Specific locus method

These contrasting characteristics make Bombyx worthy to work on in the study of mutagenesis even if it lacks several advantages of Drosophila. Although, in Bombyx a suitable sex-linked recessive lethal method is not yet available, instead, a specific locus method is conveniently used, and the work to be reviewed in this chapter was mostly performed using this method. 

Several methods have been used in the silkworm for the quantitative assessment of mutations induced by radiation and/or chemical agents. These are the specific locus method, the recessive lethal method,the dominant lethal method, and the method for detection of nondisjunction. A recessive lethal method assesses lethal mutations occurring on a whole genome and is used for the study of both spontaneously accumulated and radiation-induced lethals in the silkworm. Th system, however, is rather complicated and tedious and will not be dealt with in this article. 

The method essentially consists of mating irradiated (or treated) homozygous dominant individuals with a test strain homozygous for known 

FIGURE 1. Schematic illustration of gametogenesis of the silkworm in relation to the developmental stages in both sexes. Duration represented by a dashed line indicates a period during which differentiation of the same cell type continues but utilizable gametes are not produced, because of the shortness of time. (I, II,. . . ) First, second,. . . larval instars. Meiosis denotes meiotic metaphases I and II. 

The marker genes that have been most conveniently used in the silkworm are pink ipe, 5-0.0) and red re, 5-31.7). Both are easily detected a few days after deposition, when wild-type eggs turn from light yellow to dark brown, due to the pigmentation of serosa cells. Mutant eggs can be observed with the naked eye, but a binocular microscope of low magnification is useful for the detection of mosaic mutants. Other markers that have been used are body-color mutants of newly hatched larvae, chocolate ch, 13-9.6) and sex-linked chocolate sch, 1-21.5). Several mutants with translucent skin, os (1-0.0), od (1-49.6), oc (5-40.8), ok (5-4.7), and others, can also be utilized as markers, but these require raising of Fi larvae at least up to the 4th instar. 

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The specific-locus method is particularly useful in making comparisons of the effects of dose, dose fractionation, sex, cell stage, and other factors on the mutational response in mammalian germ cells, as well as comparisons of the relative responses to different chemicals. Most of what is known about the induction of gene mutations and small deficiencies in germ cells of mammals by radiation and chemicals has been learned in such comparative studies. 

A number of techniques have been devised for mutation studies in the mouse, but undoubtedly the most productive has been the specific-locus method, which so far has found its greatest use for the assessment of the genetical hazards of ionizing radiations to man. The method has the potential for fulfilling the same role with respect to environmental and chemical mutagens, and, as the first results already demonstrate, it is likely to provide information which will elucidate the nature of the mutation process in the mammalian germ cell and indicate some of the variables which affect it. 

The specific-locus method consists essentially of mating treated and untreated wild-type mice, either male or female, to a strain homozygous for a number of recessive genes which have readily visible phenotypic effects. The offspring produced from the cross will be wild type unless a mutation has occurred at one of the loci represented by the recessive genes, when the recessive phenotype or perhaps a new or intermediate phenotype will be seen. The system thus provides a simple means of estimating the mutation frequencies to recessive visibles, including those with lethal effects. Dominant visibles would, of course, also be detected. 

TABLE 1. Results of Experiments to Detect Chemically Induced Recessive Visible Mutations in Male Mouse Germ Cells by the Specific-Locus Method ... 

Nature of Mutations Obtained by the Specific Locus Method... 

FIGURE 2. Specific locus method in which egg-color genes are used as markers. Wild-type individuals are treated by chemicals, and after emergence they are mated to pe re partners. All Fi eggs are expected to be wild type, but when mutation occurs, pe or re individuals will appear manifesting the character on the whole body or partially, as mosaics. 

Using these strains, the mutation response to MMC was compared among strains. The mutation response was assessed by the specific locus method. The data are given in Table 6. 

For mutation detection, the specific locus method was used. Meiotic metaphase occurs around V-2. 

TABLE 10. Results of Mutagenic Activity Tests of Chemical Compounds Using the Specific Locus Method in... 

Another, and the main, purpose of mutagenicity testing is to detect the potentiality of environmental chemicals to produce mutations in germ cells that will be transmitted to the descendant generations. Drosophila can be used most conveniently for this purpose. Testing with mice provides very useful information for extrapolation to man, but it requires large-scale experiments, especially when the specific locus method is used. In contrast, this method can be used... 

The root locus method provides a very powerful tool for control system design. The objective is to shape the loci so that closed-loop poles can be placed in the. v-plane at positions that produce a transient response that meets a given performance specification. It should be noted that a root locus diagram does not provide information relating to steady-state response, so that steady-state errors may go undetected, unless checked by other means, i.e. time response. 

Selby, P.B. and Olson, W.H. (1981). Methods and criteria for deciding whether specific-locus mutation-rate data in mice indicates a positive, negative or inconclusive result. Mutation Res. 83 403-418. 

The specific-locus test is not intended for human risk estimation. The seven loci sampled may not be representative of the whole genome. To continue the example of the appendix to this chapter, a mutation rate of 0.000003 per seven loci corresponds to 0.009 for 20,000 loci, if this is taken as the haploid gene number. This is nearly 1% per zygote, not a negligible number. It would be desirable to have methods to measure mutations in the entire genome, or in an entire chromosome, such as are used in Drosophila. Such methods are being developed in mice, but cure not yet suitable for routine use. 

Ehling, U.H. Specific-locus mutations in mice, pp. 233-256. In A. Hollaender and F.J. de Serres, Eds. Chemical Mutagens Principles and Methods for Their Detection. Vol. 5. New York Plenum Press, 1978. 

Specific locus mutations in the ad-3 region result in the accumulation of a purple pigment in the mycelium as well as a requirement for adenine. Mutants are recovered on the basis of pigment accumulation. Our experience has shown that this method of selection enables us to recover not only mutants with complete blocks and partial blocks, but also mutants that are so leaky that we cannot even demonstrate an adenine requirement (de Serres, 1964). In this respect, the ad-3 system is ideal for the detection of the mutagenic activity of chemicals, because we believe that all types of mutagenic activity resulting in the structural alteration of DNA will be detected. 

D. Clive, W. G. Flamm, and J. B. Patterson, Specific-locus mutational assay systems for mouse lymphoma cells, in Chemical Mutagens Principles and Methods for Their Detection, Vol. 3 (A. Hollaender, ed.), pp. 79-103, Plenum Press, New York (1973). 

SNP genotyping is an advanced genotyping method that measmes SNPs between genomes. In an SNP, a single base pair is mutated at a specific locus, leading to the malfunction of the specific gene product and a disease... 

Two types of screening procedures are used to identify ES cell clones carrying a targeted integration of the construct DNA polymerase chain reaction (PCR) and Southern blot analysis (Southern, 1975). Both methods rely upon the specific juxtaposition of vector components and target locus sequences after homologous recombination. 

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