Isolating Mutations in Muscle or Nerve in Drosophila

Isolating Mutations in Muscle or Nerve in Drosophila

How could mutations in muscle or nerve be isolated? Since these mutations could be lethal, we ought to seek conditional mutations. That is, the mutation ought to be expressed only under special conditions, for example at elevated temperature. Suzuki developed ingenious meth-ods for the isolation of temperature-sensitive paralytic mutations. The flies we seek ought to be perfectly normal at low temperature, be paralyzed at high temperature, and recover rapidly when returned to low temperature. Undoubtedly such mutations would be exceedingly rare, and great numbers of flies would have to be screened in order to find a few candidate mutants. Such large numbers necessitated the use of tricks to eliminate the need for sorting males and females.

The first trick used an attached X chromosome. This is an inseparable pair of X chromosomes denoted as X^X. Mating males with females containing an attached X chromosome, yields the expected four types of offspring (Fig. 8.22). Both the X’X^X and YY are lethal and therefore only mutagenized males or females with the attached X chromosome result from this mating. If the attached X chromosome contains a dominant temperature-sensitive lethal mutation, the females can be killed by a brief temperature pulse, leaving only the desired, mu-tagenized males as a pure stock. The female stocks required for the first mating also can be generated by this same technique.

The second problem was the actual selection for the temperature-sen-sitive paralytics. This was done by introducing up to 10⁴ flies into a cubical box about two feet on a side. The temperature in the box was

raised, and the box was given a bang on a table top to make the flies fly upwards. Any temperature-sensitive paralytics remained at the bottom of the box. These were trapped by rotating the box so that they were

collected on a ledge. Then the flies were anesthetized by adding carbon dioxide or ether and those flies which had been able to fly fell to the bottom of the box where they were killed by the addition of detergent and acetic acid.

Like most mutant selection schemes, several additional phenotypes were found in addition to those sought. One of these was rex for rapid exhaustion. After running around for few minutes a rex mutant shud-ders a bit and falls over in a paralysis which lasts a few minutes. It can then get up and is perfectly normal for about an hour. Another mutant was the bas for bang-sensitive. The desired mutants fell into three types: para^tsfor temperature-sensitive paralytic, sts^tsfor stoned, and shi^tsfromthe Japanese word for paralyzed. The para mutation lies in the protein which forms the membrane sodium channel that is essential for nervous impulse transmission.