Isolation and characterization of a cDNA encoding a synaptonemal complex protein QIANFACHEN,RONALD E. PEARLMAN, AND PETERB. MOENS
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Department of Biology, York University. 4700 Keele Street, Downsview, Ont., Canada M3 J IP3 Received February 7 , 1992 R. E., and MOENS,P. B. 1992. Isolation and characterization of a cDNA encoding a CHEN, Q., PEARLMAN, synaptonemal complex protein. Biochem. Cell Biol. 70: 1030- 1038. A gene encoding a 65-kilodalton antigen of the rat synaptonemal complex, SC65, has been cloned by screening rat testis Agtl 1 and AZAPII cDNA expression libraries using polyclonal antibodies against rat synaptonemal complex proteins. The longest open reading frame, initiating at an ATG codon in the cDNA, encodes a protein of 431 amino acids, with a relative molecular mass of 50 000. Immunological analysis locates the SC65 gene product on the synaptonemal complex between the pairing faces of the parallel aligned cores of homologous chromosomes in spermatocytes. Of the rat tissues examined, the SC65 gene is transcribed in testis, brain, and heart at similar levels, and in the liver at a much lower level. The DNA sequence extending about 80 base pairs downstream of the translation termination codon has 93% similarity to the identifier sequence present in the rat genome in 1 x 10' - 1.5 x 10' copies and in cDNA clones of precursors of brain-specific mRNAs. The amino acid sequence encoded by the SC65 gene contains an acidic region in the C-terminal domain of the protein, potential glycosylation sites, and at least one possible phosphorylation site. The protein shows no overall similarity to proteins of known function, nor is there similarity to protein sequences present in GenBank or EMBL data bases. Key words: meiosis, synaptonemal complex, antibody, rat testis cDNA library, molecular cloning. CHEN, Q., PEARLMAN, R. E., et MOENS,P. B. 1992. Isolation and characterization of a cDNA encoding a synaptonemal complex protein. Biochem. Cell Biol. 70 : 1030-1038. Le gene SC65, codant un antigkne de 65 kilodaltons du complexe synaptonernal de rat, a ete clone en criblant les banques d'expression de cDNA (Xgtll et AZAPII) de testicule de rat a l'aide d'anticorps polyclonaux contre les proteines du complexe synaptonemal de rat. Le plus long cadre de lecture ouvert debutant par un codon ATG dans le cDNA code une proteine de 431 acides amines, de masse molCculaire relative de 50 000. Une analyse immunologique a permis de localiser le produit du gkne SC65 dans le complexe synaptonernal entre les lames apparikes des chromosomes homologues alignes parallklement dans les spermatocytes. Plusieurs tissus de rat ont ete analysb; le gkne SC65 est transcrit ti un taux semblable dans les testicules, le cerveau et le coeur, et il est transcrit a un niveau beaucoup plus faible dans le foie. La sequence de DNA s'etendant environ 80 paires de bases au-dela du codon de terminaison de la traduction est homologue a 93% avec la sequence identificatrice dont environ 1 x 10' - 1.5 x 10' copies se trouvent dans le genome de rat et prksente dans les clones de cDNA de precurseurs de RNA messagers sptcifiques du cerveau. La sequence d'acides aminks codte par le gkne SC65 comporte une region acidique dans le domaine C-terminal de la protkine, des sites potentiels de glycosylation et au moins un site possible de phosphorylation. La proteine n'a pas d'homologie globale ni avec des proteines de fonction connue, ni avec les sequences proteiques se trouvant dans les bases de donnees GenBank ou EMBL. Mots cl&s : meiose, complexe synaptonemal, anticorps, banque de cDNA de testicule de rat, clonage moltculaire.
Introduction The synaptonemal complex is at the interface between paired chromosomes at meiotic prophase in germ cells of most sexually reproducing species. The SC is of interest because its presence is correlated with important meioticspecific functions such as synapsis, recombination, and segregation (reviewed in von Wettstein et al. 1984; Moens 1987; John 1990). Identification of SC protein components is based on recognition by autoimmune antibodies (Dresser 1987), generation of mono- and poly-clonal anti-SC antibodies (Heyting et al. 1987), screening with antibodies against known proteins (Heyting et al. 1983; Spyropoulos and Moens 1984; Moens and Earnshaw 1989), and molecular genetic techniques (Hollingsworth et a/. 1990). Some of the ABBREVIATIONS: EMBL, European Molecular Biology Laboratory; SC, synaptonemal complex; kDa, kilodaltons; IPTG, isopropylthiogalactoside; BSA, bovine serum albumin; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; NBT, nitroblue tetrazolium; PMSF, phenylmethylsulphonyl fluoride; SDS-PAGE, sodium dodecyl sulfate - polyacrylarnide gel electrophoresis; PBS, phosphate-buffered saline; bp, base pair(s); CIP, calf intestinal alkaline phosphatase; FITC, fluorescein isothiocyanate; Mabs, monoclonal antibodies; DAB, diaminobenzidine. Prinled in Canada / Imprime au Canada
protein components of the SCs are specific to the SCs and the meiotic nucleus (Heyting et al. 1988; Offenberg et a/. 1991; Smith and Benevente 1992), whereas others, such as those detected by anti-topoisomerase I1 (Moens and Earnshaw 1989), or preimmune sera generally have epitopes in common with more ubiquitous proteins (Dresser 1987). In this article, we report that the SC-positive serum of a rabbit recognizes a 65-kDa polypeptide in Western blots, and with electron microscopy of immunogold-labelled, surface-spread SCs, we show that the antigen is located in the pairing zone of the SC. From clones of rat testis cDNA libraries containing sequences encoding the 65-kDa polypeptide, we determine the nucleotide sequence of the gene and demonstrate a major 2000 nucleotide transcript in testis, heart, and brain tissues. Materials and methods Zmmunoscreening of the rat testis cDNA Agtll library with antiSC antibody A Agtl 1 library of rat testis cDNA (Clontech Laboratories) was screened on nitrocellulose filters with rabbit serum immunopositive for SCs essentially as described by Snyder et al. (1987). Half a million phage recombinants were plated on Escherichia coli Y 1090 at a density of 25 x lo3phage/90-mm plate. IPTG-treated filters
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CHEN ET AL.
