A Three-Part Laboratory Exercise Using Flightless Fruit Flies
Transcription
A Three-Part Laboratory Exercise Using Flightless Fruit Flies
This article reprinted from: Chinnici, J. P. and R. Ketcham. 2008. A three-part laboratory exercise using flightless fruit flies (Drosophila melanogaster) to study modes of inheritance. Pages 127-136, in Tested Studies for Laboratory Teaching, Volume 29 (K.L. Clase, Editor). Proceedings of the 29th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 433 pages. Compilation copyright © 2008 by the Association for Biology Laboratory Education (ABLE) ISBN 1-890444-11-1 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright owner. Use solely at one’s own institution with no intent for profit is excluded from the preceding copyright restriction, unless otherwise noted on the copyright notice of the individual chapter in this volume. Proper credit to this publication must be included in your laboratory outline for each use; a sample citation is given above. Upon obtaining permission or with the “sole use at one’s own institution” exclusion, ABLE strongly encourages individuals to use the exercises in this proceedings volume in their teaching program. Although the laboratory exercises in this proceedings volume have been tested and due consideration has been given to safety, individuals performing these exercises must assume all responsibilities for risk. The Association for Biology Laboratory Education (ABLE) disclaims any liability with regards to safety in connection with the use of the exercises in this volume. The focus of ABLE is to improve the undergraduate biology laboratory experience by promoting the development and dissemination of interesting, innovative, and reliable laboratory exercises. Visit ABLE on the Web at: http://www.ableweb.org A Three-Part Laboratory Exercise Using Flightless Fruit Flies (Drosophila melanogaster) to Study Modes of Inheritance Joseph P. Chinnici1 and Robert B. Ketcham2 1 Department of Biology Box 842012 Virginia Commonwealth University Richmond, VA 23284-2012 [email protected] 2 Department of Biology University of Delaware Newark, DE 19716-2590 [email protected] Abstract: This is an inquiry-based investigation of genetic modes of inheritance using flightless Drosophila as the experimental organism. We present the three-part laboratory writeup, suitable for use as a student handout. We describe the flightless mutant strains and where they may be obtained, how to use carbon dioxide as fly anesthesia, various molecular websites for students to research the mutant genes, and helpful hints for setting up the laboratory. Practical benefits of using flightless flies include convenience in handling the organisms by inexperienced students, and reduced likelihood of flies escaping to invade other areas of the school building. Association for Biology Laboratory Education (ABLE) 2007 Proceedings, Vol. 29:127-136 Modes of Inheritance Using Drosophila 127 Introduction This objective of this exercise is for students to perform an inquiry-based investigation of genetic modes of inheritance using flightless fruit flies as the experimental organism (see Chinnici et al., 2005). We have used this exercise in introductory general education science college courses for non-science majors, and many high school advanced biology courses also use this exercise. Students learn how to anesthetize the flies, distinguish male and female fruit flies, identify unknown (to them) mutant traits by comparing mutant and wild-type flies, and determine genetic modes of inheritance for their mutant type by setting up parental, F1, and F2 generation crosses and observing the offspring of these crosses. They analyze their F2 generation data using chi-square analyses. The exercise consists of three 90-120 minute sessions each separated by two weeks (performed on days 1 [week 1], 15 [week 3], and 29 [week 5]) to allow the offspring of each generation to develop into adults. Other lab exercises may be performed on days 8 [week 2] and 22 [week 4]. Materials Here, we describe the flightless flies and where they may be purchased, how to use carbon dioxide as an alternative to “Fly Nap” for fly anesthesia, various molecular websites for students to research the different mutants, and helpful hints for setting up the laboratory. Flightless Fruit Flies Ten years ago, one of the authors (JPC) constructed the various mutant strains of flightless flies used in this exercise. The available flightless strains of Drosophila are white eyes, whiteapricot eyes, yellow body, singed wings, and cut wings (all recessive X-linked traits); Bar eyes (a dominant X-linked trait); dumpy wings, vestigial wings, scarlet eyes, sepia eyes, ebony body, apterous wings, and eyeless eyes (all autosomal recessive traits). Each of these mutant strains has the X-linked recessive trait miniature wings fixed in its genetic background. The “wildtype” or normal strain used in exercises with these mutants is the miniature wing strain. Thus, miniature wing is the genetic “standard” for all these strains. The practical benefit of using flightless flies is convenience in handling flies by typically inexperienced students, and no likelihood of flies escaping the laboratory to invade other areas of the school building. Carolina Biological Supply Company carries the flightless fruit fly kits and individual strains. Go to <carolina.com>, type in “flightless fruit flies” and select “teacher resources” for more information about use of the flies. At the end of the article Using Flightless Fruit Flies in the Genetics Teaching Lab, click on “Flightless Fruit Fly Kits” and, then, “Flightless Fruit Fly Mutants” for more information. Using CO2 As An Anesthetic As an alternative to using “Fly-Nap” for fly anesthesia, one of us (RK) uses carbon dioxide. Once a CO2 delivery system is constructed in a lab room, it provides a convenient, odor-free means for students to anesthetize flies. Flies stay "out" as long as CO2 is supplied, but they recover within minutes when removed from the CO2. Different CO2 delivery systems can vary a lot in details of 128 ABLE 2007 Proceedings Vol. 29 Chinnici and Ketcham construction. Here, we describe the system we built into RK’s teaching labs at the University of Delaware and add some comments on where the system at the University of Kentucky differs from ours. Tanks, regulators, control valves. We installed two CO2 tanks in each of our lab rooms. Each tank has its own two-stage regulator (Fisher 10-572E) and the piping from the two tanks come together in a T, where a 3-way selector valve (McMaster Carr 4373K51) allows the operator to choose which tank is in use. This makes it possible for a lab instructor to restore the CO2 supply simply by throwing the selector valve to the second tank if one tank is emptied during a class activity. Each tank has a lever-style shut off valve (McMaster Carr 4726K72) installed between its regulator and the selector valve. Piping from tank to work stations. The University of Kentucky has an ideal set up for distributing CO2 around the room. Their labs were built with gas cocks at each student seat and they converted that system to CO2. At Delaware, we had to install our piping from scratch. An early version consisting of 1/2" Tygon tubing running down each bench was workable but cumbersome. We upgraded by installing 3/4" c-PVC tubing underneath each bench, with a branch point at each student station. Each branch consists of a length of amber gas-line tubing (Fisher 14-178 2B), which steps down to aquarium airline tubing. Student stations. Our student stations use flexible silicone aquarium