The EMBO Journal vol.10 no.9 pp.2681 -2688, 1991
Isolation and characterization of cDNA clones for plant cyclins
Shingo Hata"3, Hiroshi Kouchi', Iwao Suzuka1'4 and Tetsuro Ishii2 'National Institute of Agrobiological Resources, 2-1-2 Kannondai. Tsukuba, Ibaraki 305, and 2Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305, Japan 3Present address: Faculty of Science, Himeji Institute of Technology, Kamigori, Ako-gun, Hyogo 678-12, Japan 4Present address: National Institute of Animal Health. 3-1-1 Kannondai, Tsukuba, Ibaraki 305, Japan Communicated by T.Hunt
We have isolated and sequenced a carrot cDNA and two soybean cDNAs encoding mitotic cyclin homologs. The soybean clones were derived from nearly identical cognate genes. The carrot cyclin and soybean cyclins were slightly more similar to A-type and B-type cyclins thus far defined, respectively. However, they had divergent amino acid sequences in the portion that is most highly conserved in known cyclins and we could not easily include them in either of the phylogenetic types. Since the homology between carrot and soybean cyclins was low, each of them might define a novel and distinct type. The mRNA of carrot cyclin, 1.5 kb in length, was expressed concomitant with somatic embryogenesis of cultured cells. Expression of soybean cyclin mRNAs, 1.6 kb in length, was localized in proliferating parts of seedlings. As in the case of cyclin genes of marine invertebrates, microinjection of a synthetic mRNA for the soybean cyclin induced the maturation of Xenopus oocytes. Other cyclin genes may be present because, on Southern blot analysis of soybean genomic DNA, the isolated soybean cDNA probe hybridized with additional genes under low stringency. Key words: cell proliferation/cyclin/cDNA cloning/plant
Introduction Cyclins, homologs of the fission yeast cdc]3 gene product, each comprise the regulatory subunit of a heterodimeric protein kinase (cdc2 kinase or maturation-promoting factor). Their association with the catalytic p34cdc2 subunit, a homolog of the cdc2+ gene product, is primarily required for eukaryotic cells to undergo mitosis or meiosis. In addition, their breakdown causes cells to shift to the GI phase (see Nurse, 1990; Lewin, 1990; Pines and Hunter, 1990a, for reviews). Cyclins were first detected in marine invertebrates (Evans et al., 1983; Swenson et al., 1986; Standart et al., 1987). Since then, their presence has been demonstrated in a fission yeast (Solomon et al., 1988; Goeble and Byers, 1988), fly (Lehner and O'Farrell, 1989), frog (Minshull et al., 1989) and man (Pines and Hunter, 1989). Furthermore, the presence of two distinct types of cyclins, cyclins A and B, has been reported in clam
can
Oxford University Press
(Westendorf et al., 1989), fly (Whitfield et al., 1989; Lehner and O'Farrell, 1990a), frog (Minshull et al., 1990) and man (Wang et al., 1990; Pines and Hunter, 1990b). The independently conserved A- and B-type cyclins seem to play distinct roles in the cell cycle (Lehner and O'Farrell, 1990a; Pines and Hunter, 1990b; Minshull et al., 1990). However, nothing is known about the cyclins of higher plants. Studies on the regulation of plant cell proliferation have great potential importance for plant breeding and crop yields. We have isolated and characterized both genomic clones and cDNAs for plant proliferating cell nuclear antigen (PCNA, an auxiliary protein of DNA polymerase 6; this should not be confused with cyclins (Suzuka et al., 1989, 1991). It has also been reported that plants have p34cdc2 or related proteins (John et al., 1989; Feiler and Jacobs, 1990; Hata, 1991). In the present paper, we report the isolation and characterization of cyclin cDNAs from carrot and soybean. Interestingly, they had characteristic structures. They may serve as molecular probes for research on plant cell proliferation and differentiation.
Results cDNA cloning and sequencing of plant cyclins The published amino acid sequences for mitotic cyclins have two regions showing a particularly high degree of conservation (Minshull et al., 1989; Lehner and O'Farrell, 1989; Pines and Hunter, 1989). Based on their sequences, two redundant oligonucleotides were synthesized (see Materials and methods). The probes were then used to screen cDNA libraries of differentiating carrot embryos and growing soybean root nodules. Approximately 1.2 x 105 clones of each of the libraries were screened, and one carrot clone and two soybean clones were isolated. The carrot cDNA, designated as C13-1, is 1244 bp in length and has an open reading frame for 341 amino acids (Figure la). It has two regions corresponding to oligonucleotide probes; the first and second probes had one and six mismatches, respectively. The clone seems to be truncated at its 5' end, because on Northern blot analysis the size of the transcript was found to be 1.5 kb (see Figure 3a). One soybean cDNA, designated as S13-6, is 1596 bp in size and has an open reading frame for a 454 amino acid polypeptide, the predicted molecular mass of which is 50 105 (Figure lb). The size of the cDNA excellently corresponds to that of the mRNA (see Figure 4a). Moreover, primer extension analysis showed that the S 13-6 cDNA lacks less than 25 nucleotides from the 5' end of the mRNA (data not shown). Therefore, we assume that the first ATG codon represents the translation initiation site, although no in-frame stop codon was found upstream of the open reading frame. The other soybean clone, designated as S 13-7, is a partial cDNA of 954 bp in length (Figure lb). Obviously, it encodes a close cognate of S 13-6. Regions corresponding to the -
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S.Hata et al.
