Lab 7: Genetics of Organisms

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General Overview

Tip: "One good source of information on drosophila is the drosophila manual, available from Carolina Biological. It comes with every order of fly material and is often given away free at conferences. You might call them and ask if they would be willing to send you one. The manual is an excellent source of information on many topics."
Margery Weitkamp, James Monroe High School, North Hills, California. 1/15/99

Question: "Does anyone know where I can find information about which drosophila mutations are recessive, which are dominant, autosomal, sex-linked, etc.?"

Answer 1: "I use these two references quite a bit: The virtual fly lab is excellent (ed. note: check other sites if clogged) The Drosophila Virtual Library. This directory points to Internet resources for research on the fruit fly drosophila melanogaster." (See below for these links.) Franklin M. Bell, St. Mary's Hall, San Antonio, Texas. 11/19/99 Answer 2: "When you order flies from Carolina, they send you a booklet that has some of this information. Surely the Internet also has a page for this."
Jerry Burke, St. Mary's School, Medford, Oregon. 11/19/99

Comment: "There is an unfortunate misconception that dominant must somehow be 'better' and more common. We teachers promote this somewhat with hybrid crosses in which 75 percent of the offspring express the dominant trait. Hybrid crosses are paper/pencil exercises, not nature. Furthermore, many mutations are recessive alleles (just look at the fruit fly list). Many contrary examples can be found, such as dominant Huntington's chorea. Another reason for this misconception is selection. 'Bad' dominant alleles are selected against. Huntington's remains in the gene pool because it does not express itself until after the individual has had offspring. Also, 'bad' recessive alleles can hide in their hybrid state, masked by the 'good' dominant allele. I don't think genes 'become' dominant. There are probably a lot of genes that have no alternate alleles, i.e., the trait is present in only one form (development of a heart). Any alternative would be strongly selected against and never become present in the gene pool. Dominant/recessive has no meaning here without the presence of an alternate allele. Alleles evolve because they are selected for after the mutation. Another thing to think about with dominant/recessive: Oftentimes, the dominant allele is a 'dose' of some protein that gets coded for. The recessive is the lack of that dose. Brown eyes are the expression of the pigment that masks the nonexpression, which everyone (except for Edgar and Johnny Winters) has behind it. Blood types have two possible doses (A or B), and one possible lack of a dose (recessive O). The higher incidence of O might suggest (pardon me if I 'beg the question') it is more adaptive. O blood has antibodies for both A and B, i.e., it can recognize when markers (antigens) enter the body from any other blood type. Isn't it better to be able to defend oneself against foreign invaders? Remember, this system evolved not knowing we would develop transfusion technology. I give my students the analogy of a house painter. The paint is the dominant allele, the recessive is the unpainted house. It still has color but was not acted upon by the painter. Note that this logic is sprinkled heavily with evolution concepts. If this is not done, any explanation would be less satisfying I believe. Whether right or wrong, it makes sense according to the natural selection model and even fits a genetics scheme.

You can really complicate things with epistasis (one gene's interactive effect is to allow/disallow the other gene's expression). Perhaps there is a fence around the house in the analogy above. The painter may be present (dominant allele A-), but unless the other gene is also present in its dominant form (B-), the house can't get painted! A-B- paints the house the dominant form, aaB- paints it the recessive color, A-bb or aabb does not paint the house (gate locked, painter frustrated)."
Jerry Burke, St. Mary's School, Medford, Oregon. 12/5/99

Comment: "One of the legacies of our emphasis on the particulate nature Mendelian genetics is that we sometimes get to thinking that all traits are Mendelian. I'm just reminding everyone that most traits of interest are quantitative-controlled by the interaction of any number of genes. Dominant/recessive models that help to formulate an understanding of Mendelian traits don't really enlighten much in quantitative inheritance models where gene frequencies are so important. It's really important that we make this distinction. I first realized this (not just book learning) about 12 years ago when I was trying to select some traits in fast plants that would behave in a Mendelian manner. This was before I knew Paul W. or that he had already identified some Mendelian traits. Have you ever tried to do this? Just look at any population of organisms-which physical traits are single-gene traits (Mendelian) and which are polygenic (quantitative)? I think you'll be amazed. It is not easy to screen for single gene traits-you often have to go through dozens to find what you want. That is because most of the physical traits that we would consider (anthropomorphically) important for an organism's survival are polygenic. (There are, of course, the exceptions of metabolic pathway enzymes, but I'm referring to physically expressed traits.) This is particularly important in an evolutionary sense and one of the results of emphasizing Mendel so much. Students think that all inheritance is Mendelian-if that were so (for all traits), then slow evolutionary change would be highly unlikely.

