Four full-length and one partial cDNA clones encoding four different A-type cyclins were isolated from a tobacco S-phase-specific library. The corresponding mRNAs displayed sequential appearance and disappearance during the cell cycle of highly synchronized suspension-cultured tobacco cells. Sequence analysis showed that the plant A-type cyclins can be subdivided into three distinct structural groups that are likely to be represented in every plant species. Two of the isolated tobacco cyclins belonging to the same group were highly expressed throughout S and G2 phases but showed different kinetics of induction at the G1yS transition. Another one belonging to a second group was induced at mid-S phase and expressed until mid-M phase. A similar expression pattern was previously reported for a tobacco cyclin belonging to the third group. This sequential expression of multiple A-type cyclins in one type of plant cells makes a clear distinction from the situation in animal cells in which only one A-type cyclin exists in a given species. Furthermore, the expression of the different A-type cyclin genes responded differently upon a block at mid-S phase by DNA synthesis inhibitors. These results suggest that the multiple A-type cyclins act at different steps of the plant cell cycle and, therefore, exert distinct functions. In contrast, the expression of B-type cyclins was restricted to a narrow window corresponding to the M phase. Our knowledge of the eukaryotic cell cycle has progressed considerably during the last few years, by the finding that protein kinases play a central role in the cell cycle. The activity of the protein kinases depends on the association between a cyclin-dependent serineythreonine kinase (cdk) as the catalytic moiety and a cyclin as a regulatory subunit. The activity of the complexes is further regulated through phosphorylationydephosphorylation mechanisms and binding to inhibitors (1). In animal cells, distinct cdks associating sequentially with different cyclins determine the substrate specificity of the complexes and monitor the cell cycle transitions at the G1yS and G2yM major checkpoints (2, 3). These sequential associations result from stage-specific synthesis and degradation of the cyclins while the catalytic subunit is present throughout the cell cycle. The animal cyclins have been subdivided into mitotic (Aand B-type) and G1 (C-, D-, and E-type) cyclins according to sequence homologies and expression patterns. While the G1 cyclins are involved in progression through G1 phase and at the G1yS transition, the mitotic Aand B-type cyclins are essential in progression through G2 phase and at the G2yM transition. In addition, the A-type cyclin has also been shown to play essential functions in DNA replication during S phase (4–6). It has been recently shown that the basic components of the cell cycle machinery are similar in plants and animals (7). cdk-like cDNAs and genes have been isolated from several monoand dicotyledonous plants and shown to share about 60% sequence homology with animal cdc2 and cdk2. Using polymerase chain reaction (PCR), reduced-stringency hybridization, or complementation of yeast mutants, cDNAs corresponding to plant mitotic cyclins have also been isolated from several plants (7, 8). More recently isolation of cDNAs corresponding to G1 cyclins (D-type) has been reported from two plant species (9, 10). However, due to the lack of a highly synchronizable plant system, assignment of plant cyclin expression to a particular stage of the cell cycle has only been roughly determined, essentially by using inhibitors arresting the cell cycle at different points or by differential in situ hybridization patterns. Herein we report the isolation of four full-length and one partial cDNAs corresponding to four different A-type cyclins from the highly synchronizable tobacco BY2 cells. We show that the different A-type cyclins expressed sequentially during S and G2 phases of an unperturbed cell cycle and displayed different responses toward inhibitors of DNA replication, suggesting their different functional implications during cell cycle progression. Moreover, expression of two B-type cyclins appeared to be strictly limited to the M phase. MATERIALS AND METHODS Cloning of Tobacco Cyclins. PCR fragments corresponding to mitotic cyclins were obtained from cDNA from exponentially growing suspension-cultured tobacco cells by using degenerate primers in the cyclin box corresponding to amino acid sequences M(RyS)AI(LyF)(VyIyM)DW and KYEE(Iy M)Y(AyPySyT)P. A BY2 S-phase-specific cDNA library was constructed from 1 mg of poly(A)1RNA in the vector lZAPII (Stratagene) and screened with the PCR fragments corresponding to A-type cyclins. DNA inserts were excised according to the protocol from the manufacturer and sequenced by the dideoxynucleotide method (11). Synchronization and Analysis of Cyclin Gene Expression. Synchronization of tobacco BY2 (Nicotiana tabacum L. cv. Bright Yellow 2) suspension-cultured cells was achieved by a 24-hr subculture of stationary phase cells (7 days old) in a medium containing aphidicolin (Sigma; 3–6 mgyliter) followed by extensive washes, as described (12). Measurement of DNA synthesis, mitotic index, and isolation of RNA were as in ref. 13. Total RNA (25 mg) was electrophoresed on formaldehydeyagarose gels and transferred to Hybond-Nmembranes (Amersham). Hybridization was under standard highstringency conditions (11). Transcript levels were quantified from the blots using a PhosphorImager (Molecular Dynamics). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: cdk, cyclin-dependent serineythreonine kinase. Data deposition: The sequences reported in this paper have been deposited in the GenBank data base (accession nos. X92964–X92967 and X93467). ‡To whom reprint requests should be addressed.