C13-1 C13-1
A P S N T T P E P A S K R R V V L G E I CTGCTCCATCAATGACCACACCAGAGCCAGCCTCCAAGAGGAGGGTTGTGCTGGGAGAGATT
(20)
S N N S S A V S G N E D L L C R E F E V P K C V A Q K K R K R G V K E D V G V D 63 TCGAACAATTCGAGTGCTGTTTCTGGAAATGAGGACTTGTTGTGCAGGGAGTTTGAGGTGCCCAAGTGTGTAGCACAGAAGAAAAGGAAGAGGGGTGTGAAAGAAGATGTGGGTGTTGAT
(60)
1
C13-1
F G E K F D D P Q N C S A Y V S D V Y E Y L K Q M E N E T K R R P N N N Y I E Q (100) 183 TTCGGTGAGAAATTTGATGACCCACAGATGTGTTCTGCTTATGTTTCTGATGTTTATGAGTATCTTAAGCAAATGGAGATGGAGACTAAAAGAAGACCAATGATGAATTATATTGAGCAA
C13-1
V Q K D V T S N N R G V L V D W L V E V S L E Y K L L P E T L Y L A I S Y V D R (140) 303 GTCCAAAAAGATGTGACCTCTAACATGAGAGGAGTTTTGGTGGATTGGTTGGTTGAGGTTTCCTTGGAGTACAAACTTCTTCCAGAAACTCTGTACTTGGCCATTTCGTATGTTGATAGA
C13-1
Y L S V N V L N R Q K L Q L L G V S S F L I A S K Y E E I K P K N V A D F V D I (180) 423 TACTTATCAGTAAATGTTCTGAATAGACAAAAGCTGCAACTTCTCGGTGTTTCTTCGTTTCTTATTGCCTCAAAATATGAAGAAATCAAGCCCAAGAACGTTGCAGATTTTGTTGATATT
C13-1
T D N T Y S Q Q E V V K N E A D L L K T L K F E N G S P T V K T F L G F I R A V (220) 543 ACTGATAACACGTACTCACAGCAAGAGGTAGTTAAAATGGAAGCTGATCTCCTCAAAACTCTCAAGTTTGAAATGGGAAGCCCAACAGTTAAAACCTTTCTGGGATTTATCAGAGCTGTA
C13-1
Q F N P D V P K L K F E F L A N Y L A E L S L L D Y G C L E F V P S L I A A S V (260) 663 CAAGAAAATCCTGATGTTCCTAAATTGAAGTTTGAATTCTTGGCAAATTACCTTGCAGAACTCAGTTTGTTGGACTATGGATGCCTCGAGTTTGTACCTTCTTTGATAGCTGCATCTGTA
C13-1
T F L A R F T I R P N V N P W S I A L Q K C S G Y K S K D L K E C V L L L H D L (300) 783 ACATTCCTTGCACGATTTACCATTAGACCAAATGTGMTCCTTGGAGTATAGCTTTACAAAAATGTTCTGGATATAAATCAAAGGATTTGAAAGAATGCGTTCTTCTCCTGCACGATCTG
C13-1
Q N G R R G G S L S A V R D K Y K K H K F K C V S T L S P A P E I P E S I F N D (340) 903 CAAATGGGAAGAAGAGGGGGTTCTCTGTCGGCTGTTAGAGA('AAGTACAAGAAGCATAAGTTCAAATGTGTGTCAACATTATCTCCAGCTCCAGAGATCCCTGAATCAATTTTCAATGAT
(341) V end C13-1 1023 GTTTAAGAATCCATGATGCCAATATGCTTGTACAAATCAACCGTGTGCCTGGTGCATATGATCTGAAGCAATTTGATGCTAATTTCTCCAAATTGGTCACTGTAGGGAGCTAGTTTTGTA C13-1 1143 TCTTTTATGTTTCCTACGAACAACATGGGCGTAAGTATTAGAGTGACATCCATTAGAAGTTGGATTTAGGATGTAACTAGCTTTATTTTAGTTCAGTTGAG
N A S R I V Q Q Q Q A R G E A V V G G G
S13-6
20
TTTCAATTCAAGACCAAAGAAGAATCTCTCACAAATGGCGTCAAGAATTGTTCAACAACAACAAGCCAGAGGTGAAGCAGTAGTAGGGGGAGGA
1
K Q Q K K N G V A D G R N R K A L G D I G N L A N V R G V V D A K P N R P I T R 95 AAACAACAGAAGAAGAACGGTGTTGCTGATGGAAGGAACCGCAAAGCATTGGGTGACATTGGGAATTTGGCCAATGTAAGAGGCGTTGTTGATGCCAAACCAAATCGCCCCATCACAAGG
60
S13-6
S F G A Q L L A N A Q A A A A A D N S K R Q A C A N V A G P P A V A N E G V A V 215 AGTTTTGGTGCACAATTACTTGCCAATGCACAAGCAGCAGCAGCTGCTGATAATAGCAAGAGACAAGCATGTGCTAATGTGGCTGGTCCTCCTGCCGTTGCTAATGAAGGAGTTGCGGTG
100
S13-6
A K R A A P K P V S K K V I V K P K P S E K V T D I D A S P D K K E V L K D K K 335 GCCAAAAGAGCGGCCCCCAAACCGGTTTCGAAGAAAGTGATCGTAAAACCTAAGCCTTCTGAGAAAGTCACTGACATTGATGCTAGTCCAGATAAGAAGGAGGTCCTGAAAGACAAGAAG
140
S13-6 S13-6
K E G D A N P K K K S Q H T L T S V L T A R S K A A C G I T N K P K E Q I I D I 180 455 AAGGAAGGAGATGCCAACCCCAAGAAGAAATCGCAACACACTCTTACTTCAGTGCTCACTGCTAGGAGCAAGGCAGCATGTGGCATAACAAACAAACCAAAGGAACAGATTATTGACATT
S13-6 S13-7
S13-6 S13-7
D A S D V D N E L A A V E Y I D D I Y K F Y K L V E N E S R P H D Y I G S Q P E 220 575 GATGCTTCTGATGTTGACAATGAGCTTGCTGCTGTGGAGTACATTGATGACATTTACAAGTTCTACAAACTTGTTGAGAACGAGAGCCGCCCCCACGACTATATTGGTTCACAGCCTGAG . C....A... A. A.A C ..........A 1 HinclI H (24) D I N E R N R A I L V D W L I D V H T K F E L S L E T L Y L T I N I I D R F L A V 695 ATAAATGAGAGGATGAGAGCTATTCTGGTTGATTGGCTGATAGATGTTCACACCAAGTTTGAACrTTCACTTGAGACCCTTTACTTGACCATCAATATAATTGATCGGTTTTTAGCAGTT C. C.T..A .C. 75.
