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Restricted germ cell expression of a gene encoding a novel mammalian HORMA domain-containing protein
Gene Expression Patterns 5 (2004) 257–263 www.elsevier.com/locate/modgep
Restricted germ cell expression of a gene encoding a novel mammalian HORMA domain-containing protein Stephanie A. Pangasa,b, Wei Yana,1, Martin M. Matzuka,b,c, Aleksandar Rajkovicd,* a Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA c Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA d Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza T617, Houston, TX 77030, USA b
Received 1 June 2004; received in revised form 12 July 2004; accepted 20 July 2004 Available online 11 September 2004
Abstract HORMA-domain containing proteins are involved in cell cycle regulation by interactions with chromatin. Through an in silico subtractive screen for mouse genes preferentially expressed in newborn ovaries, we identified a gene that would encode a novel mammalian HORMA domain-containing protein termed Newborn Ovary HORMA protein (Nohma). Reverse transcription polymerase chain reaction and Northern blot analysis have demonstrated a 1.7 kb transcript that is expressed exclusively in germ cells in both male and female mouse gonads. Nohma expression can be detected postnatally in male, and prenatally in female gonads, but shows a sexually dimorphic expression in adult gonads. The Nohma transcript is abundant in the adult testis but limited in the adult ovary. The mouse Nohma gene is comprised of 15 exons and maps to chromosome 3. A human ortholog was also identified. The expression pattern of Nohma suggests that it may be a critical regulatory protein in germ cell meiosis. q 2004 Elsevier B.V. All rights reserved. Keywords: Ovary; Testis; Oocyte; HORMA domain; Germ cell; Nohma
1. Results and discussion During germ cell development, a number of germ cell specific factors have been shown to play critical roles in fertility and early embryonic development (Dean, 2002; Matzuk and Lamb, 2002). To identify novel germ cell specific genes, in silico subtraction of the publicly available newborn ovary expressed sequence tag (EST) library against all other ESTs (Ko et al., 2000; Rajkovic et al., 2001; Suzumori et al., 2002) was used to identify Unigene cluster Mm.179050. All ESTs within the Nohma cluster were derived from either testis or newborn ovary EST libraries. We have subsequently named this sequence Newborn Ovary HORMA (Nohma). * Corresponding author. Tel.: C1-713-798-8360; fax: C1-713-7988410. E-mail address: [email protected] (A. Rajkovic). 1 Present address: Department of Physiology and Cell Biology, University of Nevada, Reno, NV 89557, USA. 1567-133X/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2004.07.008
Analysis of the mouse cDNA sequence corresponding to Nohma reveals an 1179 nucleotide open-reading frame predicted to encode a protein of 392 amino acids (Fig. 1). Amino acids 23–235 contain a HORMA domain, a conserved protein domain originally named for the proteins Hop1, Rev7 and MAD2 (Aravind and Koonin, 1998). Similar to the Rev7 and MAD2 proteins, no other known domains are present in the mouse NOHMA sequence. A human ortholog of Nohma also was identified in the NCBI human testis libraries. Using virtual translation and homology to mouse Nohma, human NOHMA contains an open reading frame of 1164 bp predicted to encode a protein of 387 amino acids. Amino acids 22–227 of the human sequence contain the HORMA domain and alignment with the mouse HORMA domain shows that these proteins share 89.2% amino acid identity (Fig. 2). Overall, mouse and human NOHMA are approximately 77% identical in amino acid sequence. In addition, all mouse and human NOHMA sequences contain the invariant N-terminal
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Fig. 1. Nohma cDNA and translation. The Nohma cDNA is 1385 nucleotides long and contains an open reading frame of 1179 nucleotides that would encode a protein of 392 amino acids. The amino terminus contains a HORMA domain (solid underline) but no other known protein motifs. The sequence of mouse Nohma has been deposited under GenBank accession number AY626343.
