Search

We have focused our interest on two cis-regulatory elements, named site II motif and telo box, identified within the promoter of plant proliferating cellular nuclear antigen (PCNA) and putatively involved in meristematic expression of the gene. A conserved topological association between site II motifs and telo boxes is observed in the promoter of numerous genes expressed in cycling cells, including several cell cycle-related genes and 153 Arabidopsis genes encoding ribosomal proteins. Meristematic expression of a GUS reporter gene was observed in plants under the control of Arabidopsis site II motif within a minimal promoter. This expression is strongly enhanced by addition of a telo box within this chimaeric promoter. We showed by gel retardation experiments that the site II motif is a target for several DNA-binding activities present in Arabidopsis crude cell extract and can bind a transcription factor, At-TCP20, from the Teosinte branched 1, Cycloidea, PCF (TCP)-domain protein family. In yeast two-hybrid experiments, At-TCP20 appears to be a potential partner of AtPuralpha, which was previously shown to bind telo boxes. An important consequence of this analysis is to reveal new and conserved regulatory processes concerning the regulation of plant ribosomal gene expression in cycling cells. The implication of these observations in plant-specific developmental pathways is discussed.

Referee: Dr. Philip Becraft, Zoology and Genetics/Agronomy Depts., 2116 Molecular Building, lowa State University, Ames, IA 50011 Forty-two lectin receptor kinase (lecRK)-related sequences and nine related soluble legume lectin sequences were identified in the Arabidopsis thaliana genome. The genes are scattered as a single or gathered copies at different loci throughout the five chromosomes, and four predicted lecRK probably correspond to pseudogenes. Both structural alignments and molecular modeling revealed striking similarities between the lectinlike domain of lecRK, and related A. thaliana soluble lectins and legume lectins. The hydrophobic cavity is extremely conserved, whereas most of the residues forming the monosaccharide-binding site and the bivalent cation-binding site of legume lectins are poorly conserved. LecRK should be unable to bind the simple sugars usually recognized by genuine legume lectins. Molecular modeling of the kinase domain suggests that, except for two apparently inactive rece...

The lectin receptor protein kinases (lecRKs) belong to a large gene family in Arabidopsis thaliana . As for numerous receptor-like kinases (RLKs) of plants, no physiological role has been assigned to lecRKs. To try to elucidate the role of one of them, the expression of lecRK-a1 was studied during development and in response to abiotic stimuli. Histochemical analysis of transgenic plants containing the lecRK-a1 promoter fused to the GUS reporter gene revealed an increase of the expression level of lecRK-a1 in parallel with natural senescence processes. These results were confirmed by in situ hybridization and RT-PCR experiments and appeared to be specific to this member of the lecRK-a family. The expression of lecRK-a1 is locally activated in response to wounding and is also activated in response to oligogalacturonides. LecRK-a1 seems to play an important role in both the developmental program and adaptive processes in A . thaliana.

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.

D-type cyclins are believed to regulate the onset of cell division upon mitogenic signaling. Here, the isolation is reported of a new D-type cyclin gene (CYCD4;1) of Arabidopsis thaliana (L.) Heynh. during a two-hybrid screen using the cyclin-dependent kinase CDC2aAt as bait. Transcription of CYCD4;1 can be induced by sucrose. The co-regulated expression of CYCD4;1 and CDC2aAt in starved suspension cultures upon mitogenic stimulation indicates that the formation of a complex between these two partners is important for the resumption of cell division activity. By in-situ hybridizations CYCD4;1 was shown to be expressed during vascular tissue development, embryogenesis, and formation of lateral root primordia. Expression during the latter process suggests that the induced expression of D-type cyclins by mitogenic stimuli might be one of the rate-limiting events for the initiation of lateral roots.

An Arabidopsis cDNA clone that defines a new class of plant serine/threonine receptor kinases was found to be a member of a family of four clustered genes (lecRK-a1–a4) which have been cloned, sequenced and mapped on chromosome 3. This family belongs to a large superfamily encoding putative receptors with an extracellular domain homologous to legume lectins and appears to be conserved at least among dicots. In the Columbia ecotype only the lecRK-a1 and perhaps the lecRK-a3 gene is functional, since lecRK-a2 is disrupted by a Ty-copia retroelement and lecRK-a4 contains a frameshift mutation. Structural analysis of the lecRK-a1 and lecRK-a3 deduced amino-acid sequences suggests that the lectin domain is unlikely to be involved in binding monosaccharides but could interact with complex glycans and/or with hydrophobic ligands. Immunodetection of lecRK gene products in plasma membranes purified by free-flow electrophoresis showed that the lecRK-a proteins are probably highly glycosylated integral plasma membrane components.