were incubated on plates for 8-10 h. Screenings were carried out in duplicate. All washes were done in TBST (0.05% Tween-20 in Tris-buffered saline, i.e., 50 mM Tris-HC1 (pH 8.0) and 150 mM NaCl). Filters were blocked in 1% BSA in TBST at 4OC for 30 min before incubation with antibody. Antibodies that recognized E. coli proteins were removed from the screening serum by adding E. coli lysate according to protocols supplied by Bio-Rad. Filters were incubated in the screening serum at 4OC overnight and the subsequent incubations were performed at 4°C to increase the signals. Bound antibodies were detected with an alkaline phosphatase conjugated goat anti-rabbit antibody (Bio-Rad). Reactive phage replicas were visualized by development with BCIP and NBT as described by the supplier (Promega). A total of three screenings at decreasing plaque densities were carried out until pure phage was obtained. Preparation of lysogens, fusion protein, and anti-fusion protein antibody, and affinity purification of anti-fusion protein antibody Generation of Xgtl 1 recombinant lysogens in E. coli Y1089 and preparation of fusion protein extracts from the Xgtl 1 lysogens were carried out by the procedures of Huynh et al. (1985). Lysogens were collected and purified by their temperature sensitivity (Snyder et al. 1987). Proteins from extracts of lysogens were prepared and stored in 75% ammonium sulphate as a slurry at 4OC. For preparation of fusion proteins, protein extract from 500 pL of the (NH4),S04 precipitate was centrifuged at 10 000 x g for 20 min at 4°C and resuspended in 500 pL TEP buffer (100 mM Tris-HC1 (pH 7.4), 10 mM EDTA, 1 mM PMSF diluted from a 100-mM stock in ethanol at a concentration of approximately 4 pg/pL. About 100-150 pg protein extract/well was separated in a7.5% SDS-PAGEgel(160 x 180 x 1.5 mm, 12wells). Thegel was stained by soaking it in cold 0.25 M KC1 (Hager et al. 1980) for 20 min with gentle agitation. The fusion proteins, which were identified in previous experiments using standard Coomassie blue G-250 staining, were excised from the gel, pooled, rinsed in distilled water, lyophilized for 24 h, and then ground into a fine powder which could be stored at - 80°C for long time periods. For production of anti-fusion protein antibody, the powder of fusion protein from the gels (100-200 pg of protein) was resuspended in 0.5 mL PBS and injected into a 2.5-kg New Zealand female rabbit subcutaneously. Two subsequent injections of 100 pg were given at 4 and 8 weeks. Serum was collected prior to injections and 7-10 days after each injection, and then analyzed by using immuno-DAB, immuno-gold staining, and immunoblotting techniques as described below. Proteins produced from phage were used to affinity purify antibodies as described by Snyder et al. (1987). Proteins isolated from E. coli Y1089 infected by wild-type Xgtll were used in control incubations. Screening of the rat testis cDNA AZAPII library with radioactively labelled DNA probe Hybridization of XZAPII plaques with a radioactively labelled DNA probe was performed essentially according to the suplier's protocol (Stratagene). Escherichia coli XLl-Blue cells infected with the XZAPII phage were plated, amplified in situ, and processed as described by Sambrook et al. (1989). Prehybridization and hybridization under stringent conditions used a Blotto-Hyb mix (5 x SSPE, 1% SDS, 0.5% Carnation nonfat dry skim milk powder). Hybridizing plaques were purified through at least three rounds of screening. Subcloning and DNA sequencing Restriction endonucleases and DNA modifying enzymes were purchased from several suppliers (Pharmacia, Gibco/BRL, Boehringer Mannheim, Amersham, New England Biolabs, and U.S. Biochemical Corp). Manipulations of DNA were according to Sambrook et al. (1989). The EcoRI cDNA inserts isolated from the lambda libraries were subcloned in both orientations into the
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pEMBL18 vector (Dente et al. 1983) using E. coli JMlOl as host (Hanahan et al. 1983). For DNA sequencing, single-stranded pEMBL18 DNA was prepared by superinfection of the cells containing the plasmids with f l phage IR1 (Dente et al. 1983) or VCSM13 (Stratagene). The single-stranded DNA was used as a template for nucleotide sequencing by the dideoxy chain termination method (Sanger et al. 1977) using [(Y-~~sI~ATP, the 17-bp universal primer, and the Sequenase Version 2.0 kit as recommended by the supplier (U.S. Biochemical Corp.). Both strands were sequenced for almost the entire cDNA. For short regions where both strands were not sequenced, at least two independent subclones from both phages 2 x 2 and ZAPII-1 were sequenced. Computer-assisted analysis of sequences was performed with the programs Analyseq and Seqaid. Nucleic acid and protein sequences were searched against all sequences in the GenBank and EMBL data bases using a FASTA search according to Pearson and Lipman (1988). Labelling of DNA For preparation of hybridization probes, DNA restriction fragments were isolated from agarose gels as described by Heery et al. (1990) and labelled with [(Y-"P]~ATP(3000 Ci/mM, 1 Ci = 37 GBq; Amersham) using the random priming protocol of Feinberg and Vogelstein (1983). For preparation of DNA size markers, wild-type lambda DNA was digested with the appropriate restriction enzymes. Fragments were labelled at their 3 ' termini using the Klenow fragment of DNA polymerase I of E. coli as described in Sambrook et al. (1989). For preparation of primer for the primer extension analysis, the EcoRI cDNA fragment of 1.4 kbp was purified from an agarose gel (Heery et al. 1990), digested with StuI (Fig. 5b), dephosphorylated with CIP, and labelled at its 5 ' termini with T4 polynucleotide kinase and [Y-~~P]ATP as described in Sambrook et al. (1989). The labelled AvaII-StuI fragment of 124 bp (Fig. 5b) was isolated from an agarose gel after a second digestion with AvaII, denatured, and used as primer. Isolation of total RNA and poly(A) mRNA, Northern blotting, and primer extension analysis Total RNA was isolated from rat tissues by the acid guanidinium thiocyanate - phenol - chloroform extraction method of Chomczynski and Sacchi (1987). Poly(A) mRNA was purified from the total RNA using the Pharmacia mRNA purification kit according to the supplier's directions. For Northern blotting, either total RNA or poly(A) mRNA was separated by electrophoresis through 1.25% formaldehyde agarose gels as described in Sambrook et al. (1989) and transferred to Amersham Hybond N + nylon membrane by capillary blotting. After blotting, the membrane was fixed in 0.05 N NaOH for 5 min, rinsed in 2 x SSPE, and prehybridized in a mixture of 50% formamide, 5 x SSPE, 5 x Denhardt's solution, 0.5% SDS, and 20 pg/mL of sonicated and denatured salmon sperm DNA for 1.5 h at 42°C (Sambrook et al. 1989). Hybridization was carried out by adding the labelled DNA probe (2 x lo6 cpm/mL; specific activity, 5 x 10' cpm/pg) to the prehybridization solution and incubating at the same temperature for 24 h. After washing twice (10 min each) in 2 x SSPE - 0.1 % SDS at room temperature and once in 1 x SSPE - 0.1% SDS at 60°C for 15 min, the membrane was exposed to X-ray film using intensifying screens for 20 h or longer. For mapping of the 5 ' termini of RNA, primer extension analysis was performed as described in Sambrook et al. (1989) using an AvaII-StuI restriction fragment (124 bp, Fig. 5b) labelled only at the StuI 5 ' terminus as the primer. The cDNA extended by murine reverse transcriptase was separated in an 8% acrylamide - 7.5 M urea gel, and extended products were visualized by exposing the gel to X-ray film using intensifying screens for 2 days or longer. Immunocytology Rat spermatocytes from 30-day-old Sprague-Dawley rats were
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incubated with anti-SC antibody, and visualized with NBT and BCIP as described by Heyting et al. (1988). Results Zmmunoscreening of Xg TI1 rat testis cDNA expression library with anti-SC antibody Rabbit R16 serum gave a positive immune reaction with surface-spread rat SCs at a dilution of 1: 1000, and with 30-, 33-, and 65-kDa bands on an immunoblot of a crude preparation of rat SCs separated on 10% SDS-PAGE (Fig. 1). The R16 serum was used to screen a rat testis Xgtl 1 cDNA expression library for genes encoding proteins recognized by the R16 antibodies. The Xgtll library contained 1.7 x lo6 independent phage. About 5 x 10' recombinant phage were screened and three immunoreactive phage (1x1, 1x2, and 2x2) were identified and purified.
FIG. 1. An &munoblot analysis of a crude preparation of rat SCs separated on 10% SDS-PAGE and stained with Coomassie blue (D). (A) Proteins of spermatocyte nuclei transferred to nitrocellulose membrane and stained with Coomassie blue prior to the antibody incubation. (B) Blot after incubation in Q2X2 serum (the SC-positive serum from a rabbit injected with the fusion protein produced in lysogen 2x2 from a positive phage of the cDNA library). The Q2X2 serum recognizes a 65-kDa peptide. (C) Blot after incubation in SC-negative rabbit serum. Molecular size markers are the following: myosin, 200 kDa; E. coli P-galactosidase, 116 kDa; rabbit muscle phosphorylase b, 97 kDa; BSA, 66 kDa; hen egg white ovalbumin, 45 kDa; bovine carbonic anhydrase, 30 kDa; The arrow marks the 65-kDa band recognized by anti-Q2X2 antibody.
