Characterization of a rice gene coding for a lipid transfer protein.

The cloning and sequence analysis of a gene that encodes a lipid transfer protein (LTP) from rice is reported. A genomic DNA library from Oryza sativa was screened using a cDNA encoding a maize LTP. One genomic clone containing the gene (Ltp) was partially sequenced and analyzed. The open reading frame is interrupted by an 89-bp intron. From the results of Southern hybridizations, Ltp appears to be a member of a small multigenic family. Transcripts of the corresponding gene were detected in several tissues including coleoptile, leaf, endosperm, scutellum and root. The transcription start point was determined by primer extension. The deduced amino-acid sequence of the Ltp product is shown to be homologous to LTPs from other crops.

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.

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.

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.

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.

AtE2F-a and AtDP-a, members of the E2F family of transcription factors, induce Arabidopsis leaf cells to re-enter S phase.

In eukaryotes, transcription factors of the E2F family, in addition to having a role in cell proliferation, participate in regulating apoptosis, differentiation and development. In Arabidopsis thaliana, eight gene sequences have been identified as encoding E2F or DP homologues. DP proteins form heterodimers with E2Fs. The aim of this work was to characterize the functions of three of these factors: AtE2F-a, AtE2F-b and AtDP-a. Here we report that AtE2F-a and AtE2F-b transactivate a reporter gene via an E2F consensus cis-acting element in Arabidopsis protoplasts. AtE2F-a is a more potent activator than AtE2F-b. Furthermore, co-expression of the E2F partner AtDP-a, or the DNA binding protein AtPur alpha, modulates the activation of AtE2F-a. Taken together, these results suggest that AtE2F-a, AtE2F-b and AtDP-a share features characteristic of members of the E2F family of transcription factors. Moreover, over-expression of AtE2F-a and AtDP-a can induce differentiated, non-dividing, leaf cells to re-enter S-phase. We conclude that the transcription factor AtE2F-a plays an important role in progression into S phase, which probably correlates with its capacity to stimulate transcription.

The tef1 box, a ubiquitous cis-acting element involved in the activation of plant genes that are highly expressed in cycling cells.

In Arabidopsis thaliana, the tef1 box is a cis-acting promoter element of the EF-1 alpha A1 gene involved in the activation of transcription in meristematic tissues. The initiation of root calli in transgenic Arabidopsis by 2,4-D shows that the tef1-dependent expression of the GUS reporter gene is not restricted to meristematic regions but involves all of the cycling cells. Hybridization experiments conducted using Arabidopsis cDNA clones organized in a dense array on filters, and cDNA probes prepared from cells in various states of growth, or blocked at different steps of the cell cycle, indicate that the enhanced expression of EF-1 alpha genes occurs in cycling cells at the point of entry into the cell cycle and remains constant during transit through the cycle. The analysis of several promoters of genes, other than EF-1 alpha, which are overexpressed in growing cells and involved in the processes of translation or redox regulation, reveals the presence of sequences showing partial homologies with the tef1 box. The Arabidopsis ribosomal gene srp18 and the tobacco gene thioh2, encoding a thioredoxin h, contain such sequences. Gel retardation experiments suggest that these sequences are targets for the same proteins as those that interact with the tef1 box of the Arabidopsis EF-1 alpha A1 gene. In transfected Arabidopsis protoplasts, the putative tef1 sequence thioh2 partially restores the activity of a tef1 box-less EF-1 alpha A1 promoter. These data demonstrate that the tef1 box is a ubiquitous cis-acting element involved in the transcriptional activation of plant genes that are overexpressed in cycling cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Fine structure and evolution of the rDNA intergenic spacer in rice and other cereals.

The intergenic spacer of a rice ribosomal RNA gene repeating unit has been completely sequenced. The spacer contains three imperfect, direct repeated regions of 264-253 bp, followed by a related but more highly divergent region. Detailed analysis of the sequence allows the presentation of an evolutionary scenario in which the 264-253-bp repeats are derived from an ancestral 150-bp sequence by deletion and amplification. Comparison of the rice sequence with those of maize, wheat, and rye shows that, despite considerable divergence from the ancestral sequence, several regions have been highly conserved, suggesting that they may play an important role in the structure and/or expression of the ribosomal genes.

Plant interstitial telomere motifs participate in the control of gene expression in root meristems.

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.

Isolation and characterization of an unusual repeated sequence from the ribosomal intergenic spacer of the crucifer Sisymbrium irio.

