Báo cáo Y học: Proliferating cell nuclear antigen from a basidiomycete, Coprinus cinereus Alternative truncation and expression at meiosis pptx

Proliferating cell nuclear antigen from a basidiomycete, Coprinus cinereus Alternative truncation and expression at meiosis Fumika Hamada, Satoshi Namekawa, Nobuyuki Kasai, Takayuki Nara, Seisuke Kimura, Fumio Sugawara and Kengo Sakaguchi Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Japan The primary purpose o f the present study was to investi- gate whether DNA replication at meiotic prophase also requires replication factors, especially proliferating cell nuclear antigen (PCNA). We cloned PCNA cDNAs (CoPCNA) from a cDNA library made from basidia of the basidiomycete, Coprinus cinereus. Interestingly, although CoPCNA is a s ingle-copy gene in the g enome, two dier- ent PCNA cDNA species were isolated using degenerate primers and a meiotic cDNA library, and were design ated as CoPCNA-a and CoPCNA-b. CoPCNA-b was made by truncating at speci®c sites in CoPCNA-a mRNA, 5¢-AA- GAAGGAGAAG-3¢ and 5¢-GAAGAGGAAGAA-3¢. Both of these sequ ences were present in exon IV in the genomic sequence, and interestingly the former was the same as the i nverse sequence o f t he latter. C oPCNA-a was 107 a mino acids larger t han human PCNA, and so the 107 amino-acid sequence was inserted in a loop, the so-called D 2 E 2 loop, in human PCNA. Northern blotting analysis indicated that CoPCNA was expressed not only at pre- meiotic S but also at the meiotic prophase stages such as leptotene and early zygotene, just before and when kary- ogamy occurs and the homologous chromosomes pair. Western blotting analysis using anti-(CoPCNA-a)Igre- vealed that at least two CoPCNA mRNAs before a nd after truncation w ere translated at t he meiotic prophase as CoPCNA-a and CoPCNA-b. Keywords: Coprinus cinereus; Coprinus PCNA (CoPCNA); meiotic prophase; truncating of CoPCNA mRNA; two CoPCNA species. Proliferating cell nuclear antigen (PCNA) has important roles in DNA replication and repair including nucleotide excision repair, postreplicational mismatch repair, base excision repair, apoptosis and cytosine methylation [1]. PCNA is known t o interact with DNA polymerases d and e, p21 and many other factors [2±6]. In these interactions, PCNA appears t o be t he major protein involved in determining the binding coun terpart, e.g. homologous regions of FEN-1 and p21 compete for binding to the same site on PCNA [7]. PCNA could therefore be a k ey protein in the mechanism of coordination of DNA replication and repair, and one of the key proteins controlling mitotic cell cycle progression. On the other hand, DNA synthesis has been reported to occur not only at S phase of the meiotic cell cycle, but also at meiotic prophase [8±10]. In this context, we have been interested in the role(s) of PCNA in the meiotic cell cycle, especially at meiotic prophase in which homol- ogous chromosomes pair and recombine. The purpose of this study was to investigate the roles of PCNA in meiosis. In meiosis, chromosomes co ndense f rom the dispersed state typical of interphase during early meiotic prophase, forming l ong t hin threads i n l eptotene, and eac h acq uire s a proteinaceous axial core to which the two sister chromatids are attached. Then, homologous chromosomes become aligned during zygotene, forming the synaptinemal complex and, at pachytene, nonsister chromatids of the completely paired chromosomes recombine forming t he chiasmata that become visible during diplotene. Two cell divisions follow, reductional and e quational, resulting in four gametes. According to biochemical studies of lily meiosis [8±10], a small amount of DNA replicates at zygotene, and repair synthesis of DNA occurs at pachytene. Both DNA synthe- ses occur on nonsense DNA regions on parts of t he chromosomal DNA, and the regions on the chromosomal DNA are different from each o ther; very h igh Cot se quences at zygotene and middle repetitious sequences at pachytene [8±10]. There are therefore two possible meiotic events that may require PCNA, homologous chromosome pairing at zygotene and their recombination a t pachytene. We have investigated meiosis-related protein factors using meiotic cells in the basidiomyc ete, Coprin us ci nereus [11±20]. This organism is especially well suited for studies of meiosis, because its meiotic cell cycle is long and n aturally synchronous [16,19,21±23]. Each fruiting cap is extremely rich in meiotic cells at the same stage (10 6 ±10 7 cells), and the nuclear numbers are easily observable. In the meiotic cycle, the dikaryonic cells are at premeiotic stages f rom t he S phase to leptotene, and for 6 h the beginning of the karyogamy stage (at which point the two nu clei are fused) is the zygotene stage a t which the homologous chromosomes Correspondence to K. Sakaguchi, Department of Applied Biological Science, Science University of Tokyo, 2641 Yamazaki , Noda-shi, Chiba-ken 278, Japan. Fax: + 81 471 23 9767, Tel.: + 81 471 24 1501 (ext. 3409), E-mail, kengo@rs.noda.sut.ac.jp Abbreviations: PCNA, proliferating cell nuclear ant igen; CoPCNA, PCNA cDNA. (Received 31 J uly 2 001, revised 22 October 2001, ac cepted 25 October 2001) Eur. J. Biochem. 