published_or_final_version / abstract / Botany / Doctoral / Doctor of Philosophy

<p>The classical approach to gene discovery has been to culture micro-organisms demonstrating a specific enzyme activity and then to recover the gene of interest through shotgun cloning. The realization that these standard microbiological methods provide limited access to the true microbial biodiversity and therefore the available microbial genetic diversity (collectively termed the Metagenome) has resulted in the development of environmental nucleic acid extraction technologies designed to access this wealth of genetic information, thereby avoiding the limitations of culture dependent genetic exploitation. In this work several gene discovery technologies was employed in an attempt to recover novel bacterial laccase genes (EC 1.10.3.2), a group of enzymes in which considerable biotechnological interest has been expressed. Metagenomic DNA extracted from two organic rich environmental samples was used as the source material for the construction of two genomic DNA libraries. The small insert plasmid based library derived from compost DNA consisted of approximately 106 clones at an average insert size of 2.7Kbp, equivalent to 2.6 Gbp of cloned environmental DNA. A Fosmid based large insert library derived from grape waste DNA consisted of approximately 44000 cfu at an average insert size of 25Kbp (1.1 Gbp cloned DNA). Both libraries were screened for laccase activity but failed to produce novel laccase genes. As an alternative approach, a multicopper oxidase specific PCR detection assay was developed using a laccase positive Streptomyces strain as a model organism. The newly designed primers were used to detect the presence of bacterial multicopper oxidases in environmental samples. This resulted in the identification of nine novel gene fragments showing identity ranging from 37 to 94% to published putative bacterial multicopper oxidase gene sequences. Three clones pMCO6, pMCO8 and pMCO9 were significantly smaller than those typically reported for bacterial laccases and were assigned to a recently described clade of Streptomyces bacterial multicopper oxidases.</p> <p><br /> Two PCR based techniques were employed to attempt the recovery of flanking regions for two of these genes (pMCO7 and pMCO8). The use of TAIL-PCR resulted in the recovery of 90% of the pMCO7 ORF. As an alternative approach the Vectorette&trade / system was employed to recover the 3&rsquo / downstream region of pMCO8. The complexity of the DNA sample proved to be a considerable technical challenge for the implementation of both these techniques. The feasibility of both these approaches were however demonstrated in principle. Finally, in an attempt to expedite the recovery of fulllength copies of these genes a subtractive hybridization magnetic bead capture technique was adapted and employed to recover a full &ndash / length putative multicopper oxidase gene from a Streptomyces strain in a proof of concept experiment. The StrepA06pMCO gene fragment was used as a &lsquo / driver&rsquo / against fragmented Streptomyces genomic DNA (&lsquo / tester&rsquo / ) and resulted in the recovery of a 1215 bp open reading frame. Unexpectedly, this ORF showed only 80% identity to the StrepA06pMCO gene sequence at nucleotide level, and 48% amino acid identity to a putative mco gene derived from a Norcardioides sp JS614.</p>

No description available.

<p>The classical approach to gene discovery has been to culture micro-organisms demonstrating a specific enzyme activity and then to recover the gene of interest through shotgun cloning. The realization that these standard microbiological methods provide limited access to the true microbial biodiversity and therefore the available microbial genetic diversity (collectively termed the Metagenome) has resulted in the development of environmental nucleic acid extraction technologies designed to access this wealth of genetic information, thereby avoiding the limitations of culture dependent genetic exploitation. In this work several gene discovery technologies was employed in an attempt to recover novel bacterial laccase genes (EC 1.10.3.2), a group of enzymes in which considerable biotechnological interest has been expressed. Metagenomic DNA extracted from two organic rich environmental samples was used as the source material for the construction of two genomic DNA libraries. The small insert plasmid based library derived from compost DNA consisted of approximately 106 clones at an average insert size of 2.7Kbp, equivalent to 2.6 Gbp of cloned environmental DNA. A Fosmid based large insert library derived from grape waste DNA consisted of approximately 44000 cfu at an average insert size of 25Kbp (1.1 Gbp cloned DNA). Both libraries were screened for laccase activity but failed to produce novel laccase genes. As an alternative approach, a multicopper oxidase specific PCR detection assay was developed using a laccase positive Streptomyces strain as a model organism. The newly designed primers were used to detect the presence of bacterial multicopper oxidases in environmental samples. This resulted in the identification of nine novel gene fragments showing identity ranging from 37 to 94% to published putative bacterial multicopper oxidase gene sequences. Three clones pMCO6, pMCO8 and pMCO9 were significantly smaller than those typically reported for bacterial laccases and were assigned to a recently described clade of Streptomyces bacterial multicopper oxidases.</p> <p><br /> Two PCR based techniques were employed to attempt the recovery of flanking regions for two of these genes (pMCO7 and pMCO8). The use of TAIL-PCR resulted in the recovery of 90% of the pMCO7 ORF. As an alternative approach the Vectorette&trade / system was employed to recover the 3&rsquo / downstream region of pMCO8. The complexity of the DNA sample proved to be a considerable technical challenge for the implementation of both these techniques. The feasibility of both these approaches were however demonstrated in principle. Finally, in an attempt to expedite the recovery of fulllength copies of these genes a subtractive hybridization magnetic bead capture technique was adapted and employed to recover a full &ndash / length putative multicopper oxidase gene from a Streptomyces strain in a proof of concept experiment. The StrepA06pMCO gene fragment was used as a &lsquo / driver&rsquo / against fragmented Streptomyces genomic DNA (&lsquo / tester&rsquo / ) and resulted in the recovery of a 1215 bp open reading frame. Unexpectedly, this ORF showed only 80% identity to the StrepA06pMCO gene sequence at nucleotide level, and 48% amino acid identity to a putative mco gene derived from a Norcardioides sp JS614.</p>