surface spread according to standard procedures (Counce and Meyer 1973; Dresser and Moses 1980), with modifications (Moens and Pearlman 1989). The immunostaining procedures were described previously (Moens et a[. 1987; Moens and Earnshaw 1989). Secondary antibodies were conjugated with either 5 nm colloidal gold (Sigma) for electron microscopy. or with FITC or peroxidase (Daymar Laboratories, Inc.) for light microscopy. Control samples were incubated in preimmune serum or unrelated antibodies. Immunoblotting
For immunoblotting, proteins from SCs or spermatocyte nuclei of rat were separated according to Laemmli (1970) on SDS-PAGE. After electrophoresis, the gel was frozen at - 20°C for 1 h or more. thawed at 4°C for 2 h and at room temperature for another 2 h, and renatured by washing six times in renaturing buffer (50 mM Tris-HC1,20% glycerol, pH 7.4) for 15 min (Heyting et al. 1987). The freezing and renaturing process enhanced the transfer efficiency and antigenicity of recovered protein. After renaturation, proteins were transferred to nitrocellulose membrane (Dunn 1986),
Zmmunological verification of the SC gene To determine whether the immunoreactive phage encoded SC proteins, fusion proteins were prepared from E. coli Y1089 lysogens that allowed the Xgtll protein product to accumulate to high level without cell lysis. When extracts of the lysogen of 2 x 2 were separated by SDS-PAGE, the /3-galactosidase (1 16 kDa) was replaced by a fusion protein of approximately 155 kD. Polyclonal sera were prepared against the fusion proteins produced in lysogens of 1x1, 1x2, and 2x2. These sera (Q1X 1, Q 1x2, and Q2X2) reacted with the respective fusion protein antigens but not with /3-galactosidase, indicating that the antibodies were directed against epitopes of the SC protein in the 0-galactosidase SC fusion proteins. Anti-/3-galactosidaseserum was prepared using /3-galactosidase purified from E. coli infected with wild-type Xgtl 1 to analyze /3-galactosidase on immunoblots. The sera, Q l X l and QlX2, which stained the rat SCs by immuno-FITC and immuno-gold labelling, failed to react with proteins on Western blots of SC proteins and were not analyzed further in this study. The Q2X2 serum reacted with a 65-kDa protein on the immunoblot of a preparation of rat SC proteins (Fig. 1). Quantitative analysis of the goldgrain distribution on micrographs of immuno-gold-labelled spread rat SCs (Fig. 2) indicated that the antigen was localized to the inner side of the lateral elements (Fig. 3). The 2 x 2 fusion protein, as well as the control P-galactosidase, was used to affinity purify anti-SC antibody as described in Materials and methods. The affinity-purified antibody stained the rat spread SCs by irnrnuno-FITC labelling (Fig. 4), while the affinity-purified control antibody did not. We therefore concluded that the phage 2 x 2 contained cDNA encoding an antigenically reactive portion of the 65-kDa SC protein and the gene was called SC65.' Cloning and characterization of the SC cDNA Mapping and sequencing experiments (Fig. 5a) suggested that the phage 2x2, containing a 1.O-kbp EcoRI DNA insert, did not contain the entire cDNA of SC65. T o obtain the complete SC65 cDNA, we used a 317-bp PstI fragment (317 bp, Fig. 5a) of the 2 x 2 insert as a hybridization probe to screen a second rat testis cDNA library in the vector XZAPII (Stratagene). One hybridizing phage clone, ZAPII-1, was found by restriction mapping to contain DNA overlapping the phage 2 x 2 insert. This clone contained a n '~ccessionnumber for SC65 cDNA is X65454.
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FIG. 2. Ultrastructural localization of the antigen recognized by polyclonal Q2X2 antibody. The 5-nm gold grains are located predominantly along the inner side of the lateral elements. There are very few grains on the lateral elements or to either side of the SC. The positions of the grains correspond to those reported for monoclonal anti-rat SC antibody III15B8 and differs from that of Mab II52F10 which localizes to the lateral elements (Moens et al. 1987). The preimmune serum produces a low level of background grains on and off the SCs. Bar, 0.2 pm.
insert of 2.4 kbp with an internal EcoRI site, giving 1.O- and 1.4-kbp EcoRI fragments (Fig. 5b). The EcoRI cDNA inserts from 2x2 and ZAPII-1 and restriction fragments derived from these inserts were subcloned into the pEMBL18 plasmid vector (Dente et al. 1983). For DNA sequence analysis, six overlapping plasmid clones covering the sense strand and seven overlapping plasmid clones covering the antisense strand were produced (Fig. 5c). The cDNA was completely sequenced as described in Materials and methods. From the sequence analysis and the subsequent Northern blotting experiment, we determined that the 1.4-kbp cDNA insert of the clone ZAPII-1 (Fig. 5b) contained the SC65 cDNA sequence. The SC65 cDNA sequence of 1408 bp is presented in Fig. 6. A long open reading frame of 1293 bp was found. The first methionine (ATG) codon, presumed to be the translation initiation site, was located 20 bp from the 5' end of the cDNA. The open reading frame was terminated with a TAA codon at position 1294. The derived amino acid sequence of the SC65 protein identified a relatively highly charged protein with 29.23% charged amino acids including 9.28% glutamic acid, 7.42% arginine, 6.26% aspartic acid, 3.25% histidine, and 3.02% lysine. The predicted SC65 protein was 431 amino acids in length with a size of 50 kDa. This was less than the size of 65 kDa estimated by SDSPAGE and Western blot analysis (Fig. 1). The 3 ' nontranslated region of the cDNA from ZAPII-1 was 95 bp long and lacked a putative poly(A) cleavageaddition site and an oligo(dA) tract. Primer extension analysis of rat testis poly(A) mRNA (data not shown) suggested a transcription start site about 150 bp upstream from the AvaII site (Fig. 5b). Northern analysis (see below) defined a mRNA of approximately 2000 nucleotides for SC65. This suggested a 3' untranslated region of approximately 550 nucleotides, including the poly(A) tail.
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FIG. 3. Histogram of accumulated gold grain distributions in 5-nm classes (total 198 grains) over a cross-sectional SC profile. All measurements were standardized to an SC with a central element width of 80 nm and lateral elements of 40 nm. The solid horizontal bars mark the lateral elements that are 40 nm from the SC centre at 0. Most gold grains are located between the lateral elements, indicating that the SC65 gene product occurs primarily in the pairing region of the SC.
A search of the complete GenBank and EMBL data bases with the SC65 amino acid sequence revealed no similarity with any known protein. Inspection of the amino acid sequence of the SC65 protein did, however, reveal a number of identifiable features. A strongly acidic region was present in the C-terminal domain of the protein. In addition, potential glycosylation sites (Thr-Xaa-Asn and Ser-Xaa-Asn) at positions 142,251,255,334, and 408, and a potential casein kinase I1 phosphorylation site at position 352 of the SC65 protein were found. The protein contained no obvious
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FIG. 4. Immuno-FITC labelling of whole-mount surface-spread rat SCs with the fraction of rabbit Q2X2 antibody affinity purified with 2x2 fusion protein. The affinity-purified antibody recognizes the SCs, thereby confirming that fusion protein 2 x 2 shares epitopes with SC antigens. The control buffer from filters blotted from clones that are negative for the antigen do not produce a visible FITC image. Bar, 4 pm.
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FIG. 5. (a, b) Restriction map of rat SC65 cDNA and structure of cloned inserts. The phage 2 x 2 was isolated from the Xgtll rat testis cDNA library using polyclonal serum (R16) against rat SCs. The clone ZAPII-1, isolated from the XZAPII rat testis cDNA library using the PstI restriction fragment from 2 x 2 as a probe (solid line under the map in a), overlaps the 2 x 2 insert. The solid line under the map in b indicates the DNA fragments used as hybridization probes for the Northern blotting. The solid arrow under the map in b is the DNA fragment used as primer for the primer extension analysis. (c) Fragments subcloned into the pEMBL plasmid vectors. Arrows at the ends indicate the orientation of inserts in the plasmids. The subcloned insert-carrying plasmids were used for DNA sequencing as described in the Materials and methods. The dotted line represents the sequence of the EcoRi fragment of ZAPII-1 which is not part of the SC65 cDNA. The symbols for the restriction enzymes are the following: A, AvaII; B, BgnI; E, EcoRI; P, PstI; S, StuI.
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FIG. 7. Northern blot analysis of total RNA and poly(A) mRNA from different rat tissues. The blot was probed with the EcoRI-BglII restriction fragment (Fig. 5b). The size markers are indicated to the left of the blot. A, 40 pg total RNA from the testis; B, 5 pg poly(A) mRNA from the testis; C, 5 pg poly(A) mRNA from the brain; D, 5 pg poly(A) mRNA from the heart; E, 5 pg poly(A) mRNA from the liver.
to Northern blot analysis, using probes prepared from the EcoRI-BglII restriction fragment of the ZAPII-1 DNA (Fig. 5b). Figure 7 shows an autoradiogram of a filter hybridized to the EcoRI-BgnI fragment. Two transcripts were detected with this probe, a major one of approximately 2000 nucleotides and a minor one of approximately 2400 nucleotides. Both were detected in rat testis, brain, and heart tissue at similar levels, but in liver tissue at a very low level. The origin of the 2400 nucleotide transcript was not clear although it could be a precursor of the major 2000 nucleotide transcript or a low abundance, alternatively spliced transcript.