269, 164±174 (2002) Ó FEBS 2002 pair. The chromosomes then recombine at pachytene. It is therefore possible to precisely characterize meiosis-related PCNA in relation to each of the meiotic events. In the present paper, we found Coprinus PCNA proteins with special properties from characterization of a Coprinus PCNA gene in the cells at meiotic prophase. Interestingly, unlike m ammals and yeast, the cells produced two species of Coprinus PCNA protein, although the gene is present in a single copy in the genome. Consistent with the p redicted role in meiosis, the Coprinus PCNA mRNAs were expressed at limited meiotic prophase stages. MATERIALS AND METHODS Culture of C. cinereus and collection of the fruiting bodies The basidiomycete C. cinereus (American Type Culture collection no. 56838) was used in this study. The culture methods used here were nearly identical to those described previously [21]. Culture dishes (6 cm in height and 9 cm in diameter) containing sterile horse manure were inoculated with a dikaryotic stock culture of C. cinereus on day 0. These cultures were incubated from d ay 0 to day 7 at 37 °C in total darkness and from day 7 onwards at 25 °C under a light cycle of 16 h light and 8 h dark to allow photoinduction of fruiting body formation. The light cycle started at 05:00(K + 1). Karyogamy was de®ned as the time at which 5% of all basidia had fused nuclei, and interestingly began at 04:00(K + 0), 1 h before the light was turned on. Fruiting bodies were undergoing karyog- amy from 04:00(K + 0) to 09:00(K + 5) (i.e. in late leptotene to early zygotene), they were in pachytene from 10:00(K + 6) to 11:00(K + 7), and then were undergoing division from 12:00(K + 8) to 14:00(K + 10). Under these conditions, meiotic cells all at the same stage of prophase could be readily obtained. T he fruiting caps were harvested, quickly frozen in liquid N 2 , and then stored at )80 °C until used. CDNA and cloning of Coprinus proliferating cell nuclear antigen (CoPCNA) Culture of Escherichia coli and phage, e xtraction of plasmid DNA, electrophoresis of DNA and RNA, were carried out according to the methods described previously [24] unless otherwise speci®ed. To isolate homologous PCNA cDNA, two primers were used corresponding to amino-acid motifs conserved in human PCNA, Schizosaccharomyces pombe PCNA, Droso- phila melan ogaster PCNA and Arabidopsis thaliana PCNA: sense primer (5¢-CCGGCATCAACCTGCARDSNATG GA-3¢) and antisense primer (5¢-GATCGATGT CCATCAGCTTCAYNTCRWARTC-3¢)(N A, C, G or T; R  AorG;Y CorT;W A o r T). The primers were used in PCR reactions with cDNA generated f rom poly(A)+ RNA isolated from fruiting bodies of C. cinereus as template. PCR was performed with 1 lgofthecDNAas a t emplate i n a volume of 50 lL in the presence of 2 l M of each of the two primers in a buffer containing 250 m M dNTPs (Amersham P harmacia Biotech), 10 m M Tris/HCl, pH 8.3, 50 m M KCl, 1 . 5 m M MgCl 2 , 0.05% Nonidet P-40, and 2 U of ExTaq thermostable DNA polymerase (Takara). Cycling conditions were: 94 °Cfor2min;94°Cfor30s; 50 °Cfor1min;72°C for 1 m in; 40 cycles, followed by a 10-min extension at 72 °C. The major 200-bp PCR band was subcloned into the pGEM-T vector (Promega) and sequenced. To determine the 5¢ and 3¢ termini of the gene, 5¢ and 3¢ RACE PCR was performed. The DDBJ/EMBL/GenBank accession number of the nucleotide sequence reported in this paper is AB05 6703 for the proliferating cell nuclear antigen (CoPCNA). Genomic DNA isolation and Southern hybridization analysis Genomic DNA was isolated from Coprinus myc elium tissue anddigestedwithrestrictionenzymessuchasXhoI, NdeI, EcoRI and Bam HI [25]. The DNA fragments were fraction- ated on 1% agarose gels, transferred on Hybond-N + membranes [24]. The hybridization procedure was per- formed [26]. The probe (amino-acid residues 131 to 223; see below) was l abeled with 32 P using a Multiprime DNA label kit ( Amersham Pharmacia Biotech). After prehybridization, hybridization was carried out at 42 °C f or 16 h, followed b y washing with 2 ´ NaCl/P i /EDTA, 1% SDS at 65 °Cfor 15 min, 1 ´ NaCl/P i /EDTA, 1% SDS a t 65 °C for 15 min, and 0.2 ´ NaCl/P i /EDTA, 1% SDS at 65 °C for 15 min. Search for three-dimensional