LABORATORY 7: GENETICS OF ORGANISMS
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1 LABORATORY 7: GENETICS OF ORGANISMS OVERVIEW In this laboratory you will use Drosophila melanogaster, fruit flies, to do genetic crosses. You will learn how to collect and manipulate fruit flies, collect data from F1 and F2 generations, and analyze the results from a monohybrid, dihybrid or sexlinked cross using a statistical test called the Chisquare test. OBJECTIVES Before doing this laboratory you should understand: the definitions of P1, F1 and F2 with respect to genetics how to use a Punnett square to determine the phenotypic ratios of a genetic cross chisquare analysis of data (see work sheet in Appendix D) the life cycle of the diploid organism Drosophila melanogaster After doing this laboratory you should be able to: investigate the independent assortment of two genes and determine whether the two genes are autosomal or sexlinked using a multigeneration experiment analyze data from genetic crosses using chisquare statistical analysis INTRODUCTION Drosophila melanogaster (fruit fly) is an excellent organism for genetics studies because it has simple food requirements, occupies little space, is hardy, completes its life cycle in about 12 days at room temperature, produces large numbers of offspring, may be immobilized easily for examination and sorting, and has many types of hereditary variations that can be recognized with low power magnification. Drosophila has a diploid number (2n) of chromosomes (n=4). These chromosomes can be easily extracted from the large salivary gland cells (see pp.78, Extraction of Salivary Gland Chromosomes). Much research about the genetics of Drosophila over the last 50 years has resulted in a wealth of reference literature and knowledge about hundreds of its genes. The Life Cycle of Drosophila It is important to realize that a number of factors determine the length of each stage in the life cycle. Of these factors, temperature is the most important. At room temperature (about 25 C), the complete life cycle takes 10 to 12 days. The eggs. The eggs are small ovals and have two filaments at one end. They are usually laid on the surface of the culture medium and, with practice, can be seen without any magnification. The eggs hatch into larvae after about a day. The larval stage. The wormlike larva eats almost continuously, and its black mouthparts can easily be seen moving back and forth even when the larva itself is less distinct. Larvae tunnel through the culture medium while eating; thus channels are a good indication of the successful growth of a culture. The larva sheds its skin twice as it increases in size. In the last of the larval stages, the cells of the salivary glands contain giant chromosomes, which may be seen readily under low power magnification after proper staining (see pp.78, Salivary Gland Extraction). The pupal stage. When a mature larva in a laboratory culture is about to become a pupa, it usually climbs up the side of the culture tube or onto the strip of paper provided in the culture. The last larval covering then becomes harder and darker, forming the pupa case. Through this case, the later stages of metamorphosis to an adult fly can be observed. In particular, the eyes, wings and legs can be observed. The adult stage. When metamorphosis is complete, the adult flies emerge from the pupa case. They are fragile and light in color and their wings are not fully hardened. These flies darken within a few hours and take on the normal appearance of the adult fly. They live a month or more and then die. A female will not mate until 10 to 12 hours after emerging from the pupa. Once she has mated, she stores the sperm in a specialized receptacle called a spermatheca and fertilizes her eggs while she lays them. To ensure a controlled mating, it is necessary to use virgin females.
2 Figure 7.1: The Life Cycle of Drosophila melanogaster SEXING FLIES Distinguish male flies from female flies by looking for the following characteristics. Males are usually smaller than females. Males have dark, blunt posteriors, whereas the females have lighter, pointed posteriors. The males have sex combs, which are groups of black bristles on the uppermost joint of the forelegs, whereas females do not. Figure 7.2: Female and Male Drosophila Design of the Exercise
3 This genetics experiment will be carried on for several weeks. Drosophila with well defined mutant traits will be used. Your teacher will assign a culture tube to your group. You are responsible for making observations and keeping records concerning what happens as mutant traits are passed from one generation to the next. You will be assigned one of the following crosses: 1 Monohybrid. In these crosses, the mode of inheritance can be determined when one single contrasting pair of characteristics is involved. 2 Dihybrid. In these crosses, the mode of inheritance can be determined when two pairs of contrasting characteristics are considered simultaneously. 3 Sexlinked. In these crosses, the mode of inheritance can be determined when the mutant characteristic is associated with the X chromosome. To make these experiments interesting and challenging, you will not be given any information about the type of cross given to your group nor the names of any particular mutations involved in the experiments until just before the final lab writeup. There will be separate culture tubes of the parental types (wild type and each of the 3 mutations) containing both males and females. You will study their eye color & shape, bristle number & shape, wing size & shape and antennae size & shape until you are familiar with their characteristics. Once you have become comfortable with their "normal" phenotype, it will not be difficult to identify mutations in the following generations. Before beginning the actual procedure of typing and sexing your group's tube, you should understand the long range time table for this extended experiment. Long Range Summary of For the four weeks of this experiment you will be completely responsible for the health of your fly cultures. If you understand the life cycle of Drosophila, can anesthetize and manipulate the flies without killing them, and can follow the long range experimental plan, your group should have a fun and successful four weeks. WEEK ONE: The parental flies have already mated. This first week will be used to familiarize yourself with the handling and scoring (counting the numbers of flies by gender and by type of mutation) of the flies. You will be asked to distinguish male and female wildtype flies and identify mutations. At this time you may be able to see either the eggs on the surface of the medium or the tunneling larvae in your F1 culture tube. WEEK TWO: (7 to 10 days later DAYS 710) You will score the emerging F1 generation. Because not all flies will emerge simultaneously, this count may take several days. After each count, do not return the flies to the culture tube because you will either dispose of them or use them for a cross in another culture tube. WEEK THREE: (4 to 7 days later DAYS 1417) The F1 generation has mated and laid their larvae. You will dispose of these mated F1 flies by dropping them in alcohol. You will also complete the Salivary Gland Extraction using the final stage larvae. WEEK FOUR: (7 to 10 days later DAYS 2427) You will score the emerging F2 generation. It is particularly important that you and your group members are meticulous about scoring this week. Dispose of each day's count. Although you want as large a sample size as possible, you do not want to rescore flies as this might skew the final results.
