Nearly 7000 Arabidopsis thaliana-expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.
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.
Ribonucleotide reductase (RNR) is a key enzyme involved in the DNA synthesis pathway. The RNR-encoded genes are cell cycle regulated and specifically expressed in S phase. The promoter of the RNR2 gene encoding for the small subunit was isolated from tobacco. Both in vivo and in vitro studies of the DNA-protein interactions in synchronized BY2 tobacco cells showed that two E2F-like motifs were involved in multiple specific complexes, some of which displayed cell cycle-regulated binding activities. Moreover, these two elements could specifically interact with a purified tobacco E2F protein. Involvement of the E2F elements in regulating the RNR2 promoter was checked by functional analyses in synchronized transgenic BY2 cells transformed with various RNR2 promoter constructs fused to the luciferase reporter gene. The two E2F elements were involved in upregulation of the promoter at the G1/S transition and mutation of both elements prevented any significant induction of the RNR promoter. In addition, one of the E2F elements sharing homology with the animal E2F/cell cycle-dependent element motif behaved like a repressor when outside of the S phase. These data provide evidence that E2F elements play a crucial role in cell cycle regulation of gene transcription in plants.
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.
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