airline tubing (Penn Plax STD25) and a two-valve aquarium airline gang valve (Penn Plax VN2). From the gang valve, one line is attached to a 16G 3" hypodermic needle (Fischer 14-817-103) with the tip cut off; students use this by inserting the needle into a vial or bottle to anesthetize the flies before dumping them onto a working platform. The second line from the gang valve goes to the working platform, which students use to inspect and sort flies under the dissecting microscope. Connections in the airline tubing are made using Luer fittings (Value Plastics FTLL230-1 and MTLL230-1). My choice of material for building student work platforms is floral foam. It has high resistance to gas flow but disperses the gas very uniformly. I buy bricks (9" x 3" x 41/4") at a local florist, and cut them to 1" x 3" x 23/4" blocks on a band saw (wearing a respirator is important - the dust is irritating). A channel for the gas to enter the block is created by pushing the handle of a flysorting paint brush (Carolina 17-3094) down the center of the block, starting in the center of the smallest face and extending about 7/8 of the length of the block. Each block is covered on the bottom and four sides with card stock, as a gas barrier. A hole punched into the card stock at one end accommodates a Luer fitting (FTLL230-1) that fits into the opening of the gas channel down the center of the block. The top and four sides of the block are covered with Whatman #1 filter paper, cut, folded, and taped to the bottom of the block to form a flat, smooth, gas-permeable working surface for sorting flies. Several other materials may make suitable substitutes for floral foam. I have used cellulose sponges, Styrofoam (though most Styrofoam packaging is impervious to gas), and upholsterer's foam. The University of Kentucky built working platforms using the bottom half of pipette-tip boxes covered with Mylar fabric. The CO2 in their system is dispersed by an aquarium air stone in the hollow base of the pipette-tip box. Modes of Inheritance Using Drosophila 129 Student Outline We present the three-part laboratory exercise write-up, suitable for use as a student handout, in APPENDIX A. MS-Word formatted files of these three exercises are available by emailing JPC ([email protected]). Materials and Equipment Initially, (week 1) students work in groups of two or three. Each group receives three miniature wing cultures; a “wild-type” culture containing male and female flightless flies with the miniature wing trait only; an “unknown” mutant culture with males and females possessing one mutant trait; and, a culture containing only virgin wild-type females. Each group also receives an empty culture vial (in which to place sleeping flies) and an anesthetizing chamber (an empty vial with a foam stopped through which a wand from the “Fly-Nap” kit is placed) if “Fly-Nap” is used. Each group also uses a dissecting microscope, index cards (on which sleeping flies are placed for viewing and sorting), and either toothpicks or a small artists watercolor brush (for pushing the flies around on the index cards). For the second part of the exercise (week 3), each student in the groups receives a food vial without flies, in which to place some F1 flies to generate the F2 generation. For the third part of the exercise (week 5), each student in the class will need a dissecting microscope in order to collect data from his/her vial of flies set up in week 3, as well as an individual anesthetizing chamber. Notes for the Instructor Culture Medium We use Instant Drosophila food as the culture medium for maintaining our stocks and for the experimental procedures: go to <www.carolina.com > and type in “17 3200” (for white medium) or “17 3210” (for blue medium). To prepare student fly cultures, first, we add the dry Instant Drosophila medium to a vial. Then, we sprinkle in some dry yeast (obtained from the supermarket). When adding water, we use distilled water if available, or jugs of drinking water from the supermarket. We avoid using tap water due to the risk of introducing mold into the cultures. In addition, we add a small rectangle of white paper toweling impregnated with “Tegosept” mold inhibitor to each vial. Tegosept may be ordered fro Carolina Biological Supply Co.: <https://www2.carolina.com/webapp/wcs/stores/servlet/ProductDisplay?jdeAddressId=&catalogId= 10101&storeId=10151&productId=23738&langId=-1&parent_category_rn=&crumbs=n>. We mix 10 grams of Tegosept powder into 100 ml of 100% ethyl alcohol and soak full sheets of white paper toweling, squeezing the excess fluid out, and then hanging the wet towels up on a clothesline until the alcohol evaporates. Then, we cut the paper towels into small rectangles (1 x 4 inches = 2.5 x 10 cm). We then push the “tego-strip” into the surface of the fly-food in the vial with the handle of a small artist’s watercolor brush. These “tego-strips” accomplish two purposes: protect against mold infestation, and give the larvae more surface area for pupation. 130 ABLE 2007 Proceedings Vol. 29 Chinnici and Ketcham Collecting “Virgin” Female Drosophila In the parental generation, “virgin” (previously unmated) wild-type females are crossed with mutant males. We collect the virgin females for the students to use, since the entire experiment will be ruined if the parental generation females are not virgins, a trivial reason for students to have their experiments fail. Virgin females are easy to collect, since female fruit flies cannot accept sperm from males until they are at least four hours post-emergence from their pupal cases (it takes that time for them to expel larval wastes from their seminal receptacles). So, a few days before the beginning of the exercise, we clear all the wild-type culture vials of adults at 10:00AM, then return three hours later (1:00PM) and collect any “new” females which have emerged, knowing that they are virgins. We repeat this again three hours later (4:00PM) and collect more virgin females. If needed, we do this again the following day. Then, the day before the exercise begins, we place 6-8 virgin females each in fresh food vials and have students add mutant males to these vials to begin the parental generation crosses. Alternatively, one may ask the students to collect their own virgin females, but this would be burdensome to the instructor who would have to clear the wild-type vials for each group of students three hours before they collect the virgins. In addition, in very young flies, it is more likely for inexperienced students to mis-sex the flies since the pigmentation is quite pale in newly emerged flies, increasing the chance for error. Adding virgin males to the parental cross would ruin the experiment. On days 8 (week 2) and 22 (week 4), either the instructor or the students must remove the adult flies from their vials, so that when the next generation of adults emerges several days later, they will not intermingle and mate with their parents (thus ruining the experiment). Molecular Websites for Researching the Mutant Genes Since we live in a molecular age, students should become exposed to some of the molecular aspects of fruit fly genetics. One way to incorporate some molecular biology into this exercise is to have students submit a report on some molecular aspects of the particular mutant gene they are following in their crosses. A good place for them to begin is at the “WWW Virtual Library: Drosophila” website: <http://www.ceolas.org/fly/>. Then, the student might go to the “National Center for Biotechnology Information” website for Drosophila melanogaster <http://www.ncbi.nlm.nih.gov/genome/guide/fly/> and