A modified version of the experiment that can be done at home has now been worked out by Richard LaFond of Monson, Mass. Although LaFond's apparatus is largely assembled from scrap materials and presents a deceptively simple appearance, it provides the experimenter with a powerful means for delving into an exciting aspect of genetics. LaFond writes:

"The eye of Drosophila has been found to contain two genetically controlled pigment systems, one brown and the other red. These systems were first revealed by their different solubilities. The red pigment is water-soluble but the brown is not. During the fly's early development the brown pigment appears first; the red, some hours later. The normal eye color of flies of the "wild" type, such as Oregon-R, is brick red, caused by the presence of both pigments This eye color appears when all genes are working normally.

"In the case of mutant flies that have eyes of abnormal color, such as scarlet, a gene suppresses the formation of brown pigment. Accordingly the eyes are red. Mutants with brown eyes, on the other hand, have a gene that suppresses the formation of red pigment. A cross between a mutant with scarlet eyes and one with brown eyes produces a hybrid with white eyes. In effect the pigment systems cancel out.

"It is the red pigments and other brightly fluorescing compounds that comprise the pteridines. These compounds are situated not only in the eyes of the fly but also in the ovaries and testes and in the Malpighian tubules, which act as a kidney. The relative amounts present in a specimen tend to differ at each stage of the life cycle as well as between mutants and their hybrid offspring. For this reason experiments having to do with the pteridines are open to almost limitless variation.

"One must, of course, have a stock of flies in order to conduct experiments. An easy way to collect Drosophila is to leave outdoors in a shaded area a culture bottle containing a special food lich in yeast. By careful inbreeding it is possible to develop a number of mutant strains from the wild stock. Specimens of all types also can be bought from suppliers. My initial flies were obtained from the Curator of Stocks, Division of Chemotherapy, The Institute for Cancer Research, 7701 Burholme Avenue, Philadelphia, Pa. 19111.

"Having acquired a small initial stock by capture or purchase, the experimenter then perpetuates the stock by culturing techniques. The live specimens come in small vials Adults are promptly transferred to a culture bottle, but the vials are not discarded immediately. Eggs have been laid in the food from which young flies will soon hatch.

"I use half-pint milk bottles as culture vessels. Before transferring flies to these containers each bottle is sterilized and equipped with a supply of food. A number of food preparations have been developed for culturing Drosophila. I use the recipe devised by Boris Spassky of the Rockefeller Institute. This nutrient is made by adding 194 milliliters of tap water to 29 milliliters of unsulfured molasses (Grandma's brand) and bringing the mixture to a boil in a pan. To the boiling solution are added 26 grams of regular Cream of Wheat and two grams of uniodized salt. The mixture must be stirred constantly . and cooked for about five minutes. The pan is then removed from the stove. Two milliliters of a 10 percent solution of Tegosept M, a brand of methyl-p-hydroxybenzoate, are stirred into the mixture as a preservative. The 10 percent solution is made by diluting 10 grams of the compound in 100 milliliters of 95 percent ethyl alcohol.

"To milk bottles that have been thoroughly washed and boiled in water add the food mixture to a depth of about half an inch by means of a funnel that prevents food from spattering on the glass. Wipe any condensed water from the inner wall of the bottle. Plug the opening of each bottle with an unwaxed paper cap or a tuft of cotton covered with a piece of cheesecloth. Place the bottles in a pressure cooker containing about 100 milliliters of water and boil for 30 minutes at a pressure of 15 pounds per square inch.

"After sterilization stand the capped bottles on a convenient wooden shelf or table until any large drops of water adhering to the inner wall evaporate. This step is important because in an excessively moist bottle the flies may get stuck in the food medium and drown. Excessive moisture can be removed with a sterilized paper towel. When the interior of the bottle is dry, fold a piece of sterilized paper toweling 11 inches long and 2 1/2 inches wide into quarters, so that the folded sheet measures 2 3/4 by 2 1/2 inches, and push one end below the surface of the food medium. The paper strip provides a place on which Drosophila larvae can pupate. Drosophila thrive on a fermenting medium. This is provided by sprinkling a pinch of Fleischmann's active dry yeast over the surface of the food. Each bottle is then carefully labeled with the name of the type of fly it will house and the date.

