 19, wherein the modulator suppresses transcription of an O1-180 gene.
21. The composition of claim 19, wherein the modulator enhances transcription of an O1-180 gene.
22. The composition of claim 19, wherein the modulator is a polypeptide.
23. The composition of claim 19, wherein the modulator is a small molecule.
24. The composition of claim 19, wherein the modulator is a polynucleotide sequence.
25. The composition of claim 24, wherein the polynucleotide sequence is DNA or RNA.
26. The composition of claim 24 further comprising an expression vector, wherein the expression vector comprises a promoter and the polynucleotide sequence, operatively linked.
27. A pharmaceutical composition comprising a modulator of O1-180 activity dispersed in a pharmaceutically acceptable carrier.
28. The composition of claim 27, wherein the composition inhibits O1-180 activity.
29. The composition of claim 27, wherein the composition stimulates 01-180 activity.
30. A method of modulating contraception comprising administering to an animal an effective amount of a modulator of O1-180 activity or O1-180 expression dispersed in a pharmacologically acceptable carrier, wherein said amount is capable of decreasing conception.
31. A method of enhancing fertility comprising administering to an animal an effective amount of a modulator of O1-180 activity or O1-180 expression dispersed in a pharmacologically acceptable carrier, wherein said amount is capable of increasing conception.
32. The method of claim 30 or 31, wherein the animal is female.
33. The method of claim 30 or 31, wherein the animal is male.
34. A method of screening for a modulator of O1-180 expression comprising the steps of providing a cell expressing a O1-180 polypeptide;
contacting said cell with a candidate modulator;

