Mol Gen Genet (1992) 233:10-16 © Springer-Verlag 1992

Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants Junji Hashimoto, Toshio Hirabayashi, Yuriko Hayano, Shingo Hata*, Yuko Ohashi, Iwao Suzuka 1, Takahiko Utsugi 2, Akio Toh-E 2, and Yoshiko Kikuchi 2 National Institute of Agrobiological Resources and 1 National Institute of Animal Health, Tsukuba, Ibaraki 305 2 Department of Biology, Faculty of Science, The University of Tokyo, Bunkyoku, Tokyo 113, Japan Received September 24, 1991

Summary. Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 and edc2Os-2) encoding structural homologues of the cde2+/CDC28(cde2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62% identical to CDC28 of Saccharomyces cerevisiae and much more similar to the yeast and mammalian p34 cd~2kinases than to rice R2, a edc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1, cdc2Os-2 and R2) are derived from distinct genes and are each found in a single copy per rice haploid genome. R N A blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings, ede2Os-1 could complement a temperature-sensitive yeast mutant of edc28. However, despite the similarity in structure, both cdc2Os-2 and R2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of the ode2 cell cycle kinase in rice. Key words: p34 cdc2- Oryza sativa - Protein kinase - Cell cycle control

Introduction The cdc2 protein kinase plays a central role in the regulation of the cell division cycle in eukaryotes (for reviews, see Nurse 1990; Lewin 1990; Pines and Hunter 1990a). * Present address: Faculty of Science, Himeji Institute of Technology, Kamigori, Ako-gun, Hyogo 678-12, Japan Correspondence to: J. Hashimoto

The nucleotide sequence data in this paper have been deposited in the EMBL database under accession number X60374 (cdc2Os-1) and X60375 (edc2Os-2)

In the fission yeast Schizosaccharomycespombe, cdc2 acts at two points in the cell cycle: at "Start", where it is required for entry into S phase, and then in late G2, where it regulates entry into mitosis (Nurse and Bissett 1981). This is also the case for CDC28 in Saecharomyces cerevisiae (Reed and Wittenberg 1990). The catalytic activity of cdc2 +/CDC28 proteins is regulated by changes in phosphorylation states and in association with other proteins, such as cyclins (see, for example, Minshull et al. 1990; Solomon et al. 1990; Krek and Nigg 1991). Functional homologues of the yeast cdc2+/CDC28 genes (cdc2) have been identified in human cells (Lee and Nurse 1987), chicken (Krek and Nigg 1989), Drosophila (Lehner and O'Farrell 1990; Jimenez et al. 1990) and plants (Hirt et al. 1991; Colasanti 1991; Ferreira et al. 1991). The degree of similarity in their amino acid sequences compared to those of their yeast counterparts is of the order of 60%. However, the homologues from Drosophila (Lehner and O'Farrell 1990) and J(enopus (Paris et al. 1991) failed to complement yeast mutants, in spite of their high levels of homology (i.e., 54% and 65 % identical to the cdc2 protein of S. pombe, respectively). Homologues distantly related to edc2 have been reported from yeasts (Simon et al. 1986; Uesono et al. 1987; Courchesne et al. 1989; Elion et al. 1990; Irie et al. 1991) and other organisms (Bourouis et al. 1990; Shuttleworth et al. 1990). Their levels of homology to the cdc2 proteins are less than 50 % and most have been shown to be essential for cell proliferation. An earlier study has reported on a cDNA, R2, from rice, which is distantly related to cdc2 (Hata 1991). This study showed that the EGVPSTAIR motif, found in all the cdc2 functional homologues, is only partially conserved in R2 (13 of 16 amino acids). In addition, R2 has an extra C-terminal domain of 118 amino acids compared to the functional homologues. We now describe the isolation and characterization of two cDNA clones from rice which encode proteins that are highly homologous to the ede2 protein kinase, and demonstrate that one of them can complement a temperature-sensitive cde28 mutant in S. cerevisiae.

II Materials and methods

Isolation and sequencin9 of rice cdc2 cDNAs. Degenerate oligonucleotides were synthesized based upon two conserved regions of the cdc2 protein, corresponding to the amino acid sequences GEGTYGV and WYRAPEV. Their sequences are 5'-CCCTCGAGGGIGA(A/G) GGIACITA(C/T)GGIGT-3' and 5'-GGGAATTCAC (C/T)TCIGGIGCIC(G/T)(A/G)TACCA-3'. The former primer has an XhoI site and the latter a BamHI site incorporated to facilitate cloning of PCR products. PCR was performed on a lambda ZAPII (Stratagene) cDNA library, constructed from poly(A) + R N A of cultured cells of rice (Oryza sativa L., cv. Nipponbare). Amplified products were digested with XhoI and BamHI, and ligated to XhoI+BamHI-digested pBluescript (Stratagene). Partial sequencing of several subclones showed sequences not related to cdc2. Therefore, recombinants were screened with 32p-labeled synthetic oligonucleotides based upon another conserved amino acid sequence (EGVPSTAIRE), the nucleotide sequence of which is 51-GA(A/G)GGIGTICCI(T/A)(C/G)IACIGCIAT (T/C/A)CGIGA-3'. A 491 (bp) rice cdc2 probe thus obtained was used to screen the rice cDNA library. Phage DNAs from the positive clones were excised in vivo to recover pBluescript plasmids according to the instructions supplied by Stratagene. The nucleotide sequences of both strands were determined by the dideoxy chain termination method using an automated fluorescent DNA sequencer (Model 370A, Applied Biosystems). Complementation of an S. cerevisiae cdc28 mutant. The S. cerevisaiae strains cdc28-13 (MA Ta cdc28-13 ura3 trp l leu2 His-Gal +) and cdc28-4 (MATa cdc28-4 ura3 trpl leu2 His-Gal +) were used as recipients in the complementation tests. Both strains were isolated by Reed (1980) and were provided by K. Matsumoto (Nagoya University). Each cDNA was placed under the transcriptional control of the GAL7 promoter on the multicopy expression vector pAA7, which carries URA3 as a selective marker; the vector was kindly provided by Y. Nogi (Tajima et al. 1986). cDNAs were inserted into the expression vector as BamHI-SalI fragments, replacing a BglII-SalI fragment of the plasmid pAA7 such that the cDNA coding sequences were positioned between the GAL7 promoter and the transcription terminator of the vector in the correct orientation. Yeast transformation was carried out by the alkali ion method (Ito et al. 1983), selecting Ura + transformants at 26°C on SD medium supplemented with appropriate amino acids (Sherman et al. 1986). The Ura + transformants were streaked on YPGal plates containing 10 gg/ml ethidium bromide, and on SD-Ura plates and were incubated at 34-37 ° C for 2 or 3 days. Southern and Northern blot analysis. To avoid cross hybridization, PCR fragments were prepared and used as probes. The 135bp (1-135), 226bp (901-1126) and 354bp (1091-1444) fragments were amplified from

