Mater encodes a maternal protein in mice with a leucine-rich repeat domain homologous to porcine ribonuclease inhibitor

Mammalian Genome 11, 281–287 (2000). Incorporating Mouse Genome © Springer-Verlag New York Inc. 2000 Mater encodes a maternal protein in mice with a leucine-rich repeat domain homologous to porcine ribonuclease inhibitor Zhi-Bin Tong,1,2 Lawrence M. Nelson,1 Jurrien Dean3 1 Developmental Endocrinology Branch, NICHD, National Institutes of Health, Building 10, Room 10N262, Bethesda, MD 20892-1862, USA Department of Pediatrics, Georgetown University Medical Center, Washington, D.C. 20007, USA 3 Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA 2 Received: 10 September 1999 / Accepted: 23 November 1999 Abstract. MATER (Maternal Antigen That Embryos Require) is an ooplasm-specific protein first identified as an antigen (OP1) associated with ovarian autoimmunity in mice. Its primary structure has been deduced from full-length cDNA that encodes a 125kDa protein required for progression of the mouse embryo beyond two cells. Expression of the gene encoding MATER is restricted to the oocyte, which makes it one of a growing, but still limited, number of maternal-effect genes in mammals. To further investigate the function of MATER during oogenesis and early development, we have characterized the gene and resultant protein. Mater is a single-copy gene in the genome of 129/Sv mice and is located at the proximal end of Chromosome (Chr) 7. The gene, spanning approximately 32 kbp, contains 15 exons ranging in size from 48 to 1576 bp, which together encode the 1111 amino acid MATER protein. The first five exons encode 26–27 amino acid hydrophilic repeats, and exons 8–14 encode 14 leucine-rich repeats. The threedimensional structure of the latter domain can be closely modeled on the previously determined X-ray crystallographic coordinates of porcine ribonuclease inhibitor. These characterizations of the gene and protein provide the basis for genetic investigations of MATER function in early mammalian development. Introduction Little is known about the mechanisms by which the embryonic genome is activated after fertilization in mammals. As mouse oocytes grow, they accumulate transcripts that are either translated directly into proteins or stored for activation later in oogenesis by controlled polyadenylation. During subsequent meiotic maturation and ovulation, the germ cells are transcriptionally inert, and many of the maternal transcripts are degraded (Bachvarova and De Leon 1980; Clegg and Piko 1983; Fox and Wickens 1990; Stutz et al. 1998). Although some maternal transcripts persist, the developmental program of the early mouse embryo is primarily determined by the activation of the embryonic genome that begins late in the one-cell zygote and is dominant by the two-cell stage (Bolton et al. 1984; Schultz 1993). Early embryos, in which transcription is metabolically inhibited, divide to two cells but progress no further (Flach et al. 1982). Thus, there must be well-controlled mechanisms by which maternal mRNAs are replaced by zygotic transcripts, chromatin structure of the early embryo is rendered permissive for gene expression, and the cell cycle is regulated to accommodate early development. Because of the absence of transcription prior to the formation of pronuclei in the one-cell zygote, The nucleotide sequences for the coding regions of the mouse Mater gene have been deposited in the GenBank database, Accession Numbers AF143559–AF143573. Correspondence to: Zhi-Bin Tong, E-mail: tongz@ccl.nichd.nih.gov it seems likely that many of these processes will be regulated, either directly or indirectly, by pre-existing maternal gene products. One approach to identify cell-specific proteins is to screen cDNA expression libraries with antisera from patients with autoimmune disease directed against the tissue of interest. This approach has proven powerful in characterizing immunologic targets associated with disease and in identifying novel proteins that are of considerable biologic import (Fritzler et al. 1993; Garcia-Lozano et al. 1997; Ramos-Morales et al. 1998). Such antisera can also be obtained from animal models of specific autoimmune diseases. For example, female mice thymectomized as neonates are known to spontaneously develop autoimmune disorders, and, in certain strains, the predominant autoimmune process is directed against the ovary (Kojima and Prehn 1981). Autoantibodies directed against oocyte antigens within the cytoplasm are major antiovarian antibodies during development of oophoritis in these mice and are not adsorbed by proteins of other tissues (Taguchi et al. 1980). This suggests that there is a specific antigen(s) present within the oocyte cytoplasm that is targeted by the mouse immune system after thymectomy. Recently, we have shown that a 125-kDa mouse oocyte protein is recognized by the immune sera from neonatally thymectomized female mice, but not from male and sham-operated female mice. Using the immune sera to screen an ovarian cDNA expression library, a full-length cDNA that encodes an ooplasm-specific protein was isolated (Tong and Nelson 