INTRODUCTION

Drosophila melanogaster, more commonly known as fruit fly, is an invertebrate

of approximately three millimeters in size. D. melanogaster appear in nature as both male
and female, which comes useful in genetic mating experiments. They produce many
offspring. This is only one reason for which they are considered a model organism
(Kosinski-Collins, 2014).
D. melanogaster are also fairly cheap organisms with a short, easily observable
life span of approximately 12 days (Baade, 2014). D. melanogaster are especially valued
for genetic studies because they have an easily observable phenotype of any given
genetic mechanism under study, classifying them as especially genetically amenable
organisms.
D. melanogaster carry one sex chromosome and 3 autosomal chromosomes. Their
genome is mapped out and readily available to scientists for study. Genetic screening is a
tool used in genetics that is capable of tracing specific genes and mapping out their
conserved domain among species, analyzing the similarities in their amino acid
sequences among organisms.
Ptpmeg is a dosage-dependent gene that is homologous to the gene responsible in
humans as a colon cancer tumor suppressor (Itoh et al., 1993). For example in this
experiment, GMR-Gal4:UAS-Ptpmeg/CyO, the gene that codes for protein ptpmeg, was
used as the focus of this experiment in order to determine whether when in combination
with certain male deficiency should the gene exhibit a rough eye that is suppressed or
enhanced. Gene GMR-Gal4:UAS-Ptpmeg/CyO contains the eye specific promotor,
GMR, GAL4, the gene-encoding transcription factor, UAS, a type of yeast promoter
upstream activator sequence, and the sequence to code for the protein ptpmeg, that is
dosage dependent and will subsequently exhibit either one of several ranges of rough eye
(see Figure 1) (Kina et al., 2007).
Our experiment showed one strain of our male deficiency, #23438, in
combination with a female virgin fly with gene GMR-Gal4:UAS-Ptpmeg/CyO, proved to
show that there was a resulting phenotype of enhanced rough eye. This conclusively
showed that the male deficiency deleted the suppressor of the gene, leading to this
enhanced version of the rough eye (Kang et al., 2014).


Figure 1
There are a couple of possible outcomes in the
Ptpmeg cross. There are two possible
manifestations of the rough eye phenotype in D.
melanogaster (Kosinski-Collins, 2014).

MATERIALS AND METHODS

Setting Up Experimental Crosses
Wild-type flies were observed under the dissecting microscope to be familiar with
D. melanogaster anatomy, eye color, and how to differentiate among the two sexes and a
female virgin fly from a female non-virgin fly. The organisms were consequently
dissected, as described (Kosinski-Collins, 2014).
Our male deficiency lines, labeled: 7614, 9719, 24348, and 5878, were each
compared under the microscope and transferred to new vials with wild-type (WT) female
virgin flies containing the gene as described (Kosinski-Collins, 2014).

Phenotypic Analysis of Progeny from Crosses
After anesthetization, cross progeny were examined under microscope for
phenotypic showings. The progeny were examined and counted for enhanced or
suppressed rough eye as described (Kosinski-Collins, 2014).

Scoring Progeny by Phenotype
Using websites http://www.flybase.org and http://www.ncbi.nlm.nih.gov/BLAST,
gene domains deleted by the deficiency strain Df(2L)BSC were analyzed and the
respective conserved domains of the unknowns were scored as described (Kosinski-
Collins, 2014).


RESULTS

Punnet Square for Cyo (Chromosome 2)
Def Cyo
Ptpmeg ~Ptpmeg/Def
~straight wing, unknown
eye
~Ptpmeg/Cyo
~rough eye, curly wings
Cyo ~Cyo/Def
~curly wing, wild-type eyes
~Cyo/Cyo
(homozygous lethal)

This punnet square shows the possible genotypes and phenotype manifestations
associated with the deficiencies located on Chromosome 2 in the D. melanogaster.
However, the phenotype of interest to the experiment is that outcome between ptpmeg
and the deficiency, resulting in straight wings and unknown eyes. The progeny were
subsequently analyzed for their eye phenotype, indicating whether the suppressor or
enhancer was deleted.