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.

As part of the goal to generate a detailed transcript map for Arabidopsis thaliana, 1152 single run sequences (expressed sequence tags or ESTs) have been determined from cDNA clones taken at random in libraries prepared from different sources of plant material: developing siliques, etiolated seedlings, flower buds, and cultured cells. Eight hundred and ninety-five different genes could be identified, 32% of which showed significant similarity to existing sequences in Arabidopsis and an array of other organisms. These sequences in combination with their positioning on the Arabidopsis genetic map will not only constitute a new set of molecular markers for genome analysis in Arabidopsis but also provide a direct route for the in vivo analysis of their gene products. The sequences have been made available to the public databases.

Background Short interstitial telomere motifs (telo boxes) are short sequences identical to plant telomere repeat units. They are observed within the 5' region of several genes over-expressed in cycling cells. In synergy with various cis-acting elements, these motifs participate in the activation of expression. Here, we have analysed the distribution of telo boxes within Arabidopsis thaliana and Oryza sativa genomes and their association with genes involved in the biogenesis of the translational apparatus. Results Our analysis showed that the distribution of the telo box (AAACCCTA) in different genomic regions of A. thaliana and O. sativa is not random. As is also the case for plant microsatellites, they are preferentially located in the 5' flanking regions of genes, mainly within the 5' UTR, and distributed as a gradient along the direction of transcription. As previously reported in Arabidopsis, a conserved topological association of telo boxes with site II or TEF cis-acting elements is observed in almost all promoters of genes encoding ribosomal proteins in O. sativa. Such a conserved promoter organization can be found in other genes involved in the biogenesis of the translational machinery including rRNA processing proteins and snoRNAs. Strikingly, the association of telo boxes with site II motifs or TEF boxes is conserved in promoters of genes harbouring snoRNA clusters nested within an intron as well as in the 5' flanking regions of non-intronic snoRNA genes. Thus, the search for associations between telo boxes and site II motifs or TEF box in plant genomes could provide a useful tool for characterizing new cryptic RNA pol II promoters. Conclusions The data reported in this work support the model previously proposed for the spreading of telo boxes within plant genomes and provide new insights into a putative process for the acquisition of microsatellites in plants. The association of telo boxes with site II or TEF cis-acting elements appears to be an essential feature of plant genes involved in the biogenesis of ribosomes and clearly indicates that most plant snoRNAs are RNA pol II products.

We have focused our interest on two cis-regulatory elements, named site II motif and telo box, identified within the promoter of plant proliferating cellular nuclear antigen (PCNA) and putatively involved in meristematic expression of the gene. A conserved topological association between site II motifs and telo boxes is observed in the promoter of numerous genes expressed in cycling cells, including several cell cycle-related genes and 153 Arabidopsis genes encoding ribosomal proteins. Meristematic expression of a GUS reporter gene was observed in plants under the control of Arabidopsis site II motif within a minimal promoter. This expression is strongly enhanced by addition of a telo box within this chimaeric promoter. We showed by gel retardation experiments that the site II motif is a target for several DNA-binding activities present in Arabidopsis crude cell extract and can bind a transcription factor, At-TCP20, from the Teosinte branched 1, Cycloidea, PCF (TCP)-domain protein family. In yeast two-hybrid experiments, At-TCP20 appears to be a potential partner of AtPuralpha, which was previously shown to bind telo boxes. An important consequence of this analysis is to reveal new and conserved regulatory processes concerning the regulation of plant ribosomal gene expression in cycling cells. The implication of these observations in plant-specific developmental pathways is discussed.

The promoters of Arabidopsis eEF1A genes contain a telomere motif, the telo-box, associated with an activating sequence, the tef-box. Database searches indicated the presence of telo-boxes in the 5' region of numerous genes encoding components of the translational apparatus. By using several promoter constructs we demonstrate that the telo-box is required for the expression of a beta-glucoronidase gene in root primordia of transgenic Arabidopsis. This effect was observed when a telo-box was inserted upstream or downstream from the transcription initiation site, and occurred in synergy with the tef-box. These results clearly indicate that interstitial telomere motifs in plants are involved in control of gene expression. South-western screening of a lambdaZAP library with a double-stranded Arabidopsis telomere motif resulted in characterization of a protein related to the conserved animal protein Puralpha. The possibility of a regulation process similar to that achieved by the Rap1p in Saccharomyces cerevisiae is discussed.

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.

