Microbial genetics

Many different ways to create mutants have been established. This research
demonstrates yet another variation of the promoter tagging technique that allows for a
single step selection of the putative transgenic plants that have a mutation in
constitutively expressed genes. While tomato transformants have not yet been
convincingly confirmed, tobacco transformation resulted in seven transgenic lines
showing resistance to high concentrations of kanamycin. Two transgenic lines were
further investigated and three putative promoters isolated. Transient expression analysis
of leaves transformed by particle bombardment with vectors carrying beta-glucuronidase
gene driven by these putative promoters suggests two of them to be functional. Further
investigation is needed to confirm the expression in the stably transformed plants as well
as cloning of the genes downstream of the functional promoters and research of their
functions.

Aconitase is an important enzyme in the citric Acid Cycle, is needed for maintenance of mitochondrial DNA, is a key regulator of iron in the cell, and is very sensitive to oxidative stress. We have isolatd the yeast ACO1 gene, which codes for aconitase, and randomly mutated it to create a mutant library of cells each expressing a different version of ACO1. We will select for oxidative stress resistant aconitase in S. cerevisiae by subjecting strains to successive rounds of heat shock and competitive growth against other mutants. The "winner" of this competition will then be analyzed for which version of aconitase it is expressing, which may lead to increased longevity.

In my thesis work, I attempted to construct a plasmid that would allow stable integration of genes into the Saccharomyces cerevisiae yeast genome under the control of the repressible TetO promoter. The yeast ACO1 gene was cloned under the control of the TetO operator and the tTA transactivator. This construct was inserted into yeast cells in order to observe the effects of aconitase overexpression on aging. Unfortunately, the transformed cells appeared incapable of aconitase expression as determined by glutamic acid auxptrophy, a phenotype of aconitase mutants. We have sequenced the pIT1ACO1 plasmid and have found many abnormalities in the promoter region. If the plasmid can be made to function as intended, the resulting yeast strain can be used in the future to determine if aconitase plays an important role in cellular aging.