Studies on the selection of mutants in Aspergillus nidulans

Abstract Not Provided

Adjacent single-stranded regions mediate processing of tRNA precursors by RNase E direct entry

The RNase E family is renowned for being central to the processing and decay of all types of RNA in many species of bacteria, as well as providing the first examples of endonucleases that can recognize 50 -monophosphorylated ends thereby increasing the efficiency of cleavage. However, there is increasing evidence that some transcripts can be cleaved efficiently by Escherichia coli RNase E via direct entry, i.e. in the absence of the recognition of a 50 -monophosphorylated end. Here, we provide biochemical evidence that direct entry is central to the processing of transfer RNA (tRNA) in E. coli, one of the core functions of RNase E, and show that it is mediated by specific unpaired regions that are adjacent, but not contiguous to segments cleaved by RNase E. In addition, we find that direct entry at a site on the 50 side of a tRNA precursor triggers a series of 50 -monophosphate-dependent cleavages. Consistent with a major role for direct entry in tRNA processing, we provide additional evidence that a 50 -monophosphate is not required to activate the catalysis step in cleavage. Other examples of tRNA precursors processed via direct entry are also provided. Thus, it appears increasingly that direct entry by RNase E has a major role in bacterial RNA metabolism

Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species

Genetic approaches to the study of ribosomes

Specific mutants of Escherichia coli have been used in order to investigate certain aspects of ribosome function, bio synthesis and decay. These studies show that in the function of the ribosome its various components are very interdependent and cooperative. It was possible to demonstrate that a binding site for erythromycin, which is on the 50S ribosomal subunit, could be affected by alterations in the 30S ribosomal subunit. The alterations are in ribosome proteins as well as in 16S rRNA. On the other hand, a mutational alteration, probably in the 16S rRNA, blocks ribosome maturation but at a relatively late stage. This alteration leads to cell death. The maturation cleavages of rRNAs were investigated in as train lacking RNase III, a specific enzyme which cleaves out p16 and p23 from the growing rRNA transcript. These studies suggest that at least three ribonucleases participate in the primary processing (during transcription), while at least three other ribonucleases participate in the secondary processing, which leads to the final mature rRNA molecules. When cells stop growing exponentially, decay of ribosome stakes place. By analyzing the process in mutants lacking or containing altered ribonucleases, it was possible to elucidate a mechanism for this turnover. The process starts by an endonucleolytic attack on the rRNA in the ribosomal subunits. After such an attack the ribosome disintegrates to fragments of RNA and ribosomal proteins, both of which can be further decayed to soluble material. Two endoribonucleases were identified in cell extracts which could be the enzymes which initiate this important turnover process.DAVID APIRION, Department of Microbiology and Immunology, Washington University School of Medicine, St. Louis, Missouri