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

Eur. J. Biochem. 269, 164–174 (2002) (cid:211) FEBS 2002

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

107 amino acids larger than human PCNA, and so the 107 amino-acid sequence was inserted in a loop, the so-called D2E2 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) Ig re- vealed that at least two CoPCNA mRNAs before and after truncation were translated at the meiotic prophase as CoPCNA-a and CoPCNA-b.

and

Keywords: Coprinus cinereus; Coprinus PCNA (CoPCNA); meiotic prophase; truncating of CoPCNA mRNA; two CoPCNA species.

The primary purpose of 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 single-copy gene in the genome, two di(cid:128)er- ent PCNA cDNA species were isolated using degenerate primers and a meiotic cDNA library, and were designated as CoPCNA-a and CoPCNA-b. CoPCNA-b was made by truncating at specific sites in CoPCNA-a mRNA, 5¢-AA- 5¢-GAAGAGGAAGAA-3¢. GAAGGAGAAG-3¢ Both of these sequences were present in exon IV in the genomic sequence, and interestingly the former was the same as the inverse sequence of the latter. CoPCNA-a was

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 to interact with DNA polymerases d and e, p21 and many other factors [2–6]. In these interactions, PCNA appears to be the major protein involved in determining the binding counterpart, e.g. homologous regions of FEN-1 and p21 compete for binding to the same site on PCNA [7]. PCNA could therefore be a key 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 condense from the dispersed state typical of interphase during early meiotic prophase, forming long thin threads in leptotene, and each acquires 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 the chiasmata that become visible during diplotene. Two cell divisions follow, reductional and equational, 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 the chromosomal DNA, and the regions on the chromosomal DNA are different from each other; very high Cot sequences 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 at pachytene.

We have investigated meiosis-related protein factors using meiotic cells in the basidiomycete, Coprinus cinereus [11–20]. This organism is especially well suited for studies of meiosis, because its meiotic cell cycle is long and naturally synchronous [16,19,21–23]. Each fruiting cap is extremely rich in meiotic cells at the same stage (106–107 cells), and the nuclear numbers are easily observable. In the meiotic cycle, the dikaryonic cells are at premeiotic stages from the S phase to leptotene, and for 6 h the beginning of the karyogamy stage (at which point the two nuclei are fused) is the zygotene stage at 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 antigen; CoPCNA, PCNA cDNA. (Received 31 July 2001, revised 22 October 2001, accepted 25 October 2001)

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Cycling conditions were: 94 (cid:176)C for 2 min; 94 (cid:176)C for 30 s; 50 (cid:176)C for 1 min; 72 (cid:176)C for 1 min; 40 cycles, followed by a 10-min extension at 72 (cid:176)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 AB056703 for the proliferating cell nuclear antigen (CoPCNA).

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 mammals 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 predicted role in meiosis, the Coprinus PCNA mRNAs were expressed at limited meiotic prophase stages.

Genomic DNA isolation and Southern hybridization analysis

M A T E R I A L S A N D M E T H O D S

Culture of C.cinereusand collection of the fruiting bodies

Genomic DNA was isolated from Coprinus mycelium tissue and digested with restriction enzymes such as XhoI, NdeI, EcoRI and BamHI [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 labeled with 32P using a Multiprime DNA label kit (Amersham Pharmacia Biotech). After prehybridization, hybridization was carried out at 42 (cid:176)C for 16 h, followed by washing with 2 · NaCl/Pi/EDTA, 1% SDS at 65 (cid:176)C for 15 min, 1 · NaCl/Pi/EDTA, 1% SDS at 65 (cid:176)C for 15 min, and 0.2 · NaCl/Pi/EDTA, 1% SDS at 65 (cid:176)C for 15 min.

Search for three-dimensional structure of human and CoprinusPCNA

To simulate the three-dimensional structures of the PCNAs, modeling of the human PCNA protein was compared with 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

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 day 0 to day 7 at 37 (cid:176)C in total darkness and from day 7 onwards at 25 (cid:176)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 defined 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. The fruiting caps were harvested, quickly frozen in liquid N2, and then stored at )80 (cid:176)C until used.

