Molecular characterization and outer membrane association of a Chlamydia trachomatis protein related to the hsp70 family of proteins.

One route by which Chlamydia trachomatis is internalized into host endometrial epithelial cells is receptor-mediated endocytosis. Although this implies an adhesin-receptor interaction exists, specific chlamydial surface molecules have not been identified. We are investigating potential adhesin molecules using an in vitro functional assay to select for chlamydial recombinant Escherichia coli expressing an adherent phenotype. We have previously shown that E. coli JM109(pPBW58) attaches to epithelial cells by a specific process paralleling C. trachomatis and expresses at least three plasmid-encoded proteins (18, 28, and 82 kDa; Schmiel, D. H., Knight, B. T., Raulston, J. E., Choong, J., Davis, C. H., and Wyrick, P. B. (1991) Infect. Immun. 59, 4001-4012).One route by which Chlamydia trachomatis is internalized into host endometrial epithelial cells is receptor-mediated endocytosis. Although this implies an adhesin-receptor interaction exists, specific chlamydial surface molecules have not been identified. We are investigating potential adhesin molecules using an in vitro functional assay to select for chlamydial recombinant Escherichia coli expressing an adherent phenotype. We have previously shown that E. coli JM109(pPBW58) attaches to epithelial cells by a specific process paralleling C. trachomatis and expresses at least three plasmid-encoded proteins (18, 28, and 82 kDa; Schmiel, D. H., Knight, B. T., Raulston, J. E., Choong, J., Davis, C. H., and Wyrick, P. B. (1991) Infect. Immun. 59, 4001-4012)

Silent but Not Static: Accelerated Base-Pair Substitution in Silenced Chromatin of Budding Yeasts

Subtelomeric DNA in budding yeasts, like metazoan heterochromatin, is gene poor, repetitive, transiently silenced, and highly dynamic. The rapid evolution of subtelomeric regions is commonly thought to arise from transposon activity and increased recombination between repetitive elements. However, we found evidence of an additional factor in this diversification. We observed a surprising level of nucleotide divergence in transcriptionally silenced regions in inter-species comparisons of Saccharomyces yeasts. Likewise, intra-species analysis of polymorphisms also revealed increased SNP frequencies in both intergenic and synonymous coding positions of silenced DNA. This analysis suggested that silenced DNA in Saccharomyces cerevisiae and closely related species had increased single base-pair substitution that was likely due to the effects of the silencing machinery on DNA replication or repair

Easy detection of chromatin binding proteins by the histone association assay

The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and non-chromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes

Novel features of ARS selection in budding yeast Lachancea kluyveri

<p>Abstract</p> <p>Background</p> <p>The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined.</p> <p>Results</p> <p>In this study we have isolated and characterized autonomously replicating sequences (ARSs) in <it>Lachancea kluyveri </it>- a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that <it>L. kluyveri </it>ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in <it>Saccharomyces cerevisiae</it>. Moreover, compared with <it>S. cerevisiae </it>and <it>K. lactis</it>, the replication licensing machinery in <it>L. kluyveri </it>seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all <it>S. cerevisiae </it>ARSs tested and most <it>Kluyveromyces lactis </it>ARSs. In contrast, only about half of the <it>L. kluyveri </it>ARSs function in <it>S. cerevisiae </it>and less than 10% function in <it>K. lactis</it>.</p> <p>Conclusions</p> <p>Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.</p

Tight associations between transcription promoter type and epigenetic variation in histone positioning and modification

Abstract Background Transcription promoters are fundamental genomic cis-elements controlling gene expression. They can be classified into two types by the degree of imprecision of their transcription start sites: peak promoters, which initiate transcription from a narrow genomic region; and broad promoters, which initiate transcription from a wide-ranging region. Eukaryotic transcription initiation is suggested to be associated with the genomic positions and modifications of nucleosomes. For instance, it has been recently shown that histone with H3K9 acetylation (H3K9ac) is more likely to be distributed around broad promoters rather than peak promoters; it can thus be inferred that there is an association between histone H3K9 and promoter architecture. Results Here, we performed a systematic analysis of transcription promoters and gene expression, as well as of epigenetic histone behaviors, including genomic position, stability within the chromatin, and several modifications. We found that, in humans, broad promoters, but not peak promoters, generally had significant associations with nucleosome positioning and modification. Specifically, around broad promoters histones were highly distributed and aligned in an orderly fashion. This feature was more evident with histones that were methylated or acetylated; moreover, the nucleosome positions around the broad promoters were more stable than those around the peak ones. More strikingly, the overall expression levels of genes associated with broad promoters (but not peak promoters) with modified histones were significantly higher than the levels of genes associated with broad promoters with unmodified histones. Conclusion These results shed light on how epigenetic regulatory networks of histone modifications are associated with promoter architecture