finally to <http://www.ncbi.nlm.nih.gov/sites/entrez> where one selects “Gene” for Search and then types in the mutant name (for instance, “white” ). APPENDIX B lists some results for various mutant genes. An alternative molecular approach is to go to the FlyBase website, “A Database of Drosophila Genes & Genomes” <http://www.flybase.org/>. Here, one can simply select “genes” for Data Class (or “alleles” for “cut-6”) and then type in the mutant name (for instance, “miniature”). APPENDIX C lists some results for various mutant genes or alleles. Literature Cited Chinnici, J. P., A. M. Farland, and J. W. Kent. 2005. An Inquiry-Based Investigation of Modes of Inheritance Using “Flightless” Fruit Flies. The American Biology Teacher 67:38-44. Modes of Inheritance Using Drosophila 131 About the Authors Dr. Joseph P. Chinnici received his A.B. in Biology from LaSalle University in 1965, and his Ph.D. in Biology from the University of Virginia in 1970. He has been a faculty member in the Biology Department at Virginia Commonwealth University in Richmond, VA since 1970. Currently, he is an Emeritus Associate Professor of Biology and Life Sciences at VCU. In 2001, he was awarded the Distinguished Teaching Award from the College of Humanities and Sciences at VCU. Dr. Chinnici has published over 35 papers in a variety of research and teaching journals and has been a PI or coPI on several federal and state research grants totaling over five million dollars. Dr. Robert B. Ketcham earned his undergraduate degree at Wesleyan University and his Ph.D. degree at the University of Delaware. He currently works as Laboratory Coordinator in the Department of Biology at the University of Delaware, managing the laboratory component of the non-science majors' biology class. ©2007 Joseph P. Chinnici and Robert B. Ketcham 132 ABLE 2007 Proceedings Vol. 29 Chinnici and Ketcham APPENDIX B. Molecular Websites of Interest for Drosophila Mutants http://www.ceolas.org/fly/ [The WWW Virtual Library: Drosophila. This directory points to internet resources for research on the fruit fly Drosophila melanogaster] http://www.ncbi.nlm.nih.gov/ [National Center for Biotechnology Information] http://www.ncbi.nlm.nih.gov/sites/entrez [type in Drosophila and mutant name (e.g., Drosophila white)]. Some results are listed below. If one clicks on the gene symbol, a complete description appears (the longer website at the end of each summary). white [Drosophila melanogaster] Other Aliases: Dmel_CG2759, BACN33B1.1, CG2759, DMWHITE, EG:BACN33B1.1, unnamed, w(AT)[[13]] Other Designations: white CG2759-PA Chromosome: X; Location: 3B6-3B6 Annotation: Chromosome X, NC_004354.3 (2684632..2690499, complement) GeneID: 31271 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=31271&ordinalpos=9&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum ebony [Drosophila melanogaster] Other Aliases: Dmel_CG3331, CG3331 Other Designations: ebony CG3331-PA Chromosome: 3R; Location: 93C7-93D1 GeneID: 42521 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=42521&ordinalpos=1&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum yellow [Drosophila melanogaster] Other Aliases: Dmel_CG3757, CG3757, EG:125H10.2, T6 Other Designations: yellow CG3757-PA Chromosome: X; Location: 1A5-1A5 Annotation: Chromosome X, NC_004354.3 (250542..255278) GeneID: 30980 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=30980&ordinalpos=17&it ool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum vestigial [Drosophila melanogaster] Other Aliases: Dmel_CG3830, CG3830, VG, vg21 Other Designations: vestigial CG3830-PA Chromosome: 2R; Location: 49E1-49E1 GeneID: 36421 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=36421&ordinalpos=15&it ool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum cut [Drosophila melanogaster] Other Aliases: Dmel_CG11387, CG11387, Ct, Cut, kf Other Designations: cut CG11387-PA, isoform A; cut CG11387-PB, isoform B Chromosome: X; Location: 7B4-7B6 Annotation: Chromosome X, NC_004354.3 (7503181..7570056) GeneID: 44540 Modes of Inheritance Using Drosophila 133 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=44540&ordinalpos=2&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum apterous [Drosophila melanogaster] Other Aliases: Dmel_CG8376, CG8376, LIM, S-2a, Xa, blt Other Designations: apterous CG8376-PA, isoform A; apterous CG8376-PB, isoform B Chromosome: 2R; Location: 41F8-41F8 GeneID: 35509 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=35509&ordinalpos=27&it ool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum Bar [Drosophila melanogaster] Other Aliases: FBgn0000154, BB, Bar eye, BarH1, InfraBar, Ultrabar, bar Chromosome: 1; Location: 1-57.0 GeneID: 44798 This record was discontinued. http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=44798&ordinalpos=1&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum BarH1 [Drosophila melanogaster] Other Aliases: Dmel_CG5529, BH1, Bar, Bar H1, Bar-H1, BarHI, CG5529, barH1 Other Designations: BarH1 CG5529-PA Chromosome: X; Location: 16A4-16A5 Annotation: Chromosome X, NC_004354.3 (17291534..17297312) GeneID: 32724 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=32724&ordinalpos=4&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum BarH2 [Drosophila melanogaster] Other Aliases: Dmel_CG5488, B, BH2, Bar, Bar-H2, CG5488 Other Designations: BarH2 CG5488-PA Chromosome: X; Location: 16A1-16A1 Annotation: Chromosome X, NC_004354.3 (17208614..17218195) GeneID: 32723 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=32723&ordinalpos=5&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum dumpy [Drosophila melanogaster] Other Aliases: Dmel_CG33196, CG15637, CG33196, CT35799, DP, SP460 Other Designations: dumpy CG33196-PB Chromosome: 2L; Location: 24F4-25A1 GeneID: 318824 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=318824&ordinalpos=1&it ool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum singed [Drosophila melanogaster] Other Aliases: Dmel_CG32858, CG1536, CG32858, Sn, fs(1)K418, fs(1)M45 Other Designations: singed CG32858-PA, isoform A; singed CG32858-PB, isoform B; singed CG32858-PC, isoform C Chromosome: X; Location: 7D1-7D2 Annotation: Chromosome X, NC_004354.3 (7858057..7880134) GeneID: 31717 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=31717&ordinalpos=1&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum 134 ABLE 2007 Proceedings Vol. 29 Chinnici and Ketcham scarlet [Drosophila melanogaster] Other Aliases: Dmel_CG4314, CG4314 Other Designations: scarlet CG4314-PA Chromosome: 3L; Location: 73A3-73A3 GeneID: 39836 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=39836&ordinalpos=1&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum [sepia] CG6781 [Drosophila melanogaster] Other Aliases: Dmel_CG6781 Other Designations: CG6781-PA Chromosome: 3L; Location: 66D5-66D5 GeneID: 38973 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=38973&ordinalpos=1&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum brown [Drosophila melanogaster] Other Aliases: Dmel_CG17632, CG17632, Pm, Su(w[coJ]), unnamed Other Designations: brown CG17632-PA Chromosome: 2R; Location: 59E2-59E3 GeneID: 37724 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=37724&ordinalpos=60&it ool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum eyeless [Drosophila melanogaster] Other Aliases: Dmel_CG1464, CG1464, DPax-6, EYEL, Ey, Ey/Pax6, Pax-6, Pax6, eye, l(4)33 Other Designations: eyeless CG1464-PA, isoform A; eyeless CG1464-PB, isoform B; eyeless CG1464-PC, isoform C; eyeless CG1464-PD, isoform D Chromosome: 4; Location: 102C2-102C2 Annotation: Chromosome 4, NC_004353.3 (718315..741787) GeneID: 43812 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=43812&ordinalpos=1&ito ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum Modes of Inheritance Using Drosophila 135 APPENDIX C. More Molecular Websites of Interest for Drosophila Mutants http://www.flybase.org/ [ FlyBase: A Database of Drosophila Genes & Genomes] choose “genes”, type in “miniature”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=miniature&authors=&year=&alltext=&caller=quicksearch select “m”: http://www.flybase.org/reports/FBgn0002577.html choose “genes”, type in “Bar”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=Bar&authors=&year=&alltext=&caller=quicksearch select “B-H1”: http://www.flybase.org/reports/FBgn0011758.html select “B-H2”: http://www.flybase.org/reports/FBgn0004854.html choose “genes”, type in “white”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=white&authors=&year=&alltext=&caller=quicksearch select “w”: http://www.flybase.org/reports/FBgn0003996.html choose “genes”, type in “white apricot”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbal&context=white&authors=&year=&alltext=&caller=quicksearch select “w^a”: http://www.flybase.org/reports/FBal0018195.html choose “genes”, type in “yellow”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=yellow&authors=&year=&alltext=&caller=quicksearch select “y”: http://www.flybase.org/reports/FBgn0004034.html choose “genes”, type in “cut”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=cut&authors=&year=&alltext=&caller=quicksearch select “ct”: http://www.flybase.org/reports/FBgn0004198.html choose “alleles”, type in “cut”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbal&context=cut&authors=&year=&alltext=&caller=quicksearch select “ct^6”: http://www.flybase.org/reports/FBal0001934.html choose “genes”, type in “dumpy”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=dumpy&authors=&year=&alltext=&caller=quicksearch select “dp”: http://www.flybase.org/reports/FBgn0053196.html 136 ABLE 2007 Proceedings Vol. 29 Chinnici and Ketcham choose “genes”, type in “vestigial”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=vestigial&authors=&year=&alltext=&caller=quicksearch select “vg”: http://www.flybase.org/reports/FBgn0003975.html choose “genes”, type in “scarlet”: http://www.flybase.org/reports/FBgn0003515.html choose “genes”, type in “sepia”: http://www.flybase.org/reports/FBgn0086348.html choose “genes”, type in “brown”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=brown&authors=&year=&alltext=&caller=quicksearch select “bw”: http://www.flybase.org/reports/FBgn0000241.html choose “genes”, type in “ebony”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=ebony&authors=&year=&alltext=&caller=quicksearch select “e”: http://www.flybase.org/reports/FBgn0000527.html choose “genes”, type in “eyeless”: http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=eyeless&authors=&year=&alltext=&caller=quicksearch select :ey”: http://www.flybase.org/reports/FBgn0005558.html Exercise 1. 1-1 © Joseph P. Chinnici Exercise 1 Using the Scientific Method: Fruit Fly Studies A. B. C. Introduction; Fruit Fly Life Cycle Setting up a Fruit Fly Study Determining Inheritance Patterns A. Introduction. The Scientific Method consists of observation, hypothesis, experimentation, and drawing conclusions based on the results of the experiments performed. In this exercise, we will use elements of the Scientific Method to gather information about certain variable physical traits in fruit flies. From the observations, you will be asked to form hypotheses about the inheritance of these traits and devise experiments that will test the validity of your hypotheses. We will use fruit flies in our studies because these organisms are readily available. Fruit flies have been important insect organisms for genetic research since the early 1900s; today, more is known about the genetic control of fruit fly development, behavior, and evolution than is known about humans. In order for you to better understand the experiments you will perform, please read the following sections to gain some basic knowledge of the life cycles of fruit flies. Fruit fly life cycle. Fruit flies (Drosophila melanogaster) continue to be extremely useful organisms for the study of genetics. There are various reasons for this: they are small, being easy and economical to culture in the laboratory; they breed prolifically - each female is capable of laying several hundred eggs; they have a short generation time - from egg to adult in about 10 days at 25ΕC.; they have a large number of easy to see (under low magnification) external characteristics that show genetic variation; and they have a well understood life cycle, including descriptive embryology that is now being understood at the molecular level. Fruit flies are typical insects whose life cycle shows complete metamorphosis (the young stages do not resemble the adult stage; Fig. 1-1). Mature male and female flies mate and produce fertilized eggs each of which are oval in shape and about 0.5 mm in length. Within 24 hours, each egg hatches into a tiny nearly transparent wormlike larva, highly adapted to burrowing into and eating fleshy fruit. As each larva grows, it sheds ("molts") its external skin ("exoskeleton") twice, becoming whiter and larger after each molt. Thus, the larval period consists of three stages (called "instars"); the 1-2 Exercise 1. third instar larva reaches a length of 4.5 mm. The larvae are such intensely active and voracious feeders that the culture medium in which they are crawling becomes heavily channeled and furrowed, a sure sign that the mating was successful. When the third instar larvae are mature (about 5 days after the eggs were laid), they will crawl up the sides of the container and adhere to some relatively dry surface such as the side of the bottle or the paper toweling which has been inserted in the food. The larvae then pupate, during which their outer skin hardens and darkens and internal changes occur leading to the formation of the adult body from buds of tissue within the puparium. This is similar to what occurs when caterpillars metamorphose into butterflies. About 5 days after puparium formation in Drosophila, each puparium splits open at the anterior end and either a young male or female adult emerges, pale, wet, and with soft wings folded up like a parachute on its back (dorsal surface). Within 4 hours of emergence, each adult has produced its body pigmentation, has elongated and stiffened its wings, and has sexually matured. Matings between males and females then may occur, leading to another generation of offspring. Figure 1-1. Life cycle of fruit flies (Drosophila melanogaster). Exercise 1. 1-3 B. Setting up a fruit fly study. 1. Objectives. You will be given several vials each containing a small colony of fruit flies homozygous (pure breeding) for some physical trait known to the lab instructor. One vial contains normal flies, another contains mutants. The objectives of working with these flies are: 2. A. To describe in as much detail as possible, by observation of the flies in these vials, the mutant trait. B. To set up appropriate matings to determine the mode of inheritance of the mutant trait, namely: 1. Is the mutant trait dominant or recessive? 2. Is the gene for the mutant trait located on a sex chromosome (the X chromosome) or on a non-sex chromosome (one of the autosomes)? Fruit fly morphology (Fig. 1-2) A. B. Prominent features. Fruit flies have a typical insect body arrangement of head, thorax (chest), and abdomen. 