1999). Originally designated OP1, it is now referred to as MATER (Maternal Antigen That Embryos Require), because its absence in genetically engineered mice results in a striking failure of the mouse embryo to progress beyond early cleavage stages (Tong et al., manuscript in preparation). The primary structure of MATER was deduced from a single open reading frame of the cDNA that encodes a 1111-amino acid protein with a predicted molecular mass of 125,502 Da. The recombinant protein was specifically recognized by the autoimmune sera from mice with ovarian autoimmunity. To further investigate the function of the MATER protein in oogenesis and early development, we now report characterization of the gene that encodes MATER and discuss possible functions in the transition from maternal to zygotic control of early development. Materials and methods Cloning the mouse Mater gene. With a full-length mouse MATER cDNA as a 32P-labeled probe, several genomic clones ranging from 100 to 150 kb were isolated from a mouse 129/Sv BAC library (Genome Systems, St. Louis, MO). Southern blot hybridization with 32P-labeled oligonucleotides based on the 58 and 38 ends of the cDNA were used to determine that a BAC clone harbored the intact Mater locus. After BamHI digestion, the genomic DNA fragments were subcloned into pZErO-2.1 with Zero Back- 282 Z.-B. Tong et al.: Mater encodes a maternal protein with leucine-rich repeats Fig. 1. Exon-intron map of mouse Mater. Panel A: Schematic representation of the exon-intron map of the Mater gene. DNA sequence and PCR were used to determine the number and size of the exons and introns. The 15 vertical bars represent exons as indicated by the Arabic numerals on top of each exon. The horizontal solid lines between exons represent introns. The horizontal dashed line represents the undetermined 58 end of mouse Mater gene. A scale bar (2 kbp) is shown under the map. Panel B: 58 RACE-PCR was used to determine the 58 end of mouse MATER transcripts. 58 ends of mouse MATER cDNA were synthesized from mouse ovarian total RNA by reverse transcription with a Mater exon 6-specific primer. In separate experiments (lanes 2,5), the cDNAs were amplified with a Mater exon 4-specific oligonucleotide as the reverse primer and the abridge anchor as the forward primer. All other lanes were the same as lane 2 and 5, except that either the cDNA used as template was untailed (lane 1), that the abridge anchor forward primer was absent (lane 3), or that a Mater exon 1-specific oligonucleotide was used as forward primer (lane 4) as controls. PCR products were electrophoresed on 2% agarose gel containing 0.1% ethidium bromide. The size of PCR products is indicated at the right. ground/Kan according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Additionally, the Mater exon-specific primers were used to amplify DNA fragments from the genomic BAC clone with a TaqPlus Long PCR system (Strategene, La Jolla, CA) as described below. PCR products were cloned into TA cloning vectors (Invitrogen) according to the manufacturer’s protocol and confirmed by DNA sequencing of each end. Center for Biotechnology Information at the National Library of Medicine, NIH (http://www.ncbi.nlm.nih.gov). Protein modeling was performed with Swiss-Pdb Viewer (v 3.1) and RasWin Molecular Graphics (v 2.6). Southern hybridizations. 129/Sv mouse liver genomic DNA (10 mg) were determined by sequencing the cloned genomic DNA fragments. Either DNA sequencing or polymerase chain reaction (PCR) with mouse Mater exon-specific primers were conducted to determine the size of each intron. The junction regions between exon and intron were determined by DNA sequencing. and the BAC clone DNA (2 mg) were digested with restriction enzymes, separated by 0.8% agarose gel-electrophoresis, and transferred onto a nylon membrane (Schleicher & Schuell, Keene, NH) for Southern blot hybridization. MATER cDNA was labeled (Ready-To-Go DNA dCTP Bead, Pharmacia, Inc, Piscataway, NJ) with a-[32P]dCTP (3000 Ci/mmol, ICN). After hybridization at 68°C 1 h with QuikHyb solution (Stratagene), the blots were washed with a final stringency of 0.1 × SSC, 0.1% SDS at 60°C. The hybridization signals were detected by autoradiography. Polymerase chain reactions (PCR). For rapid amplification of cDNA Genetic mapping. C57BL/6J DNA, Mus spretus DNA, and an interspe- Determination of exon-intron map. The number and sizes of the exons ends, total mouse ovarian RNA (1 mg) was used as a template for reverse transcription with a Mater exon 6-specific primer (58-GCCTCTGTCACTTCATC-38) in a 58-RACE PCR system (Life Technologies, Rockville, MD). After TdT tailing, the cDNA was amplified with abridge anchor 58-primer (58-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-38) and a Mater exon 4-specific 38-primer (58-GCCTGTTCCACTTCTTC-38). The reaction was conducted in a Perkin Elmer GeneAmp PCR System 9600: 95°C 5 min; 35 cycles of 95°C 1 min, 63°C 0.5 min, and 72°C 2 min; followed by 10 min at 72°C. The PCR products were analyzed by agarose gel electrophoresis and cloned into TA vectors for DNA sequencing. Stratagene’s TaqPlus Long PCR system was used to