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The telo-box, an interstitial telomere motif, was shown to regulate gene expression in root meristems, in synergy with a cis-acting element involved in the activation of expression of plant eEF1A genes, encoding the translation elongation factor EF1A, and of several ribosomal protein genes. We demonstrate here that the telo-box is also required for transcription activation by two other cis elements present within the promoter of genes encoding the acidic ribosomal protein rp40 and the proliferating cell nuclear antigen respectively. The control of gene expression by telo-boxes during cell cycle progression in Arabidopsis root meristems is discussed. A parallel is drawn with the function of telomeric sequences in Saccharomyces cerevisiae.

The tef box, a cis-acting element identified in promoters of several plant genes encoding components of the translation apparatus, is involved in the activation of gene expression in cycling cells. In vitro, this element mediates the formation of two protein complexes called C1 and C2. A tef-like box is also found within the intergenic transcribed spacer of several plant rRNA genes. In radish this sequence has already been described as a protein-binding site putatively involved in the regulation of rDNA expression and is sufficient for formation of C1 complexes. By using mutated tef boxes, we show that tef-dependent activation of transcription is correlated with formation of both C1 and C2 complexes in a context-dependent manner. In transient expression experiments, the activation of a minimal promoter-GUS gene fusion is associated with the formation of C2 complexes. In contrast, the ability to form C1 complexes appears to allow activation of reporter gene expression in root meristems of transgenic Arabidopsis. SDS-PAGE analysis of purified protein fractions containing either the C1 or the C2 activity indicates a complex heteromeric structure for these potential regulators. Thus, the tef box seems to be a central component of the regulation of gene transcription in distinct and overlapping developmental programs, and could be involved in co-regulation of transcription by RNA polymerases I and II.

The promoter of a gene encoding a ribosome-associated protein of 40 kDa from Arabidopsis thaliana (A-p40) was sequenced and the expression of the gene studied. A-p40 was expressed in the same organs and with the same variations as the eukaryotic elongation factor 1 alpha (eEF1A), another gene coding for a protein involved in translation Arabidopsis plants transformed with a beta-glucuronidase (GUS) gene driven by the A-p40 promoter confirm that A-p40 is expressed in actively dividing and growing cells. eEF1A promoter-GUS fusions have the same pattern of expression. Comparison of cis-acting elements from A-p40 and eEF1A revealed some common elements. A-p40 promoter deletions and transient gene expression in transfected Arabidopsis protopasts allowed the identification of trap40, a cis-acting element regulating gene expression. Gel retardation experiments indicate that eEF1A and A-p40 are regulated by different cis-acting elements. The role of such elements is discussed.

We have characterized an Arabidopsis receptor-like serine/threonine kinase gene, Ath.lecRK1 ( Arabidopsis thaliana lectin-receptor kinase), defining a new and putatively important class of plant receptor kinases. Structural features of the predicted polypeptide include an amino-terminal membrane-targeting signal sequence, a legume lectin-like extracellular domain, a single membrane-spanning domain, and a characteristic serine/threonine protein kinase domain. A recombinant protein containing the kinase domain can be autophosphorylated on a serine residue. Ath.lecRK1 is a member of a gene family of at least two closely related genes. Northern blot analysis indicates that the Ath.lecRK1 gene is weakly expressed in a variety of organs and is regulated in Arabidopsis cell suspension cultures according to the growth phase of cells. The role this new class of plant receptor kinase could play is discussed with regard to the transduction of oligosaccharide and plant hormone signals.

The sequence taAACCCTAa, or telo-box, is found at the same location within all known promoters of plant genes encoding the translation elongation factor EF-1α. An analysis of Arabidopsis DNA sequences available in databases revealed that the sequence AAACCCTAA, corresponding to 1·3 units of the plant telomeric repeat AAACCCT, is over-represented within the Arabidopsis genome. These motifs are found both within the transcribed and the untranscribed genome. The examination of their location in the available sequences as well as in selected and partially sequenced cDNA clones suggest that they are preferentially located in the 5′ region of genes. We show that a protein present in both maize and Arabidopsis cellular extracts is able to interact specifically with this motif. The putative functions associated with this sequence are discussed. We propose a model involving the telomerase to explain the spreading of this motif within the genome.

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.

