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The model genome of Arabidopsis thaliana contains a DEAD-box RNA helicase family (RH) of 58 members, i.e. almost twice as many as in the animal or yeast genomes. Transcript profiling using real-time quantitative polymerase chain reaction (PCR) has been obtained for 20 AtRHs from nine different organs. Two AtRHs exhibited plant-specific profiles associated with photosynthetic and sink organs. The other 18 AtRHs had the same transcript profile, and the levels of transcription of these 'housekeeping'AtRHs were under strict quantitative control over a large range of values. Transcript levels may be very different between the most recently duplicated genes. The master regulatory element in the definition of the transcript level is the simultaneous presence of a TATA-box and an intron in the 5' untranslated region (UTR). There is a positive and highly significant correlation between the size of the 5' UTR intron and the transcription level, as long as a characteristic TATA-box is present. Our work on the housekeeping AtRHs suggests a scenario for the evolution of duplicated genes, leading to both highly and poorly transcribed genes in the same terminal branch of the phylogenetic tree. The general evolutionary drive of the AtRH family, after duplication of a highly transcribed ancestral AtRH, was towards an alteration of the transcriptional activity of the divergent duplicates through successive events of suppression of the TATA-box and/or the 5' UTR intron.

Background . Activation of the kinase cascade (protein kinase C (PKC), tyrosine kinase (TK), and mitogen-activated protein kinase (MAPK) is a key feature of the transduction pathway, elicited by preconditioning signals and mediating their cardioprotective effects. We assessed whether such an activation occurred during cardiac operations and could thus represent a target for cardioprotective strategies. Methods . A total of 20 patients undergoing coronary artery bypass grafting surgery were studied. During the first 10 minutes of cardiopulmonary bypass (CPB), 10 were treated with sevoflurane (2.5 minimum alveolar concentration), an inhalational anesthetic that mimics preconditioning through a similar activation of the kinase cascade. Ten case-matched patients undergoing 10 minutes of sevoflurane-free CPB served as controls. Right atrial biopsies were taken before and 10 minutes after CPB and were then processed for the measurement of PKC, TK, and p38 MAPK activities by enzyme assay techniques. Troponin I was also monitored over the first 2 postoperative days. Results . Compared with pre-CPB values, PKC and p38 MAPK activities (in nanomoles per milligram of protein per minute and arbitrary units, respectively) increased significantly and to the same extent in both groups: PKC, from 20.7 ± 0.7 to 29.9 ± 3.9 in controls ( p = 0.037) and from 18.4 ± 1.1 to 23.9 ± 1.8 in sevoflurane ( p = 0.016); p38 MAPK, from 88.6 ± 8.5 to 312.9 ± 66.2 in controls ( p = 0.005) and from 114.6 ± 14.7 to 213.4 ± 51.8 in sevoflurane ( p = 0.045). Conversely, sevoflurane triggered a significant increase in TK activity (from 68.5 ± 1.4 to 83.7 ± 2.9 picomoles per milligram of protein per minute p = 0.0015) which did not occur in controls (from 67.5 ± 1.9 to 76.8 ± 4.2 picomoles per milligram of protein per minute, p = 0.09). Likewise, the peak postoperative value of troponin I was not different between controls and sevoflurane-treated patients (3.4 ± 0.6 vs 2.4 ± 0.4, p = 0.21). Conclusions . Cardiopulmonary bypass triggers an activation of the kinase cascade that is mechanistically linked to opening of potassium channels. The direct opening of these channels by the anesthetic sevoflurane does not increase kinase activation further, nor does it improve markers of cell necrosis, thus suggesting that pharmacologically targeting potassium channels may overlap the preconditioning-like effects of CPB alone.

A large collection of T-DNA insertion transformants of Arabidopsis thaliana has been generated at the Institute of Agronomic Research, Versailles, France. The molecular characterisation of the insertion sites is currently performed by sequencing genomic regions flanking the inserted T-DNA (FST). The almost complete sequence of the nuclear genome of A.thaliana provides the framework for organising FSTs in a genome oriented database, FLAGdb/FST (http://genoplante-info.infobiogen.fr). The main scope of FLAGdb/FST is to help biologists to find the FSTs that interrupt the genes in which they are interested. FSTs are anchored to the genome sequences of A.thaliana and positions of both predicted genes and FSTs are shown graphically on sequences. Requests to locate the genomic position of a query sequence are made using BLAST programs. The response delivered by FLAGdb/FST is a graphical representation of the putative FSTs and of predicted genes in a 20 kb region.

