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M Delseny

Publications and source records attributed to M Delseny.

At least 91 records · Page 5Linked to original sources

Characterization of a Radish Nuclear Gene Expressed during Late Seed Maturation.

To study long-lived mRNAs stored in radish (Raphanus sativus) seed, we have selected clones from a dry seed cDNA library by differential screening. One of these clones, p8B6, whose mRNAs are abundant in the dry seed, was characterized. This clone hybridizes to an RNA class of approximately 600 nucleotides whose accumulation begins during the desiccation phase, reaches its maximum level in the dry seed, and is no longer detectable in 12 hour old seedlings. mRNAs hybrid-selected by p8B6 encode four polypeptides, but only two are compatible with the size class of RNAs detected by Northern analysis. Three of them have previously been identified as major ;early germination' polypeptides, and their synthesis has been shown to be induced prematurely in immature embryos by a desiccation treatment. The protein deduced from the p8B6 nucleotide sequence is 9 kilodaltons in size, highly hydrophilic, rich in Gly and Glu, and contains no Cys, Trp, and lie. The amino acid sequence shares good homology with that of two recently described seed proteins: a cotton late embryogenesis abundant protein and the wheat early methionine-labeled protein. Southern blot analysis suggests that the p8B6 sequence belongs to a very small gene family. The exact function of the product encoded by p8B6 remains to be determined.

Journal Article↗

Phospholipid transfer protein: full-length cDNA and amino acid sequence in maize. Amino acid sequence homologies between plant phospholipid transfer proteins.

We have determined the primary structure of a phospholipid transfer protein (PLTP) isolated from maize seeds. This protein consists of 93 amino acids and shows internal homology originating in the repetition of (do)decapeptides. By using antibodies against maize PLTP, we have isolated from a cDNA library one positive clone (6B6) which corresponds to the incomplete nucleotide sequence. Another cDNA clone (9C2) was obtained by screening a size-selected library with 6B6. Clone 9C2 (822 base pairs) corresponds to the full-length cDNA of the phospholipid-transfer protein whose mRNA contains 0.8 kilobase. Southern blot analysis shows that the maize genome may contain several PLTP genes. In addition, the deduced amino acid sequence of clone 9C2 reveals the presence of a signal peptide. The significance of this signal peptide (27 amino acids) might be related to the function of the phospholipid-transfer protein. The amino acid sequence of maize PLTP was compared to those isolated from spinach leaves or castor bean seeds which exhibit physicochemical properties close to those of the maize protein. A high homology was observed between the three sequences. Three domains can be distinguished: a highly charged central core (around 40-60), a very hydrophobic N-terminal sequence characteristic of polypeptide-membrane interaction, and a hydrophilic C terminus. A model for plant phospholipid-transfer proteins is proposed in which the phospholipid molecule is embedded within the protein with its polar moiety interacting with the central hydrophilic core of the protein, whereas the N-terminal region plunges within the membrane in the transfer process.

Amino Acid Sequence↗

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.

Base Sequence↗

Identification and characterization of the mRNA for major storage proteins from radish.

Precursors for radish storage proteins were identified by immunoprecipitation of cell-free protein-synthesis products. The 12S globulin polypeptides (21,000-33,000 Mr) are made from four larger precursors (47,500-60,000 Mr) and the 1.7 S albumin polypeptides (7,000-12,000 Mr) are synthesized as eight products of Mr about 20,000. From a cDNA library, several clones have been identified, using heterologous cDNA probes for rapeseed cruciferin and napin, and two of them (pAE10 and pBA3) used to hybrid-select the corresponding mRNA. The napin cDNA clone selects all translation products immunoprecipitated by anti-napin antibodies whereas the cruciferin clone selects only one polypeptide among the four recognized by 12S antibodies. The 12S globulin and the 1.7S albumin message sizes were estimated to be 1950 and 800 nucleotides respectively. Experiments carried out with mRNA populations from different maturation stages suggest a sequential gene expression for the two major storage protein families. In addition two napin subfamilies can be distinguished by their expression pattern. In dry seed, napin mRNA is not detectable and the amount of cruciferin mRNA decreased considerably so that it no longer represents a major fraction of dry seed mRNA.

