DNA Base Substitutions Research Articles

Detection of single base substitutions in total genomic DNA.

Certain single base substitutions causing genetic diseases or resulting in polymorphisms linked to mutant alleles, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known. In the case of beta-thalassaemia, where 22 different single base mutations have been identified, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S1 nuclease treatment of heteroduplexes formed between wild-type and mutant DNA. Although certain single base mismatches are cleaved by S1 nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea, but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system. We show that four different human beta-thalassaemia alleles with known single base mutations can be detected with as little as 5 micrograms of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.

Methodology for the testing of food dyes for genotoxic activity: Experiments with red 2G (C.I. 18050)

A methodology for investigating genotoxicity of food colours using the fluctuation and DNA-repair assays with bacteria is described. In addition, a liquid repair test, developed to permit incorporation of microsomes and the quantitative estimation of cell viability, has been characterised with a number of positive control agents. Results obtained in these systems suggest that the food colour Red 2G induces repairable DNA damage and base-substitution mutation, but only in the presence of a rat-liver microsomal preparation. The significance of the data in the light of other toxicological information is discussed.

The rate of molecular evolution considered from the standpoint of population genetics.

The rate of amino acid substitutions in the evolution of homologous proteins is remarkably constant. Furthermore, estimated rates of amino acid substitutions based on comparisons of the alpha hemoglobin chains of various mammals with that of the carp are about the same as those based on comparisons of the carp alpha and mammalian beta or the alpha and beta chains in mammals. These uniformities are regarded as evidence for the hypothesis that a majority of amino acid substitutions that occurred in these proteins are the result of random fixation of selectively neutral or nearly neutral mutations. TWO IMPLICATIONS OF THIS POSSIBILITY ARE DISCUSSED: (a) Random gene frequency drift is playing an important role in determining the genetic structure of biological populations and (b) genes in "living fossils" may be expected to have undergone as many DNA base (and therefore amino acid) substitutions as corresponding genes (proteins) in more rapidly evolving species.

Genetic variability maintained in a finite population due to mutational production of neutral and nearly neutral isoalleles.

1. The average and the effective numbers of alleles maintained in a finite population due to mutational production of neutral isoalleles were studied by mathematical analysis and computer simulation.2. The exact formula was derived for the effective number (ne) of alleles maintained in a population of effective sizeNe, assuming that there areKpossible allelic states and mutation occurs with equal frequency in all directions. If the number of allelic states is so large that every mutation is to a new, not pre-existing, allele, we havene= 4Neu+1 − 2Neu2, whereuis the mutation rate. Thus, the approximation formula,ne= 4Neu+1, given by Kimura & Crow (1964) is valid as long as 2Neu2≪ 1.3. The formula for the average number of alleles (na) maintained in a population of actual sizeNand effective sizeNewas derived by using the method of diffusion approximation. If every mutation is to a new, not pre-existing, allele, we obtainwhereM= 4Neu. The average number of alleles as a function ofMandNis listed in Table 1.4. In order to check the validity of the diffusion approximations, Monte Carlo experiments were carried out using the computer IBM 7090. The experiments showed that the approximations are satisfactory for practical purposes.5. It is estimated that among the mutations produced by DNA base substitutions, synonymous mutations, that is, those which cause no alterations of amino acids, amount roughly to 0·2–0·3 in vertebrates.Incompletely synonymous mutations, that is, those which lead to substitution of chemically similar amino acids at a different position of the polypeptide chain from the active site and therefore produce almost no phenotypic effects, must be very common. Together with synonymous mutations, they might constitute at least some 40% of all mutations. These considerations suggest that neutral and nearly neutral mutations must be more common than previously considered.