Alfalfa Cyclins: Differential Expression during the Cell Cycle and in Plant Organs

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1 The Plant Cell, Vol. 4, , December 1992 O 1992 American Society of Plant Physiologists Alfalfa Cyclins: Differential Expression during the Cell Cycle and in Plant Organs Heribert Hirt,' Mhtyhs Mink,* Martin Pfo~ser,~ Laszlo Bogre, Jdnos Gyorgyey,* Claudia Jonak, Anton Gartner,4 Dhes Dudits,* and Erwin Heberle-Bors lnstitute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, 1030 Vienna, Austria Cell division in eukaryotes is mediated by the action of the mitosis promoting factor, which is composed of the CDC2 protein kinase and one of the various mitotic cyclins. We have recently isolated a cdc2 gene from alfalfa. Here, we report the isolation of two cyclin genes, cycms7 and cycms2, from alfalfa. The cycms2 gene shows highest similarity to type 6 cyclins. In contrast, the predicted amino acid sequence of the cycms7 gene shows similar homology scores to cyclins of all types (25 to 35%). 60th genes are expressed in dividing suspension cultured cells but cease to be expressed when the cells enter stationary phase. In synchronized alfalfa suspension cultured cells, the mrnas of cycms7 and cycms2 show maximal expression in the 62 and M phases. Transcripts of cycms2 are found only in late 62 and M phase cells, an expression pattern typical for cyclin B genes, whereas cycms7 appears with the onset of 62. This pattern indicates that alfalfa cycms7 and cycms2 belong to different classes of cyclins. In young leaves, expression of both genes is high, whereas in mature leaves no transcripts can be detected, indicating that the two cyclin genes are true cell division markers at the mrna level. In other organs, a more complex expression pattern of the two cyclin genes was found. INTRODUCTION Beach, 1988; Goebl and Byers, 1988; Hagan et al., 1988; Solomon et al., 1988), Xenopus (Minshull et al., 1989), Drosophila (Lehner and OFarrell, 1989; Whitfield et al., 1989), humans (Pines and Hunter, 1989), and recently also in carrot, soybean (Hata et al., 1991), and Arabidopsis (Hemerly et al., 1992). In this study, we describe the isolation of two partia1 cyclin tor: its activation involves physical association of ~ 3 4 cdna ~ ~ clones, ~ cycms7 and cycms2, from alfalfa. Comparison All eukaryotes have been shown to contain a 34-kD protein kinase that plays a central role in the regulation of cell division. The 34-kD protein kinase has been shown to be the product of the cdc2+/cdc28 genes in yeasts and animals (for review, see Nurse, 1990) and in various plant species (Colasanti et al., 1991; Ferreira et al., 1991; Hirt et al., 1991). The CDC2 kinase is one part of the mitosis promoting facwith cyclin proteins. In the absence of cyclins, cells cannot enter mitosis and CDC2 kinase activity is absent (Booher and Beach, 1987,1988; Minshull et al., 1989; Murray and Kirschner, 1989; Murray et al., 1989). At the end of metaphase, cyclins are destroyed and CDC2 kinase activity vanishes (Murray et al., 1989). Degradation of cyclins has been shown to be required for completion of mitosis and might be necessary for inactivation of the CDC2 kinase (Murray et al., 1989). Cyclins were originally identified in the eggs of marine invertebrates(evans et al., 1983; Swenson et al., 1986; Standart et al., 1987). On the basis of amino acid homology, cyclins have subsequently been identified in fission yeast (Booher and of the predicted cyclin protein sequences with 18 other cyclins showed that cycms2 can be classified as a type B cyclin. In contrast, cycms7 appears to