Biology 18a
Drosophila Lab Report
April 29th, 2008
Phenotypic screen of deficiency libraries implicates gene
responsible for regulation of Ptpmeg over-expressed rough eyes
in Drosophila
Jeremy Gormley
Collaborator: S. Gerad
Department of Biology, Brandeis University. Supervised by Professor Melissa Kosinski-Collins and
Teaching Assistant Yaihara Fortis
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ABSTRACT
In Drosophila, the Ptpmeg gene encodes for a protein responsible for cell
signaling and eye development. We determined which gene in Drosophila
regulates Ptpmeg expression by performing screens of various deficiency
libraries. We crossed virgin female flies over-expressing Ptpmeg with deficiency
mutants to look for an enhancement or suppression of the rough-eye phenotype
associated with the Ptpmeg virgin flies. A suppressed phenotype was identified
and a smaller deficiency mutant was then used in a second cross to determine
which gene within the deficiency caused the observed effect. Region 740 was
implicated as the region responsible for the suppressed rough eye phenotype and
the use of bioinformatics allowed us to localize and characterize possible
candidate genes whose deletion caused the suppressed phenotype. Within the
deficiency, the Vulcan gene was found to be the enhancer of the Ptpmeg rougheye phenotype. This was determined after relating the nature of Vulcan’s
conserved domain to the function of Ptpmeg. Further studies will resolve
experimental errors and analyze Vulcan directly to precisely determine how it
interacts with Ptpmeg.
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INTRODUCTION
Homologs of human genes involved in life-threatening diseases are useful to study when trying to
understand the function or regulation of their mammalian counterparts. Experiments that
purposely mutate genes to observe the ensuing effects, while not always possible in humans, are
uncontroversial when performed with other organisms and may eliminate genes’ potential risks.
Drosophila is one organism that shares many genetic and developmental aspects with human
beings, thus acting as a useful tool in genetic studies. First, Drosophila is a small species with a
short life cycle (approximately 10 days) that can be used to produce a large number of progeny
from a small number of parents (Kosinski-Collins, 2008). Second, its genome has been studied
extensively and deficiency strains – flies with part of their genomic DNA deleted – are readily
available to allow for genetic mapping of small, designated chromosome regions (Beckingham,
2005).
Another significant advantage of using drosophila stems from the effectiveness of P elements. P
elements are transposons that have been used to analyze gene function in Drosophila in vivo
(Beckingham, 2005). A Gal 4 P element, for instance, can express any gene of interest after
being inserted into genomic DNA and activating a promoter to control a gene (Beckingham,
2005). Employing Gal4, the Ptpmeg gene was over-expressed in an attempt to determine which
gene is responsible for the regulation of Ptpmeg.
Ptpmeg is an autosomal gene that encodes for cytoplasmic tyrosine phosphatase proteins which
are involved in key cellular events such as passage through the cell cycle and differentiation (Gu,
1996). In particular, Ptpmeg has found to be involved in the eye development of Drosophila and
plays an important role in cell-cell signaling that influences neuronal circuit formation in the
Drosophila brain (Whited, 2007). In humans, a homolog of Ptpmeg, PTPN3, can act as a colon
cancer tumor suppressor gene (Whited, 2007). Thus, analyzing Ptpmeg in Drosophila may help
us better understand genes like it in other organisms.
In order to determine which gene regulates Ptpmeg, female flies over-expressing wild-type
Ptpmeg would be crossed with males of various deficiency strains that cover an enhancer or
suppressor of Ptpmeg. Each Ptpmeg construct uses CyO balancers on chromosome 2 while
deficiency strains use either CyO balancers or Sb on chromosome 3. The balancers act as
dominant markers and prevent recombination between homologous chromosomes.
It was expected that the crosses would implicate the Vulcan gene as a regulator of Ptpmeg.
Vulcan potentially encodes for guanylate kinase-associate proteins (GKAP) which are known to
organize signaling proteins (Romorini, 2004). Vulcan’s influence on neuronal development
through signaling implicates it as a regulator of Ptpmeg which, as mentioned, is also involved in
cell-cell signaling. If Vulcan’s deletion produced an enhanced rough-eye phenotype, it could be
characterized as a suppressor while a suppressed rough-eye phenotype would implicate it as an
enhancer.
The first cross produced a suppressed rough-eye phenotype which was then observed again in the
second cross with a smaller deficiency. Bioinformatics was used to study annotations of the
deficiency region within the Drosophila genome, implicating Vulcan as an enhancer of Ptpmeg.
