Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/16—Masculine contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/18—Feminine contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates generally to ovary-specific genes and the proteins they encode.
• 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.
• oocytes may secrete factors which are necessary for normal granulosa cell and thecal cell function. Because oocyte growth is coordinated 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, and the process of follicular recruitment.
• 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.
• nucleoplasmin 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).
• nucleoplasmin 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, 1991; 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).
• nucleoplasmin Direct interaction of nucleoplasmin with protamine was observed in in vitro experiments, which suggest that the nucleoplasmin is bound to protamine in a 1 :1 ratio and that the polyglutamic acid tract in nucleoplasmin plays a critical role for binding to protamine (Iwata et al, 1999).
• 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 functional 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 histones H2A and H2B and along with the proteins N1/N2 that bind histones H3 and H4, can promote nucleosome assembly onto DNA (Dilworth et al, 1987; Laskey et al, 1993). Thus, 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 histones, and is required in some way for chromatin assembly.
• 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).
• 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; Nekola and Nalbandov, 1971; Salusrri et al, 1985; Vanderhyden et /., 1993; Eppig et ⁇ /., 1997a, b).
• uPA urokinase plasminogen activator
• GDF-9 Growth differentiation factor 9
• TGF- ⁇ transforming growth factor ⁇
• mice Female Gd ⁇ 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, 1996; Carabatsos et al, 1998, Elvin et al, 1999 A).
• recombinant GDF-9 affects the expression of the genes encoding hyaluronan synthase 2 (Has2), cyclooxygenase 2 (Cox2), steroid acute regulatory protein (StAR), the prostaglandin E2 receptor EP2, pentaraxin 3, LH receptor and uPA (Elvin et al, 1999B, Elvin et al, 2000).
• mice To identify key proteins in the hypothalamic-piruitary-gonadal axis, several important knockout mouse models have been generated, including four which have ovarian defects. Mice lacking 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 (Acvrl) survive to adulthood but display reproductive defects. Male mice show reduced testes size and demonstrate delayed fertility (Matzuk, et al. 1995). In contrast, female mice have a block in folliculogenesis at the early antral follicle stage leading to infertility.
• Gd ⁇ 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.
• GDF-9 functions in the reciprocal manner as an oocyte-derived growth factor which is required for somatic cell function.
• the present invention provides three ovary-specific and oocyte-specific polynucleotide sequences, O1-180 (SEQ.ID.NO.l, SEQ.ID.NO.i l, SEQ.ID.NO.12, SEQ.ID.NO.13), O1-184 (SEQ.ID.NO.3) and 01-236 (SEQ.ID.NO.5, SEQ.ID.NO.7, SEQ.ID.NO.8; SEQ.LD.NO.IO, and SEQ.ID.NO.14), the protein products they encode, fragments 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.
• the invention provides methods for detecting cell proliferative or degenerative disorders of ovarian origin and which are associated with O1-180, 01-184 or 01-236.
• the invention provides method of treating cell proliferative or degenerative disorders associated with abnormal levels of expression of O1-180, 01-184 or 01-236, by suppressing or enhancing their respective activities.
• the present invention provides a pharmaceutical composition comprising a modulator of O1-180, 01-184 and/or 01-236 expression dispersed in a pharmaceutically acceptable carrier.
• the modulator may suppress or enhance transcription of an O1-180, 01-184 and/or 01-236 gene.
• the modulator may be a polypeptide sequence, a protein, a small molecule, or a polynucleotide sequence.
• the polynucleotide sequence is DNA or RNA.
• the polynucleotide sequence is comprised in an expression vector operatively linked to a promoter.
• a further embodiment of the present invention is a pharmaceutical composition
• a pharmaceutical composition comprising a modulator of O1-180, 01-184 and/or 01-236 activity dispersed in a pharmaceutically acceptable carrier.
• the composition may inhibit or stimulate O1-180, 01-184 and/or 01-236 activity.
• the composition may be a protein, polypeptide sequence, small molecule, or polynucleotide sequence.
• 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, 01-184 and/or 01-236 activity dispersed in a pharmacologically acceptable carrier, wherein said amount is capable of decreasing conception.
• the animal may be a male or a female.
• a further embodiment is a method of enhancing fertility comprising administering to an animal an effective amount of a modulator of O1-180, 01-184 and/or 01- 236 activity dispersed in a pharmacologically acceptable carrier, wherein said amount is capable of increasing conception.
• another embodiment is a method of screening for a modulator of 01-180, 01-184 and/or 01-236 activity comprising the steps of: providing a cell expressing an Ol-l 80, 01-184 and/or 01-236 polypeptide; contacting said cell with a candidate modulator; measuring O1-180, 01-184 and/or 01-236 expression; and comparing the O1-180, 01-184 and/or 01-236 expression in the presence of the candidate modulator with the expression of 01- 180, 01-184 and/or 01-236 expression in the absence of the candidate modulator; wherein a difference in the expression of O1-180, 01-184 and/or 01-236 in the presence of the candidate modulator, as compared with the expression of O1-180, O1-184 and/or 01-236 in the absence of the candidate modulator, identifies the candidate modulator as a modulator of Ol-l 80, 01-184 and/or 01-236 expression.
• a specific embodiment of the present invention is a method of identifying compounds that modulate the activity of O1-180, 01-184 and/or 01-236 comprising the steps of obtaining an isolated 01-180, 01-184 and/or 01-236 polypeptide or functional equivalent thereof; admixing the O1-180, 01-184 and/or 01-236 polypeptide or functional equivalent thereof with a candidate compound; and measuring an effect of said candidate compound on the activity of O1-180, 01-184 and/or O1-236.
• Another embodiment is method of screening for a compound which modulates the activity of O1-180, 01-184 and/or 01-236 comprising exposing O1-180, 01-184 and/or 01-236 or a O1-180, 01-184 and/or 01-236 binding fragment thereof to a candidate compound; and determining whether said compound binds to 01-180, 01-184 and/or 01-236 or the O1-180, 01-184 and/or 01-236 binding partner thereof; and further determining whether said compound modulates Ol-l 80 or the Ol-l 80 interaction with a binding partner.
• another embodiment is a method of screening for an interactive compound which binds with O1-180, 01-184 and/or 01-236 comprising exposing a O1-180, 01- 184 and/or 01-236 protein, or a fragment thereof to a compound; and determining whether said compound bound to the O1-180, 01-184 and/or 01-236.
• Another embodiment is a method of identifying a compound that effects O1-180, 01-184 and/or 01-236 activity comprising providing a group of transgenic animals having (1) a regulatable one or more O1-180, 01-184 and/or 01-236 protein genes, (2) a knockout of one or more O1-180, 01-184 and/or 01-236 protein genes, or (3) a knock-in of one or more O1-180, 01-184 and/or 01-236 protein genes; providing a second group of control animals respectively for the group of transgenic animals; and exposing the transgenic animal group and control animal group to a potential O1-180, 01-184 and/or Ol-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.
• 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 thereof 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 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 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
• reproductive tissue is an ovary or testis.
• Other reproductive tissues may also include the uterus, vagina, oviduct, cerivx, gonads, vas deferens, prostate, seminal vesicles and epididymis.
