Stability of polyadenylic and polyadenylated ribonucleic acids in radish (Raphanus sativus) seedlings.

The stability of polyadenylic acid and polyadenylated RNA was investigated in young radish (Raphanus sativus) seedlings. We first studied the decay of poly(A) content, using a [3H]poly(U) assay, following a complete block of transcription by cordycepin (200 microgram/ml). Two lifetime classes of polyadenylic acid have been determined in these seedlings: a short-lived component with a half-life of 30 min which represents 60% of poly(A) and a more stable component with varying half-lives of which the majority range from 4-10 h and a few are considerably longer. During this period rRNA was shown to decay linearly, taking about 41 h for half of this RNA to disappear. The life-time of the other moiety of polyadenylated-RNA was analysed by continuous labelling with [3H]uridine. We have been able to demonstrate that a significant part of the mRNA molecules turns over with a half-life similar to that of the more slowly turning-over poly(A). No evidence could be obtained for rapidly turning-over messenger RNA. Thus the rapidly turning over poly(A) could correspond to a poly(A) turn-over independent of the remainder of the sequence. When labelling was very long, an apparent steady-state was reached and we determined the polyadenylated RNA content of seedlings to be 2.2% of whole cell RNA. Finally, these results were compared with those previously obtained in studying early germination of radish embryo axes. In contrast with stored mRNA which is rapidly degraded following imbibition, part of the mRNA present in 22 h old seedlings is stable for several hours.

Organization and evolution of a higher plant alphoid-like satellite DNA sequence.

Two recombinant plasmids containing, respectively, three and eight tandem repeats of a 177 base-pair (bp) element from radish nuclear DNA have been isolated. These plasmids were used as probes to investigate the organization and the copy number of this element within the genome. This sequence is present in congruent to 0.6 million copies. Restriction analysis provides evidence for sequence heterogeneity and reveals the occurrence of non-overlapping subfamilies. Nine units were sequenced and found to be remarkably conserved. However, sequences in the two clones clearly belong to two distinct subgroups. Our data suggest that these sequences evolved in a concerted manner and that homogenization mechanisms such as gene conversions certainly took place. The 177 bp sequence is made from three 60 bp blocks that are derived from a common ancestor. Exchanges between the three blocks probably occurred before they became fixed as a patchwork of short sequences, the 177 bp element. This unit of 177 bp was then amplified in several steps. The presence of such a repeated sequence can be detected in other Cruciferae when hybridizations are carried out under low stringency conditions. Direct comparison with a previously published mustard satellite DNA sequence indicates a similar organization and a 75% homology. Homology was also found with shorter regions (congruent to 60 bp) of broad bean and corn satellite DNA. Finally, homology was also found with several animal alphoid sequences, suggesting that this family also occurs in the plant genomes.

The first green lineage cdc25 dual-specificity phosphatase.

The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G(2)/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G(2)/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been coevolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages.

Characterization and evolution of napin-encoding genes in radish and related crucifers.

Three cDNA clones, encoding napin storage proteins from radish, were isolated and sequenced. They fall into two classes differing in the size of the primary translation product. Sequences of the two classes are very well conserved and they display an organization very similar to that of the homologous genes from rapeseed and Arabidopsis which have previously been described. On the basis of hybridization intensity and the number of restriction fragments, we estimate that the radish napin multigene family is represented by eight to twelve members. The use of probes specific to each subfamily demonstrates that they contribute to a similar extent to the production of napin mRNA. Analysis of the sequence data suggests that the napin ancestral genes are probably derived from successive duplication and divergence of a protogene. Comparing other available napin sequences with those of radish reveals intriguing features. Comparison of the coding sequences shows that the homology between the radish and rapeseed sequences is much higher than that between each of the four members of the Arabidopsis gene family. This would suggest that the duplications which gave rise to the different members occurred independently in the two groups of species after separation of Arabidopsis from the Brassica lineage. However, similar comparison carried out on the 3' -noncoding sequences does not support this hypothesis, but shows that slightly different duplicated genes probably already existed in the common ancestor to the three genera. This paradox can be resolved by assuming that, within each genus, coding sequences for napin-encoding genes have been considerably homogenized as a result of concerted evolution.

A pair of genes coding for lipid-transfer proteins in Sorghum vulgare.