Let me see if I can better express what I mean. Take two fast-plant traits-hairiness and yellow-green/green. For hairiness I can select for hairiness-my initial population starting at a mean number of hairs of about 3.9 hairs /first true leaf petiole and going to over 60 hairs/first true leaf petiole by the sixth generation of selection. For yellow-green/green, it is a Mendelian trait-I only get yellow-green (if homozygous recessive) or green (if homozygous dominant or heterozygous.) So for hairiness I can gradually select for a change in the population that is expressed in this trait-for yellow-green there are only two choices; it's hard for selection to do much of anything except choose between the alternatives. (The better question is why are the two alleles maintained in the population.) By the way, there are apparently about three or four genes with each gene having two or three alleles associated with the hairy trait. This gets to the nitty-gritty of this problem-just what is a trait (compared to a gene)? Natural selection works at the organismal level so traits are a conglomeration of attributes, controlled by a suite of genes. At the DNA level, natural selection is probably less important than random processes, such as drift, which probably accounts for much in the way of gene frequency change."
Brad Williamson, Olathe East High School, Olathe, Kansas. 12/5/99

Equipment and Supply Modifications

Question: "Does anyone use genetic tasting papers?" Answer: "I have a short article on taste-test papers: Hundley, Louis R. 1960. 'Taste Test Papers.' Carolina Tips, Carolina Biological Supply Co. It is a reprint that was sent to me with a order of taste-test papers. They might still have reprints available. Na benzoate tasting is a dominant trait, like PTC tasting. The author discusses the relationship between tasting PTC and tasting Na benzoate in different ways (salty, sweet, sour, bitter). He quotes a study showing that about one-quarter of a population can taste it. He also says that thiourea, chemically related to PTC, is tasted by everyone as a 'nauseating' taste."
Alexa Noble, Oak Ridge High School, Conroe, Texas. 2/9/01

Pre-Lab Preparations

Procedure Modifications

Question: "Just received my drosophila with the live already crossed! First question: I thought the students were to make the P x P cross. Having these come already crossed doesn't fit with the way I thought we were going to do this lab. I must be misunderstanding something about this lab's procedure. Second question: The labels on the vials showed that they had been initiated 11 days from today. It will be 12 days by the time I see the AP students. It seems probable that almost all of the F1s may have already hatched out and be flying around happily mating with their parents and each other. Suggestions?"

Answer: "When I do the cross we count F1s and F2s and let Carolina do the P crosses for several reasons: One is the ever unfortunate time constraint; the other is we don't have to worry about using virgins, which should be cleared out of vials every eight hours or so. The F2 generation comes from F1s self mating-so its okay that they are in the same vial (i.e., you don't need virgins). Once the F1s have emerged you can count them for about five days without worrying. After that, dispose of the vials. Place the counted F1s in new vials of food for about 3-5 days, then remove them. The flies that emerge from these crosses will be the F2s. Good luck!"
Heda O'Brien, The Bullis School, Potomac, Maryland. 2/10/99

Answer 4: "I do real flies and have found great shortcuts. I order the parent flies and make the parent cross myself. When the F1s hatch, I dispense them into vials myself and distribute the vials to the students. They wait until pupae appear, remove the F1s, look at them, and record the characteristics. Then they count F2s as they hatch. I make them work backwards and give me the genotypes and phenotypes of the parents. I always give the girls two identical crosses as backups and also to increase the numbers of flies for better ratios. It makes the time manageable for the students and the hassle is gone trying to get virgin females in that narrow window. I also use obvious phenotypes-vestigial wings, white eyes, etc."
Charlotte Freeman, Girls Preparatory School, Chattanooga, Tennessee. 2/29/00

Question: "Is it physically possible to do fruit flies with two or three classes?

Answer 1: "It is quite feasible to use the fruit flies with 57 students. I have used a portable chick incubator (Styrofoam type) and this method is quite sufficient. The temperature is easily maintained and the cost is minimal for the incubator."
Gina Sourwine, Lyme Central School, Chaumont, New York. 2/04/01

Answer 2: "I, too, had multiple sections, a large number of AP Biology students, and minimal storage space for fruit flies. I chose to use genetic corn as a substitute. Some advantages: they do not need to be anesthetized, they don't travel to the faculty lounge and/or the cafeteria, and they can be used from year to year if students handle them gently."
Fred Brown, Hall High School, West Hartford, Connecticut. 2/05/01

Extended Lab Tip: "I just ordered the three autosomal mutants (two on one chromosome and the third on another). I think they were ebony body, sepia eyes, and dumpy wings. All are described by Connecticut Valley as being easy to score for beginners. I ordered virgin females of two of the three and wild-type virgin females as well. I will tell the students that they must do the minimal number of crosses to determine which, if any, of the three are linked. They will make hypotheses and then run a chi-squared analysis. I also ordered a sex-linked yellow body for them to work with to learn to recognize the phenotype and to learn how to distinguish males from females; I also ordered also virgin females. The students will be told that they have to do enough crosses to determine whether yellow body is sex-linked or autosomal and whether it is recessive or dominant. The total for all this is five crosses, the way I figure it. The whole class of five students will have to work together to design and carry out the crosses. Each student will be responsible for following one cross.

