The cdc2 protein kinase plays a central role in control ofthe eukaryotic cell cycle of animals and yeasts. We have isolated a cDNA clone (cdc2Ms) from alfalfa (Medicago saliva L.) that is homologous to the yeast cdc2/CDC28 genes. The encoded protein is 64% identical to the yeast and mmalian counterparts and shows all the prominent structural features known from these organisms. Antibody raised against a 16-amino acid synthetic peptide with crossreactivity against p34 proteins recognized a 34-kilodalton protein in extracts ofalfalfa cells. When transferred into a fission yeast, the plant cdc2 homolog can complement a temperature-sensitive cdc2 mutant. Northern analysis revealed higher transcript levels in shoots and suspension cultures than in roots. In addition to the dominant transcript of 1.4 kilobases detected in the poly(A)+ fraction, 2.5-and 1.2-kilobase transcripts were detected in total RNA preparations from shoots or somatic embryos. Suspension cultures that were induced to form somatic embryos by an auxin (2,4-dichlorophenoxyacetic acid) showed fluctuations in transcription pattern during the induction period and embryogenesis.

Clink, a 20-kDa protein of faba bean necrotic yellows virus, a single-stranded DNA plant virus, interacts with pRB family members and a SKP1 homologue from Medicago sativa. An LxCxE motif and an F-box of Clink mediate the interactions with the respective proteins. The capacity of Clink to bind pRB correlates with its ability to stimulate viral replication. Interaction of a single protein with the cell cycle regulator pRB and SKP1, a constituent of the ubiquitin-protein turnover pathway, appears to be a novel feature. Hence, Clink may represent a new class of viral cell cycle modulators.A common strategy of DNA viruses is the creation of an environment favorable for efficient replication of their genome by subverting the cell cycle control of the host and forcing cells into DNA synthesis or S phase. In mammalian cells, a key cell cycle regulator is the retinoblastoma tumor suppressor protein pRB, which represses onset and progression into S phase by interacting with a wide range of cell cycle-related proteins. Among those are transcription factors of the E2F family that form complexes with hypophosphorylated pRB. During the G 1 /S transition, pRB is progressively phosphorylated by the action of cyclin-dependent kinases, and as a result, E2F is released from the complex and becomes available to activate the expression of S-phase-specific genes (14, 30).Oncoproteins of certain mammalian DNA tumor viruses, such as E1A of adenovirus type 6, E7 of human papillomavirus type 16, or the large T antigen protein of simian virus 40, stimulate the entry of cells into S phase by interaction with pRB through a short protein sequence comprising essentially the sequence motif LxCxE (10,45). This interaction abrogates the pRB-mediated block of cell cycle progression and may contribute to tumor formation (24). In addition, these or other viral proteins are involved in the neutralization of additional cell cycle regulators, in particular of the growth suppressor p53 (24,39). The interaction between the papillomavirus E7 and E6 proteins with pRB and p53, respectively, mediates the degradation of the latter by the ubiquitin-proteasome pathway (8,25,44). Hence, in addition to the interaction with growth suppressors, papillomaviruses make use of the protein degradation machinery to target these proteins to the 26S proteasome.Ubiquitination of proteins destined for degradation by the 26S proteasome is mediated by the action of the enzymatic complexes E1, E2, and E3 (21). E1 and E2 activate ubiquitin and catalyze the polyubiquitination of the substrate, which is thus marked for degradation by the 26S proteasome. A diverse class of complexes, the E3 ubiquitin-ligases, contains the elements of specificity for the substrates to be ubiquitinated. The

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.

Northern analysis has revealed substantial differences in mRNA accumulation of the two histone H3 gene variants represented by pH3c-1 and pH3c-11 cDNA clones. Both in partially synchronized cell suspension cultures and in protoplast-derived cells from alfalfa, Medicago varia, the maximal level of the histone H3-1 gene transcript coincided with the peak in [(3)H]thymidine incorporation. Histone H3-11 mRNA was detectable in cells throughout the period of the cell cycle studied. Various stress factors such as medium replacement, enzyme digestion of the cell wall, osmotic shock, and auxin treatment considerably increased the level of the histone H3-11 transcript. In alfalfa (Medicago sativa), the presence of H3-11 mRNA in unorganized tissues of microcallus suspension and in somatic embryos induced by auxin treatment supports the idea that this H3 variant exists in a continously active state of transcription. During embryo development, the early globular stage embryos showed increased accumulation of histone H3-11 mRNA in comparison with the later stages. The highest level of the histone H3-1 transcript was detectable 1 day after treatment of callus tissues with 2,4-dichlorophenoxyacetic acid. Somatic embryos contained appreciable levels of histone H3-1 transcripts at all stages of somatic embryo development. These observations suggest that the histone H3-1 gene belòngs to the class of replication-dependent histone genes. The histone H3-11 gene showed characteristics of a constitutively expressed replacement-type histone gene, with a specific characteristic that external factors can influence the level of gene transcription.