Discussion The complex protein composition of the SC is apparent from the number of bands in SDS-PAGE from purified SCs and the variety of SC-positive antibodies that recognize those bands in immunoblots. Of the monoclonal anti-SC antibodies generated with purified SCs, 18 react with 30to 33-kDa polypeptides, two with 55- to 66-kDa polypeptides, 13 with 120- to 130-kDa bands, and 14 with 190-kDa polypeptides (Heyting et al. 1989). The SC-specific Mabs that have been tested in detail show that the 30- to 33-kDa antigens occur in the lateral elements, a 125-kDa antigen is in the pairing region between the lateral elements, and a 190-kDa antigen occurs mainly in the lateral elements
(Heyting et al. 1989). An anti-48-kDa rat SC antigen occurs in the pairing region and is SC specific (Smith and Benevente 1992). In addition to the SC-specific proteins, the SC also has components that cross-react with antibodies against a variety of proteins. The sera of preimmune mice, rabbits, and humans frequently contain antibodies that react with SCs (Dresser 1987). In the case of SC3 (a Mab that recognizes the central element of the SC) from an autoimmune mouse, there is a cross-reaction with somatic cell cytoplasm intermediate type filaments (Dresser 1987). The presence of ubiquitous proteins in association with the SCs was further demonstrated by the positive reaction of SCs with antibodies against topoisomerase I1 (Moens and Earnshaw 1989). To gain insight into the structural and functional aspects of the SC, it is important to know the primary structure of the protein components of the SC. In the case of the HOP1 gene of Saccharomyces cerevisiae, which is required for SC assembly and recombination, the gene encodes a protein with a zinc-finger motif indicative of a DNA-binding function (Hollingsworth et al. 1990). In the rat, the cDNAs for the 125- and 190-kDa SC proteins have been isolated and sequenced (C. Heyting, personal communications). Here we report experiments using a polyclonal antibody that recognizes a 65-kDa SC protein, as well as other SC proteins, to isolate a cDNA encoding a protein component of the rat SC. Antibodies against the fusion protein produced in Xgtl 1 as well as anti-SC antibodies affinity purified with the fusion protein react with rat SCs, and the antigenic determinant in both cases is localized to the edges of the central element or pairing region of the SC near the lateral elements or chromosome cores. Western blot analysis with these same antibodies identifies a 65-kDa SC protein. The 431 amino acid sequence derived from the SC65 cDNA sequence encodes a protein that shows no similarities to sequences present in available nucleic acid and protein data bases. The predicted size of this protein is 50 kDa, less than the 65 kDa determined by SDS-PAGE and immunoblot analysis. The reason for this discrepancy is not known, but other nuclear proteins have been reported to have anomalously high molecular mass on SDS-PAGE when compared with the size predicted from analysis of the sequence of the corresponding cloned genes (Huet and Sentenac 1987; Diffley and Stillman 1989; Henry et al. 1990). The anomalous mobility can be attributed either to the effects of protein conformation on mobility, and (or) to posttranslational modification. Glycosylation is one modification that can cause a protein to migrate slower than expected on SDS-PAGE. Although glycosylation of nuclear proteins is rare, this remains a possibility to explain the anomalous mobility. Five potential N-glycosylation sites occur at positions 142,251, 255, 334, and 408 of the SC65 protein. Another identifiable feature of the SC65 protein is a potential site (Ser 352) for phosphorylation by the NII class of cyclic nucleotide independent protein kinases, or casein kinase I1 (Caizergues-Ferrer et al. 1987). This potential modification would not explain the anomalous electrophoretic mobility, but might be important for protein function. The most recognizable domain of the SC65 protein is an acidic region in the C-terminal portion of the protein. Acidic (A-) regions have been reported to be present in a number of chromosomal proteins and their possible functions have
CHEN ET AL.
been reviewed by Earnshaw (1987). T h e acidic regions in SC65 are not as high in acidic amino acids as those discussed by Earnshaw (1987), but they represent regions containing significantly higher content of Glu (E) and Asp (D) than the bulk of the protein which is 15.54% D + E. A 10-amino acid region (319-328) contains 60% D E. A 32-amino acid region (353-384) contains 50% acidic residues. This region can b e subdivided from amino t o carboxyl into stretches of 10 amino acids 70% acidic, 10 amino acids only 20% acidic, a n d 12 amino acids 58% acidic. Northern blot analysis reveals that poly(A) m R N A homologous to SC65 c D N A accumulates at similar high levels i n rat testes, brain, a n d heart tissues, b u t is present only a t a very low level in liver. Although some S C proteins are meiosis specific, some are not a n d it is therefore not unexpected that m R N A encoding a n S C protein accumulates in testis as well as in other tissues. Experiments to investigate further aspects of the tissue specificity of the SC65 m R N A a n d protein are in progress. T h e role of the brain identifier sequence in the 3 ' untranslated region of SC65 m R N A is also not clear at this time. SC65 m R N A accumulates in tissues other than brain a n d the SC65 protein has been rigorously identified in rat germ cells undergoing meiosis. More cDNA sequences encoding rat S C proteins must be identified to address detailed questions about the structure, function, a n d regulation of expression of S C proteins, but the detailed characterization of o n e S C protein here is an important step in this process.
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+
Acknowledgements This research was supported b y Natural Sciences a n d Engineering Research Council o f Canada grants t o P.B.M. a n d R.E.P. W e thank Nora Tsao a n d Barbara Spyropoulos f o r their assistance. Caizergues-Ferrer, M., Belenguer, P., Lapeyre, B., Amalric, F., Wallace, M.O., and Olson, M.O.J. 1987. Phosphorylation of nucleolin by a nucleolar type NII protein kinase. Biochemistry, 26: 7876-7883. Chomczynski, P., and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate - phenol - chloroform extraction. Anal. Biochem. 162: 156-159. Counce, S.J., and Meyer, G.F. 1973. Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole mount electron microscopy. Chromosoma, 44: 231-253.
Dente, L., Cesareni, G., and Cortese, R. 1983. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 11: 1645-1655.
Diffley, J.F.X., and Stillman, B. 1989. Similarity between the transcriptional silencer binding proteins ABFl and RAP1. Science (Washington, D.C.), 246: 1034-1038. Dresser, M.E. 1987. The synaptonemal complex and meiosis: an immunocytochemical approach. In Meiosis. Edited by P.B. Moens. Academic Press, New York. pp. 245-274. Dresser, M.E., and Moses, M. J. 1980. Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus). IV. Light and electron microscopy of synapsis and nucleolar development by silver staining. Chromosoma, 76: 1-22. Dunn, S.D. 1986. Effects of the modification of transfer buffer composition and renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal. Biochem. 157: 144-153. Earnshaw, W.C. 1987. Anionic regions in nuclear proteins. J. Cell Biol. 105: 1479- 1482.
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Feinberg, A.P., and Vogelstein, B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. Hager, D.A., and Burgess, R.R. 1980. Elution of proteins from sodium dodecyl sulfate - polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal. Biochem. 109: 76-86. Hanahan, D., and Meselson, M. 1983. Plasmid screening at high colony density. Methods Enzymol. 100: 333-342. Heery, D.M., Gannon, F., and Powell, R. 1990. A simple method for subcloning DNA fragments from gel slice. Trends Genet. 6: 137.
Henry, Y.A.L., Chambers, A., Tsang, J.S.H., Kingsman, A.J., and Kingsman, S.M. 1990. Characterization of the DNA binding domain of the yeast RAP1 protein. Nucleic Acids Res. 18: 2617-2623.
