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INFORMATION to USERS the Most Advanced Technology Has Been Used to Photo Graph and Reproduce This Manuscript from the Microfil INFORMATION TO USERS The most advanced technology has been used to photo­ graph and reproduce this manuscript from the microfilm master. UMI films the original text directly from the copy submitted. Thus, some dissertation copies are in typewriter face, while others may be from a computer printer. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyrighted material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are re­ produced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each oversize page is available as one exposure on a standard 35 mm slide or as a 17" x 23" black and white photographic print for an additional charge. Photographs included in the original manuscript have been reproduced xerographically in this copy. 35 mm slides or 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. r Accessing the World'sUMI Information since 1938 300 North Zeeb Road Ann Arbor Ml 48106 1346 USA Order N um ber 8726G95 DNA sequences and comparison of argininosuccinate synthetase genes from two methanogenic archaebacteria Morris, Christina Jane, Ph.D. The Ohio State University, 1987 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V 1. Glossy photographs or pages ____ 2. Colored illustrations, paper or print _______ __ 3. Photographs with dark background _____ 4. Illustrat'ons are poor copy _______ 5. Pages with black marks, not original copy ____ 6. Print shows through as there is text on both sides of page _________ 7. Indistinct, broken or small print on several pages S 8. Print exceeds margin requirements _______ 9. Tightly bound copy with print lost in sp in e ________ 10. Computer printout pages with indistinct print _______ 11. Page(s)_____________ lacking when material received, and not available from school or author. 12. Page(s)_____________ seem to be missing in numbering only as text follows. 13. Two pages numbered ________ , Text follows. 14. Curling and wrinkled pages ______ 15. Dissertation contains pages with print at a slant, filmed as received __________ 16. Other _____ ____ ______ University Microfilms International DNA SEQUENCES AND COMPARISON OF AR6IHIN0SUCCINATE SYNTHETASE GENES FROM TWO METHANOGEN1C ARCHAEBACTERIA DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Christina Jane Morris, B Sc , M.S. The Ohio State University 1987 Reading Committee: Approved By John N. Reeve Charles J Daniels Darrell R. Galloway Berl R. Oakley Adviser Department of Microbiology PLEASE NOTE: Copyrighted materials in this document have not been filmed at the request of the author. They are available for consultation, however, in the author's university library. These consist of pages: Cartoon in pre-pages University Microfilms International 300 N ZE EB RD . ANN AH BOB. Ml 48106 (313) 7fj 1 4 700 T o Mum and Dad ii ACKNOWLEDGEMENTS I uiould like to thank my adviser, Dr John Reeve, for his advice, encouragement and support throughout this project Thanks also go to the past and present members of Dr Reeve's and Dr Daniels’ labs for their assistance, ideas, understanding in times of need and, most of all, friendship Special thanks go to Dr Chuck Daniels for the use of his computer and office. To quote Maureen Stapleton: "I mould like to thank everyone I’ve ever met in my mhole life", but perhaps I’ll keep it short and finally thanx my family for their love, support and phone calls VITA October 4, 1959 ....................................... Born - Bristol. England. 1981 ............................................................................... B.Sc. (Honours), Queen Mary Col lege, Uni vers it y of London/ London. England. 1981-1982 ................................................. Teaching Associate. Department of Microbiology, The Ohio State University. Columbus. Ohio, USA 1982-1987 Research Associate, Department of Microbiology, The Ohio State University 1983 ............................................................................... M S The Ohio State Un i versity 1987 ............................................................................... UCLA International School on Mo1ecu 1ar Evo1ut ion Fellowship. Los Angeles, Ca1i forn i a PUBLICATIONS Morris, C.J. and JN. Reeve <1984). Functional expression of an archaebacterial gene from the methanogen Methanosarcina barken in Escherichia coli and Bacillus subtilis In, Microbial Growth on Cl Compounds. <R.L. Crawford and R S. Hanson, eds.) American Society for Microbiology, Washington, D.C. 20006. Morris, C J and J N Reeve <1985) Expression of a methanogen gene in Escherichia coli and in Bacillus subtilis In, Proceedings of the First International Symposium on Biotechnological Advances in Processing Municipal Wastes for Fuels and Chemicals <A,A Antonopoulos, ed.) Academic Press, pl75-186 Reeve, J.N., P.T Hamilton, G.S. Beckler, C.J. Morris and C H Clarke <1986). Structure of methanogen genes. Syst Appl. Microbial. 7-. 5-12 iv Reeve, J N., G.S. Beckler, J.W Brown, D.S. Cram, E.S. Haas, P.T. Hamilton, C.J. Morris, B.A, Sherf and CF. Weil <1986). Divergence of methanogens, conservation of the his 1 gene sequence in all three biological kingdoms and the status of Methanohacterium thermoautotrophicLim Proceedings of the Fifth Inter national Symposium on Growth on Cl Compounds. Haren, Netherlands, P255-260. FIELD OF STUDY Molecular Biology TABLE OF CONTENTS Page ACKNOWLEDGEMENT......................................................................................................................... ni UITA ......................................................................................................... iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREUIAT IONS x INTRODUCTION I LITERATURE REUIEW 4 Genome structure ..................................................................................................... 4 DNA-dependent RNA polymerases .............................................................. 6 Ar chaebac terial ribosomes ............................................................................... 7 7S RNA 11 Protein-encoding genes from ar'chaebacteria 11 Computer analysis of amino acid and DNA sequences ...................................................................................................................... 14 Argininosuccinate synthetase- genes and pseudogenes ....................................... 17 METHODS .................................................................................. 19 Grouith of cells ............................................................................................................ 19 Isolation of methanogen DNA ........................................................................ 19 Large scale isolation of plasmids from E, coli .................... 21 Large scale isolation of plasmids from £ subtilis 23 Small scale isolation of plasmids from E coli and £ subtilis .......................... 24 Preparation and transformation of competent cells of £ coli........................................................................................................25 Preparation and transformation of competent cells of £ subtilis 26 Restriction and ligation of plasmid DNA. .................................. 27 Agarose and polyacrylamide gel electrophoresis ................. 29 Minicell preparation and radioactive labeling of plasmid encoded polypeptides ............................................................. 29 Nick translation ............................................................................................................ 31 vi Radioactive labeling of oligomer probes 32 DNADNA hybridization with dehydrated agarose gels .............................................. 33 DNADNA hybridization with Zeta-Probe membrane. 34 Maxam and Gilbert sequencing 35 6 reaction ............... 37 G + A reaction 38 C * T reaction ....................................................................................................... 38 C reaction ...... 38 Strand cleavage reaction ........................................................................... 39 M13 sequencing. 40 RESULTS....................................................................................................................................................... 43 Construction of plasmids pET821, pET822, pET8£3, PET824 and pET825 43 Expression of methanogen derived DNA in JL suhtilis 46 Polypeptides synthesized in DS410 minicells encoded by pET821> pET823, pET824 and pET825 47 Nucleotide sequence of pET821 53 Nucleotide sequence of pET371 .................................................................. 68 Codon usage .......................... 67 Computer analysis of carbamyl phosphate synthetase genes .................................................................................................. 70 Computer analysis of argininosuccinate synthetase genes .................................................................................................. 76 Construction of an evolutionary
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