260
(64) S13-6 S13-7
K T V P R R E L Q L V G I S A N L N A S K Y E E I W P P E V N D F V C L S D R A 815 AAGACAGTTCCAAGGAGGGAACTGCAATTGGTTGGCATCAGTGCCATGCTGATGGCATCCAAATATGAAGAAATCTGGCCCCCTGAGGTTAATGACTTTGTCTGCCTCTCAGATAGGGCT ........................ G 195 ....... .............................................................
300
(104) S13-6 S13-7
Y T H E H I L T N E K T I L N K L E W T L T V P T P L V F L V R F I K A S V P D 340 935 TACACTCATGAACATATACTAACCATGGAGAAAACCATATTGAACAAGCTGGAATGGACTTTGACTGTGCCAACACCTTTAGTTTTCCTTGTTCGTTTTATCAAGGCATCAGTCCCAGAT 315. . G.GG A.T..T. T. A. G.G. A F A (144) Q
S13-6 S13-7
U E L D N N A H F L S E L G N N N Y A T L N Y C P S N V A A S A V L A A R C T L 380 1055 CAGGAGTTGGACAACATGGCTCATTTCCTGTCTGAGTTGGGAATGATGAACTACGCAACCTTAATGTACTGCCCGTCAATGGTTGCTGCCTCAGCAGTGCTTGCAGCAAGATGCACTCTG T .A. TA. T. 435. A. N (184) F E
S13-6 S13-7
N K A P F W N E T L K L H T G Y S Q E Q L N D C A R L L V G F Y S T L E N G K L 420 1175 AACAAGGCTCCTTTTTGGAATGAGACACTTAAGCTGCACACTGGTTACTCACAAGAGCAACTCATGGATTGTGCTAGACTATTGGTGGGGTTCTACTCCACTCTTGAGAATGGAAAGCTT 555 CC....C.T. ...GC. HindM (224) H L G
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Plant cyclins
S13-6
S13-7
454 R V V Y R K Y S D P Q K G A V A V L P P A K F L L P E G S A S Q H S end 1295 AGGGTTGTGTACAGAAAATATTCTGACCCTCAGAAGGGTGCTGTTGCTGTGCTCCCACCCGCTAAATTTCTTCTGCCTGAAGGTTCTGCTTCCCAACATAGTTAAAAAGAAGAAAAGG-TG G.C..A .A. ---T. C .G. ..-. 675. (257) S end
GAGAGGAGTAGT-------------CTTTTATGATTTTCAGCAAATAATCATTTGTATTTTTTTTCTTCATAAGGTACAAGAATTGATTTTTATATAAGATGCAAGTTTGTCACTTGCCA .
S13-6 S13-7
1413 791
S13-6 S13-7
1520 ATTGTCACAGCATCAAA-----CTTTTATT-TGGGGTTCACGTTCTCTGAGTGTAGTGTGCAACCAGGTTGCAGTTATATACA 902 ........AT....T.TCTAA . C... AA .CA.
T..C.G.T.GTG
. G. -AA-.....
C.. .GATAATATAGAGC .........-.-.
G.
Fig. 1. Nucleotide and deduced amino acid sequences of plant cyclin cDNAs. (a) Carrot clone C13-1. (b) Soybean clones S13-6 and S13-7. S13-7 nucleotides identical to those of S13-6 are denoted by dotted lines, and only differences in nucleotides or amino acids are noted. Dashes denote gaps. The positions corresponding to the oligonucleotide probes are indicated by solid underlines. The restriction sites for HincIl and HindIlI are indicated by wavy underlines. ( ) C,123 1 A e-on. C.1am A
eon
144--53)
KESVC,VDFGEKFDDPQMCSAYVSDVYFYI,KQMEMETKRRPMMNYIEQVQKDVTS SLPEEEKPLDREAVILTVPEYEEDIYNYLRQAEM--KNRAKPGYM-KRQTDITT **** Q
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* * K VRFI KASVPDQELDNMA VNDFVCLSDRAYTHEHILTMEKTILNKLEWTLTVPTPILVFL HT A GRPL LHFLRR SK L L I ME YT D DF YAPEVADFVT I SDNAYTKKE I LEMEQH I LKKLNFSFGRPLCLHFLRRDSKAGQVDANKHTLA
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A- and Fig. 2. Homology among plant cyclins and other cyclins. (a) Amino acid sequence comparison of C13-1 with the consensus sequences of asterisks open and Solid B. cyclin clam with and the consensus with sequences. of S13-6 A. (b) Comparison B-type cyclins, and with clam cyclin indicate distinctive amino acids belonging to the A-type and B-type consensus sequences, respectively. The conserved arnino acid 'Z' in the 'destruction box' represents either D, E or N. Gaps were introduced for optimal alignment. Sequence motifs mentioned in the text are boxed.
