Briefly, some findings in my research are as follows: (1) The genetic variants of the CA repeats in IGF1 promoter 1 can affect the activity of promoter 1, and the CA repeat showed a suppressive effect on the activity of the promoter 1 of IGF1 gene. EMSA results have shown that the CA repeats could bind to certain nuclear protein. (2) The SNPs T/C (rs5742612) and T/A (rs2288377) can also affect the activity of the promoter 1 in IGF1 gene, and the activity of C-A haplotype is significantly higher than that of T-T haplotype. EMSA results have shown that the SNP T/A (rs2288377) could bind to certain nuclear protein. (3) I developed the new dual reporter assay method to investigate the transactivation interaction between the SNP T/G (rs2071430) and C/A (rs17000900) in the MxA promorer. This new method can not only improve the detection limit for small difference between haplotypes, but also calculate the model of transactivation effect between these two SNPs. The results were better than those of traditional method, and it gave a clear-cut demonstration of the effect of interaction between these two SNPs on the activity of MxA promoter. / In addition, in the IGF1 study, the core promoter region of promoter 2 was identified through 5' deletion mutagenesis methods. Moreover, a cell-type specific mechanism of bidirectional activation of promoter was found. / Recently, more and more studies focus on gene function with the completion of the Human Genome Project. It is well known that polymorphism of human genome sequence is a common phenomenon in the human population. Specially, a lot of genetic polymorphisms, including single nucleotide polymorphisms (SNPs) and microsatellites, have been reported in the regulatory region of many genes. However, the effects of most of these genetic polymorphisms on gene expression are still unknown. The polymorphisms in the promoter can play an important role in the gene regulation. For example, some SNPs located in the transcription factor binding site (TFBS) can affect gene transcription. So, it is very necessary to directly study the effect of genetic variants on promoter transactivation activities. In this study, we studied the effect of genetic polymorphisms on gene expression through reporter gene assay, electrophoretic mobility shift assay (EMSA), and so on. And the candidate genes include insulin-like growth factor 1 (IGF1) and myxovirus resistence 1 (MxA). Some SNPs and microsatellites have been reported in the promoters of these genes. In our previous researches, we focused on the study of the association between these polymorphisms and some diseases, and it was found that a few SNPs significantly associated with relevant diseases. Based on the previous results, in my project, I developed new functional assays and also improved existing methods to analyse the functional effect of these genetic variants of promoters on transactivation activities. / by Huang, Wei. / "March 2008." / Adviser: Nelson Leung Sang Tang. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 139-145). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Lau Suk Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 128-150). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Table of content --- p.ii / List of Figures --- p.x / List of Tables --- p.xiv / Abbreviation --- p.xv / Abstract --- p.xviii / 論文摘要 --- p.xx / Chapter 1 --- Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Tetraodon nigroviridis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Genomic Sequencing Project --- p.3 / Chapter 1.1.3 --- Tetraodon nigroviridis as Study Model --- p.4 / Chapter 1.1.3.1 --- Genomic Comparison --- p.4 / Chapter 1.1.3.2 --- Gene Order and Structural Studies --- p.5 / Chapter 1.1.3.3 --- Genomic Evolution --- p.6 / Chapter 1.2 --- Transcriptional Regulation and Transcription Factors Binding Sites Prediction --- p.7 / Chapter 1.2.1 --- Transcriptional Regulation --- p.7 / Chapter 1.2.1.1 --- Chromatin Remodeling --- p.7 / Chapter 1.2.1.2 --- Locus Control Regions (LCR) and Boundary Elements --- p.8 / Chapter 1.2.1.3 --- Promoter Structure --- p.9 / Chapter 1.2.1.4 --- Transcriptional Machinery Assembly --- p.10 / Chapter 1.2.2 --- Transcription Factors and Their Binding Sites --- p.11 / Chapter 1.2.3 --- Transcription Factor Binding Site Prediction --- p.12 / Chapter 1.3 --- Acetylcholinesterase --- p.15 / Chapter 1.3.1 --- Background --- p.15 / Chapter 1.3.2 --- Regulation ofAChE --- p.17 / Chapter 1.3.2.1 --- Transcriptional Level --- p.17 / Chapter 1.3.2.2 --- Post-transcriptional Level --- p.19 / Chapter 1.3.2.3 --- Post-translational Level --- p.20 / Chapter 1.3.2.3.1 --- Oligomerization --- p.20 / Chapter 1.3.2.3.2 --- Glycosylation --- p.21 / Chapter 1.3.2.3.3 --- Phosphroylation --- p.22 / Chapter 1.3.3 --- Functions of AChE --- p.23 / Chapter 1.3.3.1 --- Hydrolysis Acetylcholine --- p.23 / Chapter 1.3.3.2 --- Embryonic Development --- p.23 / Chapter 1.3.3.3 --- Haemotopotesis and Thrombopsiesis --- p.24 / Chapter 1.3.3.4 --- Neuritogensis --- p.24 / Chapter 1.3.3.5 --- Amyloid Fibre Assembly --- p.24 / Chapter 1.3.3.6 --- Apoptosis --- p.25 / Chapter 1.3.4 --- AChE and Alzheimer's disease --- p.25 / Chapter 1.3.4.1 --- Treatment for AD Patients --- p.27 / Chapter 1.4 --- Inducible Cell Expression Systems --- p.28 / Chapter 1.5 --- Objectives --- p.32 / Chapter 2 --- Chapter 2 Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Primer Design --- p.34 / Chapter 2.2.2 --- Cell Culture --- p.34 / Chapter 2.2.3 --- Transformation --- p.35 / Chapter 2.2.4 --- Plasmids Preparation --- p.35 / Chapter 2.2.5 --- Plasmids Screening --- p.36 / Chapter 2.2.6 --- RNA Extraction --- p.36 / Chapter 2.2.7 --- Reverse Transcriptase Polymerase Chain Reaction and Construction tnAChE/pCR4 vector --- p.37 / Chapter 2.2.8 --- Genomic Analysis --- p.37 / Chapter 2.2.9 --- Protein Sequence Analysis --- p.38 / Chapter 2.2.10 --- Genomic DNA Extraction --- p.39 / Chapter 2.2.11 --- Construction of Reporter Vectors ptnAChE_565/pGL3 and ptnAChK1143/pGL3 --- p.39 / Chapter 2.2.12 --- Luciferase Assay --- p.40 / Chapter 2.2.13 --- Transcription Factors and Promoter Prediction --- p.40 / Chapter 2.2.14 --- Protein Assay --- p.41 / Chapter 2.2.15 --- AChE Activity Determined by Ellman's Method --- p.41 / Chapter 2.2.16 --- Histochemistry --- p.42 / Chapter 2.2.17 --- Protein Extraction from Tissues --- p.42 / Chapter 2.2.18 --- Construction of Bacterial Expression Vector His-MBP-tnAChEAC/pHISMAL --- p.43 / Chapter 2.2.19 --- Protein Expression in Bacterial Expression System --- p.43 / Chapter 2.2.20 --- Purification and Thrombin Cleavage of His-MBP- tnAChEAC --- p.44 / Chapter 2.2.21 --- SDS Electrophoresis --- p.44 / Chapter 2.2.22 --- Western Blotting --- p.45 / Chapter 2.2.23 --- Construction of Tet-Off Expression Vector --- p.45 / Chapter 2.2.24 --- Transient Expression of tnAChEAC --- p.46 / Chapter 2.2.25 --- Establishment of Stable Tet-Off CHO Cell Lines Overexpressing tnAChEAC --- p.47 / Chapter 2.2.26 --- MTT Assay --- p.47 / Chapter 2.2.27 --- Partial Purification of tnAChEΔC --- p.48 / Chapter 3 --- Chapter 3 Sequence Analysis of AChE Gene of Tetraodon nigroviridis --- p.49 / Chapter 3.1 --- Results --- p.49 / Chapter 3.1.1 --- Cloning of tnAChE from Tetraodon nigroviridis