A cDNA clone encoding a small GTP-binding protein, the ADP-ribosylation factor (ARF) was isolated from a cDNA library of Arabidopsis thaliana cultured cells. The predicted amino acid sequence was highly homologous to the known yeast, bovine and human ARF sequences. Southern analysis of Arabidopsis genomic DNA suggested the existence of at least two copies of ARF genes. The level of ARF mRNA was found to be nearly constant during all cell growth stages in suspension cultures.

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.

Localization of Escherichia coli RNA polymerase binding sites and initiation sites on superhelical or linear polyoma virus DNA was achieved by electron microscopy and by hybridization of short synthesis complementary RNA to polyoma DNA Hpa II fragments. These sites are located in positions 0·06, 0·25, 0·57, 0·85 and 0·98 on the physical map of polyoma DNA (Griffin et al. , 1974). With an equal ratio of enzyme to DNA, threefold less enzyme is bound to linear DNA than to superhelical DNA; however, the distribution of bound enzyme molecules in the different sites was identical on both templates. In addition, the complex formed with linear DNA is less stable than the complex formed with superhelical DNA. These findings are correlated with the decreased template activity of the linear DNA relative to superhelical DNA. Electron micrographs of complexes, obtained in the presence of a high ratio of enzyme to DNA, demonstrate the unwinding properties of the RNA polymerase.

The temporal and spatial sequence of nuclear disappearance during the terminal differentiation of lens fiber cells could be due to an impairment of the DNA repair pathways or to the appearance of an active DNA degradation process. The results presented here favor the second hypothesis. A single-stranded DNA nuclease activity and a double-stranded DNA nuclease activity have been found in chick embryo fiber cells. Moreover, there is a good correspondence between the variations of the nuclease activity and the stages of differentiation of the different samples analysed.

Yeast RNA polymerase II initiates in vitro transcription at two sites located within the vector DNA and the cloned promoter, on a recombinant plasmid DNA containing the yeast iso1 cytochrome c promoter. Both initiation sites are found within a DNA fragment hypersensitive to osmium tetroxide modification. Using a series of yeast iso1 cytochrome c promoter deletions, we have characterized an upstream DNA sequence required for optimal transcription from this site and shown in this case a correlation between osmium sensitivity and the capacity of RNA polymerase to initiate. However, perturbation of the double helix is not sufficient to generate a transcription initiation site. Insertion of 28 alternating AT residues at the EcoRV site of pBR322 generates an site hypersensitive to osmium tetroxide modification, that does not serve as a transcription start site.

Several steps in the transcription of double-stranded polyoma DNA by calf thymus RNA polymerase II(B) were analysed. Electron microscopy observations show that, as found for the Escherichia coli enzyme, the eucaryotic RNA polymerase exhibits a higher affinity for superhelical DNA as compared to linear DNA. The half-life time of complexes formed at 37 °C between the enzyme and superhelical DNA is relatively short (30 to 60 s), compared with 30 h for the E. coli enzyme under identical conditions. Binary complexes formed with linear DNA have a faster dissociation rate. The same results were obtained by using the nitrocellulose binding assay or by measuring the decay of heparin-resistant complexes. Only ternary complexes, enzyme-DNA-RNA, obtained after the formation of the first phosphodiester bond, are resistant to polyanions. These results suggest that, as for the procaryotic enzyme, RNA polymerase II has to melt locally the double-stranded DNA during initiation, but that it lacks a sigma type factor necessary to stabilise the open initiation complexes. RNA polymerase II synthesizes shorter RNA chains than E. coli RNA polymerase. The formation of RNase I resistant RNA-DNA hybrids during transcription may explain the reduced rate of chain elongation.

Yeast RNA polymerase B catalyzes an efficient abortive initiation on double-stranded DNA templates using the appropriate combination of primer and substrate. The specificity of initiation was investigated using a recombinant plasmid (pJD14 DNA) containing the structural gene for yeast alcohol dehydrogenase I (ADHI). The combination of the dinucleotide UpA and UTP was 10 fold more efficient with pJD14 DNA than with the vector pBR322 DNA to direct the synthesis of the trinucleotide UpApU. Under these conditions, stable enzyme-DNA complexes were formed and could be retained on nitrocellulose filters. Using the UpA-primed system and a short pulse of RNA synthesis, transcription complexes were located on the yeast part of pJD14 DNA as evidenced by agarose gel electrophoresis. Southern hybridization of the pulsed RNA was restricted to a region, within the yeast DNA fragment, upstream to the initial region of the ADHI gene.