"Cultures of Drosophila should not be kept more than 20 days because they may become infected with mites, which markedly decreases their abundance. Mites are minute members of the class Arachnida; an effective agent against them is benzyl benzoate. Make up a solution of this compound and mineral oil in equal proportions. Shake the solution well and spread it on the shelves supporting the culture bottles. Maintain a temperature of 72 degrees Fahrenheit in the storage room. A new generation of flies will appear in 12 to 14 days. When discarding an old culture, always wash the used bottles thoroughly in hot water and any convenient detergent and then boil them in fresh water to destroy mold spores.

"Before you count or handle individual flies you must anesthetize them. The anesthetizing apparatus consists of a peanut butter jar closed with a large cork through which the spout of a small funnel is inserted. A small sponge in the bottom of the jar is moistened with a few drops of ether. A miniature cage for holding the flies is made from the larger section of a No. 000 gelatin capsule (manufactured by Eli Lilly and Company) by perforating the gelatin about six times with a hot sewing needle. The open end of the capsule is slipped partway into or over the end of the funnel and, if it does not make a snug fit, taped in place. Flies are transferred to the anesthetizing cage by placing the mouth of the open culture bottle tightly against that of the funnel, orienting the assembly so that the bottle containing the ether is on the bottom and tapping the culture bottle. The flies will fall down through the funnel into the anesthetizing cage. I use Merck motor ether. The container in which the ether is stored must be tightly closed when not in use.

"The flies must be counted as one step in nearly all experiments. It is also often necessary to separate them by sex, type, age and so on. During such steps the anesthetized specimens are manipulated by means of a small camel's-hair brush, preferably on a smooth white surface such as white glass or a sheet of clear glass that rests on white paper. The sex of specimens is easily determined by examining inverted flies under a five-power magnifying glass. The distinguishing sexual features appear in the accompanying illustration [below].

"Normally flies remain anesthetized for five to 10 minutes. Some individuals revive sooner than others. These can be anesthetized again by inverting over them a Petri dish or other shallow container into which is fastened a small piece of paper toweling moistened with a few drops of ether. Remove specimens from the ether promptly when they stop moving. Overexposure will kill them.

"After a culture has been maintained for 20 days transfer all adults to a fresh culture bottle. Recap the old bottle and 48 hours later transfer the young flies that have hatched during the interval to a fresh culture bottle. The old flies can be used to start new cultures. Develop scrupulously clean work habits in order to avoid contaminating or mixing cultures. Specimen types can be mixed accidentally, for example, by transferring a soiled glass rod or other implement to which an egg adheres from one bottle to another.

"For separating the pteridines I use a chromatographic apparatus of the descending-paper type. Essentially it consists of a closed glass box that houses an elevated container of solvent in which the upper end of the paper is immersed. The dimensions of the apparatus are shown in the accompanying illustration [below]. A square inch of glass is cut from one corner of the close-fitting cover to provide access for transferring the solvent to the container. This opening is sealed with a thick sheet of paraffin in which a round hole about half an inch in diameter is made. The hole is then fitted with a removable stopper, which is also made of paraffin. The container for the solvent, which rests on the top platform of a removable framework, can be any convenient shallow vessel about seven inches long and an inch or two deep. I use an aluminum pan that rests on a framework made of parts from an old Erector set.

"Brackets of wire and paraffin, attached to one edge of the pan, support a slender glass rod 10 inches long over which a piece of moist filter paper is draped. In addition to serving as a support for the paper, the rod prevents a siphoning action that would cause the solvent to flow; the only kind of flow should be that due to capillary action. The upper edge of the paper strip is weighted against the bottom of the solvent container by a glass butter dish filled with sand held in place by a layer of melted paraffin. A second glass rod, attached about halfway down the framework, serves as a stop to keep the paper away from the glass housing.

"Two glass jars of about 50-milliliter capacity are placed on the lower platform of the framework. These each contain 30 milliliters of a solution that by evaporation brings about an equilibrium between the atmosphere of the chamber and the vapor content of the filter paper. One can make a 100-milliliter stock of this solution by diluting 2S.9 milliliters of 27 percent ammonium hydroxide with distilled water. The entire apparatus must be carefully leveled before use so that the surface of the solvent in the container makes a right angle with respect to the center line of the paper strip.

