BIOL. 364/564: Genetics
Laboratory: Drosophila Genetics (Revised Spring 2013)
Life Cycle
There are four distinct stages in the life cycle of the fruit fly: egg, larva, pupa and adult (Figure 1).
At 25 ˚C, a fresh culture of Drosophila melanogaster will produce new adults in two weeks: seven days in the egg and larval stages and approximately six days in the pupal stages. Adult flies may live for several weeks.
The day after the egg is laid, the larva hatches (Figure 1, top). The larva molts twice, shedding its cuticle, mouth hooks and spiracles. During the periods of growth before and after molting, the larva is called an instar. The fruit fly has three instars. The cuticle of the third instar hardens to become the puparium (pupa) and metamorphosis occurs inside. The pupa begins to darken prior to emergence of an adult fly. About one day before emergence, the folded wings appear as two dark oval bodies, and pigment in the eyes is visible through the puparium. When metamorphosis is complete, the adult
(Figure 1, bottom) emerges (ecloses) by forcing its way through the anterior end (operculum) of the puparium.
Immediately following emergence, the fly is light in color, the wings are unexpanded, and the abdomen is long. The sex of newly emerged flies is often difficult to distinguish because of light pigmentation and displacement of sexual structures toward the posterior tip of the abdomen. In a few hours the wings expand, the abdomen becomes more rotund, and color gradually darkens. Two days after emerging, a female can start laying eggs. Even virgin females lay eggs that are unfertilized and will not develop. After maturity, fruit flies remain fertile as long as they live.
Sex bristles that aid in copulation
Figure 1.
Life stages of Drosophila melanogaster and comparison of dorsal anatomy for sexes.
Genome
Drosophila has four pairs of chromosomes: the X/Y sex chromosomes and the autosomes 2, 3, and
4. The fourth chromosome is small with few expressed genes. The genome is about 165 million bases and contains an estimated 14,000 genes. The genome was (almost) completely sequenced in 2000, and analysis of the data is now mostly complete. Polytene chromosomes are the features that first put
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Drosophila in the genetics spotlight. As the fly larva grows, it keeps the same number of cells, but some of these cells need to make much more gene product. As cells get bigger, each chromosome divides hundreds of times, but all the chromatids stay attached to each other. The result is a massively thick polytene chromosome, which can easily be seen under the microscope. These chromosomes have patterns of dark and light bands that are unique for each section of the chromosome. The pattern of polytene bands tells you what part of the chromosome you are looking at. Any large deletions or other rearrangements of a chromosome can be identified, and using modern nucleic acid probes, individual cloned genes can be placed on the polytene map.
Culturing
Cultures should be kept at 20 to 25 ˚C. While the life cycle of Drosophila may be shorter at higher temperatures, those above 25 ˚C are conducive to the growth of bacteria, fungi and mites. Cultures should never be exposed to direct sunlight since this could lead to reproductive sterility.
Prepare growth medium by adding one level measure (plastic cup provided) of "Instant Drosophila
Medium" to a sterile vial. Add one and ¼ measures (plastic cups provided) of tap water to the vial and allow it to stand for a few minutes (with sponge plug in place) until liquid is absorbed completely. Add a little more water if the medium appears dry. Sprinkle about 10 grains of dry yeast on the surface of the medium. Remove excess water on vial sides by wiping with a clean paper towel. Keep vials closed whenever possible. Prepare medium immediately prior to use. To prevent dehydration, it is essential that flies have food in their vial at all times (except when anesthetizing).
Anesthetizing
Anesthetizing flies is simplified by the use of CO2 . In the main lab (Caputo 301) you need to plug in the regulator before turning on the tank. Turn on main valves at the gas tank (counter clockwise for valve at top, parallel to the copper tubing for in-line valve), attach tubing with a blunt needle to the CO2 outlet (pink) on the lab bench, then adjust the flow of gas at the bench outlet to a gentle stream. Check gas flow against your cheek to make sure it’s not too forceful. To transfer flies from one vial to another, invert the vial so that the sponge plug is facing down and insert the needle between the vial and the plug.