4 The following procedures will be used throughout the lab: SEE IMPORTANT NOTE ON FOLLOWING PAGE! FlyNap (to be used each week to anesthetize and score your flies) 1. ~ your group's culture tube ~ stereo microscope with illuminator name and symbol of mutation male female Record observations about what the flies in your culture tube are doing. Do they seem active? Are some near the bottom and some near the top? Do many appear to be dead? 2. With all your materials close at hand, uncap the small bottle of FlyNap. 3. Dip wand into the FlyNap. 4. Roll the wand's brush on the inner edge of the bottle to reduce excess FlyNap. 5. With the wand in one hand and the culture tube in the other, tap the tube gently a few times on the table. This knocks the flies to the lower portion of the tube. Have this demonstrated if you do not understand. 6. When all the flies seem to be in the lower portion of the tube, with one finger QUICKLY push the plug slightly to one side and stick the anesthetic end of the FlyNap wand into the culture vial so that the anesthetic tip is below the plug. Release the sponge. 7. Keep the culture vial upright with the wand in place. 8. While waiting for the flies to fall asleep, place the filter paper on the platform of the microscope. 9. Using a pencil, lightly draw an X, about the size of a fly, in the center of the filter paper. 10. Focus the microscope on the X. 11. Watch the flies closely and remove the plug and wand when the flies are anesthetized (2 minutes in an empty vial). When all the flies appear immobile, take the wand out and lay it on a paper towel. (You now have about 515 minutes to score your flies). 12. Empty the tube on to the filter paper. 13. Manipulate, using the paint brush, one fly into the center of the filter paper. Focus the microscope on that one fly. 14. Position the illuminator and identify its phenotype. 15. Score your flies. Fill in the appropriate table according to what week in the experiment it is. 16. Either return your flies back to the culture tube with media or dispose of them in the morgue (a jar containing 70% EtOH). How To Make Drosophila Media ~ measuring cup ~ Formula 424 Instant Drosophila Medium ~ dry viable yeast ~ cool tap water 1 Dump equal volumes of Instant Drosophila Medium and cool tap water into a vial. 2 Sprinkle a few grains (610) of dry viable yeast on top. The metabolic byproducts of yeast include CO2. Large amounts of yeast in a culture can produce enough CO2 to render Drosophila sterile or even cause death. 3 After one minute (allow media to set), flies can be introduced and the vial plugged.
5 NOTE: When returning flies to the culture tube (whether it is an "old" or "new" tube): 1 Place vial on its side. 2 Place flies AWAY FROM THE MEDIA (see below diagram). Otherwise, your flies will be in danger of "drowning." 3 Leave the vial on the back table. LET YOUR TEACHER KNOW YOUR TUBE IS THERE. Your tube will be placed in the incubator when your flies wake up. MEDIA SPONGE place flies somewhere PLUG in this area * KEEP AWAY FROM MEDIA * RECORD ALL DATA ON THE DATA SHEET WEEK ONE: EXAMINATION OF PARENTAL TYPES Goals for this lab period are: to learn how to anesthetize flies properly using FlyNap to learn how to score fly generations by distinguishing males from females and wild type from mutant strains to identify and record the mutations of each possible parental type to accurately describe your fly culture's general state of appearance There will be several stations set up, each with a stereomicroscope (dissecting microscope) and a tube of Drosophila of either wildtype or one of the three mutations. 1 Immobilize the flies using the FlyNap procedure and observe them carefully under the dissecting microscope. 2 Separate the males from the females and look for the mutation(s). 3 Identify each mutation and assign a name and symbol (of your choice). Record data in Table 7.1 on the Data Sheet. You will be asked to: (1) identify both a male and female wildtype fly and (2) identify the mutations present in each of the mutant flies. After you make your observations, return your flies to the culture tube. You will also be assigned a culture tube with F1 larvae. The F1 generation has been produced by the mating of two of the above four groups of flies (wild, mutation A, mutation B, mutation C).
