Elena Beccari

Ribosomal Protein Production in Normal and Anucleolate Xenopus Embryos: Regulation at the Posttranscriptional and Translational Levels

We have studied the regulation of ribosomal protein (r-protein) synthesis in Xenopus anucleolate mutants, which lack the genes for rRNA. The accumulation of mRNA for the two r-proteins analyzed parallels the controls up to stage 30. This mRNA is mobilized onto polysomes and is translated as in normal embryos, but r-proteins are unstable in the absence of rRNA to assemble with. A translational control of rp-mRNA distribution between polysomes and mRNPs is observed, but this is not due to an autogenous regulation by r-proteins. After stage 30 the amount of rp-mRNA declines specifically in the mutants because the transcripts are unstable. Considering the temporal correlation between this event and the onset of r-protein synthesis we suggest that an autogenous control operates at the level of transcript stability.

Expression of ribosomal protein genes and regulation of ribosome biosynthesis in Xenopus development

Studies on ribosome biosynthesis in developing Xenopus oocytes and embryos, and after microinjection of cloned ribosomal-protein genes, have revealed that the synthesis of ribosomal proteins (r-proteins) is controlled by two types of regulation: (1) a post-transcriptional regulation, operated by feedback of the r-proteins themselves, controls processing and stability of r-protein transcripts and thus the amount of the corresponding mRNA present in the cell; and (2) a translational regulation controls the efficiency of utilization of r-protein mRNA (rp-mRNA) in response to the cellular needs for new ribosomes.

Size of ribosomal DNA repeating units in Xenopus laevis: Limited individual heterogeneity and extensive population polymorphism

Purified somatic ribosomal DNAs of 45 Xenopus laevis adults have been separately analyzed by agarose gel electrophoresis after Eco RI digestion. A pattern of gene length heterogeneity, unique for each individual, has been observed. This length heterogeneity, known to involve the spacer, is limited to few different size classes (up to about 10). The analysis of ribosomal DNA purified from ovaries indicates that, during oogenesis, amplification involves preferentially, if not exclusively, only some size classes of genes. An electron microscope study of reannealed molecules of individual high molecular weight ribosomal DNA demonstrates a tandem organization of equally sized genes, forming homogeneous blocks within the nucleolar organizer.

Localization of new peptidoglycan at poles in Bacillus mycoides, a member of the Bacillus cereus group

Bacillus mycoides is a sporogenic Gram-positive soil bacillus of the B. cereus group. This bacillus, which forms hyphal colonies, is composed of cells connected in filaments that make up bundles and turn clock- or counterclockwise depending on the strain. A thick peptidoglycan wall gives the rod cells of these bacilli strength and shape. One approach used to study peptidoglycan neoformation in Gram positives exploits the binding properties of antibiotics such as vancomycin and ramoplanin to nascent peptidoglycan, whose localization in the cell is monitored by means of a fluorescent tag. When we treated B. mycoides strains with BODIPY-vancomycin, we found the expected accumulation of fluorescence at the midcell septa and localization along the cell sidewall in small foci distributed quite uniformly. Intense fluorescence was also observed at the poles of many cells, more clearly visible at the outer edges of the cell chains. The unusual abundance of peptidoglycan intermediates at the cell poles after cell separation suggests that the construction process of this structure is different from that of B. subtilis, in which the free poles are rarely reactive to vancomycin.

Transcriptional analysis of ftsZ within the dcw cluster in Bacillus mycoides

BackgroundIn Bacillus mycoides, as well as in other members of the B. cereus group, the tubulin-like protein of the division septum FtsZ is encoded by the distal gene of the cluster division and cell wall (dcw). Along the cluster the genes coding for structural proteins of the division apparatus are intermingled with those coding for enzymes of peptidoglycan biosynthesis, raising the possibility that genes with this different function might be coexpressed. Transcription of ftsZ in two model bacteria had been reported to differ: in B. subtilis, the ftsZ gene was found transcribed as a bigenic mRNA in the AZ operon; in E. coli, the transcripts of ftsZ were monogenic, expressed by specific promoters. Here we analyzed the size and the initiation sites of RNAs transcribed from ftsZ and from other cluster genes in two B. mycoides strains, DX and SIN, characterized by colonies of different chirality and density, to explore the correlation of the different morphotypes with transcription of the dcw genes.ResultsIn both strains, during vegetative growth, the ftsZ-specific RNAs were composed mainly of ftsZ, ftsA-ftsZ and ftsQ-ftsA-ftsZ transcripts. A low number of RNA molecules included the sequences of the upstream murG and murB genes, which are involved in peptidoglycan synthesis. No cotranscription was detected between ftsZ and the downstream genes of the SpoIIG cluster. The monogenic ftsZ RNA was found in both strains, with the main initiation site located inside the ftsA coding sequence. To confirm the promoter property of the site, a B. mycoides construct carrying the ftsA region in front of the shortened ftsZ gene was inserted into the AmyE locus of B. subtilis 168. The promoter site in the ftsA region was recognized in the heterologous cellular context and expressed as in B. mycoides.ConclusionsThe DX and SIN strains of B. mycoides display very similar RNA transcription specificity. The ftsZ messenger RNA can be found either as an independent transcript or expressed together with ftsA and ftsQ and, in low amounts, with genes that are specific to peptidoglycan biosynthesis.

Transcriptional Activity of the 5′ Region of the Xenopus Laevis R-Protein Gene L1

In a previous study we analyzed in detail the 5′ flanking region of the gene coding for the ribosomal protein L14 of Xenopus laevis (Carnevali et al. 1989) and found that its functionality as a promoter was abolished when shortened beyond a sequence located at −53 from the major capsite of the gene.