Plant ribosomal DNA external spacer binding factors: a novel protein binds specifically to a sequence close to the primary pre-rRNA processing site.

Sequence analyses of the ribosomal DNA (rDNA) external spacer region revealed a peculiar structure around the primary pre-rRNA processing site in radish. Using the electrophoretic mobility shift assay, DNAse I footprinting and methylation interference analysis, we have identified in radish extracts a novel nuclear factor, NF B, that recognises this region. NF B binds to a unique CGATTTTGCCCCTGA sequence located 164 bp downstream of the transcription initiation site and immediately upstream of the pre-rRNA processing site. Interestingly, this motif is flanked by four homologous sequences, including the primary pre-rRNA processing site, which are not recognised by NF B. Based on these data and the prediction that alternative hairpin loops can be formed in this region of the nascent pre-rRNA, a putative role for NF B as a factor coupling transcription and pre-rRNA processing is discussed. NF B is unique among plant and animal rDNA-binding proteins and it differs from a previously described radish factor and from other proteins that bind to plant rDNA promoters.

In vitro transcription from cauliflower mosaic virus promoters by a cell-free extract from tobacco cells.

We have studied transcription from the cauliflower mosaic virus 19S and 35S promoters in a cell-free system derived from tobacco cells in suspension culture. While a whole-cell extract is incapable of detectable transcription from these promoters, successive purification by column chromatography allows the preparation of two fractions which contain all factors necessary for transcription from the 19S promoter. In contrast, transcription from the 35S promoter leads to the accumulation of short RNAs. This accumulation can only be partially alleviated by modifying the conditions of transcription.

Detection of a potential transcription control sequence on the cauliflower mosaic virus genome by dinucleotide primed "in vitro" transcription.

The three sites of selective dinucleotide-primed "in vitro" transcription initiation on a cloned cauliflower mosaic virus DNA fragment have been localised by S1 nuclease mapping. Two of these sites lie within a region which has been shown to be essential for transcription complex formation on the viral sequences, one corresponding to a nuclease S1 hypersensitive site and the other to an imperfect repeat 100bp downstream. These sequences show striking homology with known transcription control sequences. These observations and the effect of the sequences on "in vitro" transcription raise the possibility that they may be involved in control of transcription of the viral genome.

Selective dinucleotide-primed in vitro transcription of a cloned fragment of cauliflower mosaic virus DNA is dependent on a limited region of the viral genome.

We have previously shown that plant RNA polymerase II preferentially forms ternary transcription complexes on a cloned fragment of the cauliflower mosaic virus genome in the presence of a particular dinucleotide/purine NTP combination (ApG + ATP). This preferential interaction is observed when the viral sequences are present on a discrete circular molecule. Deletion of a 205-bases-pair region abolishes this selectivity. The deleted region contains a considerable number of symmetrical or repeating elements. The use of nuclease S1 as a probe shows that this region contains a homopurine-homopyrimidine sequence which is extremely sensitive to this enzyme, indicating its capacity to adopt a non-B DNA conformation. A possible alternative structure of these sequences, which may explain the preferential interaction with the RNA polymerase, is presented.

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.

Developmental regulation of the synthesis of proteins encoded by stored mRNA in radish embryos.

Major radish (Raphanus sativus L. cv National) proteins synthesized at the beginning of germination have been characterized by their migration in two-dimensional electrophoresis.The use of 15-minute labelings shows that these proteins are encoded by stored mRNA. They undergo little or no posttranslational modification. Other proteins become detectable only after 1 hour of imbibition, and are probably encoded by newly synthesized mRNA. Comparison with proteins synthesized during embryogenesis, late germination, or with those present in dry embryos allows the classification of the proteins encoded by stored mRNA into two sets:The first set is synthesized also during late embryogenesis and is present in dry embryos. Some of these polypeptides are no longer synthesized later in germination while the others continue to be synthesized. The corresponding stored mRNAs can be considered as remnants of mRNA actively translated during embryogenesis.The second set is synthesized only during early germination. Their messengers appear during late embryogenesis although they are apparently not translated at this stage, but translation can be induced by a desiccation treatment. These polypeptides may play a particular role during early germination.

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.

Enzymatic properties of plant RNA polymerases : An approach to the study of transcription in plants.

Results obtained in the past few years in the study of the reaction mechanism of plant RNA polymerases are reviewed and discussed. They suggest that valuable information can be obtained using a highly simplified transcription system composed of purified plant enzymes and cloned genes. This type of approach may provide a starting point for the development of an in vitro transcription system. The detailed study of the fundamental enzymatic properties of the plant RNA polymerases allows a comparison with the well documented corresponding bacterial enzyme.

Selective transcription of a cloned cauliflower mosaic virus DNA fragment in vitro by soybean RNA polymerase II in the presence of dinucleotide primers.

Transcription of a cloned cauliflower mosaic virus (CaMV) DNA fragment (plasmid pCa 8) was studied at a low enzyme: DNA ratio. Preincubation with purine nucleoside triphosphates leads to essentially random transcription, while in the presence of a dinucleoside monophosphate and a purine nucleoside triphosphate in the preincubation medium certain combinations prime preferential transcription of the eucaryotic moiety of the chimeric plasmid. Characterisation of transcription primed by the most efficient combination, ApG + ATP, shows that a low enzyme: DNA ratio is absolutely essential for selective initiation. Interestingly the presence of the eucaryotic insertion is essential for the transcription of vector sequences. Analysis of RNA primed by ApG + ATP and of short chains synthesised in the presence of the GTP analogue 3'-OMeGTP shows a high degree of selectivity of transcription initiation sites. Hybridisation of primed RNA to restriction fragments of pCa8 shows that initiation occurs within a limited region of the inserted CaMV fragment.

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.

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.

Analysis of DNA associated with nucleosomes in pea chromatin.

Micrococcal nuclease digestion of chromatin from ungerminated and 48 h-germinated pea embryos yields DNA fragments which are multiples of basic units of 194-195 base pairs. Extensive digestion produces a core particle of 145 base pairs. Deoxyribonuclease I gives rise to fragments which are multiples of 10 bases upon analysis on denaturing gels. These values are comparable with those found for other plant materials. These results indicate that gross changes in nucleosomal organization do not accompany the onset of germination.

Hormonal control of transcription in higher plants.

1. Nucleolar RNA polymerase Ib obtained from auxin-treated lentil roots exhibits a higher transcriptional activity than the enzyme obtained from control roots. This difference is due to a change in the enzyme properties after auxin treatment. It is suggested that the hormonal effect is mediated by a factor that changes the molecular properties of nucleolar RNA polymerase. 2. Four fractions, alpha, beta, gamma and delta, that stimulate the activity of RNA polymerase Ib, have been extracted from lentil roots. Two of them, gamma and delta have been studied. Factor delta can stimulate nucleolar polymerase Ib and the nucleoplasmic enzyme II equally well, while factor gamma is specific for polymerase Ib. 3. The curve of UMP incorporation in vitro, with and without factors gamma or delta suggests that they are initiation factors. This conclusion is reinforced by the analysis of simultaneous incorporation of [gamma-32P]ATP and [3H]UMP in the RNAs synthesized in vitro. 4. Although the level of factor delta is independent of auxin treatment, that of factor gamma is doubled in auxin-treated roots. These results suggest that factor gamma is an auxin-induced protein that modulates the specific activity of the nucleolar RNA polymerase. 5. A general model of the mode of action of auxins at the molecular level is proposed. It integrates into a unified scheme the above results as well as those obtained by other workers.