Eur. J. Biochcm. 203, 367-371 (1992) i,FEBS 1992

Identification of carrot cDNA clones encoding a second putative proliferating cell-nuclear antigen, DNA polymerase 6 auxiliary protein Shingo HATA ', Hiroshi KOUCHI I , Yoshiyuki TANAKA', Eiichi MTNAMI ', Takashi MATSUMOTO' lwao SUZUKA3 and Junji HASHIMOTO'

' National Institute of Agrobiological Resources

' National Institute of Agro-environmenlal

Sciences National Institute ol'Anima1 Health, Ibaraki, Japan

(Received August 9, 1991) - EJB 91 1082

The proliferating cell-nuclear antigen (PCNA) plays a key role in the control of eukaryotic DNA replication. We have isolated two cross-hybridizing groups of cDNA encoding carrot homologs of PCNA. Sequence analysis and Southern-blot experiments showed that the cDNA were derived from two distinct genes. One corresponded to the typical PCNA, which is known to be highly conserved in eukaryotes from yeast to man; its mRNA is 1.2 kb in size and the calculated molecular mass of the protein is 29 kDa. The other encoded a larger PCNA homolog which has not previously been reported; the mRNA is 1.5 kb in size, the N-terminal three quarters (calculated molecular mass, 29 kDa) of the protein product is 88% identical at the amino acid level to the typical PCNA, but the protein has an extra C-terminal domain of 11 kDa. Both PCNA homologs were apparently coexpressed concomitant with somatic embryogenesis. The mRNA level of the novel homolog is 10 - 20% that of the typical PCNA in the embryos. The presence of the second putative PCNA may provide new insight into studies on the mechanism of DNA replication in eukaryotes.

The proliferating cell-nuclear antigen (PCNA) was originally discovered as a nuclear antigen in human proliferative cells [I]. Subsequently, it was identified as an auxiliary protein for DNA polymerase 6 [2, 31. PCNA plays an important role in eukaryotic DNA replication by increasing the processibility of the polymerase 6 during elongation of the leading strand [4-61. In addition, PCNA is a highly conserved protein in widely divergent species [7]. Previously, we showed that PCNA is also present in higher plants [8, 91 and that its expression is localized to the proliferating organs [lo]. So far, it has been believed that each organism contains only one molecular species of PCNA. Somatic embryogenesis in cultured carrot cells is an attractive system for investigating the mechanism of plant cell differentiation [Ill. A number of studies on the gene-expression program of somatic embryogenesis have been carried out and several genes were reported to be induced in the process [12- 141. In those studies, however, the structures and functions of the genes remained unclear. We have recently Correspondence to S. Hata, Faculty of Science, Himeji Institute of Technology, Kamigori, Ako-gun, Hyogo, Japan 678-12 Ahhrc4ution. PCNA, proliferating cell-nuclear antigen. Enzymes. DNA polymerase 6, modified T7 DNA polymerase (EC 2.7.7.7); restriction enzymes BcoRI, EcoT141, HindlII, PstI, XbuI (EC 3.1.21.4). Nore. The novel nucleotide sequence data published here have been deposited in the EMBL sequence data bank and are available under the accession numbers X62976 and X62977.

demonstrated that a mitotic cyclin gene is expressed concomitant with the embryogenesis [15]. We undertook the present investigation to determine whether or not somatic embryogenesis occurs concomitant with the induction of PCNA expression. As a first step, we isolated cDNA for carrot PCNA using PCNA clones of other plants as probes. Unexpectedly, however, the cDNA formed two cross-hybridizing groups. The results described below provide evidence for the presence of a second putative PCNA in the carrot. The mRNA of the two PCNA homologs were apparently coexpressed during embryogenesis.

