This study investigates the possibility of developing the RFLP/PCR assay as a new mutagenicity screening test for the detection and monitoring of the genetic effects of chemical pollution in the freshwater environment using <I>Xenopus laevis</I>. The two mutagens used were N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (B[a]P). MNNG- and B[a]P-induced mutations were detected at a frequency of 10<SUP>-4</SUP> in a sequence specific manner at selective recognition sites of the α<SUP>A</SUP><SUB>1</SUB>-globin, β<SUP>L</SUP><SUB>1</SUB>-globin and <I>c-myc</I> I genes during larval development. In maturing toadlets, B[a]P-induced G:C to T:A transversions at a frequency of 10<SUP>-1</SUP> at the BsiLI-site 913-917. Stage-specific differences in the incidence, frequency and time dependence of the B[a]P-induced mutations were observed during development. The nature of the effects of N-methyl-N-nitrosourea (NMU) upon <I>in vitro</I> DNA synthesis were also investigated, by determining if the mutagen induced intracellular environment could enhance the mutagenic and carcinogenic capacity of NMU through an increase in the mispairing probability of the methylated bases and simultaneous damage to the specific activity and fidelity of a DNA polymerase. The sensitivity of the PCR based assay depended on the target sequence length. The NMU damaged DNA template and polymerase acted in concert to potentiate DNA synthesis inhibition and produced dose-dependent differences in the mechanism of this inhibition. Using an error-prone activity the <I>Taq</I> polymerase was able to read through a small fraction of DNA lesions. Although no significant differences were observed in the types and frequencies of the base substitution mutations, the spectrum of susceptible nucleotide positions and miscoding potential of NMU depended on which component of the replication complex was targeted.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:637778 |
| Date | January 1995 |
| Creators | Kennerley, G. A. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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