AP Biology Lab 7 GENETICS OF ORGANISMS

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Raymond Stephens
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1 AP Biology Laboratory Dat: Nam and Priod: OVERVIEW AP Biology Lab 7 GENETICS OF ORGANISMS In this lab you will us living organism to do gntic cross3s. You will larn how to collct and manipulat th organisms, collct data from F 1 and F 2 gnrations, and analyz th rsults from a monohybrid, dihybrid or sx-linkd cross. Th procdurs that follow apply to fruit flis. OBJECTIVES Bfor doing this lab you should undrstand: Chi-squar analysis of data, and th lif cycl of diploid organisms usful in gntics studis. Aftr doing this lab you should b abl to: invstigat th indpndnt assortmnt of two gns and dtrmin whthr th two gns ar autosomal or sx-linkd using a multignrational xprimnt, and analyz th data from your gntic crosss using chi-squar analysis tchniqus. INTRODUCTION Drosophila mlanogastr, th fruit fly, is an xcllnt organism for gntics studis bcaus it has simpl food rquirmnts, occupis littl spac, is hardy, complts its lif cycl in about 12 days at room tmpratur, producs larg amounts of off spring, can b immobilizd radily for xamination and sorting, and has many typs of hrditary variations that can b obsrvd with low-powr magnification. Drosophila has a small numbr of chromosoms (four pairs). Ths chromosoms ar asily locatd in th salivary glands clls. Drosophila xists in stock culturs that can b radily obtaind from svral sourcs. Much rsarch about th gntics of Drosophila during th last 50 yars has rsultd in a walth of rfrnc litratur and a knowldg about hundrds of its gns. Th Lif Cycl of Drosophila Th Eggs. Th ggs ar small, oval shapd, and hav two filamnts at on nd. Thy ar usually laid on th surfac of th cultur mdium and, with practic, can b sn with th nakd y. Th ggs hatch into larva aftr about on day. Th Larval Stag. Th wormlik larva ats almost continuously, and its black mouth parts can asily b sn moving back and forth vn whn th larva itslf is lss distinct. Larva tunnl through th cultur mdium whil ating; thus, channls ar a good indication of th succssful growth of a cultur. Th larva shds its skin twic as it incrass in siz. In th last thr larval stags, th clls of th salivary glands contain giant chromosoms, which may b sn radily undr low powr magnification aftr propr staining. Th Pupal Stag. Whn a matur larva in a lab cultur is about to bcom a pupa, it usually climbs up th sid of a cultur bottl or onto th strip providd in th cultur bottl. Th last larval covring thn bcoms hardr and darkr, forming th pupal cas. Through this cas th latr stags of mtamorphosis to an adult fly can b obsrvd. In particular, th ys, th wings, and th lgs bcom radily visibl. 86

2 Th Adult Stag. Whn mtamorphosis is complt, th adult flis mrg from th pupal cas. Thy ar fragil and light in color and thir wings ar not fully xpandd. Ths flis darkn in a fw hours and tak on th normal apparanc of an adult fly. Thy liv a month or mor and thn di. A fmal dis not mat for about tn to twlv hours aftr mrging from th pupa. Onc sh has matd, sh stors a considrabl quantity of sprm in rcptacls and frtilizs hr ggs as sh lays thm. To nsur a controlld mating, it is ncssary to us fmals that hav not matd bfor (virgins). Figur 7.1: Th Lif Cycl of Drosophila mlanogastr It is important to raliz that a numbr of factors dtrmin th lngth of tim of ach stag in th lif cycl. Of ths factors, tmpratur is th most important. At room tmpratur (about 25 o C), th complt cycl taks tn to twlv days. 87

3 Dsign of th Exrcis This gntics xprimnt will b carrid on for svral wks. Drosophila with wll-dfind mutant traits will b assignd to you by your tachr. You ar rsponsibl for making obsrvations and kping rcords concrning what happns as mutant traits ar passd from on gnration to th nxt. You will b assignd to study a crtain mod of inhritanc using particular gntic crosss of flis having on or two mutations. Th mods of inhritanc most commonly usd ar: 1. Monohybrid. In ths xprimnts th mod of inhritanc is dtrmind whn a singl contrasting pair of charactristics is involvd. 2. Dihybrid. In ths xprimnts th mod of inhritanc is dtrmind whn two pairs of contrasting charactristics ar considrd simultanously. 3. Sx-linkd. In ths xprimnts th mod of inhritanc is dtrmind whn th mutant charactristic is associatd with th X-chromosom. To mak ths xprimnts intrsting and challnging, you will not b told th mod of inhritanc, nor th nam for th particular mutation(s) you ar studying. Study th wild typ flis (both mal and fmal) until thir phnotypic charactristics ar familiar. Flis having on or two mutations can thn b idntifid by making comparisons with th wild typ flis. Th most commonly studid mutations ar y color or shap, bristl numbr or shap, wing siz or shap, or antnna siz or shap. You should mak up your own nam for th particular mutation(s) that you idntify in your fils. Procdur 1. Obtain a vial of wild typ flis. Practic immobilizing and sxing (dtrmining th gndr of) ths flis. Examin ths flis and not th charactristics of thir ys, wings, bristls, and antnna. 2. To mak handling asir, immobiliz th flis by chilling thm. Sinc th activity lvl of th flis is dpndnt on nvironmntal tmpratur, th following stps immobiliz th flis. a. Hold th vial containing th flis at an angl and twirl it in ic for svral minuts. b. Whn th flis ar immobilizd, dump thm into a small, plastic Ptri dish containing a #1 Whatman filtr papr. c. Plac th Ptri dish on top of th ic in ordr to maintain th cool tmpratur ncssary to kp th flis immobilizd. d. Us th disscting microscop to viw th flis. Th top of th ptri dish can b on or off whn viwing. 3. Distinguish mal flis from fmal flis by liking for th following charactristics (illustratd in Figur 7.2): a. Mals ar usually smallr than fmals. b. Mals hav dark, blunt abdomns, and fmals hav lightr, pointd abdomns. c. Only th mals hav sx combs, which ar groups of black bristls on th upprmost joint of th forlgs. 88

