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Transcript
Genetics discussion
“Sex in the Brain”
Drosophila/O’Farrell – Hopefully this will help get
through a somewhat challenging paper.
Hotta and Benzer published a beautiful and
classical analysis in 1972 in which they made
mosaic flies that were half male and half female
(attached). These are gynandromorphs. They
examined their behavior in mating assays and were
able to “map” the behaviors to areas in the brain.
While really elegant and highly recommended, I
didn’t use this paper for the discussion because it
is very much dated. Nonetheless, the assigned
paper tackles a similar issue at much higher
resolution – can we define the neuronal and
genetic basis of specific behaviors?
Kimura K, Hachiya T, Koganezawa M, Tazawa T,
Yamamoto D. (2008) Fruitless and doublesex
coordinate to generate male-specific neurons
that can initiate courtship. Neuron 59: 759-69
I have included a review (Siwicki and Kravitz, 2009)
that might help get you in gear to read this paper
and I suggest that you look at the first two pages of
the review before tackling the paper.
Also the paper uses a fancy version of mosaics
(clonal analysis) in which things are turned around
so that the clones specifically activate expression
of a gene. This is call the MARCM technique. I
presented this in the last lecture and I have
attached a brief technical account of the method. It
is based on clonal loss of GAL80, which is a
repressor of GAL4.
The authors mention in an easily overlooked spot
an important detail in their descriptions of their
genotypes. The thing they describe as a fruNP21reporter has GAL4 under the control of the fru
promoter. It is not directly a reporter, but it induces
genes that are placed under UAS control (in flies
when we say UAS it is short for UASGAL4), and of
course only induces these when the fruNP21-GAL4 is
expressed and when the GAL4 is active.
Importantly, the GAL4 is only active when clones,
induced by action of heat shock induced Flip
recombinase expression, remove the GAL80
repressor (tub in tub-GAL80 refers to the tubulin
promoter which is ubiquitously expressed – e.g
GAL80 is everywhere unless a mitotic clone
removes the chromosome arm carrying this
transgene). The UAS construct that they use to
make the cells green is called UAS-mCD8-GFP.
The mCD8 fusion makes it membrane bound so
that it marks the full path of the neurons. It sounds
pretty complicated and they don’t really say why
they are doing all this just to define the cells that
express fru. The reason is that so many cells
express fru that the brain would be an indiscernible
mass of stain if they were to all express at the
same time. By making clones they can see
individual cells and their processes in each clone
and they add up the result from many clones to
define the whole.
I’ve appended the supplement to the PDF of the
paper. In addition there was a movie that I’ve
include as separate quicktime file if all the stuff
gets uploaded. The movie is nice but just for fun.
The text describing it was:
Movie S1. Male-Type Sexual Behavior Exhibited
by a tra1 Mosaic Female. A tra1 mosaic female with
red eyes follows a wild-type female with white eyes
and occasionally extends one of her wings.
Embryos of the y hs-flp/w; G13 UAS-mCD8-GFP;
tra1 ri FRT2A fruNP21/tub-Gal80 FRT2A genotype
were exposed to heat shock at 37°C for 1 hr to
induce somatic recombination. The last frame of
the video shows the brain from the same mosaic
female whose courtship behavior was recorded in
the video. Double staining of the brain with the
anti-GFP antibody and nc82 Mab showed that the
female fly had P1 neurons as a result of the tra− mediated masculinization, as shown by GFP
expression.
I hope this is enough for you go with. Try and
understand how the genetic tools were used, the
paper spends most of its energy explaining the
biology of the work.
Pat