MATERIALS AND METHODS Materials

Suspension cells of carrot (Daucus rarnta (L.) cv. Kurodagosun), initiated from hypocotyls of seedIings in the presence of 2,4-dichlorophenoxyacetic acid, were fractionated by the method of Fujimura and Komamine [16]. The proembryogenic masses were grown without 2,4-dichlorophenoxyacetic acid into somatic embryos [I 61. Poly (A)-rich RNA was isolated from a mixture of staged embryos (globular stage/heart stage/torpedo stage = 1 :2:4, fresh mass). The construction of a AZAPII cDNA library has already been described [15]. Genomic DNA was prepared [17] from a cell line, named IM-1, which was obtained from the above suspen-

368 sion cells. It was subcultured every week for more than a year after filtration through an 180-pm nylon mesh. cDNA cloning and sequencing The recombinant phages were plated with Escherichia coli XL-1 Blue, then transferred to Hybond-N membranes (Amersham; 4 x lo4 plaques/sheet). They were then screened with a mixture (equivalent numbers of molecules) of cDNA for rice PCNA and soybean PCNA [91, labeled by random priming [18]. Hybridization was performed at 37°C overnight in the mixture described [19]. The membranes were washed with 2 x buffer A (buffer A = 0.15 M NaCl, 0.015 M sodium citrate, pH 7) and 0.1% SDS at 50°C twice for 15 min each. Phage DNA in the positive clones were excised in vivo to recover pBluescript SK(-) (Stratagene). The nucleotide sequence was determined according to Sanger et al. [20] using modified T7 polymerase [21] (Sequenase version 2.0; US Biochemical).

shown in Fig. 1A. Clone 4.10 has an open reading frame for a 264-amino-acid polypeptide, the calculated molecular mass of which is z 29 kDa. Since the molecular size and structure of this protein product are very similar to those of PCNA species so far defined [7, 91, we call it ‘typical’ PCNA. Two overlapping clones, designated as P3 and 1.13, composed the other group, the composite sequence of which is shown in Fig. 1B. An open reading frame for a 365-amino-acid polypeptide was found, the predicted molecular mass for which is z 40 kDa. We designate the novel PCNA homolog as ‘larger’ PCNA. Within the overlapping region of the larger PCNA clones, the sequence of 1.13 differed from that of P3 by three nucleotides, resulting in one amino acid substitution (Fig. 1 B). The two clones may thus originate from a polymorphic allele. Southern-blot-hybridization analysis On genomic Southern blotting, probes derived from the typical and larger PCNA cDNA species detected distinct bands from each other (Fig. 2). Since both probes contain entire coding regions, we conclude that the two PCNA homologs are derived from two distinct genes.

Blot hybridization analysis For Southern blot-hybridization analysis, 2 pg carrot genomic DNA was loaded onto each lane after digestion with EcoR1, HindIII, Pstl or Xbal. The samples were fractionated in 0.8% agarose gels, then blotted onto Hybond-N membranes. The filters were hybridized with the indicated probes for 1 d at 42°C in the mixture described [19]. They were then initially washed in 6 x buffer A and 0.1 % SDS at 42°C once for 30 min, 2 x buffer A and 0.1% SDS at 42°C twice for 30 min each and 1 x buffer A and 0.1% SDS at 65°C once for 20 min, followed by a high stringency wash in 0.1 x buffer A and 0.1% SDS at 65°C once for 20 min. Northern-blot hybridization analysis was performed with 1 pg poly (A)-rich RNA in each lane as described [15, 191.

In vitro translation 1 pg each of poly (A)-rich RNA from the indicated materials was translated in a wheat germ extract [22,23] at 30°C for 1 h, using 0.06 MBq of ~-[4,5-~H]leucine (Amersham). The samples were subjected to SDSlpolyacrylamide electrophoresis [24], treated with Enhance (New England Nuclear), then fluorographed at -85°C.

