Accurate and simple sizing of primer extension products using a non-radioactive approach facilitates identification of transcription initiation sites
ABSTRACT An improved method of non-radioactive identification of transcription start sites is presented in which the use of 7-deaza dGTP in the primer extension reaction allows the product to be directly aligned to cycle sequencing traces on an automated sequencer. This removes the documented need to apply corrections for mobility differences.
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ABSTRACT: Salmonella Typhimurium isolate D23580 represents a recently identified ST313 lineage of invasive non-Typhoidal Salmonellae (iNTS). One of the differences between this lineage and other non-iNTS S. Typhimurium isolates is the presence of prophage BTP1. This prophage encodes a gtrC gene, implicated in O-antigen modification. GtrC(BTP1) is essential for maintaining O-antigen length in isolate D23580, since a gtr(BTP1) mutant yields a short O-antigen. This phenotype can be complemented by gtrC(BTP1) or very closely related gtrC genes. The short O-antigen of the gtr(BTP1) mutant was also compensated by deletion of the BTP1 phage tailspike gene in the D23580 chromosome. This tailspike protein has a putative endorhamnosidase domain and thus may mediate O-antigen cleavage. Expression of the gtrC(BTP1) gene is, in contrast to expression of many other gtr operons, not subject to phase variation and transcriptional analysis suggests that gtrC is produced under a variety of conditions. Additionally, GtrC(BTP1) expression is necessary and sufficient to provide protection against BTP1 phage infection of an otherwise susceptible strain. These data are consistent with a model in which GtrC(BTP1) mediates modification of the BTP1 phage O-antigen receptor in lysogenic D23580, and thereby prevents superinfection by itself and other phage that use the same O-antigen co-receptor. This article is protected by copyright. All rights reserved.Molecular Microbiology 01/2015; DOI:10.1111/mmi.12933 · 5.03 Impact Factor
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ABSTRACT: The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions.PLoS Genetics 06/2013; 9(6):e1003568. DOI:10.1371/journal.pgen.1003568 · 8.17 Impact Factor
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ABSTRACT: The instability of recombinant clones accommodating large or full-length viral genomes is frequently a technical challenge in RNA virus research. In an attempt to establish a rapid plasmid-based reverse genetics system that utilizes long RT-PCR technique (LRP), similar difficulty was encountered in the cloning of 9022-bp LRP amplicon. All HCV genotype 1a strains used for LRP cloning showed a remarkable difference in terms of cloning stability. Subsequent analysis revealed the predictive value of phylogenetic positions in determining the cloning stability. Putative E. coli promoters on the HCV genome might be responsible for such cloning difference. An exhaustive exploration, testing nearly one hundred cloning protocols, did not reveal a general approach that can achieve stable cloning for all HCV 1a strains. The selection of appropriate strains, guided by phylogenetic analysis, appears to be necessary prior to the construction of infectious HCV 1a clones. These observations are not only valuable for potentially establishing an HCV 1a cell culture model but also have general implications for other RNA viruses due to concern about cloning instability.Journal of virological methods 04/2013; DOI:10.1016/j.jviromet.2013.04.010 · 1.88 Impact Factor