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 G 2 phases but showed different kinetics of induction at the G 1 ͞S 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 serine͞threonine kinase (cdk) as the catalytic moiety and a cyclin as a regulatory subunit. The activity of the complexes is further regulated through phosphorylation͞dephosphorylation 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 G 1 ͞S and G 2 ͞M 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 (A-and B-type) and G 1 (C-, D-, and E-type) cyclins according to sequence homologies and expression patterns. While the G 1 cyclins are involved in progression through G 1 phase and at the G 1 ͞S transition, the mitotic A-and B-type cyclins are essential in progression through G 2 phase and at the G 2 ͞M 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 mono-and dicotyledonous plants and shown to share about 60% sequence ...

Protein-DNA interactions in the proximal region of an Arabidopsis H4 histone gene promoter were analyzed by DMS in vivo footprinting combined with LMPCR amplification. Interactions were identified over six particular sequence motifs, five of which were previously shown to bind proteins in maize histone H3 and H4 promoters and are commonly found in the corresponding regions of other plant histone gene promoters. These motifs are located within a 126 bp fragment which was previously shown to confer preferential expression in meristems of transgenic plants. The contribution of each cis-element to the overall expression level and specificity was investigated by testing individual or combined mutations in transgenic Arabidopsis plants. All five motifs behaved as positive cis-elements of unequal strength. The GCCAAT-like sequence GCCACT behaved as a strong positive cis-element but had no influence on the specificity. In contrast, the nonamer AGATCGACG and to a lesser extent the closely linked hexamer CCGTCG proved to be essential for meristem-specific expression. Involvement of the highly conserved histone-specific octamer CGCGGATC in specific expression was revealed at some stages of meristem development. Importance of these three cis-elements, nonamer, hexamer, and octamer, was further confirmed by the fact that combining mutations of two of them either abolished the promoter activity or completely modified the promoter specificity. Mutation of the fifth cis-element, a degenerate copy of the octamer, little perturbed the promoter function. However disruption of both octamers had a dramatic negative effect, thus suggesting that the two copies cooperate to achieve maximal function in the wild-type promoter, possibly by mobilizing the proliferation-specific factors binding to the nonamer and CCGTCG cis-elements.

SummaryThe regulation of histone gene expression during the cell cycle has been studied in a synchronized tobacco (BY2) cell suspension. Maximal expression was found in S phase but unlike other eukeryoti¢ organisms the amount of histone mRNA was not coupled to DNA synthesis. Using aphidicolin as an inhibitor of DNA synthesis either before or at mid-S phase an accumulation of histone mRNA= was found in the absence of any DNA synthesis which was not due to a stabilization of the mRNAs but to actual control at the transcriptional level. At the completion of unperturbed or delayed S phase, histone mRNAs were selectively degraded by a post-transcriptional mechanism requiring de novo transcription.

The activity of the Arabidopsis thalana A1 EF-1 alpha gene promoter was analyzed in transgenic Arabidopsis plants. The 5' upstream sequence of the A1 gene and several promoter deletions were fused to the beta-glucuronidase (GUS) coding region. Promoter activity was monitored by quantitative and histochemical assays of GUS activity. The results show that the A1 promoter exhibits a modular organization. Sequences both upstream and downstream relative to the transcription initiation site are involved in quantitative and tissue-specific expression during vegetative growth. One upstream element may be involved in the activation of expression in meristematic tissues; the downstream region, corresponding to an intron within the 5' non-coding region (5'IVS), is important for expression in roots; both upstream and downstream sequences are required for expression in leaves, suggesting combinatorial properties of EF-1 alpha cis-regulatory elements. This notion of specific combinatorial regulation is reinforced by the results of transient expression experiments in transfected Arabidopsis protoplasts. The deletion of the 5'IVS has much more effect on expression when the promoter activity is under the control of A1 EF-1 alpha upstream sequences than when these upstream sequences were replaced by the 35S enhancer. Similarly, a synthetic oligonucleotide corresponding to an A1 EF-1 alpha upstream cis-acting element (the TEF1 box), is able to restore partially the original activity when fused to a TEF1-less EF1-alpha promoter but has no significant effect when fused to an enhancer-less 35S promoter.