RESULTS Isolation and sequencing of cDNA for carrot PCNA homologs In a search for cDNA species for carrot PCNA homologs, the library of differentiating carrot embryos was screened under conditions of low stringency with rice PCNA and soybean PCNA as probes. 32 positive spots were detected for 4.8 x los clones. Ten recombinant phages were picked out at random and analyzed. Their nucleotide sequences and deduced amino acid sequences showed that five of them were PCNA related, while the other five clones were false positives and thus not related to PCNA. Strikingly, the PCNA-related clones could be classified into two groups. Three of the five composed one group; the nucleotide and amino acid sequences of a representative clone, designated as 4.10, are

Comparison of the deduced amino acid sequences Fig. 3 shows amino acid sequence alignment of the typical carrot PCNA, the larger carrot PCNA and the previously characterized rice PCNA [9]. At the amino acid level, the N-terminal three-quarters of the larger PCNA exhibited high similarity to the typical carrot PCNA (88% identity) and the rice PCNA (86% identity). Therefore, the two carrot PCNA homologs are very closely related to the known PCNA in other species [7,9]. At the nucleotide level, however, the above three species of PCNA are less similar (74-78% identity within the conserved region). This is why the typical and larger PCNA do not cross-hybridize under conditions of high stringency. To obtain information on the role of the extra Cterminal domain of the larger PCNA, a search of databases was carried out for similar amino acid sequences, by means of the DNASIS version 6.0 software (Hitachi). However, the extra domain of 11 kDa did not show significant similarity with any known proteins. Expression of the PCNA homologs Next we carried out Northern-blot-hybridization analysis to examine the correlation between the embryogenesis and the expression of PCNA homologs. The mRNA for both the larger PCNA (3.5 kb in size) and the typical PCNA (1.2 kb) were similary expressed at much higher levels in embryos than in proembryogenic masses or in unorganized calli (Fig. 4). These expression patterns were also similar to those of carrot mytotic cyclin [15]. Dot-blot analysis using sequentially diluted poly (A)-rich RNA samples showed that the amount of mRNA for the larger PCNA was 10 - 20% that of the typical PCNA in embryos (data not shown). DISCUSSION We have identified two genes encoding carrot PCNA homologs, one of which codes an unusually large PCNA molecule. Thus, PCNA constitute a small gene family, at least in the carrot. There may be other members of the family because the genomic Southern blots showed several bands (Fig. 2).

369 A

1

CCAM Set Leu Glu Leu Arg Leu Val Gln Gly Ser Leu Leu LYS LYS Val net ASP Ser I I e Lys ASP Leu Val Asn Asp AIa Asn Phe Asp Cys Ser Ala Thr Gly Phe Ser Leu Gln Ala let ATG TTG GAG CTC CGT CTA GTC CAG GGC AGT CTG CTG AAG AAG GTG ATG GAT TCG ATC AAG GAT CTC GTC AAC GAC GCC AAC TTC GAC TGC TCG GCC ACG GGG TTC TCG CTG CIA GCC ATG

40

6

ASP Ser Ser His Val AIa Leu Val Ala Val Leu Leu Arg Ser Glu GIY Phe Glu His Tyr Arg CYS ASP Arg Asn Ile Ser net GIy n e t Asn Leu Gly Asn net Ala Lys net Leu LYS 126 GAC TCC AGC CAC GTG GCG CTG GTG GCC GTG CTG CTC AGA TCG GAG GGC TTC GAG CAC TAG CGG TGC GAT AGG AAC ATA TCG ATG GGG ATG M T CTG GGC AAC ATG GCC M G ATG TTG MG