Heyting, C., Dietrich, A.J.J., Koperdraad, F., and Redeker, E.J.W. 1983. Do synaptonemal complexes contain actin and myosin? Chromosomes Today, 8: 316. Heyting, C., Moens, P.B., van Raamsdonk, W., Dietrich, A.J.J., Vink, A.C.G., and Redeker, E.J.W. 1987. Identification of two major components of the lateral elements of synaptonemal complexes of the rat. Eur. J. Cell Biol. 43: 148-154. Heyting, C., Dettmers, R. J., Dietrich, A. J. J., Redeker, E. J.W., and Vink, A.C.G. 1988. Two major components of synaptonemal complexes are specific for meiotic prophase nuclei. Chromosoma, 96: 325-332. Heyting, C., Dietrich, A. J. J., Moens, P.B., Dettmers, R. J., Offenberg, H.H., Redeker, E.J.W., and Vink, A.C.G. 1989. Synaptonemal complex proteins. Genome, 31: 81-87. Hollingsworth, N.M., Goetsch, L., and Byers, B. 1990. The HOP1 gene encodes a meiosis-specific component of yeast chromosomes. Cell, 61: 73-84. Huet, J., and Sentenac, A. 1987. TUF, the yeast DNA-binding factor specific for UAS,,, upstream activating sequences: identification of the protein and its DNA-binding domain. Proc. Natl. Acad. Sci. U.S.A. 84: 3648-3652. Huynh, T.V., Young, R.A., and Davis, R.W. 1985. Constructing and screening cDNA library in lambda gtlO and lambda g t l l . In DNA cloning: a practical approach. Vol. 1. Edited by D.M. Glover. IRL Press Limited, Oxford, U.K. pp. 49-78. John, B. 1990. Meiosis. Edited by P.W. Barlow, D. Bray, P.B. Green, and J.W. Slack. Cambridge University Press, Cambridge, U.K. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Milner, R.J., Bloom, F.E., Lai, C., Lerner, R.A., and Sutcliffe, J.G. 1984. Brain-specific genes have identifier sequences in their introns. Proc. Natl. Acad. Sci. U.S.A. 81: 713-717. Mitchell, P.J., and Tjian, R. 1989. Transcriptional regulation in mammalian cells by sequence-specific DNA-binding proteins. Science (Washington, D.C.), 245: 371-378. Moens, P.B. 1987. Meiosis. Edited by P.B. Moens. Academic Press. New York. Moens, P.B., and Earnshaw, W.C. 1989. Anti-topoisomerase I1 recognizes meiotic chromosome cores. Chromosoma, 98: 317-322.
Moens, P.B., and Pearlman, R.E. 1989. Satellite DNA I in chromatin loops of rat pachytene chromosomes and in spermatids. Chromosoma, 98: 287-294. Moens, P.B., Heyting, C., Dietrich, A.J. J., van Raamsdonk, W., and Chen, Q. 1987. Synaptonemal complex antigen localization and conservation. J. Cell Biol. 105: 93-103. Offenberg, H.H., Dietrich, A.J.J., and Heyting, C. 1991. Tissue distribution of two major components of synaptonemal complexes of the rat. Chromosoma, 101: 83-91.
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Pearlman, R.E., Tsao, N., and Moens, P.B. 1992. Synaptonemal complexes from DNAse-treated rat pachytene chromosomes contain (GT), and LINE/SINE sequences. Genetics, 130: 865-872.
Pearson, W.R., and Lipman, D. J. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. U.S.A. 85:
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Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Press, New York. Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467.
Smith, A., and Benevente, R. 1992. Identification of a structural protein component of rat synaptonemal complexes. Exp. Cell Res. 198: 291-297. Snyder, M., Elledge, S., Sweetser, D., Young, R.A., and Davis, R.W. 1987. Xgtll: gene isolation with antibody probes and other applications. Methods Enzymol. 154: 107-128. Spyropoulos, B., and Moens, P.B. 1984. The synaptonemal complex: does it have contractile proteins? Can. J. Genet. Cytol. 26: 776-781.
von Wettstein, D., Rasmussen, S.W., and Holm, P.B. 1984. The synaptonemal complex in genetic segregation. Annu. Rev. Genet. 18: 331-431.
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Department of Biology, York University. 4700 Keele Street, Downsview, Ont., Canada M3 J IP3 Received February 7 , 1992 R. E., and MOENS,P. B. 1992. Isolation and characterization of a cDNA encoding a CHEN, Q., PEARLMAN, synaptonemal complex protein. Biochem. Cell Biol. 70: 1030- 1038. A gene encoding a 65-kilodalton antigen of the rat synaptonemal complex, SC65, has been cloned by screening rat testis Agtl 1 and AZAPII cDNA expression libraries using polyclonal antibodies against rat synaptonemal complex proteins. The longest open reading frame, initiating at an ATG codon in the cDNA, encodes a protein of 431 amino acids, with a relative molecular mass of 50 000. Immunological analysis locates the SC65 gene product on the synaptonemal complex between the pairing faces of the parallel aligned cores of homologous chromosomes in spermatocytes. Of the rat tissues examined, the SC65 gene is transcribed in testis, brain, and heart at similar levels, and in the liver at a much lower level. The DNA sequence extending about 80 base pairs downstream of the translation termination codon has 93% similarity to the identifier sequence present in the rat genome in 1 x 10' - 1.5 x 10' copies and in cDNA clones of precursors of brain-specific mRNAs. The amino acid sequence encoded by the SC65 gene contains an acidic region in the C-terminal domain of the protein, potential glycosylation sites, and at least one possible phosphorylation site. The protein shows no overall similarity to proteins of known function, nor is there similarity to protein sequences present in GenBank or EMBL data bases. Key words: meiosis, synaptonemal complex, antibody, rat testis cDNA library, molecular cloning. CHEN, Q., PEARLMAN, R. E., et MOENS,P. B. 1992. Isolation and characterization of a cDNA encoding a synaptonemal complex protein. Biochem. Cell Biol. 70 : 1030-1038. Le gene SC65, codant un antigkne de 65 kilodaltons du complexe synaptonernal de rat, a ete clone en criblant les banques d'expression de cDNA (Xgtll et AZAPII) de testicule de rat a l'aide d'anticorps polyclonaux contre les proteines du complexe synaptonemal de rat. Le plus long cadre de lecture ouvert debutant par un codon ATG dans le cDNA code une proteine de 431 acides amines, de masse molCculaire relative de 50 000. Une analyse immunologique a permis de localiser le produit du gkne SC65 dans le complexe synaptonernal entre les lames apparikes des chromosomes homologues alignes parallklement dans les spermatocytes. Plusieurs tissus de rat ont ete analysb; le gkne SC65 est transcrit ti un taux semblable dans les testicules, le cerveau et le coeur, et il est transcrit a un niveau beaucoup plus faible dans le foie. La sequence de DNA s'etendant environ 80 paires de bases au-dela du codon de terminaison de la traduction est homologue a 93% avec la sequence identificatrice dont environ 1 x 10' - 1.5 x 10' copies se trouvent dans le genome de rat et prksente dans les clones de cDNA de precurseurs de RNA messagers sptcifiques du cerveau. La sequence d'acides aminks codte par le gkne SC65 comporte une region acidique dans le domaine C-terminal de la protkine, des sites potentiels de glycosylation et au moins un site possible de phosphorylation. La proteine n'a pas d'homologie globale ni avec des proteines de fonction connue, ni avec les sequences proteiques se trouvant dans les bases de donnees GenBank ou EMBL. Mots cl&s : meiose, complexe synaptonemal, anticorps, banque de cDNA de testicule de rat, clonage moltculaire.
Introduction The synaptonemal complex is at the interface between paired chromosomes at meiotic prophase in germ cells of most sexually reproducing species. The SC is of interest because its presence is correlated with important meioticspecific functions such as synapsis, recombination, and segregation (reviewed in von Wettstein et al. 1984; Moens 1987; John 1990). Identification of SC protein components is based on recognition by autoimmune antibodies (Dresser 1987), generation of mono- and poly-clonal anti-SC antibodies (Heyting et al. 1987), screening with antibodies against known proteins (Heyting et al. 1983; Spyropoulos and Moens 1984; Moens and Earnshaw 1989), and molecular genetic techniques (Hollingsworth et a/. 1990). Some of the ABBREVIATIONS: EMBL, European Molecular Biology Laboratory; SC, synaptonemal complex; kDa, kilodaltons; IPTG, isopropylthiogalactoside; BSA, bovine serum albumin; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; NBT, nitroblue tetrazolium; PMSF, phenylmethylsulphonyl fluoride; SDS-PAGE, sodium dodecyl sulfate - polyacrylarnide gel electrophoresis; PBS, phosphate-buffered saline; bp, base pair(s); CIP, calf intestinal alkaline phosphatase; FITC, fluorescein isothiocyanate; Mabs, monoclonal antibodies; DAB, diaminobenzidine. Prinled in Canada / Imprime au Canada
protein components of the SCs are specific to the SCs and the meiotic nucleus (Heyting et al. 1988; Offenberg et a/. 1991; Smith and Benevente 1992), whereas others, such as those detected by anti-topoisomerase I1 (Moens and Earnshaw 1989), or preimmune sera generally have epitopes in common with more ubiquitous proteins (Dresser 1987). In this article, we report that the SC-positive serum of a rabbit recognizes a 65-kDa polypeptide in Western blots, and with electron microscopy of immunogold-labelled, surface-spread SCs, we show that the antigen is located in the pairing zone of the SC. From clones of rat testis cDNA libraries containing sequences encoding the 65-kDa polypeptide, we determine the nucleotide sequence of the gene and demonstrate a major 2000 nucleotide transcript in testis, heart, and brain tissues. Materials and methods Zmmunoscreening of the rat testis cDNA Agtll library with antiSC antibody A Agtl 1 library of rat testis cDNA (Clontech Laboratories) was screened on nitrocellulose filters with rabbit serum immunopositive for SCs essentially as described by Snyder et al. (1987). Half a million phage recombinants were plated on Escherichia coli Y 1090 at a density of 25 x lo3phage/90-mm plate. IPTG-treated filters
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CHEN ET AL.