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MATERIALS AND METHODS
Experimental crosses with deficiency mutants
Drosophila from deficiency strains 1491 and 3003 were separately mated to Ptpmeg virgin flies
as described (Kosinski-Collins, 2008). All crosses were incubated at 29°C for two weeks with
parent flies transferred to new vials after one week as described (Kosinski-Collins, 2008). F1
progeny were scored as described (Kosinski-Collins, 2008). The process was repeated with the
more localized deficiency strains, 3366 and 9503, obtained after class data identified from the
first cross which part of the genome was involved in the regulation of the Ptpmeg rough-eye
phenotype.
Bioinformatics using deficiency strain
Based on class data from the second cross, FlyBase was used as described to explore the 740
deletion strain that covered the genomic region h44 - 41A3 in Drosophila (Kosinski-Collins,
2008). Genes deleted by the deficiency were investigated using NCBI’s BLAST as described
(Kosinski-Collins, 2008)
RESULTS
First cross with large deficiency strains
After the first cross was set up with large deficiency mutants of the Drosophila genome, the F1
progeny were scored for the presence of an enhanced or suppressed Ptpmeg rough-eye phenotype.
Table 1: Observed phenotypes and progeny counts for the F1 progeny of the cross between
deficiency strain 3003 (♂) and Ptpmeg virgin flies (♀)
Balancer Associated Phenotype
Curly Wings, Stubble Bristles
Curly Wings
Stubble Bristles
Eye Phenotype
Number of Progeny
WT
14
WT
14
Rough
16
Straight wings, Long Bristles
Rough
20
WT refers to wild-type eye and Rough refers to rough eye similar to the
Ptpmeg virgin females.
The phenotypes of the flies’ wings, bristles, and eyes were determined by inspection for the cross
with deficiency 3003. As shown in Table 1, 14 flies were observed with curly wings, stubble
bristles and wild-type eyes; 14 flies were observed with curly wings and wild-type eyes; 16 flies
were observed with stubble bristles and rough eyes; and 20 flies were observed with straight
wings, long bristles, and rough eyes.
Table 2: Observed phenotypes and progeny counts for the F1 progeny of the cross between
deficiency strain 1547 (♂) and Ptpmeg virgin flies (♀)
Balancer Associated Phenotype
Curly Wings
Curly Wings
Eye Phenotype
Number of Progeny
-
-
WT
21
Rough
23
Straight wings, Long Bristles
Rough
27
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WT refers to wild-type eye and Rough refers to rough eye similar to the
Ptpmeg virgin females
The phenotypes of the flies’ wings, bristles, and eyes were determined by inspection for the cross
with deficiency 1547. As shown in Table 2, 21 flies were observed with curly wings and wildtype eyes; 23 flies were observed with curly wings and rough eyes; and 27 flies were observed
with straight wings, long bristles, and rough eyes.
Table 3: Observed phenotypes and progeny counts for the F1 progeny of the cross between
deficiency strain 1491 (♂) and Ptpmeg virgin flies (♀)
Balancer Associated Phenotype
Curly Wings
Curly Wings
Eye Phenotype
Number of Progeny
-
-
WT
22
Rough
22
Straight wings, Long Bristles
Rough
40
WT refers to wild-type eye and Rough refers to rough eye similar to the
Ptpmeg virgin females
The phenotypes of the flies’ wings, bristles, and eyes were determined by inspection for the cross
with deficiency 1491. As shown in Table 3, 22 flies were observed with curly wings and wildtype eyes; 22 flies were observed with curly wings and rough eyes; and 40 flies were observed
with straight wings, long bristles, and rough eyes.
Table 4: Observed phenotypes and progeny counts for the F1 progeny of the cross between
deficiency strain 3007 (♂) and Ptpmeg virgin flies (♀)
Balancer Associated Phenotype
Curly Wings, Stubble Bristles
Curly Wings
Stubble Bristles
Eye Phenotype
Number of Progeny
WT
11
WT
11
Rough
11
Straight wings, Long Bristles
Rough
17
WT refers to wild-type eye and Rough refers to rough eye similar to the
Ptpmeg virgin females.
The phenotypes of the flies’ wings, bristles, and eyes were determined by inspection for the cross
with deficiency 3007. As shown in Table 4, 11 flies were observed with curly wings, stubble
bristles and wild-type eyes; 11 flies were observed with curly wings and wild-type eyes; 11 flies
were observed with stubble bristles and rough eyes; and 17 flies were observed with straight
wings, long bristles, and rough eyes.
A class-wide analysis was carried out on F1 progeny from various deficiency strains. As shown
in tables 1 – 4, our individual crosses did not produce an enhanced or suppressed Ptpmeg rougheye phenotype. However, a suppressed Ptpmeg rough eye phenotype was observed for the cross
with the 739 deficiency strain.
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Second cross with smaller deficiency strains
A second cross was set up with smaller deficiency strains to localize the region within the
deletion of the 739 strain responsible for the observed phenotype.
