Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 166-178).

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Heartwater is a major threat to livestock in Africa due to its high mortality rate. The intracellular nature of the causative organism, Cowdria ruminantium, makes it difficult to study, hence an effective and user-friendly vaccine has been extremely difficult to obtain. Two C. ruminantium DNA libraries have recently been constructed, the lambda GEM11 bacteriophage DNA library and the lambda ZAPII bacteriophage DNA library, and this has lead to a renewed search for protective genes that could be used as a vaccine against heartwater. In this study, several molecular techniques including PCR, cloning and sequencing were used to identify genes in the lambda GEM11 bacteriophage DNA library that code for proteins, which could be used as vaccines to protect susceptible animals against heartwater. The lambda GEM11 library was screened with a rickettsial secretory protein gene sequence, known as seeD. One positive colony was selected from which the bacteriophage DNA was isolated. The C. ruminantium DNA was amplified from the bacteriophage DNA by using PCR and C. ruminantium-specific primers. The C. ruminantium DNA was screened with Mycoplasma, bovine and Cowdria DNA probes. The amplified DNA was subeloned into two vectors and the clones were screened by restriction analysis to identify clones containing inserts. The appropriate clones were sequenced and overlapping sequences matched, ordered and aligned. Two sequences were continuous with a short sequence of unidentified bases in between. Oligonucleotide primers were designed to amplify the DNA sequence between the two contiguous sequences. This led to the identification of the entire sequence of the C. ruminantium genome contained within the bacteriophage plaque. The single contiguous sequence was analysed and the putative protein-coding sequences were obtained and compared to DNA sequences of known organisms using the BLAST program. Five open reading frames were identified with homology to genes encoding specific proteins in bacteria. Two open reading frames showed homology to the genes encoding the transporter proteins, FtsY and the ABC transporter, and three open reading frames were found to be homologous to genes encoding the essential enzymes dethiobiotin synthetase, pro lipoprotein diacylglycerol transferase and the putative NADH-ubiquinone oxidoreductase subunit. The five open reading frames encode for genes, which are essential for the normal functioning of the C. ruminantium organism. However, these open reading frames might not be effective for use in a DNA vaccine since none of the open reading frames showed homology to obvious genes that could play a role in immunity and therefore confer protection. The open reading frames can be used in mutagenesis studies to produce attenuated strains of the organism that possess mutated versions of these proteins. These attentuated strains could be used for the vaccination of cattle, and thereby confer protection against viable pathogenic C. ruminantium isolates. / AFRIKAANSE OPSOMMING: Hartwater is 'n bedreiging vir vee in Afrika weens die hoë mortaliteitssyfer verbonde aan die siekte. Die intrasellulêre aard van die organisme wat hartwater veroorsaak, Cowdria ruminantium, bemoeilik navorsing aangaande die organisme. Dit het tot gevolg dat 'n effektiewe en gebruikersvriendelike entstof moeilik bekombaar is. Daar is onlangs sukses behaal met die konstruksie van twee C. ruminantium DNA genoteke, die lambda GEM11 bakteriofaag genoteek en die lambda ZAPII bakteriofaag genoteek. Dit het gelei tot 'n herlewing in die soektog na beskermende gene, wat in 'n entstof teen hartwater gebruik kan word. In hierdie studie is verskeie molekulêre tegnieke insluitende PKR, klonering en geenopeenvolging bepaling, gebruik om gene te identifiseer in die lambda GEM11 bakteriofaag genoteek wat kodeer vir proteïene wat in entstowwe gebruik kan word as beskerming teen hartwater. Die secD geen is gebruik om die lambda GEM11 bakteriofaag genoteek te sif. Een positiewe plaak is gevind waarna die DNA uit die bakteriofaag plaak geïsoleer en die C. ruminantium DNA vanuit die bakteriofaag plaak geamplifiseer is deur gebruik te maak van PKR en spesifieke C. ruminantium inleiers. Die C. ruminantium DNA is gesif met Mycoplasma, bees en Cowdria radioaktief gemerkte DNA peilers. Die C. ruminantium DNA is vervolgens in twee vektore gekloneer. Die klone is gesif deur middel van restriksie analise. Die DNA volgorde van die klone is bepaal en twee ononderbroke sekwense is geïdentifiseer met 'n gaping in die middel tussen die twee sekwense. Oligonukleotied inleiers is daarna ontwerp om die geenopeenvolging van die gaping tussen die twee sekwense te vul. Hierdeur kon die volledige geenopeenvolging van die genoom van C. ruminantium wat in die lambda GEM 11 bakteriofaag plaak voorkom, bepaal word. Hierdie volledige geenopeenvolging is vervolgens geanaliseer en die oop leesrame wat daarin voorkom geïdentifiseer. Vyf leesrame is gevind om homologie met gene wat kodeer vir proteïene wat in bakterieë voorkom, te toon. Twee leesrame het homologie met die gene wat kodeer vir transport proteïene, FtsYen die ABC transporter getoon, en drie leesrame het homologie met gene wat kodeer vir die essensiële ensieme detiobiotin sintetase, prolipoproteïen diasielgliserol transferase en die NADHubikinoon oksidoreduktase subeenheid getoon. Dié vyf leesrame het die potensiaal om as entstowwe gebruik te word aangesien al vyf leesrame kodeer vir gene wat 'n belangrike rol speel in die oorlewing van die C. ruminantium organisme. Alhoewel die leesrame moontlik nie so effektief sal wees in 'n DNA entstof nie, toon dit potensiaal om in mutasieeksperimente gebruik te word. Organismes wat die gemuteerde weergawe van die geen besit sal nie-funksionele proteïene produseer, wat 'n invloed kan hê op die normale fisiologiese funksies van die organisme en dus sal lei tot 'n minder virulente organisme. Die geattenueerde organisme kan moontlik gebruik word om diere te immuniseer en daardeur immuniteit aan diere lewer wat beskerming sal bied teen patogeniese C. ruminantium isolate.