were incubated on plates for 8-10 h. Screenings were carried out in duplicate. All washes were done in TBST (0.05% Tween-20 in Tris-buffered saline, i.e., 50 mM Tris-HC1 (pH 8.0) and 150 mM NaCl). Filters were blocked in 1% BSA in TBST at 4OC for 30 min before incubation with antibody. Antibodies that recognized E. coli proteins were removed from the screening serum by adding E. coli lysate according to protocols supplied by Bio-Rad. Filters were incubated in the screening serum at 4OC overnight and the subsequent incubations were performed at 4°C to increase the signals. Bound antibodies were detected with an alkaline phosphatase conjugated goat anti-rabbit antibody (Bio-Rad). Reactive phage replicas were visualized by development with BCIP and NBT as described by the supplier (Promega). A total of three screenings at decreasing plaque densities were carried out until pure phage was obtained. Preparation of lysogens, fusion protein, and anti-fusion protein antibody, and affinity purification of anti-fusion protein antibody Generation of Xgtl 1 recombinant lysogens in E. coli Y1089 and preparation of fusion protein extracts from the Xgtl 1 lysogens were carried out by the procedures of Huynh et al. (1985). Lysogens were collected and purified by their temperature sensitivity (Snyder et al. 1987). Proteins from extracts of lysogens were prepared and stored in 75% ammonium sulphate as a slurry at 4OC. For preparation of fusion proteins, protein extract from 500 pL of the (NH4),S04 precipitate was centrifuged at 10 000 x g for 20 min at 4°C and resuspended in 500 pL TEP buffer (100 mM Tris-HC1 (pH 7.4), 10 mM EDTA, 1 mM PMSF diluted from a 100-mM stock in ethanol at a concentration of approximately 4 pg/pL. About 100-150 pg protein extract/well was separated in a7.5% SDS-PAGEgel(160 x 180 x 1.5 mm, 12wells). Thegel was stained by soaking it in cold 0.25 M KC1 (Hager et al. 1980) for 20 min with gentle agitation. The fusion proteins, which were identified in previous experiments using standard Coomassie blue G-250 staining, were excised from the gel, pooled, rinsed in distilled water, lyophilized for 24 h, and then ground into a fine powder which could be stored at - 80°C for long time periods. For production of anti-fusion protein antibody, the powder of fusion protein from the gels (100-200 pg of protein) was resuspended in 0.5 mL PBS and injected into a 2.5-kg New Zealand female rabbit subcutaneously. Two subsequent injections of 100 pg were given at 4 and 8 weeks. Serum was collected prior to injections and 7-10 days after each injection, and then analyzed by using immuno-DAB, immuno-gold staining, and immunoblotting techniques as described below. Proteins produced from phage were used to affinity purify antibodies as described by Snyder et al. (1987). Proteins isolated from E. coli Y1089 infected by wild-type Xgtll were used in control incubations. Screening of the rat testis cDNA AZAPII library with radioactively labelled DNA probe Hybridization of XZAPII plaques with a radioactively labelled DNA probe was performed essentially according to the suplier's protocol (Stratagene). Escherichia coli XLl-Blue cells infected with the XZAPII phage were plated, amplified in situ, and processed as described by Sambrook et al. (1989). Prehybridization and hybridization under stringent conditions used a Blotto-Hyb mix (5 x SSPE, 1% SDS, 0.5% Carnation nonfat dry skim milk powder). Hybridizing plaques were purified through at least three rounds of screening. Subcloning and DNA sequencing Restriction endonucleases and DNA modifying enzymes were purchased from several suppliers (Pharmacia, Gibco/BRL, Boehringer Mannheim, Amersham, New England Biolabs, and U.S. Biochemical Corp). Manipulations of DNA were according to Sambrook et al. (1989). The EcoRI cDNA inserts isolated from the lambda libraries were subcloned in both orientations into the
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pEMBL18 vector (Dente et al. 1983) using E. coli JMlOl as host (Hanahan et al. 1983). For DNA sequencing, single-stranded pEMBL18 DNA was prepared by superinfection of the cells containing the plasmids with f l phage IR1 (Dente et al. 1983) or VCSM13 (Stratagene). The single-stranded DNA was used as a template for nucleotide sequencing by the dideoxy chain termination method (Sanger et al. 1977) using [(Y-~~sI~ATP, the 17-bp universal primer, and the Sequenase Version 2.0 kit as recommended by the supplier (U.S. Biochemical Corp.). Both strands were sequenced for almost the entire cDNA. For short regions where both strands were not sequenced, at least two independent subclones from both phages 2 x 2 and ZAPII-1 were sequenced. Computer-assisted analysis of sequences was performed with the programs Analyseq and Seqaid. Nucleic acid and protein sequences were searched against all sequences in the GenBank and EMBL data bases using a FASTA search according to Pearson and Lipman (1988). Labelling of DNA For preparation of hybridization probes, DNA restriction fragments were isolated from agarose gels as described by Heery et al. (1990) and labelled with [(Y-"P]~ATP(3000 Ci/mM, 1 Ci = 37 GBq; Amersham) using the random priming protocol of Feinberg and Vogelstein (1983). For preparation of DNA size markers, wild-type lambda DNA was digested with the appropriate restriction enzymes. Fragments were labelled at their 3 ' termini using the Klenow fragment of DNA polymerase I of E. coli as described in Sambrook et al. (1989). For preparation of primer for the primer extension analysis, the EcoRI cDNA fragment of 1.4 kbp was purified from an agarose gel (Heery et al. 1990), digested with StuI (Fig. 5b), dephosphorylated with CIP, and labelled at its 5 ' termini with T4 polynucleotide kinase and [Y-~~P]ATP as described in Sambrook et al. (1989). The labelled AvaII-StuI fragment of 124 bp (Fig. 5b) was isolated from an agarose gel after a second digestion with AvaII, denatured, and used as primer. Isolation of total RNA and poly(A) mRNA, Northern blotting, and primer extension analysis Total RNA was isolated from rat tissues by the acid guanidinium thiocyanate - phenol - chloroform extraction method of Chomczynski and Sacchi (1987). Poly(A) mRNA was purified from the total RNA using the Pharmacia mRNA purification kit according to the supplier's directions. For Northern blotting, either total RNA or poly(A) mRNA was separated by electrophoresis through 1.25% formaldehyde agarose gels as described in Sambrook et al. (1989) and transferred to Amersham Hybond N + nylon membrane by capillary blotting. After blotting, the membrane was fixed in 0.05 N NaOH for 5 min, rinsed in 2 x SSPE, and prehybridized in a mixture of 50% formamide, 5 x SSPE, 5 x Denhardt's solution, 0.5% SDS, and 20 pg/mL of sonicated and denatured salmon sperm DNA for 1.5 h at 42°C (Sambrook et al. 1989). Hybridization was carried out by adding the labelled DNA probe (2 x lo6 cpm/mL; specific activity, 5 x 10' cpm/pg) to the prehybridization solution and incubating at the same temperature for 24 h. After washing twice (10 min each) in 2 x SSPE - 0.1 % SDS at room temperature and once in 1 x SSPE - 0.1% SDS at 60°C for 15 min, the membrane was exposed to X-ray film using intensifying screens for 20 h or longer. For mapping of the 5 ' termini of RNA, primer extension analysis was performed as described in Sambrook et al. (1989) using an AvaII-StuI restriction fragment (124 bp, Fig. 5b) labelled only at the StuI 5 ' terminus as the primer. The cDNA extended by murine reverse transcriptase was separated in an 8% acrylamide - 7.5 M urea gel, and extended products were visualized by exposing the gel to X-ray film using intensifying screens for 2 days or longer. Immunocytology Rat spermatocytes from 30-day-old Sprague-Dawley rats were
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incubated with anti-SC antibody, and visualized with NBT and BCIP as described by Heyting et al. (1988). Results Zmmunoscreening of Xg TI1 rat testis cDNA expression library with anti-SC antibody Rabbit R16 serum gave a positive immune reaction with surface-spread rat SCs at a dilution of 1: 1000, and with 30-, 33-, and 65-kDa bands on an immunoblot of a crude preparation of rat SCs separated on 10% SDS-PAGE (Fig. 1). The R16 serum was used to screen a rat testis Xgtl 1 cDNA expression library for genes encoding proteins recognized by the R16 antibodies. The Xgtll library contained 1.7 x lo6 independent phage. About 5 x 10' recombinant phage were screened and three immunoreactive phage (1x1, 1x2, and 2x2) were identified and purified.
