Browsing by Subject "Sequence Analysis, DNA"

Now showing 1 - 3 of 3
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    Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation
    (American Society for Microbiology, 2016-09-19) Muramatsu, Matthew K.; Brothwell, Julie A.; Steinman, Barry D.; Putman, Timothy E.; Rockey, Daniel D.; Nelson, David E.; Department of Microbiology & Immunology, IU School of Medicine
    Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells.
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    Evaluation of the Genetic Basis of Familial Aggregation of Pacemaker Implantation by a Large Next Generation Sequencing Panel
    (Public Library of Science (PloS), 2015) Celestino-Soper, Patrícia B. S.; Doytchinova, Anisiia; Steiner, Hillel A.; Uradu, Andrea; Lynnes, Ty C.; Groh, William J.; Miller, John M.; Lin, Hai; Gao, Hongyu; Wang, Zhiping; Liu, Yunlong; Chen, Peng-Sheng; Vatta, Matteo; Department of Medical and Molecular Genetics, IU School of Medicine
    BACKGROUND: The etiology of conduction disturbances necessitating permanent pacemaker (PPM) implantation is often unknown, although familial aggregation of PPM (faPPM) suggests a possible genetic basis. We developed a pan-cardiovascular next generation sequencing (NGS) panel to genetically characterize a selected cohort of faPPM. MATERIALS AND METHODS: We designed and validated a custom NGS panel targeting the coding and splicing regions of 246 genes with involvement in cardiac pathogenicity. We enrolled 112 PPM patients and selected nine (8%) with faPPM to be analyzed by NGS. RESULTS: Our NGS panel covers 95% of the intended target with an average of 229x read depth at a minimum of 15-fold depth, reaching a SNP true positive rate of 98%. The faPPM patients presented with isolated cardiac conduction disease (ICCD) or sick sinus syndrome (SSS) without overt structural heart disease or identifiable secondary etiology. Three patients (33.3%) had heterozygous deleterious variants previously reported in autosomal dominant cardiac diseases including CCD: LDB3 (p.D117N) and TRPM4 (p.G844D) variants in patient 4; TRPM4 (p.G844D) and ABCC9 (p.V734I) variants in patient 6; and SCN5A (p.T220I) and APOB (p.R3527Q) variants in patient 7. CONCLUSION: FaPPM occurred in 8% of our PPM clinic population. The employment of massive parallel sequencing for a large selected panel of cardiovascular genes identified a high percentage (33.3%) of the faPPM patients with deleterious variants previously reported in autosomal dominant cardiac diseases, suggesting that genetic variants may play a role in faPPM.
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    Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond
    (Landes Bioscience, 2014) Mundade, Rasika; Ozer, Hatice Gulcin; Wei, Han; Prabhu, Lakshmi; Lu, Tao; Department of Pharmacology and Toxicology, IU School of Medicine
    Many biologically significant processes, such as cell differentiation and cell cycle progression, gene transcription and DNA replication, chromosome stability and epigenetic silencing etc. depend on the crucial interactions between cellular proteins and DNA. Chromatin immunoprecipitation (ChIP) is an important experimental technique for studying interactions between specific proteins and DNA in the cell and determining their localization on a specific genomic locus. In recent years, the combination of ChIP with second generation DNA-sequencing technology (ChIP-seq) allows precise genomic functional assay. This review addresses the important applications of ChIP-seq with an emphasis on its role in genome-wide mapping of transcription factor binding sites, the revelation of underlying molecular mechanisms of differential gene regulation that are governed by specific transcription factors, and the identification of epigenetic marks. Furthermore, we also describe the ChIP-seq data analysis workflow and a perspective for the exciting potential advancement of ChIP-seq technology in the future.