clone Mater genomic fragments per the manufacturer’s protocol. Briefly, either Materpositive BAC clone DNA (0.5 mg) or 129/Sv mouse liver genomic DNA (1 mg) was amplified by TaqPlus Long polymerase mixture (Taq2000 DNA polymerase and Pfu DNA polymerase) with Mater exon-specific forward and reverse oligonucleotide primers. After initially denaturing at 95°C 5 min, the reaction was carried out at 95°C 0.5 min, 62°C 0.5 min, and 72°C 5 min for 35 cycles, followed by an extension for 10 min at 72°C. Genomic and cDNA PCR products were examined by agarose electrophoresis and cloned into the TA cloning vector. DNA sequencing and analysis. A dRhodamine terminator cycle se- quencing kit was used for DNA sequencing per the manufacturer’s instruction (PE Applied Biosystems, Foster City, CA). Cycling sequencing was conducted by PCR (25 cycles of 96°C 10 sec, 50°C 5 sec, and 60°C 4 min). Electrophoresis with 5% Long Ranger gels was carried out on the ABI Prism 377 DNA Sequencer. As necessary, sequence was verified with a DMSO-modified dideoxy chain termination method (Seto 1990) with [35S]dATP (Amersham, Arlington Heights, IL) and the Sequence Sequencing Kit (US Biochemical, Cleveland, OH). Analysis of the DNA and the deduced protein utilized software available at the ExPASy Molecular Biology Server of the Swiss Institute for Bioinformatics (http://www. expasy.ch), The Sanger Centre (http://www.sanger.ac.uk), and the National cific back-cross panel of DNA (BSS) were purchased from The Jackson Laboratory (Bar Harbor, Maine). The panel consisted of genomic DNA from 94 mice obtained by crossing (C57BL/6JEi × SPRET/Ei)F1 females to SPRET/Ei males (Rowe et al. 1994). In each PCR, one pair of Mater exon-specific oligonucleotides served as primers (exon 3-specific: 58AGCAATCTTGAAAGCACG-38; exon 4-specific: 58-GCCTGTTCCACTTCTTC-38) to detect the sequence polymorphisms between parental strain DNA, while the other pair of primers specific to mouse Zp3 were used as an internal control (58-primer, 58-CAGATGAGGTTTGAGGCCACAG-38; 38-primer, 58-CAGAGTCTGGGAATTGCACAGC-38). The conditions for PCR were the same as those used for 58 RACE (above). Results Mouse Mater gene locus. A 129/Sv mouse genomic BAC library was screened with full-length MATER cDNA to isolate a clone with ∼120-kb fragment containing the intact Mater gene locus. To characterize the organization of Mater, the number and sizes of the exons and introns were determined by DNA sequencing and PCR (Fig. 1A, Table 1). Fifteen exons were identified that encoded the 1111-amino acid MATER protein. Their sizes ranged from 48 to 1576 bp with intervening introns ranging from 0.6 to 3.7 kb (Table 1). Immediate flanking sequences of each exon conformed with the border element consensus sequences (AG. . . .GT) for exon-intron splice sites (Breathnach and Chambon 1981). In addition, there were eight nucleotides of the 58 non-coding region within exon 1, and 106 nucleotides of the 38 non-coding region within exon 15. Therefore, the mouse Mater gene locus spanned ∼32 kb DNA and was composed of 15 exons and 14 introns. Z.-B. Tong et al.: Mater encodes a maternal protein with leucine-rich repeats 283 Table 1. Position and size of exons and introns of the mouse Mater gene. Exon Position Length (bp)a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1–78 79–156 157–234 235–315 316–393 394–441 442–2017 2018–2188 2189–2356 2357–2527 2528–2698 2699–2869 2870–3040 3041–3211 3212–3447 78 78 78 81 78 48 1576 171 168 171 171 171 171 171 236 a b Intron Length (bp)b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 600a 2900 601a 1350 2430 3700 2570 2150 2670 2280 1330 2100 2200 1720 The length was determined by DNA sequencing. The length was determined by PCR products identified by sequencing both ends. 58 RACE-PCR was used to determine the 58 end of the Mater gene. Mouse ovarian total RNA was used as a template to synthesize single-stranded cDNA by reverse transcription with a Mater exon 6-specific oligonucleotide used as a primer. With the 58 RACE abridge anchor primer and one Mater exon 4-specific primer, two PCR products for the 58 ends of MATER cDNA were amplified (Fig. 1B, lanes 2,5). The longer PCR product (429 bp) was consistently fainter than the other (378 bp), which may reflect relative abundance, in vivo, of the two underlying transcripts. After subcloning into TA vectors, the sequences of the two PCR products were determined (data not shown). The sequences were identical from exon 1 to exon 4, but were different at their 58 ends (119 nt and 68 nt, respectively). As a control, one DNA fragment (309 bp, lane 4) was amplified with MATER exon-1 specific oligonucleotide as the forward primer instead of the 58 RACE anchor abridge primer. These data suggested that there were two alternative transcription start sites present at the mouse Mater gene locus, the longer being less abundant than the shorter. However, the different 58 ends reflect noncoding regions of the two transcripts, and both have identical coding region and 38UTRs. Therefore, the two transcripts appeared to encode the same MATER protein. Coding regions of the Mater gene. The amino acid