measuring O1-180 expression; and comparing said O1-180 expression in the presence of said candidate modulator with the expression of O1-180 in the absence of said candidate modulator;
wherein a difference in the expression of O1-180 in the presence of said candidate modulator, as compared with the expression of O1-180 in the absence of said candidate modulator, identifies said candidate modulator as a modulator of 01-180 expression.
35. A method of identifying compounds that modulate the activity of O1-180 comprising the steps of:
obtaining an isolated O1-180 polypeptide or functional equivalent thereof;
admixing the O1-180 polypeptide or functional equivalent thereof with a candidate compound; and measuring an effect of said candidate compound on the activity of O1-180.
36. A method of screening for a compound which binds with O1-180 comprising:
exposing a O1-180 protein, or a fragment thereof to a candidate compound; and determining whether said compound binds to the O1-180 protein or fragment therof.
37. A method of identifying a compound that modulates O1-180 activity comprising (a) providing a transgenic animal having (1) one or more regulatable O1-180 genes, (2) a knock-out of one or more O1-180 genes, or (3) a knock-in of one or more O1-180 genes;
(b) providing a control animal respectively for transgenic animal in step (a);
(c) exposing the transgenic animal and control animal to a potential O1-180-modulating compound and (d) comparing the transgenic animal and the control animal group and determining the effect of the O1-180-modulating compound on the infertility or fertility in the transgenic animal as compared to the control animal.
38. A method for detecting the binding interaction of a first peptide and a second peptide of a peptide binding pair, comprising:
(i) culturing at least one eukaryotic cell under conditions to detect a selected phenotype or the absence of such phenotype, wherein the cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a DNA binding domain of a transcriptional activation protein;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation domain of a transcriptional activation protein; wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and c) a reporter element activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter element prevents exhibition of a selected phenotype;
(ii) detecting the ability of the test peptide to interact with O1-180 by determining whether the test peptide affects the expression of the reporter element which prevents exhibition of the selected phenotype, wherein said first or second peptide is an O1-180 peptide and the other peptide is a test peptide.
39. A method of identifying binding partners for O1-180 comprising the steps of:
exposing the protein to a potential binding partner; and determining if the potential binding partner binds to O1-180.
40. A pharmaceutical composition comprising a modulator of O1-236 expression dispersed in a pharmaceutically acceptable carrier.
41. The composition of claim 40, wherein the modulator suppresses transcription of an O1 -236 gene.
42. The composition of claim 40, wherein the modulator enhances transcription of an O1-236 gene.
43. The composition of claim 40, wherein the modulator is a polypeptide.
44. The composition of claim 40, wherein the modulator is a small molecule.
45. The composition of claim 40, wherein the modulator is a polynucleotide sequence.
46. The composition of claim 45, wherein the polynucleotide sequence is DNA or RNA.
47. The composition of claim 45 further comprising an expression vector, wherein the expression vector comprises a promoter and the polynucleotide sequence, operatively linked.
48. A pharmaceutical composition comprising a modulator of O1-236 activity dispersed in a pharmaceutically acceptable carrier.
49. The composition of claim 48, wherein the composition inhibits O1-236 activity.
50. The composition of claim 48, wherein the composition stimulates O1-236 activity.
51. A method of modulating contraception comprising administering to an animal an effective amount of a modulator of O1-236 activity dispersed in a pharmacologically acceptable carrier, wherein said amount is capable of decreasing conception.
52. A method of enhancing fertility comprising administering to an animal an effective amount of a modulator of O1-236 activity dispersed in a pharmacologically acceptable carrier, wherein said amount is capable of increasing conception.
53. The method of claim 51 or 52, wherein the animal is female.
54. The method of claim 51 or 52, wherein the animal is male.
55. A method of screening for a modulator of O1-236 expression comprising the steps of:
providing a cell expressing an O1-236 polypeptide contacting said cell with a candidate modulator;
measuring O1-236 expression; and comparing said O1-236 expression in the presence of said candidate modulator with the expression of O1-236 in the absence of said candidate modulator;
wherein a difference in the expression of O1-236 in the presence of said candidate modulator, as compared with the expression of O1-236 in the absence of said candidate modulator, identifies said candidate modulator as a modulator of O1-236 expression.
56. A method of identifying compounds that modulate the activity of O1-236 comprising the steps of:
obtaining an isolated O1-236 polypeptide or functional equivalent thereof;
admixing the O1-236 polypeptide or functional equivalent thereof with a candidate compound; and measuring an effect of said candidate compound on the activity of O1-236.
57. A method of identifying binding partners for O1-180 comprising the steps of exposing the protein to a potential binding partner; and determining if the potential binding partner binds to O1-180.
58. A method of identifying a compound that modulating O1-236 activity comprising (a) providing a transgenic animal having (1) one or more regulatable O1-236 genes, (2) a knock-out of one or more O1-236 genes, or (3) a knock-in of one or more O1-236 genes;
(b) providing a control animal respectively for transgenic animal in step (a);
and (c) exposing the transgenic animal group and control animal group to a potential O1-236-modulating compounds; and (d) comparing the transgenic animal and the control animal and determining the effect of the compound on infertility or fertility in the transgenic animal as compared to the control animal.
59. A method of detecting a binding interaction of a first peptide and a second peptide of a peptide binding pair, comprising:
(i) culturing at least one eukaryotic cell under conditions suitable to detect the selected phenotype; wherein the cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined transcriptional activation domain of a transcriptional activation protein;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation protein transcriptional activation domain; wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and c) a reporter element activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter element produces a selected phenotype;
(ii) detecting the binding interaction of the peptide binding pair by determining the level of the expression of the reporter element which produces the selected phenotype;
wherein said first or second peptide is an O1-236 peptide and the other peptide is a test peptide.
60. A detecting the binding interaction of a first peptide and a second peptide of a peptide binding pair, comprising:
(i) culturing at least one yeast cell under conditions to detect a selected phenotype or the absence of such phenotype, wherein the yeast cell comprises;
a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a DNA binding domain of a transcriptional activation protein;
b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation domain of a transcriptional activation protein; wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and c) a reporter element activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter element prevents exhibition of a selected phenotype;
(ii) detecting the ability of the test peptide to interact with O1-236 by determining whether the test peptide affects the expression of the reporter element which prevents exhibition of the selected phenotype, wherein said first or second peptide is an O1-236 peptide and the other peptide is a test peptide.
61. A method of identifying binding partners for O1-236 comprising the steps of:

exposing the protein to a potential binding partner; and determining if the potential binding partner binds to O1-236.
CA002483323A 2002-04-26 2003-04-23 Contraceptive targets Abandoned CA2483323A1 (en)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
PCT/US2002/013245 WO2002088314A2 (en) 2001-04-27 2002-04-26 Ovary-specific genes and proteins
USPCT/US02/13245 2002-04-26
US41126202P 2002-09-17 2002-09-17
US60/411,262 2002-09-17
US43416502P 2002-12-17 2002-12-17
US60/434,165 2002-12-17
US43978103P 2003-01-13 2003-01-13
US60/439,781 2003-01-13
US44216403P 2003-01-23 2003-01-23
US60/442,164 2003-01-23
PCT/US2003/012720 WO2003091400A2 (en) 2002-04-26 2003-04-23 Contraceptive targets

Publications (1)

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CA2483323A1 true CA2483323A1 (en) 2003-11-06

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Country Status (4)

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EP (1) EP1572924A2 (en)
AU (1) AU2003228675A1 (en)
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