80

CYS Ala GIY Asn ASP ASP lle Ile Thr Ile LYS Ala ASP ASP GIY Ser ASP Thr Val Thr Phe net Phe Glu Ser Pro Thr Gln Asp Lys Ile Ala ASP Phe Glu net Lys Leu net ASP 120 246 TGT GCG GGG AAT GAT GAT ATC ATC ACT ATT AAG GCT GAT GAT GGC AGT GAT ACT GTC ACT TTT ATG TTT GAA TCT CCC ACA CAA GAT MG ATT GCT GAC TTT GAA ATG M A CTC ATG GAC Ile ASP Ser Glu His Leu Gly IIe Pro Glu Ala Glu Tyr His Ala Ile Val Arg net Pro Ser AIa GIu Phe Ala Arg Ile Cys Lys Asp Leu Ser Ser I l e Gly Asp Thr Val Val Ile 16b 366 ATT GAT AGC G M CAT CTT GGA ATT CCA G M GCG G M TAC CAT GCT ATA GTT CGA ATG CCC TCA GCT GAG TTT GCT AGO ATA TGT AAG GAT CTT AGT AGC ATT GGT GAT ACA GTT GTC ATA 486

Ser Val Thr LYS Glu Gly Val Lys Phe Ser Thr Arg Gly Asp Ile Gly Thr Ala Asn Ile Val Cys Arg Gln Asn Thr Thr Val Asp Lys Pro Glu GIu Ala Thr Val I l e GIu net Asn 200 TCT GTG ACC A M OM GGT GTC AAG TTC TCA ACA AGA GGT GAT ATC GGA ACT GCA AAT ATT GTG TGC AGG CAG AAT ACC ACA GTA GAC AAF CCA GAG GAG GCC ACT GTG ATA GAG ATG M T

Glu Pro Val Ser Leu Thr Phe Ala Leu Arg Tyr net Asn Ser Phe Thr LYS Leu Ser Pro Leu Ser Ser Thr Val Thr Ile Ser Leu Ser Ser Glu Leu Pro Val Val Val Glu Tyr LYS 240 606 GM CCA GTT TCG TTG ACA TTT GCA TTG AGG TAC ATG AAT TCC TTT ACA AAG CTA AGC CCC TTG TCA AGC ACT GTT ACC ATT AGC CTA TCT TCT GAG CTT CCT GTT GTG GTT GAG TAT M G Ile Ala Glu net Gly Tyr IIe Arg Phe Tyr Leu Ala Pro Lys Ile Glu Glu Glu GIu Asp Glu Ser Lys Pro end 726 ATT GCT G M ATG GGT TAC ATA CGG TTC TAC TTG GCT CCC A M ATT GAA GAG GM GAA GAT GM AGC A M CCT TGAMTGTTACMTTGCAMATTATTGCMTTTTATGACTAGGTGTTTGTGTATGCTGMGAC

264

861 TGCM

B +pdl

1

TGCMTTCA~GCMCCMGA

net Leu G I U Leu Arg Leu Val Gln GIY Gly Leu Leu Lys LYS Val Leu Glu Ser I I e Lys Asp Leu Val Asn Asp Ale. Asn Phe Asp Cys Ser AIa Ser Gly Phe Ser Leu Gln Ala net 22 ATG CTG GAG CTA AGA TTA GTA CAG GGG GGT TTG CTC M G M A GTT CTT GAA TCA ATC AAA GAC CTC GTG AAT GAC GCT M T TTT GAT TGC TCG GCA TCT GGG TTT TCT CTA C M GCC ATG Lf

40

l.fS

ASP Ser Ser His Val Ala Leu Val Ala Val Leu Leu Arg Ser Glu Gly Phe GIu His Tyr Arg Cys Asp Arg Asn Ile Ser net GIy net Asn Leu GIy Asn net Ala Lys net Leu Arg 142 GAT TCG AGT CAC GTG GCG CTC GTG GCC GTG CTG CTC CGT TCA GAG GGG TTT GAG CAT TAT CGA TGT GAT CGG M C ATT TCA ATG GGG ATG AAC TTG GGG AAT AT6 GCT AAG ATG TTG AGG

