Claude Bardet

The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1α: molecular cloning, characterization and expression

SummaryThe gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1α) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the Al gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 by DNA fragment extending from −982 to −634 by upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1α F1 gene and of several highly expressed plant genes.

Modular organization and developmental activity of an Arabidopsis thaliana EF-1α gene promoter

The activity of the Arabidopsis thalana A1 EF-1α gene promoter was analyzed in transgenic Arabidopsis plants. The 5′ upstream sequence of the A1 gene and several promoter deletions were fused to the β-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-1α 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-1α upstream sequences than when these upstream sequences were replaced by the 35S enhancer. Similarly, a synthetic oligonucleotide corresponding to an A1 EF-1α upstream cis-acting element (the TEF1 box), is able to restore partially the original activity when fused to a TEF1-less EF1-α promoter but has no significant effect when fused to an enhancer-less 35S promoter.

In Vivo Interference with AtTCP20 Function Induces Severe Plant Growth Alterations and Deregulates the Expression of Many Genes Important for Development

AtTCP20 is a transcription factor belonging to the Arabidopsis (Arabidopsis thaliana) TCP-P subfamily, characterized by its capacity to bind to site II motifs (TGGGCY). Our aim was to understand the role of AtTCP20 in plant development. The expression pattern of a translational fusion of PromTCP20:CDS20∷GUS∷GFP suggested a function for AtTCP20 in several plant organs and stages of development. The role of AtTCP20 was challenged in planta by inducing expression of AtTCP20 proteins fused with either a transcriptional activator domain (VP16) or a repressor domain (EAR). Expression of both modified proteins led to severe developmental phenotypes. In-depth analysis suggested that AtTCP20 may participate in the regulation of cell expansion, cell division, and cell differentiation. Gene expression profiling in roots and hypocotyls revealed that 252 genes were down-regulated in both organs after induction of the AtTCP20∷EAR repressor gene. Site II motifs (TGGGCY) were underrepresented in their promoters. Conversely, GG(A/T)CCC sequences related to binding sites identified for TCP proteins in rice (Oryza sativa) were overrepresented, and a TCP20 fusion protein was shown to bind to these sequences in vitro. Gene ontology indicated that many targeted genes were involved in cell wall biogenesis and modification during expansion and also encoded numerous transcription factors controlling plant development. Our results are consistent with the previous proposal that AtTCP20 is involved in cell division and growth coordination. Moreover, they further suggest that AtTCP20 also contributes to cell expansion control and indicate a different involvement of this protein in plant morphogenesis depending on the organ and the developmental stage.

cDNA cloning and expression of an Arabidopsis GTP-binding protein of the ARF family

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 four members of the gene family encoding the Arabidopsis thaliana translation elongation factor EF-1α are actively transcribed

The gene family encoding the Arabidopsis thaliana translation elongation factor contains four genes, A1-A4 [1]. The genes A1, A2 and A3 are clustered within a 10 kb fragment. At present, the spacial relationship of gene A4 with the other three genes is unknown. Here, we report the complete sequence of genes A2, A3 and A4 together with the sequence of the intergenic region between A2 and A3 (Fig. 1). The genes A2 and A3 are organized in the same polarity; therefore, this intergenic region is particularly interesting since, within a 2 kb fragment, it must contain sequences involved in the termination and in the initiation of transcription of A3 and A2 genes, respectively. Relevant features found in this region are described in Fig. 1. Based upon transient expression and S 1 mapping experiments, we have recently shown that the gene A1 is actively transcribed [1]. In order to determine whether the genes A2, A3 and A4 are also transcribed we have analysed EF-lc~ cDNA clones isolated from a 2gt 11 cDNA library prepared from mRNA of an Arabidopsis thaliana cell suspension culture. Sequencing of these cDNA clones and comparison with the sequence of genomic clones allowed us to show that the four genes are actively transcribed and to localize one or several polyadenylation sites for each of them (Fig. 1). The fact that the four genes appear to be transcribed in a cell suspension culture does not exclude that they could be differentially expressed during the development as has been reported for Drosophila EF-I ~ genes [2].

In synergy with various cis-acting elements, plant insterstitial telomere motifs regulate gene expression in Arabidopsis root meristems

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.

An Arabidopsis cDNA encoding a 33-kilodalton laminin receptor homolog.

The major glycoprotein component of animal cell basement membranes, laminin, is involved in a variety of cellular activities, including cell adhesion, differentiation, and mitogenesis, that are mediated by the interaction of laminin with specific cell-surface receptors. A laminin-binding protein with an apparent molecular mass of 68 to 72 kD was first characterized in mammalian tumor cells and considered as “the laminin receptor” (Liotta et al., 1986; Wewer et al., 1986). Severa1 putative cDNA clones encoding this protein have been isolated from mammals (Yow et al., 1988; Rao et al., 1989; Van den Ouweland et al., 1989; Grosso et al., 1991). A11 the clones contained an open reading frame coding for a highly conserved polypeptide with a calculated molecular mass of 33 kD. Independently, a cDNA encoding an identical polypeptide was isolated from mouse tumor cells (Makrides et al., 1988), but the expressed protein, named factor p40, was shown to be a component of the translation machinery (Auth and Brawerman, 1992). Recently, DNA-deduced amino acid sequences exhibiting homology with the previously characterized 33-kD ”laminin receptor” were identified from hydra (Keppel and Schaller, 1991), Drosophila (M.B. Melnick, T.B. Chou, and N. Perrimon, accession No. M90422), and yeast (J. Miles and T.G. Formosa, accession No. M88277). We have isolated a cDNA clone (At lrhl) from a cDNA library of Arabidopsis thaliana cultured cells showing a striking homology to the laminin receptor cDNAs (Table I). Nucleotide homology of Atlrhl with the above-mentioned DNA sequences was restricted to the first two-thirds of the coding sequence, with highest homology to the human cDNA (66% identity). In the same way, the Atlrhl deduced polypeptide, 298 residues long, showed strong homology with the deduced protein sequences of other species from amino acids 12 to 222. In this region, mouse, human, and Drosophila polypeptides showed the highest homology to the plant sequence, corresponding to 60 to 62% identity and 78 to 79% similarity accounting for the conservative substitutions. No significant homology was found in the carboxy-terminal region from amino acids 223 to 298, apart from a relatively high conserved content in Trp. It is an intriguing feature that a11

Characterization and properties of heteromeric plant protein complexes that interact with tef cis-acting elements in both RNA polymerase II-dependent promoters and rDNA spacer sequences

Abstract 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.