The flies should quickly fall onto the plug and remain anesthetized for 30 seconds. Failure to invert the vial could cause the flies to fall into the growth medium and become trapped. To screen phenotypes, carefully tap the vial with your finger to move anesthetized flies onto a platform anesthetizer to which
CO2 is flowing. The flies should remain asleep on the platform until the CO2 is turned off. Be careful not to break the platform surface by hitting it with a vial or puncturing it with a probe.
More permanent immobilization may be achieved with Fly Nap (Carolina Biological). You should not use this until screening your testcross progeny. Since this substance may be volatile, it should be used under the fume hood. Transfer adult flies using CO2 to a designated empty vial (labeled ‘Fly
Nap’) and promptly cap it with the original sponge plug. Dip the absorbent end of the anesthetic wand into the Fly Nap and remove excess. Tap the culture vial on the lab bench to knock flies to the bottom.
With one finger, push the plug slightly to one side. Quickly insert the wand with Fly Nap it into the culture vial so that the anesthetic is below the plug. Try to prevent the wand from touching the vial sides since it dissolves plastic. Keep the culture vial upright with the wand in place until the flies stop moving (EXACTLY FIVE MINUTES). Some flies may exhibit light trembling of the legs and/or wings immediately after being anesthetized but this should stop in one to two minutes. This procedure is only effective if your timing is impeccable, otherwise the flies may never wake up. As you transfer flies out of the vial, make sure they don’t make contact with any residual Fly Nap that may be on the side of the vial.
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Sorting and Selecting Flies
Place anesthetized flies on an anesthetizing platform or, if subdued with Fly Nap, on a white index card and observe under a dissecting microscope. Use a dissecting probe or paint brush to move the flies gently by pushing them. Pale-colored flies with incompletely expanded wings have just emerged from the pupal case and may be difficult to screen for several hours. Phenotypic characterization of immature flies (as well as very old flies) is extremely difficult, especially when pigmentation or wing-size traits are in question. Sexing of newly emerged flies should be done very carefully. Often novices mistake males and females at this stage. If you are not able to have your instructor check the sexes of newly emerged flies, it is safer not to use them as virgins. Flies that are to be discarded should be dropped into the "morgue" (a beaker filled with mineral oil) while still under the influence of anesthesia.
Female Drosophila can store and use sperm from a single insemination for the major portion of their reproductive life, thus it is necessary to select virgin females for genetic crosses. Males need not be virgin. Males more than eight hours old can successfully mate with (inseminate) female fruit flies of any age. Therefore, it is extremely important that all adult flies be removed (cleared) from a culture that is to be used for the collection of virgin females.
The procedure for virgin collection follows. When pupae appear to be ready for emergence, clear all adult flies (those with legs and wings) from the culture vessel as early in the morning as practical
(7:30 - 9:30 am is good). The flies tend to emerge in greater numbers during the early part of the day.
Return within eight hours (NO LONGER) and separate newly emerged males and females into separate vials (make sure they have food). Keeping them isolated in a culture vial (with food) for more than six days before use may test the virginity of the females. If larvae appear in the vials containing "virgin" females, discard the impostors and try again. Always exercise extreme caution when collecting virgins so that you can have confidence in your crosses.
Sexing
When selecting flies for genetic crosses, it is absolutely essential that the sex of each fly be properly determined. The sex of Drosophila is most reliably distinguished through examination of the genital organs with magnification. A male fly has a genital arch that is a clamp-like structure on the ventral posterior tip of the male's abdomen (Figure 2). In mature males the genital arch is covered by dark bristles (lighter in newly emerged males), which do not occur in the female. This arch (ring of bristles) can be seen even in pale, newly emerged males; however, in these younger males the arch is often shifted posteriorly and located at the extreme tip of the body. In older flies, the dorsal tip of the abdomen is darker (because segment bands are fused) and more rounded in males than in females (in which distinct bands occur at the tip of the abdomen). Generally male flies are smaller than females of
Figure 2.
