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The Notch developmental pathway is used to send chemical
signals between adjacent cells during development. The signal can
either induce or inhibit the development of the target cell. Several
proteins involved in the pathway such as mastermind (MAM) and Suppressor
of Hairless (SuH) have already been identified and are well studied.
It is possible that other genes not yet discovered may influence
the function of the Notch pathway. Using transposable EP elements
to over express random genes at the wing margin of Drosophila melanogaster
we may identify genes that may play a role in the Notch developmental
pathway in addition to those currently being studied. The current
screen yielded several interesting results. Several products of
the screen showed enhancements to the control nicked-wing phenotype
with satisfactory penetrance and were crossed to determine the chromosome
on which the EP element landed. In the future the DNA of the flies
with enhanced wing phenotypes can be sequenced to pinpoint the location
of the EP element.
The Notch and Delta proteins are both essential proteins
that play a role in developing the nervous system and many other
structures of Drosophila melanogaster. Once a proneural cell has
formed, the Notch and Delta proteins work to laterally inhibit neuroblast
development in adjacent cells through chemical signaling. The Delta
protein acts as the signal and Notch receives the signal. When Notch
is activated by Delta the proneural genes of the cell are inhibited
and the cell will no longer become a neuroblast. A similar situation
exists at the developing wing margin where the Notch signaling is
required for the induction of key target genes (Wolpert et al. 1998).
Figure 1 below graphically shows the processes involved in the Notch
developmental pathway. Notch and Delta interaction has several consequences.
First presenilin a protease enxymatically cleaves the Notch protein
inside the target cell creating Notch intracellular (Notch IC).
Notch IC then interacts and forms a complex with the MAM and SuH
proteins. This complex binds to the promoter region of the target
genes such as E(spl) in the nervous system and vestigial at the
wing margin. MAM has been identified as a protein necessary for
normal function of the Notch developmental pathway. The Mam cDNA
is 6333 nucleotides in length. The protein produced from this region
is believed to contain three chemically significant regions including
1 basic region and 2 acidic regions (Smoller et al. 1990). Findings
suggest that the basic region of the protein physically associates
with the Notch IC protein found within cells following interaction
between Delta and Notch receptors. Through gene manipulation genes
that code for truncated versions the MAM protein have been inserted
into the genome of various strains of Drosophila melanogaster
(see figure 2). These truncations retain the basic region of the
protein but lack more carboxy segments that contain the acidic regions.
It has been shown previously that expression of MAM truncations
can elicit dominant negative phenotypes (Helms et al. 1999). For
example expression across the wing margin produces nicked wing phenotypes.
These truncations are expressed via the Gal4-UAS system. When the
yeast protein Gal-4 a transcriptional activator binds to the UAS
region it activates downstream loci. The Gal-4 complex binds to
the EP region of the DNA which contains the UAS sequence and promotes
transcription of the adjacent gene. Using the enzyme transposase
scientists have successfully moved DNA around the Drosophila melanogaster
genome. One strain of flies used in the current genetic screen is
the EP+ strain. The EP element inserted into the genome contains
the UAS regulatory sequence upstream from a promoter region. Theoretically
if the EP promoter were to land next to a gene that influences the
Notch pathway and the gene was over expressed then differences in
wing phenotypes could be observed in the form of either suppression
or an enhancement to the phenotype created using the truncated MAM
protein. The final cross of the screen uses flies with the C96-Gal4
gene and the UAS-MamH gene on the same chromosome. Gal-4 works to
drive the UAS-MamH gene along with the UAS region of the EP element
and promotes the expression of the loci. We compare the control
nicked wing phenotype to that which occurs when the EP element drives
a random gene. Alterations in the wing phenotype may reflect expression
of a new notch pathway locus.
The screen is run through a series of genetic crosses.
The first cross uses virgin female flies with the Epw+ gene found
on each X chromosome. These females are crossed with males that
are W- and have the gene for the transposase enzyme inserted onto
one of their autosomes. After twelve days of incubation the males
produced from the cross are selected for and the females are discarded.