The reason I chose to bite the bullet and pay for virgins is that I have found that it is one of the major stumbling blocks over the years, given their inexperience and demanding schedules. Even if I empty vials in the morning, it is difficult for them to get in and separate males/females without killing them and without too much time passing (so that they can be sure the females are still virgins). We do talk in class about why it is important that the females be virgins but that it does not matter for the males. By the way, Connecticut Valley does not advertise that they offer virgin yellows-bodied females because they have lower viability and are hard to collect. But they will do so, upon special request."
Barbara R. Beitch, Hamden Hall Country Day School, Hamden, Connecticut. 2/7/01

Question: "Can someone please recommend a convenient, safe alternative to anesthetizing flies without FlyNap? I know you can freeze them or use ether, but isn't there something that allows you to use carbon dioxide?"

Answer 1: "If I remember correctly, you can put the flies into an empty vial with a stopper. In another vial with a one-hole stopper fitted with a piece of flexible tubing, place a little water and an Alka-Seltzer tablet. Run the other end of the tubing between the stopper and the wall of the vial of flies. This introduces the carbon dioxide into the fly vial. The problem with this technique is that the flies don't stay out long but since carbon dioxide is heavier than air, you are supposed to be able to lay the tubing from your Alka-Seltzer vial down near your plate/tray of flies and keep them out since the carbon dioxide should settle into the plate/tray and stay there. Alternatively, I know teachers who have frozen water in pie plates and then set the plate of flies down on the ice as a secondary way of keeping the flies out. Carolyn Schofield (I think it was her) recommends freezing water in butter tubs and then laying a piece of filter paper down on top of that and putting your flies on top of the filter paper. It doesn't matter how wet the filter paper gets, the flies can still be whisked off with a paint brush. The Alka-Seltzer technique is messy so you need to try it first. You need to get the right amount of water in the vial so that you are introducing only carbon dioxide- not bubbles-into the tube."
Tricia Glidewell, Marist School Atlanta, Georgia. 5/15/01

Answer 2: "This is a little messy, but try the following: Float the lid of a small (approx. 25 mm) petri dish in a larger (50mm+) petri dish that is full of water, then freeze it. I only tried this once at a workshop I attended, but I remember that the cold keeps the flies 'out' longer. I think we used something like a piece of dry ice in water hooked up to a Pasteur pipette to produce the carbon dioxide to put the flies to sleep."
Mary Wuerth, Tamalpais High School, Mill Valley, California. 5/15/01

Answer 3: "You can use Alka-Seltzer in a vial connected with tubing to another vial to anesthetize. It is somewhat messy and sometimes the flies wake up. I find that freezing them in an empty vial (no food) overnight does it and students can easily determine the characteristics of the flies without the hazards and smells of the chemicals."
Ginger Nathanson, Sparta High School, Sparta, New Jersey. 5/15/01

Answer 4: "Virtual fly lab is now a pay site, maintained by Benjamin Cummings and Pearson Education. It is part of a larger package of online labs that you can subscribe to either entirely or in part. I believe you can buy individual subscriptions or 'lab packs' for each of the 12 labs in the series. We used the fly lab this year and it comes with a nice teacher's guide and student handouts. The ISBN for fly lab is 0-8053-6527 and the telephone number is (800) 824-7799."
Frank Bell, Saint Mary's Hall, San Antonio, Texas. 5/24/01

Answer 5: " I bought a license for the fly lab this year, but I was disappointed that they had made significant changes to the lab and its interface with students. In my opinion they took an outstanding resource and made it mediocre. It's a shame that the original is no longer available."
Bruce Faitsch, Guilford High School, Guilford, Connecticut. 5/24/01

Answer 6: "Oh, I can't help responding to my favorite soapbox. As a biology teacher I cannot resist the opportunity to use live models. Living fruit flies over three generations is an ideal creature for teaching biology in all its glory. Students learn so much more than genetics with all the potential for disaster: mold, slime, crustiness, lost flies, complaining neighbors (threatening music teachers that blame all insects, bacteria, the world's ills on biologists!), impossible statistics, realization that not all data are clean (except in physics), appreciation for the care of living things, patience, and new found respect and fond regard for lowly dipterans. Also, without raising flies, how can your students look at third instar salivary glands or sperm of heroic dimension (approaches length of the fly itself)?"
Jerry Burke, St. Mary's School, Medford, Oregon. 5/24/01

Trouble Shooting

Question: "How do you determine the degrees of freedom for a problem?"