1. Head. Prominent features on the head are the antennae, the mouth parts (called the proboscis), the large compound eyes (each consisting of about 800 individual facets), a triangular arrangement of three simple eye spots (called ocelli), and bristles. 2. Thorax. Prominent features are three pairs of walking legs, one pair of wings for flying, a pair of globular structures called halteres (analogous to the second pair of wings found in most other insects), a triangular region on the posterior dorsal surface called the scutellum, and many bristles. 3. Abdomen. Consists of a number of segments; the external genitalia and anal region are located at the posterior tip. Distinguishing males from females (Figure 1.2). Male and female Drosophila may be distinguished from each other very easily by a number of clear-cut differences. 1. Sex combs. Males possess sex-combs, a group of about 10 stout, black bristles towards the front of the first pair of legs. Females lack sex-combs. 1-4 Exercise 1. Figure 1-2: Fruit fly morphology. 2. External Genitalia. The external genitalia of males and females have obvious differences. Males have a series of brown to black Exercise 1. 1-5 external structures related to sperm transfer, but females do not. This is a striking difference even in extremely young flies. 3. 3. Number of Abdominal Segments. Females have 7-8 visible abdominal segments whereas males have 5-6. Except in very young flies, the tip of the abdomen is elongated in the female and somewhat more rounded in the male. As females age, their abdomens become swollen with maturing eggs. 4. Abdominal Pigmentation. In normal flies (but not all mutant stocks), the abdominal pigment pattern is sufficiently distinct in the two sexes to permit their identification on this basis without use of a microscope. The most posterior 3-4 segments in males are solid black, whereas each segment in the female bears a transverse narrow black band. In newly emerged adults, however, the abdominal pigmentation has not yet darkened. Experimental Procedures. A. B. Sorting out males and females. 1. Obtain a vial of normal flies ("wild type", vial labeled "+") and an empty vial. Anesthetize the flies using "Flynap" solution, following directions given by your lab instructor. 2. When the flies are no longer moving, place them onto a white plate and view them under a dissecting microscope. 3. Using a toothpick, push each fly into one of two groups, forming a group of females on the left side of the plate, and a group of males on the right side. When finished, have the student sitting next to you verify the accuracy of your sorting. If you are in doubt about your accuracy, ask the lab instructor for help. 4. When finished, leave about 10 males and 10 females on the plate and discard the other flies by dumping them into the jar of motor oil (the "fly morgue") on each table. Determining and describing the abnormal phenotype in the mutant fly colony. 1. Anesthetize the flies from the vial marked "M__", and place the sleeping flies onto the white plate. Be careful not to overdose the flies, since dead flies are not useful for mating purposes. 2. As you did for the normal flies, sort the mutant flies into male and female groups. 3. Compare the mutant males with normal males, and the normal 1-6 Exercise 1. females with the mutant females. Determine which physical trait differs consistently between normal and mutant flies. In as much detail as possible (including drawings if you desire), describe the mutant trait in the space below. FULL DESCRIPTION OF MUTANT TRAIT FROM VIAL "M_____" How does it differ from the normal (+) flies? C. Setting up a fly mating. 1. After determining and describing the mutant trait, place 10 of the healthiest looking mutant males in a clean vial. 2. Place these males into the vial marked "VIRGIN + FEMALES." Your lab instructor will demonstrate the proper procedure for this. This vial contains 5 or 6 normal females collected shortly after they emerged from their pupal cases before they became sexually mature. Thus, these females have never mated previously. Be very careful not to let the virgin females escape!! Fill in the M___ designation on the vial label. 3. Turn the vial upside down and gently tap the vial until the sleeping males fall onto the stopper. Then, lay the vial on its side until the males revive, after which you should stand the vial upright. 4. Then, discard all remaining normal and mutant flies remaining on the plate by dumping them into the fly morgue. Exercise 1. 1-7 5. Write your name on the labels of the following vials: - The "+" vial, containing eggs from normal flies. - The "M___" vial, containing eggs from mutant flies. - The "VIRGIN + FEMALE x M___" vial, containing adult normal virgin females and the mutant males you just put in. Now, Place a rubber band around the three vials and put them into the constant temperature incubator. 6. C. Check your vials next week to see the larvae and pupal cases. Also, you must remove any adults from these vials next week to prevent the offspring that will emerge from comingling with the parents. In two weeks time, enough offspring (the F1 generation) will have emerged to permit the next stage of this experiment. Determining Inheritance Patterns. The purpose of the fruit fly mating is for you to scientifically determine two important aspects of the inheritance of the mutant trait, namely whether it is a dominant or recessive trait and whether it is associated with the chromosomes that determine gender (the X and Y chromosomes) or the chromosomes that are common to both male and female flies (the autosomes). There are four realistic possibilities: autosomal dominant, autosomal recessive, X-linked dominant, and X-linked recessive (why couldn't the mutant gene be located in the Y chromosome?) Use the information on the following pages to predict the outcomes of your mating for each of these possibilities. 1. 1. Autosomal dominant inheritance. 2. Autosomal recessive inheritance. 3. X-linked dominant inheritance. 4. X-linked recessive inheritance. Autosomal dominant inheritance. If the mutant trait shows autosomal dominant inheritance, the mutant gene is designated "A", and all homozygotes (AA) and heterozygous (Aa) flies would display the mutant trait. Only the homozygous recessive (aa) flies would be normal. Fill in the following chart, predicting the phenotypes and ratios of flies in the F1 and F2 generations. P generation: (all parents are homozygotes) 1-8 Exercise 1. phenotypes: normal females x mutant males genotypes: ______________ 9 ____________ phenotypes: ______ females x _______ males genotypes: ______________ 9 _____________ F1 generation: F2 generation: (Use a Punnett square to determine the expected results) sperm eggs F2 FEMALES PHENOTYPES GENOTYPES F2 MALES RATIOS PHENOTYPES GENOTYPES RATIOS Exercise 1. 