Thesis (Ph. D.)--Illinois State University, 1986. / Title from title page screen, viewed July 13, 2005. Dissertation Committee: David P. Brunner (chair), Herman E. Brockman, Arlan G. Richardson, H. Tak Cheung, Lynne Lucher. Includes bibliographical references (leaves 167-183) and abstract. Also available in print.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 113-135). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Wisconsin--Madison, 2003. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (p. 103-116).

Strains of the budding-yeast Kluyveromyces lactis that produce killer-toxin contain two linear dsDNA plasmids, kl (8.9 Kb) and (13.4 Kb). The genes of kl are preceded by no known gene expression signals and transcripts are probably not polyadenylated. Classical and molecular genetic techniques have been used to attempt to investigate the mechanisms of replication, partitioning and gene expression utilised by these novel elements. Such studies have led to the preparation of a number of vectors and strains of use in this and other analyses. Data or presented which indicate the killer plasmids exist within the cytoplasm of the yeast cell, and probably do not require the nuclear RNA polymerases I, II or III for transcription of their genes. A preparation procedure has been developed for the linear plasmids, and plasmid k2, thought to encode factors necessary for the maintenance or expression of k1, has been cloned. Sequence analysis of 7,7 kb of plasmid k2 revealed the presence of several open reading frames (ORFs) organised in a similar manner to those of plasmid k1. One of these is predicted to encode a product with homology to two different subunits found within several DNA-directed RNA polymerases. Another ORF potentially encodes a polypeptide with homology to a DNA helicase encoded by, and required for accurate transcription of, the cytoplasmic poxvirus vaccinia. These two k2 products may be components of a killer plasmid-specific cytoplasmic gene expression system. Possible mechanisms of killer plasmid replication, partitioning and gene expression are discussed.

A 5.1 kbEcoR I fragment from Pseudomonas putida TOL plsmid pDKR1, carrying the xylE and xylL genes, was inserted into pBR 325 and transformed into E. coli. The xylE region, coding for catechol 2,3-dioxygenase, was subjected to Maxam-Gilbert sequencing reactions.

The Helicoverpa armigera stunt virus (HaSV) is a member of a family of small nonenveloped (+) ssRNA insect viruses currently known as the Tetraviridae. This family is unique in terms of the T=4 quasi-symmetry of its capsid particles and the unusually narrow host range and tissue tropism. Assembly of tetraviral particles has been well characterised and involves the combination of 240 copies of a single capsid precursor protein (VCap) into a procapsid followed by autoproteolytic cleavage to yield the major (β) and minor (γ) capsid subunits within the mature particle. HaSV has two genomic RNAs, RNA 1 encoding the replicase and RNA 2 encoding VCap and p17, the ORF of which lies upstream of and overlaping with the 5’ end of the VCap ORF. Prior to this study, Vlok (2009) used a plasmid expression system to study RNA packaging in HaSV VLPs assembled in Spodoptera frugiperda 9 (Sf9) cells co-expressing p17 and VCap. The study showed that the p17 ORF was required for the packaging of RNA 2 during capsid assembly but it was unclear whether p17 expression was required for packaging. In addition, expression from the transfected plasmids was sub-optimal affecting both the yield of VLPs and the detection of p17. The aim of this study was to use the plasmid system to test whether p17 expression was required for plasmid-derived VLP RNA packaging and then develop a baculovirus-mediated system to test this hypothesis. By using a plasmid in which the start codon of p17 was mutated, it was shown that p17 expression was required for RNA 2 packaging into plasmid-VLPs. For the baculovirus system, four recombinant baculoviruses based upon the pFastBac Dual expression system, were constructed. These included Bac20, expressing wild type RNA 2, Bac21, RNA 2 with p17 silenced, Bac23, RNA 2 and p17 expressed on a separate transcript and Bac24, RNA 2 with p17 silenced plus p17 expressed on a separate transcript. Assembly of VLPs was more efficient using the baculovirus expression system and p17 expression was observed in cells infected with Bac20, Bac23 and Bac24, but not Bac21. In contrast to the plasmid-VLPs, bac-VLPs did not require p17 for the encapsidation of RNA 2. In addition to RNA 2, Bac23 and Bac24 packaged the p17 mRNA transcribed separately from RNA 2. This insinuated that bac-VLPs may be packaging RNA non-selectively. It was proposed that p17 may play a role in packaging in an RNA-limiting environment (plasmid system) but functioned differently when viral RNA was in excess (baculovirus system). This data points to the importance of developing a replication system for the analysis of the packaging pathways of these viruses and this study has laid down the foundations for such a system in which RNA 1 and RNA 2 can be introduced into a single cell by means of a single recombinant virus.