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oligonucleotides were found in both clones; the first and second probes had two and three mismatches, respectively. Sequence comparison of the coding regions of the soybean cDNAs revealed 95 % identity at both the nucleotide and the amino acid level. The nucleotide homology was only 74% in the 3' noncoding region. Of note among the differences are the deletion of one amino acid and the different usage of termination codons, suggesting selective pressure at the protein level. The two soybean cDNAs cross-hybridized under high stringency, but the carrot cDNA, C13-1, did not hybridize at all to the soybean cDNAs even under low stringency (data not shown). Relationships among the cyclins Figure 2 shows the comparison of the amino acid sequences of plant cyclins with the consensus sequences of A- and Btype cyclins reported in the latest papers (Minshull et al., 1990; Glotzer et al., 1991). The figure also shows the structures of cyclins of clam, in which both types were identified for the first time (Swenson et al., 1986; Westendorf et al., 1989). Carrot clone C 13-1 was more similar to clam cyclin A (37 % identity) than clam cyclin B (32%) in the C-terminal two-thirds of the polypeptides. Although its putative recognition site of ubiquitin-
conjugating degradation system (destruction box), RVVLGEISN, is B-type (Glotzer et al., 1991), it has 30 distinctive amino acids belonging to the A-consensus sequence (solid asterisks in Figure 2a), as opposed to 17 amino acids belonging to the B-consensus sequence (open asterisks). It is noteworthy that the central portion of the cyclin box, KYEEIKPKN, is unusual among the known cyclins. As shown in Figure 2b, a small region of soybean clone S13-6 had the B-type motif of 'destruction box' (Glotzer et al., 1991). The C-terminal two-thirds of 513-6 was more homologous to that of clam cyclin B (40% identity) than to that of clam cyclin A (31 %), although the numbers of distinctive amino acids are the same (19 each for the Aand B-consensus sequences). The LVFLVRFIK sequence diverged from the hallmark motif, LHFLRRXSK, which is strictly conserved in all other B-type cyclins (Minshull et al., 1989; Pines and Hunt, 1987; Pines and Hunter, 1989). The central portion of the cyclin box, KYEEIWPPE, was also. The above sequence homology was not high enough to assign them to any known type; for example, A-type cyclins of clam and fly are 45 % identical in the conserved two-thirds of the proteins, and their B-type cyclins are 47% identical in this region (Lehner and O'Farrell, 1990a). In addition, C 13-1 and S 13-6 are only 32% identical in the conserved region. Therefore, C13-1 and S13-6 might define novel and distinct types of cyclins. We tentatively call C 13-1 and S 13-6 'A-like cyclin' and 'B-like cyclin', respectively. uncommon
Expression of the cyclins Somatic embryogenesis of carrot cells (Steward et al., 1958) has been an attractive system for research on plant development. It is accompanied with the localized and rapid cell proliferation. To investigate the correlation between the embryogenesis and cyclin expression, we carried out an RNA blot hybridization experiment. As shown in Figure 3a, the mRNA of carrot A-like cyclin, 1.5 kb in size, was expressed at a much higher level in the somatic embryos 2684
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Isolation and characterization of cDNA clones for plant cyclins
Shingo Hata"3, Hiroshi Kouchi', Iwao Suzuka1'4 and Tetsuro Ishii2 'National Institute of Agrobiological Resources, 2-1-2 Kannondai. Tsukuba, Ibaraki 305, and 2Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305, Japan 3Present address: Faculty of Science, Himeji Institute of Technology, Kamigori, Ako-gun, Hyogo 678-12, Japan 4Present address: National Institute of Animal Health. 3-1-1 Kannondai, Tsukuba, Ibaraki 305, Japan Communicated by T.Hunt
We have isolated and sequenced a carrot cDNA and two soybean cDNAs encoding mitotic cyclin homologs. The soybean clones were derived from nearly identical cognate genes. The carrot cyclin and soybean cyclins were slightly more similar to A-type and B-type cyclins thus far defined, respectively. However, they had divergent amino acid sequences in the portion that is most highly conserved in known cyclins and we could not easily include them in either of the phylogenetic types. Since the homology between carrot and soybean cyclins was low, each of them might define a novel and distinct type. The mRNA of carrot cyclin, 1.5 kb in length, was expressed concomitant with somatic embryogenesis of cultured cells. Expression of soybean cyclin mRNAs, 1.6 kb in length, was localized in proliferating parts of seedlings. As in the case of cyclin genes of marine invertebrates, microinjection of a synthetic mRNA for the soybean cyclin induced the maturation of Xenopus oocytes. Other cyclin genes may be present because, on Southern blot analysis of soybean genomic DNA, the isolated soybean cDNA probe hybridized with additional genes under low stringency. Key words: cell proliferation/cyclin/cDNA cloning/plant
Introduction Cyclins, homologs of the fission yeast cdc]3 gene product, each comprise the regulatory subunit of a heterodimeric protein kinase (cdc2 kinase or maturation-promoting factor). Their association with the catalytic p34cdc2 subunit, a homolog of the cdc2+ gene product, is primarily required for eukaryotic cells to undergo mitosis or meiosis. In addition, their breakdown causes cells to shift to the GI phase (see Nurse, 1990; Lewin, 1990; Pines and Hunter, 1990a, for reviews). Cyclins were first detected in marine invertebrates (Evans et al., 1983; Swenson et al., 1986; Standart et al., 1987). Since then, their presence has been demonstrated in a fission yeast (Solomon et al., 1988; Goeble and Byers, 1988), fly (Lehner and O'Farrell, 1989), frog (Minshull et al., 1989) and man (Pines and Hunter, 1989). Furthermore, the presence of two distinct types of cyclins, cyclins A and B, has been reported in clam
can
Oxford University Press
(Westendorf et al., 1989), fly (Whitfield et al., 1989; Lehner and O'Farrell, 1990a), frog (Minshull et al., 1990) and man (Wang et al., 1990; Pines and Hunter, 1990b). The independently conserved A- and B-type cyclins seem to play distinct roles in the cell cycle (Lehner and O'Farrell, 1990a; Pines and Hunter, 1990b; Minshull et al., 1990). However, nothing is known about the cyclins of higher plants. Studies on the regulation of plant cell proliferation have great potential importance for plant breeding and crop yields. We have isolated and characterized both genomic clones and cDNAs for plant proliferating cell nuclear antigen (PCNA, an auxiliary protein of DNA polymerase 6; this should not be confused with cyclins (Suzuka et al., 1989, 1991). It has also been reported that plants have p34cdc2 or related proteins (John et al., 1989; Feiler and Jacobs, 1990; Hata, 1991). In the present paper, we report the isolation and characterization of cyclin cDNAs from carrot and soybean. Interestingly, they had characteristic structures. They may serve as molecular probes for research on plant cell proliferation and differentiation.