1-9 2. Autosomal recessive inheritance. If the mutant trait shows autosomal recessive inheritance, the normal gene is designated "B", and all homozygotes (BB) and heterozygous (Bb) flies would display the normal trait. Only the homozygous recessive (bb) flies would be mutant. Fill in the following chart, predicting the phenotypes and ratios of flies in the F1 and F2 generations. P generation: (all parents are homozygotes) phenotypes: normal females x mutant males genotypes: ______________ 9 ____________ phenotypes: ______ females x _______ males genotypes: ______________ 9 _____________ F1 generation: F2 generation: (Use a Punnett square to determine the expected results) sperm eggs F2 FEMALES PHENOTYPES 3. GENOTYPES F2 MALES RATIOS PHENOTYPES GENOTYPES RATIOS X-linked dominant inheritance. If the mutant trait shows X-linked 1-10 Exercise 1. dominant inheritance, the mutant gene is designated "XA", and all flies that are XAXA and XAXa females and XAY males would display the mutant trait. Only XaXa females and XaY males would be normal. Fill in the following chart, predicting the phenotypes and ratios of flies in the F1 and F2 generations. P generation: (all parents are homozygotes) phenotypes: normal females x mutant males genotypes: ______________ 9 ____________ phenotypes: ______ females x _______ males genotypes: ______________ 9 _____________ F1 generation: F2 generation: (Use a Punnett square to determine the expected results) sperm eggs F2 FEMALES PHENOTYPES 4. GENOTYPES F2 MALES RATIOS PHENOTYPES GENOTYPES RATIOS X-linked recessive inheritance. If the mutant trait shows X-linked recessive inheritance, the normal gene is designated "XB", and all flies that are Exercise 1. 1-11 XBXB and XBXb females and XBY males would display the normal trait. Only XbXb females and XbY males would be mutant. Fill in the following chart, predicting the phenotypes and ratios of flies in the F1 and F2 generations. P generation: (all parents are homozygotes) phenotypes: normal females x mutant males genotypes: ______________ 9 ____________ phenotypes: ______ females x _______ males genotypes: ______________ 9 _____________ F1 generation: F2 generation: (Use a Punnett square to determine the expected results) sperm eggs F2 FEMALES PHENOTYPES GENOTYPES F2 MALES RATIOS PHENOTYPES GENOTYPES RATIOS Exercise 2. 2-1 © Joseph P. Chinnici Exercise 2 Fruit Flies: Collecting and Analyzing Data A. B. Interpreting Data: the Chi-Square Test Collecting Fruit Fly Data: 1. Eliminate Hypotheses 2. Setting up the F2 Generation A. Interpreting data: the Chi-Square test. Scientists make use of statistical analyses to determine which of several competing explanations is better supported by the actual data obtained from an experiment. One especially useful statistical tool for analyzing the results of genetic crosses is called Chi-Square Analysis (X2). It is based on some basic principles of probability (the chance that something will happen under certain circumstances) and the fact that expected results do not occur exactly as predicted due to chance whenever real experiments are performed. Theoretical ratios are based on hypotheses. For example, if two heterozygotes for a dominant trait mate, we expect a 3:1 ratio of phenotypes among the offspring, or, if one tosses a coin into the air 1000 times, we theoretically expect the coin to land headsup 500 times and tails-up 500 times. Observations will be very close to the expected hypothesized ratios if large samples are taken and the hypothesis is correct. In smaller samples, chance variations may occur that give a ratio apparently much different than the expected one. For example, if one tosses a coin into the air only 10 times, there is a good chance of it landing heads-up 8 times and tails-up twice. The observed ratio of 4:1 apparently is considerably different from the expected ratio of 1:1. In such cases, one must decide whether this difference is caused by accidental chance alone or whether the hypothesis is wrong (maybe the coin is "loaded" so that one side is heavier than the other). Likewise, it is important for a geneticist to know the degree to which actual results may differ from expected results due to chance rather than due to a wrong hypothesis or some error in the way the experiment was performed. Knowing this, the scientist can decide whether or not the results support the hypothesis. Coin Tossing. When tossing coins, we expect that the coin will land heads-up 50% of the time. This seldom happens exactly unless we toss the coin a very large number of times, which would eliminate chance fluctuations (called "sampling error"). Since actual experiments involve a limited number of observations, some sampling error is expected and the results will not be exactly as expected. We could propose a "null hypothesis" stating that nothing (gravity or the way the coins are tossed) has an effect Exercise 2. 2-2 on their landing heads or tails. If so, then how much deviation from the expected 1:1 ratio can occur before the null hypothesis is rejected (something is making the coins fall heads-up more than tails-up, for instance)? In most biological experiments, the null hypothesis is rejected when the deviation is so large that it could be accounted for by chance less than 5 percent of the time (a "significant deviation"). Statistics can never provide absolute proof of the validity of a hypothesis, but may set limits to our uncertainty of its correctness. If the coin tossing experiment is based on small numbers, large deviations from expected ratios occur quite often due to chance alone. But as the sample size increases, the deviation should become smaller so that if the sample was infinite in size, we would obtain an exact 1:1 ratio with no deviation at all. Degrees of Freedom. We toss a coin into the air. If it does not land heads-up, it must land tails-up. Although there are two sides to a coin, it has only one "choice" as to which side is up. Or, when we put our shoes on, if we put one shoe on the right foot first, then we must then put the other shoe on the left foot. Thus, given two possibilities, there is only one free choice or "degree of freedom." In general, we have n-1 degrees of freedom (df) in assigning numbers at random to n classes within an experiment. Thus, if there are four possible phenotypic combinations possible (say, in a 9:3:3:1 dihybrid ratio situation), there are three degrees of freedom in assigning an organism to one of these (if we choose not to place it in the first, second, or third group, we must place it in the fourth category). For most genetics situations, the number of degrees of freedom will be one less than the number of phenotypic classes. Chi-Square Test (X2). The chi-square test enables an experimenter to convert the amount of deviation from expected values into the probability of such differences occurring by chance. This test takes into account the size of the sample tested and the number of variables (degrees of freedom). The question we try to answer with the X2 test is "How small can the deviations be to be probably attributed to chance alone?" The formula for X2 is: chi-square = Σ (O - E) 2 /E where Σ = the grand total of the squared deviations (observed number minus expected number, O-E)2 divided by the expected number (E) for each class. The value of chi-square may then be converted into the probability (P) that the deviation is due to chance by using the table below for the proper number of degrees of freedom. Exercise 2. 2-3 Chi-Square Distribution Table. __________________________________________________________ Probability that deviation is due Numbers of Degrees of Freedom to chance alone 1 2 3 4 5 __________________________________________________________ these values 0.95 (95%) 0.004 0.10 0.35 0.71 1.15 support the 0.70 (70%) 0.15 0.71 1.42 2.20 3.00 hypothesis 0.50 (50%) 0.46 1.39 2.37 3.36 4.35 under 0.30 (30%) 1.07 2.41 3.66 4.88 6.06 consideration 0.10 (10%) 2.71 4.60 6.25 7.78 9.24 __________________________________________________________ don't support 0.05 ( 5%) ** 3.84 5.99 7.82 9.49 11.07 hypothesis 0.01 ( 1%) ** 6.64 9.21 11.34 13.28 15.09 (P= or <.05) 0.001(0.1%) ** 10.83 13.82 16.27 18.47 20.52 __________________________________________________________ ** Observed results are significantly different from the expected results. The chi-square test has two important limitations. First, it must be used only for the numerical data itself, never on any percentages or ratios derived from the data. Second, it cannot be used for experiments where the expected number in any phenotypic class is less than 5. Examples of using Chi-Square analysis. Suppose you observe that, from a mating, 9 fruit fly offspring are males and 3 are females. Can we say with scientific confidence that the data display a 3:1 ratio and not a 1:1 ratio? Let's see if a Chi-Square analysis will reject the hypothesis that the results support a 1:1 ratio. Phenotype Classes observed number=O expected number=E deviation (O - E) deviation2 (O - E)2 (O - E)2 E Males 9 2 x 12=6 (9-6)= 3 [3]2= 9 9/6= 1.5 Females 3 2 x 12=6 (3-6)=-3 [3]2= 9 9/6= 1.5 12 12 0 totals → 18 X2 = 3.0 How many degrees of freedom do we have? _______ What is the probability that this deviation from the expected 1:1 ratio is due to chance? ______ Is the 1:1 ratio hypothesis supported or should we reject it? Explain. Here, we see the effect of a too-small sample size leading to an inability to Exercise 2. 