be equally distantly related to the type A, B, and G1 cyclins. RNA gel blot analysis with synchronized alfalfa cell suspension cultures showed cell cycle-dependent expression of both cyclin genes in the G2 and M phases. During the growth cycle of a batch suspension culture, the two genes were expressed during exponential growth phase but not in stationary phase. In the plant, cycms7 and cycms2 were only found to be expressed in organs with dividing cells. To whom correspondence should be addressed. Current address: lnstitute of Plant Physiology, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, POB 521, Hungary. lnstitute of Botany, University of Agriculture and Forestry, Gregor- Mendelstr. 33, A-1190 Vienna, Austria. Current address: lnstitute of Molecular Pathology, Dr. Bohrgasse 3, A-1030 Vienna, Austria. RESULTS lsolation and Sequencing of Cyclin cdnas from Alfalfa To isolate cyclins of type A and B, we screened 80,000 colonies of an alfalfa (Medicago sariva) cdna library made from

2 1532 The Plant Cell suspension cultured cells that were induced to form somatic embryos with redundant oligonucleotides encoding the conserved amino acid sequences K-Y-E-E-M(I)-Y-P for the cyclin A and B type and I-L-V(I)-D-W-L-V for the cyclin B class (see Methods for the nucleotide sequences). Positive colonies were isolated and rescreened according to the method of Hanks et al. (1988) at a temperature of 58OC in order to distinguish between false positives with a fit of less than 16 nucleotides. Only one colony was found to hybridize under these conditions to the I-L-V(I)-D-W-L-V oligonucleotide. DNA was prepared from this clone and digested with Pstl. Upon gel electrophoresis, a 750-bp insert was observed. Because the insert contains GC tails at both ends, it was impossible to perform sequence analysis with this clone. A 400-bp BamHl fragment was subcloned into the vector ptz19u and sequenced from both sides. Sequencing was also possible after removal of a 5'200-bp Xbal fragment from the original clone. The complete sequence is shown in Figure 1A. The sequence at position 751 to 771 shows a fit of 18 out of the 20 nucleotides to the redundant oligonucleotide probe of the cyclin B class (see above). However, at the amino acid levei, only five of the encoded seven amino acids fit to the highly conserved cyclin B box (the encoded sequence reads I-L-V-D-C-L-L instead of I-L-V-D-W-L-V). The clone contains a nontranslated region of 132 nucleotides before the first ATG, which is followed by a putative sequence of 211 amino acids. However, there is no stop codon or any indication of a poly(a) tail in the clone. This is surprising because the library was constructed by oligo(dt) priming of the poly(a)+ RNA. Rescreening of 250,000 colonies of the same library and 1,000,000 of another cdna library, made from alfalfa somatic embryos, resulted in the isolation of two colonies that were found to be identical to the above described clone. Screening of a third cdna library made from suspension cultured alfalfa cells that were induced to form somatic embryos resulted in the isolation of one clone of 1.4 kb. The cdna sequence is shown in Figure 18 and was found to be very different from that of CycMsl. A 5'truncated open reading frame of 984 nucleotides was identified and appears to encode a different cyclin that is designated cycms2. Interestingly, in contrast to CycMsl, the cycms2 clone contains a cyclin B box (nucleotide position 313 to 333) which fits much better to the animal sequences. Rescreening the different libraries yielded no other hybridizing colony than the one indicated. These