Results cDNA cloning and sequencing of plant cyclins The published amino acid sequences for mitotic cyclins have two regions showing a particularly high degree of conservation (Minshull et al., 1989; Lehner and O'Farrell, 1989; Pines and Hunter, 1989). Based on their sequences, two redundant oligonucleotides were synthesized (see Materials and methods). The probes were then used to screen cDNA libraries of differentiating carrot embryos and growing soybean root nodules. Approximately 1.2 x 105 clones of each of the libraries were screened, and one carrot clone and two soybean clones were isolated. The carrot cDNA, designated as C13-1, is 1244 bp in length and has an open reading frame for 341 amino acids (Figure la). It has two regions corresponding to oligonucleotide probes; the first and second probes had one and six mismatches, respectively. The clone seems to be truncated at its 5' end, because on Northern blot analysis the size of the transcript was found to be 1.5 kb (see Figure 3a). One soybean cDNA, designated as S13-6, is 1596 bp in size and has an open reading frame for a 454 amino acid polypeptide, the predicted molecular mass of which is 50 105 (Figure lb). The size of the cDNA excellently corresponds to that of the mRNA (see Figure 4a). Moreover, primer extension analysis showed that the S 13-6 cDNA lacks less than 25 nucleotides from the 5' end of the mRNA (data not shown). Therefore, we assume that the first ATG codon represents the translation initiation site, although no in-frame stop codon was found upstream of the open reading frame. The other soybean clone, designated as S 13-7, is a partial cDNA of 954 bp in length (Figure lb). Obviously, it encodes a close cognate of S 13-6. Regions corresponding to the -
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S.Hata et al.
C13-1 C13-1
A P S N T T P E P A S K R R V V L G E I CTGCTCCATCAATGACCACACCAGAGCCAGCCTCCAAGAGGAGGGTTGTGCTGGGAGAGATT
(20)
S N N S S A V S G N E D L L C R E F E V P K C V A Q K K R K R G V K E D V G V D 63 TCGAACAATTCGAGTGCTGTTTCTGGAAATGAGGACTTGTTGTGCAGGGAGTTTGAGGTGCCCAAGTGTGTAGCACAGAAGAAAAGGAAGAGGGGTGTGAAAGAAGATGTGGGTGTTGAT
(60)
1
C13-1
F G E K F D D P Q N C S A Y V S D V Y E Y L K Q M E N E T K R R P N N N Y I E Q (100) 183 TTCGGTGAGAAATTTGATGACCCACAGATGTGTTCTGCTTATGTTTCTGATGTTTATGAGTATCTTAAGCAAATGGAGATGGAGACTAAAAGAAGACCAATGATGAATTATATTGAGCAA
C13-1
V Q K D V T S N N R G V L V D W L V E V S L E Y K L L P E T L Y L A I S Y V D R (140) 303 GTCCAAAAAGATGTGACCTCTAACATGAGAGGAGTTTTGGTGGATTGGTTGGTTGAGGTTTCCTTGGAGTACAAACTTCTTCCAGAAACTCTGTACTTGGCCATTTCGTATGTTGATAGA
C13-1
Y L S V N V L N R Q K L Q L L G V S S F L I A S K Y E E I K P K N V A D F V D I (180) 423 TACTTATCAGTAAATGTTCTGAATAGACAAAAGCTGCAACTTCTCGGTGTTTCTTCGTTTCTTATTGCCTCAAAATATGAAGAAATCAAGCCCAAGAACGTTGCAGATTTTGTTGATATT
C13-1
T D N T Y S Q Q E V V K N E A D L L K T L K F E N G S P T V K T F L G F I R A V (220) 543 ACTGATAACACGTACTCACAGCAAGAGGTAGTTAAAATGGAAGCTGATCTCCTCAAAACTCTCAAGTTTGAAATGGGAAGCCCAACAGTTAAAACCTTTCTGGGATTTATCAGAGCTGTA
C13-1
Q F N P D V P K L K F E F L A N Y L A E L S L L D Y G C L E F V P S L I A A S V (260) 663 CAAGAAAATCCTGATGTTCCTAAATTGAAGTTTGAATTCTTGGCAAATTACCTTGCAGAACTCAGTTTGTTGGACTATGGATGCCTCGAGTTTGTACCTTCTTTGATAGCTGCATCTGTA
C13-1