Brain --- p.49 / Chapter 3.1.2 --- "Comparative genomic analysis of tnAChE with Human, Rat, Mouse, Takifugu rubripes, ZebrafishAChE" --- p.49 / Chapter 3.1.3 --- Primary Sequence Analysis --- p.52 / Chapter 3.1.4 --- Promoter and Transcriptional Factors Predictedin tnAChE Promoter Region --- p.60 / Chapter 3.1.4.1 --- Promoter Region Analysis In Silico --- p.60 / Chapter 3.1.4.2 --- Promoter Activity Analysis --- p.76 / Chapter 3.2 --- Discussion --- p.78 / Chapter 4 --- Characterization of tnAChE in Prokaryotic and Eukaryotic Tet-Off Inducible Expression System --- p.91 / Chapter 4.1 --- Results --- p.91 / Chapter 4.1.1 --- AChE Expresses in Tetraodon nigroviridis --- p.91 / Chapter 4.1.2 --- Expression of recombinant tnAChE in Bacterial Expression System --- p.94 / Chapter 4.1.2.1 --- Construction of His-MBP-tnAChEΔC/pHISMAL Construct --- p.94 / Chapter 4.1.2.2 --- His-MBP-tnAChEAC Expression in E. coli Strains BL21 (DE) and C41 --- p.94 / Chapter 4.1.3 --- Expression of tnAChEAC in Mammalian Expression System --- p.99 / Chapter 4.1.3.1 --- Construction of tnAChEAC/pTRE2hgyo Mammalian Expression Vector --- p.99 / Chapter 4.1.3.2 --- Transient Expression of tnAChEAC --- p.99 / Chapter 4.1.3.3 --- Establishment of Tet-Off CHO Cells Stably Expressing the Inducible tnAChEAC --- p.101 / Chapter 4.1.3.4 --- Characterization of Tet-Off tnAChEAC Stably Transfected Cell Clones --- p.103 / Chapter 4.1.3.5 --- Effect of Over Expressed tnAChEAC on cell viability --- p.103 / Chapter 4.1.3.6 --- Partial Purification of tnAChEAC from Stably Transfected Cells --- p.107 / Chapter 4.1.3.7 --- tnAChE and tnAChEAC in Different pH Values --- p.112 / Chapter 4.1.3.8 --- Kinetic Study of tnAChEAC --- p.112 / Chapter 4.1.3.9 --- Inhibition of AChE Activity of Partial Purified tnAChEAC by Huperzine --- p.112 / Chapter 4.2 --- Discussion --- p.116 / Chapter 4.2.1 --- Bacterial Expression System --- p.116 / Chapter 4.2.2 --- Expression of tnAChEΔC in Mammalian System --- p.119 / Chapter 5 --- General Discussion --- p.124 / Chapter 5.1 --- Summaries --- p.124 / Chapter 5.2 --- Further works --- p.126 / Chapter 6 --- References --- p.128 / Appendix 1 internet software and database used in this project --- p.151 / Appendix 2 tnAChE mRNA sequence --- p.152 / Appendix 3 ptnAChE-1143 sequence --- p.154 / Appendix 4 Six open reading frame translation of ptnAChE-1143 --- p.156

by Kwong-Kwok Wong. / Thesis (Ph.D)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 171-183. / TITLE PAGE --- p.I / ABSTRACT --- p.II / STATEMENT --- p.V / ACKNOWLEGEMENTS --- p.VI / ABBREVIATIONS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XIII / LIST OF FIGURES --- p.XVI / Chapter Chapter 1. --- Introduction and Literature Review --- p.1 / Chapter I . --- Introduction --- p.1 / Chapter A. --- General introduction --- p.1 / Chapter B. --- Purpose of study --- p.5 / Chapter II. --- Literature review --- p.6 / Chapter A. --- Global control of aerobic-anaerobic shift --- p.6 / Chapter B. --- Identified anaerobiosis-inducible genes --- p.8 / Chapter C. --- Genetics of anaerobic regulation --- p.15 / Chapter i. --- Redox control --- p.15 / Chapter ii. --- DNA conformation --- p.15 / Chapter iii. --- fnr (oxrA) regulatory gene --- p.16 / Chapter iv. --- narL gene --- p.18 / Chapter v. --- Other regulatory genes --- p.19 / Chapter vi. --- Proposed FNR and NarL recognition sequences --- p.20 / Chapter D. --- future prospect --- p.23 / Chapter Chapter 2. --- Isolation of Anaerobiosis-inducible Promoters --- p.25 / Chapter I . --- Introduction --- p.25 / Chapter A. --- Properties of promoter-probe plasmid pKK232.8 --- p.26 / Chapter