• 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, comprising: 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; wherem 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 phen
• another embodiment is a method of identifying binding partners for O1-180, 01-184 and/or 01-236 comprising the steps of: exposing the protein to a potential binding partner; and determining if the potential binding partner binds to Ol-l 80, 01-184 and/or 01-236.
• the present invention provides key in vitro and in 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 us them as reagents to evaluate potential contraceptives to block 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 these signaling pathways that are associated with some forms of human infertility or gynecological cancers or other cancers associated with reproductive tissues.
• 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 of new contraceptives.
• FIGURE 1 Multi-tissue Northern blot analysis of ovary-specific genes.
• Northern blot analysis was performed on total RNA using O1-180, 01-184, and 01-236 probes. These gene products demonstrate an ovary-specific pattern (OV, ovary; WT, wild-type; -/-, Gd ⁇ knockout) as shown. 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.
• 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), 01-184 ( Figures 2C-2D) and 01-236 ( 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 arows).
• FIGURES 3 A and 3B In situ hybridization analysis of 01-236 in mouse ovaries. In situ hybridization was performed using probe 01-236 (partial Npm2 cDNA fragment). Brightfield analysis ( Figure 3A) and darkfield analysis ( Figure 3B) of the 01-236 mRNA in the same adult ovary sections. The probe demonstrates specific expression in all growing oocytes. Oocyte-specific 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.
• FIGURE 4 Npm2 cDNA representation. Schematic representation of the mouse Npm2 cDNA sequence (984 bp) and two of the clones isolated from the mouse ovary cDNA libraries. The original 01-236 probe (749 bp) is shown at the top and encompasses the entire Npm2 open reading frame. The open reading frame (solid box) is 621 bp and the 5' UTR and 3' UTR sequences (thin lines) are 155 bp and 205 bp, respectively. The polyA sequences are not depicted. Clone 236-1 (Npm2) was isolated from the wild-type ovary cDNA library and clone 236-3 was isolated from the Gd ⁇ knockout ovary cDNA library.
• FIGURE 5 Amino acid sequence conservation among Xenopus laevis (SEQ.JD.NO.15), mouse (SEQ.LD. NO.6), and human (SEQ.ID.NO.9) NPM2 proteins. Using the NCBI blast search tools and Megalign software, comparison of mouse (m), human (h), and Xenopus laevis NPM2 amino acid sequences reveals high identity (amino acids highlighted in blue). Spaces between the amino acids indicate gaps to aid in the alignment.
• FIGURE 6A and FIGURE 6B Structure of the mouse Npm2 gene ( Figure 6A). Two overlapping recombinant lambda clones (236-13 and 236-14), isolated from a mouse 129/SvEv library, are shown at the top, and a schematic enlargement of the Npm2 gene is also depicted. Open boxes represent untranslated regions and solid black boxes represent protein coding regions. The 236-13 insert is -19.0 kb and 236-14 insert is -21.0 kb. The entire contig is -37 kb. All 9 exons of the Npm2 gene are encompassed on a single 6.9 kb Xbal (X) fragment as shown.
• X Xbal
• FIGURES 7A and 7B Mouse Npml gene (SEQ ID NO: 7) and amino acid sequences (SEQ.LD.NO.6).
• Uppercase letters represent sequence identity with the Npml cDNA sequences; non-transcribed 5' and 3' sequences and intron sequences are shown in lowercase.
• the predicted transcription initiation codon, the termination codon, and the polyadenylation signal sequence are all underlined. Numbers along the left side represent the amino acids.
• the underlined and bolded "T” in codon 36, the bolded "c” for amino acid 26, and the underlined and bolded "C: in the 3' UTR sequence indicate differences between the cDNA and gene sequences. Arrows indicate where the 01-236 fragment initiates and ends in the cDNA sequence.
• FIGURE Chromosomal localization of the mouse Npml gene. (Top)
• 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 D14Mit203 and D14Mit32. 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 assignment was unambiguous.
• FIGURES 9A-9H Analysis of Npm2 mRNA and NPM2 protein in mouse ovaries and early embryos. In situ hybridization was performed using probe 01-236 (partial Npm2 cDNA fragment). Brightfield analysis ( Figure 9A) and darkfield analysis (Figure 9B) of the 01-236 mRNA in the same adult ovary sections.
• Figure 9C Immunohistochemistry of ovaries from a 5-week old mouse stained for NPM2 in the nuclei (bright red) of oocytes from type 3 (arrow) to antral follicles.
• Figure 9D In preovulatory GVB oocytes induced by luteinizing hormone (hCG), NPM2 is evenly stained in the cytoplasm (arrow).
• FIGURES 10A-10C Gene targeting construct for a knockout of Npm2 and genotype analysis of offspring from heterozygote intercrosses.
• Figure 10A The targeting strategy used to delete exon 2, exon 3, and the junction region of exon 4.
• PGK-hprt and MCl-tk expression cassettes are shown. Recombination were detected by Southern blot analysis using 5' and 3' probes.
• B BamHI; Bg, Bgl II; P, Pst I).
• Figure 10B Southern blot analysis of genomic DNA isolated from mice generated from intercrosses of Npm2 +/" mice.
• FIGURES 11 A-l IF. Histological analysis of ovaries from wild-type, Npml +/ ⁇ , and Npml '1' mice.
• FIG. 11 A-l ID Immunohistochemistry of ovaries from 6-week old mice stained for Npml in the nuclei of oocytes (Figure 11A and Figure 11C for Npml +/' ovaries; FIGUE 11B and FIGURE 11D for Npm2 ' ovaries).
• Figure 11E-11F PAS (Periodic acid Schiff)/hematoxylin staining of ovaries from 12 week old mice wild-type ( Figure HE) and Npm2 ⁇ / ⁇ ( Figure 1 IF) ovaries. Arrows show large antral follicles; "CL” denote corpora lutea.
• FIGURES 12A-12D In vitro culture of eggs and fluorescent-labeling of DNA from fertilized eggs from Npm2 '/' 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 24 and 48 hours of culture.
• Figures 12A, 12C Most of the eggs from wild-type mice divided to form two cell embryos by 24 h; some of two cell embryos progressed to the four cell stage after 48 h of culture.
• Figures 12B, 12D Very few eggs from Npm2 " mice cleaved into two cell embryos; no four cell embryos were detected after 48 hours of culture. Some developmentally abnormal or apparently apoptosed embryos from Npm2 ⁇ ' mice were detected.
• FIGURES 13A-13F Localization of O1-180 in mouse ovaries.
• Expression of Ol-l 80 in PMSG-treated wild-type ( Figures 13 A and 13B) and Gd ⁇ knockout ( Figures 13C-13F) ovaries was analyzed by in situ hybridization with a specific antisense probe.
• the expression of Ol-l 80 gene was detectable at early primary follicle stage (type 3a) through ovulatory follicle stage, but not in primordial follicles in wild-type ovaries.
• Gd ⁇ knockout ovaries the follicle numbers was increased per unit volume due to the arrest of follicle development at primary follicle stage, more O1-180 positive signal were detected in each section.