Table 5: Observed phenotypes and progeny counts for cross between deficiency strain 9503 (♂)
and Ptpmeg/Cy (♀)
Balancer Associated Phenotype
Curly Wings
Curly Wings
Eye Phenotype
Number of Progeny
-
-
WT
19
Rough
52
Straight wings, Long Bristles
Rough
27
WT refers to wild-type eye and Rough refers to rough eye similar to the
Ptpmeg virgin females.
The phenotypes of the flies’ wings, bristles, and eyes were determined by inspection for the cross
with deficiency 9503. As shown in Table 5, 19 flies were observed with curly wings and wildtype eyes; 52 flies were observed with curly wings and rough eyes; and 27 flies were observed
with straight wings, long bristles, and rough eyes.
Table 6: Observed phenotypes and progeny counts for cross between deficiency strain 3366 (♂)
and Ptpmeg/Cy (♀)
Balancer Associated Phenotype
Curly Wings, Stubble Bristles
Curly Wings
Stubble Bristles
Eye Phenotype
Number of Progeny
WT
9
WT
17
Rough
8
Straight wings, Long Bristles
Rough
6
WT refers to wild-type eye and Rough refers to rough eye similar to the
Ptpmeg virgin females.
The phenotypes of the flies’ wings, bristles, and eyes were determined by inspection for the cross
with deficiency 3366. As shown in Table 6, 9 flies were observed with curly wings, stubble
bristles and wild-type eyes; 17 flies were observed with curly wings and wild-type eyes; 8 flies
were observed with stubble bristles and rough eyes; and 6 flies were observed with straight
wings, long bristles, and rough eyes.
A class-wide analysis was carried out on F1 progeny from the second. Once again, as shown in
table 5 and 6, our individual results did not produce a suppressed Ptpmeg rough-eye phenotype.
After gathering class data, a suppressed Ptpmeg rough eye phenotype was found in the cross with
the 740 deficiency strain.
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Observed eye phenotype of Drosophila
The different eye phenotypes observed were photographed for comparison.
Figure 1: Ptpmeg Rough-Eye Phenotype in Drosophila
Drosophila exhibiting wild-type eyes (left) compared to Drosophila with Ptpmeg rough eyes
Figure 2: Suppression of Ptpmeg Rough-eye Phenotype in Drosophila
Drosophila with Ptpmeg rough eyes (left) compared to Drosophila with suppressed Ptpmeg rough eyes
As shown in Figure 1, the Ptpmeg rough eye phenotype is darker than the wild type eyes with less
defined ommatidium. The suppressed Ptpmeg rough-eye phenotype, as shown in Figure 2, is
lighter and resembles the wild type eyes.
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Bioinformatics
After identifying the 740 deficiency strain as the one associated with the suppressed Ptpmeg
rough eye phenotypes, the deleted region was analyzed using FlyLab. The 740 deficiency strain,
Df(2R)M41A8, was found to cover the h44 – 41A3 region of the genome on the right arm of
chromosome 2. The common genes in the 739 and 740 deficiency strains were analyzed and their
functions, if known, were determined.
Table 5: Gene Information deleted by deficiency strain Df(2R)M41A8
Number of Gene
Name of Gene
Function
Gene 1
vlc (Vulcan)
Gene 2
Nipped-A
unknown
transcription regulatory activity, protein
kinase activity
Gene 3
Nipped-B
transcription regulatory activity
Gene 4
p120 ctn
binding, signal transduction
Four common genes (Vulcan, Nipped-A, Nipped-B, and p120 ctn) were found in the
overlapping regions between strains 739 and 740. Vulcan, as shown in Table 5, was the only
gene found to have an unknown function and was thus characterized using NCBI’s BLAST
sequencing program.
Table 6: Conserved Domains of unknown genes of Df(2R)M41A8
Number of Gene
Name of
Gene
Gene 1
vlc (Vulcan)
Conserved Domain
guanylate kinase-associated protein
(GKAP)
Vulcan was characterized by the presence of a conserved domain. As shown in Table 6, a
conserved domain associated with guanylate kinase-associate protein (GKAP) was detected.
DISCUSSION
When the F1 progeny from the crosses were scored, flies with straight wings and long bristles
corresponded to the Ptpmeg over Deficiency (Ptpmeg/Def) genotype since the phenotypes
associated with Sb and CyO balancers were absent. These progeny were of interest to observe
the effect of the Deficiency on the Ptpmeg rough eyes. As Tables 1-4 demonstrate, 20 F1
Ptpmeg/Def progeny were observed for the cross with deficiency 3003; 27 for the cross with
1547; 40 for the cross with 1491; and 17 for the cross with 3007. All of these F1 progeny with
straight wings and straight bristles showed no enhancer or suppressor but only the same rough
eyes that the Ptpmeg virgin female flies exhibited. Class data showed that crosses with
deficiency strain 739 revealed a suppressed phenotype. This implicated an enhancer within the
deleted region that regulates Ptpmeg’s signaling activity involved in eye development.
