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.
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
suppressed phenotype. Within the deficiency, the vulcan gene was found to be
the enhancer of the Ptpmeg rough-eye phenotype due to the nature of its
conserved domain. Further studies will experiment with Vulcan directly to
examine 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 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). They naturally act as mutagens by inserting themselves into genomic
DNA and, furthermore, allow for the expression of any gene of interest in the Drosophila
genome. This is achieved by inserting a Gal4 P element into the genomic DNA which will then
activate a promoter that controls the gene of interest (Beckingham, 2005). Employing Gal4, an
attempt was made to determine the gene responsible for the regulation of the ptpmeg gene in
Drosophila by over expressing 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 a first cross would produce either a suppressed or enhanced rough-eye phenotype.
The same phenotype was expected after a second cross with a smaller deficiency in order to
narrow down the region responsible for the observed effect. The specific gene that regulates
ptpmeg was to be identified after using Bioinformatics to study annotations of the Drosophila
genome.
It was expected that the vulcan gene would regulate ptpmeg. While the gene’s function is
unknown, its conserved domain expression influences ptpmeg activity. The vulcan gene
potentially encodes for guanylate kinase-associate proteins (GKAP) which organize postsynaptic
density (PSD) proteins that include signaling proteins (Romorini, 2004). Vulcan’s
influence on neuronal development implicates it as a regulator of ptpmeg which, as mentioned, is
also involved in cell-cell signaling. Vulcan’s deletion was thus expected to influence ptpmeg
expression and lead to the presence of a suppressed rough eye phenotype.
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MATERIALS AND METHODS
Experimental crosses with deficiency mutants
Flies from deficiency strain 1491 and 3003 were separately mated to GMR-Gal4.UAS-Ptpmeg2F
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 rougheye 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 (Kosinski-Collins, 2008). Genes
deleted by the deficiency were investigated using NCBI’s BLAST as described (Kosinski-Collins,
2008)
RESULTS
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 cross between deficiency strain 3003 (♂)
and Ptpmeg/Cy (♀)
Balancer Associated
Balancer
Phenotype
Eye Phenotype
Number of Progeny
Genotype
Curly Wings, Stubble
Balancer I+
Bristles
Balancer II+
WT
14
Balancer I+
Balancer IIBalancer IBalancer II+
Balancer IBalancer II-
Curly Wings
WT
14
Stubble Bristles
Straight wings, Straight
Bristles
Rough
16
Rough
20
Balancer I is CyO and balancer II is Sb. WT refers to wild-type eye and
Rough refers to rough eye similar to the Ptpmeg virgin females.
Table 2: Observed phenotypes and progeny counts for cross between deficiency strain 1547 (♂)
and Ptpmeg/Cy (♀)
Balancer
Associated
Balancer
Phenotype
Eye Phenotype
Number of Progeny
Genotype
Balancer I+
Balancer II+
Balancer I+
Balancer IIBalancer IBalancer II+
Balancer IBalancer II-
-
-
-
Curly Wings
WT
21
Curly Wings
Straight wings, Straight
Bristles
Rough
23
Rough
27
Balancer I is CyO and balancer II is CyO. WT refers to wild-type eye and
Rough refers to rough eye similar to the Ptpmeg virgin females
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Table 3: Observed phenotypes and progeny counts for cross between deficiency strain 1491 (♂)
and Ptpmeg/Cy (♀)
Balancer Associated
Balancer
Phenotype
Eye Phenotype
Number of Progeny
Genotype
Balancer I+
Balancer II+
Balancer I+
Balancer IIBalancer IBalancer II+
Balancer IBalancer II-
-
-
-
Curly Wings
WT
22
Curly Wings
Straight wings, Straight
Bristles
Rough
22
Rough
40
Balancer I is CyO and balancer II is CyO. WT refers to wild-type eye and
Rough refers to rough eye similar to the Ptpmeg virgin females.
Table 4: Observed phenotypes and progeny counts for cross between deficiency strain 3003 (♂)
and Ptpmeg/Cy (♀)
Balancer Associated
Balancer
Phenotype
Eye Phenotype
Number of Progeny
Genotype
Curly
Wings,
Stubble
Balancer I+
Bristles
Balancer II+
WT
11
Balancer I+
Balancer IIBalancer IBalancer II+
Balancer IBalancer II-
Curly Wings
WT
11
Stubble Bristles
Straight wings, Straight
Bristles
Rough
11
Rough
17
Balancer I is CyO and balancer II is Sb. WT refers to wild-type eye and
Rough refers to rough eye similar to the Ptpmeg virgin females.
