Browsing by Subject "cells"
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Item A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, RustinA Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.Item The Role of Calcineurin and NFAT in the Regulation of Insulin Gene Transcription(2001-12-01) Lawrence, Michael C.; Richard Easom; Julian Borejdo; Ladislav DoryLawrence, Michael C., The Role of Calcineurin and NFAT in the Regulation of Insulin Gene Transcription. Doctor of Philosophy (Biomedical Sciences), December 2001, 185 pp., 41 illustrators, references, 222 titles. In an effort to understand glucose and hormone regulated insulin gene transcription elicited by increased intracellular calcium, a novel pathway has been identified. This pathway involves the calcium/calmodulin-dependent phosphatase 2B (calcinuerin) and nuclear factor activated T-cells (NFAT), which in the studies herein, have been determined to up-regulate insulin gene transcription in response to glucose and glucagon-like peptide-1 (GLP-1) in pancreatic β-cells. Three NFAT elements within the first 410 base pairs of the rat I insulin gene promoter were first identified, two of which are conserved (by presence and location) among mammals including dogs, mice, and humans. The presence of NFAT in rat insulinoma β-cells (INS-1) and rat pancreatic islets was detected by immumobotting, immunofluorescence, and RT-PCR. Electrophoretic mobility shift assays displayed NFAT-specific DNA-binding activity that could be competed with unlabeled NFAT probe when incubated with INS-1 cells or rat islet nuclear extracts and shifted with extracts pre-incubated in the presence of either anti-calcineurin or anti-NFAT antibodies. Transfection experiments with either the -410 rat I (rIsnI-Luc) or the NFAT-Luc promoter-reported showed increased promoter activity when stimulated by glucose or cell depolarization (increases intracellular calcium) and displayed a synergistically enhanced response when co-stimulated with glucose and GLP-1. The GLP-1 induced responses were mimicked by forskolin and concentration-dependently inhibited by each of two selective but distinct protein kinase A (PKA) inhibitors, H-89 and myristoylated PKI (14-22) amide. The selective calcineurin-inhibitor FK506, as well as the chelatin of intracellular Ca2+ by BAPTA, also abolished the effects of high glucose and GLP-1. Moreover, co-transfection experiments with a constitutively active form of calcineurin and the promoter-reporters (rISnI-Luc and NFAT-Luc) showed increased reporter activity over controls. Furthermore, two-point base pair mutations in any of the three identified NFAT sites within the rat insulin I promoter resulted in a significant (p [less than] 0.05) reduction in the combined effect of glucose and GLP-1. These studies establish the presence of NFAT in insulin-secreting cells, its ability to bind elements within the insulin gene promoter, and show that glucose and GLP-1 synergistically enhance NFAT-mediated insulin gene transcription by PKA- and calcineurin-dependent pathways in pancreatic β-cells.Item YY1 Mediated Competitve Regulation: A Governing Principle Behind Phenotype-Specific Gene Expression in Vascular Smooth Muscle Cells(2005-07-01) Roberts, Leslie Don; Stephen R. Grant; Neeraj Agarwal; Peter B. RavenLeslie Don Roberts, YYI Mediated Competitive Regulation: A governing principle behind phenotype-specific gene expression in vascular smooth muscle cells. Doctor of Philosophy (Biomedical Science), July 2005. The vascular smooth muscle cell (VSMC) lacks the typical phenotypic restriction that limit most cell-types to expressing a single phenotype, as a result, these cells are uniquely suited to wound repair, as well as, exacerbating several vascular disease-states. While much is known regarding the specific transcription factors that drive phenotype-specific gene expression the mechanisms that regulate the transition between phenotype-specific gene programs remain poorly defined. To further explore these mechanisms, we sought to better understand how VSMCs stably express their default contractile-specific gene program despite the inherent instabilities of their environment. This study explored the regulatory implications of a yet undescribed regulatory domain, that resides with a high-frequency in the promoters of the most contractile-specific gene. These domains, which we term dual regulatory domains (DRD), orient the core binding site for the transcriptional repressor Ying Yang-1 in close proximity to, or overlapping with, the core binding site for a variety of transcriptional activators. This study specifically examines the regulatory implications at two DRD where YY1 competes with the transcriptional activators C/EBPβ (C/CAAT-enhancer binding protein beta). Our findings demonstrate: i.) YY1 acts as a dominant, negative, regulator of the smooth muscle myosin heavy chain (SM-MHC) gene promoter; ii.) YY1 binds to, and repressing from, multiple sites within the regulatory context of this promoter; and iii.) The transactivation potential of C/EBPβ completes with transrepressive potential of YY1 for regulatory control over SM-MHC promoter activity and does so in a stoichiometric-dependent fashion. These findings argue that the relative concentrations of YY1 define the effective dose required of specific transcriptional activators to complete with and override the repressive effect of YY1, and by doing so, directly dictate which genes will be expressed.