Download 1 - UCSF Biochemistry

You are studying a chromosome II gene yfg because it encodes a transcription factor
that interests you. You probe for the expression of yfg (in situ hybridization) and find that
is expressed in a group of dorsal cells distributed in a diamond pattern and group of
ventral cells in a spade pattern in each segment of the embryo, and when you look at the
wing disc of wandering larvae you find that your gene is expressed in a group of cells in
a club shaped pattern (these patterns of expression are detected by in situ hybridization
to RNA). Twenty-five kb of sequence upstream of yfg lack any other annotated genes.
You look in the genomic sequence of twelve other Drosophila species
(http://flybase.org/blast/) and find that there are a number of stretches of 15 to 45 bp of
perfect conservation of sequence within these upstream sequences. These tiny islands
perfect conservation are clustered into three archipelagos each about 3 kb in size
separated by several kb of non conserved sequence. You probe for the pattern of
expression in Drosophila pseudoobscura (one of the sequenced species that you looked
at) and you find expression in the embryonic diamonds and spades resembling what you
saw in D. melanogaster, but the wing disc expression is now in the shape of a heart.
Descriptions of sensory organ distribution in D. melanogaster and D. pseudoobscura
mention a heart like distribution in wings of D. pseudoobscura and a club like distribution
in D. melanogaster. You look at the sensory organ distribution in larvae and find a
segmentally repeated diamond pattern on the dorsal side and a segmentally repeated
spade pattern on the ventral side. A late larval lethal mutation mapping close to yfg was
reported to have behavioral defects and you find that it lacks the ventral sensory organs.
As you answer these questions you can assume
- that you have done screens to isolate mutants in the structural gene and in the
various regulatory regions of yfg.
- that all the mutations are lethal when homozygous except for ones that only alter
the pattern of expression of yfg in the adult wing, and that these mutations alter
the distribution of sensory organs in the wing.
- that you have a deletion that covers yfg, both structural and regulatory regions.
- That you have stocks of your lethal alleles over a balancer e.g. CyO
And for your answers: please be fairly specific; if you use constructs, flies, crosses and
analyses, indicate what they are but feel free to invent markers, promoters etc.
(1) You are intrigued! How would you test the hypothesis that each archipelago of
conserved sequences acts autonomously to direct expression of yfg in each of its
suit like shapes?
(2) How would you test whether expression of yfg is sufficient to direct formation of
sensory organs?
(3) How would you determine whether the change in the distribution of yfg in the
wings (i.e. club pattern in D. melanogaster and a heart pattern of D.
pseudoobscura) was due to differences in cis-acting elements?
(4) How do you expect the mutations in the structural gene and in the regulatory
regions to behave in complementation tests (a) with the deletion and (b) when
tested against each other?
(5) Given that mutation of the structural gene is lethal to the fly, how would you test
whether yfg function is required for sensory organ formation?