80

CYS Ala GIY Asn ASP ASP Ile Val Thr Met LYS Ala ASP ASP ASP Gly ASP Val Ile Thr Phe net Phe Glu Ser Pro Thr Gln Asp Lys Ile Ser Asp Phe Glu net Lys Leu net ASP 120 262 TGT GCT 606 AAT GAT GAC ATT GTT ACG ATG M A GCT GAT GAT GAT GGT GAT GTT ATC ACT TTT ATG TTC GAA AGC CCG ACT CAA GAC AAG ATT TCT GAT TTT GAG ATG AAG CTG ATG GAT I l e ASP Ser Glu HIS Leu G ~ YI l e Pro Glu Ser Glu Tyr Glu Ala I l e Val Arg net Pro Ser Ala GIu Phe Ala Arg Ile Cys Lys Asp Leu Ser Thr Ile Gly Asp Thr Val Val Ile 160 382 ATT GAT AGT GAG CAT CTA GGG ATT CCA GAA TCT GAA TAT GAA GCT ATT GTG AGG ATG CCA TCT GCT GAA TTT GCT AGG ATC TGC AAA GAC CTG AGT ACC ATC GGT GAT ACT GTT GTG ATA

Ser Val Thr LYS Glu Gly Val LYS Phe Ser Thr Arg GI? ASP Ile Gly Thr AIa Asn l i e Val CYS Arg Gln Asn Thr Ser Val Asp Lys Pro GIu ASP Ala Thr Ile lie GIu net Gin 200 502 TCT GTG ACG M G GAA CGT GTG M G TTC TCC ACA CGA GGT GAT ATT GGG ACT GCA AAT ATT GTT TGC AGG CAG M C ACT TCT GTG GAC AAG CCA GAA GAT GCT ACT AT1 ATT GAG ATG C M Glu Thr Val Ser Leu Thr Phe AIa Leu Arg Tyr net Asn Ser Phe Thr LYS Ala Thr Pro Leu Ala Asn GIn Val Thr Ile Ser Leu Ser Ser Glu Leu Pro Val Val Val Glu Tyr LYS 240 622 GAA ACA GTC TCA TTG ACA TTT GCG CTG AGG TAT ATG M T TCT TTT ACA MG GCA ACT CCT CTG GCA M T CAA GTG ACC ATC AGC CTA TCA TCT GAA CTT CCA GTG GTG GTT GAG TAC AAA Ile Ala Glu net Gly Tyr I l e Arg Tyr Tyr Leu A18 Pro LYS Ile Glu Glu GIu ASP GIu A h AIa Asn Tyr AIa Gln Pro Ala Gln Asn Ser Ala Ala Ala AIa Thr Ser ASR Asn Gly 280 742 ATC GCA GAG ATG GGC TAC ATC AGA TAT TAT TTG GCT CCA M A ATT GAA GAG GAG GAC GAG GCT GCA AAT TAT GCA CAG CCT GCA C M AAC TCT GCT GCA GCC GCC ACA TCA AAC AAT GGA Val Thr Lys LYS Asn GIu Gly Asn Asn LYS Val Asp Ser Lys LYS Arg AIa Ile Lys Ser GIu Phe Val Asp Asp Ser GIu Ala AIa Thr Asp Ala Gln Pro Gln AIa Lys Ala Lys Thr LYS 320 862 ACT MG M A M T GAG GOT M T AAC M A GTT GAT TCC M G M A AGO GCA ATA M G AGC GAA TTT GTT GAT GAC AGT GAG GCT GCA ACA GAT GCG C M CCT CAG GCT AAG GCT A M ACA AM C TG Thr Glu Ala Gly Glu ASP ASP Asp Val Glu Val net ASP Thr LYS Pro LYS Asn Glu Pro Asp Asp Gly Asp Glu Val Met Glu Thr Lys Pro Lys Thr Glu Ser Asn Gly Glu Val Glu 360 982 ACC GAA GCT GGA GAA GAT GAC GAT GTT GAA GTA ATG GAC ACC MG CCA MG M T GAA CCT GAT GAT GGT GAT GAA GTA ATG GAA ACC AAG CCA AAG ACT GM AGC AAT GGT G M GTG G M Val net Asp IIe GIu end

