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Subcloning, Purification and Characteriration of the GLY7 Gene

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Subcloning, Purification and Characteriration of the GLY7 Gene Subcloning, purification and characteriration of the GLY7 gene product of Saccharomyces cerevisiae Ghoiarn Hossien Kiani Amineh A Thesis submitted in conformity with the requirements for the Degree of Master of Science Graduate Department of Molecular and Medical Genetics University of Toronto O Copyright 1997 by G. H. Kiani National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON KI A ON4 Ottawa ON KIA ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distnbute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/f%n, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur consente la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othexwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Subcloning, purification and characterization of the GLY7 gene product of Saccharomyces cerevisiae Gholarn Hossien Kiani Amineh, Master of Science, 1997 University of Toronto, Department of Microbiology ABSTRACT In Saccharomyces cerevisiae the inactivation of both the cytosolic and mitochondrial isozymes of serine hydroxymethyltransferase. as well as a third gene. designated GLYI, are necessary to yield glycine auxotrophs. The functional characterization of the GLY7 gene was approached by assaying for threonine aldolase activity catalyzing the conversion of threonine to glycine and acetaldehyde using the GLYI enzyme purified on a nickel column. No such activity was obtained with a coupled assay to alcohol dehydrogenase using either the purified HIS-tagged GLYI enzyme or the nickel column-purified HIS-tagged SHMT2. In the pursuit of showing that the expression of the GLYl gene might be up-reguiated by threonine and down-regulated by glycine. a yeast shuttle vector in which the promoter and the 5' coding region of the GLY? gene were fused to a reporter gene (Lac 2) was used to transform the yeast strain YM 22 to leucine prototrophy. The transformed yeast cells were grown in the presence and absence of L-threonine in minimal medium. A p-galactosidase assay was done on the crude extracts. A more than 1.5 times increase in P- galactosidase (P-gai) activity was observed for the yeast cells grown in the presence of threonine than in its absence, an increase that is not conclusive for regulation by threonine. Similarly, the results of the above assay with or without the glycine supplement indicate that GLYI gene is not down-regulated by glycine. We next examined the hypothesis that GLYI may be part of the "glyoxylate anaplerotic pathway" coding for an enzyme with a glyoxylate arninotransferase activity. It had been previously reported that a serl yeast strain (which has a mutation in the phosphoserine aminotransferase gene, SER?, an enzyme required for serine synthesis from glucose) could grow without serine on a non-fermentable carbon source (such as ethanol or acetate) due to the induction of the glyoxylate pathway. The glyoxylate can be transaminated to glycine which can be used to make serine using the glycine cleavage system and mitochondrial SHMT enzymes. To characterize the possible aminotransferase activity of the GLYI protein, a yeast shuttle vector containing the entire GLYl gene sequence was used to transform the serf yeast strain to uracil prototrophy. The serl yeast transformants grew on glucose but the over-production of GLYI on a multicopy plasmid did not stimulate the growth on acetate, suggesting the enzyme is not part of the glyoxylate anaplerotic pathway. To confirm the growth study results, a different genetic approach, gene disruption. was ernployed based on the reasoning that if the GLYl gene were involved in the glyoxylate pathway, its disruption would prevent the growth of the serl yeast on acetate in the absence of serine. GLYl gene disruption experiments were done on the ser7 yeast utilizing direct gene replacement and mating strategies. The GLYl gene replacement test was done using a glyl::URA3 disruption plasmid which was previously used to inactivate GLYl (where the GLYl promoter, part of the 5' non-coding region and the first three arnino acids of the gene are replaced by a 1.1 kb URA3 fragment). The ser7 cells were transformed with the above plasmid and the transformants were selected for uracil prototrophy. A number of uracil prototrophs were obtained: these were shown by Southern blot analysis to be wild type at the GLY7 locus. The GLY7 gene disruption was attempted using a mating strategy in which the ser7 yeast strain YM14: a was mated with the yeast YM11: a gly7::URAd. The resultant dipioids sporulated and the spores were plated ont0 rich YPD plates to reach their haploid state. The haploids were