structure of human and Coprinus PCNA To simulate the three-dimensional structures of the PCNAs, modeling o f the human PCNA protein was compared w ith the CoPCNA-a trimer based on the data for the human PCNA protein obtained by computer analysis. Computer analysis was performed with INSIGHT II binding site analysis (Molecular Simulations Inc., San Diego, CA, USA, 1999) [27±29]. RNA isolation and northern hybridization analysis Total RNA was prepared from caps of C. cinereus at meiotic prophase using Trizol (Gibco-BRL) according to the manufacturer's protocol. RNA s amples were fractionated on 1 .2% agarose- formaldehyde gels [30]. Total RNA (20 lg) from the caps at each meiotic stage and from the somatic tissue were loaded into each lane. The agarose gel was staine d with ethidium bromide and blotted overnight in 20 ´ NaCl/P i / EDTA onto Hybond-N + membranes (Amersham Phar- macia Biotech). The two probes (amino acids 131 t o 223 for both CoPCNA-a and CoPCNA-b,and227to268for CoPCNA-a alone; see below) were labeled with 32 P, then hybridized as described f or Southern analysis. Preparation of riboprobes and in situ hybridization Riboprobes for in situ hybridization were labeled with digoxigenin-11-rUTP using a DIG RNA Labeling Kit (Boehringer Mannheim) according to the manufacturer's protocol. The riboprobes used were a mino acids 131 to 223 for both CoPCNA-a and CoPCNA-b, and residues 227 to 268 for CoPCNA-a alone; see below. The riboprobes were subjected to mild alkaline hydrolysis b y h eating at 60 °Cfor 53 min in 0.2 M carbonate/bicarbonate buffer and used at a Ó FEBS 2002 Alternative truncating of Coprinus PCNA mRNA (Eur. J. Biochem. 269) 165 concentration of 2 mgámL )1 the fruiting c aps were ®xed overnight at 4 °C with a mixture of 4% (w/v) paraformal- dehyde and 0.25% (v/v) glutaraldehyde in 50 m M sodium phosphate buffer ( pH 7.2). The ®xed tissues were dehydrated in a series o f xylene and ethanol and embedded in paraf®n. Embedded tissues were sectioned at a thickness of 5 lm, and placed on microscope slides precoated with poly L -lysine. Sections were deparaf®nized with xylene a nd rehydrated through a graded ethanol series. They were subsequently pretreated with 10 mg ámL )1 of proteinase K in 100 m M Tris/HCl, pH 7.5, a nd 50 m M EDTA at 37 °C for 30 min, dehydrated in a graded ethanol series, and d ried under vacuum for 2 h. Hybridization and detection of hybridized riboprobes were performed [31]. Overexpression and puri®cation of CoPCNA protein CoPCNA coding region was ampli®ed using the 5¢ sense primer 5¢-GGAATTCCATATGCTTGAAGCCAAACT CGCAG-3¢ and 3¢ antisense primer 5¢-CGAGCTCGGG TCGTCACCAATCTTAGGTGCG-3¢, a nd cloned into pET21a (Novagen). The plasmid constructs were intro- duced into BL21(DE3)pLysS (Novagen). Transformed E. coli were grown at 37 °Cin2´ yeast/ tryptone medium with 1% glucose and 50 mg ámL )1 amp- icillin. Cells were grown to a D 600 of 0.8. Recombinant protein synthesis was induced by addition of 1 m M IPTG, and after 3 h the cells we re harvested by centrifugation. The cell pellets were resuspended in liquid N 2 andstoredat )80 °C. The cell pellets were resuspended in lysis buffer (20 m M Tris/HCl, pH 6.5, 10% glycerol, 500 m M NaCl, 5m M imidazole), containing 5 m M 2-mercaptoethanol and the protease inhibitors phenylmethanesulfonyl ¯uoride (1 m M ), leupeptin (1 l M ) and pepstatin A (1 l M ). Cells were lysed by a ddition of 1 mgámL )1 of lysozyme and stirred on ice for 30 min, then sonicated and Triton X-100 was added to 0.1%. Insoluble material was removed by centrifugation at 15 000 r.p.m. for 15 min. Proteins were loaded onto a 1- mL HiTrap Chelat ing column (Amersham Pharmacia Biotech). The column w as washed successively with buffer A (20 m M Tris/HCl, pH 6.5, 10% glycerol, 500 m M NaCl, 0.02% NP-40) containing 5 m M imidazole. The bound proteins were eluted with buffer A containing 400 m M imidazole. Fractions of proteins were identi®ed by SDS/PAGE, pooled and dialyzed. The dialysate w as loaded onto a Mono Q HR5/5 column (Amersham Pharmacia Biotech) equilibrate d with buffer B (50 m M Tris/HCl pH 6.5, 10% glycerol, 2 m M EDTA, 5 m M 2-mercaptoeth- anol). After washing, fractions were collected with 40 mL of alineargradientof0±0.7 M NaCl in buffer B. The protein concentrations were determined using a Bio-Rad protein assay kit with c-globulin as the standard. Immunological analysis and immuno¯uorescence microscopy A polyclonal antibody against t he CoPCNA -a protein was raised in rabbit u sing the puri®ed proteins. Western blotting analysis was carried out [32]. Anti-(rabbit IgG) Ig conju- gated with alkaline phosphatase (Promega) was used as a s econdary antibody with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates of alkaline phosphatase. Immunostaining o f Coprinus fruiting caps was carried o ut [33]. The paraf®n sections of the fruiting caps described above i n the in situ hybridization section were u sed. The cells were incubated for 1 h with the antibody against each of the CoPCNA p roteins. The antibodies against each of the CoPCNA proteins were diluted at 1 : 100 before use. The cells were the n treated for 1 h with FITC- conjugated anti- (rabbit IgG) Ig (Sigma Chemical Co.) conjugated with Alexa ¯uoro 568, which was diluted 1 : 1000 as a secondary antibody. T hen, the cells were also stained with a solution of 20 lgáL )1 4¢,6-diamido-2-phenylindole dihydrochloride n-hydrate (DAPI) for 5 min The specimens were examined under a ¯uorescence microscope (Olympus BH-2). Immunoscreening Using previously described rabbit anti-CoPCNA Ig, immunoscreening was carried out with kZAPII cDNA library from the c aps at e ach meiotic stage of C. cine reus. From the i solated plaques, plasmid DNA was p repared by the in vivo excision protocol using the ExAssist/SOLR system (Stratagene). RESULTS AND DISCUSSION Isolation and characterization of PCNA homologous cDNA in Coprinus meiocytes As described in t he introduction, proliferating cell nuclear antigen (PCNA) might play some role(s) in the meiotic cell cycle, especially at meiotic prophase. We report here that i n a basidiomycete, C. cinereus , PCNA message was speci®- cally expressed in meiotic prophase stages at which homol- ogous chrom osomes p air (zygotene) and recombine (pachytene). In lily microsporocytes small amounts of DNA synthesis were required for synaptinemal complex formation (zygotene), and for recombination b etween homologous DNAs (pachytene) [8]. The former synthesis was replication-type, and the latter was repair-type. These observations indicate that accessory proteins of DNA synthesis such as PCNA may have important roles in meiosis-speci®c biochemical e vents. We ®rst tried to i dentify the PCNA homolog in C. cine reus, and then investigated the meiotic stage-speci®c transcription. Unexpectedly, we found the production of two species of PCNA protein in C. cinereus . To isolate the Coprinus PCNA homolog, t wo d egenerate PCR primers (see Materials and methods) were used for PCR with cDNA produced from poly(A)+ RNA isolated from fruiting bodies of C. cine reus at meiotic prophase as the template. As the c DNA clones obtained were all incomplete in length, we attempted to isolate full-length cDNA by 5¢ and 3¢ RACE. Fortunately, we were able to obtain the 5¢ and 3¢ ends. The DDBJ/EMBL/GenBank accession number of the nucleotide sequence reported in this paper i s AB056703 for the proliferating cell nuclear antigen (CoPCNA). A polyclonal antibody against CoPCNA-a protein was raised in a rabbit using the puri®ed protein. W estern blotting analysis revealed that the anti-CoPCNA Ig recog- nized the CoPCNA-a protein (48 kDa) and one more protein with M r of 42 kDa (Fig. 1). To investigate the 42-kDa protein in more detail, we screened a cDNA clone 166 F. Hamada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 for the 42-kDa protein using an anti-(CoPCNA)-a Ig, and succeeded in cloning it. Interestingly, the cDNA clone for the 42-kDa protein was a truncated CoPCNA-a cDNA, a nd it was tentatively designated as CoPCNA-b. CoPCNA-a and Co PCNA-b were completely sequenced, and were 1104- and 975-bp in length, respectively. The molecular masses of CoPCNA-a and CoPCNA-b wereshowntobe48-and 42-kDa on SDS/PAGE, respectively, and were slightly larger than those e xpected from the amino-acid sequences. As human PCNA is a highly acidic protein with a pI of 4.5 [34] and a calculat ed molecular mass of 28.8 kDa, and that ran as an approximately 36-kDa protein on SDS/PAGE, CoPCNA-a and CoPCNA-b might behave in a manner similar to human PCNA. The Coprinus gen omic DNA isolated was truncated with restriction e nzymes including Xho I,NdeI,EcoRI and BamHI (Fig. 2). Southern hybridization analysis o f a sequence i n common between CoPCNA -a and CoPCNA-b (amino-acid residues 131 to 2 23 in Fig. 3A) revealed that as each of the t runcated products indicated a single band, each was single-copy gene (Fig. 2 ). These results str ongly suggested that the two CoPCNA species were transcribed from one CoPCNA gene, a nd produced by splicing alternat ively. These Coprinus PCNA cDNA sequences encoded prod- ucts of 368 and 325 amino-acid residues, respectively (Fig. 3A). We d esignated them as CoPCNA-a (368 resi- dues) and CoPCNA-b (325 residues), respectively. These molecules were 107 and 64 amino acids larger than human PCNA, respectively (Fig. 3B). The amino-acid sequen ces were very similar between CoPCNA-a and