Thesis (Ph. D.)--University of California, San Diego, 2002. / Vita. Includes bibliographical references (leaves 146-152).

Thesis (M.S.)--Bowling Green State University, 2008. / Document formatted into pages; contains vi, 48 p. : ill. Includes bibliographical references.

Thesis (Ph.D. in Biochemistry) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 118-126). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

No description available.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Bacteriophage ë has been used as a model replicon system for forty years. While the basic ë replication initiation scheme has been elucidated for several decades, many aspects of the mechanisms are unclear. I wished to study two unanswered issues in ë replication initiation. </p><p>Replication initiation of E. coli and ë each depend upon a protein generally called a licensing factor, which brings the DnaB helicase protein to the origin site to begin DNA synthesis. The licensing factors are the products of host gene dnaC and ë gene P. The synthesis of P from ë DNA in an E. coli cell can competitively interfere with DnaC activity needed for E. coli replication initiation. I wished to learn more about what happens to a host cell when exposed to extended P expression. Previous studies in this laboratory suggested that i) the continuous expression of P was tolerated by a subset of exposed cells and that ii) host defects mapping to dnaB could suppress the effect of extended P expression (P-lethality). I used DNA sequencing to determine if these suppressor mutations were within dnaB. I screened known host mutations for their influence on P-lethality. In summary: E. coli strains with GrpD55 and GrpA80 defects were found to each have two point mutations within their dnaB genes. I was unable to isolate mutations within P that suppressed P-lethality and instead obtained regulatory mutations preventing wild type P expression. Two of these sequenced mutations showed that a cI[Ts] lambda repressor was reverted to cI wild type, blocking P expression at all assay temperatures. P-lethality was reversible in cells exposed to P for up to five hours, causing me to suggest that P-Interference be used in place of the term P-lethality. A non-inducible allele of lexA prevented P-mediated cellular filamentation and enhanced P-Interference. This suggests that induction of the SOS response helps cells to tolerate extended P expression. A host strain containing a defective ClpXP protease significantly enhanced cellular sensitivity to P-Interference. This suggests an important role for the ClpXP chaperone-protease complex in degradation of P and cellular resistance to P expression. I present models to explain the P-Interference Phenotype.</p><p>Recent reports have re-opened the possibility that the tO-oop-pO element influences ë DNA replication initiation. I have also been investigating this possibility. I found that a plasmid with tO-oop-pO (the terminator, nucleotide sequence and promoter for OOP RNA) and orië DNA sequence was inhibitory to the development of repë phages, and designated this the Inhibition Phenotype (IP). In pursuing the mechanism for this inhibition, I mutated the tO-oop-pO and orië elements. I found that the expression of the 77nt OOP RNA transcript and the presence of four 18 base pair repeats (iterons) within orië were required for the IP. I isolated spontaneous phage mutants, resistant to the IP. I determined that singly infected cells were sensitive to the IP but that multiply infected cells escaped the IP. I propose that the IP to repë phage development is directed to the initial or theta mode of ë replication initiation. I found that the theta-mode of ë replication initiation can be bypassed, likely via recombination between multiple phage genomes within a singe cell. I propose models to explain the IP and also suggest a role for OOP RNA in the regulation of ë DNA replication.

The bacteriophage lambda tail protein gpK is required for tail assembly. The activity of the protein can be found at the assembling tail tip and is believed to be localized to this structure. GpK is a 27 kDa protein that has sequence identity to two families of proteins: the Mov34 family of peptidases and the NlpC/P60 family of peptidoglycan endopeptidases. Point substitutions and complementation data confirm that gpK possesses each of these domains and that they can function in trans. When the Mov34 domain is inactivated tail assembly is disrupted whereas when the NlpC/P60 domain is inactivated tails assemble but are inactive. Evidence is presented here that the C-terminal domain possesses lytic activity in isolation but not when part of the full-length protein.