Total RNA was prepared from caps of C. cinereus at meiotic prophase using Trizol (Gibco-BRL) according to the manufacturer’s protocol.

CDNA and cloning of Coprinusproliferating cell nuclear antigen (CoPCNA)

Culture of Escherichia coli and phage, extraction of plasmid DNA, electrophoresis of DNA and RNA, were carried out according to the methods described previously [24] unless otherwise specified.

RNA samples 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 stained with ethidium bromide and blotted overnight in 20 · NaCl/Pi/ EDTA onto Hybond-N+ membranes (Amersham Phar- macia Biotech). The two probes (amino acids 131 to 223 for both CoPCNA-a and CoPCNA-b, and 227 to 268 for CoPCNA-a alone; see below) were labeled with 32P, then hybridized as described for Southern analysis.

and

primer

antisense

Preparation of riboprobes and insituhybridization

To isolate homologous PCNA cDNA, two primers were used corresponding to amino-acid motifs conserved in human PCNA, Schizosaccharomyces pombe PCNA, Droso- phila melanogaster PCNA and Arabidopsis thaliana PCNA: sense primer (5¢-CCGGCATCAACCTGCARDSNATG GA-3¢) (5¢-GATCGATGT CCATCAGCTTCAYNTCRWARTC-3¢) (N (cid:136) A, C, G or T; R (cid:136) A or G; Y (cid:136) C or T; W (cid:136) A or T). The primers were used in PCR reactions with cDNA generated from poly(A)+ RNA isolated from fruiting bodies of C. cinereus as template. PCR was performed with 1 lg of the cDNA as a template in a volume of 50 lL in the presence of 2 lM of each of the two primers in a buffer containing 250 mM dNTPs (Amersham Pharmacia Biotech), 10 mM Tris/HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.05% Nonidet P-40, and 2 U of ExTaq thermostable DNA polymerase (Takara).

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 amino 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 by heating at 60 (cid:176)C for 53 min in 0.2 M carbonate/bicarbonate buffer and used at a

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Immunostaining of Coprinus fruiting caps was carried out [33]. The paraffin sections of the fruiting caps described above in the in situ hybridization section were used. The cells were incubated for 1 h with the antibody against each of the CoPCNA proteins. The antibodies against each of the CoPCNA proteins were diluted at 1 : 100 before use. The cells were then treated for 1 h with FITC-conjugated anti- (rabbit IgG) Ig (Sigma Chemical Co.) conjugated with Alexa fluoro 568, which was diluted 1 : 1000 as a secondary antibody. Then, the cells were also stained with a solution of 20 lgÆL)1 4¢,6-diamido-2-phenylindole dihydrochloride n-hydrate (DAPI) for 5 min The specimens were examined under a fluorescence microscope (Olympus BH-2).

concentration of 2 mgÆmL)1 the fruiting caps were fixed overnight at 4 (cid:176)C with a mixture of 4% (w/v) paraformal- dehyde and 0.25% (v/v) glutaraldehyde in 50 mM sodium phosphate buffer (pH 7.2). The fixed tissues were dehydrated in a series of xylene and ethanol and embedded in paraffin. Embedded tissues were sectioned at a thickness of 5 lm, and placed on microscope slides precoated with poly L-lysine. Sections were deparaffinized with xylene and rehydrated through a graded ethanol series. They were subsequently pretreated with 10 mgÆmL)1 of proteinase K in 100 mM Tris/HCl, pH 7.5, and 50 mM EDTA at 37 (cid:176)C for 30 min, dehydrated in a graded ethanol series, and dried under vacuum for 2 h. Hybridization and detection of hybridized riboprobes were performed [31].

Immunoscreening

Overexpression and purification of CoPCNA protein

Using previously described rabbit anti-CoPCNA Ig, immunoscreening was carried out with kZAPII cDNA library from the caps at each meiotic stage of C. cinereus. From the isolated plaques, plasmid DNA was prepared by the in vivo excision protocol using the ExAssist/SOLR system (Stratagene).