then screened for uracil prototrophy and serine auxotrophy. No such hapioid was obtained using the mating technique; however, during one of these mating experiments, a number of cells were encountered which were serine auxotrophic and uracil prototrophic. These cells were tested for the GLYl gene disruption using Southern blot analysis. The results of Southern analysis indicated that these cells were in fact serl/serl &gIyl::URA3/GLY? diploids. A number of tetrad dissection studies were performed on the above diploids with results indicating that the disruption of the GLY? gene in the serf background of the yeast strain YM 14 is lethal. Acknowledgments I would like to thank Dr. Andy Bognar for giving me the opportunity to work in his laboratory as well as guiding me and advising me throughout the course of this research. I would like to express my appreciation to the members of rny committee, Dr.J. Campbell and Dr. C. Lingwood for their moral and scientific support and assistance upon preparing this thesis. 1 would like to thank Dr. J.B. McNeil and Dr.Ron Pearlman for providing me with the necessary cell strains and plasmids to rnake this work possible. Finally, I would like to take this opportunity to express my utmost appreciation to my parents and my brother for their moral and financial support. Dedication I would like to dedicate this thesis to my parents. Esmat and Ali Kiani, to my brother Hamid Kiani and to my wife, Nathalie Kiani. vii Table of Contents Page ABSTRACT ii ACKNOWLEDGEMENTS v DEDICATION vi TABLE OF CONTENTS vi i LET OF ABBREVIATIONS xii LIST OF TABLES xvi i LIST OF FIGURES xviii INTRODUCTION: Overview of folate mediated one-carbon metabolism in eukaryotes Subcellular distribution of fotates Sources of one-carbon units Folate-binding proteins One-carbon metabolism is compartmentalized in eukaryotes The cytoplasmic pathways The rnitochondrial pathways A general overview of SHMT A general overview of the glycine cleavage system Glycine synthesis in eukaryotes General amino acid control in yeast Transmembrane proteins Use of synthetic peptides in raising antibody to a protein Gene fusion for purpose of expression Goals of this project MATERIALS AND METHODS: Materials Media Buffers Stock solutions and reagents Methods Routine procedures Isolation of plasmid DNA Restriction digests Agarose gel electrophoresis Ligations Preparation of fresh competent E-coli cells using calcium chloride Transformation of plasmid DNA into competent cells SDS-PAGE PCR protocol for the amplification of the GLYl gene PCR programs Protein assay Protein purification using IMAC Plcm transduction Threonine aldolase assays Assay of P-galactosidase in yeast Preparation of ssDNA for yeast transformation Yeast transformation Isolation of yeast genomic DNA Gene disruption Southern blot analysis Yeast rnating Tetrad anal ysis RESULTS: The coding region of the GLYl gene was successfully amplified The GLYl amplicons with 5' Ndel site and 3' BamHl site were subcloned into pET 15b expression vector The GLYl protein was successfully expressed in BL21 cells Most of the GLYI protein expressed in BL21 cells is produced in an insoluble state in the form of inclusion bodies Construction of CJ/~A-BL21 by Pl cm transduction of GS 459 E.coli cell strain as donor and BL 21 as recipient The glycine auxotrophy of gly~-BL 21 could be weakly over- corne provided these cells are transformed with the PG construct and induced by IPTG The GLYl protein waç expreççed in gly~=BL 21 and purified using irnmobilized ion affmity chromatography on the HIS-BIND resin Anti-GLY1 antibody could not be raised against the purified GLYl protein Yeast cytoplasrnic SHMT was also expressed in gly~-BL 21 and purified on nickel column utilizing IMAC Threonine aldolase assays of GLYl protein Glyoxylate anaplerotic pathway is functional in yeast and can provide the serl mutant yeast strain with serine when grown on a non-fermentable carbon source without serine supplements Overexpression of the GLYl gene and relief of serine Auxotrophy in a serl yeast strain Genetic probing of the possible regulation of the GLYl gene expression by threonine and glycine in a yeast setting 100 Disruption studies of the GLYl gene IO1 Tetrad analysis of ser?/serl;gly?::URA3/GLYIYM 14 diploids 104 The GLYI gene lacks the Gcn4p consensus sequence 106 Hydrophobicity analysis of the GLYl protein 109 DISCUSSION AND CONCLUSIONS III 1 FUTURE DiRECTlONS Raising antibody against the GLYI protein A more sensitive threonine aldolase assay The lethality hypothesis Transmembrane protein identity confirmation of the GLYl 1 protein 120 I APPENDICES 121 1 REFERENCES 133 sii Abbreviations Ab - antibody ac - activity ADE3 - the gene encoding cytoplasmic trifunctional Cl-THF-synthase enzyme in S.
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