• Figures 13A, 13C and 13E brightfield analysis of the ovaries
• Figures 13B, 13D and 13F corresponding darkfield analysis of the same ovary sections.
• Figures 13E and 13F were high power magnification of the same sections shown in Figures 13C and 13D.
• FIGURE 14 Structure of the O1-180 (SEQ.ID.NO.i l) gene and O1-180
• FIG.ID.NO.12 SEQ.ID.NO.12 pseudogene.
• Diagrams representing the O1-180 pseudogene and the O1-180 gene are shown at the top along with unique restriction endonucleases sites which were important in constructing the linear map shown at the bottom. Exons and introns are drawn to scale. Boxes denote exons, hatched regions denote protein coding portions and the solid regions denote the untranslated portions. Lines connecting boxes denote introns.
• Ol-180ps O1-180 pseudogene; O1-180: O1-180 gene; B: BamHI; S: Sail; X: Xhol; [0040]
• FIGURES 15A and 15B Comparison of O1-180 gene and O1-180 pseudogene.
• FIGURE 16 Maps of mouse chromosome 5, showing the position in centiMorgan (cM) of the marker best linked to O1-180 gene (A) and its related pseudogene (B) (data and maps generated at the Jackson Laboratory Bioinformatics Server).
• FIGURE 17 Gene targeting constructs for Ol-l 80. The targeting strategy used to delete exon 1. PGK-hprt and MCl-tk expression cassettes are shown.
• FIGURE 18 Northern blot analysis of O1-180 mRNA expression in multiple tissues.
• FIGURE 19 Western blot analysis of recombinant O1-180.
• FIGURES 20A-20F Immunostaning of O1-180 in mouse ovaries. Anti- Ol -180 polyclonal antibodies (made in goats) were used for IHC to detect the expression of Ol- 180 in mouse ovary sections.
• Figures 20A- Figure 20D are wild-type ovaries;
• Figure 20E- Figure 20F are Gd ⁇ knockout ovaries.
• Figure 20b is a negative control with normal goat serum. The Ol-l 80 protein was localized specifically to the cytoplasm of mouse oocytes and zygotes but disappears after this point. Staining indicates the location of the Ol-l 80 protein.
• FIGURES 21A AND 21B In vitro culture of O1-190 mouse embryos.
• FIGURE 21 A shows embryos cultured from Ol-180 +/" mice and
• Figure 21B shows embryos cultred from Ol-l 80 " " mice.
• Ml 6 medium On the third day of in vitro culture in Ml 6 medium, most control embryos progressed to the morula or blastocyst stage, while zygotes in O1-180 knockout mice still remained at the one-cell or two-cell stage.
• FIGURE 22 This figure shows a comparison of the human and mouse Ol-l 80 proteins. The differences are underlined. The proteins have a similarity of 91.3%. DETAILED DESCRIPTION OF THE INVENTION
• animal refers to a mammal, such as human, non- human primates, horse, cow, elephant, cat, dog, rat or mouse, hi specific embodiments, the animal is a human.
• 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 are 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') 2 , 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).
• binding protein refers to proteins that demonstrate binding affinity for a specific ligand. 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. sub. r or its soluble receptor
• ception refers to the union of the male sperm and the ovum of the female; fertilization.
• contraceptive 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 immunocontraceptive 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.
• DNA is defined as deoxyribonucleic acid.
• 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.
• 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.
• expression includes both transcription of a gene and translation of mRNA into a gene product.
• expression only includes transcription of the nucleic acid encoding genes of interest.
• the term “therapeutic construct” may also be used to refer to the expression construct or transgene.
• the present invention utilizes the expression construct or transgene as a therapy to treat infertility.
• the present invention utilizes the expression construct or transgene as a "prophylactic construct" for contraception.
• the "prophylactic construct” is a contraceptive.
• 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 infra.
• 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.
• genomic sequences e.g., genomic sequences, cDNA sequences and engineered segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins and mutant.
• native gene refers to a gene as found in nature with its own regulatory sequences
• 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.
• Fertility refers to the quality of being productive or able to conceive. Fertility relates to both male and female animals.
• hyperproliferative disease is defined as a disease that results from a hype roliferation 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.
• 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.
• 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.
• 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 know 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.
• nucleotide is defined as a chain of nucleotides.
• nucleic acids are polymers of nucleotides.
• nucleic acids and polynucleotides as used herein are interchangeable.
• nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
• polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
• recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
• polynucleotides include mutations of the polynucleotides, include but are not limited to, mutation of the nucleotides, or nucleosides by methods well known in the art.
• polypeptide is defined as a chain of amino acid residues, usually having a defined sequence.
• polypeptide is interchangeable with the terms “peptides” and “proteins”.
• 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.
• 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.
• RNA is defined as ribonucleic acid.
• RNA interference or "iRNA” is an RNA molecule that is used to inhibit a particular gene of interest.
• under transcriptional control or "operatively linked” is defined as the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
• nucleoplasmin 2 (01-236 or NPM2)
• xNPM2 mammalian ortholog of Xenopus laevis nucleoplasmin
• NPM2 human gene, Npm2 mouse gene, and Xnpm2 Xenopus gene; NPM2 protein in all species).
• Human nucleophosmingene gene maps to human chromosome 5q35, encodes a 294 amino acid protein, and has orthologs in mouse (Npml, also called B23, accession # Q61937) and Xenopus laevis (Xnpml or N038 accession # X05496).
• Mouse nucleoplasmin/nucleophosmin 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 # AF081280).
• 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).
• the Npm2 cDNA sequences have been used by the inventors to obtain the mouse Npm2 gene and the human NPM2 cDNA and gene and also map these genes.
• mice lacking Npm2 have defects in fertility due to abnormalities in early post-fertilization cleavage events.
• the discovery of the mammalian homolog of the most abundant nuclear protein in Xenopus laevis oocytes and eggs is important for a clear understanding of oogenesis, fertilization, and post-fertilization development in mammals and possibly also to define further oocyte factors which are necessary in mammalian cloning experiments.
• nucleoplasmin influences its function. Comparison of the forms of nucleoplasmin from the oocyte (i.e., in the ovary) versus egg (i.e., after ovulation and ready for fertilization) demonstrate dramatic differences in the level of phosphorylation. Xenopus laevis egg nucleoplasmin is substantially larger than the oocyte form, migrating -15,000 daltons larger on SDS-PAGE due to phosphorylation differences (Sealy et al, 1986).
• Nucleoplasmin has -20 phosphate groups/protein in the egg compared to ⁇ 10 phosphate groups/proteins in the oocyte, and an egg kinase preparation can modify the oocyte nucleoplasmin so it resembles the egg form (Gotten et al, 1986). Functionally, this hyperphosphorylation of nucleoplasmin stimulates its nuclear transport (Vancurova et al, 1995) and also results in a more active form, leading to increased nucleosome assembly (Sealy et al, 1986) and sperm decondensation (Leno et al, 1996).
• a hyperphosphorylated form of nucleoplasmin is also present during the early stages of Xenopus laevis embryogenesis where it is believed to play some function during the rapid cell cycles and DNA replication (Burglin et al, 1987).
