Chromatography of Drosophila Eye Pigments Jeffrey Jian September 30, 2011 Genetics Lab Lab Partners: Heejoo Kim,

Aneeqa Kabir, Megan Bosi, Vashish Amarjit

Introduction The objective of this experiment was to observe the chromatographic indices towards the genetic makeup of linked traits in the fruit fly species Drosophila melanogaster. To do so, individuals that displayed certain physical characteristics were obtained to have their ommatidia pigments, which were used for sight, isolated and run using the paper chromatography process. The qualitative results of the paper chromatography as a result of the flies' phenotypes were then analyzed for their genotypes. Drosophila was used for this experiment due to its compact nature in both physical structure and genomic size. Its genetic code has been completely mapped and thus many of its genes, such as those for its eyes and wings, have been identified. If a particular gene has been identified, then its respective variations can be observed, and it was thus the variation among the population that was physically isolated for and analyzed in this experiment. Pigments for eye coloration, specially the colors red, brown, sepia, and white, were isolated for both male and female individuals. Because the gene for eye color in Drosophila are linked to the X chromosome - that is, the gene is not found on the Y chromosome in males - it would be rudimentary to identify males that expressed one gene over another. In this situation, having males that only expressed the recessive allele eases identification. Materials and Methods 1. Using a pencil, lightly drew a line 1.75cm from the bottom of each of the two filter papers. Folded the papers in half so that the crease was perpendicular to the pencil line's direction. 2. Lightly made four evenly spaced pencil marks along the pencil lines. On the opposite direction, labeled them with four separate names: 1. WT female and WT male (WT = wild-type) 2. BR female and BR male (BR = brown) 3. SE female and SE male (SE = sepia) 4. W female and W male (W = white) 3. Anesthetized the fruit flies as they were in separate breeding containers; added sufficient anesthesia for the larvae, as they were more resistant than the adults The anesthesia was comprised of triethyl amine with fragrances The larvae showed resistance to the anesthetic 4. Removed a portion of the fruit flies from each breeding container and transferred them into four separate petri dishes (after anesthetizing them) 5. For the first petri dish (containing white-eyed Drosophila individuals), moved it under a dissection microscope and removed two of each gender using forceps 6. For the first male, aligned its head with the corresponding tick mark on the filter paper and, using the blunt end of the scalpel, crushed only the head to isolate the ommatidia pigments 7. Repeated step (6) to isolate the pigments of the second male 8. Repeated steps (6) and (7) to isolate the pigments of the two white-eyed Drosophila females The females' eye colors were red due to heterozygosity even though the males were white-eyed The fruit flies are expected to be twitching occasionally; this is not a signal to stop the isolation

9. Repeated steps (6) through (8) to isolate each of the visual pigments for the remaining sepia, wild-type, and brown-eyed fruit flies If, at any time, the fruit flies did twitch enough and regain consciousness to flip over, stopped the isolation and replaced the petri dish cover to prevent the flies from escaping 10. When completed isolating the pigments, covered each of the filter papers with aluminum foil to prevent light energy from degrading pigments 11. After the isolation step, moved the filter papers into the chromatography chamber that already had the 30mL solution inside; placed the filter papers in a square-like shape to minimize contact The chromatography solution was comprised of isopropanol and distilled water Place the filter papers so that the pencil line, and not the labels, were closest to the chromatography solution 12. Started the 60 minute timer the moment when both filter papers were immersed in chromatography solution, keeping track of the solvent front's movement 13. After the timer expired, dried the filter papers for 60 minutes before viewing results under a UV light Wore goggles in a dark room to observe the stains under UV light for protection; viewed the results for a maximum of 1 minute per period Results Upon initial observation, both of the sepia bands had more pronounced colors in both intensity and abundance than that of the wild type (figure 1). The sepia bands' column direction was also greatly angled towards the right. Additionally, the extraction of sepia pigments was not as clean as the other bands, as evident by there being two distinct streaks for the females' pigments. Similarly, the wild-type pigment extraction produced enough pigments for the bands to distinctly move to the chromatogram's end (figure 2). While both sepia pigments and wildtype pigments exhibited bands that were completely visible, the white and brown pigments did not have bands that manifested themselves on the filter paper. Although white pigments extracted from male flies did not appear (figure 1), both male and female brown pigments were not visible at all. An assessment of each pigment's abundance relative to that of wild-type was also compiled (figure 3). For the white pigment, of which only the female sex's pigments were observed on the chromatogram, its blue bands were in abundance of similar quantity to that of sepia female pigments. Sepia female's pigments were comparatively scarce to wild-type female's, while the yellow band was far more pronounced (figure 1). As aforementioned, the brown pigment exhibited no visible pigments for analysis, though the faint light-blue streak was still visible. The problem with this streak was that there was only one visible, for the female sex, while the male sex did not exhibit a streak. In terms of pteridine structures and ommochrome structures produced, drosopterins were just barely observed, though they could also have manifested because of the solid residue, which obscured the view. Many of the other pteridine structures did not appear in the UV lighting, and comparisons with a predetermined band order revealed that the observed bands were highly deviant.

