Detection of Mutations in Drosophila

Mutations induced due to radiations or due to chemicals could be broadly of two types - (i) lethal mutations, (ii) visible mutations. Both these types could be located either on sex chromosomes or on autosomes (autosomes are chromosomes other than sex chromosomes). Different methods for detection of sex linked lethals, sex linked visibles and autosomal mutations are available.

Detection of sex linked lethals
The ClB method. This method involves use of a ClB stock which carries (i) an inversion in heterozygous state to work as crossover suppressor (C), (ii) a recessive lethal (l) on X-chromosome in heterozygous state, and (iii) a dominant marker, Barred (B)for the barred eye (narrow eye). One of the two X-chromosomes in a female fly carried all these three features and the other X-chromosome was normal. Male flies irradiated for induction of mutations were crossed to ClB females. Male progeny receiving ClB X-chromosome will die (Fig. 21.3).

The ClB female flies obtained in progeny can be detected by barred phenotype. These are crossed to normal males. In the next generation 50% of males receiving ClB X-chromosome will die. The other 50% males will receive X-chromosome, which may or may not carry the induced mutation. In case lethal mutation was induced no males will be observed. On the other hand, if no lethal mutation was induced, 50% males will survive. Thus, the ClB method was the most efficient method for detecting sex linked lethal mutations. This has since been improved upon and therefore, has been replaced by Muller-5 method.

MuIIer-5 method. This method makes use of a Muller-5 Drosophila stock, which carries two marker genes, dominant 'Bar' (barred eye) and recessive 'apricot', but does not carry a lethal as in CIB. Moreover, Muller-5 contains a more complex inversion and therefore, has a better crossover suppressor than in ClB. As shown in Figure 21.4, in F₂ generation, 50% males are Muller-5 in phenotype and remaining 50% are wild type. If a lethal mutation is induced in X-chromosome of irradiated male no wild type males would appear in F₂ generation. Therefore, absence of wild type males in F₂ is an indication of an induced lethal mutation. Using different doses of irradiation and working out the corresponding frequencies of lethal mutations, a linear relationship could be observed between the radiation dose and mutation frequency (Fig. 21.5). When different kinds of radiations were used, it was found that alpha (α) particles gave the lowest mutation frequencies (.0084 per individual per 1000 roentgen) and beta (β), gamma (γ) and hard X-rays gave the highest frequency (.029).

Detection of sex linked visible mutations Attached X-method. The methods described in the preceding section were meant for detection of sex linked lethals. For detection of sex linked visible mutations, Muller-5 and attached X-chromosomes were used. The attached X females (XXY) have a special advantage. When these females are crossed to an irradiated male, X-chromosome of irradiated male goes either to superfemale daughters or to the sons. Since in sons there is a single X-chromosome, any visible induced mutation will immediately express itself and can be easily scored (Fig. 21.6).

Detection of autosomal mutations (lethal or visible)
Balanced lethal system and detection of mutations. Detection of mutations on autosomes (chromosomes other than sex chromosomes) makes use of a balanced lethal stock. Balanced lethal stock is one which carries recessive lethal alleles on two specific homologous chromosomes, their normal allele being present on its homologue; consequently homozygous individuals will not survive. The concept of balanced lethal would be better understood using a specific example. For detection of visible mutations in Drosophila, a stock carried the dominant genes Cy (curly wing)and L (lobed eye)on one chromosome and Pm (plum eye i.e. brownish eye) on the other homologue so that the organism can be designated as Cy L/Pm. Phenotype of the stock which was heterozygous for Cy, L and Pm, was curly, lobe and plum. If such individuals are crossed among themselves, progeny is always heterozygous, because each of the two homologous chromosomes carried recessive lethal genes (Fig. 21.7). Through the presence of inversion, crossing over was also suppressed, so that Cy and/or L may not be transferred to the chromosome carrying Pm and vice versa.

For detection of an autosomal mutation, Cy L/Pm stock is crossed to irradiated fly. From F₁, Cy L flies were again backcrossed to Cy LIPm in order to obtain curly, lobe (heterozygotes) male and female flies carrying the same autosome to be tested. Such Cy L heterozygotes when crossed among themselves would give 1CyL/CyL : 2 CyL/++ : 1 ++/++. Since CyL homozygotes are lethal, phenotypic ratio will be 2 : 1. However, if lethal mutation was induced, only curly will appear. If a recessive visible mutation was induced, this will appear in homozygous state in one-third of the progeny (Fig. 21.8).