sequence deduced from the Mater genomic clone had seven polymorphisms, two of which were highly conserved when compared with that deduced from the sequence of the original MATER cDNA (Fig. 2). Presumably these polymorphisms between NIH Swiss and 129/Sv mice have no functional sequelae, and both mouse lines have normal oogenesis and fertility. The exon map of Mater showed a striking symmetry with the first five exons containing 78 or 81 nucleotides, sufficient to encode a 26- or 27-amino acid polypeptide. Additionally, seven exons (8–14) at the 38 end of Mater were either 171 or 168 nucleotides in length and encoded 56 or 57 amino acid polypeptides (Fig. 1, Table 1). These repeats suggested partial gene duplication through evolution and implied a functional role for these domains. When the entire polypeptide chain of MATER was compared with itself by Dotplot analysis, the presence of repeat motifs at the amino- (1–128 amino acids) and carboxyl- (671–1068 amino acids) termini was evident by the parallel but offset lines in these regions (Fig. 3A). Alignment of the polypeptides encoded by each of the first five exons revealed a highly conserved hydrophilic repeat (Fig. 3B), the sequence of which had a low degree of homology (20–25% identity) with several cytoskeletal components including actin, neurofilaments, and microtubules. Alignment of the polypeptides encoded by exons 8–14 did not initially appear to be as well conserved (Fig. 3C), but further analysis revealed a Fig. 2. Amino acid polymorphisms in MATER. The primary structure of the 1111-amino acid MATER protein shown on the first line was deduced from the coding regions of the Mater gene isolated from a 129Sv genomic library. The amino acid sequence, represented with single letter code, is numbered on the right. For comparison, the amino acid sequence deduced from a cDNA clone derived from NIH Swiss mice (Tong and Nelson 1999) is shown on the second line. Dashes represent identities between the NIH Swiss and 129Sv mice; the seven polymorphism at positions 7 (Asp → Glu), 42 (Leu → Gly), 87 (Lys → Arg), 256 (Pro → Leu), 977 (Gln → His), 1003 (Asn → Ser), and 1004 (Asn → Ser) are indicated by E, G, R, L, H, S and S, respectively. repeated leucine-rich motif that was not in register with the exon repeat (see below). A hydropathy plot of the 1111-amino acid MATER protein confirmed the presence of a hydrophilic amino terminus and indicated a short, hydrophilic carboxyl terminal tail (Fig. 4A). With no evidence of a signal peptide to direct it into a secretory pathway, no membrane-spanning domains, and no nuclear translocation signals, it appeared likely that the MATER was present within the cytoplasm. MATER also contained an ATPase-associatedactivities domain (amino acids 192–404), which has been implicated in a variety of essential cellular functions that may be mediated by the domain’s ability to anchor proteins or participate in the multi-subunit proteasome complex (Confalonieri and Duguet 1995). In addition, there was a particularly striking region near the carboxyl terminus (amino acids 671–1068) that contained 14 tandem leucine-rich repeats (Fig. 4B). These repeats correspond to the canonical sequence, LxxLxLxxN/CxL, that was first reported in porcine ribonuclease inhibitor and is critical for protein-protein interactions (Hofsteenge et al. 1988; Kajava 1998). The three-dimensional structure of this motif has been determined at 2.0–2.4 Å resolution for three protein complexes: porcine ribonuclease inhibitor/RNase A, human placental RNase inhibitor/angiogenin, and the U2B9/U2A8 spliceosomal complex (Kobe and Deisenhofer 1995; Papageorgiou et al. 1997; 284 Fig. 3. Repeated exons encode protein repeats. Panel A: Dotblot analysis in which the amino acid sequence of MATER was compared with itself (1–1111 amino acids), and identities were indicated by a dot. The left-toright center diagonal line reflects the direct correspondence of the two copies of the same protein. The shorter diagonal lines parallel, but offset from the center, reflect repeated sequences in the amino- and carboxyltermini. Panel B: Each of the first five exons encoded 23–27 amino acids that were aligned using Pileup (GCG Wisconsin Package). Amino acid identities are indicated by white letters and a dark background; similarities are indicated by a grey background. Exon numbers are indicated at left. Panel C: Exons 8 through 14 each encoded 56–57 amino acids which were aligned and portrayed as in Panel B. Price et al. 1998). The initial leucines form a b-strand that is followed by a turn and then an a-helix (Fig. 4B). Taking advantage of the similarity of the three leucine-rich repeat structures and the homology between the approximately 400 amino acid leucine-rich repeats in MATER and ribonuclease inhibitor, Swiss Model software was used to predict the leucine-rich repeat domain in MATER. The resultant theoretical structure (Fig. 4C) matched well with that reported for porcine ribonuclease inhibitor and suggested that the leucine-rich repeats formed a non-globular domain in MATER shaped like a horseshoe. The 14 b-strands would form an internal b-sheet with