"Chromatograms are made on strips of Whatman No. 1 chromatography paper cut four inches wide and 22 inches long. My paper was bought from Howe and French, 99 Broad Street, Boston, Mass. 02110. It comes in sheets 18 3/4 inches long by 22 1/2 inches wide. As an aid in placing specimens uniformly on the paper I draw a light pencil line squarely across each strip at a distance of 6 3/4 inches from one end and divide the line into five equal intervals by four light pencil dots. The material to be analyzed is placed on these dots.

"To prepare for the analysis of adult flies first anesthetize selected specimens of the same age. Age is an important factor because the concentration of pteridines in the flies varies during the life cycle. The concentration also differs substantially between the head and the body. Moreover, the chromatograms of males differ from those of females.

"One begins a typical experiment by severing the heads of 10 flies with a razor blade and squashing the material onto the paper with the end of a glass rod. I always reserve the right-hand dot for the control specimen, which is prepared by applying to the dot the heads of 10 Oregon-R wild-type flies. If the control specimen fails to separate as anticipated, the chromatogram is discarded. The control also provides a convenient cross-check for estimating the amounts of pteridines in other specimens in relation to those naturally present in the wild type.

"When one is making chromatograms of fly bodies rather than heads, one must take care to separate all head tissue cleanly. A small amount of head tissue can contain more pteridines than a whole body, hence even a tiny fragment can seriously distort a body chromatogram. The bodies are boiled in water for three minutes to coagulate the protein and thus facilitate the chromatographic separation. After boiling they are placed on paper toweling to remove excess water. Five bodies are applied to each spot. All spots must be dried at room temperature before the chromatographic paper is placed in the apparatus. In addition, a pencil notation is made next to each spot that. includes its descriptive initials-such as p for plum eye, st for scarlet or brightred eye, v for vermilion eye, w for white-eye mutants, bw for brownish eye, w^a for white-apricot eye-together with the date and time.

"After the paper strip has dried, the solvent pan is placed on the upper platform of the framework. The strip is then draped over the upper glass rod and anchored in place by the butter dish. The loose end of the strip is threaded between the lower glass rod and the framework so that it hangs freely suspended. Paper clips of the pinch type are attached to the bottom edge as weights. The ammonium hydroxide containers are then put on the lower platform. Now the entire assembly is placed in the glass housing, covered by the g]ass top, draped with a cloth that excludes light and left undisturbed for two hours. During this interval the vapor content of the paper reaches equilibrium with the atmosphere of ammonia released by the ammonium hydroxide. Good separations cannot be expected if the interval of equilibration is stinted.

"When equilibration is complete, remove the cloth cover and the paraffin plug and insert into the solvent container a rubber tube equipped with a small plastic funnel. This is used to transfer 230 milliliters of developing solvent into the tray. The solvent consists of 360 milliliters of 1-propanol (n-propyl alcohol), 45 milliliters of 7 percent aqueous ammonia and 135 milliliters of distilled water. Finally, remove the rubber tubing, reinsert the paraffin plug promptly, cover the apparatus to exclude light and maintain the room temperature at 68 degrees F. for 20 hours. At the end of this interval remove the chromatogram from the apparatus. Handle the paper by its ends and suspend it upside down for drying at room temperature. Return the solvent and equilibrating solutions to their respective storage bottles.

"When the chromatogram has dried, it can be examined under an ultraviolet lamp. If all has gone well, the characteristic fluorescent bands will appear [see Figure 1]. Caution: Never look directly at the bulb of an ultraviolet lamp. The rays are injurious to the eyes. Protective goggles should always be worn when working with ultraviolet radiation. Dual lamps that emit ultraviolet at wavelengths of 2,537 and 3,660 angstrom units and operate from regular house current are available from the Edmund Scientific Co. of Barrington, N.J. Pteridines that emit blue or violet light glow with greatest brilliance when they are irradiated at a wavelength of 3,660 angstroms; those that emit reds or yellows appear brightest when they are irradiated at a wavelength of 2,537 angstroms.