89-

365

1102 GTT ATG GAT ATT GAG TAGTTTTCCAGCTGMCATTATCATTTCTATTACTAGTMTTTTCCCAAGTAGAMCTMMMCCTTTTTAGTGAAGCTMTATTTAAGTTCTAGGTGATGTCTATTGTCATTTTGTGTAGTCTATATGACCCTTTGG 1256 GAGCTAGATCTGCTAGGCACGGCATATATGATGCTTATATTCTAACTCTAG

1,23

Fig. 1. Nucleotide and deduced amino acid sequences of carrot cDNA clones for PCNA homologs. (A) Clone 4.10 for ‘typical’ PCNA; (B) the composite sequences of clones P3 and 1.13 for ‘larger’ PCNA. The ends of the cDNA clones are indicated in (B). Within the overlapping region, sequences agree a t all positions except for nucleotides 891,908 and 909. Nucleotides of 1.I3 that are different to those of P3 are shown below the line and the different amino acid at codon 296 is shown above.

However, the pattern tends to be complicated by the introns within the genes. Further study is necessary to clarify how many copies of the PCNA-like gene are present on the carrot genome. Of obvious importance is an understanding of the biological functions of the two PCNA homologs. The two molecular species may play the same role in DNA replication. However, it is also possible that they play slightly different roles. Another possibility is that the larger PCNA has exactly the same function as that of the typical one after being processed by a protease. Unfortunately, however, we do not yet have a

good system for resolving this problem. There has been no report of an in vitro DNA replication system in plants, even the presence of a plant S-like DNA polymerase has been described only recently [25].One feasible approach to assess their roles will be the use of a replication system of simian virus 40 in vitro [4-61. Since the structures of PCNA species in eukaryotes are highly conserved, plant PCNA may exhibit some activity in a mammalian system. This will be an important subject of investigation in the future. In carrot, somatic embryogenesis is accompanied by localized and rapid cell proliferation [I I]. We have shown that

A

B

A 1 2 3 4

1 2 3

1 2 3 4

R 1 2 3

C 1

2

3

kb -50 kO

-

-

19.3

-

7.7 6.2 4.3 3.5 2.7

-

1.9 1.5

-

0.9

-

0.4

-

1 . 5 I+

19 kD

Fig. 2. Southern-blot-hybridization analysis. Carrot genomic DNA samples (2 pg/lane), digested with EcoRI (lane I), Hind111 (lane 2), PsrI (lane 3) or XhuI (lane 4), were probed with entire cDNA fragments of typical PCNA 4.10 (A) and larger PCNA P3 (B). A phage i-EcoT141 digest served as molecular size markers.

TCP

1

1.CP

1

RP

1

TCP

51

LCP

51

RP

51

TCP

101

LCP

LO1

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- 3 3 kD

Fig. 4. Expression of the larger PCNA (A) and the typical PCNA (B) in carrot somatic embryos. 1 pg of each of poly (A)-rich RNA from proembryogenic masses (lane l), embryos (globular stage/heart stage/ torpedo stage = 1 :2:4, fresh mass; lane 2) and unorganized calli (lane 3) was probed with the 32P-labeled cDNA fragment of clone 1 .I3 (1 x lo6 cpm/ml hybridization mixture), then washedunda conditions of high stringency The sheet was reprobed with the 32Plabeled clone 4.10 fragment under the same conditions as above. Autoradiography was carried out for 5 d (A) and overnight (B). To confirm the integrity of each RNA preparation, in vitro translation was carried out and the 3H-labeled proteins were analyzed (C).