• the high percentage of serine and threonine residues in frog and mammalian NPM2 suggest a similar role of phosphorylation of mammalian nucleoplasmin 2 in mammalian eggs. Phosphorylation could act to regulate when NPM2 acts, making it inactive until the critical time (i.e., histon addition to maile and femal pronuclei or during transcriptional arrest).
• casein kinase II specifically interacts with nucleoplasmin and phosphorylates it, and an inhibitor of casein kinase II can block nuclear transport of Xenopus laevis nucleoplasmin (Vancurova et al, 1995).
• casein kinase II phosphorylation sites are conserved betweenfrog nucleoplasmin2 (Serl25 and Serl77), mouse NPM2 (Thrl23 and Serl84), and human NPM2 (Thrl27 and Serl91).
• a casein kinase II-NPM2 interaction in vivo could be predicted in mammals.
• the present invention provides three novel proteins, Ol-l 80
• the invention provides a method for detection of a cell proliferative or degenerative disorder of the ovary, which is associated with expression of O1-180, 01-184 or 01-236.
• the invention provides a method for treating a cell proliferative or degenerative disorder associated with abnormal expression of Ol- Ol-l 80, 01-184, 01-236 by using an agent which suppresses or enhances their respective activities.
• O1-180, 01-184 and 01-236, as well as fragments and derivatives thereof, will also possess biological activities that will make them useful as diagnostic and therapeutic reagents.
• GDF-9 is an oocyte-expressed gene product which has a similar pattern of expression as O1-180, 01-184, and 01-236. It has been shown that mice lacking GDF-9 are infertile at a very early stage of foUicular development, at the one-layer primary follicle stage (Dong, et al). These studies demonstrate that agents which block GDF-9 function would be useful as contraceptive agents in human females. Since O1-180, 01-184, and 01-236 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 would also be infertile. Thus, blocking the function of any or all of these gene products would result in a contraceptive action.
• inhibin 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 foUicular fluid. Because of its suppression of FSH, inhibin has been advanced as a potential contraceptive in both males and females. O1-180, 01- 184 and 01-236 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, 01-184, 01-236 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, 01-184 and 01-236 may be useful as indicators of developmental or reproductive anomalies in prenatal screening procedures.
• MuUerian inhibiting substance (MIS or anti-MuUerian 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, 01-184 and 01-236 may function similarly and may, therefore, be targets for anti-cancer agents, such as for the treatment of ovarian cancer.
• O1-180, 01-184 and 01-236 proteins, 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, 01-184 and 01-236 and agonists and antagonists thereof can be used to identify agents which enhance fertility (e.g., increase the success of in vivo or in vitro fertilization) in a mammal.
• assays of these or related oocyte-expressed gene products can be used in diagnostic assays for 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).
• these 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 in vitro.
• Ol-l 80, 01-184 and/or 01-236 play a role in oocyte maturation, they may be useful targets for in vitro fertilization procedures, e.g., in enhancing the success rate.
• the present invention relates to O1-180 (SEQ.ID.NO.2, SEQ.ID.NO.16), O1-184 (SEQ.ID.NO.4) and 01-236 (SEQ.JX NO 6, SEQ.ID.NO.9) polypeptides, proteins, or agents thereof.
• 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 Ol-l 80, 01-184 or 01-236 with proteolytic enzymes, known as proteases, can produce a variety of N-terminal, C-terminal and internal 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.NO. 2, SEQ.ID.NO. 4, SEQ.ID.NO.
• 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.
• substantially pure refers to O1-180, 01-184 and 01-236 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 Ol-l 80, 01-184 and 01-236 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 Ol-l 80, 01-184 and O1-236 polypeptides can also be determined by amino-terminal amino acid sequence analysis.
• 01-180, 01-184 and 01-236 polypeptides include functional fragments of the polypeptides, as long as their activities remain. Smaller peptides containing the biological activities of O1-180, 01-184 and 01-236.
• the polypeptides of the invention include the disclosed sequences and conservative variations thereof.
• conservative variation denotes the replacement of an amino acid residue by another, biologically similar residue.
• 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.
• 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 polypep-tide.
• 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.
• a derivative of a compound comprising amino acids in a sequence corresponding to the sequence of O1-180, 01-184 or 01-236 need not comprise a sequence of amino acids that corresponds exactly to the sequence of Ol-l 80, 01-184 or 01-236, so long as it retains a measurable amount of the activity of the O1-180, 01-184 or 01-236.
• a O1-180, 01-184 or 01-236 mutant may be generated and tested for O1-180, 01-
• O1-180, 01-184 or 01-236 mutants may also be synthesized to reflect a O1-180, Ol-
• O1-180, 01-184 or 01-236 mutants may be used as antagonists to inhibit or enhance fertitlity.
• O1-180, 01-184 or 01-236 mutants may be used as potential contraceptive compositions and/or fertility enhancement compostions.
• the invention also provides polynucleotides encoding the Ol-l 80
• SEQ.ID.NO.l SEQ.ID.NO.il, SEQ.ID.NO.13 and SEQ.ID.NO.12
• O1-184 SEQ.ID.NO.3
• 01-236 SEQ.ID.NO.5, SEQ.ID.NO.7, SEQ.TD.NO.8; SEQ.ID.NO.10, and SEQ.ID.NO.14 proteins and fragments and derivatives thereof.
• These polynucleotides include DNA, cDNA and RNA sequences which encode O1-180, 01-184 or 01-236.
• polynucleotides encoding all or a portion of O1-180, 01-184 and/or 01-236 are also included herein, as long as they encode a polypeptide with the activity of O1-180 (SEQ.ID.NO.l, SEQ.ID.NO.il, SEQ.ID.NO.13 and SEQ.ID.NO.12), 01-184 (SEQ.ID.NO.3) or 01-236 (SEQ.ID.NO.5, SEQ.ID.NO.7, SEQ.ID.NO.8; SEQ.ID.NO.10, and SEQ.ID.NO.14).
• Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides.
• polynucleotides of O1-180 (SEQ.ID.NO.l, SEQ.ID.NO.il, SEQ.ID.NO.13 and SEQ.ID.NO.12), 01-184 (SEQ.ID.NO.3) or O1-236 (SEQ.ID.NO.5, SEQ.ID.NO.7, SEQ.ID.NO.8; SEQ.ID.NO.10, and SEQ.ID.NO.14) may be subjected to site- directed mutagenesis.
• the polynucleotide sequences for O1-180, O1-184 and 01-236 also includes antisense sequences.
• the polynucleotides of the invention include sequences that are degenerate as a result of the genetic code.
• nucleotide sequences 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-l 80, 01-184 and 01-236 polypeptides encoded by the nucleotide sequences are functionally unchanged.
• DNA sequences of the invention can be obtained by several methods.
• 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.
• the O1-180, 01-184 and 01-236 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.
• 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.
• 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).
• DNA sequences encoding 01-180, 01-184 and 01-236 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.
• genomic DNA isolates are 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.
• 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).
• a cDNA expression library such as lambda gtl 1
• Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of 01- 180, 01-184 and/or 01-236 cDNA.