arginine and central glutamate residues (Fig. 2) that were originally described for HORMA domains (Aravind and Koonin, 1998). A conserved proline is found at position 28 in the five sequences shown in Fig. 2; however, some HORMA-domain containing proteins contain other residues at this site (Aravind and Koonin, 1998). Based on database analysis, the Nohma gene maps to chromosome 3 at 3 F2.1. The gene is comprised of 15 exons with 14 intervening introns. The initiator methionine ATG is located in exon 2 at position 73 and the stop codon in exon 16 at position 1249. The HORMA domain spans introns 3 through 10. Stretches of nucleotides identical to portions of the full-length Nohma sequence are also located on chromosomes 6 and 15, but since these sequences lack introns they are likely pseudogenes. The human NOHMA ortholog maps to human chromosome 1q21.3 and is syntenic with the mouse Nohma chromosome 3 location. The defining characteristic of HORMA domain-containing proteins appears to be an interaction with chromatin, particularly chromatin associated with DNA adducts,
double strand-DNA breaks, or non-attachment to the mitotic spindle (Aravind and Koonin, 1998). Many are critical regulatory proteins for mitosis and meiosis. For example, human MAD2 is a mitotic spindle checkpoint protein that interacts with the anaphase promoting complex (APC) to inhibit APC activity until all of the chromosomes are aligned on the metaphase plate (Fang et al., 1998). In mice, null mutations in Mad2 cause chromosomal missegregation and subsequent embryonic death at 6.5 days postcoitus (Dobles et al., 2000) and Mad2 heterozygous mice are prone to tumors (Michel et al., 2001). In addition to its function during the mitotic cell cycle, it has also been suggested that MAD2 has a role during metaphase I of meiosis (Wassmann et al., 2003). The HORMA domain of mouse and human NOHMA are most similar to Saccharomyces cerevisiae Hop1p with 28% amino acid similarity. Yeast mutants of HOP1 have defects in chromosomal condensation, synapsis and recombination (Hollingsworth and Byers, 1989; Loidl et al., 1994). Yeast Hop1p binds double-strand breaks during meiotic prophase
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Fig. 2. HORMA domain alignment. The HORMA domains from mouse and human NOHMA were aligned with the known HORMA domains of Saccharomyces cerevisiae Hop1 and Rev7 proteins and human MAD2. The conserved residues between mouse and human are shown in bold. Mouse NOHMA and human NOHMA are 89% identical within the HORMA domain. Both are most closely similar to yeast Hop1p. The invariant arginine, glutamine, and valine residues are indicated by asterisks. A conserved proline (arrow) at position 28 is indicated. In addition, four of the five sequences contain a conserved histidine residue in the C-terminal region of the domain (arrowhead). The GenBank accession number for amino acid sequences used for alignment were: AY626343 (mouse NOHMA), amino acids 23–227; AY626344 (human NOHMA), amino acids 22–235; J04877 (yeast Hop1p), amino acids 18–254; BC000356 (human MAD2), amino acids 12–201; U07228 (yeast Rev7p), amino acids 1-207.
in association with the synaptonemal complex (Kironmai et al., 1998; Schwacha and Kleckner, 1994). While Hop1p homologs have been identified in Arabidopsis thaliana and Caenorhabditis elegans, no homologous mammalian gene has been cloned. Mutations in C. elegans and A. thaliana Hop1p homologs also disrupt meiosis (Caryl et al., 2000; Zetka et al., 1999), suggesting that Hop1p is a critical meiotic protein whose function has been conserved in crown eukaryotes. We examined the tissue distribution of Nohma by Northern analysis. By this method, adult mouse tissues demonstrate a 1.7 kb transcript predominantly expressed in the testes but which also has a low but detectable level of expression in the 14-day ovary (Fig. 3A). No other tissue appears to express Nohma. To further characterize the Nohma expression pattern in the male, we analyzed RNA during postnatal development of the testis (days 5, 10, 15, 20, 35) and in the adult (60 d). At birth and until day 5, the seminiferous tubules of the testis contain only mitotically active, undifferentiated type A1 spermatogonia.
Primary spermatocytes, which undergo meiosis, do not appear until postnatal day 8–10. Expression of Nohma in the testis is detectable by Northern blotting analysis at 10 days postpartum and continues during postnatal gonadal development as well as in the adult (Fig. 3B). Thus, Nohma expression in the testis coincides with the onset of meiosis I and is further suggestive of a role for NOHMA during meiosis I in male germ cells. Very little Nohma cDNA can be detected from adult ovary RNA by reverse transcription polymerase chain reaction (RT-PCR) but the Nohma transcript is present in embryonic, newborn, 2 day, and 9 day ovaries (Fig. 3C). The sexually dimorphic expression patterns may reflect the differences between the start of meiosis in males and females, since meiosis begins embryonically in females but postnatally in males. However, the expression of Nohma in the female also is detected at embryonic day 12.5 (E12.5)—a time of mitotic proliferation (Fig. 3C). Meiosis in the female germ cells begins at approximately E13.5–E14.5 (Pepling and Spradling, 2001). Therefore, it is possible that Nohma
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Fig. 3. RNA expression profile of mouse Nohma. (A) Northern analysis of RNA from multiple tissues. 15 mg of tissue-specific total RNA was analyzed in each lane. All tissues are from adult mice except the ovary RNA, which was collected from a juvenile (14 d) female mouse. Nohma expression is only detected in gonadal tissues. The membrane was hybridized with radiolabeled Nohma or 18S rRNA probes. 18S rRNA served as a loading control. (B) Northern blot for postnatal expression of Nohma in the testis. Expression could be detected by Northern blotting after day 10 postpartum (10 d) and expression continues through adulthood (60 d). (C) RT-PCR for Nohma expression during female gonadal development. cDNA was amplified from embryonic days E12.5 to E17.5 and newborn, 2 day (2 d) and 9 day (9 d) postpartum using Nohma specific primers. The signal from adult ovaries was almost undetectable. Primers specific for actin were used to verify equivalent amounts of input RNA.
