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In the following study, I have investigated aberrant integration events of the maize Activator/Dissociation ( Ac/Ds) family of transposable elements (TEs) in Arabidopsis. The purpose of the study was twofold: (i) to identify sequence modifications associated with aberrant transposition that are informative regarding the mechanism of Ac/Ds transposition; and (ii) to extend our understanding of the mechanisms by which class II TEs can influence genome structure. This work focuses on a large inversion identified on chromosome II. A lone Ds element comprises one breakpoint of the inversion and the second breakpoint is composed solely of Arabidopsis sequences. The analysis of the sequence modifications present at both breakpoints indicates that the event was precipitated by the abortive transposition of Ds. This is the first event of its kind identified for an Ac/Ds and the event defines a novel mechanism by which these TEs can induce change within a genome. Further, the presence of deletions at both termini of the implicated Ds suggests that the transposition of Ac/Ds involves fully excised intermediates. To obtain further support for this model, a population of Arabidopsis individuals harboring Ds excision events was screened for reintegrated elements. Several integrations were analyzed at the sequence level and compared to wild-type integration sites. While no genome rearrangements were detected, a number of integrations display small deletions within both the Ds termini and the DNA flanking the elements. These results are consistent with the presence of fully excised Ac/Ds intermediates. Further, the results suggest that dissolution of the transposase active complex at different points in the transposition process will result in the formation of distinct aberrant transposition products. During the characterization of the inversion, a novel Arabidopsis TE family, FARE, was identified. The FARE elements are foldback transposons, a heterogeneous and poorly characteri

There are 30-35 copies of the retrotransposon Ty in the haploid genome of most laboratory strains of Saccharomyces cerevisiae. Ty elements are 5.9 kb long, have LTRs of 340 bp and produce a genomic RNA that is 5.7 kb in length, through which it transposes. They contain two ORFs; the TYA gene of the class I element Tyl-15 is 1,319 nucleotides long whilst its TYB gene, which possesses the consensus sequences of retroviral acid proteases, reverse transcriptases and integrases, is 3,984 nucleotides long. TYB overlaps TYA by 38 nucleotides at its 5' end and is expressed as a TYA;TYB fusion protein. In an attempt to asign some in vivo functions to the Ty encoded proteins, the whole transcriptional unit of Tyl-15 was overexpressed in yeast. Electron micrographs of the overexpressing cells revealed an abundance of cytoplasmic, ~60 nm virus-like particles (Ty-VLPs). These contain the 5.7 kb Ty RNA and have an associated reverse transcriptase activity. Their major protein is p2 (48 kD), which is a proteolytic derivative of the primary translation product of TYA, pi (50 kD), a protein that is also assembled into particles. A truncated pi, encoded by a TYA gene shortened by 60 codons at its 3' end, still aggregates into particles. Furthermore, the addition of α2-IFN (20 kD) onto its carboxy terminus, to produce a pl-IFN fusion protein of ~68 kD, does not interfere with its particle forming properties. The hybrid VLPs are easy to purify and elicit an antibody response in rabbits to the Ty and IFN epitopes. A series of fragments spanning Tyl-15 were inserted into the packaging analysis vector, pMA924. The association of the resulting PGK-Ty-IFN hybrid RNAs with Ty-VLPs, in the presence of abundant Ty encoded proteins, was analysed by Northern blotting and used to assess the packaging capabilities of Ty RNA. Unexpectedly, RNA sequences that, direct specific packaging are produced from regions throughout Tyl-15. The signals corresponding to TYA are probably located within two regions of the genomic RNA, 5' to nucleotide 236 and 3' to nucleotide 737; those corresponding to TYB are in at least two, currently undefined, areas. The TYB gene of the class II element Tyl-17 overlaps TYA by 44 nucleotides. The fusion of α2-IFN cDNA fragments into all three reading phases of TYB followed by a Western blot analysis demonstrated that, like the TYB gene of Tyl-15, it too is expressed as a TYA:TYB fusion protein.

The Rhizobium-legume symbiosis is an opportunistic association between two symbiotic partners that results in the formation of the root nodule. The process depends on the expression of a number of plant and bacterial genes that are considered critical for the establishment and maintainance of the symbiotic state. The merits of a mutational approach to the analysis of symbiosis have been recognized for several years and transposon Tn5 mutagenesis of Rhizobium has led to the identification of several symbiotic genes. This study describes the use of Tn5 mutagenesis for the isolation of symbiotically defective mutants of Bradyrhizobium japonicum. Two classes, auxotrophic and cell surface-altered mutants defective in nodule formation, have been characterized. In B. japonicum USDA 122, histidine auxotrophs that are defective in nodulation have been studied. The mutagenized DNA region has been cloned and the wild-type DNA region isolated by hybridization and complementation. In B. japonicum 61A76, Tn5-induced cell surface-altered mutants have been isolated by selecting for bacteriophage resistance. Several parameters have been used to demonstrate alterations in cell surface components. It has been shown that the Tn5 insertion is not the primary cause of the mutation in two of the characterized mutants. Complementation tests have led to the isolation of a wild-type DNA-containing cosmid, pPS23A, that overcomes the symbiotic defect in one of the mutants. Analysis of the cell surface showed a partial restoration of surface components in the complemented mutant.

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

Thesis (Ph.D.)--University of Iowa, 2009. / Thesis supervisor: Debashish Bhattacharya. Includes bibliographical references (leaves 34-37).

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

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