A class-wide analysis was carried out on F1 progeny from various deficiency strains. As shown
in tables 1 – 4, our individual results 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. 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.
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Table 5: Observed phenotypes and progeny counts for cross between deficiency strain 9503 (♂)
and Ptpmeg/Cy (♀)
Balancer
Associated
Balancer
Phenotype
Eye Phenotype
Number of Progeny
Genotype
Balancer I+
Balancer II+
Balancer I+
Balancer IIBalancer IBalancer II+
Balancer IBalancer II-
-
-
-
Curly Wings
WT
19
Curly Wings
Straight wings, Straight
Bristles
Rough
52
Rough
27
Balancer I is CyO and balancer II is CyO. WT refers to wild-type eye and
Rough refers to rough eye similar to the Ptpmeg virgin females.
Table 6: Observed phenotypes and progeny counts for cross between deficiency strain 3003 (♂)
and Ptpmeg/Cy (♀)
Balancer Associated
Balancer
Phenotype
Eye Phenotype
Number of Progeny
Genotype
Curly
Wings,
Stubble
Balancer I+
Bristles
Balancer II+
WT
9
Balancer I+
Balancer IIBalancer IBalancer II+
Balancer IBalancer II-
Curly Wings
WT
17
Stubble Bristles
Straight wings, Straight
Bristles
Rough
8
Rough
6
Balancer I is CyO and balancer II is Sb. WT refers to wild-type eye and
Rough refers to rough eye similar to the Ptpmeg virgin females.
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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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.
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.
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The genes within this region were analyzed and, as shown in Table 5, their functions, if known,
were determined. Genes of unknown function were characterized by the presence of conserved
domains.
Table 5: Gene Information deleted by deficiency strain Df(2R)M41A8
Number of Gene
Name of Gene
Function
Gene 1
vlc
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
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, 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. As shown in Table 6, a conserved
domain associated with guanylate kinase-associate protein (GKAP) was detected.
DISCUSSION
When the F1 progeny were scored from the first cross, our individual results were already known
to be unviable. While setting up the cross, the males and females from each of the 1491, 3003,
3007, and 1547 deficiency strains were not separated so both sexes were added into the same vial
along with the Ptpmeg virgin flies. For this reason, the progeny were not analyzed for an
enhanced or suppressed Ptpmeg rough-eye phenotype and the progeny counts were not compared
to the expected ratios. Nevertheless, the flies were observed but the ptpmeg/Deficiency flies’
phenotypes, as Tables 1 - 4 demonstrate, show no enhancer or suppressor covered by any of the
deficiency strains from this first cross. Instead, class data showed that crosses with deficiency
strain 739 revealed a suppressed phenotype and, thus, an enhancer that regulates ptpmeg’s
signaling activity involved in eye development was within the deleted region.
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 within or overlapping the implicated 739 strain. A 1:4
ratio was expected for the 3003 deficiency strain with Sb balancers while only a 1:3 ratio was
expected for the 9503 strain with CyO balancers since the progeny with CyO/Cyo genotypes
would die due to homozygous balancers. The progeny count did not conform to these ratios, as
shown in Tables 5 and 6, however, suggesting that the deficiencies may have fatally harmed the
development of the flies. It is also possible that incubation conditions did not allow for proper
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 . Once again, however, Tables 5 and 6 suggest
that our individual crosses did not produce a suppressed Ptpmeg rough-eye phenotype. Class
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data, however, implicated deficiency strain 740 since its cross with Ptpmeg flies resulted in the
phenotype for Ptpmeg/Def progeny.
The Vulcan gene was identified, through Bioinformatics, as the enhancer of ptpmeg activity due
to the nature of its conserved domain. Initially, the enhancer 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. Since vulcan’s conserved
domain, as shown in Table 8, is associated with regions that encode for guanylate kinaseassociated proteins (GKAPs), it potentially influences ptpmeg protein expression via proteinprotein binding domains as expected rather than gene level expression. Furthermore, since
GKAPs are known to organize signaling proteins, Vulcan appears to be necessary for proper
neuronal transmission which implicates the gene’s affiliation with ptpmeg which is necessary for
proper neuronal development. Ptpmeg may not have the ability to control cell-cell signaling
pathways without the influence of Vulcan on axonal activity.
Since vulcan’s gene function is not known, however, the study 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
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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