Françoise Grellet

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.

GASA, A GIBBERELLIN-REGULATED GENE FAMILY FROM ARABIDOPSIS THALIANA RELATED TO THE TOMATO GAST1 GENE

A multiple gene family of at least four members, related to a GA-stimulated transcript (GAST1) from tomato, was characterized in Arabidopsis thaliana by analysing four related cDNAs, named GASA1 to GASA4. The corresponding peptides display comparable structural features: (1) a putative signal peptide of 18 to 23 residues; (2) a highly divergent hydrophilic region of about 22 amino acids; (3) a conservative 60 amino acid C-terminal domain containing 12 cysteines. This organization has also bean shown in two related peptides from tomato, GAST1 found in shoots and RSI-1 found in early lateral roots. Southern blot hybridization patterns showed single-copy genes for all four members of the GASA family. Accumulation of the various transcripts, monitored by northern blot hybridization, indicated that the various genes are expressed differentially in plant organs. Specific mRNAs were mostly detected in flower buds and immature siliques in the case of GASA1, in siliques and dry seeds in the case of GASA2 and 3, and in growing roots and flower buds in the case of GASA4. At least two of the GASA genes are activated in GA-deficient mutant ga5, as early as 4 to 8 h after spraying with 50 μM GA3. The complex patterns of expression and regulation of the various genes suggest that the related peptides are involved in a developmental regulation process in Arabidopsis.

Two different Em-like genes are expressed in Arabidopsis thaliana seeds during maturation

Using a radish cDNA probe, we have isolated and characterized two genomic clones from Arabidopsis thaliana (GEA1 and GEA6) encoding two different proteins that are homologous to the “Early methionine-labelled” (Em) protein of wheat. GEA1 differs from GEA6 and Em clones of wheat in that a sequence coding for 20 amino acid residues is tandemly repeated 4 times. These two genomic clones correspond to two genes named AtEm1 and AtEm6. Sequencing of several cDNA clones showed that both genes are expressed. The transcription start site was determined for both genes by RNase mapping. The site of polyadenylation is variable and there is no obvious consensus sequence for polyadenylation at the 3′ ends of the genes. mRNA corresponding to GEA6 is present only in nearly dry and dry seeds, whereas that corresponding to GEA1 appears in immature seeds and is maximum in dry seeds. No expression of either gene could be detected in leaf, stem, or floral buds. Expression of both genes could be detected in immature seeds when the siliques were incubated with abscisic acid (ABA), demonstrating that both genes are ABA responsive. However, examination of the 5′ upstream region does not reveal any extensive homology, suggesting that regulation of the two genes differs. In situ hybridization with a GEA1 probe demonstrated that the expression of this gene is essentially located in the provascular tissues of the cotyledons and axis of the dry seed as well as in the epiderm and outer layers of the cortex in the embryo axis.

Identification of the most represented repeated motifs in Arabidopsis thaliana microsatellite loci

The major simple sequence repeats present in the Arabidopsis genome were identified by Southern hybridizations with 49 oligonucleotide probes matching all the possible combinations of motifs up to 4 nucleotides long. The method used allowed us to perform all the hybridizations under the same temperature conditions. A good correlation was observed with the data obtained from database analysis, indicating that the method can be useful for identifying the major classes of microsatellite loci in species for which few or no sequence data are available. AG/CT, AAG/CTT, ATG/CAT and GTG/CAC are the major motifs present in the Arabidopsis genome that can be used as convenient probes to isolate microsatellite loci by screening libraries. AAG/CTT is the more frequent of these motifs, and its relative frequency in Arabidopsis is much higher than averagely found in the plant kingdom. About 8% of the cDNA clones from an immature silique library contains AG/CT, AAG/CTT or ATG/CAT microsatellite loci. Several microsatellite loci were isolated by screening genomic and cDNA libraries. Twenty-six tri-nucleotide loci were PCR amplified from four different ecotypes, and polymorphism was observed for 12 of them; 10 loci showing two alleles and 2 loci showing three alleles.

A CHLOROPHYLL SYNTHETASE GENE FROM ARABIDOPSIS THALIANA

During the course of an Arabidopsis thaliana genome sequencing project, we identified a gene, G4, with a derived amino acid sequence showing homology to the product of the Rhodobacter capsulatus bchG locus which is involved in the esterification of bacteriochlorophyllide with geranylgeraniol. The relationship between this gene and bchG was confirmed by the isolation and analysis of a corresponding full-length cDNA. Comparison of genomic and cDNA sequences indicated that the gene is made up of 14 exons, some of them being very short. Southern and Northern analyses showed that this sequence represents a single-copy gene and its transcript is detected only in green or greening tissues. Both homologies and expression data suggest that this gene encodes a chlorophyll synthetase, one of the last enzymes of chlorophyll biosynthesis, and thus represents a new example of a nuclear gene encoding an enzyme of this pathway in higher plants.

The Arabidopsis thaliana cDNA sequencing projects.

© 1997 Federation of European Biochemical Societies

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.

Late Embryogenesis Abundant (LEA) protein gene regulation during Arabidopsis seed maturation

Summary This paper reviews part of our studies on gene regulation during the Arabidopsis seed maturation phase. Essentially, three complementary strategies have been used. The first one consisted in identifying genes expressed during this period by random sequencing of EST from a dry seed cDNA library and by comparing their frequency with that in an immature cDNA library. The second strategy focused on the detailed analysis of the expression of a specific group of genes coding for the class I LEA proteins, the Em genes, and analysis of their promoter. Finally we evaluated the expression of a number of LEA gene in various regulatory mutants, including abi3, lec1 and abi5 Altogether, our results illustrate the complexity of expression patterns and the interaction of various factors to define several distinct regulatory pathways.

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.

Histone content of germinating pea embryo chromatin decreases as DNA replicates

MODIFICATIONS in the expression of genetic information in eukaryotes are probably controlled by changes in the proteins associated with chromatin1. Unfortunately, in most of the differentiating systems which have been studied these changes are barely detectable, so that we do not know either which changes occur or when they occur. As a part of a more general study of the molecular developmental biology of seed germination we have investigated changes in chromatin of germinating pea embryo axes. Some particularly interesting features of this biological system make it useful for studying developmental changes in chromatin structure. First, it is quite easy to obtain large numbers of homogeneous seeds. Second, germination constitutes a switch from a dormant, arrested state to a new developmental programme. It is thus possible to study the sequence of molecular events which underlie the transition from a completely inactive genome to an active one. Third, this reactivation of the genome is accompanied by two waves of synchronous mitoses which can be considiered as temporal markers. Finally, the germinating embryo constitutes a set of differentiating tissues. To our knowledge, no animal system shares all these features together. The results which are described in the following sections demonstrate that a considerable loss of histone per DNA unit is associated with this reactivation of the genome during germination and that most of these changes take place as DNA replicates.