transcription precedes its translation or protein function, or alternatively, that NOHMA may have roles in both mitosis and meiosis. In situ hybridization on ovaries from mice at postnatal day 7, shows that Nohma localizes to oocytes clustered in germ cell cysts, as well as those in primordial and primary follicles (Fig. 4A and D). No expression was detectable in oocytes from adult ovaries (Fig. 4C). In juvenile and adult testes, Nohma localizes to spermatocytes but not spermatogonia or Sertoli cells (Fig. 4G–L). These data support findings from the in silico subtraction and indicate a restricted germ cell expression of Nohma. In summary, we have identified and characterized the expression pattern of a novel gene that would encode a HORMA domain-containing protein. Nohma expression is restricted to male and female germ cells and Nohma is the first mammalian germ cell-specific HORMA gene to be described. Nohma is expressed in female oocytes during pre- and post-natal development but its expression level decreases during adulthood. In the testis, Nohma is expressed in spermatocytes during early postnatal development and into adulthood. Likely, NOHMA plays a key role in meiosis in both male and female germ cells.
Targeted disruption of this gene will be necessary to evaluate the role of NOHMA during germline development in both males and females.
2. Experimental procedures 2.1. In silico subtraction In silico subtraction was performed as previously described (Rajkovic et al., 2001; Suzumori et al., 2002). Briefly, an electronic database subtraction was carried out between the 7577 ESTs from a murine newborn ovary cDNA library against the entire collection of murine ESTs in the NCBI database. 100 ESTs were present in the newborn mouse ovary library and absent in all others. Of the 26 that were chosen for subsequent analysis, seven ESTs, including Mm.179050, were confirmed to be gonad specific. 2.2. Nucleotide sequence and protein analysis Mouse Nohma and human NOHMA sequences were determined by database searches in combination with
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Fig. 4. In situ hybridization for Nohma in ovaries and testes. Panels (A,B,D,E) show hybridization of antisense Nohma riboprobes to 7 d ovaries (Panels (A,D), darkfield; Panels (B,E) are the corresponding brightfield images, respectively). Panels (D,E) are shown at a higher magnification. Nohma is restricted to oocytes contained in germ cell cysts (GC) around the periphery of the ovary, and in oocytes from primordial follicles (PF) and primary follicles (PrF). No expression was detected in the adult ovary (Panel C,F, darkfield and brightfield, respectively). Panel (G–I) show hybridization of Nohma riboprobes to a 15 d testis section (Panel G,H, darkfield and brightfield, respectively). Expression is visible in spermatocytes (SP) in the seminiferous tubules. Panel I is a high magnification image of Panel G and silver grains in the emulsion above the tissue indicate the in situ hybridization signal. Silver grains are most dense over spermatocytes (SP). No silver grains can be seen over Sertoli cells (SC) or spermatogonia (SG). Expression is also detectable in spermatocytes in the adult testis (Panel J–L) (Panel J,K, darkfield and brightfield, respectively). Panel (L) is a higher magnification of Panel (J) showing silver grains over the pachytene spermatocytes (Ps) but not in round spermatids (Rsd) or spermatogonia (SG).
