Frog Anatomy Brochure

8/10/2019 Frog Anatomy Brochure

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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 maleand 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 givengenetic mechanism under study, classifying them as especially genetically amenable

organisms.

D. melanogastercarry one sex chromosome and 3 autosomal chromosomes. Their

genome is mapped out and readily available to scientists for study. Genetic screening is atool 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 combinationwith 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 promoterupstream 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, incombination 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 thisenhanced version of the rough eye (Kang et al., 2014).

Figure 1There are a couple of possible outcomes in thePtpmeg cross. There are two possible

manifestations of the rough eye phenotype inD.

melanogaster (Kosinski-Collins, 2014).


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MATERIALS AND METHODS

Setting Up Experimental CrossesWild-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 afemale virgin fly from a female non-virgin fly. The organisms were consequentlydissected, 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) femalevirgin flies containing the gene as described (Kosinski-Collins, 2014).

Phenotypic Analysis of Progeny from Crosses

After anesthetization, cross progeny were examined under microscope forphenotypic showings. The progeny were examined and counted for enhanced or

suppressed rough eye as described (Kosinski-Collins, 2014).

Scoring Progeny by PhenotypeUsing websiteshttp://www.flybase.organdhttp://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 CyoPtpmeg ~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 manifestationsassociated with the deficiencies located on Chromosome 2 in theD. 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 orenhancer was deleted.
http://www.flybase.org/http://www.flybase.org/http://www.flybase.org/http://www.ncbi.nlm.nih.gov/BLASThttp://www.ncbi.nlm.nih.gov/BLASThttp://www.ncbi.nlm.nih.gov/BLASThttp://www.ncbi.nlm.nih.gov/BLASThttp://www.flybase.org/


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Punnet Square for Tb (Chromosome 3)

+/Def +/Tb

Ptpmeg/+ ~Ptpmeg/+; +/def

~straight wing, long bodies,unknown eyes

~Ptpmeg/+; +/Tb

~straight wings, tubbybodies, 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 manifestationsassociated with the deficiencies located on Chromosome 3 in theD. 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 thepresence 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 femalevirgin flies containing the gene and the male deficiency line 9719.


Balancer-

Associated

Phenotype


Progeny

% Progeny

Curly Wings Rough 5 50

Curly Wings Wild-Type 1 10

Straight Wings Rough Eye 4 40

Straight Wings Wild-Type --------- 0The 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.


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Balancer-

Associated

Phenotype


Progeny

% Progeny

Curly Wings Rough 5 33.3

Curly Wings Wild-Type 5 33.3Straight 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.


Balancer-

Associated

Phenotype


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 thatthere 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


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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 activity27 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 domainsdeleted by the deficiency strain Df(2L)BSC, and the domains respective functions as

taken fromhttp://www.flybase.org.
http://www.flybase.org/http://www.flybase.org/http://www.flybase.org/http://www.flybase.org/


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Conserved domain of unknown genes of Df(2L)BSC

3 CG14933 Kazal Type Serine Protease


6 CR43936 Unknown function

14 CG16964 Unknown function

15 CR43314 Unknown function18 CG34163 Protein coding






39 CG6746 PTPLA; Protein tyrosine

phosphatase-like activity

42 CG6766 PRKCSH; Glucosidase II

beta subunitlike protein

47 CR44590 Unknown function50 CR44589 Unknown function


From smaller male deficiency, 24348, these were the indicated gene functions ofthe unknown domains that were deleted by the deficiency strain Df(2L)BSC, as taken

fromhttp://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 deficiencystrain 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 deficiencystrain 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 tableConserved 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
http://www.ncbi.nih.gov/BLASThttp://www.ncbi.nih.gov/BLASThttp://www.ncbi.nih.gov/BLASThttp://www.ncbi.nih.gov/BLAST


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REFERENCES

Baade, J. 2014.Lecture 1: Using Drosophila as a model organism. January 14. (fromBiology 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 geneencoding a cytosolic protein tyrosine phosphatase homologous to cytoskeletal-associated

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 phosphatasePTPMEG 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 LaboratoryManual. Brandeis University, pp. 18-55.

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Frog Anatomy Brochure