Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (M. Sc.)--Carleton University, 1992. / Also available in electronic format on the Internet.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 191-206).

The restriction endonuclease Hpa I cleaves wild type T7 DNA into nineteen fragments. These Hpa I fragments were inserted into the ampicillin resistance gene of the plasmid pBR322. The poly A - poly T method of construction was chosen in order to insure single T7 DNA inserts. Two hundred fifty clones were identified as carrying T7 DNA segments by colony hybridization with ³²P T7⁺ DNA. From these, clones carrying specific areas of the T7 genome were identified by various methods described in this thesis. Clones carrying Hpa I fragments E and G were identified by hybridization with Southern filters of electrophoressed Hpa I digested T7 DNA. Clones carrying most of gene 5 and parts of gene 4 were identified by hybridization with a purified restriction fragment carrying those genes (Hpa I fragment D). Clones containing parts of genes 10 and 11 were identified by marker rescue tests. Marker rescue "spot tests" were developed as a fast screening procedure for T7 genes and rely on generalized recombination between the T7 segment carried by the plasmid and the infecting phage DNA. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The complete detection and genome sequencing of resistance plasmids is required to understand the co-occurrence of resistance genes on plasmids and their transmission, which is necessary to mitigate the spread of drug resistant bacterial infections. One group of multi-drug resistance plasmids of interest are the New Delhi Metallo--lactamase (NDM) associated plasmids. Bacteria pathogens with NDM genes are resistant to a broad range of beta-lactam antibiotics, including carbapenems, a mainstay for treating bacterial infections. We sequenced clinical isolates collected from patients that failed to respond to antibiotic treatments in the Hamilton-Niagara community using Next Generation Sequencing (NGS) technology, which is the standard technique for sequencing bacterial genomes. I analyzed their assembled genomes via the development of a new resistance plasmid prediction bioinformatics pipeline: RGI:Mobilome. RGI:Mobilome predicted NDM bearing plasmids in 15 isolates, along with other resistance genes on plasmids. However, the NDM plasmid predictions of all 15 isolates were fragmented due to incomplete genome assemblies, caused by repetitive sequences within bacterial genomes and use of Illumina sequencing technology. However, plasmid predictions greatly improved when we leveraged the high nucleotide accuracy of Illumina reads and the structural resolving power of long reads generated with the Oxford Nanopore Technology (ONT) to produce ‘hybrid’ genome assemblies. From the 15 putative hybrid-assembly NDM plasmids, one plasmid was a complete match to a known pKP-NDM1 plasmid, seven plasmids were “variants” of known and well-characterized plasmids, and the remaining seven plasmids were “distant homologs” of known and well-characterized plasmids, suggesting previously undescribed NDM-associated plasmids in our community. This thesis project reveals the diversity of NDM plasmid types within the Hamilton-Niagara community, which is valuable to epidemiologists and public health practitioners to devise actionable plans required to mitigate the spread of multi-drug resistance NDM plasmids and for clinicians to treat infections caused by NDM positive strains. / Thesis / Master of Science (MSc) / The spread of antimicrobial resistance often arises from the exchange of resistance plasmids between bacterial species in their immediate environment. One resistance gene carried on plasmids is the New Delhi Metallo-beta-lactamase (NDM) gene, which confers resistance to almost all beta-lactam antibiotics. To control the spread of NDM plasmids, we need to detect them completely. Using genome sequencing, we analyzed clinical isolates from patients in hospitals within the Hamilton-Niagara community for NDM plasmids. From our results, we detected one complete match to a known NDM plasmid. We also detected seven NDM-associated plasmids that are close variants of known NDM plasmids, plus another seven NDM-associated plasmid that we classified as ‘novel’ since they are widely different from known NDM plasmids. This thesis reveals the diverse NDM plasmids within the clinics of the Hamilton-Niagara community, which should guide public health workers to develop policies to mitigate the spread of resistance NDM plasmids.

Physical and restriction mapping of the salicylate catabolic plasmid SAL from Pseudomonas putida strain PpG 2119 was carried out by standard multiple restriction analysis and by cross hybridization studies using radioactively labeled restriction fragment probes. The total numbers of fragments produced, their respective sizes, the arrangement of the restriction fragments on the plasmid and the map locations of the enzyme recognition sites for Hpal, Xhol, Dral and Smal are given.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: Turlough M. Finan.