"Chromatograms should be examined and evaluated as soon as they dry because the fluorescent compounds tend to fade with time. As a convenience in scoring and recording the results I prepare a table in advance. Symbols designating all specimens are listed in a column that extends down the left edge of the page [see Figure 7]. A similar row across the top of the page lists the several pteridines: DRO for drosopterins that fluoresce orange-red; XAN for xanthopterin, green; SP for sepiapterin, yellow; AHP for 2-amino-4-hydroxypteridine, blue; BIO for biopterin, blue; ISO for isoxanthopterin, violet, and RFL for riboflavinlike compounds mixed with sepiapterin, yellow. The apparent brilliance of each band as judged by eye is then recorded by means of plus and minus symbols, as shown in the accompanying illustration [Figure 8].

"Isoxanthopterin is present in the bodies of both male and female flies but is found in much larger amounts in male bodies because it is concentrated in the testes. This difference in concentration, as disclosed by the chromatogram, immediately identifies the sex of most specimens. Certain mutants, however, such as the rosy-1 (ry^l), rosy-2 (ry²) and maroon-like (ma-l), do not produce detectable quantities of isoxanthopterin. Rosy-2 males can still be distinguished by an abnormally large amount of 2-amino-4-hydroxypteridine, the precursor compound in the formation of isoxanthopterin.

"Interesting changes in the amounts of the pteridines with time can be observed by doing chromatography of larvae, young and old pupae and hatched flies of various ages. Young pupae are brown and translucent, whereas older pupae are opaque. Larvae that have begun to climb onto the paper in the culture bottle and up the wall of the bottle are removed with a glass rod and applied directly to the paper. I chromatographed five larvae, five pupae and 10 heads of sepia mutants one day old. The larvae show a very weak fluorescence that does not seem to indicate any particular pteridine. Young pupae display xanthopterin, together with a substance I could not identify that fluoresces in the blue portion of the spectrum. There was also a hint of yellow sepiapterin. Xanthopterin and sepiapterin appear in substantial amounts during the late pupal stage. "The experiments I found most interesting involved crossing different eye color mutants (and also crossing such mutants with flies of the wild type) and then chromatographing the offspring. Virgin females must be used for making controlled crosses because females can store the sperm from one insemination for a large part of their reproductive lives. To collect virgin females, clear a culture bottle of all flies. Search the paper carefully for adults that may be hidden in its folds. Use a bottle that contains many pupae from which new flies will soon hatch. From this bottle collect females within 10 hours of the time they hatch. (After 10 hours they will mate with males, although they do not lay eggs for two days.) Transfer the virgins to a fresh culture bottle placed on its side so that they will not stick to the food at the bottom. Males of any age and desired type are then anesthetized for transfer. Several pairs of males and females are placed in a fresh culture bottle and labeled with the description of the cross and the date. When larvae begin to climb up the wall of the bottle and onto the paper, the adults are removed.

"Always remember when chromatographing young flies of any type that the relative amounts of the pteridines change with the age of the specimen. Use specimens that are approximately the same age. I usually select flies that are 12 days old. To age specimens, clear a culture bottle of adults, collect the young as they hatch during an interval of an hour or so and then transfer the young to a fresh, dated culture bottle. Chromatograph after 12 days.

"An important consideration in interpreting the chromatograms of the mutants and the crosses is whether the characteristic pattern is the result of the major gene or genes under consideration or merely a reflection of the specimen's genetic background. Genes other than those assumed by the experimenter can modify the amounts of pteridines in the fly. For example, it has been shown that the amount of isoxanthopterin is influenced by many genes because there is less variation in the concentration of this compound in inbred stocks of flies than in flies that have been collected in nature and mass-cultured for maximum heterozygosity, or variation of genes.

"In my experiments I have assumed that the obvious differences between chromatograms of the various mutants are associated with a major gene. Although I chromatographed many flies, I made no attempt to exclude the possibility of background effects by crossing mutants with flies of the wild type and then re-isolating the stock after six or seven backcross generations to compare the chromatographic results with those of the original strain. This step would be essential, however, in a quantitative study designed to reveal the influences of genetic background."

Bibliography

EXPERIMENTS IN GENETICS WITH DROSOPHILA. Monroe W. Strickberger. John Wiley & Sons, Inc., 1962.

FRACTIONATING THE FRUIT FLY. Ernst Hadorn in Scientific American, Vol. 206, No. 4, pages 100-110; April, 1962.

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