B. --- Properties of promoter-probe plasmid pFZYl --- p.28 / Chapter II. --- Materials and methods --- p.30 / Chapter A. --- Bacterial strains and plasmids --- p.30 / Chapter B. --- Media --- p.30 / Chapter C. --- Solutions --- p.31 / Chapter D. --- Small scale prepartaion of plasmid DNA --- p.32 / Chapter E. --- Large scale preparation of plasmid DNA --- p.33 / Chapter F. --- Digestion of DNA with restriction endonucleases --- p.35 / Chapter G. --- Analysis of DNA samples with agarose gel electrophoresis --- p.36 / Chapter H. --- Dephosphorylation of DNA fragments --- p.37 / Chapter I. --- Partial digestion of Chromosomal DNA with restriction enzyme Sau3A. --- p.37 / Chapter J. --- Ligation of DNA --- p.38 / Chapter K. --- Preparation of competent cells --- p.38 / Chapter L. --- Transformation --- p.40 / Chapter M. --- Chloramphenicol resistance levels test for promoter clones with plasmid pKK232.8 --- p.41 / Chapter N. --- Preparation of crude cell extract for chloramphenicol acetyltransferase (CAT) assays --- p.41 / Chapter O. --- CAT assay --- p.42 / Chapter P. --- Protein assay --- p.42 / Chapter Q. --- β-galactosidase assay --- p.43 / Chapter III . --- Results --- p.45 / Chapter A. --- Molecular cloning of anaerobiosis-inducible promoters with promoter-probe plasmid pKK232.8 --- p.45 / Chapter B. --- Molecular cloning of anaerobiosis-inducible promoters with promoter-probe plasmid pFZYl --- p.54 / Chapter IV. --- Summary and Discussion --- p.70 / Chapter A. --- Cloning with promoter-probe plasmid pKK232.8 --- p.70 / Chapter B. --- Cloning with promoter-probe plasmid pFZYl --- p.71 / Chapter C. --- Number of anaerobiosis inducible promoters --- p.73 / Chapter Chapter 3. --- Subcloning and Sequencing --- p.74 / Chapter I . --- Introduction --- p.74 / Chapter II. --- Materials and methods --- p.74 / Chapter A. --- Bacterial strains and bacteriophages --- p.74 / Chapter B. --- Preparation of M13mp RF plasmid --- p.75 / Chapter C. --- DNA sequencing by the chain termination method --- p.75 / Chapter D. --- Polymerase chain reaction (PCR) for the amplification of DNA fragments cloned in plasmid pFZYl --- p.79 / Chapter E. --- Using Exonuclease III to construct unidirectional deletions to generate nested clones --- p.80 / Chapter F. --- Direct gel electrophoresis --- p.81 / Chapter G. --- C-testiscreening for the orientation of insert in M13 phage --- p.81 / Chapter III . --- Results --- p.82 / Chapter A. --- Subcloning and sequencing of pFE29 and pFE117 --- p.82 / Chapter B. --- Subcloning and sequencing of pHSKl --- p.90 / Chapter C. --- Subcloning and sequencing of pHSK8 --- p.100 / Chapter D. --- "Subclonig and sequencing of pFSl, pFS22 and pFS3 4" --- p.109 / Chapter IV. --- Summary and Discussion --- p.113 / Chapter A. --- Trimming down size of DNA fragments to smaller fragments which still contained anaerobiosis-inducible promoters --- p.113 / Chapter B. --- Nucleotide sequencing --- p.113 / Chapter C. --- Sucloning and sequencing strategy --- p.115 / Chapter Chapter 4. --- Expression of Anaerobiosis-inducible Promoters --- p.120 / Chapter I . --- Introduction --- p.120 / Chapter II. --- Materials and methods --- p.122 / Chapter A. --- Bacterial strains and phages --- p.122 / Chapter B. --- Media --- p.125 / Chapter C. --- Transformation in Salmonella typhimurium --- p.125 / Chapter D. --- Genetic techniques --- p.126 / Chapter III. --- Results --- p.129 / Chapter A. --- Expression of Escherichia coli qlpT promoter --- p.129 / Chapter B. --- "Expression of Salmonella typhimurium anaerobiosis-inducible promoters cloned in pHSK8, pFS22 and pFS34" --- p.134 / Chapter IV. --- Summary and