Brassica↗

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.

Base Composition↗

Changes in Protein Synthesis in Rapeseed (Brassica napus) Seedlings during a Low Temperature Treatment.

Changes induced by cold treatment in young rapeseed (Brassica napus) seedlings were investigated at the molecular level. Following germination at 18 degrees C for 48 hours, one half of the seedlings was transferred to 0 degrees C for another 48 hour period, the other half being kept at 18 degrees C as a control. Newly synthesized proteins were labeled for the last 6 hours of incubation with [(35)S]methionine. The different polypeptides were separated by two-dimensional electrophoresis in polyacrylamide gels. Newly synthesized proteins were revealed by fluorography. Protein synthesis clearly continues at 0 degrees C and some polypeptides preferentially accumulate at this temperature. On the other hand, synthesis of several others is repressed while many are insensitive to cold treatment. Similar changes are also observed when mRNA is prepared from cold treated seedlings, translated in vitro in a reticulocyte cell free system and compared with the products of mRNA extracted from control samples. Among the genes which are repressed we identified the small subunit of ribulose 1,6-bisphosphate carboxylase. These changes are also detectable after shorter treatments.

Journal Article↗

In vitro synthesis of a plant phospholipid transfer protein: a study by high performance liquid chromatography.

In order to study the biosynthesis of a plant phospholipid transfer protein (PLTP), poly (A)+RNAs have been prepared from maize seedlings and translated in vitro with a rabbit reticulocyte lysate. The newly synthesized proteins were then separated by fast protein liquid chromatography (FPLC) followed by SDS-PAGE or by high performance liquid chromatography (HPLC) coupled to a radioactivity detector monitor. It has been showed that a radioactive band comigrating with a 14C methylated pure PLTP was detected by SDS-PAGE. This result, confirmed by direct radioactive monitoring, indicates that PLTP is actively synthesized in vitro.

Carrier Proteins↗

Characterization of Radish (Raphanus sativus) Storage Proteins.

Radish (Raphanus sativus cv Rond rose à bout blanc Vilmorin) seeds, as other cruciferae oil seeds, contain two major types of storage protein aggregates which can be separated by gel filtration into 12 and 1.7 Svedberg fractions. These two fractions have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, and two bidimensional gel electrophoresis systems. These results were compared with those obtained with rapeseed storage proteins. Radish 12 Svedberg particles are made of a series of nine major polypeptides ranging from 33 to 30 kilodaltons. These polypeptides present charge heterogeneity. The 12 Svedberg particle is made of six subunits approximately 55 kilodaltons. Each subunit is a couple of two polypeptides linked by a disulfide bridge. The 1.7 Svedberg particle has a simpler composition. It is made of two polypeptides of 10 and 12 kilodaltons and smaller peptides of approximately 7 kilodaltons. Twelve and 1.7 Svedberg particles also differ in their amino acid composition, the 1.7 Svedberg being particularly rich in glutamic acid and proline. Its components are basic. The organization of the rapeseed storage protein is similar but more complex.

Journal Article↗

Methylation Pattern of Radish (Raphanus sativus) Nuclear Ribosomal RNA Genes.

The methylation pattern of radish Raphanus sativus nuclear rDNA has been investigated using the Hpa II, Msp I, and Hha I restriction enzymes. The presence of numerous target sites for these enzymes has been shown using cloned rDNA fragments. A large fraction of the numerous rDNA units are heavily methylated, being completely resistant to Hpa II and Hpa I. However, specific sites are constantly available in another fraction of the units and are therefore unmethylated. The use of different probes allowed us to demonstrate that hypomethylated sites are present in different regions. Major hypomethylated Hha I sites have been mapped in the 5' portion of 25S rRNA coding sequence. Among the hypomethylated fraction, different methylation patterns coexist. It has been possible to demonstrate that methylation patterns are specific for particular units. The Hha I pattern of rDNA in tissues of different developmental stages was analyzed. Evidence for possible tissue specific differences in the methylation pattern is reported.