CoPCNA-b (Fig. 3A). The CoPCNA-b polypeptide lacked the amino- acid residues from 227 to 269 of CoPCNA-a (Fig. 3). Database searches with the BLASTX program [35] revealed the gene of CoPCNA-a, which showed the highest degree of homology to PCNAs from other organisms, was to have 34.2% identity to the human PCNA protein, 32.7% to Arabidopsis PCNA protein, and 41.5% to S. pombe PCNA protein (Fig. 3A). We searched for homology a mong CoPCNA and PCNAs from other organisms, human, Arabidopsis or S. pombe using the BLASTX program. The human, Arabidopsis and S. pombe PCNA proteins lacked a polypeptide of amino-acid residues from residues 195±305 CoPCNA-a (Fig 3A,B) and the a mino-acid sequence f rom 184 to 195 of them did n ot have homology with C oPCNA. This site (184±195) was in a loop called the D 2 E 2 loop in human PCNA (Fig. 3B). Interestingly, the polypeptide of amino-acid residues 184 to 305 o f CoPCNA-a contained three nuclear localization signal (NLS) peptides, PEKKKIK, KKRKKK and PAKKAKT (boxes in Fig. 3A), which w ere not present in the o ther eukaryotic species, and the polypeptide of CoPCNA-b also had a n NLS peptide (PAKKAKT). The eukaryotic PCNAs newly synthesized in the cytoplasm m ust move into the nucleus with the other PCNA-binding proteins that have the s ignal. However, CoPCNA-a alone (and perhaps C oPCNA-b alone) may be able to move into the nucleus. Characterization of PCNA homologous gene in Coprinus genome, and of alternative truncation sites of CoPCNA-a mRNA To characterize the splicing process, we also cloned the genomic Coprinus PCNA sequence. We used 1104 bp of full-length cDNA for CoPCNA- a, and succeeded in 131 kDa 96 M 44 CoPCNA-α CoPCNA-β 35 17.8 Fig. 1. Immunoblotting analysis of CoPCNA in the fruiting bodies during meiosis. Aliquots of 30 lg of the proteins extracted f rom caps of C. cinereus at meiotic prophase were subjected to Western blotting analysis using anti-(CoPCNA-a) Ig. Numbers indicate the position and size of the protein standard. The a rrowheads represent th e p osi- tions of CoPCNA-a and CoPCNA-b. XhoI NdeI EcoRI BamHI Fig. 2. Southern analysis of CoPCNA. Total Coprinus genomic DNA was digested with XhoI, NdeI, EcoRI, or BamHI and hybridized with the common sequence used as a 32 P-labeled cDNA probe for amino- acid residues 131±223 for both CoPCNA-a and CoPCNA-b shown in Fig. 2A. Ó FEBS 2002 Alternative truncating of Coprinus PCNA mRNA (Eur. J. Biochem. 269) 167 obtaining the full-length genomic clone. The restriction enzyme map and the relationship between the gene and cDNA are summarized in Fig. 4A. Careful comparisons of the deduced amino-acid sequenc- es with those from other organisms suggested that the g ene consists of six exons and ®ve introns, and also c ontains a 1104-bp ORF (Fig. 4A). The nucleotide sequence data reported in t his p aper appear in the DDBJ/EMBL/ GenBank nucleotide sequence database with the accession number AB056703. As shown in Fig. 4A, the complete PCNA gene sequence wascomparedwiththetwocDNAs,CoPCNA-a and CoPCNA-b. The alternative splicing site in the gene was i n exon IV for CoPCNA-a, suggesting that for CoPCNA-b, the CoPCNA-a mRNA truncation d id not occur by splicing, but by another mechanism, because the truncation occurred i n the exon that has no i ntron. Therefore, we called it Ôalternative truncationÕ in the later part of this report. We searched for and found the s pecial sequence sites in the CoPCNA gene for truncation. The mRNA sequences Fig. 3. (A) Alignment of the predicted amino-acid sequences of CoPCNA-a and CoPCNA-b w ith those of S. pombe, Ar abidop sis and human, and (B) truncation derivatives of C oPCNA- a and CoPCNA-b p rote ins. In (A), asterisks i ndicat e amino -acid id en tity com mon to all ®ve sequ ences, and dots indicate amino-acid identity between ®ve of the sequences. The open boxes indicate nuclear localization signals. ( B) Proteins were designated as CoPCNA-a (368 residues) and CoPCNA-b (325 residues), respectively. The polypeptide of CoPCNA-b lacked amino-acid residues from 227 to 269 in CoP CN A -a. T he site (184±195) in human PCNA was in a loop called the D 2 E 2 loop. CoPCNA-a and C oPCNA-b had insertion s of 107 and 64 aminoacids,inthisD 2 E 2 loop, respectively. 