groups of cDNA for PCNA will be valuable for histological studies, involving in situ hybridization, on carrot embryogenesis. MLELRLVQGSLLKKVMDSIKDLVNDANFUCSATGFSLQAMDSSHVALVAV ********* ***** *************** ***************** Finally, we would like to point out that a second PCNA MLELRLVQGGLLKKVLESIKDLVNDANFU~SASGFSLQAMDSSHVALVAV ********* ******* * ** *****x* ************a*** MLELRLV9GSLLKKVLEAIRELVTUANFDCSGTGFSLQAMUSSHVALVAL homolog may also be present in species other than carrot. Leibovici et al. [27] found a longer mRNA for PCNA during LLRSEGFEHYRCDRNISMGMNLGNMAKMLKCAGNDDIITIKADDGSU~VT **************:***a*****:**** ******* * **** * * oogenesis of the amphibian, Xenopus luevis. They also detected LLRSEGFEHYRCDRNISMGMNLGNMAKMLRCAGNDDIVTMKADDDGDVIT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * a protein of 43 kDa that cross-reacts with anti-PCNA serum. LLRSEGFEHYRCDRNLSMGMNLNNMAKMLRCAGNDDIITIKADDGSDTVT These observations might indicate the presence of a larger FMFESPTQDKIADFEMKLMDIDSEHLGIPEAEYHAIVRMPSAEFARICKU * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * PCNA in Xenopus. In this respect, it is interesting to note the FMFESPTQUKISDFEMKLMUIDSEHLGIPESEYEAIVRMPSAEFARICKD * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * recent identification of processed pseudogenes in human and FMFESPNQDKIADFEMKLMDIDSEHLGIPDSEYQAIVRMPSSEFSRICKD mouse genomes [28, 291, which may be generated by reverse transcription of the typical PCNA mRNA. The carrot larger LSSIGDTVVISVTKEGVKFSTRGDIGTANIVCRQNTTVUKPEEATVIEMN ** ................................. ***** ** *** PCNA might be generated in a similar process, except that it LSTIGDTVVlSVTKEGVKFSTRGOIGTA”TSVDKPEDATlIEMQ * * ***** ************ ************* remained functional. The polymerase-chain reaction [30] will LSSIGDTVIISVTKEGVKFSTAGDIGTANIVCRQNKTVUKPEDATIIEMQ be an effective method for detecting novel PCNA homologs EPVSLTFALRYMNSFTKLSPLSSTVTISLSSE~PVVVEYKIAEMGYIRF~ * *************** ** ........................ * in other species, The second putative PCNA may provide a ETVSLTFALRYMNSFTKATPLANQVTISLSSELPVVVEYKIAEMGYI~~~ * * * . . . . . . . . . . . . . . . . . . . . . . . . . * new insight for studies on DNA replication in eukaryotes. EPVSLTFALRYMNSFTKASPLSEQVTISLSSELPVVVEYKIAEMGYIRFY

TCP

151

L.CP

151

lip

151

TCP

201

I.CV

aoi

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201

TCP

251

LAPKIEEEEDESKP

I C P

251

LAPKIEEEDEAANYAQPAQNSAAAATSNNGTKKNEGNNKV

RP

251

LAPKIEEDEEMKS

* * * * * * I * * * * * *

******%******I**

********

*******

*

( 2 9 1 - 3 ~ 5 :

Fig.3. Alignment of amino acids of the typical carrot PCNA (TCP, upper line), the larger carrot PCNA (LCP, middle line) and the rice PCNA (RP, lower line; 191). Identical amino acids arc indicated by asterisks

the expression of mRNA for both the PCNA homologs is induced during the process. The typical PCNA was expressed at a higher level than the larger one. Smith et al. [26] identified a nuclear protein of 4.5 kDa that is expressed in correlation with embryogenesis. Interestingly, they discussed that the protein might be a carrot homolog of PCNA. However, the molecular mass of the protein was even larger than those of known PCNA. The discovery of the second larger PCNA may explain this discrepancy. It is possible that different PCNA homologs are used in different embryogenic programs. Therefore, in addition to the carrot mitotic cyclin cDNA [1.5], the two

We are thankful to Drs T. Moriuchi, T. Murata and Y . Egawa for discussions and Mrs I. Muramatsu for the excellent technical assistance. This work was supported by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan.

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