Under conditions of nitrogen limitation leguminous plants can form associations with soil bacteria of the genus Rhizobium culminating in the formation of symbiotic root nodules. In such nodules rhizobia convert atmospheric nitrogen into ammonia, which then serves as nitrogen source for the plant. Mutual recognition of the symbiotic partners is central to the establishment of the Rhizobium-legume symbiosis. An important characteristic of this recognition process is its specificity. Recent research has shown that rhizobia produce species-specific extracellular lipo-oligosaccharides known as Nod factors which elicit, in the appropriate plant host, a variety of symbiosis-related events such as root hair deformation, cortical cell divisions and nodule formation [1]. However, host-specificity also appears to be determined by the plant partner. In 1989, Diaz et al. [2] showed that white clover roots carrying a pea lectin gene were susceptible to infection and nodulation by a pea-specific Rhizobium strain which normally cannot nodulate white clover. This finding supported an earlier hypothesis [3, for reviews see 4-6] that plant lectins (carbohydrate-binding proteins) might be determinants of host-specificity through their specific binding to bacterial polysaccharides. As a means of determining which symbiotic events might be mediated by lectins, we have adopted the complementary approach of studying lectin gene expression throughout the various successive stages of the Rhizobium-legume interaction. For this purpose, we have characterized the three members of a lectin gene family from the model legume Medicago truncatula. Each of the three lectin promoters was fused to the E. coli gusA reporter gene (encoding sglucuronidase = GUS) and these constructions were introduced into alfalfa (M. varia) plants using an Agrobacterium tumefaciens transformation approach.

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.

In Arabidopsis thaliana, the activation process of the A1 EF-1 alpha gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5' non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5' intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 alpha promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 alpha genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.

International audience; In A. thaliana the translation elongation factor EF-1 alpha is encoded by a small multigenic family of four members (A1-A4). The A1 gene promoter has been dissected and examined in a transient expression system using the GUS reporter gene. Deletion analysis has shown that several elements are involved in the activation process. One cis-acting domain, the TEF 1 box, has been accurately mapped 100 bp upstream of the transcription initiation site. This domain is the target for trans-acting factors identified in nuclear extracts prepared from A. thaliana. Homologies are found between the TEF 1 box and sequences present at the same location within the A2, A3 and A4 promoters. This observation, together with those obtained from gel retardation assays performed using DNA fragments from the A4 promoter, suggest that the activation process mediated by the TEF 1 element is conserved among the A. thaliana EF-1 alpha genes. Analysis of nearly full length cDNA clones has shown that in addition to a single intron located within the coding region, the A1 gene contains a second intron located within the 5' non coding region. Such an intron is also present within the A2, A3 and A4 genes. This 5' intervening sequence appears to be essential to obtain a maximum GUS activity driven by the A1 gene 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.

The specific activity of yeast RNA polymerases A or B, when complexed with P37 cofactor, compares favorably with that of E. coli RNA polymerase. The stimulation is observed only with double-stranded DNA but does not result from DNase action. The Km for nucleotide substrates and the optimal conditions of transcription are not modified. P37 stimulates RNA synthesis by ternary transcription complexes in the presence of poly(rI) which prevents reinitiations. The RNA chain length, estimated by 5' end labeling or sedimentation, is increased in the presence of P37. On the other hand, the trinucleotide synthesis, which reflects the chain initiation reaction, is not affected. Therefore, the cofactor appears to act at the elongation step of RNA synthesis.

Summary The study of the mechanism of action of the hypoglycemic biguanides (dimethyl and phenethyl biguanides) known as neoglycogenesis inhibitors, lead us to investigate this metabolic pathway on various cellular and acellular kidney cortex systems where, in the absence of glycogen, free glucose is obtained. o 1 On cellular systems: cortex slices, whatever substrates were employed, biguanide inhibition of neoglycogenesis has been observed. A selective stimulation by Mn++ at the level of reactions from pyruvate to triose-phosphates has been demonstrated. 2 With regard to acellular systems, studies of cortex homogenate have shown the absolute Mn++ requirement for glucose formation from substrates below the triose phosphate level. A relation between Mn++ and stimulation of neoglycogenesis occuring during fasting is demonstrated and a biguanide Mn++ interaction (as one explanation for there inhibitory action) is postulated. An action of biguanides on glucose-6-phosphatase activity is also indicated. 3 Experiments on kidney cortex cytosol concerning the reaction from malate to phosphoenol pyruvate (in presence of fluoride, an inhibitor of enolase) or to glucose-6-phosphate (in the absence of fluoride) confirm the importance of an Mn++-PEP carboxykinase regulation and inhibition by biguanides at this level.