FIG. 1. An &munoblot analysis of a crude preparation of rat SCs separated on 10% SDS-PAGE and stained with Coomassie blue (D). (A) Proteins of spermatocyte nuclei transferred to nitrocellulose membrane and stained with Coomassie blue prior to the antibody incubation. (B) Blot after incubation in Q2X2 serum (the SC-positive serum from a rabbit injected with the fusion protein produced in lysogen 2x2 from a positive phage of the cDNA library). The Q2X2 serum recognizes a 65-kDa peptide. (C) Blot after incubation in SC-negative rabbit serum. Molecular size markers are the following: myosin, 200 kDa; E. coli P-galactosidase, 116 kDa; rabbit muscle phosphorylase b, 97 kDa; BSA, 66 kDa; hen egg white ovalbumin, 45 kDa; bovine carbonic anhydrase, 30 kDa; The arrow marks the 65-kDa band recognized by anti-Q2X2 antibody.
surface spread according to standard procedures (Counce and Meyer 1973; Dresser and Moses 1980), with modifications (Moens and Pearlman 1989). The immunostaining procedures were described previously (Moens et a[. 1987; Moens and Earnshaw 1989). Secondary antibodies were conjugated with either 5 nm colloidal gold (Sigma) for electron microscopy. or with FITC or peroxidase (Daymar Laboratories, Inc.) for light microscopy. Control samples were incubated in preimmune serum or unrelated antibodies. Immunoblotting
For immunoblotting, proteins from SCs or spermatocyte nuclei of rat were separated according to Laemmli (1970) on SDS-PAGE. After electrophoresis, the gel was frozen at - 20°C for 1 h or more. thawed at 4°C for 2 h and at room temperature for another 2 h, and renatured by washing six times in renaturing buffer (50 mM Tris-HC1,20% glycerol, pH 7.4) for 15 min (Heyting et al. 1987). The freezing and renaturing process enhanced the transfer efficiency and antigenicity of recovered protein. After renaturation, proteins were transferred to nitrocellulose membrane (Dunn 1986),
Zmmunological verification of the SC gene To determine whether the immunoreactive phage encoded SC proteins, fusion proteins were prepared from E. coli Y1089 lysogens that allowed the Xgtll protein product to accumulate to high level without cell lysis. When extracts of the lysogen of 2 x 2 were separated by SDS-PAGE, the /3-galactosidase (1 16 kDa) was replaced by a fusion protein of approximately 155 kD. Polyclonal sera were prepared against the fusion proteins produced in lysogens of 1x1, 1x2, and 2x2. These sera (Q1X 1, Q 1x2, and Q2X2) reacted with the respective fusion protein antigens but not with /3-galactosidase, indicating that the antibodies were directed against epitopes of the SC protein in the 0-galactosidase SC fusion proteins. Anti-/3-galactosidaseserum was prepared using /3-galactosidase purified from E. coli infected with wild-type Xgtl 1 to analyze /3-galactosidase on immunoblots. The sera, Q l X l and QlX2, which stained the rat SCs by immuno-FITC and immuno-gold labelling, failed to react with proteins on Western blots of SC proteins and were not analyzed further in this study. The Q2X2 serum reacted with a 65-kDa protein on the immunoblot of a preparation of rat SC proteins (Fig. 1). Quantitative analysis of the goldgrain distribution on micrographs of immuno-gold-labelled spread rat SCs (Fig. 2) indicated that the antigen was localized to the inner side of the lateral elements (Fig. 3). The 2 x 2 fusion protein, as well as the control P-galactosidase, was used to affinity purify anti-SC antibody as described in Materials and methods. The affinity-purified antibody stained the rat spread SCs by irnrnuno-FITC labelling (Fig. 4), while the affinity-purified control antibody did not. We therefore concluded that the phage 2 x 2 contained cDNA encoding an antigenically reactive portion of the 65-kDa SC protein and the gene was called SC65.' Cloning and characterization of the SC cDNA Mapping and sequencing experiments (Fig. 5a) suggested that the phage 2x2, containing a 1.O-kbp EcoRI DNA insert, did not contain the entire cDNA of SC65. T o obtain the complete SC65 cDNA, we used a 317-bp PstI fragment (317 bp, Fig. 5a) of the 2 x 2 insert as a hybridization probe to screen a second rat testis cDNA library in the vector XZAPII (Stratagene). One hybridizing phage clone, ZAPII-1, was found by restriction mapping to contain DNA overlapping the phage 2 x 2 insert. This clone contained a n '~ccessionnumber for SC65 cDNA is X65454.
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FIG. 2. Ultrastructural localization of the antigen recognized by polyclonal Q2X2 antibody. The 5-nm gold grains are located predominantly along the inner side of the lateral elements. There are very few grains on the lateral elements or to either side of the SC. The positions of the grains correspond to those reported for monoclonal anti-rat SC antibody III15B8 and differs from that of Mab II52F10 which localizes to the lateral elements (Moens et al. 1987). The preimmune serum produces a low level of background grains on and off the SCs. Bar, 0.2 pm.
insert of 2.4 kbp with an internal EcoRI site, giving 1.O- and 1.4-kbp EcoRI fragments (Fig. 5b). The EcoRI cDNA inserts from 2x2 and ZAPII-1 and restriction fragments derived from these inserts were subcloned into the pEMBL18 plasmid vector (Dente et al. 1983). For DNA sequence analysis, six overlapping plasmid clones covering the sense strand and seven overlapping plasmid clones covering the antisense strand were produced (Fig. 5c). The cDNA was completely sequenced as described in Materials and methods. From the sequence analysis and the subsequent Northern blotting experiment, we determined that the 1.4-kbp cDNA insert of the clone ZAPII-1 (Fig. 5b) contained the SC65 cDNA sequence. The SC65 cDNA sequence of 1408 bp is presented in Fig. 6. A long open reading frame of 1293 bp was found. The first methionine (ATG) codon, presumed to be the translation initiation site, was located 20 bp from the 5' end of the cDNA. The open reading frame was terminated with a TAA codon at position 1294. The derived amino acid sequence of the SC65 protein identified a relatively highly charged protein with 29.23% charged amino acids including 9.28% glutamic acid, 7.42% arginine, 6.26% aspartic acid, 3.25% histidine, and 3.02% lysine. The predicted SC65 protein was 431 amino acids in length with a size of 50 kDa. This was less than the size of 65 kDa estimated by SDSPAGE and Western blot analysis (Fig. 1). The 3 ' nontranslated region of the cDNA from ZAPII-1 was 95 bp long and lacked a putative poly(A) cleavageaddition site and an oligo(dA) tract. Primer extension analysis of rat testis poly(A) mRNA (data not shown) suggested a transcription start site about 150 bp upstream from the AvaII site (Fig. 5b). Northern analysis (see below) defined a mRNA of approximately 2000 nucleotides for SC65. This suggested a 3' untranslated region of approximately 550 nucleotides, including the poly(A) tail.
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FIG. 3. Histogram of accumulated gold grain distributions in 5-nm classes (total 198 grains) over a cross-sectional SC profile. All measurements were standardized to an SC with a central element width of 80 nm and lateral elements of 40 nm. The solid horizontal bars mark the lateral elements that are 40 nm from the SC centre at 0. Most gold grains are located between the lateral elements, indicating that the SC65 gene product occurs primarily in the pairing region of the SC.