the a-helices on the external circumference. If the analogy with the other proteins with similar structures pertains, then the internal b-sheet on MATER would be available for interactions with other proteins, perhaps ribonucleases, within the oocyte’s cytoplasm. Genetic mapping of the single-copy mouse Mater gene. We reasoned that if Mater was a single copy within the genome, there would be no restriction fragment polymorphisms present between the BAC clone and isogenic genomic DNA. Thus, Southern blot hybridization was applied to compare the restriction enzyme digestion patterns between the Mater BAC clone and 129/Sv genomic DNA. With a 32P-labeled, full-length MATER cDNA probe, the hybridization patterns were identical for restriction fragments after digestion with BamHI, EcoRI, and HindIII (Fig. 5). After digestion with PstI, one fragment (∼5 kbp) was absent in the BAC Z.-B. Tong et al.: Mater encodes a maternal protein with leucine-rich repeats clone DNA, and the ∼18-kbp fragment had a stronger signal in comparison with genomic DNA. This reflected a junction fragment including parts of Mater gene and BAC vector. We concluded from these data that there was only one copy of Mater in the mouse genome. A C57BL/6J × Mus spretus interspecific backcross panel (Rowe et al. 1994) was used to localize Mater in the mouse genome. A genomic polymorphism between the parental strains (C57BL/6J and Mus spretus) was determined at the Mater locus with PCR. In these experiments, a pair of Mater exon-specific primers amplified a PCR product (721 bp) from C57BL/6J and 129/Sv, but not Mus spretus DNA (Fig. 6A). The failed PCR amplification of the spretus DNA was attributed to DNA polymorphisms at the 58-primer region between parental strains of DNA. The DNA sequence corresponding to the 58-primer at C57BL/6J and 129/Sv mouse genomic DNA was the same as the 58-primer sequence found in NIH Swiss mouse (58-AGCAATCTTGAAAGCACG-38), while the DNA sequence at this region for Mus spretus was 58-AGCAATCTTGAAAGACTG-38. Both had identical sequences corresponding to the 38-primer (58-GCCTGTTCCACTTCTTC-38). To monitor the PCR, we included an internal control using the primers specific for the mouse Zp3 gene (Rankin et al. 1998), which effectively amplified a 238-bp fragment in both parental strains. The control Zp3 fragment (238 bp) was detected in all 96 DNA samples including the two parental strains, but the Mater fragment (701 bp) was present in only 53 DNA samples (Fig. 6). In a haplotype analysis with 94 animals, the Mater locus mapped to the proximal end of mouse Chr 7, within 4 cM of the centromere (Fig. 6). This region is syntenic with human Chr 19q13, a region that does not, as yet, contain known disease loci. Discussion The fully grown oocyte is the largest cell in mammals, and as a result of active transcription during oogenesis, mouse oocytes accumulate 90 pg of poly(A)+ RNA (Bachvarova et al. 1985). During meiotic maturation and subsequent ovulation, the maternal genome is transcriptionally inert (Flach et al. 1982), and most maternal transcripts are deadenylated and/or degraded (Paynton et al. 1988) prior to, or concomitant with, the activation of the zygotic genes at the two-cell stage (Pratt et al. 1983). Virtually nothing is known about the molecular basis of this maternal to embryonic transition, which involves the degradation of oocyte, but not zygotic transcripts, the activation of the embryonic genome, and the induction of cell cycle progression. In seeking candidate maternal proteins involved in these processes, antisera from an experimental mouse model of autoimmune oophoritis have been used to isolate MATER, a 125-kDa oocyte-specific protein (Tong and Nelson, 1999). With the cDNA that encodes MATER, a BAC clone that contains the Mater locus has been isolated from a 129/Sv genomic library. The Mater gene is single-copy in the mouse genome and contains 15 exons that span 32 kbp at the proximal end of mouse Chr 7. The deduced 1111-amino acid protein is polymorphic at seven residues compared with the MATER protein sequence in NIH Swiss mice. Each of the first five exons encodes a 26- to 27-amino acid hydrophilic repeat. Exon 7, by far the largest, encodes the 524-amino acid core of MATER, and this central region is followed by 14 leucine-rich repeats encoded by exons 8–14. The correspondence of the Mater exon map with the Nterminal hydrophilic repeat and the C-terminal leucine-rich repeat in the MATER protein is quite striking. While the hydrophilic repeat has only low homology with cytoskeletal proteins in the databases, the leucine-rich repeat has been identified in a number of proteins that have been arranged in families (for review see Kajava 1998). Three proteins with leucine-rich repeats most similar to MATER have been co-crystallized with the proteins to which they bind, and their three-dimensional structure has been deter- Z.