CoPCNA coding region was amplified using the 5¢ sense primer 5¢-GGAATTCCATATGCTTGAAGCCAAACT CGCAG-3¢ and 3¢ antisense primer 5¢-CGAGCTCGGG TCGTCACCAATCTTAGGTGCG-3¢, and cloned into pET21a (Novagen). The plasmid constructs were intro- duced into BL21(DE3)pLysS (Novagen).

R E S U L T S A N D D I S C U S S I O N

Isolation and characterization of PCNA homologous cDNA in Coprinusmeiocytes

As described in the introduction, proliferating cell nuclear antigen (PCNA) might play some role(s) in the meiotic cell cycle, especially at meiotic prophase. We report here that in a basidiomycete, C. cinereus, PCNA message was specifi- cally expressed in meiotic prophase stages at which homol- ogous chromosomes pair (zygotene) and recombine (pachytene). In lily microsporocytes small amounts of DNA synthesis were required for synaptinemal complex formation (zygotene), and for recombination between 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-specific biochemical events. We first tried to identify the PCNA homolog in C. cinereus, and then investigated the meiotic stage-specific transcription. Unexpectedly, we found the production of two species of PCNA protein in C. cinereus.

Transformed E. coli were grown at 37 (cid:176)C in 2 · yeast/ tryptone medium with 1% glucose and 50 mgÆmL)1 amp- icillin. Cells were grown to a D600 of 0.8. Recombinant protein synthesis was induced by addition of 1 mM IPTG, and after 3 h the cells were harvested by centrifugation. The cell pellets were resuspended in liquid N2 and stored at )80 (cid:176)C. The cell pellets were resuspended in lysis buffer (20 mM Tris/HCl, pH 6.5, 10% glycerol, 500 mM NaCl, 5 mM imidazole), containing 5 mM 2-mercaptoethanol and the protease inhibitors phenylmethanesulfonyl fluoride (1 mM), leupeptin (1 lM) and pepstatin A (1 lM). Cells were lysed by addition of 1 mgÆmL)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 Chelating column (Amersham Pharmacia Biotech). The column was washed successively with buffer A (20 mM Tris/HCl, pH 6.5, 10% glycerol, 500 mM NaCl, 0.02% NP-40) containing 5 mM imidazole. The bound proteins were eluted with buffer A containing 400 mM imidazole. Fractions of proteins were identified by SDS/PAGE, pooled and dialyzed. The dialysate was loaded onto a Mono Q HR5/5 column (Amersham Pharmacia Biotech) equilibrated with buffer B (50 mM Tris/HCl pH 6.5, 10% glycerol, 2 mM EDTA, 5 mM 2-mercaptoeth- anol). After washing, fractions were collected with 40 mL of a linear gradient of 0–0.7 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 immunofluorescence microscopy

To isolate the Coprinus PCNA homolog, two degenerate PCR primers (see Materials and methods) were used for PCR with cDNA produced from poly(A)+ RNA isolated from fruiting bodies of C. cinereus at meiotic prophase as the template. As the cDNA 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 is AB056703 for the proliferating cell nuclear antigen (CoPCNA).

A polyclonal antibody against CoPCNA-a protein was raised in a rabbit using the purified protein. Western blotting analysis revealed that the anti-CoPCNA Ig recog- nized the CoPCNA-a protein (48 kDa) and one more protein with Mr of 42 kDa (Fig. 1). To investigate the 42-kDa protein in more detail, we screened a cDNA clone

A polyclonal antibody against the CoPCNA-a protein was raised in rabbit using the purified proteins. Western blotting analysis was carried out [32]. Anti-(rabbit IgG) Ig conju- gated with alkaline phosphatase (Promega) was used as a secondary antibody with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates of alkaline phosphatase.