A search of the complete GenBank and EMBL data bases with the SC65 amino acid sequence revealed no similarity with any known protein. Inspection of the amino acid sequence of the SC65 protein did, however, reveal a number of identifiable features. A strongly acidic region was present in the C-terminal domain of the protein. In addition, potential glycosylation sites (Thr-Xaa-Asn and Ser-Xaa-Asn) at positions 142,251,255,334, and 408, and a potential casein kinase I1 phosphorylation site at position 352 of the SC65 protein were found. The protein contained no obvious
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BIOCHEM. CELL BIOL. VOL. 70, 1992
FIG. 4. Immuno-FITC labelling of whole-mount surface-spread rat SCs with the fraction of rabbit Q2X2 antibody affinity purified with 2x2 fusion protein. The affinity-purified antibody recognizes the SCs, thereby confirming that fusion protein 2 x 2 shares epitopes with SC antigens. The control buffer from filters blotted from clones that are negative for the antigen do not produce a visible FITC image. Bar, 4 pm.
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P
BP P
FIG. 5. (a, b) Restriction map of rat SC65 cDNA and structure of cloned inserts. The phage 2 x 2 was isolated from the Xgtll rat testis cDNA library using polyclonal serum (R16) against rat SCs. The clone ZAPII-1, isolated from the XZAPII rat testis cDNA library using the PstI restriction fragment from 2 x 2 as a probe (solid line under the map in a), overlaps the 2 x 2 insert. The solid line under the map in b indicates the DNA fragments used as hybridization probes for the Northern blotting. The solid arrow under the map in b is the DNA fragment used as primer for the primer extension analysis. (c) Fragments subcloned into the pEMBL plasmid vectors. Arrows at the ends indicate the orientation of inserts in the plasmids. The subcloned insert-carrying plasmids were used for DNA sequencing as described in the Materials and methods. The dotted line represents the sequence of the EcoRi fragment of ZAPII-1 which is not part of the SC65 cDNA. The symbols for the restriction enzymes are the following: A, AvaII; B, BgnI; E, EcoRI; P, PstI; S, StuI.
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FIG. 7. Northern blot analysis of total RNA and poly(A) mRNA from different rat tissues. The blot was probed with the EcoRI-BglII restriction fragment (Fig. 5b). The size markers are indicated to the left of the blot. A, 40 pg total RNA from the testis; B, 5 pg poly(A) mRNA from the testis; C, 5 pg poly(A) mRNA from the brain; D, 5 pg poly(A) mRNA from the heart; E, 5 pg poly(A) mRNA from the liver.
to Northern blot analysis, using probes prepared from the EcoRI-BglII restriction fragment of the ZAPII-1 DNA (Fig. 5b). Figure 7 shows an autoradiogram of a filter hybridized to the EcoRI-BgnI fragment. Two transcripts were detected with this probe, a major one of approximately 2000 nucleotides and a minor one of approximately 2400 nucleotides. Both were detected in rat testis, brain, and heart tissue at similar levels, but in liver tissue at a very low level. The origin of the 2400 nucleotide transcript was not clear although it could be a precursor of the major 2000 nucleotide transcript or a low abundance, alternatively spliced transcript.
Discussion The complex protein composition of the SC is apparent from the number of bands in SDS-PAGE from purified SCs and the variety of SC-positive antibodies that recognize those bands in immunoblots. Of the monoclonal anti-SC antibodies generated with purified SCs, 18 react with 30to 33-kDa polypeptides, two with 55- to 66-kDa polypeptides, 13 with 120- to 130-kDa bands, and 14 with 190-kDa polypeptides (Heyting et al. 1989). The SC-specific Mabs that have been tested in detail show that the 30- to 33-kDa antigens occur in the lateral elements, a 125-kDa antigen is in the pairing region between the lateral elements, and a 190-kDa antigen occurs mainly in the lateral elements
(Heyting et al. 1989). An anti-48-kDa rat SC antigen occurs in the pairing region and is SC specific (Smith and Benevente 1992). In addition to the SC-specific proteins, the SC also has components that cross-react with antibodies against a variety of proteins. The sera of preimmune mice, rabbits, and humans frequently contain antibodies that react with SCs (Dresser 1987). In the case of SC3 (a Mab that recognizes the central element of the SC) from an autoimmune mouse, there is a cross-reaction with somatic cell cytoplasm intermediate type filaments (Dresser 1987). The presence of ubiquitous proteins in association with the SCs was further demonstrated by the positive reaction of SCs with antibodies against topoisomerase I1 (Moens and Earnshaw 1989). To gain insight into the structural and functional aspects of the SC, it is important to know the primary structure of the protein components of the SC. In the case of the HOP1 gene of Saccharomyces cerevisiae, which is required for SC assembly and recombination, the gene encodes a protein with a zinc-finger motif indicative of a DNA-binding function (Hollingsworth et al. 1990). In the rat, the cDNAs for the 125- and 190-kDa SC proteins have been isolated and sequenced (C. Heyting, personal communications). Here we report experiments using a polyclonal antibody that recognizes a 65-kDa SC protein, as well as other SC proteins, to isolate a cDNA encoding a protein component of the rat SC. Antibodies against the fusion protein produced in Xgtl 1 as well as anti-SC antibodies affinity purified with the fusion protein react with rat SCs, and the antigenic determinant in both cases is localized to the edges of the central element or pairing region of the SC near the lateral elements or chromosome cores. Western blot analysis with these same antibodies identifies a 65-kDa SC protein. The 431 amino acid sequence derived from the SC65 cDNA sequence encodes a protein that shows no similarities to sequences present in available nucleic acid and protein data bases. The predicted size of this protein is 50 kDa, less than the 65 kDa determined by SDS-PAGE and immunoblot analysis. The reason for this discrepancy is not known, but other nuclear proteins have been reported to have anomalously high molecular mass on SDS-PAGE when compared with the size predicted from analysis of the sequence of the corresponding cloned genes (Huet and Sentenac 1987; Diffley and Stillman 1989; Henry et al. 1990). The anomalous mobility can be attributed either to the effects of protein conformation on mobility, and (or) to posttranslational modification. Glycosylation is one modification that can cause a protein to migrate slower than expected on SDS-PAGE. Although glycosylation of nuclear proteins is rare, this remains a possibility to explain the anomalous mobility. Five potential N-glycosylation sites occur at positions 142,251, 255, 334, and 408 of the SC65 protein. Another identifiable feature of the SC65 protein is a potential site (Ser 352) for phosphorylation by the NII class of cyclic nucleotide independent protein kinases, or casein kinase I1 (Caizergues-Ferrer et al. 1987). This potential modification would not explain the anomalous electrophoretic mobility, but might be important for protein function. The most recognizable domain of the SC65 protein is an acidic region in the C-terminal portion of the protein. Acidic (A-) regions have been reported to be present in a number of chromosomal proteins and their possible functions have
CHEN ET AL.
been reviewed by Earnshaw (1987). T h e acidic regions in SC65 are not as high in acidic amino acids as those discussed by Earnshaw (1987), but they represent regions containing significantly higher content of Glu (E) and Asp (D) than the bulk of the protein which is 15.54% D + E. A 10-amino acid region (319-328) contains 60% D E. A 32-amino acid region (353-384) contains 50% acidic residues. This region can b e subdivided from amino t o carboxyl into stretches of 10 amino acids 70% acidic, 10 amino acids only 20% acidic, a n d 12 amino acids 58% acidic. Northern blot analysis reveals that poly(A) m R N A homologous to SC65 c D N A accumulates at similar high levels i n rat testes, brain, a n d heart tissues, b u t is present only a t a very low level in liver. Although some S C proteins are meiosis specific, some are not a n d it is therefore not unexpected that m R N A encoding a n S C protein accumulates in testis as well as in other tissues. Experiments to investigate further aspects of the tissue specificity of the SC65 m R N A a n d protein are in progress. T h e role of the brain identifier sequence in the 3 ' untranslated region of SC65 m R N A is also not clear at this time. SC65 m R N A accumulates in tissues other than brain a n d the SC65 protein has been rigorously identified in rat germ cells undergoing meiosis. More cDNA sequences encoding rat S C proteins must be identified to address detailed questions about the structure, function, a n d regulation of expression of S C proteins, but the detailed characterization of o n e S C protein here is an important step in this process.
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