2-4 statistically determine whether a 3:1 ratio or a 1:1 ratio is the "true" result. Now, suppose you observe that 90 flies are males and 30 flies are females. With this larger sample size, can we say with scientific confidence that the data display a 3:1 ratio and not a 1:1 ratio? Let's see if another Chi-Square analysis will reject the hypothesis that the results support a 1:1 ratio. Fill in the blanks to complete the analysis. Phenotype Classes observed number=O Green leaf 90 White leaf 30 totals → 120 expected number=E deviation (O - E) deviation2 (O - E)2 (O - E)2 E 120 X2 = How many degrees of freedom do we have? _______ What is the probability that this deviation from the expected 1:1 ratio is due to chance? ______ Is the 1:1 ratio hypothesis supported or should we reject it? Explain. B. Collecting fruit fly data. Introduction. Two weeks ago, you set up a mating involving virgin normal ("wild type") females and mutant males. Last week, you removed the parental flies from the vial so that they would not mate with the F1 flies when they emerge. Today, you will examine the F1 flies that have emerged, set up a mating between the F1 males and F1 females, and eliminate any hypotheses about the inheritance of the mutant trait that no longer are supported by your observations. As part of lab Exercise 2, you turned in a description of your mutant flies. Your lab instructor has by now returned Part B of Lab Report 2, commenting on the accuracy of your description of the mutant trait in your flies. If your description of the trait was accurate, simply copy the description below. If your description was innacurate, reexamine the mutant flies and give a new description of the trait below, for your lab Exercise 2. 2-5 instructor to check again. Below, briefly redescribe the mutant trait (M_____; refer back to Exercise 1, or look at the flies in your mutant male x mutant female vial: Examining the F1 generation flies. Retrieve your group of 3 vials from the constant temperature incubator. Perform the following only after your lab instructor has demonstrated the correct procedure for transferring flies for observation: 1. Remove about half the flies from the vial marked "Virgin + Females x Mutant males" by quickly transferring them into a clean vial stoppered with a foam plug containing a small brush containing a small amount of "Flynap" anesthesia. 2. Do not overexpose the flies to Flynap since dead flies are of little further use. When the flies have stopped moving about, remove the stopper and let the flies fall onto a white plate. 3. Place the plate with flies onto the stage of a dissecting microscope and, using a toothpick, sort the flies into a group of females on the left and a group of males to the right. 4. Examine each group of flies, noting the presence or absence of the mutant trait, and whether any change has occurred in the appearance of the mutant trait between the P and F1 flies. Fill in the table on the next page. SEX OF THE FLIES PHENOTYPE: NORMAL OR MUTANT COMMENTS ABOUT THE MUTANT TRAIT Exercise 2. 2-6 FEMALES MALES Setting up the F1 x F1 crosses. 1. Obtain two fresh food vials from your lab instructor. 2. Label each vial as follows: F1 female x F1 male (M date on each label. 3. Insert the anesthetized flies you just examined into one vial, stopper the vial, and lay it on its side until the flies awaken. 4. Transfer the remaining flies from the Virgin + Females x Mutant males vial into the second fresh vial, following the instructions of your lab instructor. You now have two vials of flies of the identical F1 x F1 cross. In two weeks time, you will be able to collect a considerable amount of data from the F2 generation produced by these matings. 5. Next week, check your vials to see the larvae and pupal cases. Also, you must remove any adults from these vials next week to prevent the offspring that will emerge from co-mingling with the parents. In two weeks time, enough offspring (the F2 generation) will have emerged to permit definite determination of the mode of inheritance of the mutant trait and statistical analysis of the results. ) and write your name and Interpretation of the F1 generation data. Refer back to Exercise 1, which describes the four possible inheritance patterns for mutant traits in fruit flies. Based on the F1 data from your cross, is the mutant trait in Exercise 2. 2-7 your flies inherited as a dominant or as a recessive? Explain. Can you rule out either that the mutant trait is X-linked or autosomal in chromosomal location? Elaborate. So, cross out from the list below any mode of inheritance that no longer applies for your mutant trait: AUTOSOMAL DOMINANT AUTOSOMAL RECESSIVE X-LINKED DOMINANT X-LINKED RECESSIVE Note below anything unusual about the inheritance of your mutant trait: 2-8 Exercise 2. Exercise 3 3-1 © Joseph P. Chinnici Exercise 3 Fruit Flies: Collecting and Analyzing Data A. B. C. E. Introduction Collecting Fruit Fly Data Chi Square Analysis of Fruit Fly Data Eliminating or Supporting Hypotheses Writing a Lab Report A. Introduction. D. During Exercise 1, you were given a vial of fruit flies displaying a mutant phenotype. By comparing various physical characteristics of normal flies with those of the mutants, you wrote a description of the mutant phenotype, which your lab instructor checked for accuracy [If the description was inaccurate, you re-described the trait during Exercise 2.] Also during Exercise 1, you set up a P generation mating involving normal females and mutant males. A week later, you removed the parents. During Exercise 2, you observed the phenotypes of the F1 generation flies and noted whether your observations supported dominant or recessive inheritance and autosomal or X-linked inheritance. Also, during Exercise 2, you set up two vials of the F1 x F1 mating. Last week, you removed the F1 flies. Today, you will observe the F2 generation flies from the two vials set up during Exercise 2, analyze the data using the Chi-Square test, and determine or verify the exact mode of inheritance for your mutant fly trait. Also, your lab instructor will give you some guidance in writing a scientific lab report of the entire fruit fly experiment. B. Collecting fruit fly data: the F2 generation. Retrieve your vials from the constant temperature incubator. Perform the following only after your lab instructor has demonstrated the correct procedure for transferring flies for observation: 1. Remove all adult flies from one of the F1 x F1 vials by quickly transferring them Exercise 3. 3-2 into a clean vial stoppered with a foam plug to which a small wand dipped in "Flynap" anesthesia is attached. 