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M

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Fig. 1. Immunoblotting analysis of CoPCNA in the fruiting bodies during meiosis. Aliquots of 30 lg of the proteins extracted from 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 arrowheads represent the posi- tions of CoPCNA-a and CoPCNA-b.

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, and it was tentatively designated as CoPCNA-b. CoPCNA-a and CoPCNA-b were completely sequenced, and were 1104- and 975-bp in length, respectively. The molecular masses of CoPCNA-a and CoPCNA-b were shown to be 48- and 42-kDa on SDS/PAGE, respectively, and were slightly larger than those expected from the amino-acid sequences. As human PCNA is a highly acidic protein with a pI of 4.5 [34] and a calculated 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.

Arabidopsis PCNA protein, and 41.5% to S. pombe PCNA protein (Fig. 3A). We searched for homology among 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 amino-acid sequence from 184 to 195 of them did not have homology with CoPCNA. This site (184–195) was in a loop called the D2E2 loop in human PCNA (Fig. 3B).

The Coprinus genomic DNA isolated was truncated with restriction enzymes including XhoI, NdeI, EcoRI and BamHI (Fig. 2). Southern hybridization analysis of a sequence in common between CoPCNA-a and CoPCNA-b (amino-acid residues 131 to 223 in Fig. 3A) revealed that as each of the truncated products indicated a single band, each was single-copy gene (Fig. 2). These results strongly suggested that the two CoPCNA species were transcribed from one CoPCNA gene, and produced by splicing alternatively.

Interestingly, the polypeptide of amino-acid residues 184 to 305 of CoPCNA-a contained three nuclear localization signal (NLS) peptides, PEKKKIK, KKRKKK and PAKKAKT (boxes in Fig. 3A), which were not present in the other eukaryotic species, and the polypeptide of CoPCNA-b also had an NLS peptide (PAKKAKT). The eukaryotic PCNAs newly synthesized in the cytoplasm must move into the nucleus with the other PCNA-binding proteins that have the signal. However, CoPCNA-a alone (and perhaps CoPCNA-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

These Coprinus PCNA cDNA sequences encoded prod- ucts of 368 and 325 amino-acid residues, respectively (Fig. 3A). We designated 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 sequences 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

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

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 32P-labeled cDNA probe for amino- acid residues 131–223 for both CoPCNA-a and CoPCNA-b shown in Fig. 2A.

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obtaining the full-length genomic clone. The restriction enzyme map and the relationship between the gene and cDNA are summarized in Fig. 4A.

As shown in Fig. 4A, the complete PCNA gene sequence was compared with the two cDNAs, CoPCNA-a and CoPCNA-b. The alternative splicing site in the gene was in exon IV for CoPCNA-a, suggesting that for CoPCNA-b, the CoPCNA-a mRNA truncation did not occur by splicing, but by another mechanism, because the truncation occurred in the exon that has no intron. Therefore, we called it (cid:212)alternative truncation(cid:213) in the later part of this report.

We searched for and found the special sequence sites in the CoPCNA gene for truncation. The mRNA sequences

Careful comparisons of the deduced amino-acid sequenc- es with those from other organisms suggested that the gene consists of six exons and five introns, and also contains a 1104-bp ORF (Fig. 4A). The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/ GenBank nucleotide sequence database with the accession number AB056703.

Fig. 3. (A) Alignment of the predicted amino-acid sequences of CoPCNA-a and CoPCNA-b with those of S. pombe, Arabidopsis and human, and (B) truncation derivatives of CoPCNA-a and CoPCNA-b proteins. In (A), asterisks indicate amino-acid identity common to all five sequences, and dots indicate amino-acid identity between five 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 CoPCNA-a. The site (184–195) in human PCNA was in a loop called the D2E2loop. CoPCNA-a and CoPCNA-b had insertions of 107 and 64 amino acids, in this D2E2loop, respectively.