Discussion --- p.137 / Chapter A. --- A pair of divergent promoters were both regulated by anaerobiosis and glucose. --- p.137 / Chapter B. --- fnr(oxrA) dependent and independent promoters --- p.137 / Chapter C. --- Effect of nitrate on anaerobiosis expression. --- p.138 / Chapter Chapter 5. --- Analysis of Anaerobiosis-inducible Promoter-containing DNA sequences and Final Discussion --- p.141 / Chapter I. --- Analysis of anaerobiosis-inducible promoter-containing DNA sequences --- p.141 / Chapter A. --- "Search for initiation codon, conserved ""-10"" and ""-35"" regions" --- p.141 / Chapter B. --- Search for FNR binding sites and NarL binding sites. --- p.151 / Chapter C. --- Homology search among the promoter sequences of all anaerobiosis-inducible genes. --- p.156 / Chapter II. --- Final Discussions. --- p.164 / Chapter A. --- Summary of the properties of the sequenced and characterized promoters cloned in this study --- p.164 / Chapter B. --- Further studies. --- p.167 / REFERENCES --- p.168

Existing methods to systematically characterize sequence-intrinsic activity of promoters are limited by relatively low throughput and the length of sequences that could be tested. Here we present Survey of Regulatory Elements (SuRE), a method to assay more than a billion DNA fragments in parallel for their ability to drive transcription autonomously. In SuRE, a plasmid library is constructed of random genomic fragments upstream of a barcode and decoded by paired-end sequencing. This library is transfected into cells and transcribed barcodes are quantified in the RNA by high-throughput sequencing. By computationally analyzing the resulting data using generalized linear models, we succeed in delineating subregions within promoters that are relevant for their activity on a genomic scale, and making accurate predictions of expression levels that can be used to inform minimal promoter reporter construct design. We also show how our approach can be extended to analyze the differential impact of single-nucleotide polymorphisms (SNPs) on gene expression.

Rat selenoprotein W (SeW) promoter activity was investigated using different concentrations of cadmium, copper, and zinc. Two fragments (404bp and 1265bp) of the SeW promoter, containing a single metal response element (MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter plasmid. The constructs were transfected into cultured rat C6 (glial) and L8 (myoblast) cells and promoter activity measured by means of luciferase reporter gene fused to the SeW promoter fragments in the reporter plasmid. With post-transfection exposure of these cell lines to these metals, copper and zinc, but not cadmium, significantly increased promoter activity of the unmutated 1265bp (not 404bp) construct (p<0.05) only in the C6 cells. Mutation of the MRE sequence abolished promoter response to metal exposure but did not eliminate promoter activity. The results suggest that SeW expression in glial cells can be increased on exposure to copper and zinc and that this response is dependent on the MRE sequence present in the SeW promoter. To understand transcriptional regulation of the SeW gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter, and antibodies, we were able to show that the specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as the MRE. However, the MRE, GRE, AP-1 and LF-A1 did not yield any specific binding. Although, competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore the SeW gene may be a target for Sp1 whose interaction with the SeW promoter may activate or repress the transcription of SeW. / Graduation date: 2003

No description available.