2. You may overexpose the flies to Flynap since you no longer need the flies for mating purposes, and dead flies will not awaken and fly off. After the flies have been thoroughly anesthetized, remove the stopper and let the flies fall onto a white plate or index card. 3. Place the plate with flies onto the stage of a dissecting microscope and, using a toothpick, sort the flies into a group of females on the left and a group of males to the right. 4. Examine each group of flies, and further subdivide the flies (if necessary) into groups of normal females, mutant females, normal males, and mutant males. Count the number of flies in each group and record these data in the table at the top of the next page. 5. Repeat steps 1-4 for the second F1 x F1 vial. C. Chi Square analysis of fruit fly data. To statistically support the likelihood that the data you collected represents either a 3:1 ratio or a 1:1 ratio, perform the following Chi-Square analyses: 1. Refer to the table on the next page. Compare the total number of females to the total number of males [use the data from the TOTALS (B) row]. What would you expect the sex ratio to be from this mating? Explain your answer. Data from F2 generation: Exercise 3 PHENOTYPES 3-3 FEMALES vial 1 + vial 2 = total MALES vial 1 + vial 2 = total NORMAL + = + = MUTANT + = + = TOTALS (B) NOTE ANY DIFFERENCES IN THE MUTANT PHENOTYPES OF FEMALES AND MALES TOTALS (A) Exercise 3. 3-4 Perform the Chi Square analysis of the sex ratio data (from TOTALS (B) row). A ChiSquare distribution table is given below for your convenience. Chi-Square Distribution Table. __________________________________________________________ Probability that deviation is due Numbers of Degrees of Freedom to chance alone 1 2 3 4 5 __________________________________________________________ these values 0.95 (95%) 0.004 0.10 0.35 0.71 1.15 support the 0.70 (70%) 0.15 0.71 1.42 2.20 3.00 hypothesis 0.50 (50%) 0.46 1.39 2.37 3.36 4.35 under 0.30 (30%) 1.07 2.41 3.66 4.88 6.06 consideration 0.10 (10%) 2.71 4.60 6.25 7.78 9.24 __________________________________________________________ don't support 0.05 ( 5%) ** 3.84 5.99 7.82 9.49 11.07 hypothesis 0.01 ( 1%) ** 6.64 9.21 11.34 13.28 15.09 (P= or <.05) 0.001(0.1%) ** 10.83 13.82 16.27 18.47 20.52 __________________________________________________________ ** Observed results are significantly different from the expected results. Chi-Square analysis of the sex ratio data: Phenotype Classes observed number=O expected number=E deviation (O - E) deviation2 (O - E)2 (O - E)2 E Females Males totals → Degrees of Freedom: ______; Probability that deviation is due to chance: ______ Do your data support your hypothesis regarding sex ratio? If there is a disagreement, how might this be explained? 2. Compare the total number of normal flies to the total number of mutant flies [use Exercise 3 3-5 the data from the TOTALS (A) column]. What would you expect the normal : mutant ratio to be from this mating? Explain your answer. Perform, in the table below, the Chi Square analysis of the normal : mutant ratio data. Chi-Square analysis of the normal : mutant ratio: Phenotype Classes observed number=O expected number=E deviation (O - E) deviation2 (O - E)2 (O - E)2 E Normal Mutant totals → Degrees of Freedom: ______; Probability that deviation is due to chance: ______ Do your data support your hypothesis regarding the normal:mutant ratio? If there is a disagreement, how might this be explained? 3. Compare the total number of normal female flies, normal male flies, mutant female flies, and mutant male flies [use the data from the table]. Exercise 3. 3-6 What would you expect the ratios of the four categories of flies to be to be from this mating? Explain your answer. Perform, in the table below, the Chi Square analysis of the data. Phenotype Classes observed number=O expected number=E deviation (O - E) deviation2 (O - E)2 (O - E)2 E Normal females Normal males Mutant females Mutant males totals → Degrees of Freedom: ______; Probability that deviation is due to chance: ______ Do your data support your hypothesis regarding the normal:mutant ratio? If there is a disagreement, how might this be explained? D. Eliminating or supporting hypotheses. Exercise 3 3-7 From the data you collect for the F2 generation, you should be able to eliminate all but one of the four original hypotheses about the mode of inheritance for your mutant trait. Recall from Exercise 1 that the four hypotheses are: autosomal dominant, autosomal recessive, X-linked dominant, and X-linked recessive. Based on all your data (F1 and F2 generations), give the reasons you are able to either reject or support each of the following modes of inheritance: Autosomal Dominant: Autosomal Recessive: X-linked Dominant: X-linked Recessive: E. Writing a Lab Report. Exercise 3. 3-8 One important method of communication used by scientists to tell others about the research they do is the journal article or research paper. Many scientific journals are published on a monthly or quarterly schedule, and each contains a number of research papers. In these papers, the scientists involved in doing the research write about their studies using a standard format of Introduction, Methods and Materials, Results, Discussion, and Conclusion: 1. First, the authors introduce the topic they are researching by talking about previous studies that have been published and the objectives of their study (i.e., why their studies are important). 2. Then, they describe the experiments they have performed, giving the methods and materials used in enough detail that some other scientist could repeat the study. 3. Next, the results of the experiment are fully presented, along with appropriate statistical analyses. 4. These results then are discussed as to their significance and meaning, including how the results might suggest additional experiments that might be done. 5. Finally, the authors reach conclusions based on the results of their study and the discussion of their results. Most research papers also include a references of bibliography section in which previous research studies relevant to the present study are cited as to journal volume, page numbers, and year of publication. Your Report. As part of Exercise 3, you are to write up the results of your fruit fly study based loosely on the research paper style just described. Follow the directions given below, and any additional information given by your lab instructor. Be sure to use a computerized word processing program to prepare your report. Your research report must contain the following sections: 1. Title page: Your name, your partner’s name(s), mutant designation (M_____), lab section, lab instructor's name, title of your research. 2. Objectives: the objectives of the research project. What were you, as an amateur scientist, trying to find out? 3. Methods and Materials: the organisms used, what was done (the techniques), when each step was performed, etc. 4. Results: It is best to present the data and statistical analyses in the form of tables similar or identical to the ones in this manual. You are encouraged to photocopy the data and statistical tables from Exercises 1, 2, or 3 for this section. Exercise 3 3-9 Include statistical analyses of F2 generation data (chi-square analyses of sex ratio and phenotypic ratio data). 5. Discussion: Discuss whether the results either support or do not support each of the four potential modes of inheritance. 6. Conclusions: Give what conclusions you reached about the correct mode of inheritance, and what further studies you would suggest that might lead to additional information about the mutant trait. 3-10 Exercise 3.