Accurate and simple sizing of primer extension products using a non-radioactive approach facilitates identification of transcription initiation sites

Department of Biology and the Hull York Medical School, University of York, United Kingdom.
Journal of microbiological methods (Impact Factor: 2.03). 04/2010; 81(3):256-8. DOI: 10.1016/j.mimet.2010.03.021
Source: PubMed


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.

12 Reads
  • Source
    • "One TSS was upstream of gtrA*, and the second upstream of gtrC BTP1 (Fig. 5A). The former is the same TSS previously identified for the gtr LT2_I operon (STM0559-7) in S. Typhimurium strain LT2 that is phase variable (Broadbent et al., 2010b). However, as shown earlier, in strain D23580, transcription from this promoter region is not controlled by phase variation. "
    [Show abstract] [Hide abstract]
    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; 96(2). DOI:10.1111/mmi.12933 · 4.42 Impact Factor
  • Source
    • "It has been demonstrated that TATA-box containing genes often contain several transcription start sites [13]. We observed one major transcription start site within the KIAA1199 5′-flanking region based on 5′ primer extension analysis and 5′RLM RACE experiment, which are common approaches used to determine transcription start sites [18]. Our analysis using the CAGE database, which is primarily used to locate transcription start sites in the genome [13], further confirmed our observation, although two neighboring guanines (G) upstream of the dominant transcription start site were also predicted to be potential transcription start points, but with less probability. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Emerging evidence has demonstrated that upregulated expression of KIAA1199 in human cancer bodes for poor survival. The regulatory mechanism controlling KIAA1199 expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human KIAA1199 promoter in terms of regulation of KIAA1199 gene expression. A 3.3 kb fragment of human genomic DNA containing the 5'-flanking sequence of the KIAA1199 gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic KIAA1199 promoter containing a TATA-box close to the transcription start site. A combination of 5'-primer extension study with 5'RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human KIAA1199 gene. Bioinformatics analysis suggested that the 1.4 kb KIAA1199 promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for KIAA1199 gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these cis- and trans-acting elements in controlling KIAA1199 gene expression. Finally, we found that upregulated KIAA1199 expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of KIAA1199 expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling KIAA1199 gene expression in human cancer.
    PLoS ONE 09/2012; 7(9):e44661. DOI:10.1371/journal.pone.0044661 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 · 4.42 Impact Factor
Show more