-B. Tong et al.: Mater encodes a maternal protein with leucine-rich repeats 285 Fig. 4. Structural motifs in MATER. Panel A: A hydropathy plot (Kyte and Doolittle 1982) of MATER indicated hydrophilic repeats at the amino (1–128 amino acids) and carboxyl (1070–1111 amino acids) termini. The amino-terminal hydrophilic repeats, a motif associated with ATPase activities (192–404 amino acids), and a series of leucine-rich repeats (671–1068 amino acids) near the carboxyl terminus are indicated below. Panel B: The 14 leucine-rich repeats encoded by exons 8–15 were 28–29 amino acids in length. Each repeat had a sequence that can be modeled into a b-strand followed by a turn and then an a helix. The defining motif is indicated by bold letters and a grey background. Panel C: With Swiss Model and Rasmol, the structure of the MATER leucine-rich repeats was modeled based on the previously determined three-dimensional structure of porcine ribonuclease inhibitor. The predicted structure of this region is that of a non-globular domain shaped as a horseshoe with a b-sheet forming the inner aspect, and the alpha helices forming the outer circumference. Fig. 5. Southern blot hybridization for restriction enzyme analysis of the mouse Mater gene locus. DNA from the Mater BAC (A, 2 mg) and 129/Sv mouse liver (B, 10 mg) was electrophoresed and transferred to a membrane after digestion with BamHI (lane 1), EcoRI (lane 2), HindIII (lane 3), and PstI (lane 4). The blots were probed with 32P-labeled full-length MATER cDNA, and hybridization signals were detected by autoradiography. Molecular sizes of DNA are indicated on the left of each panel. mined. The first two complexes involve porcine and human ribonuclease inhibitors binding to known members of the ribonuclease superfamily (Hofsteenge et al. 1988; Kobe and Deisenhofer 1995), and the third involves U2A8 binding to U2B9 in an RNA spliceosome (Papageorgiou et al. 1997). In each case the 28–29 amino acid, leucine-rich repeats form a non-globular domain in which the b-sheets and a-helices are arranged in a semicircle, with the a-helices on the outside and the b-sheets on the inside exposed to solvent. The LxxLxLxxN/CxL repeat is part of the b-sheet with considerable diversity in adjacent sequences. The parallel b-sheets are the primary participants in the protein-protein interaction, and the inhibitors seem to engulf the much smaller ribonuclease domains to effectively inhibit their enzymatic activity. The function of MATER remains to be determined, but preliminary analyses of mice in which the Mater gene has been inactivated indicate that the MATER protein is required for embryonic development beyond two cells (Tong et al., manuscript in preparation). A similar block at the two-cell stage has been observed in embryos in which transcription has been metabolically inhibited with a-amanatin. As noted above, maternal transcripts are degraded during meiotic maturation and ovulation. If this involves the activation of a ribonuclease just prior to ovulation, the maternal ribonuclease would have to be inhibited to preserve zygotic transcripts, including those required for embryonic gene expression. MATER, with its putative ribonuclease inhibitor domain and cytoplasmic location, would be well positioned to bind to the maternal ribonuclease(s) and prevent degradation of zygotic transcripts. In mice lacking MATER, the maternal ribonuclease activity would persist and could account for the observed block at the two-cell stage. However, other possibilities abound, including a direct or indirect for MATER in the activation of the embryonic genome or in the de-repression of the second cell division. These and other possible functions are currently under investigation. While many genes are expressed in mammalian germ cells, surprisingly few oocyte-specific genes or gene products have been described. Some genes, such as those that encode the three zona pellucida proteins (Epifano et al. 1995) or FIGa, an oocytespecific bHLH transcription factor (Liang et al. 1997), are required for folliculogenesis, fertilizations, and passage down the oviduct. Others that are also expressed in male germ cells, such as GDF-9, appear to have oocyte-specific function as judged by the phenotype in mice lacking the gene product (Dong et al. 1996; Fitzpatrick et al. 1998; McGrath et al. 1995). Still other genes are transcribed only in oocytes, but it remains unclear whether the proteins deduced from the nucleic acid sequence of the transcripts are actually translated (West et al. 1996). Comparisons of functional elements in these and additional oocyte-specific promoters may provide insights into mechanisms that regulate gene expression within female germ cells. Two MATER-specific RT-PCR products, one fainter than the other, were consistently observed, which suggests either alternative splicing of upstream exons or an alternative transcriptional start site, or both. Alternative transcription is not uncommon for mammalian genes (Grandien et al. 1997; Klein et al. 1998; Or- 286 Z.