• DNA sequences encoding Ol-l 80, 01-184 or 01-236 can be expressed in vitro 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.
• the O1-180, 01-184 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, 01-184 or 01-236 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 baculo virus- 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, 01-184 or 01-236 can be expressed in either prokaryotes or eukaryotes.
• Hosts can include microbial, yeast, insect and mammalian organisms.
• 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.
• Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
• the host is prokaryotic, such as E coli
• competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well known in the art.
• MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired.
• Eukaryotic cells can also be co-transformed with DNA sequences encoding the O1-180, 01-184 or 01-236 cDNA sequences of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the neomycin resistance gene.
• Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein, (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
• a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
• Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention may be carried out by conventional means induding preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
• the invention includes antibodies immunoreactive with 01 - 180, 01 - 184 or 01-236 polypeptides or functional fragments thereof.
• Antibodies which consists 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 O1-180, 01-184 or 01-236.
• cell-degenerative disorder denotes the loss of any type of cell in the ovary, either directly or indirectly.
• GDF-9 there is a block in the growth of the granulosa cells leading to eventual degeneration (i.e., death) of the oocytes (Dong et al, 1996). This death of the oocyte appears to lead to differentiation of the granulosa cells.
• GDF-9 no normal thecal cell layer is formed around the follicles.
• 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 01-180, 01-184, or 01-236.
• O1-180, 01-184, or 01-236 Absence or misexpression of O1-180, 01-184, or 01-236 could result in defects in the oocyte/egg leading to the inability of the egg to be fertilized by spermatozoa. Alternatively, embryos may not develop or halt development during the early stage of embryogenesis or show defects in fertilization secondary to absence of these oocyte derived factors. Therefore, O1-180, 01-184 or 01-236 compositions may be employed as a diagnostic or prognostic indicator of infertility in general. More 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, 01-184 or 01-236 expression.
• One embodiment of the instant invention comprises a method for detecting variation in the expression of O1-180, 01-184 or 01-236. This may comprise determining the level of O1-180, 01-184 or 01-236 expressed, or determining specific alterations in the expressed product. In specific embodiments, alterations are detected in the expression of 01- 180, 01-184 or 01-236.
• 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.
• 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.
• the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification.
• the identified product is detected.
• the detection may be performed by visual means (e.g., ethidium bromide staining of a gel).
• the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).
• 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 inco ⁇ orated herein by reference) and PCRTM- SSCP.
• ASO allele-specific oligonucleotide
• dot blot analysis e.g., 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 inco ⁇ orated herein by
• 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).
• Antibodies can be used in characterizing the O1-180, 01-184 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.
• 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 like also may be used in connection with the present invention.
• 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.
• labels and methods of labeling 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. [0115] 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 fluorescent, which can react with specific anti-hapten antibodies.
• the detectably labeled antibody is given a dose which is diagnostically effective.
• 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.
• 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.
• the dosage of detectably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the mdividual. 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.
• 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.
• a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which may readily be detected by conventional gamma cameras.
• 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 (DTP A) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.
• DTP A diethylenetriaminepentacetic acid
• EDTA ethylenediaminetetraacetic acid
• Typical examples of metallic ions which can be bound to the monoclonal antibodies of the invention are In, Ru, Ga, 68 Ga, 72 As, 89 Zr and 201 Ti.
• the monoclonal antibodies of the invention can also be labeled with a paramagnetic isotope for pu ⁇ oses of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR).
• MRI magnetic resonance imaging
• ESR electron spin resonance
• any conventional method for visualizing diagnostic imaging can be utilized.
• gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI.
• Elements which are particularly useful in such techniques include 157 Gd, 55 Mn, 162 Dy, 55 Cr and 56 Fe.
• cell-proliferative disorder or hype ⁇ roliferative disorder denotes malignant as well as non-malignant cell populations which often appear to differ from the surrounding tissue both mo ⁇ hologically and genotypically.
• the Ol-l 80, 01-184 and 01-236 polynucleotides that are antisense molecules are useful in treating malignancies of the various organ systems, particularly, for example, the ovaries.
• any disorder which is etiologically linked to altered expression of O1-180, 01-184 or 01-236 could be considered susceptible to treatment with a Ol-l 80, 01-184 or 01-236 suppressing reagent, respectively.
• the invention provides a method for detecting a cell proliferative disorder of the ovary which comprises contacting an anti-Ol-180, 01-184 or 01-236 antibody with a cell suspected of having an O1-180, 01-184 or 01-236 associated disorder and detecting binding to the antibody.
• the antibody reactive with Ol-l 80, 01-184 or 01-236 is labeled with a compound which allows detection of binding to O1-180, 01-184 or 01-236, respectively.
• an antibody specific for an O1-180, 01-184 or 01-236 polypeptide may be used to detect the level of O1-180, 01-184 or 01-236, respectively, in biological fluids and tissues.
• a 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 foUicular fluid.
• the level of O1-180, 01-184 or 01-236 in the suspect cell can be compared with the level in a normal cell to determine whether the subject has an O1-180, 01-184 or Ol-236-associated cell proliferative disorder.
• the subject is human.
• the antibodies of the invention can be used in any subject in which, it is desirable to administer in 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.
• the antibodies in these immunoassays can be detectably labeled in various ways.
• 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.
• RIA radioimmunoassay
• ELISA sandwich 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.
• O1-180, 01-184 and 01-236 Due to the expression of O1-180, 01-184 and 01-236 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, 01-184 and 01-236 could play a role in regulation of the menstrual cycle and, therefore, could be useful in various contraceptive regimens.
• O1-180, 01-184, 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.
• 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.
• 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 01-236 are maternal effect genes since they function in processes of early embryogenesis.
• 01-236 or NPM2 may play a role in in chromatin remodeling during early embryoonic development.
• 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).
• oocytes can efficiently remodel not only sperm nuclei during fertilization, but also somatic cell nuclei.
• the inventors have contemplated the role of NPM2 in nuclear transfer cloning (Zuccotti et al, 2000).
• NPM2 (encoded by 01-236) is a critical factor in mammalian oocytes for chromatin remodeling during early embryonic development.
• supplementing enucleated oocytes with NPM2 may facilitate cloning by nuclear transfer technologies.
• the monoclonal antibodies of the invention can be used in vitro and in vivo to monitor the course of amelioration of an O1-180, O1-184 or Ol-236-associated disease in a subject.
• a particular therapeutic regimen aimed at ameliorating the O1-180, 01-184 or Ol-236-associated disease is effective.
• ameliorate denotes a lessening of the detrimental effect of the Ol- 180, 01-184 or Ol-236-associated disease in the subject receiving therapy.
• 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.
• a cell-proliferative disorder is associated with the expression of Ol-l 80, 01-184 or 01-236
• nucleic acid sequences that interfere with the expression of O1-180, 01-184 or 01-236, respectively, at the translational level can be used.
• This approach utilizes, for example, antisense nucleic acids or ribozymes to block translation of a specific O1-180, 01-184 or 01-236 mRNA, either by masking that mRNA with an antisense nucleic acid or by cleaving it with a ribozyme.