Comparison of posterior ventral anatomy in females and males
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the same strain, but size alone is not reliable for sorting sexes. Careful examination of the front legs will reveal sex combs on the males but not on the females (Figure 1). You should use all of these criteria when evaluating the sex of your flies to make sure you have flies of the gender you desire.
Mating and Counting
The first mating between two distinct pure-breeding strains is designated a parental cross (P1 or
P2). The progeny of parental crosses comprise the first filial generation or F1. The generation resulting from an F1 X F1 cross is called the F2. Testcross progeny result from a cross of F1 flies with homozygous recessive (often mutant) parents.
Optimally, at least four virgin females and four males of the appropriate types should be placed in each mating vial (a couple of extras won't hurt), and each mating should be set up in duplicate. Do not allow anesthetized flies to become trapped in the growth medium. If the medium is moist, place the vial on its side and place sleeping flies on the plastic side until they recover, then turn the vial upright as soon as possible. Three or four days after a cross is started, the parents should be transferred to new vials. Make sure that each new vial contains at least one healthy female. For P1 and P2 crosses a total of four healthy mating vials should provide plenty of F1 progeny. With testcross and F1xF1 crosses, you should transfer parents of each cross to a third vial after another three or four days if possible. This will provide three sets of duplicate vials for each cross. Seven days after transfer of adults to the final vial, place the adults (parents) in the morgue. This step precludes breeding between generations and removes parents before progeny are screened.
Progeny should begin to emerge ten to fourteen days after a cross is initiated. Females generally emerge before males. Be sure to label your vials with the first date of emergence. Anesthetize the progeny and count them every day for about eight days. Counts made for a period of less than eight days may omit individuals with slow developmental rates. Also, some mutant characteristics such as wing shape and body color are more difficult to see when the flies are newly emerged. Counting for more than eight days, greatly increases the risk of including flies from the next generation. Be sure to collect F1 virgins as necessary. Only count flies that were alive before you anesthetized them; dead flies are usually dehydrated making pigmented traits such as body color and eye color difficult to ascertain.
Tips for Success with Drosophila melanogaster
Label all vials carefully by writing information on the colored tape provided (do not write directly on the vial). Be sure to include your group section/number on a piece of red tape. Do not use that color tape for any other purpose. Include the characteristics of the adult flies added (ex. purebreeding ywm females and f males), a description of the vial's purpose (ex. P1 cross or Testcross or
Stock). The information on each vial should be sufficient enough for your instructor to know exactly what it is and its purpose.
Vials with no identifying labels may be confiscated.
Keep the lab clean and organized at all times . Close food bag(s) tightly when not in use to prevent stray flies and mites from contaminating the food. Never introduce moisture into the food bags, and keep the same measuring cup inside the bag to prevent contamination. When adding water to the medium, use a clean cup from the tray near the sink and make sure that it is clean. Keep vial plugs sterile by placing them into the vials as soon as they are removed from the bag. Keep all vial and plug bags tightly closed. Mites have been a serious problem in the lab recently. You should dispose of any cultures more than four weeks old by anesthetizing the adults, putting them in the morgue, and then placing the vials into the freezer (in fly room) overnight to kill all larvae. The next day, return to the lab and clean vials and plugs. Empty and thoroughly wash all vials when you are done with them.
Remove tape and scrub out all debris with a bottlebrush. Place clean vials on the drying racks above
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the sinks. Clean foam plugs by rinsing with warm water until they appear clean, wring out excess moisture and place them in the drying basket to the right of the sink. Before you use an anesthetizing platform, wipe it thoroughly with a paper towel that has been sprayed to moisten it with 70% ethanol.
