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PREDICTIVE MUTAGENESIS AND CARCINOGENESIS: EFFECTS OF VARIATIONS IN THE CONDITIONS OF DNA SYNTHESIS UPON THE IN VITRO ACCURACY OF THE DNA REPLICATION APPARATUS OF BACTERIOPHAGE T4

Razzaki, Tashmeem Fatima (1985) PREDICTIVE MUTAGENESIS AND CARCINOGENESIS: EFFECTS OF VARIATIONS IN THE CONDITIONS OF DNA SYNTHESIS UPON THE IN VITRO ACCURACY OF THE DNA REPLICATION APPARATUS OF BACTERIOPHAGE T4. PhD thesis, University of Karachi, Karachi.

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Abstract

Using an in vitro DNA replication system constituted from purified T4 proteins; we have investigated effects of various perturbations in conditions of DNA synthesis on frequencies of deoxynucleotide misinsertion and subsequent excision. Under standard conditions of synthesis, the T4 replication complex (consisting of proteins coded by T4 genes 32, 41, 43, 44, 45, 61 and 62) replicates phage X174 DNA with high fidelity. In vitro replication accuracy closely paralleles that seen in vivo. Accuracy can be experimentally altered by introducing appropriate deoxynucleotide pool imbalances. Taking advantage of this technique one can raise the sensitivity of 3n assay system in which replication is allowed to proceed past an ambercodon and error frequency is measured from base mispairings leading to generation of the wild type codon. Both transition and transverison mutations :an be measured and increases as well as decreases in accuracy can be estimated. Using slightly mutagenic pool imbalances we measured reversion frequencies of X174 mutants amber 3 (AT → GC transition) and amber 16 which reverts by transversion (AT + TA and AT + CG). It was found that variations in temperature (20 to 45°C), pH (from 7 to 9.5), the concentrations of potassium acetate (from 0 to 150 mM), Mg++ (5 to 30 mM), and all 4 dNTPs together (from 20 micromolar to 2 mM Each) had no effect on the rates of formation of mispairs leading to either transitions or transversions. Also, the addition of deoxynucleoside monophosphates (Up to 3 mM each), sodium pyrophosphate (up to 3 mM), and spermidine (up to 6 mM) did not affect the accuracy of phage T4 replication apparatus in vitro The rate of DNA synthesis was affected greatly by these perturbations (more than IO-fold), but the accuracy remained unchanged. Only replacement of TTP by 5-bromodeoxyuridine triphosphate or of Mg++ by Mn++ was found to be mutagenic. No antimutagenic conditions were observed.

Item Type:Thesis (PhD)
Uncontrolled Keywords:carcinogenesis, mutagenesis, dna synthesis, dna replication system, t4 proteins; deoxynucleotide pool
Subjects:Biological & Medical Sciences (c) > Biological Sciences(c1) > Microbiology(c1.8)
ID Code:1264
Deposited By:Mr. Muhammad Asif
Deposited On:22 Jan 2007
Last Modified:04 Oct 2007 21:05

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