Approximately five genes coding for lipid-transfer proteins (LTP) can be detected in Sorghum vulgare by DNA blots using a specific genomic probe. Two of these genes have been identified and sequenced. The two genes (ltp1 and ltp2) code for very similar (91.8% identity) proteins, they are separated by approx. 4 kb of DNA and their open reading frames may be read in the same direction. The gene (ltp1) located upstream has an intron placed in the same position already described for other ltp in maize and rice. Gene ltp2 has no intron. cDNAs corresponding to ltp1 have been identified in a 6-day-old plantlet library, but not for ltp2. The results of the comparison between the two sequences indicate the presence of a gap between the two genes in their promoter region. LTP seem to be coded for in plants by a small family of genes. At least in sorghum, two of its components are tightly clustered in the same genomic region.

Rice lipid transfer protein (LTP) genes belong to a complex multigene family and are differentially regulated.

Several cDNA clones encoding three different lipid transfer proteins (LTPs) have been isolated from rice (Oryza sativa L.) in order to analyse the complexity, the evolution and the expression of the LTP gene family. The mature proteins deduced from three clones exhibited a molecular mass of 9 kDa, in agreement with the molecular mass of other LTPs from plants. The clones were shown to be homologous in the coding region, while the 3' non-coding regions diverged strongly between the clones. The occurrence of at least three small multigene families encoding these proteins in rice was confirmed by Southern blot analysis. When compared with each other and with LTPs from other plants, the cluster including rice LTPs and other cereal LTPs indicated that these genes duplicated rather recently and independently in the different plant phyla. The expression pattern of each gene family was also investigated. Northern blot experiments demonstrated that they are differentially regulated in the different tissues analysed. Components such as salt, salicylic acid and abscisic acid were shown to modulate Ltp gene expression, depending on tissues and gene classes, suggesting a complex regulation of these genes.

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.

Multiple mRNA coding for phospholipid-transfer protein from Zea mays arise from alternative splicing.

We have isolated a novel cDNA coding for maize phospholipid-transfer protein. The cDNA sequence is similar to the first one obtained by Tchang et al. [J. Biol. Chem. 263 (1988) 16849-16855] differing only by a mslal number of nucleotide substitutions and insertions. One of these insertions is 74 bp long and is flanked by consensus intron splicing sequences. The protein coded by the two cDNA has identical amino acids except in the C terminus. This difference derived from the presence of the 74-bp insert. The possible existence of an alternative splicing mechanism that could introduce heterogeneity in the sequence of these proteins is proposed.

The Arabidopsis thaliana cDNA sequencing projects.

Nearly 30000 Arabidopsis thaliana EST (Expressed Sequence Tags) have been produced by a French-American consortium. Despite redundancy, these sequences tag about half of the expected Arabidopsis genes. Approximately 40% of the non-redundant EST can be assigned a putative function by simple homology search. This programme allowed the identification of a large number of genes which would have been very difficult to isolate by other classical techniques. It considerably stimulated many areas of plant biology by the rapid discovery a large number of genes, by revealing multigene families and by allowing the analysis of differential expression of the different members. Finally this programme facilitated construction of physical maps of the chromosomes and opened the way for complete sequencing of the Arabidopsis genome and comparative mapping of the major plant crops.

The Arabidopsis thaliana cDNA sequencing projects.

Nearly 30000 Arabidopsis thaliana EST (Expressed Sequence Tags) have been produced by a French and an American consortium. Despite redundancy these sequences tag about half the expected Arabidopsis genes. Approximately 40% of the non-redundant EST can be assigned a putative function by simple homology search. This programme allowed the identification of a large number of genes which would have been very difficult to isolate by other classical techniques. It considerably stimulated many areas of plant biology by the rapid discovery a large number of genes, by revealing multigene families and by allowing the analysis of differential expression of the different members. Finally this programme facilitated construction of physical maps of the chromosomes and opened the way for complete sequencing of the Arabidopsis genome and comparative mapping of the major plant crops.

Mutations in the fatty acid elongation 1 gene are associated with a loss of beta-ketoacyl-CoA synthase activity in low erucic acid rapeseed.

Low erucic acid rapeseed (LEAR) is characterised by a near absence of very long chain fatty acids in the seed oil which has been correlated with a lack of acyl-CoA elongation activity. Here we show that the absence of acyl-CoA and ATP-dependent elongation activities in microsomes isolated from LEAR embryos is associated with an absence of beta-ketoacyl-CoA synthase activity encoded by the Bn-fatty acid elongation 1 (FAE1) genes. Size exclusion chromatography of solubilised microsomes revealed the presence of a high molecular mass acyl-CoA elongase complex in high erucic acid rapeseed which was absent in microsomes isolated from LEAR seeds. Although transcripts for the Bn-FAE1 genes were detected in LEAR embryos, immunoblots using antisera raised against the beta-ketoacyl-CoA synthase indicated an absence of this protein. Comparison of the deduced amino acid sequences of immature embryo cDNAs reveals that LEAR alleles of Bn-FAE1 encode variant beta-ketoacyl-CoA synthase proteins.