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sequencing of the following ESTs from American Type Culture Collection (Manassas, VA, USA): L0269H11, L0274C01, L0208F05, and L0215F06. Full-length Nohma was used to determine the amino acid sequence and was characterized using the programs EXPASy, PSORT, Pfam and ScanProsite. Multiple sequence alignment and homology were determined using ClustalW (http://www. ebi.ac.uk/clustalw/). 2.3. Experimental animals Mice were maintained according to the NIH Guide for the Care and Use of Laboratory Animals. Prenatal days were counted as the number of days following the identification of a vaginal plug (E0.5). Postnatal days were counted as number of days following birth. All tissues were collected from C57BL/6/129SvEv (hybrid strain) mice for RNA analysis or in situ hybridization. 2.4. Northern analysis Northern analysis was performed as previously described (Yan et al., 2002). 15 mg of total RNA was fractionated on 1.2% formaldehyde-agarose gels and transferred onto nylon membranes (Hybond-N, Amersham Pharmacia, Arlington Heights, IL). Radioactive probes were synthesized using (a-32P) deoxy (d)-ATP using the Strip-EZ kit (Ambion Inc., Austin, TX). After hybridization, membranes were exposed to film. Blots were stripped and reprobed for 18S rRNA as a loading control. 2.5. RT-PCR Total RNA was isolated using RNA-STAT (Leedo Medical Laboratories, Houston, TX). Five micrograms of total RNA was reverse transcribed using the Superscript system (Invitrogen Technologies, Rockville, MD). PCR was performed using the primers 5 0 GTCCCAACACCT TTTCACA-3 0 AND 5 0 -AGACACATCAAGTTCAGATG3 0 which amplify a 341 bp product and span exon 10 to exon 13. PCR was carried out for 25–30 cycles at 94 8C for 30 00 (denaturation), 60 8C for 30 00 (annealing) and 72 8C for 30 00 (extension). Amplification using actin-specific primers was used as a control for equivalent RNA levels. Water only (no template) and no reverse transcriptase controls were included for each PCR (data not shown). 2.6. In situ hybridization In situ hybridization was performed as previously described (Elvin et al., 1999). Briefly, ovaries were fixed in 4% paraformaldehyde and embedded in paraffin. Five micrometers sections were hybridized to 35S-labeled sense and antisense riboprobes as described (Rajkovic et al., 2002). Signal was detected by autoradiography using
NTB-2 emulsion (Eastman Kodak, Rochester NY) and tissue was counterstained with hematoxylin.
Acknowledgements This work has been supported by grants HD00849, AAOGF, HD01426 and a Basil O’Connor Starter Scholar Research Award (5-FY02-266) from the March of Dimes Birth Defects Foundation to A. Rajkovic and NIH grants HD33438 and HD42500 to M. Matzuk. S. Pangas is a Postdoctoral Fellow from the Center for Reproductive Biology Training Grant HD007165. W. Yan was supported by a Postdoctoral fellowship from the Ernst Schering Research Foundation.
References Aravind, L., Koonin, E.V., 1998. The HORMA domain: a common structural denominator in mitotic checkpoints, chromosome synapsis and DNA repair. Trends Biochem. Sci. 23, 284–286. Caryl, A.P., Armstrong, S.J., Jones, G.H., Franklin, F.C., 2000. A homologue of the yeast HOP1 gene is inactivated in the Arabidopsis meiotic mutant asy1. Chromosoma 109, 62–71. Dean, J., 2002. Oocyte-specific genes regulate follicle formation, fertility and early mouse development. J. Reprod. Immunol. 53, 171–180. Dobles, M., Liberal, V., Scott, M.L., Benezra, R., Sorger, P.K., 2000. Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell 101, 635–645. Elvin, J.A., Yan, C., Wang, P., Nishimori, K., Matzuk, M.M., 1999. Molecular characterization of the follicle defects in the growth differentiation factor9-deficient ovary. Mol. Endocrinol. 13, 1018–1034. Fang, G., Yu, H., Kirschner, M.W., 1998. The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Leukemia 12, 960–969. Hollingsworth, N.M., Byers, B., 1989. HOP1: a yeast meiotic pairing gene. Genetics 121, 445–462. Kironmai, K.M., Muniyappa, K., Friedman, D.B., Hollingsworth, N.M., Byers, B., 1998. DNA-binding activities of Hop1 protein, a synaptonemal complex component from Saccharomyces cerevisiae. Mol. Cell Biol. 18, 1424–1435. Ko, M.S., Kitchen, J.R., Wang, X., Threat, T.A., Wang, X., Hasegawa, A., et al., 2000. Large-scale cDNA analysis reveals phased gene expression patterns during preimplantation mouse development. Development 127, 1737–1749. Loidl, J., Klein, F., Scherthan, H., 1994. Homologous pairing is reduced but not abolished in asynaptic mutants of yeast. J. Cell Biol. 125, 1191–1200. Matzuk, M.M., Lamb, D.J., 2002. Genetic dissection of mammalian fertility pathways. Nat. Med, 8 Suppl. 2002;, S33–S40. Michel, L.S., Liberal, V., Chatterjee, A., Kirchwegger, R., Pasche, B., Gerald, W., et al., 2001. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 409, 355–359. Pepling, M.E., Spradling, A.C., 2001. Mouse ovarian germ cell cysts undergo programmed breakdown to form primordial follicles. Dev. Biol. 234, 339–351. Rajkovic, A., Yan, M.S.C., Klysik, M., Matzuk, M., 2001. Discovery of germ cell-specific transcripts by expressed sequence tag database analysis. Fertil. Steril. 76, 550–554. Rajkovic, A., Lee, J.H., Yan, C., Matzuk, M.M., 2002. The ret finger protein-like 4 gene, Rfpl4, encodes a putative E3 ubiquitin-protein ligase expressed in adult germ cells. Mech. Dev. 112, 173–177.