6 WEEK TWO: F1 COUNT Goals for this week are: to accurately describe your fly culture Day 1 scoring (use some flies for F1 cross) to cross the F1 generation using 6 mating pairs to learn the proper procedure for disposing of the flies (alcohol morgue) Day 2 scoring 1 Describe the general health of your culture below. 2 Follow the above FlyNap procedure and score all the flies which have emerged for two days. Record your results in Table 7.2 on the Data Sheet. 3 Make a fresh culture tube following the "How to Make Drosophila Media" procedure. 4 Place 6 mating pairs of flies from this F1 generation into a fresh culture tube. In a few days, the F2 generation will emerge. 5 Drop flies in the morgue after they have been counted and the 6 mating pairs have been set aside. 6 Place the tube with the emerging F1 generation and the tube with the new mating pairs in the incubator. Make sure they are labeled with the generation, your group name and your symbols for the flies inside. WEEK THREE: F1 DISPOSAL Goals for this week are: to accurately describe the appearance of your culture to dispose of F1 generation to check over your data set to complete the chisquare worksheet in Appendix D. Turn in at the start of WEEK FOUR. 1 Examine your culture carefully for eggs (on the surface of the media) or tunnels within the media. 2 Once these observations are made, dispose of the F1 flies. WEEK FOUR: F2 COUNT This is the final and most important week of the experiment. Your group MUST BE meticulous about the scoring over the next 4 or 5 days. Record your results in Table 7.3 on the Data Sheet. You should, as a group, decide on who will do the count on which of the next few days. If a weekend falls within this time, it will not skew your results. However, the person assigned to score just after the weekend will have a long session in front of him/her. It would be easier on the group, and better for your results, if two people share the task of scoring after any hiatus. Dispose of the flies in alcohol after you have scored them (DO NOT return counted flies to the culture tube). Goals for this week are: to score the emerging F2 generation to finish analysis of results NOTE: An important detail to keep in mind while filling out Tables 7.2 and BE SURE TO DISTINGUISH BETWEEN OBSERVED MUTATIONS!
7 WEEK 3: DATA SHEET your assigned culture WEEK ONE: EXAMINATION OF PARENTAL TYPES Description: Table 7.1: Week One (you need to fill in the first column and have the second two columns checked off and initialed by your teacher after you have identified them) ~ your group's culture tube ~ stereo microscope with il name and symbol of mutation male female wild type (+) WEEK TWO: F1 COUNT Description: ~ your group's culture tube ~ stereo microscope with illuminator name and symbol of mutation male female wild type (+) Table 7.2: Emerging F1 generation: WEEK THREE: F1 DISPOSAL Description: WEEK FOUR: F2 COUNT Table 7.3: Emerging F2 generation ~ your group's culture tube ~ stereo microscope with illuminator name and symbol of mutation male female wild type (+) date mutation(s) observed number of males number of females date name of mutation number of males number of females
8 ANALYSIS OF RESULTS 1 From your results, define your cross: Sex linked or autosomal? Dominant or recessive mutation? Monohybrid or dihybrid? 2 Refer to the text book and review Punnett squares. In the space below, construct two Punnett squares that describe your cross. They should reflect your expected results of (1) the parental and (2) the F1 crosses. 3 According to these squares (the Punnett squares from #2), what are the expected ratios for the genotypes and phenotypes of the F1 and F2 generations? Be sure to clearly indicate the trait(s) each number represents. 4 Quantitatively, do your results deviate from what was expected? If so, explain why. 5 Are the deviations between the observed and expected results for the F2 phenotypic ratio within the limits expected by chance? To answer this question properly you must use the chi square statistical analysis. Report below your χ 2 value as well as your probability, p. AS ALWAYS, SHOW ALL CALCULATIONS!!! χ 2 = p = ~ your group's culture tube ~ stereo microscope with illuminator 1 What was your Null Hypothesis for this experiment? Do you accept or reject it on the basis of your χ 2 value? 2 Was it necessary for the females of the parental (P) generation to be virgins when they were mated? Why or why not? 3 Was it necessary for the females of the F1 generation to be virgins when they were mated? Why or why not? 4 Why were the adult flies removed from the culture tubes after weeks 2 and 4?
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