• 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, 01-184 or Ol-236-producing cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus- Sakura, 1988).
• 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.
• 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.
• the present invention also provides gene therapy for the treatment of cell proliferative or degenerative disorders which are mediated by Ol-l 80, 01-184 or 01-236 proteins. Such therapy would achieve its therapeutic effect by introduction of the respective Ol- 180, 01-184 or 01-236 cDNAs or Ol-l 80, 01-184, or 01-236 antisense polynucleotide into cells having the proliferative or degenerative disorder. Delivery of O1-180, 01-184, or 01-236 cDNAs or antisense O1-180, 01-184 or 01-236 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.
• RNA virus such as a retrovirus
• retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
• MoMuLV Moloney murine leukemia virus
• HaMuSV Harvey murine sarcoma virus
• MuMTV murine mammary tumor virus
• RSV Rous Sarcoma Virus
• 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.
• 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 ⁇ 2, 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.
• NTH 3T3 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.
• O1-180, 01-184, or 01-236 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 in vitro and in vivo.
• RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et ⁇ /1981).
• liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
• the foUowing 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).
• 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 cations.
• lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingohpids, 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.
• 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.
• RES reticulo-endothelial system
• Active targeting 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.
• a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
• the surface of the targeted delivery system may be modified in a variety of ways.
• lipid groups can be inco ⁇ orated 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.
• the present invention also contemplates the use of O1-180, 01-184 or Ol- 236 and active fragments, and nucleic acids coding therefore, in the screening of compounds for activity in either stimulating O1-180, 01-184 or 01-236, overcoming the lack of O1-180, 01- 184 or 01-236 or blocking or inhibiting the effect of an O1-180, 01-184 or 01-236 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.
• the invention is to be applied for the screening of compounds that bind to the O1-180, 01-184 or 01-236 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 pennitting determining of binding.
• the assay may measure the inhibition of binding of O1-180, 01-184 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, 01-184 or 01-236, binding partner or compound) is labeled.
• 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.
• Purified O1-180, 01-184 or 01-236 can be coated directly onto plates for use in the aforementioned drug screening techniques.
• non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase.
• fusion proteins containing a reactive region may be used to link the Ol-l 80, 01- 184 or 01-236 active region to a solid phase.
• Various cell lines containing wild- type or natural or engineered mutations in O1-180, 01-184 or 01-236 gene can be used to study various functional attributes of O1-180, 01-184 or 01-236 and how a candidate compound affects these attributes.
• Methods for engineering mutations are described elsewhere in this document, as are naturally-occurring mutations in Ol-l 80, 01-184 or 01-236 that lead to, contribute to and/or otherwise cause infertility.
• the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell.
• culture may be required.
• the cell may then be examined by virtue of a number of different physiologic assays.
• molecular analysis may be performed in which the function of Ol-l 80, 01-184 or 01-236, or related pathways, may be explored.
• yeast two-hybrid analysis is performed by standard means in the art with the polypeptides of the present invention, i.e., O1-180, 01-184 or
• 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.
• 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.
• reporter genes can be utilized, including ⁇ - galactosidase, HIS3, ADE2, or URA3.
• 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.
• alternative DNA-binding domain/activation domain components may be used, such as LexA.
• any activation domain may be paired with any DNA binding domain so long as they are able to generate transactivation of a reporter gene.
• 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.
• a two hybrid system is utilized wherein protein-protein interactions are detected in a cytoplasmic-based assay.
• 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).
• hSos 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°C of a mutant yeast cdc25H.
• Vectors such as pMyr and pSos
• 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 inco ⁇ orated by reference).
• a method of screening for a peptide which interacts with O1-180, 01-184 or 01-236 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, 01-184 or O1-236, respectively, wherein the at least part of Ol-l 80, Ol-l 84 or 01-236 respectively, is fused to a DNA activation domain.
• the assay for interaction between the test peptide and the Ol-l 80, 01-184 or 01-236 polypeptide or fragment thereof by assaying for interaction between the DNA binding domain and the DNA activation domain.
• the assay for interaction between the DNA binding and activation domains may be activation of expression of ⁇ -galactosidase.
• Recombinant O1-180, 01-184 or 01-236 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, 01-184 or 01-236 can be phosphorylated with 32 [P] or used unlabeled and detected with streptavidin or antibodies against the tags. . lambda.
• gtl lcDNA expression libraries are made from cells of interest and are incubated with the recombinant Ol-l 80, 01-184 or 01-236, washed and cDNA clones which interact with Ol-l 80, 01-184 or 01-236 isolated. Such methods are routinely used by skilled artisans. See, e.g., Sambrook (supra).
• 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, 01-184 or 01-236. 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.
• 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, 01-184 or 01-236 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 inco ⁇ orated 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).
• Another alternative method is isolation of proteins interacting with the 01- 180, 01-184 or 01-236 directly from cells. Fusion proteins of O1-180, 01-184 or 01-236 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-l 80, 01-184 or 01-236 are eluted specifically from the beads and analyzed by SDS-PAGE. Binding partner primary amino acid sequence data are obtained by microsequencing.
• 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.
• Another alternative method is immunoaffinity purification.
• Recombinant O1-180, 01-184 or 01-236 is incubated with labeled or unlabeled cell extracts and immunoprecipitated with anti- O1-180, 01-184 or 01-236 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 art may be used prior to microsequencing.
• Yet another alternative method is screening of peptide libraries for binding partners.
• Recombinant tagged or labeled O1-180, 01-184 or 01-236 is used to select peptides from a peptide or phosphopeptide library which interact with the O1-180, 01-184 or 01-236. Sequencing of the peptides leads to identification of consensus peptide sequences which might be found in interacting proteins.
• the present invention also encompasses the use of various animal models.
• any identity seen between human and other animal Ol-l 80, 01-184 or 01-236 provides an excellent opportunity to examine the function of O1-180, 01-184 or 01-236 in a whole animal system where it is normally expressed.
• By developing or isolating mutant cells lines that fail to express normal Ol-l 80, 01-184 or 01-236 one can generate models in mice that enable one to study the mechanism of Ol-l 80, 01-184 or 01-236 and its role in oogenesis and embryonic development.
• 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 pu ⁇ oses, including but not limited to oral, nasal, buccal, rectal, vaginal or topical.
• administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
• systemic intravenous injection regional administration via blood or lymph supply and intrarumoral injection.
• Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Such criteria include, but are not limited to, increased fertility, decreased fertility or contraception.
• transgenic animals are produced which contain a functional transgene encoding a functional Ol-l 80, 01-184 or O1-236 polypeptide or variants thereof.
• Transgenic animals expressing O1-180, 01-184 or 01-236 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 Ol-l 80, 01- 184 or 01-236.
• Transgenic animals of the present invention also can be used as models for studying disease states.
• an O1-180, 01-184 or 01-236 transgene is introduced into a non-human host to produce a transgenic animal expressing an 01- 180, 01-184 or 01-236.