Thesis (Ph. D.)--University of Idaho, 2006. / Abstract. "July 2006." Includes bibliographical references. Also available online in PDF format.

pC221 belongs to a family of staphylococcal plasmids, including pT181, pS194, pC223, pUB112 and pCW7 . All possess open reading frames with 70-80% homology to the pC221 rapD gene. REP D has sequence specific topoisomerase activity at the pC221 origin (or iD) which is thought to be involved in replication initiation. DNase I footprinting has been carried out, showing that REP D binds to a region of oriD downstream of the nick site. The pattern of DNase I cleavage suggests that REP D contacts one face of the DNA helix, which may be bent around the protein. Extracts of S. aureus support incorporation of radioactive dNTPs into pC221 in the presence of REP D. Labelling with a[32P] dATP shows that replication initiates within the region containing oriD and proceeds in the direction expected for elongation of a 3' OH generated by nicking at oriD. With supercoiled DNA, REP D initiates replication of other members of this plasmid family in Vitro. However, with relaxed DNA, REP D is specific for oriD, suggesting that a change in the DNA, stabilised by supercoiling of the DNA or by binding of REP D, may be required for nicking. Of three inverted repeat sequences (ICRI, II & III) at the origin, ICRII has the greatest predicted hairpin stability and is almost totally conserved. Nicking takes place within the loop of this proposed hairpin. Disruption of base pairing within this hairpin has been investigated by mutagenesis of cloned oriD and using oligonucleotides based on the ICRII sequence. These experiments show that the 3' side of ICRII is more important for nicking than the 5' side. This is in agreement with footprinting data which shows that REP D binds the 3' side of ICRII, along with the whole of ICRIII. However, there is no evidence for hairpin formation at ICRII being required for nicking.

