Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 67-73). Also available via the Internet.

Thesis (M.S.)--University of Wisconsin--Madison, 1982. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 79-91).

Thesis (Ph. D.)--University of Hong Kong, 1994. / Includes bibliographical references (leaves 165-187).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 171-186).

Thesis (Ph. D.)--University of Idaho, 2005. / Also available online in PDF format. Abstract. "December 2005." Includes bibliographical references.

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

R1162 is efficiently mobilized during conjugation by IncP-1 plasmids such as RK2 and R751. Transfer is terminated when the transferred strand, linearized at the 38 base-pair origin of transfer (oriT), is recircularized by the plasmid encoded protein MobA. For strand rejoining, MobA covalently linked to the 5' end of the strand rejoins the ends by a reversible transesterification reaction. The minimal oriT fragment of R1162 contains a highly conserved 12 base region (core) including the cleavage site and a ten base imperfect inverted repeat (IR) that is not highly conserved. From those oligonucleotides with a partially degenerate oriT core base sequence, the subpopulations that are bound by MobA, cleaved and rejoined by this protein and support termination of transfer were identified. Both the IR and the adjacent core bases TAA, are needed for tight binding to MobA, whereas the location of the dinucleotide YG determines the site of strand cleavage. At the IR MobA stabilizes duplex DNA during gel electrophoresis and binds weakly to oligonucleotides lacking the outer arm of the inverted repeat, supporting a model where secondary structure at IR provides a duplex region needed for binding during termination (Zhang and Meyer 1995). Significantly altered IR sequences did allow strong binding to MobA yet completely different IR sequences did not, indicating the IR serves a structural role for binding with low base specificity. A 184 residue aminoterminal MobA fragment capable of binding and cleaving oriT was identified by using phage display and partial enzymatic digestion of the protein. No smaller fragments that could bind or cleave oriT were identified. An active nucleoprotein intermediate consisting of MobA covalently linked to the 5' end of the cleaved oriT was used to show that a single molecule of MobA is sufficient to carry out all the DNA processing steps during transfer. / text

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The aim of this study was to develop a genetic transformation system for the plant pathogenic fungus Rhizoctonia solani (teliomorph, Thanatephprus cucumeris Frank [Donk]). The availability of a transformation system would allow us to study gene exchange, epidemiology, and to use techniques such as gene disruption or gene silencing to investigate the role of fungal enzymes in pathogenesis. The approach adopted was to use Agrobacterium tumefaciens to transform the fungus as reports in the literature suggested that this was the most efficient and easiest method to use. As a preliminary test, Fusarium oxysporum was transformed using a binary vector (pBINAN) containing a hygromycin resistance gene under control of an ascomycete promoter and terminator. Hygromycin resistant transformants were obtained after co-incubation of fungal conidia with the bacterium. The presence of the transgene was confirmed by analysis of DNA. The number of transformants depended on the genetic background of the A tumefaciens. Strains AGLO or AGRO gave higher numbers of transformants compared to LBA4404. No transformants were obtained when the hygromycin gene was under control of a basidiomycete (pBINHL1), or a plant (CaMV35S) promoter. Since the basidiomycete promoter used in pBINHL1 originates from Ustilago maydis, the vector was tested by transformation of Ustilago cynodontis. Stable transformants of U cynodontis were obtained with this vector. A series of experiments were carried out on transformation of R. solani mycelium. Both the protoplast and the Agrobacterium transformation methods were tested. Parameters affecting protoplast production and regeneration were examined. Protoplast production varied with the age of the mycelium, with the osmotic stabilizer used, and with time of treatment with protoplasting enzymes. Regeneration of protoplasts was also affected by the osmotic stabilizer and the growth medium. Transformation of several isolates from different anastomosis groups (AG) was attempted by inducing protoplasts to take up DNA using polyethyleneglycol. Two plasmids were used; (1) pAN7-1 containing the resistance gene under control of an ascomycete promoter, and (2) pHL-1 in which the resistance gene is under control of a basidiomycete promoter. No transformants were obtained. Attempts were then made to transform mycelium and protoplasts using A tumefaciens. The experiments used both mycelium and protoplasts as the recipient. A number of small resistant colonies were obtained using binary plasmid (pBINHL1) in which mycelium was transformed with the resistance gene was driven by the basidiomycete promoter. On transfer to fresh medium these colonies would grow to about 2cm diameter, and then stop growing. On a second transfer to fresh medium they failed to show any growth. No resistant colonies were obtained from A. tumefaciens transformation of protoplasts. To improve transformation efficiency, a vector was constructed in which the hygromycin resistance gene was fused to an R solani laccase promoter sequence. No resistant colonies were obtained using this vector. Further experiments were carried out using a hygromycin resistance gene specially modified for expression in basidiomycetes by the insertion of artificial introns in the 5’ and 3’ untranslated regions, and a number of AT to CG conversions in the coding region. Most of the recipient isolates gave transformants with the unstable resistance phenotype. However, one AG 6 isolate gave transformants with a stable resistance phenotype. Of six transformants recovered from this isolate, five were shown by PCR and southern blotting to contain the transgene. In four of these transformants the resistance phenotype was stable in the absence of selection.