• 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 inco ⁇ orated herein by reference
• Brinster et al, 1985 which is inco ⁇ orated herein by reference in its entirety
• Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition eds., Hogan, Beddington, Costantimi and Long, Cold Spring Harbor Laboratory Press, 1994; which is inco ⁇ orated herein by reference in its entirety.
• transgenic animals and cell lines derived from such animals may find use in certain testing experiments.
• transgenic animals and cell lines capable of expressing wild-type or mutant O1-180, 01-184 or O1-236 may be exposed to test substances. These test substances can be screened for the ability to enhance wild-type 01- 180, 01-184 or 01-236 expression and or function or impair the expression or function of mutant O1-180, 01-184 or 01-236.
• compositions - expression vectors, virus stocks, proteins, antibodies and drugs - in a form appropriate for the intended application.
• this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
• 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.
• 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.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
• the use of such media and agents for pharmaceutically active substances is well know 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 inco ⁇ orated into the compositions.
• 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, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
• 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.
• 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.
• 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.
• a coating such as lecithin
• surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium stearate, and gelatin.
• Sterile injectable solutions are prepared by inco ⁇ orating 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.
• dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• 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.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion 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 inco ⁇ orated into the compositions.
• the polypeptides of the present invention may be inco ⁇ orated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
• a mouthwash may be prepared inco ⁇ orating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
• the active ingredient may be inco ⁇ orated 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.
• a paste dentifrice may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
• 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.
• 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.
• solutions 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.
• the solution 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.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
• one dosage could be dissolved in 1 ml of isotonic NaCl 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 FDA Office of Biologies standards.
• Ovaries from Gd ⁇ knockout mice are histologically very different from wild-type ovaries due to the early block in folliculogenesis.
• one layer primary follicles are relatively enriched in Gd ⁇ knockout ovaries and abnormal foUicular 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 Gd ⁇ knockout ovaries.
• Clones O1-180, 01-184, and 01-236 were oocyte-specific and expressed in oocytes of primary (one-layer) preantral follicles through ovulation ( Figure 2).
• the 01-236 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 01-236 mRNA persisted through the antral follicle stage. Interestingly, the oocyte-specific. expression pattern of the 01-236 gene product paralleled the expression of other oocyte-specific genes which the inventors have studied including Gd ⁇ (McGrath et al, 1995) and bone mo ⁇ hogenetic protein 15 (Dube et al, 1998).
• Wild-type ovary and Gd ⁇ 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 are useful to define the possible function and cellular localization of the novel protein.
• important domains e.g., signal peptide sequences, transmembrane domains, zinc fingers, etc.
• Example 1 The 01-236 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-9 deficient ovaries as per manufacturer's instructions and as in Dube et al, 1998). In brief, approximately 300,000 clones of either wild-type or GDF-9 knockout mouse ovary cDNA libraries were hybridized to [alpha- P] dCTP random-primed probes in Church's solution at 63°C. Filters were washed with 0.1X Church's solution and exposed overnight at - 80°C.
• the 01-236 cDNA fragment Upon primary screening of the mouse ovarian cDNA libraries, the 01-236 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 form a 984 bp overlapping contig (excluding the polyA sequences) and encode a 207 amino acid open reading frame (Figure 4). Including the polyA tail, this sequence approximates the 1.0 kb mRNA seen by Northern blot analysis suggesting that nearly all of the 5' UTR sequence had been isolated.
• 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 5). Thus, mammalian NPM2 was predicated to translocate to the nucleus where it would primarily function.
• NPM2 conserved between NPM2 and nucleplasmin 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.
• 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).
• Multiple putative phosphorylation sites were predicted from the NPM2 and nucleoplasmin sequences. Several putative phosphorylation sequences that were conserved between the two proteins are shown in Figure 5.
• nucleoplasmin Phosphorylation of nucleoplasmin was 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. Thus, 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.
• Npml cDNAs (clone 236-1) was used to screen a mouse 129/SvEv genomic library (Stratagene) to identify the mouse Npm2 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 Npml was encoded by 9 exons and spans -6.6 kb ( Figures 6 and 7 A and 7B (SEQ ID NO: 7A)). Two moderate size introns (introns 4 and 5) contributed the majority of the gene size.
• 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 have been 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.
• the inventors Using the gene sequences obtained above, the inventors generate a targeting vector to mutate the O1-180, 01-184 and 01-236 genes in embryonic stem (ES) cells. These targeting vectors are 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 O1-180, 01-184 and 01-236 genes.
• ES embryonic stem
• the 01-184 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 01-184 or express a mutant form of the polypeptide.
• Hybridization was carried out at 50-55°C with 5x10 6 cpm for each riboprobe per slide for 16 hours in 50% deionized formamide/0.3 M NaCl/20 mM Tris-HCl (pH 8.0)/5 mM EDTA/10 mM NaPO 4 (pH8.0)/10% dextran sulphate/lxDenhardts/0.5 mg/ml yeast RNA.
• High stringency washes were carried out in 2xSSC/50% formamide and 0.1X 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.
• the Npm2 gene product was oocyte-specific ( Figures 9A and 9B).
• the probe demonstrates specific expression in all growing oocytes. Oocyte-specific 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. The "sense" probe does not detect a signal for this oocyte-specific gene.
• NPM2 protein The subcellular localization of NPM2 protein was determined by immunohistostaining of mouse ovaries with anti-NPM2 antibodies.
• the cDNA encoding the full-length mouse NPM2 protein was amplified by PCR to introduce a BamHI site before the start codon and a Xhol site before the stop codon. This PCR fragment was cloned into pET-23b(+)(Novagen) to produce a His-tagged NPM2 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.
• Ovaries were fixed in 4% paraformaldehyde in PBS for 2 h, processed, embedded in paraffin, and sectioned at 5 ran 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 section 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 Ramon, CA).
• mice were injected with PMSG and hCG to induce superovulation, the NPM2 protein redistributes in the oocytes of antral follicles after germinal vesicle breakdown.
• preovulatory GVB oocytes the NPM2 was evenly distributed in the cytoplasm of the oocyte ( Figure 9D).
• a targeting vector for Npm2 was constructed to delete exons 2 and 3 and the splice junction of exon 4.
• the deletion targeting vector contains from left to right, 2.2 kb of 5' Npm2 homology, a PGK-hprt expression cassette, 4.6 kb of 3'Npm2 homology and an MCl-tk (thymidine kinase) expression cassette.
• the linearized Npm2 targeting vector was electroporated into AB2.1 ES cells.
• ES cell clones were selected in Ml 5 medium containing HAT (hypoxanthine, aminopterine and thymidine and FIAU [l-(2'-deoxy-2'-fluoro-B-D- arabinofuranosyl)-5'-iodouracil]. Culturing of ES cells and collection and injection of blastocysts have been previously described by Matzuk et al, 1992. For genomic Southern blot analysis, Bglll-digested DNA was transferred to GeneScreen Plus nylon membrane and probed with an external 190 bp PCR synthesized fragment corresponding to exon 9 sequence (3' probe).
• An internal 200 bp PCR synthesized fragment (49 bp exon 1 plus 150 bp 5' upstream sequence) was also used to distinguish the wild-type and Npm2 null (Npr ⁇ 2tmlZuk) alleles when D ⁇ A was digested with BamHI.