For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. Tissue- or signal-responsive promoters are in high demand in practical plant biotechnology. The present study sought to characterise the activities of two promoters in sugarcane, namely the UBI (ubiquitin) promoter and the SUC-1 promoter (UBI linked in tandem to the cauliflower mosaic virus 35S promoter). It was hypothesised that the activity of UBI would be maintained or even increased under conditions of environmental stress, since it is well documented that ubiquitin is a stress-related protein. A further hypothesis was that SUC-1 might enhance overall gene expression since the CaMV 35S component is a constitutive promoter widely and successfully used in plant transformation. Plants of the sugarcane variety NC0310, containing the cry1A(c) (Bt) gene from Bacillus thuringiensis, were used as models in a system in which the plants were stressed by withholding water supply in a controlled manner. Since large numbers of clones of both transgenic and wild-type plants were needed for the water stress and expression experiments, three micropropagation techniques, namely, shoot tip-, callus- and node culture, were optimised and compared. The objective was to propagate genetically stable plants rapidly. Compared to shoot tip culture, node and callus culture proved slow and inefficient. Shoot tip culture was thus chosen as the most suitable for the regeneration of experimental material. Relative Water Content (RWC) determination, leaf elongation measurements and Infra Red Gas Analysis (IRGA) were compared in order to find the most appropriate method of measuring plant water status. In addition to being destructive, no observable differences were evident between the control (non-stressed) and water-stressed plants when using RWC as a measure. Results obtained from leaf elongation measurements compared favourably to the more sophisticated IRGA readings, showing that leaf elongation is as sensitive a measure of water stress. On the basis of preliminary studies with untransformed plants using the latter two techniques, water regimes for stress-induction in the final experiments were designed. Leaf elongation measurements, which are simple and non-destructive, were ultimately chosen to measure plant water status. In the final water stress experiment non-transgenic NCo310 and clonal populations of six transformants were used (three containing the UBI promoter; three the SUC-1 promoter). Exactly half of the plants of each type were stressed by withholding water supply, while the other half (controls) were watered manually twice a day. Leaf elongation measurements were made at the same time daily on the third youngest leaf of 6 plants from each population per treatment. At the same time, leaf samples were taken daily for molecular analysis. The stress regime led to marked differences in leaf elongation between control and water-stressed plants. In terms of physiological response (leaf rolling and senescing), plants containing the SUC-1 promoter appeared least affected. The reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridisation were used to assay UBI and SUC-1 activity. RT-PCR revealed that both promoters drove Bt gene expression in controls and experimentals throughout the stress period, although differences in signal intensity were not observed. The extent of expression occurring in each type of plant was revealed in Northern blots probed with two genic sequences (1) the transgene and (2) sugarcane EST ME42, homologous to heat shock protein 82 in rice. Individual transformants showed overall levels of transgene expression that were variable, possibly due to insert position in the plant genome, as well as variations in relation to the application of stress. SUC-1 seemed superior to UBI in terms of driving transgene expression under stressful environmental conditions, since UBI promoter activity appeared to decrease under stress, while SUC-1 promoter activity remained constant. In addition to the expected 2.0 kb Bt transcript, transcripts of smaller than expected size were also obtained, leading to the suggestion of premature polyadenylation signals in the coding region of the wild-type Bt234 gene. Upon inspection of the transgene sequence, a number of motifs rarely present in plant genes were observed, namely A/T rich sequences, ATTTA motifs and numerous potential polyadenylation sites. / Thesis (M.Sc.)-University of Natal, Durban, 1999.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 1999. / Includes bibliographical references.

Thesis (M.Sc.)--York University, 2007. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 129-130). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR32034

The specific goal of this study was to construct and test transcriptional fusions of zein promoters and a yeast reporter gene that will serve as part of a two plasmid system that will allow for the identification of maize transcriptional regulators of zein genes. Zein genes are expressed coordinately and tempO~ly during endosperm development and are controlled at the transcriptional level (Pedersen et aL, 1980; Kodryzcki et al. t 1989). The accumulation of zein proteins in the endosperm presents an ideal model system to study plant gene regulation. These proteins are synthesized only in the endosperm tissue, and their concentration in the endosperm determine the nutritional quality of the seed. Because of the coordinate and temporal regulation of zein gene transcription, there is a strong likelihood that there exists positive regulatory elements of zein gene expression during early endosperm development. We know that the control of storage protein gene expression is mediated by regulatory elements in the endosperm of maize seeds. It has been shown that the recessive mutation opaque-2 (02) specifically reduces the 22,000 zein polypeptide .. Schmidt et at (1990) and Aukennan et at (1991) show that the wild-type 02 encodes a protein containing a basic leucine zipper domain / Master of Science