A recombinant plasmid containing a 433 base pair (bp) Bam HI fragment from Sisymbrium irio genomic DNA was isolated and characterized. This fragment was shown to be a ribosomal intergenic spacer (IGS) sequence which is reiterated up to six times in the IGS and extends close to the 5' end of the 18S rRNA gene. The nucleotide sequence of the cloned element is composed of 10-11 40 bp blocks that are probably derived from a common ancestor. The presence of a similar sequence can be detected in the DNA of another Sisymbrium species and in Matthiola incana. Homology was also found with the last 43 nucleotides of the radish IGS 3' end, suggesting that there is possibly a common ancestral nucleotide motif in cruciferous IGS sequences. The cloned element hybridises to RNA transcripts, indicating that the S. irio IGS repetitive sequence is at least partially transcribed during the pre-rRNA transcription process.

A nuclear protein fraction binding to dA/dT-rich sequences upstream from the radish rDNA promoter [corrected].

The external spacer (ES) of rRNA nuclear genes (rDNA) contains the sequences that control rDNA transcription initiation and enhancement. The ES is also characterized in most species by the presence of multiple repeated elements. In higher plants very few data are available on the cis- and trans-acting elements which control rDNA transcription. Using electrophoretic mobility shift assays (EMSA) it is shown that nuclear extracts from young radish leaves (NER) contain a protein fraction which binds to specific sequences in the radish ES. DNase I footprinting analysis allows mapping of the NER protein binding to dA/dT homopolymer stretches and to a 13-bp dA/dT-rich short repeat, found both in the seven approximately 100 bp repeat regions (located -1077 to -740 from transcription initiation site) and the region (-120 to -55) containing the putative promoter for rDNA transcription initiation. Whether this ES binding is due to a single or several different proteins is not known. So far, protein(s) binding to dA/dT-rich regions of a plant rDNA ES has not yet been described. Whether it is a specific RNA polymerase I transcription factor(s) or plays a more general role in genome expression remains to be elucidated.

Genome DNA sequencing around the EF-1 alpha multigene locus of Arabidopsis thaliana indicates a high gene density and a shuffling of noncoding regions.

In Arabidopsis thaliana, EF-1 alpha proteins are encoded by a multigene family of four members. Three of them are clustered at the same locus, which was positioned 24 cM from the top of chromosome 1. A region of DNA spanning 63 kb around these locus was sequenced and analyzed. One main characteristic of the locus is the mosaic organization of both genes and intergenic regions. Fourteen genes were identified, among which only four were already described, and other unidentified are most likely present. Functionally diverse genes are found at close intervals. Exon and intron distribution is highly variable at this locus, one gene being split into at least 20 introns. Several duplications were found within the sequenced segment both in coding and noncoding regions, including two gene families. Moreover, a sequence corresponding to the 5' noncoding region of the EF-1 alpha genes and harboring a 5' intervening sequence is duplicated and found upstream of several genes, suggesting that noncoding regions can be shuffled during evolution.

Structural and transcriptional characterization of the external spacer of a ribosomal RNA nuclear gene from a higher plant.

A lambda recombinant phage, carrying a radish rDNA fragment spanning the complete external spacer and its borders, has been isolated and characterized by sequencing. The fragment is 2911 bp long and includes 486 bp of the 3' end of the 25S rRNA sequence, 2349 bp of spacer and the first 76 bp of the 5' end of the 18S rRNA sequence. The spacer can be divided into three regions: two unique domains flanking a 830-bp region of repeated sequences. Seven repeats ranging from 80 to 103 bp can be recognized. They are separated by short arrays of 12-21 adenylic residues. Each repeat slightly differs from the others by single-nucleotide changes or short deletions. Examination of single-nucleotide changes common to two units suggests that a duplication arose during the evolution of this sequence. The repeated region was subcloned and used as a probe to demonstrate that it is highly species-specific: in stringent conditions it does not cross-hybridize with the spacer of ribosomal genes from closely related species such as Brassica. Transcription products, starting or finishing within the spacer sequence, were mapped by northern blotting, primer extension and S1 mapping. Two major precursors were identified starting respectively at positions 2095 and 2280. The region surrounding the start at 2095 presents extensive homology with an analogous region in maize, rye, mung bean, Xenopus and tse-tse fly. However, longer transcripts can be detected. Several 3' ends downstream of the 25S terminus were also observed. Taken together these results indicate that rDNA transcription and pre-rRNA processing in plants are more complex than anticipated from previous studies.

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs.

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.