S.A. Pangas et al. / Gene Expression Patterns 5 (2004) 257–263 Schwacha, A., Kleckner, N., 1994. Identification of joint molecules that form frequently between homologs but rarely between sister chromatids during yeast meiosis. Cell 76, 51–63. Suzumori, N., Yan, C., Matzuk, M.M., Rajkovic, A., 2002. Nobox is a homeobox-encoding gene preferentially expressed in primordial and growing oocytes. Mech. Dev. 111, 137–141. Wassmann, K., Niault, T., Maro, B., 2003. Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes. Curr. Biol. 13, 1596–1608.
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Yan, W., Rajkovic, A., Viveiros, M.M., Burns, K.H., Eppig, J.J., Matzuk, M.M., 2002. Identification of Gasz, an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper. Mol. Endocrinol. 16, 1168–1184. Zetka, M.C., Kawasaki, I., Strome, S., Muller, F., 1999. Synapsis and chiasma formation in Caenorhabditis elegans require HIM-3, a meiotic chromosome core component that functions in chromosome segregation. Genes Dev. 13, 2258–2270.
Restricted germ cell expression of a gene encoding a novel mammalian HORMA domain-containing protein Stephanie A. Pangasa,b, Wei Yana,1, Martin M. Matzuka,b,c, Aleksandar Rajkovicd,* a Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA c Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA d Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza T617, Houston, TX 77030, USA b
Received 1 June 2004; received in revised form 12 July 2004; accepted 20 July 2004 Available online 11 September 2004
Abstract HORMA-domain containing proteins are involved in cell cycle regulation by interactions with chromatin. Through an in silico subtractive screen for mouse genes preferentially expressed in newborn ovaries, we identified a gene that would encode a novel mammalian HORMA domain-containing protein termed Newborn Ovary HORMA protein (Nohma). Reverse transcription polymerase chain reaction and Northern blot analysis have demonstrated a 1.7 kb transcript that is expressed exclusively in germ cells in both male and female mouse gonads. Nohma expression can be detected postnatally in male, and prenatally in female gonads, but shows a sexually dimorphic expression in adult gonads. The Nohma transcript is abundant in the adult testis but limited in the adult ovary. The mouse Nohma gene is comprised of 15 exons and maps to chromosome 3. A human ortholog was also identified. The expression pattern of Nohma suggests that it may be a critical regulatory protein in germ cell meiosis. q 2004 Elsevier B.V. All rights reserved. Keywords: Ovary; Testis; Oocyte; HORMA domain; Germ cell; Nohma
1. Results and discussion During germ cell development, a number of germ cell specific factors have been shown to play critical roles in fertility and early embryonic development (Dean, 2002; Matzuk and Lamb, 2002). To identify novel germ cell specific genes, in silico subtraction of the publicly available newborn ovary expressed sequence tag (EST) library against all other ESTs (Ko et al., 2000; Rajkovic et al., 2001; Suzumori et al., 2002) was used to identify Unigene cluster Mm.179050. All ESTs within the Nohma cluster were derived from either testis or newborn ovary EST libraries. We have subsequently named this sequence Newborn Ovary HORMA (Nohma). * Corresponding author. Tel.: C1-713-798-8360; fax: C1-713-7988410. E-mail address: [email protected] (A. Rajkovic). 1 Present address: Department of Physiology and Cell Biology, University of Nevada, Reno, NV 89557, USA. 1567-133X/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2004.07.008
Analysis of the mouse cDNA sequence corresponding to Nohma reveals an 1179 nucleotide open-reading frame predicted to encode a protein of 392 amino acids (Fig. 1). Amino acids 23–235 contain a HORMA domain, a conserved protein domain originally named for the proteins Hop1, Rev7 and MAD2 (Aravind and Koonin, 1998). Similar to the Rev7 and MAD2 proteins, no other known domains are present in the mouse NOHMA sequence. A human ortholog of Nohma also was identified in the NCBI human testis libraries. Using virtual translation and homology to mouse Nohma, human NOHMA contains an open reading frame of 1164 bp predicted to encode a protein of 387 amino acids. Amino acids 22–227 of the human sequence contain the HORMA domain and alignment with the mouse HORMA domain shows that these proteins share 89.2% amino acid identity (Fig. 2). Overall, mouse and human NOHMA are approximately 77% identical in amino acid sequence. In addition, all mouse and human NOHMA sequences contain the invariant N-terminal
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Fig. 1. Nohma cDNA and translation. The Nohma cDNA is 1385 nucleotides long and contains an open reading frame of 1179 nucleotides that would encode a protein of 392 amino acids. The amino terminus contains a HORMA domain (solid underline) but no other known protein motifs. The sequence of mouse Nohma has been deposited under GenBank accession number AY626343.