No description available.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

No description available.

Geok-yen Yeo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 225-233). / Title page --- p.i / Members of Thesis Advisory Committee --- p.ii / Abstract --- p.iii -iv / Acknowledgments --- p.v / Dedication --- p.vi / Table of Contents --- p.vii -xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-31 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Fermentation --- p.1 / Chapter 1.3 --- Growth in Escherichia coli --- p.3 / Chapter 1.3.1 --- Aerobic growth in Escherichia coli --- p.3 / Chapter 1.3.2 --- The regulation of enzyme synthesis during cell metabolism --- p.7 / Chapter 1.3.3 --- Anaerobic growth in E. coli --- p.8 / Chapter 1.3.4 --- Anaerobic regulation by the transcriptional regulator Fnr --- p.12 / Chapter 1.3.5 --- "The case for ""Pasteur Control Proteins"" (PCP)" --- p.13 / Chapter 1.4 --- The family of alcohol dehydrogenases : An overview --- p.15 / Chapter 1.4.1 --- Molecular characteristics of alcohol dehydrogenases --- p.17 / Chapter 1.4.2 --- Residue conservation in alcohol dehydrogenases --- p.24 / Chapter 1.4.3 --- The effect of amino acid substitution on substrate specificity --- p.25 / Chapter 1.5 --- Alcohol dehydrogenases in bacteria --- p.28 / Chapter 1.5.1 --- Alcohol dehydrogenase in E. coli --- p.28 / Chapter 1.6 --- Aims of this study --- p.30 / Chapter CHAPTER 2 --- MATERIALS & METHODS --- p.32 -90 / Chapter 2.1 --- Bacterial strains --- p.32 / Chapter 2.2 --- Plasmids --- p.32 / Chapter 2.2.1 --- "Low copy number plasmid, pTJS75Km" --- p.32 / Chapter 2.2.2 --- "High copy number plasmid, pUC18" --- p.33 / Chapter 2.3 --- Bacterial culture media and solutions --- p.39 / Chapter 2.3.1 --- Luria Bertani (LB) medium --- p.39 / Chapter 2.3.2 --- L-Broth + MOPS --- p.39 / Chapter 2.3.3 --- "R medium, containing Triphenyltetrazolium chloride-ethanol (TTC-EtOH)" --- p.40 / Chapter 2.3.4 --- SOB and SOC media --- p.41 / Chapter 2.3.5 --- M9 Glucose medium --- p.42 / Chapter 2.3.6 --- Terrific Broth (TB) --- p.42 / Chapter 2.3.7 --- Rich Broth (RB) --- p.43 / Chapter 2.3.8 --- Antibiotic solutions --- p.43 / Chapter 2.4 --- Restriction endonucleases and other enzymes --- p.44 / Chapter 2.5 --- Isolation of chromosomal DNA --- p.45 / Chapter 2.5.1 --- Preparation of chromosomal DNA by spooling --- p.45 / Chapter 2.5.2 --- Preparation of chromosomal DNA by cesium chloride density gradient --- p.48 / Chapter 2.6 --- Isolation of plasmid DNA --- p.50 / Chapter 2.6.1 --- Large-scale preparation of plasmid by CsCl density gradient --- p.50 / Chapter 2.6.2 --- Small-scale preparation of plasmid DNA --- p.54 / Chapter 2.6.2. --- A Boiling method --- p.54 / Chapter 2.6.2. --- B Alkaline Lysis method --- p.55 / Chapter 2.6.3 --- Preparation of plasmid DNA by Qiagen columns --- p.56 / Chapter 2.7 --- Purification of DNA --- p.59 / Chapter 2.7.1 --- Ethanol precipitation --- p.59 / Chapter 2.7.2 --- Concentration and desalting using Centricon columns --- p.59 / Chapter 2.7.3 --- Purification of DNA by Geneclean procedure --- p.61 / Chapter 2.8 --- DNA cloning techniques --- p.63 / Chapter 2.8.1 --- Restriction endonuclease digestion --- p.63 / Chapter 2.8.2 --- Agarose-ethidium bromide gel electrophoresis --- p.65 / Chapter 2.8.2. --- A Gel loading buffer --- p.66 / Chapter 2.8.2. --- B Electro-elution of DNA --- p.67 / Chapter 2.8.3 --- Size fractionation --- p.68 / Chapter 2.8.3. --- A Salt gradient fractionation --- p.68 / Chapter 2.8.3. --- B Sucrose gradient --- p.70 / Chapter 2.8.4 --- Dephosphorylation of restriction-enzyme digested vector plasmid using calf intestinal phosphatase (CIP) --- p.71 / Chapter 2.8.5 --- Ligation of vector and insert --- p.72 / Chapter 2.8.6 --- Preparation of competent cells --- p.73 / Chapter 2.8.7 --- DNA transformation --- p.75 / Chapter 2.8.7.A --- By heat shock --- p.75 / Chapter 2.8.7.B --- By electroporation --- p.75 / Chapter 2.9 --- Screening for adhC transformants --- p.78 / Chapter 2.9.1 --- Screening for adhC clones --- p.78 / Chapter 2.9.2 --- Screening for pUC18 transformants --- p.79 / Chapter 2.10 --- Confirmation of adhC clones --- p.80 / Chapter 2.10.1 --- Reproduction of red colonies on R plates and antibiotic resistance --- p.80 / Chapter 2.10.2 --- T7 phage test for E. coli strains --- p.80 / Chapter 2.10.3 --- Plasmid size determination --- p.82 / Chapter 2.10.4 --- Re-transformation into E. coli host strains --- p.82 / Chapter 2.10.5 --- Physiological study of adhC clones --- p.83 / Chapter 2.10.6 --- Alcohol dehydrogenase assay --- p.84 / Chapter 2.11 --- The dye-binding method of protein determination --- p.87 / Chapter 2.12 --- Special procedures --- p.88 / Chapter 2.12.1 --- Generation of adh clones with deletions --- p.88 / Chapter 2.12.2 --- Sequencing reactions --- p.89 / Chapter CHAPTER 3 --- RESULTS: PART I Cloning and Restriction Mapping of the adhC mutation in a low copy number plasmid vector --- p.91 -122 / Chapter 3.1 --- Introduction: Cloning strategy --- p.91 / Chapter 3.2 --- Cloning of the adh mutation from strain CC2807B (an ADH overproducing mutant strain) in pTJS75Km --- p.93 / Chapter 3.2.1 --- Construction of the 'HK' clones --- p.93 / Chapter 3.3 --- Restriction mapping of the adh clones --- p.101 / Chapter 3.4 --- Subcloning the adhC insert --- p.110 / Chapter 3.4.1 --- Construction of plasmid pHK14 --- p.110 / Chapter 3.4.2 --- Construction of plasmid pHK15 --- p.115 / Chapter 3.4.3 --- Construction of plasmid pSS22 --- p.121 / Chapter 3.5 --- Remarks concerning the clones --- p.121 / Chapter CHAPTER 4 --- RESULTS:PART II Cloning and Sequencing of the adhC mutation in a high copy number plasmid vector --- p.123 -148 / Chapter 4.1 --- Introduction --- p.123 / Chapter 4.1.1 --- Choice of sequencing strategy --- p.123 / Chapter 4.1.2 --- An attempt to eliminate clone instability --- p.124 / Chapter 4.2 --- Subcloning of adh insert in pUC18 --- p.125 / Chapter 4.2.1 --- Study of adh clone EPR --- p.125 / Chapter 4.2.2 --- Re-construction of plasmid pEPR ( = pEE5) --- p.126 / Chapter 4.2.3 --- Construction of plasmids pEH2 and pEH3 --- p.127 / Chapter 4.2.4 --- Construction of a nested deletion library --- p.138 / Chapter CHAPTER 5 --- RESULTS : PART III Sequencing of the Mutation --- p.149 -177 / Chapter 5.1 --- Nucleotide sequencing --- p.149 / Chapter 5.2 --- Sequencing of the cloned adhC gene insert --- p.150 / Chapter 5.3 --- Analysis of the sequenced DNA by DNASIS computer software --- p.151 / Chapter 5.3.1 --- Search for codons associated with initiation and termination of transcription using the open reading frame (ORF) search --- p.151 / Chapter 5.3.2 --- Translation of the nucleotide sequence at the open reading frame (start 223 - end 2896) --- p.152 / Chapter 5.4 --- Search for DNA sequence homology with known DNA sequences --- p.152 / Chapter 5.4.1 --- Sequence homology of the structural gene (nucleotide # 223- #28%) : Two nucleotide changes revealed in DNA sequence of the structural gene adhE of Escherichia coli --- p.153 / Chapter 5.4.2 --- adhC mutation is due to changes in two amino acids --- p.153 / Chapter 5.4.3 --- The DNA sequence 5' of the mutated structural gene (upstream sequence) --- p.155 / Chapter 5.4.4 --- The DNA sequence 3' of the mutated structural gene (downstream sequence) --- p.156 / Chapter 5.5 --- Comparisons between the computer-predicted properties of the mutant and wild-type protein --- p.156 / Chapter 5.5.1 --- Prediction of the alcohol dehydrogenase protein secondary structure by the Robson Method --- p.156 / Chapter 5.5.2 --- Isoelectric point prediction --- p.156 / Chapter CHAPTER 6 --- RESULTS : PART IV Comparative Studies of Alcohol Dehydrogenase Expressionin adhC Strains and Clones --- p.178 -203 / Chapter 6.1 --- Introduction --- p.178 / Chapter 6.1.1 --- Basis for the alcohol dehydrogenase assay --- p.178 / Chapter 6.1.2 --- Choice of assay method --- p.179 / Chapter 6.1.3 --- Points to consider for ADH assay --- p.179 / Chapter 6.2 --- General growth characteristics of bacterial strains --- p.181 / Chapter 6.2.1 --- Plate cultures --- p.181 / Chapter 6.2.2 --- Overnight liquid cultures --- p.183 / Chapter 6.2.3 --- Batch liquid cultures --- p.183 / Chapter 6.2.4 --- ADH activity of strain CC2807B --- p.190 / Chapter 6.2.5 --- Comparison of ADH activity --- p.192 / Chapter 6.3 --- Investigating the mutated ADH enzyme --- p.197 / Chapter 6.3.1 --- Oxygen inactivation of the mutated enzyme --- p.197 / Chapter 6.3.2 --- Thermostability of the mutated enzyme --- p.201 / Chapter CHAPTER 7 --- DISCUSSION --- p.204 -220 / Chapter 7.1 --- Cloning of the adhC mutation --- p.204 / Chapter 7.1.1 --- Instability of clones in plasmid vector pUC18 --- p.204 / Chapter 7.1.2 --- Eliminating 'toxic' genes adjacent to adh locus --- p.207 / Chapter 7.1.3 --- Cloning in pTJS75Km low copy number vector --- p.208 / Chapter 7.2 --- DNA sequence of the adhC clones --- p.211 / Chapter 7.2.1 --- The basis for sequencing pUC 18-derived clones --- p.211 / Chapter 7.2.2 --- Homology to known alcohol dehydrogenases (ADH) sequences --- p.213 / Chapter 7.3 --- Findings concerning the adhC mutation --- p.217 / Chapter 7.3.1 --- How amino acid substitutions may affect an enzyme --- p.217 / Chapter 7.3.2 --- Physiological aspects of the bacterial cell due to the mutated enzyme --- p.218 / Chapter 7.4 --- Conclusions --- p.220 / APPENDICES --- p.221 -224 / REFERENCES --- p.225 -233