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Features of the Rho-dependent transcription termination polar element within the hisG cistron of Salmonella typhimurium
No full textPrevious genetic analysis showed that the polar effects of mutations in the hisG cistron of Salmonella typhimurium are dependent on the presence of a single putative transcription termination element within the hisG gene. In fact, all proximal mutations causing translation termination are strongly polar, whereas distal ones are not. The element was mapped by isolating mutations able to relieve the polar phenotype, and they were found to be small deletions in the region downstream of the translational stop codon (M. S. Ciampi and J. R. Roth, Genetics 118:193-202, 1988). In this study, we analyzed the his-specific RNAs synthesized in vivo in different strains harboring the polar frameshift hisG2148 mutation. The nature of the polarity effects is clearly transcriptional, since shorter RNA molecules were produced. When the hisG2148 mutation was transferred in a rho background or in strains harboring the small distal deletions, an increase in readthrough transcription was observed. The transcriptional termination element was characterized in more detail by performing high-resolution S1 nuclease mapping experiments. This analysis showed that (i) termination or exonucleolytic degradation following termination produced transcripts with heterogeneous 3' ends; (ii) this process is dependent on the transcription termination factor Rho, since relief of termination occurs in a rho background; and (iii) the element appears to function as a transcription terminator, at least to some extent, even in the course of active translation of the hisG cistron
Did the parasitoid Pnigalio mediterraneus (Hymenoptera: Eulophidae) track the invasion of the horse chestnut leafminer?
No full textHow communities of natural enemies, such as parasitoids, adapt to the range expansion of their hosts or the arrival of a novel invasive host is an important question in invasion biology. Do parasitoids track the expansion of their hosts from their shared native range? Do they locally adapt both behaviorally and physiologically to the arrival of a novel species by shifting hosts? Few studies have addressed those questions, yet they are important to develop efficient methods to manage invasive species. Here we focus on Pnigalio mediterraneus Ferri,re and Delucchi (Hymenoptera: Eulophidae), an important parasitoid of two major agricultural and ornamental pests, the olive fruit fly Bactrocera oleae Rossi (Diptera: Tephritidae) and the horse chestnut leafminer Cameraria ohridella Deschka & Dimic (Lepidoptera: Gracillariidae). C. ohridella recently invaded Europe starting from the Southern Balkans, whereas B. oleae has been associated since the Quaternary with wild olives in the Mediterranean, where it largely spread after the domestication of cultivated olives. We used two markers, the ribosomal spacer ITS2 and the mitochondrial gene COI. Although the ITS2 dataset provided little variation and no phylogeographic signal, analysis of mtDNA of 188 individuals of P. mediterraneus from 54 European localities allowed us to identify 53 haplotypes. Both nucleotide and haplotype diversity were higher for Mediterranean samples, and from samples reared from B. oleae. The statistical parsimony network identified one haplotype as the most frequent, ancestral and mainly associated with C. ohridella. Our findings suggest that P. mediterraneus locally host switched to C. ohridella from other hosts in the Balkans and later tracked the horse chestnut leafminer invasion over Europe. Therefore both host-tracking and ecological sorting could explain the current distribution of P. mediterraneus haplotypes
Structure and function of the Salmonella typhimurium and Escherichia coli K-12 histidine operons
No full textWe have determined the complete nucleotide sequence of the histidine operons of Escherichia coli and of Salmonella typhimurium. This structural information enabled us to investigate the expression and organization of the histidine operon. The proteins coded by each of the putative histidine cistrons were identified by subcloning appropriate DNA fragments and by analyzing the polypeptides synthesized in minicells. A structural comparison of the gene products was performed. The histidine messenger RNA molecules produced in vivo and the internal transcription initiation sites were identified by Northern blot analysis and S1 nuclease mapping. A comparative analysis of the different transcriptional and translational control elements within the two operons reveals a remarkable preservation for most of them except for the intercistronic region between the first (hisG) and second (hisD) structural genes and for the rho-independent terminator of transcription at the end of the operon. Overall, the operon structure is very compact and its expression appears to be regulated at several levels
Alternative patterns of his operon transcription and mRNA processing generated by metabolic perturbation
No full textPrevious studies have shown that the expression of the his operon of Salmonella typhimurium is regulated at the level of transcription initiation, transcription elongation and RNA processing. We have analyzed his RNA in both prototrophic strains or strains harboring regulatory and auxotrophic mutations grown under a variety of metabolic conditions that lead to differential expression of the operon. Under some of these conditions, there is an increase in the amount of prematurely released his-specific RNA, resulting in modulation of the relative amount of full-length transcripts. Under the same metabolic conditions, there is also a modulation of RNA processing events that generate a very stable RNA species comprising the five distal cistrons. These effects appear to be due to perturbation of the translation process caused by alterations in the intracellular pool of initiator transfer RNA
In vivo analysis of the mechanisms responsible for strong transcriptional polarity in a "sense" mutant within an intercistronic region
No full textWe have studied a very unusual strong polar mutant in the intercistronic barrier between the second (hisD) and third (hisC) cistrons of the histidine operon of Salmonella typhimurium to obtain further insights into the molecular mechanisms leading to transcription termination within cistrons. We have performed a detailed transcriptional analysis in vivo and have found that the his mRNA in this polar mutant is reduced in size as a result of premature termination of transcription at a cryptic Rho-dependent site within the proximal region of the hisC cistron
Gene structure in the histidine operon of Escherichia coli. Identification and nucleotide sequence of the hisB gene.