T F L A R F T I R P N V N P W S I A L Q K C S G Y K S K D L K E C V L L L H D L (300) 783 ACATTCCTTGCACGATTTACCATTAGACCAAATGTGMTCCTTGGAGTATAGCTTTACAAAAATGTTCTGGATATAAATCAAAGGATTTGAAAGAATGCGTTCTTCTCCTGCACGATCTG
C13-1
Q N G R R G G S L S A V R D K Y K K H K F K C V S T L S P A P E I P E S I F N D (340) 903 CAAATGGGAAGAAGAGGGGGTTCTCTGTCGGCTGTTAGAGA('AAGTACAAGAAGCATAAGTTCAAATGTGTGTCAACATTATCTCCAGCTCCAGAGATCCCTGAATCAATTTTCAATGAT
(341) V end C13-1 1023 GTTTAAGAATCCATGATGCCAATATGCTTGTACAAATCAACCGTGTGCCTGGTGCATATGATCTGAAGCAATTTGATGCTAATTTCTCCAAATTGGTCACTGTAGGGAGCTAGTTTTGTA C13-1 1143 TCTTTTATGTTTCCTACGAACAACATGGGCGTAAGTATTAGAGTGACATCCATTAGAAGTTGGATTTAGGATGTAACTAGCTTTATTTTAGTTCAGTTGAG
N A S R I V Q Q Q Q A R G E A V V G G G
S13-6
20
TTTCAATTCAAGACCAAAGAAGAATCTCTCACAAATGGCGTCAAGAATTGTTCAACAACAACAAGCCAGAGGTGAAGCAGTAGTAGGGGGAGGA
1
K Q Q K K N G V A D G R N R K A L G D I G N L A N V R G V V D A K P N R P I T R 95 AAACAACAGAAGAAGAACGGTGTTGCTGATGGAAGGAACCGCAAAGCATTGGGTGACATTGGGAATTTGGCCAATGTAAGAGGCGTTGTTGATGCCAAACCAAATCGCCCCATCACAAGG
60
S13-6
S F G A Q L L A N A Q A A A A A D N S K R Q A C A N V A G P P A V A N E G V A V 215 AGTTTTGGTGCACAATTACTTGCCAATGCACAAGCAGCAGCAGCTGCTGATAATAGCAAGAGACAAGCATGTGCTAATGTGGCTGGTCCTCCTGCCGTTGCTAATGAAGGAGTTGCGGTG
100
S13-6
A K R A A P K P V S K K V I V K P K P S E K V T D I D A S P D K K E V L K D K K 335 GCCAAAAGAGCGGCCCCCAAACCGGTTTCGAAGAAAGTGATCGTAAAACCTAAGCCTTCTGAGAAAGTCACTGACATTGATGCTAGTCCAGATAAGAAGGAGGTCCTGAAAGACAAGAAG
140
S13-6 S13-6
K E G D A N P K K K S Q H T L T S V L T A R S K A A C G I T N K P K E Q I I D I 180 455 AAGGAAGGAGATGCCAACCCCAAGAAGAAATCGCAACACACTCTTACTTCAGTGCTCACTGCTAGGAGCAAGGCAGCATGTGGCATAACAAACAAACCAAAGGAACAGATTATTGACATT
S13-6 S13-7
S13-6 S13-7
D A S D V D N E L A A V E Y I D D I Y K F Y K L V E N E S R P H D Y I G S Q P E 220 575 GATGCTTCTGATGTTGACAATGAGCTTGCTGCTGTGGAGTACATTGATGACATTTACAAGTTCTACAAACTTGTTGAGAACGAGAGCCGCCCCCACGACTATATTGGTTCACAGCCTGAG . C....A... A. A.A C ..........A 1 HinclI H (24) D I N E R N R A I L V D W L I D V H T K F E L S L E T L Y L T I N I I D R F L A V 695 ATAAATGAGAGGATGAGAGCTATTCTGGTTGATTGGCTGATAGATGTTCACACCAAGTTTGAACrTTCACTTGAGACCCTTTACTTGACCATCAATATAATTGATCGGTTTTTAGCAGTT C. C.T..A .C. 75.
260
(64) S13-6 S13-7
K T V P R R E L Q L V G I S A N L N A S K Y E E I W P P E V N D F V C L S D R A 815 AAGACAGTTCCAAGGAGGGAACTGCAATTGGTTGGCATCAGTGCCATGCTGATGGCATCCAAATATGAAGAAATCTGGCCCCCTGAGGTTAATGACTTTGTCTGCCTCTCAGATAGGGCT ........................ G 195 ....... .............................................................
300
(104) S13-6 S13-7
Y T H E H I L T N E K T I L N K L E W T L T V P T P L V F L V R F I K A S V P D 340 935 TACACTCATGAACATATACTAACCATGGAGAAAACCATATTGAACAAGCTGGAATGGACTTTGACTGTGCCAACACCTTTAGTTTTCCTTGTTCGTTTTATCAAGGCATCAGTCCCAGAT 315. . G.GG A.T..T. T. A. G.G. A F A (144) Q
S13-6 S13-7
U E L D N N A H F L S E L G N N N Y A T L N Y C P S N V A A S A V L A A R C T L 380 1055 CAGGAGTTGGACAACATGGCTCATTTCCTGTCTGAGTTGGGAATGATGAACTACGCAACCTTAATGTACTGCCCGTCAATGGTTGCTGCCTCAGCAGTGCTTGCAGCAAGATGCACTCTG T .A. TA. T. 435. A. N (184) F E
S13-6 S13-7
N K A P F W N E T L K L H T G Y S Q E Q L N D C A R L L V G F Y S T L E N G K L 420 1175 AACAAGGCTCCTTTTTGGAATGAGACACTTAAGCTGCACACTGGTTACTCACAAGAGCAACTCATGGATTGTGCTAGACTATTGGTGGGGTTCTACTCCACTCTTGAGAATGGAAAGCTT 555 CC....C.T. ...GC. HindM (224) H L G
2682
...........
