Thesis (Ph. D.)--Cornell University, August, 2007. / Vita. Includes bibliographical references (leaves 165-176).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 70-79).

abstract: Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than deoxyribose nucleic acid (DNA) sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. In 2011, Haynes et al. showed that a synthetic regulator called the Polycomb chromatin Transcription Factor (PcTF), a fusion protein that binds methylated histones, reactivated an artificially-silenced luciferase reporter gene. These synthetic transcription activators are derived from the polycomb repressive complex (PRC) and associate with the epigenetic silencing mark H3K27me3 to reactivate the expression of silenced genes. It is demonstrated here that the duration of epigenetic silencing does not perturb reactivation via PcTF fusion proteins. After 96 hours PcTF shows the strongest reactivation activity. A variant called Pc2TF, which has roughly double the affinity for H3K27me3 in vitro, reactivated the silenced luciferase gene by at least 2-fold in living cells. / Dissertation/Thesis / Masters Thesis Biological Design 2019

Quorum sensing is a mechanism used by many proteobacteria to regulate expression of target genes in a population-dependent manner. The quorum sensing system of Vibrio fischeri activates the luminescence (lux) operon when the autoinducer signaling molecule (N-3-oxohexanoyl homoserine lactone) is recognized and bound by the activator protein LuxR. LuxR subsequently binds to the lux box centered at à 42.5 bp upstream of the transcription initiation site and activates transcription from the lux operon promoter, resulting in the emission of light at high cell densities. LuxR consists of 250 amino acids arranged into an N-terminal (regulatory) domain and a C-terminal (activation) domain, and is thought to function as an ambidextrous activator capable of making multiple contacts with the alpha and sigma subunits of RNA polymerase (RNAP). Published work describing the results of alanine scanning mutagenesis performed on the C-terminal domain of LuxR (residues 190-250) has identified residues (K198, W201 and I206) that appear to play a role in positive control of transcription initiation. Additional mutagenesis of residues 180-189 has been undertaken via a three-primer or four-primer PCR-based method in this study. Variants of LuxR were screened for their ability to activate luciferase production and to repress transcription from an artificial promoter, and production of full-length LuxR was measured, in an attempt to identify additional positive control variants. No additional positive control variants were found in this study. Work has also been undertaken to identify intergenic suppressors between positive control variants of LuxR and the RNAP alpha subunit, RpoA. Starting with a recombinant Escherichia coli strain encoding the lux operon and LuxR variant I206E, a random chemical mutagenesis was performed on a vector encoding RpoA. Following transformation of the mutated plasmids encoding RpoA, high throughput luminescence assays were used to identify isolates with phenotypes brighter than the control. Isolation of an intergenic suppressor will confirm the existence of protein-protein interactions between LuxR and RpoA within the transcription initiation complex. The ability of other LuxR family members to establish productive protein-protein interactions with RNAP necessary for transcription initiation was also examined. LuxR homologues EsaR of Pantoea stewarti ssp. stewartii, a repressor of known function, and ExpR of Erwinia carotovora subsp. carotovora were also analyzed for their ability to activate the lux operon, as well as to repress transcription from an artificial promoter containing the lux box. / Master of Science

No description available.

No description available.

Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, <i>Vibrio fischeri</i>. In <i>V. fischeri</i>, the luminescence or <i>lux</i> genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the <i>lux</i> operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the <font face = "symbol">a</font>CTD of RNAP in LuxR-dependent transcriptional activation of the <i>lux</i> operon promoter has been investigated. The effect of seventy alanine substitution variants of the <font face = "symbol">a</font> subunit was determined <i>in vivo</i> by measuring the rate of transcription of the <i>lux</i> operon via luciferase assays in recombinant <i>Escherichia coli</i>. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by <i>in vitro</i> assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxR<font face = "symbol">D</font>N, was used for <i>in vitro</i> studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxR<font face = "symbol">D</font>N, and fourteen residues in the <font face = "symbol">a</font>CTD were identified as having negative effects on the rate of transcription from the <i>lux</i> operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxR<font face = "symbol">D</font>N-dependent activation <i>in vivo</i> and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type <font face = "symbol">a</font>CTD is capable of interacting with the <i>lux</i> DNA fragment tested, all five of the variant forms of the <font face = "symbol">a</font>CTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that <font face = "symbol">a</font>CTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the <i>lux</i> operon during quorum sensing. / Master of Science