results indicate high instability andlor very low abundance of these types of mrnas. To determine the copy number of the CycMsl and cycm.92 genes in alfalfa, DNA gel blot analysis was performed with genomic DNAs from M. sativa. Hybridization with a 32Plabeled cycms7 probe, as shown in Figure 2A, revealed the presence of two bands in Hindlll-digested total DNA (Figure 2A, lane 1) but only one band in Bglll-digested DNA (Figure 2A, lane 2). When the same DNAs were probed with a radiolabeled cycms2 probe, as shown in Figure 26, the Hindlll digest revealed hybridization to one band (Figure 28, lane 1) but to four bands in Bglll-digested DNA (Figure 28, lane 2). Because alfalfa is an autotetraploid species, these data indicate the occurrence of one copy per haploid genome for both cyclin genes. A Zs":C AAC TIlC TCT CAC TGT TTC TCT CTT CCA m TrT TTC AAG GRT CTT CAT TTT CRC CCC CTT CTT CTT TTA GGA TCT TCC ATT TTT TTI TGT uzj TTG TGT TTC ATT 'M"I TTT TTC ATT GAA GAA AAA ATG M G m TCT GAG GIG AAG AAT GTI TCT AAC AAC CCC ACA AAT m H K ~ S E E I N Y S N N P T N F G M GGA GGG TTA GAT TCT AGA AAG GTT UX: GAA AAC AGI AGA GCI TTG GGT GTG ATT AAT E G G L O S R K Y C E N R R A L G V I N CAG AAT TTG GTT GTG CAA GGA CGT CCT TAT CCT TGT GTT GTI AAC AAG AGG GCA TTG TCA O N L Y V C G R P Y P C Y Y N X R A L S GAG IGA AAT GAT GTT TGT GAG AAG AM C M GCG GAT CCG GTI CAT CGI CCC ATC ACT AGG E R N D V C E K K Q A D P V H R P I T R AGG TTT GCT GCA AAG ATT GCT AGE ACA A&A ACA TCT U T GCT GIG GGA ACT ACC AAA AGG P ~ A A X I A S T X T S N A E G T T K R TCA AAC TTG GCA AAA TCA AGT TCA AAT GGA VIT CGA GAT Tpc ATA m GTT GAT GAT GAA S N L A K S S S N G F G O T I F Y D D E CAC AAC CCA GTG GAC GAT CAG CCA GTG CCA ATG CCT TTA GAG CAA ACA GAA CCA ATC CAT H K P V E D O P V P I A L E Q T E P I H ACT GAA TCA GAT CAG ATG GAG OM GTI GAG ATG GAG GAT ATC ATG GAA GAG CCT CTI ATG S E S D Q H E C V E I E D I I E E P V I GAC ATI GAC ACC CCT GAT GCA AAT CIC CCT CTI CCA GTT CCT OM TAT ATI G14 GAT CTI D I D T P D A N D P L A V A E Y I E D L TAC TCT TAC TAC AGI AAA GTT GAG AGT ACT AGC TGT GTI TCA CCA AAC TAT li% GCA CAG Y S ~ Y R R V E S T S C Y S P N Y H A Q 750 C M TTT GAC ATT AAT GAA AGG ATG AGG GCT ATA CTG GTT GAC xc TTA Tn; Q F D l N E R l R A l L V D C L L cyati AAT TCG AAT GAG 'M"I GGA AAC Tpc ATC GCC ATP GAT GAT OM CTC M G TIA CCA GM GAC N S N E F G N F I A I D D E L K L P E O C M CCA GAG CCT ATG ACT TTG OIG CAT ACT GAA CCA ATG CAT AGT GAT CCA CTT GAG ATG ~ P E P * T L E H T E P * H ~ D P L E u GUL GAG CTT GAG ATG GAG GAT ATA GAG uill GAG ATG A m TX GAC ATT GAT AGC TGT GAT E E V f M E D I E G C M I L D l D S C D GCA M C M T TCT CTI GCT GTI GTC GAG TAT ATT GAA GAT CTC CAT GCT TAC TAC AGA M G k W N S L A V V E Y I E D L H A Y I R K ATT GAG TAT CTI GGC TGT GTI TCA C U ACC TAC ATG GAT GM CAA CTI GAC C M AAT GAG I E Y L C C V S P T Y M D E Q L D L N E A% ATG AGG GCT ATA CTA GlT GAC TGG CTG A m GAG GTT CAT GAC AAA TTC GAC CTC ATG R M R I I L V D Y L I E V H D K F D L M CAA GAG ACA CTG TIT CTI ACA GTT M T CTC ATC GAC AGA TIC TX GCT AAG CAG AAT GTT ~ E T L F L T ~ N L ~ D R F L A K Q N V CTI AGA AAG AAA CTT CAG CTG 01T GGT CTT GTT GCC ATG CTI TX GCA TGC AAG TAT GAG V R R K L Q L V G L V I R L L A C X T E G M GTT TCT GTG CCT GTC GlT WG GAT CTG ATC CAT ATA GCT GAC AGA GCT TAC ACA AGG ~ ~ S ~ P ~ V S D L I H I A D R ~ Y T AAA GAC CTC M AAG TTG ATG CTC M ACT CAG TAT AAC TCT CTT ATT G ATG GAG C CTC ATG K D I L E M ~ K L I L N T L O Y N M S L CCA ACT GCT TIT GTT TTC ATG AGA AGG 277 CTG AAG GCT GCT CAA GCT CAC luu AAA CTI P T A Y Y F M R R F L K ~ A a I D r, K L GAG CTG CTG GCT m Tpc TTG GTT GIC CTA TCT CTG GTG GM TAT GAG I\TG