Plant cyclins
S13-6
S13-7
454 R V V Y R K Y S D P Q K G A V A V L P P A K F L L P E G S A S Q H S end 1295 AGGGTTGTGTACAGAAAATATTCTGACCCTCAGAAGGGTGCTGTTGCTGTGCTCCCACCCGCTAAATTTCTTCTGCCTGAAGGTTCTGCTTCCCAACATAGTTAAAAAGAAGAAAAGG-TG G.C..A .A. ---T. C .G. ..-. 675. (257) S end
GAGAGGAGTAGT-------------CTTTTATGATTTTCAGCAAATAATCATTTGTATTTTTTTTCTTCATAAGGTACAAGAATTGATTTTTATATAAGATGCAAGTTTGTCACTTGCCA .
S13-6 S13-7
1413 791
S13-6 S13-7
1520 ATTGTCACAGCATCAAA-----CTTTTATT-TGGGGTTCACGTTCTCTGAGTGTAGTGTGCAACCAGGTTGCAGTTATATACA 902 ........AT....T.TCTAA . C... AA .CA.
T..C.G.T.GTG
. G. -AA-.....
C.. .GATAATATAGAGC .........-.-.
G.
Fig. 1. Nucleotide and deduced amino acid sequences of plant cyclin cDNAs. (a) Carrot clone C13-1. (b) Soybean clones S13-6 and S13-7. S13-7 nucleotides identical to those of S13-6 are denoted by dotted lines, and only differences in nucleotides or amino acids are noted. Dashes denote gaps. The positions corresponding to the oligonucleotide probes are indicated by solid underlines. The restriction sites for HincIl and HindIlI are indicated by wavy underlines. ( ) C,123 1 A e-on. C.1am A
eon
144--53)
KESVC,VDFGEKFDDPQMCSAYVSDVYFYI,KQMEMETKRRPMMNYIEQVQKDVTS SLPEEEKPLDREAVILTVPEYEEDIYNYLRQAEM--KNRAKPGYM-KRQTDITT **** Q
MR
IL
DW
MR
IL
DWLVEV
*
* * DF
* I
DR
V
L
Y
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V
A
LQLVGVT
DI
* KYEEM
*
* *
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* * *** * F LLQET
Q
YM
K
K
E
Y R
I
Y
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NMRGVLVDWLVEVSLEYKLLPETLYLAI SYVDRYLSVNVLNRQKLQLLGVSSFLIASIEE A KYEEI
3-
con. am
Y
LE
Y
6,3-142
A
E C 1 A C
* * E
*
* IY
* SEY
* P
DS
013l'-1 o rn. ,-1am, A
N
APSMTTPEPASKR[RVVLGSNSSAVSGNEDLLLCREFEVPKC R ALGVI N MSQ.PFALHHDGENQMQRRGKMNTRSNGLSGQKRAALGVITNQVNQQVR QPSRAAKPKSSEF
o n.
B
* ALGZI
R
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EEYKL
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P
S
L
L
ME
I
YT
* * GRPL
*
*
*
*
* LHFLRR
HT
SK
B (-on. C i ?, A corn. C am A
TDNTYSQQEVVKMEADLLKTLKFEMGSPTVKTFILGF I RAVQENPDVPKLKF K F PT LK L FD Q LRME TDD Y YPP V EF YPPDVKEFAYI TDDTYTSQQVLRMEHL I LKVLTFDVAVPTTNWFCEDFLKSCDADD--KLKS
B 5- o n. C 1 3 -1 A eon.
* *** * * ** Y E Y W L L PS IAA AKYLMEL K LSBL1,DYGCLE -FVPSL I AASVTFLARFT I RPNVNPWS I ALQKCSGYKSKDLKE EFLANYLAE T Y W LA A Y PS EL L -- LTMFLTElTLI DMDAYLKYLPSITAAAALCLARYSLGIE--PWPQNLVKKTGYEIGHFVD
Clam A B
C
PIVADFVD I
onr.
** ** * S KY V V L,LLHD.,QMGRRGGSLSAVRDKYKKHKF'KCVSTLSPAPEIPESIFNDV V EKY Y QA A L
C:3-1
C
A e-on. C Lam A
CLKDLHKTSLGAESHQQQAVQEKYKQDKYHQVSDFSKNPVPHNLALLAL
A S B
* ALGVI
conr-,. 13-6 eon.
Clam B
S1:3-e6 eonr.
57-171
Clam B
63-146
con.
S13-6 B
con.
Clam A
B
eon.
S1 3-H B eon. C'am B
MR
ccit).