Answer: "The number of degrees of freedom depends on the number of phenotypic classes, which, in turn, depends on the way the cross is done: white female x wildtype males gives, in the F2, one wild-type female: one white female : one wild-type male : one white male. Therefore, there are four phenotypic classes and three degrees of freedom. When the cross is done the other way: wild-type female x white male, one gets, in the F2, two wild-type females : one wild-type male : one white male. Therefore, three phenotypic classes and two degrees of freedom. I hope this makes sense."
Dwayne A. Wise, Mississippi State University, Mississippi State, Mississippi. 1/4/01

Question: "On page 87 of the lab book, it reads 'If the calculated chi-square value is greater than or equal to the critical value from the table, then the null hypothesis is rejected.' Can I assume, then, that if the calculated chi-square value is LESS than or equal to the critical value from the table, then the null hypothesis is accepted? My kids are getting chi-squares of, say, 0.00875. With one degree of freedom, does this mean that the null hypothesis is correct?"

Answer 1: "Most statisticians would say that you 'fail to reject the null hypothesis' if chi-square is less than critical. I use this arcane language because I feel that it emphasizes the idea that science rarely proves anything (contrary to the popular media), but instead only disproves the other possible ideas. That way we can get off scot-free when some cherished idea (Central Dogma, humans have 100,000 genes) has to be trashed when we get more complete data. Hope my thoughts help."
David Hall, Martin County High School, Stuart, Florida. 4/11/01

Answer 2: "A chi-square greater than the critical value indicates that the null hypothesis is rejected. This means that the real ratio is very different from the expected ratio, and unlikely to have happened by chance, thus most likely the expected ratio (null hypothesis) is incorrect. A chi-square less than the critical value leads to the conclusion that the null hypothesis cannot be rejected, which is slightly different from being accepted. It may be that it cannot be rejected (proven false) due to small sample size, large variation in the data, etc. The distinction is that it may be possible to prove something false, but impossible to prove something true. Example: If someone says 'I always tell the truth,' you only need to show one example of a lie to prove it false, but a thousand examples of telling the truth, while providing substantial evidence, cannot prove it is true. To summarize: In proper statistical language you cannot say that the null hypothesis is correct, you must say it cannot be rejected. Nevertheless, a chi-square less than the critical value provides evidence that the null hypothesis is probably correct."
Gerry Rau, Lincoln American School, Taichung, Taiwan. 4/11/01

Alternative Lab Ideas

Question: "I am running the virtual fly lab instead of AP #7 and was wondering if anyone had suggestions of which crosses to test. I am not a drosophila person at all and do not know which genes are sex linked or how each trait is dominant."

Answer 1: "Try some of the following: White eyes, prune eyes-sex-linked recessive Dumpy wings, vestigial wings, cinnibar eyes-chromosome 2 recessive Sepia eyes, ebony body, spineless-chromosome 3 recessive Eyeless, shaven-chromosome 4 recessive. You can mix or match to present linkage, independent assortment, crossing over, etc. Avoid curly, asristapedia, and some of the others. They are homozygous dominant lethal. The phenotype is only expressed in the heterozygote, so there are no pure strains. I drove a few of my 'smarter' students crazy with them."
Israel Solon, Greenhill School, Dallas, Texas. 1/14/99

Answer 2: "I had my classes do the live flies as well as the virtual fly lab. Be aware that many of the crosses can be lethal-I suggest you choose some, run them yourself, figure out the genetics before you assign them. Otherwise, you and the students will have a difficult time. You will also need to make each cross in reverse by sex to check for the sex-linked one. It is great-wonderful to do chi squares. My students could appreciate the virtual fly after hours of microscope work. Good luck."
Pam Kitchens, Harlem High School, Marlem, Georgia. 1/14/99

Tip: "Two great genetics programs that I believe are available for PCs are Catlab and Birdbreed. Catlab is lots of fun because the inheritance of the various coat patterns in the program is something the students have seen (and wondered about) in their own pets. Birdbreed concerns the genetics of parakeets."
Anne Soos, Stuart Country Day School, Princeton, New Jersey. 6/28/99

Question: "I have never used fly genetics software/Web sites and am really curious now. I have always felt that nothing could replace the highs and lows from actually using the flies and experiencing the media crashing down and wiping out the population and getting the less that perfect data and then running the chi square test and finding out it really was a 3:1 ratio. My students love the ownership of their cultures and, while I've learned ways to short cut it such as doing big flasks of parent crosses as a class demonstration and then giving each of them their own F1 vials, they still are responsible for getting to the lab and getting the F1 flies out before the F2s hatch and then getting the F2s counted before the next generations emerge. I hate for them to lose this responsibility and hands on experience... but again it would be nice to get it done quickly and easily. Has anyone else decided that technology is really the way to go rather than hands on?"