168 F. Hamada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 around the truncating sites in the CoPCNA gene are summarized in Fig. 4 B,C. CoPCNA-b mRNA appeared to be produced from the CoPCNA-a mRNA by truncation a t speci®c sites; i.e. 5¢-AAGAAGGAGAAG-3¢ and 5¢-GA AGAGGAAGAA-3¢ (Fig. 4B). Both of the sequences at which truncation occurred were present in exon IV in the CoPCNA-a genomic sequence, and interestingly the f ormer sequence was the i nverse of the l atter (Fig. 4C). Both of the 5¢ and 3¢ truncation s ites were also special repeat sequences (Fig. 4B,C), s uggesting the existence of a special truncation system that is slightly different from the normal splicing process. Isolation of the recombinant CoPCNA-a homologue protein, and modeling of the three-dimensional structure of CoPCNA-a by computer simulation To characterize the CoPCNAs in more detail, the recom- binant CoPCNA proteins were overexpressed and puri®ed. Extracts from the E. coli cells contained a six-histidine Fig. 4. Genomic structure and sequence sites. (A) G enomic structur e o f CoPCNA . T he thin lines in the cDNA c lone represent ¯anking and i ntro n sequences, and the thick lines at both ends of the cDNA clone rep resen t 5¢ and 3¢UTR sequences. E xons in the cDNA c lone are indicated by open boxes. B oth of the sequences in w hich truncation occurred were present in Exon IV in the CoPCNA-a geno mic gene. (B) T h e special sequence sites in the CoPCNA gene for truncation. The m RNA sequences around the truncation sites in the CoPCNA ge ne are s ummarized. CoPCNA-b mRNA appeared to be prod uc ed from CoPCNA-a mRNA by truncation at special sites. (C) The special sequence sites were 5¢-AAGAAGGAGAAG-3¢ and 5¢-GAAGAGGAAGAA-3¢. Interestingly, the former sequence is the inverse of the latter. Ó FEBS 2002 Alternative truncating of Coprinus PCNA mRNA (Eur. J. Biochem. 269) 169 C-terminal-tagged CoPCNA-a fusion protein. The CoP- CNA-a protein was p uri®ed to near homogeneity by chromatography on His bind resin, and Mono Q HR5/5 column (see Materials a nd methods). Figure 5A,B show the results of S DS/PAGE and Sephacryl S 300 gel ®ltration chromatography of the Mono Q HR5/5 fraction. The molecular mass of the recombinant CoPCNA-a protein monomer was 48 kDa (Fig. 5A), and in vivo, the protein was p resent as a 150-kDa trimer (Fig. 5B), similar to human PCNA. To simulate the three-dimensional structures of t he PCNAs, modeling of the human PCNA protein was compared to the CoPCNA-a trimer model based on the data for the human PCNA protein obtained by computer analysis. F igure 6 shows a 3D computer generated possible structure for the CoPCNA-a trimer, superimposed with the structure of the human PCNA trimer. Interestingly, both proteins could be completely superimposed, except the human sequence from Ser186 to Glu191. The CoPCNA-a monomer had an inserted polypeptide of 107 amino-acid residues between Ser186 to Glu191 in the human PCNA monomer. These 107 amino-acid residues (a DEK-rich peptide site) must protrude beyond the monomer core protein, although we could not simulate this because the other PCNAs have no such DEK-rich peptide site. The 107 amino-acid residues correspond to the amino-acid residues inserted into the polypeptide sequence of human PCNA depicted in Fig. 3. Therefore, amino acids 227±270 in CoPCNA-b must also be forced out of the core protein. The protruding peptide site is behind the site binding to p21, FEN-1 and DNA polymerase d suggesting that it has no important role(s) in the PCNA structure [36,37]. Timing of events of Coprinus meiosis, and Northern hybridization and Western blotting analyses of CoPCNAs We investigated the meiotic stage-speci®c transcription as described below, after the precise timing of events of experimentally controlled Coprinus meiosis. To test whether the CoPCNA gene is expressed at the time of zygotene, pachytene or both, total RNA was extracted from the basidia taken from the s ynchronous cultures every 1 h after induction of meiosis, and hybridization with the common to both C oPCNA-a and CoPCNA-b as a probe (residues 131 to 223 in Fig. 3A) was performed (Fig. 7A). Both forms of the transcripts were v ery strongly expressed in the mycelium tissues (mitotic cells; see Fig. 7A), and at premeiotic S phase (PreS) in which the genomic DNA replicates (Fig. 7A). The transcripts began to accu- mulate markedly at the stages before karyogamy (before K + 1), and b ecame most abundant from 0 to 1 h after the lights were turned on (K + 0 to K + 1). Then, the signal rapidly faded until 2 h after late leptotene, and became strong again at middle zygotene (K + 4). The signal completely disappeared at late zygotene (K + 5), and then, the t ranscript a ppeared again at a moderate level at middle pachytene (K + 6) (Fig. 7A). The transcripts were also moderately detected at diplotene and diakines is (K + 8). In meiosis, the transcripts were also strongly expressed in the basidia at middle zygotene, and again at middle pachytene (Fig. 7 A). In a ll cases in w hich the s ignal was positive, one band was observed (Fig. 7A) because the mRNA signals for CoPCNA-a and CoPCNA-b appeared not to be a ble to b e separated from