The mechanism of gene regulation used by Gram-negative bacteria during quorum sensing is well understood in the bioluminescent marine bacterium Vibrio fischeri. The cell-density dependent activation of the luminescence (lux) genes of V. fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl) homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. LuxR, a 250 amino acid polypeptide, binds to a site known as the lux box centered at position -42.5 relative to the luxI transcriptional start site. During transcriptional activation of the lux operon, LuxR is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli sigma 70 subunit of RNAP in LuxR-dependent transcriptional activation of the luxI promoter has been investigated. Rich in basic amino acids, this conserved portion of sigma 70 is likely to be surface-exposed and available to interact with transcription factors bound near the -35 element. The effect of 16 single and 2 triple alanine substitution variants of sigma 70 between amino acid residues 590 and 613, was determined in vivo by measuring the rate of transcription from a luxI-lacZ translational fusion via b-galactosidase assays in recombinant E. coli. In vitro work was performed with LuxRDN, the autoinducer-independent C-terminal domain (amino acids 157 to 250) of LuxR because purified, full length LuxR is unavailable. Single-round transcription assays were performed in the presence of LuxRDN and 19 variant RNAPs, one of which contained a C-terminally truncated sigma 70 subunit devoid of region 4. Results indicate that region 4 is essential for LuxRDN-dependent luxI transcription with two specific amino acid residues, E591 and K597, having negative effects on the rate of LuxRDN-dependent luxI transcription in vivo and in vitro. None of the residues tested were identified as having any effect on LuxR-dependent luxI transcription in vivo. These findings suggest that region 4.2 is most likely to be in close proximity to LuxR when bound to the luxI promoter. However, unlike the situation found for other ambidextrous activators, no single residue within region 4.2 of sigma 70 may be critical by itself for LuxR-dependent during transcriptional activation. / Master of Science

<I>Vibrio fischeri</I>, a symbiotic bioluminescent bacterium, serves as one of the best understood model systems for a mechanism of cell-density dependent bacterial gene regulation known as quorum sensing. During quorum sensing in <I>V. fischeri</I>, an acylated homoserine chemical signal (autoinducer) is synthesized by the bacteria and used to sense their own species in a given environment. As the autoinducer levels rise, complexes form between the autoinducer and the N-terminal domain of a regulatory protein, LuxR. In response to autoinducer binding, LuxR is believed to undergo a conformational change that allows the C-terminal domain to activate transcription of the luminescence or <I>lux</I> operon. To further understand the mechanism of LuxR-dependent transcriptional activation of the <I>lux</I> operon, PCR-based site-directed mutagenesis procedures have been used to generate alanine-substitution mutants in the C-terminal forty-one amino acid residues of LuxR, a region that has been hypothesized to play a critical role in the activation process. An <I>in vivo</I> luminescence assay was first used to test the effects of the mutations on LuxR-dependent activation of the <I>lux</I> operon in recombinant <I>Escherichia coli</I>. Luciferase levels present in cell extracts obtained from these strains were also quantified and found to correlate with the luminescence results. Eight strains encoding altered forms of LuxR exhibited a "dark" phenotype with luminescence output less than 50% and luciferase levels less than 50% of the wildtype control strain. Western immunoblotting analysis with cell extracts from the luminescence and luciferase assays verified that the altered forms of LuxR were expressed at levels approximately equal to wildtype. Therefor, Low luminescence and luciferase levels could be the result of a mutation that either affects the ability of LuxR to recognize and bind its DNA target (the <I>lux</I> box) or to establish associations with RNA polymerase (RNAP) at the <I>lux</I> operon promoter necessary for transcriptional initiation. A third <I>in vivo </I>assay was used to test the ability of the altered forms of LuxR to bind to the <I>lux</I> box (DNA binding assay/repression). All of the LuxR variants exhibiting the "dark" phenotype in the luminescence and luciferase assay were also found to be unable to bind to the <I>lux</I> box in the<I> </I>DNA binding assay. Therefore, it can be concluded that the alanine substitutions made at these positions affect the ability of LuxR to bind to the <I>lux</I> box in the presence and absence of RNA polymerase. Another class of mutants exhibited wildtype phenotypes in the luminescence and luciferase assays but were unable to bind to the <I>lux</I> box in the DNA binding assay. The alanine substitutions made at these amino acid residues may be making contacts with RNAP that are important for maintaining the stability of the DNA binding region of LuxR. Alanine substitutions made at these positions have a defect in DNA binding at the promoter of the <I>lux</I> operon only in the absence of RNAP. None of the alanine substitutions made in the C-terminal forty-one amino acids of LuxR were found to affect activation of transcription of the <I>lux</I> operon without also affecting DNA binding. Taken together, these results support the conclusion that the C-terminal forty-one amino acids of LuxR are important for DNA recognition and binding of the <I>lux</I> box rather than positive control of the process of transcription initiation. / Master of Science