This helps to destroy mites and remove any remaining eggs or larvae. After you are done, repeat this process and also wipe off the surfaces of the microscope stage and the bench top with alcohol. Never try to outsmart fruit flies by collecting virgins more than eight hours after clearing vials. You are the one that will suffer when repeating all crosses for a second time. Please try not to allow flies to escape! A laboratory full of fruit flies increases chances for contamination of crosses and does not enhance our popularity with colleagues.
Please cooperate with your colleagues!
Group projects are successful only if everyone does their fair share. If your group discovers it cannot work together effectively, tell your instructor about it immediately. It is possible for individuals to get permission to write the fly assignments on their own, but advanced permission is necessary if you want to take this approach.
Strains and Potential Crosses
Stock cultures of pure-breeding strains with some of the following traits will be used.
Wild-type (WT) flies : red eyes, gray bodies, smooth bristles, long straight wings parallel to body that extend well beyond the tip of the abdomen.
Eye color mutants: scarlet ( st) eyes are brighter red than wild type, brown ( bw ) eyes are more dull, and white ( w ) eyes lack pigmentation. More than one genotype can result in white eyes.
Body color mutants: yellow ( y ) has pale bands, ebony ( e ) is dark, chocolate is brown and lacks bands.
Wing length mutants: miniature ( m ) wings are shorter than wild type (Figure 3)
Bristle shape mutants: forked ( f ) bristles are bent or split and shorter than wild type (Figure 3.); singed
(sn) bristles appear misshapen, as if they have been touched with a match.
A)
B) m
+
f sn
Figure 3.
Comparison of A) miniature wings ( m ) accompanied by ebony body and white or brown eyes, and
B) forked ( f ) or singed ( sn) bristles to wild type (+). In panel A, the male has white eyes and ebony body while the female has brown eyes and ebony body.
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Make sure you can recognize each of the traits effectively. Once you set up your crosses, the progeny could have any combination of phenotypes, so you’ll have to be able to easily distinguish the mutant and wild-type traits. The difference between miniature and wild-type wings , and between yellow and wild-type body color can be difficult to distinguish, especially in young flies.
These traits should not be evaluated until wings are fully extended and color has developed (~8 hr).
General Procedures: Stock Cultures, Crosses, Screening
Set up a stock culture of each strain that you receive by transferring pure-breeding adults from the original vials into a new vial with food. Transfer adults to new vials with fresh food again every 14 days to generate a second stock vial for each strain. These will be critical if you need to collect virgins of pure-breeding strains for testcrosses with F1 flies, and for verification of phenotypes. Check on your stocks routinely to make sure they are healthy and no mites are visible.
Do not keep any vials for more than four weeks.
Begin to collect virgin females and males from each of the original vials containing the two mutant pure-breeding strains. These will be needed for crosses that will be set up in lab during the next few weeks. The pure-breeding males don’t have to be virgins, but you’ll want to be absolutely certain they are males from the correct pure-breeding stock.
Set up crosses in duplicate vials to avoid loss due to contamination. Label vials containing all crosses carefully to avoid any confusion. Be sure to transfer parents to new vials every 3 – 4 days, but keep the original vials. Transfer all parents to the morgue before progeny begin to emerge.
As the progeny begin to emerge, note this date on the vial. Each group should count at least
100 progeny flies from each of the two reciprocal crosses, and each group member will be asked to count 250 progeny flies from the mapping testcross. The counting can be done over a period of eight days after the progeny first start to emerge. As you screen flies, record the number of each phenotype and whether each fly is male or female. Keep the results of each of your crosses separate (they may be different if sex-linkage is involved).
A data table like the one on page 7 can be used to keep track of your results. If you need virgin
F
1 females for use in testcrosses, remember to collect them as you screen the F1 progeny. For all crosses, you can use chi-square analysis to compare the numbers in different phenotypic categories to those expected based on your hypotheses.
