Question

You are given a mutator strain of E. coli. Describe an experiment to phenotypically differentiate whether the causative mutation for high mutation rates in this strain is in DNA photolyase OR UvrB.

Answer 1

Answer:

Determination of the causative mutation for high mutation rates in mutated E coli strain is in DNA photolyase OR UvrB by analysis of gene-specific repair:

1. In this method the cells are going to be rinsed in PBS (phosphate buffered saline ) and prelabelled using 3H- thymidine and 0.1 μM thymidine about 20 hours. Later the growing bacterial cells are going to be irradiated with ultra violet light (30 J/m²) and associated lysis finally they must be incubated for 24 hours in the presence of 5-bromodeoxyuridine + 5-fluorodeoxyuridine with conc. 10uM and 1uM respectively.

2. Later DNA need to be separated and subjected to purification finally using cesium chloride we have to do isolation of both replicated fragments and parent DNA fragments. Now the we can assess the frequency of UV-induced cyclobutane pyrimidine dimmers associated in restriction fragments using specific incision by T4 endonuclease V.

3. Now we can assess the frequency of pyrimidone photoproducts finally by the removal of cyclobutane pyrimidine dimmers using DNA photolyase. Finally the DNA essentially need to be subjected to considerable incubation in the presence of UvrABC excinuclease complex associated with Escherichia coli.

4. Performing electrophoresis of the final samples using alkaline agarose gel materials finally DNA essentially need to be transferred to Hybond N+ membranes later considerably hybridized with gene-specific probes.

Using filters such as InstantImagerTM phenotypically we can determine the mutation induced altered numbers of both cyclobutane pyrimidine dimmers & 6-4 pyrimidone photoproducts in individual restriction fragment using calculation of relative band intensities of full-size restriction fragments. Now further poisson distribution ration we can determine the intensity of mutation associated with both DNA photolyase or UvrABC excinuclease in the MUTATED e Coli strain.