3 males from the first cross are placed in a vial and introduced
to 4 virgin females with the W1118 gene on each of their X chromosomes
and are allowed to mate freely. The vials for these crossed are
incubated and can be scored in the period between 12-15 days. When
scoring this cross eye color is important. Males are selected for
that have non-mottled w+ eyes. The male fly's eye color is recorded
and each male fly found with this phenotype is assigned a number
and letter according to which vial it came from. The eye color selected
for indicates that the EP element moved from the X chromosome to
an autosome in the Drosophila melanogaster genome and is
worth further investigation. These males are placed in their own
vial. They are introduced to three virgin females who have the W-
gene on both of their X chromosomes and C96-RH2/Sb on chromosome
3. This cross is incubated and scored at 12 days. Flies from this
cross are scored for their wing phenotype. Any changes from the
usual phenotype will result in further testing. If an interesting
wing phenotype is encountered a male with the EPw+ and stubble genes
is mated with 4 W1118 virgin females. This cross will map the location
of the hop to either chromosome 2 or chromosome 3. Another follow
up cross involves taking a male with the enhanced wing phenotype
and crossing him with C96RH2/Sb. This cross will determine if the
enhancement or suppression is genuine and not due to chromosomal
breakdown or a new background mutation.
By the conclusion of the experiments 10 week period
over 450 individual hops have been scored. Out of the hops scored
4 have produced enhancements with acceptable phenotype penetrance.
Each was retested as described in the methods section. The first
of these vials 1053A contained 10 flies with enhancements out of
49 total flies from the original set of crosses. After analyzing
the vial it was subjected to a retest. The results of the retest
also produced interesting results. The enhancement showed up again
in these flies. The enhancement was viewed on both stubble bristled
flies along with normal bristled flies. In the retest 56.9% of the
flies showed some enhancement. Scoring the results of the retest
using the W1118 virgin females assisted in mapping the hop to either
chromosome 2 or 3. The vial had stubble males with white colored
eyes only suggesting that the hop landed on the second chromosome.
Vial 1073B also showed promising enhancements. 28 flies out of a
total of 129 showed some enhancement. When retested however the
enhancement did not show up in the progeny. Vial 1078B showed similar
results to 1073B. The vial had 20 enhanced flies out of a total
of 98. Again when retested the enhancement did not reappear in the
resulting progeny. Vial 1207B had 21 enhancements out of a total
of 75 flies. This vial showed the most prominent penetrance. We
are awaiting the results of the follow up crosses.
Vial 1053A has yielded positive results that after further testing
may lead to the discovery of a new enhancer. The original penetrance
was near the theoretical maximum and the enhanced wing phenotype
reappeared after the test cross. The results of the test cross suggest
that the EP hop landed on the second chromosome. Flies with enhanced
wings were found from the test cross that had both normal and stubble
bristles. The stubble gene is found on the third chromosome of the
drosophila genome of the C96RH2/Sb virgin females used in the cross.
The male we used for the retest also had a stubble gene on its third
chromosome but lacked C96-Gal4. In the test cross both stubble and
non-stubble flies displayed varying degrees of enhancement. In order
for there to be stubble and non-stubble flies with the phenotype
the EP hop would have to have landed on the second chromosome and
not the third chromosome. The results of the retest with W1118 virgins
helped to affirm our belief that the hop landed on chromosome 2.
This vial will most likely undergo further testing. Some of the
vials failed to produce hops that passed the retest. Two possible
reasons exist for the failure for these enhancements to pass. First
and most likely is a spontaneous mutation within the C96RH2/Sb stock
retained in the lab. The stock is several years old and consists
of thousands of flies. If one of these flies underwent a spontaneous
mutation and its genes were passed on through the stock itís
possible that the mutation caused and enhancement. This enhancement
would be passed down through the progeny and could affect the results
of the genetic screen. Another possibility that is less likely but
is still a possibility is genetic duplication. A duplication of
either the C96-Gal4 gene or the UAS-MamH gene could produce an enhancement
to the control phenotype. A duplication of either of these genes
could possibly cause the control phenotype to appear more severe
than before because they are the essential elements driving the
system. Further testing on vial 1053A will reveal if the EP element
is producing a genuine enhancement. If this proves to be true then
the DNA of the strain of flies can be sequenced and the EP elements
location within the genome can be identified. After identification
of the EP loci downstream genes can be analyzed for their possible
role in causing the enhancement.
This material is based on work supported by the National Science
Foundation under Grant No. CHE- 0316076 and the SURE program of
Emory University.
Much of the genetic code of different types of animals is still
a mystery to scientists. The amount of information contained within
is vast and it will take a long time to understand it. Our experiment
attemtps to find new genes of fruit flies that we do not understand.
Specifically we are looking for genes that help the fly develop
into its adult form.
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