Chlamydia trachomatis antigens in enteroendocrine cells and macrophages of the small bowel in patients with severe irritable bowel syndrome

<p>Abstract</p> <p>Background</p> <p>Inflammation and immune activation have repeatedly been suggested as pathogentic factors in irritable bowel syndrome (IBS). The driving force for immune activation in IBS remains unknown. The aim of our study was to find out if the obligate intracellular pathogen <it>Chlamydia </it>could be involved in the pathogenesis of IBS.</p> <p>Methods</p> <p>We studied 65 patients (61 females) with IBS and 42 (29 females) healthy controls in which IBS had been excluded. Full thickness biopsies from the jejunum and mucosa biopsies from the duodenum and the jejunum were stained with a monoclonal antibody to <it>Chlamydia </it>lipopolysaccharide (LPS) and species-specific monoclonal antibodies to <it>C. trachomatis </it>and <it>C. pneumoniae</it>. We used polyclonal antibodies to chromogranin A, CD68, CD11c, and CD117 to identify enteroendocrine cells, macrophages, dendritic, and mast cells, respectively.</p> <p>Results</p> <p><it>Chlamydia </it>LPS was present in 89% of patients with IBS, but in only 14% of healthy controls (p < 0.001) and 79% of LPS-positive biopsies were also positive for <it>C. trachomatis </it>major outer membrane protein (MOMP). Staining for <it>C. pneumoniae </it>was negative in both patients and controls. <it>Chlamydia </it>LPS was detected in enteroendocrine cells of the mucosa in 90% of positive biopsies and in subepithelial macrophages in 69% of biopsies. Biopsies taken at different time points in 19 patients revealed persistence of <it>Chlamydia </it>LPS up to 11 years. The odds ratio for the association of <it>Chlamydia </it>LPS with presence of IBS (43.1; 95% CI: 13.2-140.7) is much higher than any previously described pathogenetic marker in IBS.</p> <p>Conclusions</p> <p>We found <it>C. trachomatis </it>antigens in enteroendocrine cells and macrophages in the small bowel mucosa of patients with IBS. Further studies are required to clarify if the presence of such antigens has a role in the pathogenesis of IBS.</p

Diversity of Eukaryotic DNA Replication Origins Revealed by Genome-Wide Analysis of Chromatin Structure

Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers) positions nucleosomes adjacent to the origin to promote replication origin function

Chlamydial Pre-Infection Protects From Subsequent Herpes Simplex Virus-2 Challenge in a Murine Vaginal Super-Infection Model

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) genital tract co-infections have been reported in humans and studied in vitro but the clinical consequences are unknown. Limited epidemiologic evidence suggests that these co-infections could be more severe than single infections of either pathogen, but the host-pathogen interactions during co-infection remain uncharacterized. To determine whether disease progression and/or pathogen shedding differs between singly-infected and super-infected animals, we developed an in vivo super-infection model in which female BALB/c mice were vaginally infected with Chlamydia muridarum (Cm) followed later by HSV-2. Pre-infection with Chlamydia 3 or 9 days prior to HSV-2 super-infection conferred significant protection from HSV-2-induced neurologic disease and significantly reduced viral recovery compared to HSV-2 singlyinfected controls. Neither protection from mortality nor reduced viral recovery were observed when mice were i) super-infected with HSV-2 on day 27 post Cm; ii) infected with UV-irradiated Cm and super-infected with HSV-2; or iii) azithromycin-treated prior to HSV-2 super-infection. Therefore, protection from HSV-2-induced disease requires active infection with viable chlamydiae and is not observed after chlamydial shedding ceases, either naturally or due to antibiotic treatment. Thus, Chlamydia-induced protection is transient and requires the continued presence of chlamydiae or their components. These data demonstrate that chlamydial pre-infection can alter progression of subsequent HSV-2 infection, with implications for HSV-2 transmission from co-infected humans