UBC Theses and Dissertations
Nucleoside triphosphate pools in cultures of Escherichia coli. Mychajlowska, Lydia
Nucleotide pools in a synchronized culture of Escherichia coli B/r/l oscillate as a function of age. Purine nucleotides showed a gradual increase from zero age to the time of subsequent division, with a maximal 50% increase immediately prior to division. In contrast, pyrimidine nucleotides underwent a diamatic increase of about 50% in the first half of the generation cycle, declining at a time coincident with the termination of a round of DNA replication. A second 50 - 70% increase started at the time of the onset of DNA replication and continued towards cell division, as did the purine. The fluctuation of pyrimidines between zero age and the middle of the division cycle suggests a functional relationship between pyrimidine pool fluctuations and the regulation of DNA replication. Nucleotide pools decrease immediately in the presence of chloramphenicol to 10% of the control concentrations, and overshoot 50 - 70% in restoration of protein synthesis. Feedback inhibition of carbomyl phosphate synthesis (which is required for pyrimidine biosynthesis) by excess arginine may explain the fluctuations of nucleotide pools in the presence of chloramphenicol. Immediate depletion of nucleotide pools could be du to a very rapid turnover of nucleotide biosynthetic enzymes. The depletion of precursor pools, may explain the inability of a cell to reinitiate DNA replication in the absence of protein synthesis. In a comparison experiment, however, nucleotide pools in a temperature-sensitive initiator mutant were seen to accumulate at non-permissive temperature. In this case, protein synthesis occurred but initiation of DNA synthesis could not take place. This confirms the current hypothesis that a functional initiator protein is required for reinitation. Nucleotide pools in the presence of nalidixic acid dropped slightly and although no DNA synthesis occurred, pools showed no accumulation. This suggested a secondary effect of the inhibitor. Experiments prior to the pool analyses showed the importance of balanced growth in such studies. The cell size distribution was seen to be a more valid criterion than exponential increase in numbers. Exposure to cold temperatures was seen to upset balanced growth for at least one generation.
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