cdc2Os-1, cdc2Os-2 and R2 cDNA clones, respectively. The fragments were labeled by the random primer method (Feinberg and Vogelstein 1983). Genomic D N A from rice seedlings was digested with restriction enzymes, fractionated on an 0.8 % agarose gel, and transferred to nylon membranes (Hybond-N, Amersham). Total RNAs were isolated from cultured rice cells in the early logarithmic growth phase, from primary shoots and roots of 4-day-old rice seedlings, and from leaves of 14-day-old rice seedlings. The samples of R N A were separated on 1.2 % agarose gels containing formaldehyde and transferred to Hybond-N membranes. Hybridization was carried out as described (Sambrook et al. 1989). Results

Clonin9 of cdc2-homologous cDNAs from rice To isolate a full-length cDNA homologous to cdc2, the 491 bp fragment isolated with the third oligonucleotide probe (see Materials and methods) was used as a probe to screen a cDNA library constructed with poly (A) + R N A from cultured rice cells. Two positive phage clones were isolated out of 240 000 plaques screened. Sequence analysis of these cDNA clones revealed extended open reading frames (cdc2Os-1, Fig. 1 ; cdc2Os-2, Fig. 2). The putative proteins deduced from these ORFs have sequences characteristic of cdc2 protein kinases (Hanks et al. 1988). For example, the ATP binding region (GEGTYG, amino acids 11-16), the stretch of the most highly conserved 16 amino acids (EGVPSTAIREISLLKE, amino acids 42-57) and the domains typical of protein kinases (HRDLKPQN, amino acids 125-132 in cdc2Os-1, 124-131 in cdc2Os-2; DFG, amino acids 146-148 in cdc2Os-1,145-147 in cdc2Os-2) are present in both clones. Figure 3 shows an alignment of the putative protein sequences of the two clones with those of cdc2 from fission yeast, budding yeast, maize and human, and with R2, a cDNA clone previously isolated from rice. At the amino acid level, the two rice homologues (cdc2Os-1 and cdc2Os-2) are 61.6% identical to CDC28 of S. cerevisiae. cdc2Os-1 and cdc2Os-2 are closely related to each other (83.3% identical), cdc2Os-1 is highly homologous to the maize cdc2 (93.5% identical), while the homology between cdc2Os-2 and the maize cdc2 is 84.2%.

Complementation of S. cerevisiae cdc28 In order to determine whether these two cDNA clones, cdc2Os-1 and cdc2Os-2, and R2 are functional as cdc2 kinase genes, we introduced the DNAs into a temperature-sensitive cdc28 mutant of budding yeast, and tested for their ability to complement the mutant phenotype. Each D N A was placed under the control of the GAL7 promoter on the multicopy vector pAA7, which carries the URA3 as a selective marker (Tajima et al. 1986). Ura ÷ transformants were selected at the permissive temperature (26 ° C) and cells were streaked on

12

'~

~

~

~t~

~

c9

~

~

~

. . . . . . . .

(.9

,~

¢-9 ~.~

--dl--

(J

(J

,,ere-

~

~

CZ

C9

~

~'~

~,

(:~1

~

~-

~

~

~

~-

"~

o~

~

~

t'-~D

~

~

(=

! o! . . . . . . . . . . . . . . . ~.9

CJ

(.9

(.5

~--

~--

(.9

~'~

u'~

~

~

~,

~-

~_

~

~

(.9

~-

I--

~.9

~--

I--

~

~

C£

~

~1--

gl. ¢.J

.J

-J

...I I--

u.~-

...I

0

¢.n (-~

z~:l:

I--o

..J~-

(:~

~

~

h.-

c.J (J

w~c

m

(.9

o

~,

c9

a:

co

~

~

c9

~-

o

I--

(.9

(.9

~-

(J

(.9

h-

(.9

~.J

(.9

~J

c9

(J

o

(.9

(J

;-

E

o

(.J

~

o

(J

;--

l--

~E

(.9

(9

(.J

(.J

(.9

l-

~-

~

I--

~_

:=:(J

c.)

(J

(J

(.9

h-

c~

(.9

h-

(J

~.J

~::

(.9

(.9

cJ

(.9

~

o

~J

~-

h-

h-

C5

(J

(J

~--

(J

(.9