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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

around the truncating sites in the CoPCNA gene are summarized in Fig. 4B,C. CoPCNA-b mRNA appeared to be produced from the CoPCNA-a mRNA by truncation at specific 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 former sequence was the inverse of the latter (Fig. 4C). Both of the 5¢ and 3¢ truncation sites were also special repeat sequences (Fig. 4B,C), suggesting the existence of a special truncation

To characterize the CoPCNAs in more detail, the recom- binant CoPCNA proteins were overexpressed and purified. Extracts from the E. coli cells contained a six-histidine

Fig. 4. Genomic structure and sequence sites. (A) Genomic structure of CoPCNA. The thin lines in the cDNA clone represent flanking and intron sequences, and the thick lines at both ends of the cDNA clone represent 5¢ and 3¢UTR sequences. Exons in the cDNA clone are indicated by open boxes. Both of the sequences in which truncation occurred were present in Exon IV in the CoPCNA-a genomic gene. (B) The special sequence sites in the CoPCNA gene for truncation. The mRNA sequences around the truncation sites in the CoPCNA gene are summarized. CoPCNA-b mRNA appeared to be produced 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.

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Timing of events of Coprinusmeiosis, and Northern hybridization and Western blotting analyses of CoPCNAs

C-terminal-tagged CoPCNA-a fusion protein. The CoP- CNA-a protein was purified to near homogeneity by chromatography on His bind resin, and Mono Q HR5/5 column (see Materials and methods). Figure 5A,B show the results of SDS/PAGE and Sephacryl S 300 gel filtration 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 present as a 150-kDa trimer (Fig. 5B), similar to human PCNA.

We investigated the meiotic stage-specific 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 synchronous cultures every 1 h after induction of meiosis, and hybridization with the common to both CoPCNA-a and CoPCNA-b as a probe (residues 131 to 223 in Fig. 3A) was performed (Fig. 7A).

Both forms of

To simulate the three-dimensional structures of the 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. Figure 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].

the transcripts were very 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 became 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 transcript appeared again at a moderate level at middle pachytene (K + 6) (Fig. 7A). The transcripts were also moderately detected at diplotene and diakinesis (K + 8). In meiosis, the transcripts were also strongly expressed in the basidia at middle zygotene, and again at middle pachytene (Fig. 7A). In all cases in which the signal was positive, one band was observed (Fig. 7A) because the mRNA signals for CoPCNA-a and CoPCNA-b appeared not to be able to be separated from each other because of their size. When a

A

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Fig. 5. SDS/PAGE of purified CoPCNA-a protein (A) and determination of its molecular mass by gel filtration chromatography (B). (A) SDS/PAGE analysis of purified CoPCNA-a after Mono Q column chromatography. The purified CoPCNA-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 left of the panel. (B) After Mono Q column chromatography, the purified CoPCNA-a was loaded onto the S300 column. The arrow indicates the position at which CoPCNA-a was found. The molecular mass of the protein in the peak was 150 kDa.

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the stages at which the homologous chromosomes 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 forms of CoPCNA is also translated in mitosis (Fig. 7B).

Subcellular localization of CoPCNA during meiotic cell division

it was concluded that

cDNA probe that could recognize only CoPCNA-a was used (amino-acid residues 227 to 268 in Fig. 3A) the mRNA signal was also the same size as that for CoPCNA-a (data not shown). As the majority of the basidia at K + 0 and K + 1 were in PreS or leptotene to early zygotene, not pachytene, during this period as determined from fluores- cent microscopic observation of the monokaryonic nuclei and from electron microscopic observation of the presence the of synaptinemal complex, CoPCNAs were discontinuously expressed at PreS at which the genomic DNA replicates, at leptotene at which the axial core in each of the chromosomes is formed, at zygotene at which the homologous chromosomes pair, and at pachytene at which the zygotene-paired chromosomes recombine. Diplotene and diakinesis are the stages at which the pachytene-recombined chromosomes disjunct.