No full textThe bifunctional enzyme imidazoleglycerolphosphate dehydratase and histidinolphosphate phosphatase is encoded by the hisB gene. The fourth gene of the histidine operon, hisB, was cloned and mapped on a 2,300 base pair DNA fragment. In the present study we report the complete nucleotide sequence of the hisB gene of Escherichia coli. The gene is 1,068 nucleotides long and codes for a protein of 355 amino acids with an apparent molecular weight of 39,998 daltons. The protein product(s) of the hisB region of both Salmonella typhimurium and E. coli were identified by subcloning and expression in an in vitro translation system. In both organisms the hisB gene directed the synthesis of a single protein with an apparent molecular weight of 40,500 daltons, consistent with the data derived from the nucleotide sequence analysi
Rickettsia Symbionts Cause Parthenogenetic Reproduction in the Parasitoid Wasp Pnigalio soemius (Hymenoptera: Eulophidae)
No full textBacteria in the genus Rickettsia are intracellular symbionts of disparate groups of organisms. Some Rickettsia strains infect vertebrate animals and plants, where they cause diseases, but most strains are vertically inherited symbionts of invertebrates. In insects Rickettsia symbionts are known to have diverse effects on hosts ranging from influencing host fitness to manipulating reproduction. Here we provide evidence that a Rickettsia symbiont causes thelytokous parthenogenesis ( in which mothers produce only daughters from unfertilized eggs) in a parasitoid wasp, Pnigalio soemius (Hymenoptera: Eulophidae). Feeding antibiotics to thelytokous female wasps resulted in production of progeny that were almost all males. Cloning and sequencing of a fragment of the 16S rRNA gene amplified with universal primers, diagnostic PCR screening of symbiont lineages associated with manipulation of reproduction, and fluorescence in situ hybridization ( FISH) revealed that Rickettsia is always associated with thelytokous P. soemius and that no other bacteria that manipulate reproduction are present. Molecular analyses and FISH showed that Rickettsia is distributed in the reproductive tissues and is transovarially transmitted from mothers to offspring. Comparison of antibiotic-treated females and untreated females showed that infection had no cost. Phylogenetic analyses of 16S rRNA and gltA gene sequences placed the symbiont of P. soemius in the bellii group and indicated that there have been two separate origins of the parthenogenesis-inducing phenotype in the genus Rickettsia. A possible route for evolution of induction of parthenogenesis in the two distantly related Rickettsia lineages is discussed
Processing of a polycistronic mRNA requires a 5' cis element and active translation
No full textWe have characterized a major processed species of mRNA in the his operon of Salmonella typhimurium. In vivo and in vitro analyses of the his transcripts from wild-type and mutant strains using S1 nuclease protection assays, measurements of RNA stability, deletion mapping, gel retardation, and in vitro translation assays demonstrate that the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis-acting element and translation of the cistron immediately downstream of the 5' end of the processed species. The cistrons contained in this segment are also independently transcribed from an internal promoter which is maximally active in the absence of readthrough transcription from the primary promoter
Alternative patterns of his operon transcription and mRNA processing generated by metabolic perturbation.
No full textPrevious studies have shown that the expression of the his operon ofSalmonella typhimurium is regulated at the level of transcription initiation, transcription elongation and RNA processing. We have analyzed his RNA in both prototrophic strains or strains harboring regulatory and auxotrophic mutations grown under a variety of metabolic conditions that lead to differential expression of the operon. Under some of these conditions, there is an increase in the amount of prematurely released his-specific RNA, resulting in modulation of the relative amount of full-length transcripts. Under the same metabolic conditions, there is also a modulation of RNA processing events that generate a very stable RNA species comprising the five distal cistrons. These effects appear to be due to perturbation of the translation process caused by alterations in the intracellular pool of initiator transfer RNA
Processing of a polycistronic mRNA requires a 5' cis element and active translation.
No full textWe have characterized a major processed species of mRNA in the his operon of Salmonella typhimurium. In vivo and in vitro analyses of the his transcripts from wild-type and mutant strains using S1 nuclease protection assays, measurements of RNA stability, deletion mapping, gel retardation, and in vitro translation assays demonstrate that the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis-acting element and translation of the cistron immediately downstream of the 5' end of the processed species. The cistrons contained in this segment are also independently transcribed from an internal promoter which is maximally active in the absence of readthrough transcription from the primary promoter
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