CTG AAC m E L V I F F L V D L S L Y E Y E M L K F CCA CCA TCC TTG GTI GCT GCT GCT GCT CTC TAC ACA GCT C M TGC ACT GTC AGT GGT TTC P P S L V A A A A V Y T I Q C T Y S G P AAA CAC TGG AAC AAG ACA TGT GAG TCG CAC ACI UIC TAC TCA GAA GAT CAG CTG TIG GAG K H W N K T C E Y H T N I S E D Q L L E TGC TCT ATG CTC ATG CTT GGA TIC CAC CAG AAA GCC GGA GCC ujc A&A CTG >.2k GGA GTG C S ~ L H V G F H Q K A G A G K L T G V CAT AGG AAG TAT GGC TCA GCA AAA TTI AGC Tpc ACCT GCT AAA TGT GAA CCA GCA TGT TlT H R K I G S A K I S F T A K C E P A C F CTI CTG GAG AAC AAG AAC CAG CCA TAA GAA GAG AGC CAG CCA TGT ATA GCT TIT CTT ACA L L E N K W Q P AAC AAT ACC ATA AGT GTG CTG CTG ATT TTG UX: GGT TIA AGA AGA ACA AAA CAT AAA GAG C M CTT TGG TTG CTT TGT ACT TIT CTC AAG A M TIC TGT TAA GTI ACT m ATT CTI TGG TCA TTI TTA TlT TIG TTG TTC ATG Tpc CAT AGG TGA ACA TAA TM m CAG AAC m m ATT ATG TCT TCT GTA TTT TCT CTT TGA ACA AAA TAA TGG GAT ATA GAC ACA ATT m GTC TAT crc ccc m vr TGA TGC m TAT AAC TTG mr =li TGC AAC TTC AAT TCA ATT TTA CCT TTA AAA A M AAA AAA AAA AA Figure 1. Nucleotide Sequences of Alfalfa cycms7 and cycms2 cdnas and Their Predicted Arnino Acid Sequences. (A) cycms7. The EMBL accession nurnber is X (e) cycms2. The EMBL accession number is X Therefore, we conclude that the cycmsl and cycms2 genes are both present as single copy genes in alfalfa. The cycms2 Gene Encodes a Member of the Type B Cyclins, but cycmsl Cannot Be Classified into Any Cyclin Class Alignment of the predicted protein sequences from cycmsl and cycms2 revealed 65% identity over 111 amino acids.

3 Alignment of the predicted alfalfa cyclin protein sequences with Differential Expression of Alfalfa Cyclins 1533

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5 Differential Expression

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7 Differential Expression of Alfalfa Cyclins 1537 Genomic DNA Gel Blot Analysis Ten micrograms of DNAof M. sativa was digested with Hindlll or Bglll and separated on 0.7 /o agarose gels. After blotting, the nylon filters were hybridized to randomly 32P-labeled fragments of cycms7 or cycms2. RNA Extraction and RNA Gel Blot Analysls Suspension cultured cells of M. varia or plant tissue of M. sativa was frozen in liquid nitrogen and extracted by grinding in a mortar as described (Cathala et al., 1983). RNA gel blot analysis was performed according to Gyorgyey et al. (1991). Probes for hybridization were the coding regions of cycms7 and cycms2 cdna clones or the coding region of a phosphoprotein phosphatase 2A cdna clone from alfalfa as a control. All fragments were 32P-labeled by random priming (Feinberg and Vogelstein, 1983). ACKNOWLEDGMENTS This work was supported by Grant No. S 6004 BIO from the dsterreichischer Fonds zur Forderung der Wissenschaften and the Austrian Ministry of Science and Research. Received August 3, 1992; accepted October 12, REFERENCES Booher, R., and Beach, D. (1987). lnteraction between cdcl3+ and cdc2+ in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2+ protein kinase. EMBO J. 6, Booher, R., and Beach, D. (1988). lnvolvement of cdcl3+ in mitotic control in Schizosaccharomycespombe: Possible interaction of the gene product with microtubules. EMBO J. 7, Cathala, G., Savouret, J.-F., Mendez, E., West, B.L., Karin, M., Martlal, J.A., and Baxter, J.D. (1983). A method for isolation of intact, translationally active ribonucleic acid. DNA 2, Colasantl, J., Tyers, M., and Sundaresan, V. (1991). lsolation and characterization of cdna clones encoding a functional ~34 C' homologuefmnzeamays. Proc. Natl. Acad. Sci. 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