-B. Tong et al.: Mater encodes a maternal protein with leucine-rich repeats Fig. 6. Genetic mapping and chromosomal localization of the mouse Mater gene. Parental strains of DNA (C57BL/6J and Mus spretus) and an interspecific backcross BSS DNA panel were used for genetic mapping. Panel A: DNA polymorphism of mouse Mater. Top, one pair of mouse Mater exon-specific primers effectively amplified DNA (701 bp) from mouse genomic DNA of C57BL/6J (lanes 1,4) and 129/Sv (lanes 3,6), but failed to amplify Mus spretus mouse genomic DNA (lanes 2,5). A pair of Zp3 primers used as internal controls to monitor the PCR amplified a 238-bp DNA from all three strains. Mouse DNA: C57BL/6J (lanes 1,4,7), Mus spretus (lanes 2,5,8), and 129/Sv (lanes 3,6,9). Primers: Mater-specific primers alone (lanes 1–3), both Mater- and Zp3-specific primers (lanes 4–6), and Zp3-specific primers alone (lanes 7–9). Bottom, nine examples of the PCR of the 94 DNA samples of back-cross progeny animals ([C57B1/6J × SPRET/Ei)F1] × SPRET/Ei) with both Mater and Zp3 primers. Molecular sizes of DNA markers are indicated on the left. Panel B: Haplotype analysis of BSS DNA panel. Filled and open boxes indicate the presence of the C57B1/6J and Mus spretus alleles, respectively. Previously mapped loci around Mater are indicated on the left. The number at the bottom of each column of squares indicates the number of progeny with the particular haplotype. Panel C: Schematic representation of part of mouse Chr 7. The centromere is an open circle at the top of the vertical line. Dashed lines represent undetermined distances. The Mater locus and the surrounding loci are listed on the right. Scale bar (3 cM) is shown as a vertical line on the right. zechowski et al. 1997). For example, human estrogen receptor (hER) gene is transcribed from three different promoters to yield three different mRNA isoforms with unique 58 untranslated regions (58UTRs), but identical coding regions (Grandien et al. 1997). Although these mRNA isoforms vary in their tissue distributions and efficiency of transcription and translation, they are translated into the same protein to function in the particular tissue. Like the hER gene, the two mouse MATER mRNA isoforms have an identical coding region and 38 UTR. It will be interesting to characterize the promoter(s) that direct gene expression exclusively in the oocytes. If different, the function of each promoter might be related to specific stages of oocyte growth and development. Cloning and characterization of mouse Mater locus provide us with a new determinant not only to study oocyte development and maternal effects on embryogenesis, but also to investigate the pathogenesis of ovarian autoimmunity. We have shown that MATER is an autoantigen and a predominant target of autoreactive B-cells in mice with autoimmune oophoritis. Although this autoimmune ovarian disease is mediated by pathogenic CD4 T lymphocytes (Sakaguchi et al. 1982; Smith et al. 1991), autoreactive T- and B-cells can share a common antigen target for the organ-specific autoimmunity (Kaufman et al. 1993; Tisch et al. 1993). Induction of specific immune tolerance to the organspecific inciting antigen can ameliorate the entire spectrum of autoimmune reactions in the particular organ (Alderuccio et al. 1993; Kaufman et al. 1993; Tisch et al. 1993). The characterization of the Mater gene provides a reagent to determine whether transgenesis can be used to induce immune tolerance to the MATER protein and prevent autoimmune oophoritis, analogous to results reported on the b-subunit of H/K ATPase that initiates autoimmune gastritis (Alderuccio et al. 1993). Acknowledgments. We appreciate constructive discussions with Dr. Carolyn Bondy and the guidance of Drs. Craig Hyde and Andrey Kajava on the analysis of the molecular structures. We are also grateful to Ms. Lucy Rowe and Mary Barter of The Jackson Laboratory for help with the haplotype analysis. We thank Mr. Keith Zachman for DNA sequencing on the ABI Prism 377 Sequences. References Alderuccio F, Toh BH, Tan SS, Gleeson PA, van Driel IR (1993) An autoimmune disease with multiple molecular targets abrogated by the transgenic expression of a single autoantigen in the thymus. J Exp Med 178, 419–426 Bachvarova R, De Leon V (1980) Polyadenylated RNA of mouse ova and loss of maternal RNA in early development. Dev Biol 74, 1–8 Bachvarova R, De Leon V, Johnson A, Kaplan G, Paynton BV (1985) Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes. Dev Biol 108, 325–331 Bolton VN, Oades PJ, Johnson MH (1984) The relationship between cleavage, DNA replication, and gene expression in the mouse 2-cell embryo. J Embryol Exp Morphol 79, 139–163 Breathnach R, Chambon P (1981) Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 50, 349–383 Clegg KB, Piko L (1983) Poly(A) length, cytoplasmic adenylation and synthesis of poly(A)+ RNA in early mouse embryos. Dev Biol 95, 331–341 Confalonieri F, Duguet M (1995) A 200-amino acid ATPase module in search of a basic function. Bioessays 17, 639–650 Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N et al. (1996) Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531–535 Epifano O, Liang L-F, Familari M, Moos MC, Jr., Dean J (1995) Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development 121, 1947–1956 Fitzpatrick SL, Sindoni DM, Shughrue PJ, Lane MV, Merchenthaler IJ et al. (1998) Expression of growth differentiation factor-9 messenger ribonucleic acid in ovarian and nonovarian rodent and human tissues. Endocrinology 139, 2571–2578 Flach G, Johnson MH, Braude P, Taylor RAS, Bolton VN (1982) The transition from maternal to embryonic control in the 2-cell mouse embryo. EMBO J 1, 681–686 Fox CA, Wickens MP (1990) Poly(A) removal during oocyte maturation: a default reaction selectively prevented by specific sequences in the 38 UTR of certain maternal mRNAs. Genes Dev 4, 2287–2298 Fritzler MJ, Hamel JC, Ochs RL, Chan EK (1993) Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex. J Exp Med 178, 49–62 Garcia-Lozano JR, Gonzalez-Escribano MR, Wichmann I, Nunez-Roldan A (1997) Cytoplasmic detection of a novel protein containing a nuclear Z.