arginine and central glutamate residues (Fig. 2) that were originally described for HORMA domains (Aravind and Koonin, 1998). A conserved proline is found at position 28 in the five sequences shown in Fig. 2; however, some HORMA-domain containing proteins contain other residues at this site (Aravind and Koonin, 1998). Based on database analysis, the Nohma gene maps to chromosome 3 at 3 F2.1. The gene is comprised of 15 exons with 14 intervening introns. The initiator methionine ATG is located in exon 2 at position 73 and the stop codon in exon 16 at position 1249. The HORMA domain spans introns 3 through 10. Stretches of nucleotides identical to portions of the full-length Nohma sequence are also located on chromosomes 6 and 15, but since these sequences lack introns they are likely pseudogenes. The human NOHMA ortholog maps to human chromosome 1q21.3 and is syntenic with the mouse Nohma chromosome 3 location. The defining characteristic of HORMA domain-containing proteins appears to be an interaction with chromatin, particularly chromatin associated with DNA adducts,
double strand-DNA breaks, or non-attachment to the mitotic spindle (Aravind and Koonin, 1998). Many are critical regulatory proteins for mitosis and meiosis. For example, human MAD2 is a mitotic spindle checkpoint protein that interacts with the anaphase promoting complex (APC) to inhibit APC activity until all of the chromosomes are aligned on the metaphase plate (Fang et al., 1998). In mice, null mutations in Mad2 cause chromosomal missegregation and subsequent embryonic death at 6.5 days postcoitus (Dobles et al., 2000) and Mad2 heterozygous mice are prone to tumors (Michel et al., 2001). In addition to its function during the mitotic cell cycle, it has also been suggested that MAD2 has a role during metaphase I of meiosis (Wassmann et al., 2003). The HORMA domain of mouse and human NOHMA are most similar to Saccharomyces cerevisiae Hop1p with 28% amino acid similarity. Yeast mutants of HOP1 have defects in chromosomal condensation, synapsis and recombination (Hollingsworth and Byers, 1989; Loidl et al., 1994). Yeast Hop1p binds double-strand breaks during meiotic prophase
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Fig. 2. HORMA domain alignment. The HORMA domains from mouse and human NOHMA were aligned with the known HORMA domains of Saccharomyces cerevisiae Hop1 and Rev7 proteins and human MAD2. The conserved residues between mouse and human are shown in bold. Mouse NOHMA and human NOHMA are 89% identical within the HORMA domain. Both are most closely similar to yeast Hop1p. The invariant arginine, glutamine, and valine residues are indicated by asterisks. A conserved proline (arrow) at position 28 is indicated. In addition, four of the five sequences contain a conserved histidine residue in the C-terminal region of the domain (arrowhead). The GenBank accession number for amino acid sequences used for alignment were: AY626343 (mouse NOHMA), amino acids 23–227; AY626344 (human NOHMA), amino acids 22–235; J04877 (yeast Hop1p), amino acids 18–254; BC000356 (human MAD2), amino acids 12–201; U07228 (yeast Rev7p), amino acids 1-207.