Po Lai See. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 142-152). / Abstracts in English and Chinese. / TITLE PAGE --- p.p.1 / ACKNOWLEGDEMENTS --- p.p.2 / ABSTRACT --- p.p.3 / 摘要 --- p.p.6 / TABLE OF CONTENTS --- p.p.9 / LIST OF FIGURES AND TABLES --- p.p.16 / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter 1.1 --- Estrogen and Estrogen Receptors and its Action --- p.p.18 / Chapter 1.1.1 --- Estrogen --- p.p.19 / Chapter 1.1.2 --- Estrogen Receptors --- p.p.19 / Chapter 1.1.3 --- Structural Differences between ERa and ERp --- p.p.21 / Chapter 1.1.4 --- Functional Differences --- p.p.22 / Chapter 1.1.5 --- Effects of Selective Estrogen Receptor Modulators --- p.p.22 / Chapter 1.1.6 --- Estrogen works --- p.p.23 / Chapter 1.1.7 --- Estrogen Receptors and Breast Cancer --- p.p.24 / Chapter 1.2 --- Flavonoids： Properties and Biological Activities --- p.p.25 / Chapter 1.2.1 --- Chemical Structure and Classification of flavonoids --- p.p.25 / Chapter 1.2.2 --- Biological Properties and Action Mechanism of Flavonoids… --- p.p.27 / Chapter 1.2.3 --- Flavonoids and breast cancer prevention --- p.p.27 / Chapter 1.3 --- Aims and Scopes of Investigation --- p.p.29 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Chemicals --- p.p.30 / Chapter 2.1.1 --- Flavonoids --- p.p.30 / Chapter 2.1.2 --- Plasmids --- p.p.30 / Chapter 2.2 --- Mammalian cell culture --- p.p.31 / Chapter 2.2.1 --- Maintenance of cells --- p.p.31 / Chapter 2.2.2 --- Preparation of cell stock --- p.p.32 / Chapter 2.2.3 --- Cell recovery from liquid nitrogen stock --- p.p.32 / Chapter 2.3 --- Identification of estrogenic activity in flavonoids --- p.p.33 / Chapter 2.3.1 --- Steady Glo Luciferase Assay --- p.p.33 / Chapter 2.3.2 --- The Biorad Protein Assay kit (a modified Bradford method). --- p.p.33 / Chapter 2.4 --- Viability Assay --- p.p.34 / Chapter 2.5 --- ERE Luciferase reporter gene assay --- p.p.35 / Chapter 2.5.1 --- Transient transfect ion of cell using lipofectamine PLUS reagent --- p.p.36 / Chapter 2.5.2 --- Dual Luciferase Assay --- p.p.37 / Chapter 2.6 --- ERα competitive binding ASSAY --- p.p.37 / Chapter 2.7 --- Apoptotic death assay --- p.p.38 / Chapter 2.8 --- Semi-quantitative RT-PCR Assay --- p.p.40 / Chapter 2.8.1 --- "Isolation of RNA using TRIzol® Reagent (Life Technology,USA) " --- p.p.40 / Chapter 2.8.2 --- Quantitation of RNA --- p.p.41 / Chapter 2.8.3 --- First strand cDNA synthesis --- p.p.41 / Chapter 2.8.4 --- PCR reactions --- p.p.43 / Chapter 2.9 --- Flow Cytometry Analysis --- p.p.43 / Chapter 2.10 --- Total triglyceride and cholesterol measurement --- p.p.44 / Chapter 2.10.1 --- Determination