Reciprocal crosses: Dominance of alleles, autosomal or sex-linked genes
Reciprocal crosses between two pure-breeding (homozygous) mutant strains (as in the two examples outlined below) are the best way to determine dominance for mutant and wild-type alleles of a trait, and to determine if a gene is sex-linked or autosomal. Examples of two different reciprocal crosses are outlined below. The details for your crosses will depend on your goal and will be outlined in more detail by your instructor.
P1: Ebony White Miniature Singed virgin females x Wild-type males
P2: Wild-type virgin females x Ebony White Miniature Singed males
OR
P1: Yellow White Miniature Forked virgin females x Wild-type males
P2: Wild-type virgin females x Yellow White Miniature Forked males
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Complementation crosses: Are two phenotypes controlled by the same or two different genes?
If two strains share the same phenotype (for example, both have white eyes), it is important to set up crosses between the two unknown strains (as follows) to see if one or two genes are involved.
P3-a: White strain1 virgin females x White strain 2 males
P3-b: White strain 2 virgin females x White strain 1 males
Testcrosses: linkage of genes on chromosomes
F
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virgin females can be crossed with pure-breeding recessive males to determine if multiple genes are linked or assorting independently. You would need to do testcrosses and count substantial numbers of progeny if you want to map linked genes and determine their order.
Testcross: F1 females x pure-breeding recessive males
If there are no pure-breeding strains that express the recessive form for all of your traits, you will have to improvise. When genes are X-linked, screening the male progeny can provide information about linkage even thought the female siblings express the dominant traits inherited with their father's X chromosome. Explain how you will deal with this in your proposal.
Sample Data Collection Table for F1 or Testcross Results
Using this type of table will make it much easier for your group members to keep track of the flies each person screens. The table should be attached firmly to your lab notebook and each entry in the table should be initialed to indicate the contributor.
Table 1. Tally Sheet for Progeny Screened from Cross
Number observed
Phenotype Observed Sex (Include initials and date above each column) Total
Wild type M
Wild type F
Ebony, white, miniature, singed M
Ebony, white, miniature, singed F
Ebony, white
Ebony, white
Miniature, singed
Miniature, singed
M
F
M
F
Note: Phenotype descriptions may be added or deleted. Those shown as examples may not be applicable to the experiments that you perform to answer questions about your traits.
NOTE: Your group will not be the first to investigate these traits in Drosophila . You can learn what to expect for genes controlling similar traits at . Enter the candidate gene symbol in the text box with ‘Species: Dmel only’ and ‘Search: ID/Symbol/Name’. Sort through the list to find the best match to your symbol and click on it to obtain information. You should also research your traits using Google Scholar and looking in the Drosophila Manual that is available in the fly room.
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Make sure you use formats for tables and legends that are appropriate for publication in a scientific journal.
Titles for tables belong on top; they start with 'Table #' and succinctly describe the table contents, but details about parts of the table should be explained using footnotes.
Figures are diagrams and flowcharts; the title of a figure should be included in its legend as the first phrase beginning with 'Figure #'. The figure title is followed by sentences that further describe all symbols and other content in the figure. Examples of good formats for figures and tables follow.
Figure 2. CEN4 deletions in HRM3 plasmids. Representative HRM3 -containing plasmids were subjected to restriction and sequence analysis to determine the region deleted in YCp50. All deletions remove the three CDE elements of
CEN4 . Short (1-4 bp) regions of homology that originally flanked the deletions occur at the site of plasmid rejoining.
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Sample Chi square Analysis
The following table shows an example of Chi square analysis for four possible phenotypes involving two different genes. Your tables should include all possibilities for three genes at a time and, thus, will be much larger.
Chi square ( x
2 )

(Obs – Exp)
2
Exp
Table 1. Comparison of progeny observed to those expected if the Aa and Bb genes are unlinked .