in association with the synaptonemal complex (Kironmai et al., 1998; Schwacha and Kleckner, 1994). While Hop1p homologs have been identified in Arabidopsis thaliana and Caenorhabditis elegans, no homologous mammalian gene has been cloned. Mutations in C. elegans and A. thaliana Hop1p homologs also disrupt meiosis (Caryl et al., 2000; Zetka et al., 1999), suggesting that Hop1p is a critical meiotic protein whose function has been conserved in crown eukaryotes. We examined the tissue distribution of Nohma by Northern analysis. By this method, adult mouse tissues demonstrate a 1.7 kb transcript predominantly expressed in the testes but which also has a low but detectable level of expression in the 14-day ovary (Fig. 3A). No other tissue appears to express Nohma. To further characterize the Nohma expression pattern in the male, we analyzed RNA during postnatal development of the testis (days 5, 10, 15, 20, 35) and in the adult (60 d). At birth and until day 5, the seminiferous tubules of the testis contain only mitotically active, undifferentiated type A1 spermatogonia.
Primary spermatocytes, which undergo meiosis, do not appear until postnatal day 8–10. Expression of Nohma in the testis is detectable by Northern blotting analysis at 10 days postpartum and continues during postnatal gonadal development as well as in the adult (Fig. 3B). Thus, Nohma expression in the testis coincides with the onset of meiosis I and is further suggestive of a role for NOHMA during meiosis I in male germ cells. Very little Nohma cDNA can be detected from adult ovary RNA by reverse transcription polymerase chain reaction (RT-PCR) but the Nohma transcript is present in embryonic, newborn, 2 day, and 9 day ovaries (Fig. 3C). The sexually dimorphic expression patterns may reflect the differences between the start of meiosis in males and females, since meiosis begins embryonically in females but postnatally in males. However, the expression of Nohma in the female also is detected at embryonic day 12.5 (E12.5)—a time of mitotic proliferation (Fig. 3C). Meiosis in the female germ cells begins at approximately E13.5–E14.5 (Pepling and Spradling, 2001). Therefore, it is possible that Nohma
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Fig. 3. RNA expression profile of mouse Nohma. (A) Northern analysis of RNA from multiple tissues. 15 mg of tissue-specific total RNA was analyzed in each lane. All tissues are from adult mice except the ovary RNA, which was collected from a juvenile (14 d) female mouse. Nohma expression is only detected in gonadal tissues. The membrane was hybridized with radiolabeled Nohma or 18S rRNA probes. 18S rRNA served as a loading control. (B) Northern blot for postnatal expression of Nohma in the testis. Expression could be detected by Northern blotting after day 10 postpartum (10 d) and expression continues through adulthood (60 d). (C) RT-PCR for Nohma expression during female gonadal development. cDNA was amplified from embryonic days E12.5 to E17.5 and newborn, 2 day (2 d) and 9 day (9 d) postpartum using Nohma specific primers. The signal from adult ovaries was almost undetectable. Primers specific for actin were used to verify equivalent amounts of input RNA.
transcription precedes its translation or protein function, or alternatively, that NOHMA may have roles in both mitosis and meiosis. In situ hybridization on ovaries from mice at postnatal day 7, shows that Nohma localizes to oocytes clustered in germ cell cysts, as well as those in primordial and primary follicles (Fig. 4A and D). No expression was detectable in oocytes from adult ovaries (Fig. 4C). In juvenile and adult testes, Nohma localizes to spermatocytes but not spermatogonia or Sertoli cells (Fig. 4G–L). These data support findings from the in silico subtraction and indicate a restricted germ cell expression of Nohma. In summary, we have identified and characterized the expression pattern of a novel gene that would encode a HORMA domain-containing protein. Nohma expression is restricted to male and female germ cells and Nohma is the first mammalian germ cell-specific HORMA gene to be described. Nohma is expressed in female oocytes during pre- and post-natal development but its expression level decreases during adulthood. In the testis, Nohma is expressed in spermatocytes during early postnatal development and into adulthood. Likely, NOHMA plays a key role in meiosis in both male and female germ cells.
Targeted disruption of this gene will be necessary to evaluate the role of NOHMA during germline development in both males and females.