e ach other because o f their size. When a 1000 BA 100 100kDa 150 10 90807060 63 5040 retention volume (ml) MW(kDa) 75k 50k 37k Fig. 5. SDS/PAGE of puri®ed CoPCNA-a protein (A) and determination of i ts molecular mass by g el ®ltration chromatography (B). (A) SDS/PAGE analysis of pu ri®ed C oP CNA-a after M ono Q column chromatography. T he pu ri® ed C oPCNA-a was fractioned by 12.5% SDS polyacrylamide gel electrophoresis. The gel was stained with CBB. Standard marker proteins are indicated by arrows to the l eft of the panel. (B) After Mono Q column chromatograp hy, the puri®ed Co PCNA-a was loaded onto t he S 300 column . The arrow indicates the po sition a t w hich CoP C NA-a was found. The molecular mass of the protein in the peak w as 150 kDa. 170 F. Hamada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 cDNA probe that could recognize only CoPCNA-a was used (am ino-acid residues 227 to 268 in Fig. 3A) the mRNA signal was also the same size as th at for CoPCNA-a (data not shown). As the majority of the basidia at K + 0 and K + 1 w ere in PreS or leptote ne to early zygotene, not pachytene, during this period as determined from ¯uores- cent microscopic observation of the monokaryonic nuclei and from electron microscopic observation of the presence of synaptinemal co mplex, it was concluded that t he CoPCNAs were discontinuously expressed at P reS at which the genomic DNA replicates, at leptotene at which the axial core in each of the chromosomes is formed, at zygotene at which t he homologous chromosomes pair, and at p achytene at which the zygotene-paired chromosomes recombine. Diplotene and diakinesis are the stages at which the pachytene-recombined chromosomes d isjunct. We raised a polyclonal antibody against the recombinant CoPCNA-a protein in rabbit. As shown in F ig. 7B, there were two signals on immunoblots, coinciding with the molecularmassvaluesofCoPCNA-a (48 k Da) and CoP- CNA-b (42 kDa). The antibody recognized both of the CoPCNA protein species. The two alternatively truncated species of CoPCNA mRNAs were translated to the same extent. Figure 7B shows the results of Western blotting analysis using the anti-(CoPCNA-a)Iginthemeioticcell cycle. Strong signals were always observed in the basidia through leptotene to M2. The miner bands in SDS/PAGE of CoPCNA-b might be partial degradation products of the CoPCNA-b protein (Fig. 7B). As compared with the results of northern hybridization analysis, t he proteins seemed to have longer half-lives, suggesting that CoPCNA-a and CoPCNA-b are present at all meiotic prophases including the stages at which the homologous chromosome s repli- cate, condense, pair, recombine, and disjunct. Each of CoPCNA-a and CoPCNA-b also strongly signaled in the mycelium tissues (mitotic cells), indicating that both f orms of CoPCNA is also translated in mitosis (Fig. 7B). Subcellular localization of CoPCNA during meiotic cell division The results of northern h ybridization and Western blotting clearly indicated that CoPCNAs were expressed and trans- lated at the meiotic prophase stages. However, as the fruiting caps used as the meiotic tissues contain some somatic cells, it was possible that all or some of the CoPCNAs were present in t he somatic cells. Therefore, t o con®rm that all of the CoPCNAs came from the meiotic cells, the in distributions were investigated by in situ hybridization using CoPCNA cRNA and in situ immuno- ¯uorescence staining. Figure 8A shows ¯uorescent images of their transcript expression during meiotic division by in situ hybridization with the CoPCNA cRNAs (amino-acid residues 131±223 for both CoPCNA-a and CoPCNA-b, and residues 227 to 268 for CoPCNA-a alone in Fig. 3A) and standard epi¯uorescence microscopy. In situ hybridiza- tion was performed using digoxigenin-labeled antisense CoPCNA cRNAs as the probes on paraf®n sections of the fruiting caps as described in Materials and methods. W hen digoxigenin-labeled sense CoPCNA cRNAs were used a s probes, n o gene-speci®c hybridization signals were detected (Fig. 8A, NC). We were able to cle arly visual the CoPCNA mRNAs in the fruiting caps (Fig. 8A). The tissues densely stained by D API on t he surface o f the gillus corresponded to Fig. 6. A model of CoPCNA (blue) was generated by homology modeling with the structure of human PCNA (green). The locations of Ser186 and Glu191 in the D 2 E 2 loop are shown i n r ed. Both p roteins c ould b e com pletely sup erimposed, except t he hu man se quence from S er186 to Glu191. The CoPCNA-a mo nomer had an inserted polypep tide of  107 a mino-a cid r esidues b etween S er18 6 to Glu1 9 1 in the human PCNA monome r. These 107 amin o-acid residues m ust protrude b eyo nd the mo nomer core pro tein . The p rotruding peptide site is behind t he site bind ing to p21, FEN-1 and DNA polymerase d su ggestin g that it has no important ro le(s) in the CoPCNA structure. Ó FEBS 2002 Alternative truncating of Coprinus PCNA mRNA (Eur. J. Biochem. 