of the total cholesterol --- p.p.45 / Chapter 2.10.2 --- Determination of the total triglyceride --- p.p.46 / Chapter 2.11 --- Manipulation of DNA and RNA --- p.p.46 / Chapter 2.11.1 --- Transformation of DH5α --- p.p.46 / Chapter 2.11.2 --- Mini preparation of plasmid DNA --- p.p.47 / Chapter 2.11.3 --- Preparation of plasmid DNA using QIAGEN-tip 100 midi-prep kit --- p.p.48 / Chapter 2.11.4 --- Preparation of plasmid DNA using QIAGEN-tip 10000 Giga-prep kit --- p.p.49 / Chapter 2.11.5 --- Ethanol preparation of DNA and RNA --- p.p.50 / Chapter 2.11.6 --- Agarose gel electrophoresis of DNA --- p.p.51 / Chapter 2.12 --- Statistical methods --- p.p.52 / Chapter CHAPTER 3 --- Estrogenic and antiproliferative activities on MCF-7 breast cancer cells by flavonoids / Chapter 3.1 --- Introduction --- p.p.53 / Chapter 3.2 --- Results --- p.p.56 / Screening of phytoestrogens for estrogenic activities on MELN cells --- p.p.56 / Cell proliferation activity of phytoestrogens on MCF-7 and MDA-MA231 cells --- p.p.59 / Estrogenic and antiestrogenic activity of phytoestrogens on ERα or erβ transfected hepg2 cells --- p.p.64 / Chapter 3.3 --- Discussion --- p.p.73 / Chapter Chapter 4 --- interaction of baicalein with estrogen receptors / Chapter 4.1 --- Introduction --- p.p.76 / Chapter 4.2 --- Results --- p.p.78 / Estrogen receptor competition assay --- p.p.78 / ERE-Luciferase gene reporter assay --- p.p.82 / Chapter 4.3 --- Discussion --- p.p.88 / Chapter Chapter 5 --- baicalein and genistein display differential actions on er transactivation / Chapter 5.1 --- Introduction --- p.p.90 / Chapter 5.2 --- Results --- p.p.92 / Estrogenic and antiestrogenic activities of genistein and baicalein on ER transactivation --- p.p.92 / Chapter 5.3 --- Discussion --- p.p.105 / Chapter CHAPTER 6 --- APOPTOTIC EFFECTS OF BAICALEIN ON MCF-7 AND MDA-MB-231 CELL LINES / Chapter 6.1 --- Introduction --- p.p.107 / Chapter 6.2 --- Results --- p.p.111 / ER POSITIVE MCF-7 AND ER NEGATIVE MDA-MB-231 cell death assay --- p.p.111 / "Bcl-2, Bax and PS2 mRNA expression " --- p.p.116 / Arrest at sub G1 phase of MCF-7 by baicalein --- p.p.124 / Chapter 6.3 --- Discussion --- p.p.127 / Chapter CHAPTER 7 --- BAICALEIN CAN REDUCE INTRACELLULAR cholesterol and triglceride / Chapter 5.1 --- Introduction --- p.p.129 / Chapter 5.2 --- Results --- p.p.130 / Baicalein has beneficial effect on lipid metabolism --- p.p.130 / Chapter 5.3 --- Discussion --- p.p.139 / Chapter chapter 8 --- Summary --- p.p.140 / BIBLIOGRAPHY --- p.p.142 / APPENDIX 1 ABBREVIATIONS --- p.p.153 / APPENDIX 2 PRIMER LISTS --- p.p.156 / APPENDIX 3 REAGENTS AND BUFFERS --- p.p.157