Vicki L. Wong
University of Kentucky
Lexington, KY
Mentor: Dr. James Courtright

Normally, cells unable to synthesize protein or RNA can survive many hours without loss of ability to form colonies. Even if this restriction is imposed by starving the cells of an amino acid or of a purine they are unable to synthesize. However, an interference with DNA synthesis by depriving the cells of their supply of thymine results in a different response. After about 30-40 minutes, thymine-starved cells lose their capacity to form colonies. This progressive loss of colony forming ability is called thymineless death, or TLD. This effect can be seen in cells in which thymine synthesis is sufficiently blocked by metabolic inhibitors such as sulphanilamide, aminopterin and its analog trimethoprim, or fluorodeoxyruridine. The causes of this effect still remain unknown. Only one fact is known for certain: if the rates of RNA and protein synthesis are severely restricted, then the rate of loss of viability is also greatly reduced. 

Furthermore, for reasons still only partially known, i.e. activation of an alternative pathway, E. coli mutants that require thymine can grow in the presence of aminopterin and thymine much better than thymine-independent strains, even when there is a sufficient supply of thymine. Aminopterin inhibits thymidine kinase at high concentrations thus high concentrations are required to produce mutants in this way. An adjustment to the procedure of isolating thymine-requiring mutants was the replacement of aminopterin with trimethoprim. Aminopterin and trimethoprim have very similar chemical structures but aminopterin has an extended side group that makes it less soluble than trimethoprim; therefore, trimethoprim was just easier to work with. 

For the purpose of inducing thymineless death, thyA requiring mutants were first derived from HfrH, an E.coli wild type, through replica plating. Once thymine mutants were isolated, they were purified by restreaking for single colonies on nutrient agar + thymine (NA + thy) plates. Three mutant strains were studied: #2, #7, and #11. Only one strain (#11) was chosen to be used in the following experiments. The strain was chosen because it was observed to be the most susceptible to thymineless death. 

Viability of this #11 mutant was tested by starving it in M9, minimal medium, without thymine for one hour. Depriving the mutant of thymine results in thymineless death. After starvation, the mutant was treated with varying amounts of enoxacin, a soluble quinolone antibiotic for 30 minutes. Enoxacin is known to generate ~70 kilobase fragments upon SDS solubilization and protease treatment. After enoxacin treatment, cell suspensions were diluted and plated on NA + thymine. Results show that more enoxacin added to killing of cells thus lower viability compared to less amounts of enoxacin. 

Once a death rate pattern was established, the next question emerged: is there a change in chromosome morphology in thyA mutant cells when treated with enoxacin? The #11 mutant was grown in three different conditions (complete medium containing thymine, in minimal medium containing thymine and then minimal medium lacking thymine) in order to determine chromosome cleavage patterns.