Characterization of the determinants of economically important phenotypes showing complex inheritance should lead to more effective use of genetic resources. This study was conducted to determine the number, genome location and effects of QTLs determining malting quality and agronomic traits in the two North American barley quality standards. Using a doubled haploid population of 140 lines from the cross of Harrington x Morex, agronomic phenotype and malting quality data sets from nine and eight environments, respectively, and a 107-marker linkage map, QTL analyses were performed using simple interval mapping and simplified composite interval mapping procedures. Thirty five QTLs were associated either across environments or in individual environments, with five grain and agronomic traits (yield, kernel plumpness, test weight, heading date and plant height). Thirteen QTLs were associated with five malting quality traits (grain protein percentage, soluble/total protein ratio, ��-amylase activity, diastatic power and malt extract percentage). QTLs for multiple traits were coincident. The loci controlling inflorescence type [vrsl on chromosome 2 (2H) and int-c on chromosome 4 (4H)] were coincident with QTLs affecting all traits except heading date and malt extract percentage. The largest effect QTLs -for yield, kernel plumpness test weight, plant height grain protein percentage, S/T ratio, and diastatic power- were coincident with the vrsl locus. QTL analyses were conducted separately for each sub-population (six-rowed and two-rowed). Ten new QTLs were detected in the sub-populations. There were significant interactions between the vrsl and int-c loci for plant height, grain protein percentage, and SIT protein ratio. Positive transgressive segregants were found for all agronomic traits. They were more prevalent in the six-rowed sub-population, indicating that more favorable alleles were fixed in the two-rowed parent. Results suggest that this mating of two parents representing different germplasm groups caused a disruption in the balance of traits involved in malting quality, which resulted in no progeny carrying all favorable alleles and therefore surpassing the quality of either parent. This study describes some of the genetic determinants of agronomic and malting quality traits in a two-rowed x six-rowed cross and it is a first step toward the further characterization and manipulation of these determinants. / Graduation date: 2001

Graduation date: 1993

No description available.

Copies of author's previously published articles inserted. / Includes bibliographies. / iii, 277, [81] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this research is to investigate the phylogeny of isolates of the intestinal protozoan parasite Giardia intestinalis using molecular systematic techniques. Most of the isolates used in this study are propagated by the infection of suckling mice. Isolates are characterised allozymically and their genetic relationships are inferred using clustering methods. Seven lineages of isolates are identified, five comprising animal-derived G. intestinalis, and two comprising human-and animal-derived G. intestinalis. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1997?

Thesis (MDip (Medical Technology))--Cape Technikon, 1993 / The use of genetic marker testing to resolve cases of disputed paternity, is well established. The number and range of systems used depends on the expertise of the laboratory, and for this reason various laboratories offer different systems. Standard testing includes tests in the following genetic marker systems: human leukocyte antigen (tissue) typing; red cell blood groups; and red cell enzyme and serum protein testing. The Provincial Laboratory for Tissue Immunology currently offers a range of 16 genetic marker systems capable of excluding >99% of falsely accused men. Following the discovery DNA polymorphisms, particularly VNTR DNA polymorphisms, and the commercial availability of VNTR DNA probes, PLTI decided to offer this service to our clients. This study was the initial phase in the establishment of a VNTR DNA typing laboratory and covered the determination of inter-and intra-gel accuracy and precision, selection of restriction enzyme/probe combination, and evaluation and comparison of the results of 100 disputed paternity cases tested using both standard and VNTR DNA typing. Of the 100 cases tested, in 33 cases, the putative father was excluded using standard testing. These exclusions were confirmed using VNTR DNA typing, and, furthermore, an additional two exclusions of paternity were shown using only VNTR DNA typing. In another two cases of disputed paternity, the exclusions obtained using standard tests required further confirmation. VNTR DNA typing convincingly excluded both falsely accused putative fathers. The VNTR DNA typing laboratory now functions as an integral part of the disputed paternity service. Due to the cost and time involved in VNTR DNA typing it is reserved at this stage for: those cases which require further confirmation of the results of standard testing; when the probability of paternity is low (<99.7%); or when a specific request is made.

published_or_final_version / Pathology / Master / Master of Philosophy