The results of northern hybridization 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 the somatic cells. Therefore, to confirm 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- fluorescence staining. Figure 8A shows fluorescent 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 epifluorescence microscopy. In situ hybridiza- tion was performed using digoxigenin-labeled antisense CoPCNA cRNAs as the probes on paraffin sections of the fruiting caps as described in Materials and methods. When digoxigenin-labeled sense CoPCNA cRNAs were used as probes, no gene-specific hybridization signals were detected (Fig. 8A, NC). We were able to clearly visual the CoPCNA mRNAs in the fruiting caps (Fig. 8A). The tissues densely stained by DAPI on the surface of the gillus corresponded to

We raised a polyclonal antibody against the recombinant CoPCNA-a protein in rabbit. As shown in Fig. 7B, there were two signals on immunoblots, coinciding with the molecular mass values of CoPCNA-a (48 kDa) 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) Ig in the meiotic cell 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, the proteins seemed to have longer half-lives, suggesting that CoPCNA-a and CoPCNA-b are present at all meiotic prophases including

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 D2E2loop are shown in red. Both proteins could be completely superimposed, except the human sequence from Ser186 to Glu191. The CoPCNA-a monomer had an inserted polypeptide of (cid:25) 107 amino-acid residues between Ser186 to Glu191 in the human PCNA monomer. These 107 amino-acid residues must protrude beyond the monomer 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 CoPCNA structure.

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the Coprinus meiotic tissues (Fig. 8B). As important 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 in Fig. 8A, 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 that 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.

section on Western blotting analysis, intense signals for CoPCNA were also detected through the meiotic cells at leptotene to M1 similarly to the results of in situ hybridization (Fig. 8B). 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 first 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- deficient mutants are required to obtain further informa- tion, and more detailed investigation of the phenotype of the including studies of genetic mutants will be necessary, recombination frequency and the morphology of the synaptinemal complex. The project to knock out the gene has been attempted.

To confirm the results of in situ hybridization described above, fluorescence analyses of their distributions during meiotic division by in situ indirect immunofluorescence staining and standard epifluorescence microscopy were performed (Fig. 8B). Using the antibody described in the

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. Each lane contained 20 lg of total RNA isolated from caps of C. cinereus at premeioticS (lane1), leptotene & zygotene (K + 0 to K + 5, lanes 2–7), pachytene (K + 6, K + 7, lanes 8, 9) diplotene and diakinesis (K + 8, lane 10), M1 (K + 9, lane 11) and mycelium (lane 12). The blot was probed with 32P-labeled DNA (amino-acid residues 131–223 for both CoPCNA-a and CoPCNA-b as shown in Fig. 3A) (top panel). Similar amounts of RNA were loaded in each lane as confirmed by ethidium bromide staining (lower panel). (B) Western analysis of CoPCNA protein expression. Aliquots of 30 lg of the proteins extracted from caps of C. cinereus at premeioticS (lane 1), leptotene and zygotene (K + 0 to K + 5, lanes 2–7), pachytene (K + 6, K + 7, lanes 8, 9) diplotene and diakinesis (K + 8, lane 10), M1 (K + 9, lane 11), M2 (K + 10, lane 12) and mycelium (lane 13). They were subjected to Western blotting analysis using anti-(CoPCNA-a) Ig. Top panel shows CoPCNA-a, lower panel shows CoPCNA-b.

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Alternative truncating of Coprinus PCNA mRNA (Eur. J. Biochem. 269) 173

B

A

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CoPCNA-α CoPCNA-β

anti-CoPCNA-α

DAPI

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K+2

K+2

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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 riboprobes labeled with digoxigenin–UTP. One probe hybridized with both CoPCNA-a and CoPCNA-b, while the other was specific for CoPCNA-a. The right panels show hybridization for both CoPCNA-a and CoPCNA-b. The left panels show signals for CoPCNA-a alone. NC; negative control. (B) Localization of CoPCNA-a and CoPCNA-b with anti-(CoPCNA-a) Ig. Sections from fruiting tissue were stained with anti-(CoPCNA-a) Ig. Nuclei were counterstained with DAPI (left panels). NC, negative control.

Similarly, there have been no previous reports of two PCNA gene products produced by alternative 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 interest and remain to be elucidated.

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 University for technical advice with immunostaining.

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