S13-_6 L (o0 r, Clam F>
YM
K
K
*
*
Q
DI
** DRFLS
V
* R
A
KLQLVG
A
* KYEEI
RMRAILVDWL,IDVHTKFELSLETLYLTINI IDRFLAVKTVPRRELQLVGISAMLMAS KYEEMI A LQLVGVT V I DR F LLQET MR IL DW Q
KMRAILIDWLCQVHHRFHLLQETLYLTVAIIDRLLQESPVPRNKLQLVGVTSMLIASKYEEM
* * K VRFI KASVPDQELDNMA VNDFVCLSDRAYTHEHILTMEKTILNKLEWTLTVPTPILVFL HT A GRPL LHFLRR SK L L I ME YT D DF YAPEVADFVT I SDNAYTKKE I LEMEQH I LKKLNFSFGRPLCLHFLRRDSKAGQVDANKHTLA
* YPP WPP Y P
* * EF
V
*
EL
Q
Y
TDD
* Y P
L
** LRME
LK
FD
L
* LA
** A
* * PT
* *
F
* T Y
W
* L
HFLSELGMMNYATLMYCPBSM'VAASAV- LAARCTLNKAPFWNETLKLHTGYSQEQL MDCARL,L PS
KKYLME L
L
IAA
L
W
Y
Y E
KYLMEL-TITEYDIMVQYLPSKIAAAALCLSMKLLDSTH--WTETLTHYSSYCEKSLVST-MQKL *
A
* Y
* * ETL L
* * * DWLVEV EEYKL
IL
* A c o .,. S1:3 - 6 B c o r.~ Clam B9
E
Y R
KPKEQIIDIDASDVDNELAAVEYIDDIYKFY-KLVENESRP-HDYIGSQPEINE E Y LE I Y Y SEY P D D RVLLNVQNIDANDKENPQLVSEYVNDIYD-YMRDLEGKYPIRHNYLENQE-ITG
B
A
I
Y
V
con.
*
*
*
* A
N
IGNILANVRGVVDAKPNR MRSRIVQQQQARGEAVVGGGKQQKKNGVADGRNiRKA= R ALGZI N MTTRAASANLANARMMHNMEEAC,HMANLKGRPHTSHASQRNTLGDIGNQVSAITISDVPRKD
A
EKY
QA
Y
* V
QKIRVVYRKYSDPQKGAVAVLPPAKFLLPEGSASQHBS 'hvCFYSTLEN( ,V
KY
S
AS'LVIKAENSK'.TAVHTKYSSSKFMKISKLAALKSPLVKELAAASV
A- and Fig. 2. Homology among plant cyclins and other cyclins. (a) Amino acid sequence comparison of C13-1 with the consensus sequences of asterisks open and Solid B. cyclin clam with and the consensus with sequences. of S13-6 A. (b) Comparison B-type cyclins, and with clam cyclin indicate distinctive amino acids belonging to the A-type and B-type consensus sequences, respectively. The conserved arnino acid 'Z' in the 'destruction box' represents either D, E or N. Gaps were introduced for optimal alignment. Sequence motifs mentioned in the text are boxed.
2683
S.Hata et al.
oligonucleotides were found in both clones; the first and second probes had two and three mismatches, respectively. Sequence comparison of the coding regions of the soybean cDNAs revealed 95 % identity at both the nucleotide and the amino acid level. The nucleotide homology was only 74% in the 3' noncoding region. Of note among the differences are the deletion of one amino acid and the different usage of termination codons, suggesting selective pressure at the protein level. The two soybean cDNAs cross-hybridized under high stringency, but the carrot cDNA, C13-1, did not hybridize at all to the soybean cDNAs even under low stringency (data not shown). Relationships among the cyclins Figure 2 shows the comparison of the amino acid sequences of plant cyclins with the consensus sequences of A- and Btype cyclins reported in the latest papers (Minshull et al., 1990; Glotzer et al., 1991). The figure also shows the structures of cyclins of clam, in which both types were identified for the first time (Swenson et al., 1986; Westendorf et al., 1989). Carrot clone C 13-1 was more similar to clam cyclin A (37 % identity) than clam cyclin B (32%) in the C-terminal two-thirds of the polypeptides. Although its putative recognition site of ubiquitin-
conjugating degradation system (destruction box), RVVLGEISN, is B-type (Glotzer et al., 1991), it has 30 distinctive amino acids belonging to the A-consensus sequence (solid asterisks in Figure 2a), as opposed to 17 amino acids belonging to the B-consensus sequence (open asterisks). It is noteworthy that the central portion of the cyclin box, KYEEIKPKN, is unusual among the known cyclins. As shown in Figure 2b, a small region of soybean clone S13-6 had the B-type motif of 'destruction box' (Glotzer et al., 1991). The C-terminal two-thirds of 513-6 was more homologous to that of clam cyclin B (40% identity) than to that of clam cyclin A (31 %), although the numbers of distinctive amino acids are the same (19 each for the Aand B-consensus sequences). The LVFLVRFIK sequence diverged from the hallmark motif, LHFLRRXSK, which is strictly conserved in all other B-type cyclins (Minshull et al., 1989; Pines and Hunt, 1987; Pines and Hunter, 1989). The central portion of the cyclin box, KYEEIWPPE, was also. The above sequence homology was not high enough to assign them to any known type; for example, A-type cyclins of clam and fly are 45 % identical in the conserved two-thirds of the proteins, and their B-type cyclins are 47% identical in this region (Lehner and O'Farrell, 1990a). In addition, C 13-1 and S 13-6 are only 32% identical in the conserved region. Therefore, C13-1 and S13-6 might define novel and distinct types of cyclins. We tentatively call C 13-1 and S 13-6 'A-like cyclin' and 'B-like cyclin', respectively. uncommon
Expression of the cyclins Somatic embryogenesis of carrot cells (Steward et al., 1958) has been an attractive system for research on plant development. It is accompanied with the localized and rapid cell proliferation. To investigate the correlation between the embryogenesis and cyclin expression, we carried out an RNA blot hybridization experiment. As shown in Figure 3a, the mRNA of carrot A-like cyclin, 1.5 kb in size, was expressed at a much higher level in the somatic embryos 2684
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a FI
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