Chemical Mutagenesis

The ENU mutagenesis has been applied to many organisms and should be applicable to Nothobranchius. However, this requires the demonstration of efficiency of ENU mutagenesis in this species. Such results would form the basis for pursuing large-scale saturation mutagenesis in Nothobranchius to identify mutant fish with extended lifespan. To identify longevity mutants of Nothobranchius, the male Nothobranchius should be mutagenized with ENU and mated with normal females generating progeny with many heterozygous mutations representing either a gain of function or a loss of function. The specific mutation rate could be estimated by using phenotypic pigmentation patterns. To estimate a specific locus mutation rate, mutagenized males from a blue strain are mated with a red strain partner. The number of fish expressing these traits when they are homozygous in the progeny will predict the efficiency of ENU mutagenesis and the mutation rate. In ENU mutagenesis of zebrafish, the specific locus mutation frequency is as high as 0.9-1.3 x 10-3 (Solnica-Krezel et al., 1994). With the mutation rate of 1.27 x 10-3 if the distribution of mutations in the genome could be approximated by Poisson distribution, about 87% of the genes would be detected with an average of two mutations per locus, and for this approximately 1600 genomes must be screened to obtain one mutation for a given locus. Given the established methods in zebrafish, one would expect that high mutation rates will be achieved in Nothobranchius. The following scheme as a screen for selection of long-lived mutant fish is proposed: first, females from ENU mutagenized F1 progeny are used that generate gynogenetic diploids from each of these females. From this progeny, males and females are separated and kept in large tanks. This will prevent further breeding, although the density of the fish should be taken into consideration. Since the longest survivor in Nothobranchius is about 10 months, fish are selected for 12 months as lifespan. This gives approximately 20% longer life. The long-lived F2 diploid survivors are used to generate F3 homozygous. The survivorship curves of wild-type progeny of the two strains are compared to the survivorship curves of the above homozygous mutant progeny. The shift of the mutant progeny lifespan curve to the right compared to controls will confirm the longevity mutant. This homozygous progeny is then bred to a different strain of Nothobranchius, which would be polymorphic for many loci for mapping the mutant locus. Once the long-lived mutants are isolated for identification of the gene responsible for the phenotype, it should be possible to map the longevity mutant locus by using linkage methods. Since large chunks of DNA are syntenic in the Nothobranchius genome when compared to the zebrafish genome, the maps and the synteny of genes in zebrafish as well as those from mammals should be useful to predict candidate genes once a marker linked to the longevity phenotype is identified. For this, approximately 250 markers that are spaced approximately 10 cM and spanning the entire genome are needed for mapping longevity mutants. Since a large number of zebrafish EST sequences and a significant amount of genomic sequence from fugu fish as well as zebrafish are already available, it should be possible to choose a number of conserved EST marker primers in order to identify polymorphism that span the entire Nothobranchius genome. Based upon the existing linkage maps of zebrafish, the marker (EST) primers located approximately 10 cM can be selected. It should then be possible to amplify the total RNA using the above primers to find polymorphisms between two strains of Nothobranchius by SSCP or by DNA sequencing.

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