Phenotype
(list all possibilities)
Observed
Number a
Expected
Number b
Exp
(Obs – Exp)
2
Dominant for A & B
Recessive for a & b
140
135
125
125
1.8
0.8
Dominant A, recessive for b
Recessive for a, Dominant for B
Totals
110
115
500
125
125
500
1.8
0.8 x
2
= 5.2 a Only males were counted because the X chromosome from the male parent carried an f+ allele that was expressed in all female progeny, thus masking the allele for the bristle gene that was inherited from the heterozygous mother.
b
Numbers were calculated based on map distances (here 50 mu is used for the hypothesis of no linkage) and gene orders (here unlinked) shown in Figure # (note: this would be a figure that simply showed the map according to the literature for all four genes with the distances indicated).
Once the Chi square ( x 2 ) is calculated, you will need to refer to a table that gives degrees of freedom (DF) and probabilities (p) associated with each Chi square value. When using Chi square analysis with phenotypic categories and making a comparison of expectations and observations, the degrees of freedom (DF) will equal the number of phenotypic categories minus 1. In the example , there are four phenotypes so DF = 4 - 1 = 3. Find 3 in the column under DF, then go across that row to find the x 2 value that you calculated. Since 5.2 is between 2.366 and 6.251, this means the probability is less than 0.5 and greater than 0.1 that the differences between your observations and the expected values are due to chance. If you use a x
2 critical value of 0.05, then you can conclude that your results do not differ significantly from expectations if your x 2 value is equal to or less than the value in the column beneath 0.05. If your x 2 value is greater than the value shown in this column, then your results differ significantly from expectations. State the probability associated with your Chi square value in your discussion and comment on its significance.
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Obtain a bound notebook and clearly label the outside so that it can be easily identified as your property. Include your group symbol on the cover, write names and numbers of all group members on the inside cover so that you can contact each other readily. Keep the notebook in the laboratory where you work with the flies at all times so that all group members have access to it. Every time a member of the group does anything (such as clearing a vial, transferring flies from one vial to another, disposing of flies, etc.), details of the activity should be entered in your notebook so that everyone knows what you did and when.
Write very clearly and legibly so that anyone familiar with biology can pick up your notebook and understand what you’ve done. Include enough detail so that another genetics students could repeat exactly what you did. Be sure that you’re accurate, identify vials clearly,outline crosses very carefully. When progeny are counted, make sure that you indicate precisely which cross produced them as well as the sex and phenotype for each individual. Make tables and charts for entering data so they are clearer. Computer generated tables can be pasted into the notebook as long as they are secure. If you observe anything (unusual or not), include your observations in the notebook. For example, if some strains of flies develop more slowly than others and will take longer to emerge to adulthood, then this could impact results when progeny are screened. Also, noting that the flies in one vial don’t appear as healthy as those in another could lead you to choose them as parents rather than their sickly counterparts. Often in science, discoveries are made simply because someone was observant. As you collect data, think about what they mean and ideas for future analysis. Include a summary of your results and draw conclusions about what the results mean for your hypotheses.
Literature Citations
Correctly cite any sources of information used in your reports, including. textbooks and journal articles. You should use the system in which references are cited by numbers that correspond to the order that they appear in your paper. Please use the following format (adapted from Molecular
Genetics and Genomics) for your Literature Citations (bibliography. Be sure you follow the examples exactly; journal editors are very picky. Note differences between listings for book chapters and journals.
1) Aguilera A, Klein HL (1988) Genetic control of intrachromosomal recombination in
Saccharomyces cerevisiae . I. Isolation and genetic characterization of hyper-recombination mutations. Genetics 119:779-790
2) Barnes WM (1978) DNA sequence from the histidine operon control region: seven histidine codons in a row. Proc Natl Acad Sci USA 75:4281-4285
3) Björk GR (1995)
Biosynthesis and function of modified nucleosides in tRNA.
In: Söll D,
RajBhandary UL (eds) tRNA: Structure, biosynthesis, and function. ASM Press,
Washington D C, pp 165-205
4) Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual (2 nd
edn).
Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York
5) Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors.
Proc Natl Acad Sci USA 74:5463-5467
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