Discussion When sexing the flies, it was observed that the white-eyed flies had females with red eyes and males with white eyes. Because the eye pigment trait is x-linked, it is likely that the females were heterozygous for the gene - that is, they carried one dominant allele and one recessive allele. The males, however, possessed the unexpressed Y chromosome, and so had eye pigment determined purely by their lone X chromosome, which contained the recessive allele. Moreover, the "white" pigments leaned slightly toward being characterized as "ivory," but that was likely due to the dissection microscope's light source. In all of the extractions, there were small traces of residual solids from the flies on the filter paper. In the results, a possible explanation for the white pigmented fruit flies' unusual band appearance would be that the male flies, of which the sex chromosomes carried the recessive gene, produced bands that were white to begin with, and thus did not appear on the filter paper. The females, however, were heterozygous for the gene, carried both the recessive and dominant alleles, and thus appeared on the filter paper. Nonetheless, the females only carried half of the dominant alleles that expressed the wild-type phenotype, so they did not produce as much pigment as a homozygous dominant individual would have, resulting in their chromatogram band being much shorter. It was noticed that there were tremendous differences between what was actually observed on the chromatograms as opposed to initial predictions. During the sexing procedure, the sepia-eyed flies were quite similar to the brown-eyed flies, except that the brown-eyed flies had pigments of a darker hue. It was predicted that the brown-eyed flies' chromatogram would produce similar results to the sepia-eyed flies', but the results were vastly different. The difference lay in the fact that brown-eyed flies produced pigments that were not soluble in the paper because they followed the ommochrome pathway instead of the pteridine pathway. Comparing the results with the pathway manual provided, the colors observed were extremely different in the order that they appeared in. The manual's color bands, from the bottom up, began with orange, led into violet-blue and green-blue, followed by yellow, returned to blue, and concluded with yellow. Observed results were closest to the orange-yellow-blue pattern, excluding the violet and green-blues. Assuming that enzymatic mutations did not exist, then the only logical conclusion would be that the orange remained at the filter paper's bottom and was extremely diluted to the filter paper's upper edge, at which point the variations of blue manifested themselves. Because this experiment was largely qualitative, most improvements would simply be directed towards exactly quantifying the data obtained. Pigment degradation at low concentrations - only two individuals of each sex were isolated - was relatively high, and thus the method of preventing it by using aluminum foil as a cover could still be improved. Pigment purity was another issue because while the ommatidia of the Drosophila individuals were isolated, their crania were also crushed by the scalpel. Lastly, a significant error manifested when the gel was allowed to run beyond the time allotted, likely causing further bands to cluster at the edge and unavailable for observation.

Appendix

Figure 1: Chromatogram of sepia and white pigmented fruit flies

Figure 2: Chromatogram of wild-type and brown pigmented flies.

Assessment Level of Pigmentation present/absent default levels present/absent abundant/scarce present/absent abundant/scarce present/absent abundant/scarce Orange Yellow Blue Fly Type wild type sepia brown white

present present present normal normal normal present present present abundant normal abundant absent absent absent none none none present present present normal abundant abundant

Figure 3: Assessment of pigment abundance in chromatograms