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 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
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ABSTRACT: The O-antigen of Salmonella lipopolysaccharide is a major antigenic determinant and its chemical composition forms the basis for Salmonella serotyping. Modifications of the O-antigen that can affect the serotype include those carried out by the products of glycosyltransferase operons (gtr), which are present on specific Salmonella and phage genomes. Here we show that expression of the gtr genes encoded by phage P22 that confers the O1 serotype is under the control of phase variation. This phase variation occurs by a novel epigenetic mechanism requiring OxyR in conjunction with the DNA methyltransferase Dam. OxyR is an activator or a repressor of the system depending on which of its two binding sites in the gtr regulatory region is occupied. Binding is decreased by methylation at Dam target sequences in either site, and this confers heritability of the expression state to the system. Most Salmonella gtr operons share the key regulatory elements that are identified here as essential for this epigenetic phase variation.Molecular Microbiology 07/2010; 77(2):337-53. DOI:10.1111/j.1365-2958.2010.07203.x · 5.03 Impact Factor