Punnet Square for Tb (Chromosome 3)
+/Def +/Tb
Ptpmeg/+ ~Ptpmeg/+; +/def
~straight wing, long bodies,
unknown eyes
~Ptpmeg/+; +/Tb
~straight wings, tubby
bodies, rough eyes
Cyo, + ~Cyo/+; +/def
~curly wings, long bodies,
wild-type eyes
~Cyo/+; +/Tb
~curly wings, tubby bodies,
wild-type eyes

This punnet square shows the possible genotypes and phenotype manifestations
associated with the deficiencies located on Chromosome 3 in the D. melanogaster. Two
phenotypes associated with this chromosome were tubby bodies(Tb) and stubble bristle
(Sb). However, the phenotype of interest to the experiment is that outcome between
Ptpmeg/+ with +/Def because the subsequent phenotype would be indicative of the
presence of an enhancer and/or a suppressor. The progeny were subsequently analyzed
for their eye phenotype, indicating whether the suppressor or enhancer was deleted.



Table for Cross with 9719
Balancer-
Associated
Phenotype
Eye Phenotype Number of
Progeny
Number of
Progeny (5)
Curly Wings Rough 8 32
Curly Wings Wild-Type 12 48
Straight Wings Rough 5 20
Straight Wings Wild-Type --------- 0
The data collected here shows a scoring of the progeny between that of the female
virgin flies containing the gene and the male deficiency line 9719.


Table for Cross with 7614
Balancer-
Associated
Phenotype
Eye Phenotype Number of
Progeny
% Progeny
Curly Wings Rough 5 50
Curly Wings Wild-Type 1 10
Straight Wings Rough Eye 4 40
Straight Wings Wild-Type --------- 0
The data collected here shows a scoring of the progeny between that of the female
virgin flies containing the gene and the male deficiency line 7614.





Table for Cross with 24348
Balancer-
Associated
Phenotype
Eye Phenotype Number of
Progeny
% Progeny
Curly Wings Rough 5 33.3
Curly Wings Wild-Type 5 33.3
Straight Wings Rough Eye 5 33.3
Straight Wings Wild-Type ------- 0
The data collected here shows a scoring of the progeny between that of the female
virgin flies containing the gene and the male deficiency line 24348.

Table for Cross with 5878
Balancer-
Associated
Phenotype
Eye Phenotype Number of
Progeny
% Progeny
Curly Wings Rough --------- ---------
Curly Wings Wild-Type --------- ---------
Straight Wings Rough Eye --------- ---------
Straight Wings Wild-Type --------- ---------
The data collected here shows a scoring of the progeny between that of the female
virgin flies containing the gene and the male deficiency line 5878. This chart shows that
there were no progeny to score from this cross. The female virgin flies and the male flies
did mate and their larvae showed no signs of significant development. Most likely, the
cross resulted in a homozygous lethal and sterile progeny.

Gene information deleted by deficiency strain Df(2L)BSC (continued to next page)

1 CG44007 3-5 cyclic AMP phosphodiester activity
2 CG31704 Serine type endopeptidase inhibitor activity
3 CG14933 UNKNOWN
4 CG14934 Alpha glucosidase activity
5 CG42751 UNKNOWN
6 CR43936 UNKNOWN
7 CG42486 Protein coding
8 CG14935 Alpha glucosidase activity
9 CG44008 Serine type endopeptidase inhibitor activity
10 CG16960 Olfactory receptor activity
11 CG5006 Olfactory sense receptor
12 CG16961 Olfactory sense receptor
13 CG16963 Calcium ion binding (structural constituent of eye lens)
14 CG16964 UNKNOWN
15 CR43314 UNKNOWN
16 CR42938 Chaeta development
17 CG16965 Alpha, alpha-trehalasce activity
18 CG34163 UNKNOWN
19 CG16969 Cytoplasmic microtubule organization
20 CG31866 Transcription coactivator activity
21 CG31864 Metabolic activity
22 CG12264 Cysteine desulferase activity
23 CG5279 G-protein coupled receptor activity
24 CR44588 UNKNOWN
25 CR44587 UNKNOWN
26 CG5304 High affinity glutamate transmembrane transport activity
27 CG6756 P-P bond hydrolysis protein (in mitochondria)
28 CG6785 UNKNOWN
29 CG6792 Metal ion binding
30 CG12317 Amino acid transmembrane transporter
31 CG7061 Rab GTPase activator activity
32 CG14947 UNKNOWN
33 CR44591 UNKNOWN
34 CG6716 RNA polymerase II distal enhancer
35 CG6686 Neurogenesis
36 CG5202 Histone methyl transferase activity
37 CG18789 Metal ion binding
38 CG31865 Transcription coactivator activity
39 CG6746 UNKNOWN
40 CG6734 Spingomyelin phosphodiesterase activator
41 CG18788 Transmembrane protein
42 CG6766 UNKNOWN
43 CG6770 Response to oxidative stress
44 CG14945 Glycosylphosphagylinostinol diacylglycerolase activity
45 CG5317 Structural constituent of ribosome
46 CG14946 Oxidoreductase activity
47 CR44590 UNKNOWN
48 CG12314 Dorsal appendage formation
49 CR18787 Metal ion binding
50 CR44589 UNKNOWN
51 CG34164 UNKNOWN
From smaller male deficiency, 24348, these were the indicated gene domains
deleted by the deficiency strain Df(2L)BSC, and the domains respective functions as
taken from http://www.flybase.org.
Conserved domain of unknown genes of Df(2L)BSC
3 CG14933 Kazal Type Serine Protease
5 CG42751 Protein coding
6 CR43936 Unknown function
14 CG16964 Unknown function
15 CR43314 Unknown function
18 CG34163 Protein coding
24 CR44588 Unknown function
25 CR44587 Unknown function
28 CG6785 Protein coding
32 CG14947 Protein coding
33 CR44591 Unknown function
39 CG6746 PTPLA; Protein tyrosine
phosphatase-like activity
42 CG6766 PRKCSH; Glucosidase II
beta subunit like protein
47 CR44590 Unknown function
50 CR44589 Unknown function
51 CG34164 Protein coding
From smaller male deficiency, 24348, these were the indicated gene functions of
the unknown domains that were deleted by the deficiency strain Df(2L)BSC, as taken
from http://www.ncbi.nih.gov/BLAST.