-B. Tong et al.: Mater encodes a maternal protein with leucine-rich repeats localization sequence by human autoantibodies. Clin Exp Immunol 107, 501–506 Grandien K, Berkenstam A, Gustafsson JA (1997) The estrogen receptor gene: promoter organization and expression. Int J Biochem Cell Biol 29, 1343–1369 Hofsteenge J, Kieffer B, Matthies R, Hemmings BA, Stone SR (1988) Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats. Biochemistry 27, 8537– 8544 Kajava AV (1998) Structural diversity of leucine-rich repeat proteins. J Mol Biol 277, 519–527 Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GS et al. (1993) Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 366, 69–72 Klein M, Pieri I, Uhlmann F, Pfizenmaier K, Eisel U (1998) Cloning and characterization of promoter and 58-UTR of the NMDA receptor subunit epsilon 2: evidence for alternative splicing of 58-non-coding exon. Gene 208, 259–269 Kobe B, Deisenhofer J (1995) A structural basis of the interactions between leucine-rich repeats and protein ligands. Nature 374, 183–186 Kojima A, Prehn RT (1981) Genetic susceptibility to post-thymectomy autoimmune diseases in mice. Immunogenetics 14, 15–27 Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105–132 Liang L-F, Soyal SM, Dean J (1997) FIGa, a germ cell specific transcription factor involved in the coordinate expression of the zona pellucida genes. Development 124, 1939–1949 McGrath SA, Esquela AF, Lee SJ (1995) Oocyte-specific expression of growth/differentiation factor-9. Mol Endocrinol 9, 131–136 Orzechowski HD, Richter CM, Funke-Kaiser H, Kroger B, Schmidt M et al. (1997) Evidence of alternative promoters directing isoform-specific expression of human endothelin-converting enzyme-1 mRNA in cultured endothelial cells. J Mol Med 75, 512–521 Papageorgiou AC, Shapiro R, Acharya KR (1997) Molecular recognition of human angiogenin by placental ribonuclease inhibitor—an X-ray crystallographic study at 2.0 A resolution. EMBO J 16, 5162–5177 Paynton BV, Rempel R, Bachvarova R (1988) Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse. Dev Biol 129, 304–314 Pratt HPM, Bolton VN, Gridgeon KA (1983) The legacy from the oocyte and its role in controlling early development in the mouse embryo. Ciba Found Symp 98, 197–227 287 Price SR, Evans PR, Nagai K (1998) Crystal structure of the spliceosomal U2B9-U2A8 protein complex bound to a fragment of U2 small nuclear RNA. Nature 394, 645–650 Ramos-Morales F, Infante C, Fedriani C, Bornens M, Rios RM (1998) NA14 is a novel nuclear autoantigen with a coiled-coil domain. J Biol Chem 273, 1634–1639 Rankin T, Tong Z-B, Castle PE, Lee E, Gore-Langton R et al. (1998) Human ZP3 restores fertility in Zp3 null mice without affecting orderspecific sperm binding. Development 125, 2415–2424 Rowe LB, Nadeau JH, Turner R, Frankel WN, Letts VA et al. (1994) Maps from two interspecific backcross DNA panels available as a community genetic mapping resource. Mamm Genome 5, 253–274 Sakaguchi S, Takahashi T, Nishizuka Y (1982) Study on cellular events in postthymectomy autoimmune oophoritis in mice. I. Requirement of Lyt-1 effector cells for oocytes damage after adoptive transfer. J Exp Med 156, 1565–1576 Schultz RM (1993) Regulation of zygotic gene activation in the mouse. Bioessays 15, 531–538 Seto D (1990) An improved method for sequencing double stranded plasmid DNA from minipreps using DMSO and modified template preparation. Nucleic Acids Res 18, 5905–5906 Smith H, Sakamoto Y, Kasai K, Tung KS (1991) Effector and regulatory cells in autoimmune oophoritis elicited by neonatal thymectomy. J Immunol 147, 2928–2933 Stutz A, Conne B, Huarte J, Gubler P, Volkel V et al. (1998) Masking, unmasking, and regulated polyadenylation cooperate in the translational control of a dormant mRNA in mouse oocytes. Genes Dev 12, 2535– 2548 Taguchi O, Nishizuka Y, Sakakura T, Kojima A (1980) Autoimmune oophoritis in thymectomized mice: detection of circulating antibodies against oocytes. Clin Exp Immunol 40, 540–553 Tisch R, Yang XD, Singer SM, Liblau RS, Fugger L et al. (1993) Immune response to glutamic acid decarboxylase correlates with insulitis in nonobese diabetic mice. Nature 366, 72–75 Tong Z-B, Nelson LM (1999) A mouse gene encoding an oocyte antigen associated with autoimmune premature ovarian failure. Endocrinology 140, 3720–3726 West MF, Verrotti AC, Salles FJ, Tsirka SE, Strickland S (1996) Isolation and characterization of two novel, cytoplasmically polyadenylated, oocyte-specific, mouse maternal RNAs. Dev Biol 175, 132–141