The DEAD box RNA helicase (RH) proteins are homologs involved in diverse cellular functions in all of the organisms from prokaryotes to eukaryotes. Nevertheless, there is a lack of conservation in the splicing pattern in the 53 Arabidopsis thaliana (AtRHs), the 32 Caenorhabditis elegans (CeRHs) and the 29 Drosophila melanogaster (DmRHs) genes. Of the 153 different observed intron positions, 4 are conserved between AtRHs,CeRHs, and DmRHs, and one position is also found inRHs from yeast and human. Of the 27 different AtRHstructures with introns, 20 have at least one predicted ancient intron in the regions coding for the catalytic domain. In all of the organisms examined, we found at least one gene with most of its intron predicted to be ancient. In A. thaliana, the large diversity inRH structures suggests that duplications of the ancestralRH were followed by a high number of intron deletions and additions. The very high bias toward phase 0 introns is in favor of intron addition, preferentially in phase 0. Results from this comparative study of the same gene family in a plant and in two animals are discussed in terms of the general mechanisms of gene family evolution.

In the sequences released by the Arabidopsis Genome Initiative (AGI), we discovered a new and unexpectedly large family of orphan genes (127 genes by 01.08.99), named AtPCMP. The distribution of the AtPCMP genes on the five chromosomes suggests that the genome of Arabidopsis thaliana contains more than 200 genes of this family (1% of the whole genome). The deduced AtPCMP proteins are characterized by a surprising combinatorial organization of sequence motifs. The amino-terminal domain is made of a succession of three conserved motifs which generate an important diversity. These proteins are classified into three subfamilies based on the length and nature of their carboxy-terminal domain constituted by 1–6 motifs. All the motifs characterized have an important level of conservation in both sequence and spacing. A specific signature of this large family is defined. The presence of ESTs in databases and the detection of clones in A. thaliana cDNA libraries indicate that most of the genes of this family are expressed. The absence of similar sequences outside the plant kingdom strongly suggests that this unusually large orphan family is unique to plants. Features, the genesis, the potential function and the evolution of this plant combinatorial and modular protein family are discussed.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25 000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

The Arabidopsis Genome Initiative has released up to now more than 80% of the genome sequence of Arabidopsis thaliana. About 70% of the identified genes have at least one paralogue. In order to understand the biological function of individual genes, it is essential to study the structure, expression and organization of the entire multigene family. A systematic analysis of multigene families, made possible by the amount of genomic sequence data available, provides important clues for the understanding of genome evolution and plasticity. In this paper, four multigene families of A. thaliana are characterized, namely LCAD, HD-GL2, LGT and MYST. Members of HD-GL2 and LCAD have already been reported in plants. The LGT genes specify proteins containing motifs of glycosyl transferase. No plant genes similar to the LGT genes have been reported to date. The novel MYST family, most likely plant-specific, encodes proteins with no identified function. Sequencing and in silico analysis led to the characterization of 29 novel genes belonging to these four gene families. The organization, structure and evolution of all the members of the four families are discussed, as well as their chromosome location. Expression data of some of the paralogues of each family are also presented.

International audience; The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

Biological and computer-assisted analyses of a 25 kb fragment from Arabidopsis thaliana chromosome IV led to the characterization of two multigene families and three novel orphan genes, not previously described. The first gene family named AtMO1-4 encodes monooxygenases, related to the prokaryotic salicylate hydroxylases. The second gene family contains three members, two on the analysed 25 kb fragment and one on chromosome I. The latter three genes lack introns and are homologous to the previously studied Glycine max src2 gene which is overexpressed at low temperature. Gene expression and primary structure of the deduced proteins are described and compared. Three genes of unknown function, showing tissue specific expressions, are characterized on the 25 kb fragment. Full length or partial cognate cDNAs have been sequenced for all the genes studied.

The arabinose-sensitive ara1-1 mutant of Arabidopsis is deficient in arabinose kinase activity. A candidate for the ARA1 gene, ISA1, has been previously identified through the Arabidopsis genome sequencing initiative. Here we demonstrate that (1) the ARA1 gene coincides with ISA1 in a positional cloning strategy; (2) there are mutations in the ISA1 gene in both the ara1-1 mutant and an intragenic suppressor mutant; and (3) the ara1-1 and suppressor mutant phenotypes can be complemented by the expression of the ISA1 cDNA in transgenic plants. Together these observations confirm that ISA1 is the ARA1 gene. ARA1 is a member of the galactose kinase family of genes and represents a new substrate specificity among this and other families of sugar kinases. A second gene with similarities to members of the galactose kinase gene family has been identified in the EST database. A 1.8 kb cDNA contained an open reading-frame predicted to encode a 496 amino acid polypeptide. The GAL1 cDNA was expressed in a galK mutant of Escherichia coli and in vitro assays of extracts of the strain expressing GAL1 confirmed that the cDNA encodes a galactose kinase activity. Both GAL1 and ARA1 cross-hybridise at low stringency to other sequences suggesting the presence of additional members of the galactose kinase gene family.