F1 progeny from the second cross with deficiency strains 3366 and 9503 were analyzed for the
same suppressed Ptpmeg rough-eye phenotype since the strains used in the second cross covered
a smaller region of the Drosophila genome overlapping the implicated 739 strain. As Tables 5
and 6 demonstrate, 27 F1 Ptpmeg/Def progeny were observed for the cross with deficiency 9503
and 6 for the cross with 3366. Once again, however, these flies with straight wings and long
bristles did not produce a suppressed Ptpmeg rough-eye phenotype. Class data, however,
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implicated deficiency strain 740 since its cross with Ptpmeg flies resulted in suppressed rougheye phenotype for the Ptpmeg/Def progeny.
For both the first and second set of crosses, a 1:4 ratio was expected for the F1 progeny from
crosses with deficiency strains 3003, 3007, and 3366 which had Sb balancers. Only a 1:3 ratio
was expected for the 1547, 1491, and 9503 strain which had CyO balancers, however, since the
progeny with CyO/Cyo genotypes would die due to homozygous balancers. Yet, the progeny
count did not conform to these ratios in any cross, suggesting that the deficiencies likely fatally
harmed the development of the flies. If the experiment were to be changed to include less
harmful balancers, the counts may have more accurately represented the expected genotypic
ratios.
Experimental errors might have also influenced the progeny count. Incubation conditions may
not have allowed for proper fly development. An incubation temperature set too high, for
example, may have made female flies sterile, affecting the progeny count. It is also still possible
that some female flies from the deficiency strains erroneously entered the vials.
Through Bioinformatics, the Vulcan gene was identified as the enhancer of Ptpmeg due to the
suppression of the Ptpmeg rough-eye phenotype. Vulcan was expected to regulate protein
function level rather than gene level expression since the Ptpmeg constructs over-expressed
Ptpmeg with a recombinant promoter, introduced with P elements. After analyzing Vulcan’s
conserved domain, as shown in Table 6, it is evident that, since Vulcan may encode for guanylate
kinase-associated proteins (GKAPs), it indeed may potentially influence Ptpmeg via proteinprotein binding domains which affect protein expression. Furthermore, since GKAPs are known
to organize signaling proteins, Vulcan may be involved in the same neuronal transmissions
necessary for proper eye formation as Ptpmeg. Thus, without Vulcan, Ptpmeg did not have the
full ability to control cell-cell signaling pathways and axonal activity to maintain the Ptpmeg
rough-eye phenotype. Furthermore, the findings support the hypothesis that an observed
suppressed rough-eye phenotype implicates Vulcan as an enhancer and, while it is possible that
other factors may have also contributed to a reduction in the rough-eye phenotype, the experiment
verified Vulcan’s connection to Ptpmeg.
While Vulcan was identified as the enhancer present in the deleted region of deficiency 740,
Vulcan’s gene function remains known. This lends to the need for a further examination of the
regulation of Ptpmeg. Future experiments may include studying the effects that Vulcan’s deletion
in the genome has on neuronal development of the fly, in addition to determining whether the
genes of known function within deficiency 740 may also regulate Ptpmeg in some way that
compliments Vulcan’s function.
REFERENCES:
Kosinski-Collins, M. Spring 2008. Biology 18a Laboratory Manual. Brandeis University,
Waltham, MA.
Beckingham, K. M., Armstrong, J. D., Texada, M. J., Munjaal, R., and Baker, D. A.
2005. Drosophila Melanogaster – The Model Organism of Choice for the Complex
Biology of Multi-cellular Organisms. Gravitational and Space Biology 18: 17 – 30.
Gu, M., and Majerus, P. W. 1996. The Properties of the Protein Tyrosine Phosphatase
PTPMEG. The Journal of Biological Chemistry 271: 27751–27759
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Whited, J. L., Robichaux, M. B., Yang, J. C., and Garrity, P. A. 2007. Ptpmeg is required
for the proper establishment and maintenance of axon projections in the central brain of
Drosophila. Development 134: 43 - 53
Romorini, S., Piccoli, G., Jiang, M., Grossano, P., Tonna, N., Passafaro, M., Zhang, M.,
and Sala, C. 2004. A Functional Role of Postsynaptic Density-95-Guanylate KinasAssociate Protein Complex in Regulating Shank Assembly and Stability to Synapses. The
Journal of Neuroscience. 24: 9391 - 9404
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