DISCUSSION

Crossing female virgin flies expressing ptpmeg from the gene GMR-Gal4:UAS-
Ptpmeg/CyO with male deficiency strains and subsequently scoring their progeny
according to associated balancer (in this case, Cyo) and the eye phenotype, the deficiency
strain 24348 was shown to have the enhanced rough eye phenotype expressed 66.6% of
the time (refer to Table for Cross with 24348). In Mendelian genetics, the deficiency
strain 24348, located on chromosome 2, was to produce a fourth of each possible
phenotype in its F1 generation (Kosinski-Collins, 2014). From the Table for Crosses..
for 9719, 7614, and 24348, rough eye phenotype was the most prevalent in the progeny
for which the male deficiencys balancer contained chromosome 2 for Cyo.
Furthermore, deficiency strain 24348 was analyzed via bioinformatics
programming for the gene functions of the genes deleted domains in order to seek
whether genes suppressor would have been deleted as a result of over-expression of
rough eye in the eye phenotype. The conserved domain of the unknown gene showed
gene CG6746 as functioning in the genome as a protein tyrosine-phosphotase (see table
Conserved Domain of Unknown Genes of Df(2L)BSC). Those of the known genes did
not show possibly functions to suppress ptpmeg suppression activity (see table












REFERENCES

Baade, J. 2014. Lecture 1: Using Drosophila as a model organism. January 14. (from
Biology 18a) Brandeis University, Waltham, Massachusetts.

Itoh, F., Ikuta, S., Hinoda, Y., Arimura, Y., Ohe, M., Adachi, M., Ariyama, T., Inazawa,
J., Imai, K., & Yachi, A. 1993. Expression and chromosomal assignment of PTPH1 gene
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proteins. International Journal of Cancer 55(6): 947-951.

Kang, J., Yeom, E., Lim, J., Choi, K. W.. 2014. Bar Represses dPax2 and
Decapentaplegic to Regulate Cell Fate and Morphogenetic Cell Death in Drosophila Eye.
Public Library of Science 9(2):e88171.

Kina, S., Tezuka, T., Kusakawa, S., Kishimoto, Y., Kakizawa, S., Hashimoto, K.,
Ohsugi, M., Kiyama, Y., Horai, R., Sudo, K., Kakuta, S., Iwakura, Y., Iino, M., Kano,
M., Manabe, T., Yamamoto, T.. 2007. Involvement of protein-tyrosine phosphatase
PTPMEG in motor learning and cerebellar long-term depression. European Journal of
Neuroscience 26(8):2269-2278.

Kosinski, M. C., & Baade, J. 2014. Biology 18a Introductory Biology Laboratory
Manual. Brandeis University, pp. 18-55.