Journal Article↗

Detection of sequences with Z-DNA forming potential in higher plants.

Sequences of alternating purine-pyrimidine residues with Z-DNA forming potential have been detected in the nuclear DNA of two higher plant species: wheat and radish. Poly (dG-dT) and poly (dG-dC) stretches have been detected by hybridization of the corresponding nick-translated probes to Southern blots. These stretches are scattered throughout the genome and some of them belong to moderately repeated sequence families interspersed with other DNA sequences.

Cell Nucleus↗

Isolation and characterization of faithful and altered clones of the genomes of cauliflower mosaic virus isolates Cabb B-JI, CM4-184, and Bari I.

Full-length genomes of cauliflower mosaic virus (CaMV) isolates Cabb B-JI, CM4-184, and Bari I have been cloned in the SalGI site of plasmid pAT 153. The cloned DNAs were characterized by restriction mapping and infectivity assays. All the sites present in the virion DNAs were found in the cloned DNAs. Comparison of restriction maps with those of DNA from two other isolates which have been recently completely sequenced revealed a close relationship among the different isolates. Some of the clones appear to be faithful copies of the viral genomes and these viral inserts are infectious when inoculated into turnip plants. Various clones with deletions in the CaMV DNA have been isolated and characterized. Some of them may correspond to deletions naturally occurring in a subpopulation of the virus whereas others occurred during cloning. None of the deleted fragments are infectious when inoculated into plants. Strikingly, all the deletions overlap one or two of the specific single-stranded breaks characteristic of caulimoviruses, suggesting that sequences surrounding the breaks are not dispensable.

Chromosome Mapping↗

Adaptation of ribonucleic Acid metabolism to anoxia in rice embryos.

Rice (Oryza sativa var. Cigalon) is a plant which can adapt to very stringent anoxic conditions. It has previously been shown that during the adaptation period the energy charge can be used as a marker for metabolic activity. We have studied RNA metabolism during this period and correlated it with changes in the energy charge. Uptake of labeled precursor, UTP-specific activity, and incorporation were measured. Immediately after transfer to anaerobic conditions, the UTP pool size is reduced and the overall rate of incorporation drops. During adaptation, the rate of incorporation increases and stabilizes at about half of its value in aerobic conditions. Analysis of RNA shows that rRNA and mRNA are synthesized and that the processing of ribosomal RNA precursor is altered. Polyribosomes are present throughout the adaptation period although their amount is reduced during the first hour of anoxia. Changes in poly(A) content were noticed, indicating that some mRNA are rapidly degraded. Taken together, the results show that the RNA metabolism can be modulated during adaptation to anoxia in a parallel manner with energy charge changes.

Journal Article↗

Electron microscopic mapping of wheat germ RNA polymerase II binding sites on cloned CaMV DNA.

The binding sites of wheat germ RNA polymerase II were mapped on the cloned CaMV genome by observation of enzyme-linear DNA complexes by electron microscopy. Twelve sites are observed. Three of them are relatively stable in the presence of heparin and are found at positions 8-9, 21-23, and 41-44 map units on the physical map of the genome. These positions correspond to AT-rich regions of the viral genome which contain potential promoter sites. These results are discussed with reference to current information on the structure and expression of the CaMV genome.

Binding Sites↗

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.

Deoxyadenosines↗

Effect of the protein-synthesis-initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide on ribonucleic acid synthesis in radish seedlings.

2-(4-Methyl-2,6-dinitroanilino)-N-methylpropionamide, an inhibitor of initiation of protein synthesis in plants, selectively alters rRNA synthesis. Ribosomal genes are transcribed in the absence of protein synthesis, but processing of the transcripts is impaired. This results in an accumulation of the 2.3 X 10(6)-dalton pre-rRNA. Synthesis of polyadenylated RNA is not affected by the drug.

Aniline Compounds↗