Answer 1: "I guess that I am prejudiced in favor of the virtual fly lab because my first experience using real flies was such a nightmare. We were working on a 4 x 4 block schedule that left my students so busy that they couldn't come in outside of class time unless it was a Saturday (and sometimes not even then). Very often, the F1 and F2 generations were working on F3 by the time the students could come in to look at the flies. We didn't have FlyNap, and had no money with which to buy it, so we were using the old stick-em-in-the-refrigerator-and-hope-they-don't-die method. They never stay asleep long enough to count them and transfer them into new cultures. I had drosophila buzzing me the rest of the year. I had one student in another class who must have been using a fruit-scented lotion or something, because she was always being bothered by the flies. When I found the virtual fly lab, it was a dream come true. Not only could I, blind as a bat, actually see what the flies were supposed to look like, but I didn't have flies in my classroom in perpetuity."
Jo Ann Burman, Andress High, El Paso, Texas. 9/20/99

Answer 2: "Having taught genetics for more than two decades, I can certainly relate to people's complaints (nobody yet mentioned infestation of the fly cultures by mites or ants). I have always given students three unknown cultures and a wild type, and they are expected to spend the semester determining the genotypes of the unknown flies. In the process, they learn much more than genetics: how to deal with contamination, infestations, 'weak' stocks (e.g., crosses with a vestigial wing never return the expected 3:1 ratio in the F2 because homozygous vgvg flies are not very healthy) and, most of all, the responsibility of caring for a living creature. Now comes virtual fly lab and, I must say, it is very good. The problem with the 'old' way was that students had no data to analyze until the end of the term, so that sometimes the lights were out permanently. VFL gives one a way to ask the student to deal with data-the results of monohybrid and dihybrid crosses, and even test cross mapping data, early in the term. I vote with a hearty hurrah for using the two together; real flies and virtual flies. Seems like an unbeatable combination.

By the way, for people using real flies, you can avoid some of the problems with overlapping generations with judicious use of a lower temperature; just have the student keep the flies at room temperature for a few days. This slows down the developmental rate and postpones the next generation."
Dwayne A. Wise, Mississippi State University, Mississippi State, Mississippi. 9/22/99

Question: "Do any of you use the virtual fly lab? Having decimated about two AP Biology classes and smelled up my lab with Fly Nap, I'm ready for something cleaner and less time consuming."

Answer 1: "The address is listed under See also. (Ed. note: Check a search engine if this address does not work; they are often clogged. Several mirror sites are available.) My students used it this year. In the end, I think I like the program we have here called 'Hyperfly Advanced' better. It gives more types of inheritance. The virtual fly lab beats Hyperfly, however, in being able to do dihybrid (or any polyhybrid) crosses. Either one is faster than the real thing, and less messy. But I admit, too, that the experience of the real thing is valuable."
Paula Petterson, Kent Place School, Summit, New Jersey. 2/29/00

Answer 2: "I use the virtual fly lab along with the real flies and the students seem to find doing the virtual lab first helpful and fun. The Web site is very easy to navigate and the instructions are clear, though it will take the students a few minutes (about 5-10 for most of mine) to get the hang of it. I did run through a quick demonstration of a cross for them before we started."
Robert Dennison, Jersey Village High School, Houston, Texas. 2/29/00

Answer 3: "The virtual fly lab is a Web site maintained by California State University, Los Angeles. It allows students to design drosophila crosses and then immediately see the results. It shows the students what the flies look like but, unlike the actual flies, the picture is large enough to see without a magnifying glass. The program will also do the chi-square analysis of the data for the students. The students can perform a drosophila lab that would normally take about two weeks in a single class period. I like it because my eyesight is too bad to help the students work very well with flies, but the virtual fly lab solves that problem by making the pictures large enough for me to see. Another advantage is that no flies can escape and spend the rest of their lives buzzing you and your students (we never had access to FlyNap and always used the refrigerator and ice bath method)."
Jo Ann Burman, Andress High School, El Paso, Texas. 1/18/99

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