269) 171 the Coprinus meiotic tissues (Fig. 8B). A s i mportant meiotic stages, leptotene (K + 1), early to late zygotene (K + 2 and K + 5), pachytene (K + 6 and K + 7) and M1 to tetrads (K + 9) were selected. As shown i n Fig. 8 A, the CoPCNA gene was strongly expressed in meiotic tissues at leptotene to M1. Both CoPCNA-a and CoPCNA-b were expressed to the same extent through the meiotic prophase stages (Fig. 7B). These results indicated t hat CoPCNA was always expressed in the meiotic tissues. These spatial expression patterns did not agree well with the results of northern hybridization and Western blot analysis. To con®rm the results of in situ hybridization described above, ¯uorescence analyses of their distributions during meiotic division by in situ indirect immuno¯uorescence staining and standard epi¯uorescence microscopy were performed (Fig. 8B). Using the antibody described in the section on Western blotting analysis, intense signals for CoPCNA were also detected through the meiotic cells at leptotene to M1 similarly t o t he results of in situ hybridization (Fig. 8 B). These results of northern and Western blotting analyses indicated that CoPCNAs were transcribed and translated in the meiotic cells at the meiotic prophase stages. To our knowledge, this is ®rst report indicating that the PCNA gene was expressed at meiotic prophase stages, or that the meiosis-related events in which homologous DNA molecules pair required the PCNA protein. CoPCNA- de®cient mutants are required to obtain further informa- tion, and m ore detailed investigation of the phenotype of t he mutants will be necessary, including studies of genetic recombination f requency a nd the morphology o f t he synaptinemal complex. The project to knock out the gene has been attempted. Fig. 7. Expression patterns of the CoPCNA-a and CoPCNA-b at various periods in meiosis. (A) Northern blot analysis of the CoPCNA gene expression. E ach lane contained 20 lg o f total RNA i solated from caps o f C. cinereus at premeioticS (lane1), leptotene & zygotene (K + 0 to K + 5, lanes 2±7), p achytene (K + 6, K + 7, lanes 8, 9) diplote ne and diakinesis (K + 8 , lane 10), M 1 (K + 9, lane 11 ) and mycelium (lane 12). The b lot was prob ed with 32 P-labeled D NA (amino-acid residues 131±223 for both CoPCNA-a and CoPCNA- b as sh own in Fig. 3A) (top panel). Similar amounts of RNA w ere loaded in each lane as c on®rmed by ethidium bromide staining (lower panel). (B) We stern analysis of CoPCNA protein e xp ression. Aliquots of 30 lg o f the proteins extracted f rom caps of C. cinereus at premeioticS ( lane 1), leptotene and zygotene (K + 0 to K + 5, lanes 2±7), p achytene (K + 6, K + 7, lanes 8, 9) diplotene and diakinesis (K + 8, lane 10), M1 (K + 9, lane 11), M 2 (K + 10, lane 12) and mycelium ( lane 13) . The y were subjected to Western b lottin g an alysis using anti-(CoPCNA-a)Ig.ToppanelshowsCoPCNA-a, l ower pane l shows CoPCNA-b. 172 F. Hamada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Similarly, there have been no previous reports of two PCNA gene products produced by alter native truncation of exon. Recently, two types of DNA polymerase e have been found, the original and a form cleaved by caspase-3 [38]. Therefore, in a similar manner multiple species of PCNA may be required for normal meiosis. Their roles are of interestandremaintobeelucidated. ACKNOWLEDGEMENTS We thank Dr T. Kamada of Okayama University for helpful discussions with immunological analysis. We also thank Dr M. E. Zolan, her lab members, and Dr M. Celerin of Indiana U niversity for technical advice with immunostaining. A B CoPCNA-α K+1 K+2 K+6 K+9 NC CoPCNA-β CoPCNA-α DAPI K+1 K+2 K+7 K+5 K+9 NC anti-CoPCNA-α Fig. 8. Localization of CoPCNA-a and CoPCNA-b. (A) Localization of CoPCNA-a and CoPCNA- b mRNA by in situ hybridization. The fruiting tissues were sectioned and probed with two CoPCNA antisense riboprob es l abeled with digoxigenin±UTP. One probe hybrid ized with both CoPCNA-a and CoPCNA-b, w hile the other was speci®c for CoPCNA-a. The right panels sh ow hybridization for both CoPCNA- a and CoPCNA-b. The l eft panels show signals for CoPCNA-a alone. NC; negative control. (B) Localization of CoPCNA-a and C oPCNA-b with anti-(CoPCNA-a) Ig. Sections from fru iting tissue w ere stained wi th anti-(CoPCNA-a) I g. Nuclei were counterstained with DAPI (left panels). N C, negative c ontrol. 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Keywords: Coprinus cinereus;