A chloroplastic RNA-binding protein is a new member of the PPR family.

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963-4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Airabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621-2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.

Characterization of a gene encoding an abscisic acid-inducible type-2 lipid transfer protein from rice.

The cloning and sequence analysis of a novel gene that encodes a type 2 non-specific lipid transfer-like protein (LTP) from rice is reported. Sequence analysis revealed an ORF encoding a protein showing characteristics of the LTP proteins. However, rice LTP2 is more similar to heterologous LTPs than to rice LTP1, supporting the existence of two distinct families of plant LTPs. Ltp2 mRNA is accumulated only in mature seeds. In vegetative tissues, mRNA was only detected after treatment with abscisic acid (ABA), mannitol or NaCl. Transient expression experiments that the 61 nucleotides upstream of the TATA box, containing two ACGT boxes and the motif I, are sufficient for ABA responsiveness of the Ltp gene.

The distribution of T-DNA in the genomes of transgenic Arabidopsis and rice.

Almost all the nuclear genes of four Gramineae (maize, wheat, barley, rice) and pea are located in DNA fractions covering only a 1-2% GC range and representing between 10 and 25% of the different genomes. These DNA fractions comprise large gene-rich regions (collectively called the 'gene space') separated by vast gene-empty, repeated sequences. In contrast, in Arabidopsis thaliana, genes are distributed in DNA fractions covering an 8% GC range and representing 85% of the genome. Here, we investigated the integration of a transferred DNA (T-DNA) in the genomes of Arabidopsis and rice and found different patterns of integration, which are correlated with the different gene distributions. While T-DNA integrates essentially everywhere in the Arabidopsis genome, integration was detected only in the gene space, namely in the gene-rich, transcriptionally active, regions of the rice genome. The implications of these results for the integration of foreign DNA are discussed.

Effect of the protein synthesis inhibitor cycloheximide on RNA synthesis in radish seedlings.

The effect of cycloheximide on rRNA and poly (A) (+) RNA synthesis is studied in radish seedlings. When used at a concentration of 50 microng/ml cycloheximide selectively blocks rRNA synthesis without altering poly (A) (+) RNA synthesis. Processing of the 2.3 X 10(6) dalton pre-rRNA is severely impaired. This observation should help in studies of mRNA metabolism in plants. When the concentration of cycloheximide is lowered, it is possible to completely block protein synthesis without preventing RNA synthesis. This implies that the effect of cycloheximide on RNA synthesis is probably more complex than usually assumed.

Genetic variability and evolution of the Schistosoma genome analysed by using random amplified polymorphic DNA markers.

The usefulness of random amplified polymorphic DNA markers (RAPD) was assayed in an attempt to discriminate among species, strains and individuals within the genus Schistosoma. Depending on the species, 40-50 arbitrary decamer oligonucleotides were used as primers to amplify total DNA by the polymerase chain reaction (PCR). An important polymorphism was observed among 5 species, allowing a phylogenetic tree to be outlined. These differences can be used for rapid and accurate identification. A limited but easily detectable polymorphism was revealed among 3 strains of a single species (Schistosoma mansoni). Minor differences were observed among individuals of a single strain. A RAPD marker allows sexual discrimination between individuals from the terminal spined-egg species group. Although a limited number of strains have been examined, the results already indicate clearly that RAPD markers constitute a powerful tool for the analysis of genetic variability. This new tool will considerably extend the information available from morphology, isozyme and limited restriction fragment length polymorphism data and opens the way to genetic analysis of these species.

Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana.

The Arabidopsis genome contains at least 18 genes encoding members of the 70-kilodalton heat shock protein (Hsp70) family, 14 in the DnaK subfamily and 4 in the Hsp110/SSE subfamily. While the Hsp70s are highly conserved, a phylogenetic analysis including all members of this family in Arabidopsis and in yeast indicates the homology of Hsp70s in the subgroups, such as those predicted to localize in the same subcellular compartment and those similar to the mammalian Hsp110 and Grp170. Gene structure and genome organization suggest duplication in the origin of some genes. The Arabidopsis hsp70s exhibit distinct expression profiles; representative genes of the subgroups are expressed at relatively high levels during specific developmental stages and under thermal stress.

Progress in Arabidopsis genome sequencing and functional genomics.

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 25000 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.