Thesis (MScMedSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The plasmid pAL5000 is a mycobacterial plasmid isolated from Mycobacterium fortuitum. It is a low copy number plasmid, which replicates in both fast growing (e.g. M. smegmatis) and slow growing (e.g. M. bovis BCG) mycobacteria. Most mycobacterial-E. coli shuttle vectors utilise the pAL5000 origin of replication. The minimum replicon consists of ORF1 (RepA), ORF2 (RepB) and the origin of replication. Dr W.R. Bourn created an E. coli-mycobacterial vector based on the pAL5000 origin of replication (pORI) and then subjected it to semi-random mutagenesis. A high copy number mutant was identified (pHIGH) and the causative mutation was tentatively identified as a 3bp deletion situated just upstream of repB. This work describes the further characterisation of the mutant plasmid. Firstly, it was shown by retransforming M. smegmatis with both the original and mutant plasmids (pORI and pHIGH), that the mutation causing the increased copy number was plasmid-encoded and not on the chromosome. Following this, it was demonstrated by simple subcloning of the region that carries the 3 bp deletion, that other pAL5000-based vectors could be converted to high copy number. In addition to this, the subcloned region was sequenced and the nature of the mutations was confirmed. The subcloning experiment confirmed that the 3bp deletion caused the high copy number phenotype. Following this, the exact copy number of pHIGH and the relative increase in copy number was determined. From this, the copy number of pORI could also be determined. The plasmid pHIGH has a copy number of approximately 54, compared to the 8 of pORI (a relative increase by a factor of 7). Because it is important for researchers to know the characteristics of the vectors that they use, especially the influence it will have on its host, stability tests and growth curves were also performed. It was seen that the higher copy number did not markedly increase the stability, however, this is because pORI is already extremely, and unexpectedly, stable in the host M. smegmatis. According to the growth curves, the increased copy number has little effect on the growth of the host M. smegmatis. Possible mechanisms for the increased copy number were then investigated. By using a promoter probe vector, the possible existence of a promoter situated between the two open reading frames of pAL5000 (repA and repB) was investigated. It was thought that the mutation might have created, or changed an existing promoter, situated between repA and repB. The results showed, however, that in both pORI and pHIGH there might be a very weak promoter upstream of repB, but the mutation did not cause any change that was measurable by the method that was used. A further possibility was that the mutation caused a change in the RNA secondary structure, which might then have an effect on the translational efficiency of RepB. It was found that the 3bp deletion in pHIGH causes a change in the local RNA secondary structure around the ribosomal binding site and the start codon, when compared to pORI (wild type). This change may cause the translation initiation rate of RepB to be different between pHIGH and pORI. Ultimately it would lead to a different ratio of RepA and RepB in the cell. / AFRIKAANSE OPSOMMING: Die plasmied pAL5000 is ‘n mikobakteriele plasmied wat vanuit Mycobacterium fortuitum gei'soleer is. Dit is ‘n lae kopie-getal plasmied wat in beide vinnig groeiende (bv. M. smegmatis) en stadig groeiende (bv. M. bovis BCG) mikobakteriee kan repliseer. Die meeste mikobakteriele-E. coli shuttle vektore gebruik die pAL5000 oorsprong van replisering. Die minimum replikon bestaan uit ORF1 (RepA), ORF2 (RepB) en die oorsprong van replisering. Dr. W.R. Bourn het ‘n E. coli-mikobakteriele vektor gemaak wat gebaseer is op die pAL5000 oorsprong van replisering (pORI), en dit onderwerp aan semi-random mutagenese. ‘n Hoë kopie-getal mutant is gei'dentifiseer (pHIGH) en die mutasie hiervoor verantwoordelik was tentatief gei'dentifiseer as ‘n 3bp delesie, net stroomop van repB. Die projek beskryf die verdere karakterisering van die mutante plasmied. Eerstens, deur M. smegmatis te hertransformeer met die plasmied DNA (pORI en pHIGH), is dit bewys dat dit mutasie wat die toename in kopie-getal veroorsaak, deur die plasmied gekodeer word, en dat dit nie ‘n mutasie op die chromosoom is nie. Hierna is dit deur eenvoudige subklonering bewys dat die gedeelte wat die 3bp delesie dra, ander pAL5000-gebaseerde vektore ook kan verander in ‘n hoër kopie-getal. Die sub-klonerings eksperiment het ook bewys dat die 3 bp delesie die oorsaak is vir die hoë kopie-getal fenotipe. Volgende is die presiese kopie-getal van pHIGH en die relatiewe toename in kopiegetal bepaal. Die kopie-getal van pORI kon vanaf hierdie data bepaal word. Die plasmied pHIGH het ‘n kopie-getal van ongeveer 54 in M. smegmatis, in vergelyking met die 8 van pORI (‘n relatiewe toename met ‘n faktor van 7). Aangesien dit vir navorsers belangrik is om die eienskappe van die vektore wat hulle gebruik, te ken, en veral die invloed wat dit op die gasheer sal hê, is stabiliteits toetse, en groeikurwes gedoen. Die hoër kopie-getal het nie die stabiliteit werklik verbeter nie, maar dit is omdat pORI alreeds uiters stabiel is in die gasheer M. smegmatis. Volgens die groeikurwes het die toename in kopie-getal ‘n minimale effek op die groei van die gasheer M. smegmatis. Moontlike meganismes vir die hoër kopie-getal is ook ondersoek. Die moontlike bestaan van ‘n promoter tussen die twee oop-leesrame van pAL5000 (repA en repB) is ondersoek deur gebruik te maak van ‘n “promoter probe” vektor. Die mutasie kon moontlik ‘n promoter geskep het, of ‘n bestaande een tussen repA en repB verander het. Die resultate het gewys dat daar in beide pORI en pHIGH moontlik ‘n baie swak promoter stroomop van repB is, maar die mutasie het nie enige veranderinge veroorsaak wat meetbaar was met die metode wat gebruik is nie. ‘n Verdere moontlikheid was dat die mutasie ‘n verandering in die RNA sekondere struktuur kon veroorsaak het, en dit mag ‘n effek hê op die translasie effektiwiteit van RepB. Daar is gevind dat, in vergelyking met pORI, het die 3bp delesie in pHIGH ‘n verandering in die lokale RNA sekondere struktuur rondom die ribosomale bindings posisie en die begin-kodon veroorsaak. Die verandering mag veroorsaak dat die translasie inisiasie tempo van RepB verskillend is vir pORI en pHIGH. Uiteindelik sal dit lei tot ‘n heeltemal ander verhouding van RepA en RepB in die sel.