• a PCR-synthesized probe containing the 137 bp exon 2 sequence was used to verify that exon 2 was absent in mice homozygous for the Npm2tmlZuk allele when D ⁇ A was digested with Pstl.
• a single correctly targeted ES cell clone (named Npr ⁇ 2-118-Bll) was expanded, and ES cells were injected into C57B1 6 blastocysts to obtain chimeric mice which ultimately produced C57B16/129/SvEv hybrid and 129/SvEv inbred FI progeny.
• 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 10A). Outside 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. Thus, this vector generated an Npm2 null allele.
• FI heterozygous (Npm2tmlZuk/+; herein called Npm2 +/ ⁇ ) mice were viable and fertile, and were intercrossed to investigate the developmental consequences of ⁇ PM2 absence.
• mice genotyped as Npm2 homozygotes lacked Npm2 a cDNA probe that hybridized to exon 2 of the wild-type Npm2 gene was used for Southern blot analysis. As shown ( Figure 10C), this probe failed to detect any signal in DNA derived from homozygous (Npm2 ⁇ / ⁇ ) mice in which exon 2 had been deleted. Furthermore, Npm2 immunohistochemical analysis was performed on Npm2 homozygotes and controls. Whereas the expression of NPM2 protein was noted in the ovaries from the heterozygous controls ( Figure 11A and 11C), no protein was detected in oocytes in the homozygote ovaries ( Figure 11B and 11D). This confirmed that the Npr ⁇ 2tmlZuk mutation was a null allele and that Npm2 homozygotes were completely lacking ⁇ PM2 protein.
• mice 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.
• mice Immature (21-24 day old) females were injected intraperitoneally with 5 IU PMSG (pregnant mare serum gonadotropin) followed by injection of 5 IU hCG (human chorionic gonadotropin) to induce superovulation as described (Hogan et al, 1994; Matzuk et al, 1996).
• the injected mice were mated to wild-type male mice. Eggs were harvested the next morning from the oviducts of these mice. Cumulus cells were removed from the eggs by using 0.3 mg/ml hyaluronidase in M2 medium (Sigma). Eggs were cultured in Ml 6 Medium (Sigma) covered with light paraffin oil in a humidified 37°C incubator with an atmosphere of 5% CO 2 and 95% air (Hogan et al, 1994).
• a ZAP-express mouse ovary cDNA library was screened to isolate the full- length O1-180 cDNA. Excluding the polyA tail, the full-length O1-180 cDNA is about 1.3kb, and encodes an open reading frame from nucleotides 26 to 1108.
• the Ol-l 80 cDNA is 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 O1-180 cDNA ORE consists of 361 amino acids, with a molecular mass of 40 kDa.
• mouse genomic ⁇ Fix II phage library generated from mouse 129SvEv strain was screened with the full length Ol-l 80 cDNA. Twelve independent ⁇ recombinant clones were isolated; eight of which were identified as unique clones and were further characterized by subcloning, Southern blot analysis, and sequencing.
• C57 mouse tissues including brain, heart, lung, spleen, liver, small intestine, stomach, kidney, uterus, testis, and ovary, were used as templates. Consistent with the Northern blot analysis, Ol- 180 cDNA was amplified exclusively in the ovary; while the related gene cDNA was not detectable in any of the tissues. This indicated that the related gene isolated from the mouse genomic ⁇ Fix II phage library was a pseudogene.
• a BAG 129SvJ mouse genomic library was screened by PCR with two sets of Ol-l 80 gene-specific primers, and only one BAC clone was isolated.
• 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 Ol-l 80 is unknown, the inventors designed the reverse primers from the 3 '-untranslated region of the murine sequence to minimize the risk of coamplification of the hamster homologues (Makalowski and Boguski, 1998).
• O1-180 gene specific primers were (SEQ.ID.NO.20) 5'- CTAGAAAAGGGGACTGTAGTCACT-3' forward, and (SEQ.ID.NO.21) 5'- TGCATCTCCCACACAAGTCTTGCC-3' reverse; pseudo Ol-l 80 gene specific primers were (SEQ.ID.NO.22) 5'-CTAGAAAAGGGGACTATAGGCACC-3' forward, and (SEQ.ID.NO.23) 5'-TGCATCTCTCACACAAGTGTTGCT-3' reverse. Specificity of the two sets of primers was tested with A23 hamster DNA and 129 mouse DNA.
• PCR reactions were performed in 15 ⁇ l final volume, containing l ⁇ l of each panel DNA, 1.25u of Taq platinum DNA polymerase (Gibco, Rockville, MD), companion reagents (0.25mM dNTPs, 1.5mM MgC12, lxPCR buffer), and 0.4 ⁇ M of each primer.
• An initial denaturation step of 4 min at 94°C 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.
• O1-180 protein The subcellular localization of O1-180 protein was determined by immunohistostaining of mouse ovaries with anti-Ol-180 antibodies.
• the cDNA encoding the full-length mouse Ol-l 80 protein was amplified by PCR to introduce a BamHI site before the start codon and a Xhol site before the stop codon. This PCR fragment was cloned into pET-23b(+)(Novagen) to produce a His-tagged Ol-l 80 protein and sequenced to confirm the absence of mutations. The recombinant Ol-l 80 protein was purified as described in the pET System Manual (Novagen). Two goats were immunized with the purified His-tagged O1-180 to produce specific and high affinity antibodies.
• Ovaries were fixed in 4% paraformaldehyde in PBS for 2 h, processed, embedded in paraffin, and sectioned at 5 urn thickness.
• Goat anti- O1-180 polyclonal antiserum was diluted 1 :2000 in Common Antibody Dilute (BioGenex).
• the pre-i mune goat serum from the same goat was used as a control. All section 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 Ramon, CA).
• Figure 20 shows immunostaning of O1-180 in mouse ovaries. As shown in Figure 20, the Ol-l 80 protein was localized specifically to the cytoplasm of mouse oocytes and zygotes but disappeared after this point.
• a targeting vector to mutate the O1-180 gene was constructed from the isolated sequences ( Figure 17). To study the role of O1-180 in mammalian oocyte development and early embryo development, the inventors disrupted the mouse Ol-l 80 locus using ES cell technology. The targeting vector was constructed to delete exon 1 which contains the translation initiation codon. Thus, this vector generated an Ol-l 80 null allele. Example 20 Loss of Ol-l 80 results in female infertility and subfertility
• Ol-l 80 " zygotes developed to 2-cell stage and none of them developed to a 4-cell stage by day
• Two hybrid screen is used to elucidate or characterize the function of a protein by identifying other proteins with which it interacts.
• the protein Ol-l 80 or 01-236 or 01-184 is made into a chimeric protein, which contains a DNA binding domain of GAL4 along with the DNA for the protein of interest. Plasmids containing nucleotide sequences which express this chimeric protein are transformed into eukaryotic 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 protein of interest (O1-180, 01-236 or 01-184) 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 Ol-l 80, 01-236 or 01-184 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.
• Senapathy, P., et al Methods Enzymol 183, 252-278.

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