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

Interestingly, although CoPCNA is a single-copy gene in the genome, two di€er-ent PCNA cDNA species were isolated using degenerate primers and a meiotic cDNA library, and were designated

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 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€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 speci®c sites in CoPCNA-a mRNA,

5¢-AA-GAAGGAGAAG-3¢ and 5¢-GAAGAGGAAGAA-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

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 pre-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

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 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±107cells), 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)

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

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

Culture ofC 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 day 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 The fruiting caps were

harvested, quickly frozen in liquid N2, and then stored at

)80 °C until used

CDNA and cloning ofCoprinus proliferating 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 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 melanogaster PCNA and Arabidopsis thaliana PCNA:

sense primer (5¢-CCGGCATCAACCTGCARDSNATG

GA-3¢) and antisense primer (5¢-GATCGATGT

CCATCAGCTTCAYNTCRWARTC-3¢) (N ˆ A, C, G

or T; R ˆ A or G; Y ˆ C or T; W ˆ 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 lMof

each of the two primers in a buffer containing 250 mM

dNTPs (Amersham Pharmacia Biotech), 10 mMTris/HCl,

pH 8.3, 50 mMKCl, 1.5 mMMgCl2, 0.05% Nonidet P-40,

and 2 U of ExTaq thermostable DNA polymerase (Takara)

Cycling conditions were: 94 °C for 2 min; 94 °C for 30 s;

50 °C for 1 min; 72 °C for 1 min; 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 AB056703 for the proliferating cell nuclear antigen (CoPCNA)

Genomic DNA isolation and Southern hybridization analysis

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 with32P using a Multiprime DNA label kit (Amersham Pharmacia Biotech) After prehybridization, hybridization was carried out at 42 °C for 16 h, followed by washing with 2 ´ NaCl/Pi/EDTA, 1% SDS at 65 °C for

15 min, 1 ´ NaCl/Pi/EDTA, 1% SDS at 65 °C for 15 min, and 0.2 ´ NaCl/Pi/EDTA, 1% SDS at 65 °C for 15 min Search for three-dimensional structure of human andCoprinus PCNA

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 withINSIGHT IIbinding 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 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 with32P, then hybridized as described for Southern analysis

Preparation of riboprobes andin 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 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 °C for

53 min in 0.2Mcarbonate/bicarbonate buffer and used at a

concentration of 2 mgámL)1the fruiting caps were ®xed

overnight at 4 °C with a mixture of 4% (w/v)

paraformal-dehyde and 0.25% (v/v) glutaralparaformal-dehyde in 50 mMsodium

phosphate buffer (pH 7.2) The ®xed tissues were dehydrated

in a series of 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 and

rehydrated through a graded ethanol series They were

subsequently pretreated with 10 mgámL)1of proteinase K

in 100 mMTris/HCl, pH 7.5, and 50 mMEDTA at 37 °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]

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¢, and cloned into

pET21a (Novagen) The plasmid constructs were

intro-duced into BL21(DE3)pLysS (Novagen)

Transformed E coli were grown at 37 °C in 2 ´ yeast/

tryptone medium with 1% glucose and 50 mgámL)1

amp-icillin Cells were grown to a D600 of 0.8 Recombinant

protein synthesis was induced by addition of 1 mMIPTG,

and after 3 h the cells were harvested by centrifugation The

cell pellets were resuspended in liquid N2 and stored at

)80 °C The cell pellets were resuspended in lysis buffer

(20 mM Tris/HCl, pH 6.5, 10% glycerol, 500 mM NaCl,

5 mMimidazole), containing 5 mM2-mercaptoethanol and

the protease inhibitors phenylmethanesulfonyl ¯uoride

(1 mM), leupeptin (1 lM) and pepstatin A (1 lM) Cells

were lysed by addition 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 Chelating column (Amersham

Pharmacia Biotech) The column was washed successively

with buffer A (20 mM Tris/HCl, pH 6.5, 10% glycerol,

500 mMNaCl, 0.02% NP-40) containing 5 mMimidazole

The bound proteins were eluted with buffer A containing

400 mMimidazole Fractions of proteins were identi®ed 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 mMEDTA, 5 mM

2-mercaptoeth-anol) After washing, fractions were collected with 40 mL of

a linear gradient of 0±0.7MNaCl 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 the CoPCNA-a protein was

raised in rabbit using 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 secondary antibody with nitroblue tetrazolium and

5-bromo-4-chloro-3-indolyl phosphate as substrates of

alkaline phosphatase

Immunostaining of Coprinus fruiting caps was carried out [33] The paraf®n 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 ¯uoro 568, which was diluted 1 : 1000 as a secondary antibody Then, 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 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)

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 inCoprinus meiocytes

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 speci®-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-speci®c biochemical events We ®rst tried to identify the PCNA homolog in C cinereus, 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, 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 puri®ed 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

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

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

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 resiresi-dues), 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 theBLASTXprogram [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 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)

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 inCoprinus genome, and of alternative truncation sites ofCoPCNA-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 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.

XhoI Nde

I

EcoR

I BamH I

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.

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 suggsequenc-ested that the gene

consists of six exons and ®ve 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

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 Ôalternative truncationÕ 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

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 ®ve sequences, 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 CoPCNA-a The site (184±195) in human PCNA was in a loop called the D 2 E 2 loop CoPCNA-a and CoPCNA-b had insertions of 107 and 64 amino acids, in this D 2 E 2 loop, respectively.

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

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 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

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) Genomic structure of CoPCNA The thin lines in the cDNA clone represent ¯anking 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.

C-terminal-tagged CoPCNA-a fusion protein The

CoP-CNA-a protein was puri®ed 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 ®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 present as a 150-kDa trimer (Fig 5B), similar to human

PCNA

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]

Timing of events ofCoprinus 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 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 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

1000

B A

100

100kDa

150

10

90 80

70

50 40

retention volume (ml)

75k

50k 37k

Fig 5 SDS/PAGE of puri®ed CoPCNA-a protein (A) and determination of its molecular mass by gel ®ltration chromatography (B) (A) SDS/PAGE analysis of puri®ed CoPCNA-a after Mono Q column chromatography The puri®ed 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 puri®ed 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.

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

¯uores-cent microscopic observation of the monokaryonic nuclei

and from electron microscopic observation of the presence

of synaptinemal complex, it was concluded that the

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

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

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

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 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 When digoxigenin-labeled sense CoPCNA cRNAs were used as probes, no gene-speci®c 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

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 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  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.

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

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 similarlyto the results of insituhybridization (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 ®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 more detailed investigation of the phenotype of the mutants will be necessary, including studies of genetic recombination frequency and the morphology of the 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 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 32 P-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 con®rmed 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.

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

A C K N O W L E D G E M E N T S

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.

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 riboprobes labeled with digoxigenin±UTP One probe hybridized with both CoPCNA-a and CoPCNA-b, while the other was speci®c 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.