2. Experimental procedures 2.1. In silico subtraction In silico subtraction was performed as previously described (Rajkovic et al., 2001; Suzumori et al., 2002). Briefly, an electronic database subtraction was carried out between the 7577 ESTs from a murine newborn ovary cDNA library against the entire collection of murine ESTs in the NCBI database. 100 ESTs were present in the newborn mouse ovary library and absent in all others. Of the 26 that were chosen for subsequent analysis, seven ESTs, including Mm.179050, were confirmed to be gonad specific. 2.2. Nucleotide sequence and protein analysis Mouse Nohma and human NOHMA sequences were determined by database searches in combination with
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Fig. 4. In situ hybridization for Nohma in ovaries and testes. Panels (A,B,D,E) show hybridization of antisense Nohma riboprobes to 7 d ovaries (Panels (A,D), darkfield; Panels (B,E) are the corresponding brightfield images, respectively). Panels (D,E) are shown at a higher magnification. Nohma is restricted to oocytes contained in germ cell cysts (GC) around the periphery of the ovary, and in oocytes from primordial follicles (PF) and primary follicles (PrF). No expression was detected in the adult ovary (Panel C,F, darkfield and brightfield, respectively). Panel (G–I) show hybridization of Nohma riboprobes to a 15 d testis section (Panel G,H, darkfield and brightfield, respectively). Expression is visible in spermatocytes (SP) in the seminiferous tubules. Panel I is a high magnification image of Panel G and silver grains in the emulsion above the tissue indicate the in situ hybridization signal. Silver grains are most dense over spermatocytes (SP). No silver grains can be seen over Sertoli cells (SC) or spermatogonia (SG). Expression is also detectable in spermatocytes in the adult testis (Panel J–L) (Panel J,K, darkfield and brightfield, respectively). Panel (L) is a higher magnification of Panel (J) showing silver grains over the pachytene spermatocytes (Ps) but not in round spermatids (Rsd) or spermatogonia (SG).
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sequencing of the following ESTs from American Type Culture Collection (Manassas, VA, USA): L0269H11, L0274C01, L0208F05, and L0215F06. Full-length Nohma was used to determine the amino acid sequence and was characterized using the programs EXPASy, PSORT, Pfam and ScanProsite. Multiple sequence alignment and homology were determined using ClustalW (http://www. ebi.ac.uk/clustalw/). 2.3. Experimental animals Mice were maintained according to the NIH Guide for the Care and Use of Laboratory Animals. Prenatal days were counted as the number of days following the identification of a vaginal plug (E0.5). Postnatal days were counted as number of days following birth. All tissues were collected from C57BL/6/129SvEv (hybrid strain) mice for RNA analysis or in situ hybridization. 2.4. Northern analysis Northern analysis was performed as previously described (Yan et al., 2002). 15 mg of total RNA was fractionated on 1.2% formaldehyde-agarose gels and transferred onto nylon membranes (Hybond-N, Amersham Pharmacia, Arlington Heights, IL). Radioactive probes were synthesized using (a-32P) deoxy (d)-ATP using the Strip-EZ kit (Ambion Inc., Austin, TX). After hybridization, membranes were exposed to film. Blots were stripped and reprobed for 18S rRNA as a loading control. 2.5. RT-PCR Total RNA was isolated using RNA-STAT (Leedo Medical Laboratories, Houston, TX). Five micrograms of total RNA was reverse transcribed using the Superscript system (Invitrogen Technologies, Rockville, MD). PCR was performed using the primers 5 0 GTCCCAACACCT TTTCACA-3 0 AND 5 0 -AGACACATCAAGTTCAGATG3 0 which amplify a 341 bp product and span exon 10 to exon 13. PCR was carried out for 25–30 cycles at 94 8C for 30 00 (denaturation), 60 8C for 30 00 (annealing) and 72 8C for 30 00 (extension). Amplification using actin-specific primers was used as a control for equivalent RNA levels. Water only (no template) and no reverse transcriptase controls were included for each PCR (data not shown). 2.6. In situ hybridization In situ hybridization was performed as previously described (Elvin et al., 1999). Briefly, ovaries were fixed in 4% paraformaldehyde and embedded in paraffin. Five micrometers sections were hybridized to 35S-labeled sense and antisense riboprobes as described (Rajkovic et al., 2002). Signal was detected by autoradiography using
NTB-2 emulsion (Eastman Kodak, Rochester NY) and tissue was counterstained with hematoxylin.
Acknowledgements This work has been supported by grants HD00849, AAOGF, HD01426 and a Basil O’Connor Starter Scholar Research Award (5-FY02-266) from the March of Dimes Birth Defects Foundation to A. Rajkovic and NIH grants HD33438 and HD42500 to M. Matzuk. S. Pangas is a Postdoctoral Fellow from the Center for Reproductive Biology Training Grant HD007165. W. Yan was supported by a Postdoctoral fellowship from the Ernst Schering Research Foundation.
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