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Isolating new alleles
You are going to conduct a screen for new homozygous lethal alleles of the 2nd chromosome gene
stubby which encodes for a kinase that has been shown to be important for body size: embryos that are
homozygous mutant for this gene are extremely stubby and do not hatch; these mutants are therefore
considered homozygous lethal.
For this screen you are limited to using the following lines:
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A stock of wild-type flies (+/+)
L/Cyo: a stock with dominant 2nd chromosome marker L (lobed eyes) over a 2nd chromosome
balancer
stubby(1)/Cyo-GFP: a stock with the only available allele of stubby: a homozygous lethal allele
of stubby over another 2nd chromosome balancer (stubby(1) contains a point mutation in the
kinase domain that severely weakens the kinase function)
With these tools, draw your mutagenesis/crossing scheme to 1) isolate new homozygous lethal alleles
of stubby and 2) create a stock of your new homozygous lethal allele.
Figure A
You suddenly remember that in your F2s from a parallel cross of another isolated male you saw stubby
adults, and you suspect that this might be due to another non-lethal mutant allele over stubby(1) that
had arisen when you mutagenized the males. However when you go back to isolate this mutation, you
find that these flies have drowned in the food.
You decide to carry out the screen again, this time for homozygous viable mutants with a stubby
phenotype; this time you are limited to using only the following lines:
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y+/y+: a stock homozygous for the dominant 2nd chromosome insertion y+, in a y- background
(remember y- flies are yellow, so the y+ insertion makes them black again)
L/Cyo: a stock with dominant 2nd chromosome marker L (lobed eyes) over a 2nd chromosome
balancer (these flies are yellow (y-))
stubby(1)/Cyo-GFP: a stock with the only available allele of stubby: a homozygous lethal allele
of stubby over another 2nd chromosome balancer (these flies are yellow (y-))
With these tools, draw the first cross of your mutagenesis/crossing scheme.
Figure B
Luckily, in your F1 generation you again find non-curly, black adult flies that have stubby bodies. You
suspect that these stubby flies might have a homozygous-viable allele of stubby.
Draw out the rest of your crossing scheme to 1) isolate your new homozygous viable stubby allele and
2) create a stock of your new homozygous viable stubby allele.
Figure C
You sequence your new homozygous viable allele of stubby and find that it has a point mutation in one
of the domains thought to be structurally important. You test the enzymatic activity of the kinase
encoded by this allele and find that it does indeed have more activity than the enzyme encoded by
stubby(1) but less activity than wild type. This suggests that your new allele slightly weakens the
function of the protein such that mutants still are stubby, but are at least able to grow to adulthood,
while the kinase domain point mutation in stubby(1) severely weakens the function of the kinase such
that the mutants cannot survive to adulthood.