Biomedical subjects
Publications and source records attributed to R Coppolecchia.
We have isolated a cDNA clone encoding eIF4A from Schizosaccharomyces pombe. The deduced protein sequence is similar in length and sequence to other eIF4A proteins and exhibits highest similarity with the mammalian eIF4A protein. Hybridization with genomic DNA reveals two eIF4A genes located on two different chromosomes.
We have isolated a gene, STM1, which encodes a new translation initiation factor from Saccharomyces cerevisiae. The gene acts, if present on a multicopy plasmid, as a suppressor of a temperature-sensitive mutation in eIF-4A. The single copy STM1 gene is not essential, but disruption causes a slow growth phenotype. Analysis of polysomes from a strain carrying a disrupted stm1 allele shows a clear defect in translation initiation as shown by a strong reduction in polysomes and an increase in the monosomes. Sequence analysis revealed interesting features of the putative Stm1 protein. Comparison of the entire protein sequence with databanks showed some similarity with the human eIF-4B protein. The Stm1 protein has potential RNP1 and RNP2 motifs characteristic for RNA-binding proteins. The protein also contains six highly conserved direct repeats of 21-26 amino acids and one partial repeat.
During an early stage of endospore formation in Bacillus subtilis, the cell divides asymmetrically into two compartments that follow different developmental paths. The differential expression of genes in these two compartments is controlled in part by the production of compartment-specific transcription factors, sigma G and sigma K. It is not known how sigma G accumulation is restricted to one of the two compartments, the forespore. However, the observations that sigma F directs transcription of the structural gene for sigma G and that sigma F activity can be modified by the product of a gene, spoIIAB, has led us to investigate the role of spoIIAB during sporulation. We have isolated mutants that carry deletion alleles of spoIIAB. Electron microscopic examination of these mutants revealed that these mutations blocked endospore formation at an early stage before septation and caused extensive cell lysis. The spoIIAB deletion alleles caused hyperexpression of genes that are normally expressed exclusively in the forespore compartments of sporulating wild-type cells, whereas these alleles reduced expression of other genes, including spoIIE, which is expressed before septation in wild-type cells. These observations confirm that spoIIAB is essential for sporulation and are consistent with models in which the product of spoIIAB plays a role in regulating the timing and/or compartment specificity of sigma F- and sigma G-directed transcription.
The sporulation operon spoIIA of Bacillus subtilis consists of three cistrons called spoIIAA, spoIIAB, and spoIIAC. Little is known about the function of spoIIAA and spoIIAB, but spoIIAC encodes a sigma factor called sigma F, which is capable of directing the transcription in vitro of genes that are expressed in the forespore chamber of the developing sporangium. We now report that the products of the spoIIA operon constitute a regulatory system in which SpoIIAA is an antagonist of SpoIIAB (or otherwise counteracts the effect of SpoIIAB) and SpoIIAB is, in turn, an antagonist of SpoIIAC (sigma F). This conclusion is based on the observations that (i) overexpression of spoIIAB inhibits sigma F-directed gene expression, (ii) a mutation in spoIIAB stimulates sigma F-directed gene expression, (iii) a mutation in spoIIAA blocks sigma F-directed gene expression, and (iv) a mutation in spoIIAB relieves the block in sigma F-directed gene expression caused by a mutation in spoIIAA. The SpoIIAA/SpoIIAB/SpoIIAC regulatory system could play a role in controlling the timing of sigma F-directed gene expression and/or could be responsible for restricting sigma F-directed gene expression to the forespore chamber of the sporangium.
In some media, Bacillus subtilis can maintain a prolonged stationary growth phase; however, in other media, nutrient depletion triggers a complex differentiation that culminates in production of a dormant endospore. This differentiation requires the expression of many genes. We found that during the stationary phase in media in which the cells do not form endospores and do not normally express these sporulation-essential genes, a recessive mutation in spoIIAB caused increased transcription of a set of genes essential for sporulation. Evidently, the wild-type product of spoIIAB acts during the stationary phase to prevent expression of additional sporulation-specific genes.
The synthesis of nucleases and proteins specific for competence development have been studied in four different Bacillus subtilis competence-deficient mutants. The nuclease analysis showed that two DNA-binding-deficient mutants were impaired in three nuclease activities involved in binding and entry of donor DNA. The other two strains did not show any reduction in nuclease activities. Two-dimensional gel electrophoresis of the proteins, synthesized during competence development, revealed that all four mutants are lacking several competence-specific polypeptides. Our data show that these com mutations have a strong pleiotropic effect, which could be due to a block in the metabolic pathway leading to competence development.