4 Figur 7.2: Fmal and Mal Drosophila 4. Obtain a vial containing pairs of xprimntal flis. Rcord th cross numbr of th vial. This numbr will srv as a rcord as to which cross you hav obtaind. Ths flis ar th parntal gnration (P) and hav alrady bn matd. Th fmals should hav alrady laid ggs on th surfac of th cultur mdium. Th ggs (or mayb larva now) rprsnt th first filial, F 1, gnration and will b mrging from thir pupal cass in about a wk. 5. First wk (today). Immobiliz and rmov th adult flis. Obsrv thm carfully undr th disscting microscop. Sparat th mals from th fmals and look for th mutation(s). Not whthr th mutations(s) is/ar associatd with th mals or th fmals. Idntify th mutation(s) and giv it/thm a mad-up nam and symbol. Rcord th phnotyp and symbol in Tabl 7.1. Th findings should b confirmd by your tachr. 6. Plac th parnts in th morgu, a jar containing alcohol or baby oil. Labl th vial containing th ggs or larva with symbols for th mating. For xampl, if a spia-yd fmal is crossd with a wild-typ mal, th labl could b spia fmal X wild mal. Also b sur to labl th vial with your nam and th dat, Plac th vial in a warm location. 7. Scond Wk. Bgin by obsrving th F 1 flis. Immobiliz and xamin all th flis. Rcord thir sx and th prsnc or absnc of th mutation(s) (as obsrvd in th parntal flis) in Tabl 7.1. Considr th conclusions that can b drawn from ths data. Plac 5 or 6 pairs of F 1 flis in a frsh cultur bottl and th rst of th flis in th morgu For this cross th fmals nd not b virgins. Labl th nw vial F 1 X F 1. Also, labl th vial with symbols dnoting th cross, th dat and your nam. 8. Third Wk. Rmov th F 1 flis from th vials and plac thm in th morgu. Th F 2 gnration ar th ggs and/or th larva in th vial. Plac th vial back in th warm location. 89

5 9. Fourth Wk. Bgin rmoving th F 2 flis. Rcord thir sx and th prsnc or absnc of th mutant phnotyps (as obsrvd in th parntal flis in Tabl 7.2). Th mor F 2 flis collctd, th mor rliabl th data will b. You may hav to collct flis ovr a 3- or 4-day priod. Try to collct at last 200 flis. 10. To analyz your data, you will nd to larn how to us th chi-squar tst. Go to th Statistical Analysis Sction to rviw this tchniqu. Tabl 7.1: F 1 Gnration Data Dat: Phnotyp and Symbol Fmals Mals Tabl 7.2: F 2 Gnration Data Dat: Phnotyp and Symbol Fmals Mals Analysis of Rsults 1. Dscrib and nam th obsrvd mutation(s). 2. Writ a hypothsis that dscribs th mod of inhritanc of th trait(s) that you studid. This is your null hypothsis (as dscribd in th Statistical Analysis Sction). 3. Rfr to th txtbook and rviw Punntt squars. In th spac blow construct two Punntt squars to prdict th xpctd rsults of both th parntal and F 1 crosss from your null hypothsis. Parntal Cross F 1 Cross 90

6 4. Rfr to th Punntt squars abov. In th box blow rcord th xpctd ratios for th gnotyps and phnotyps of th F 1 and F 2 crosss in th xprimnt. F 1 Expctd Gnotypic Ratio Expctd Phnotypic Ration F 2 5. Do th actual rsults dviat from what was xpctd? If so, xplain how. 6. From th rsults, dscrib your cross. Is th mutation sx-linkd or autosomal? Is th mutation dominant or rcssiv? Is th cross monohybrid or dihybrid? 7. Ar th dviations for th phnotypic ratio of th F 2 gnration with th limits xpctd by chanc? To answr th qustion, statistically analyz th data using th chi-squar analysis. Calculat th chi-squar statistic for th F 2 gnration in th chart blow. Rfr to th critical valus of th chi-squar (! 2 ) distribution tabl. (Tabl 7.5) to dtrmin th p (probability valu) that is associatd with your! 2 statistic. Phnotyp # Obsrvd (o) # Expctd () (o-) (o-) 2 (o-) 2 a. Calculat th chi-squar valu for ths data. 1. How many dgrs of frdom ar thr? 2. chi-squar (! 2 ) =! 2 = 3. Rfrring to th critical valus chart, what is th probability valu for this data? b. According to th probability valu, can you accpt or rjct your null hypothsis? Explain why. Discussion 1. Why was it ncssary for th fmals of th parntal gnration to b virgins? 91

7 2. Why was it not ncssary to isolat virgin fmals for th F 1 cross? 3. Why wr th adult flis rmovd from th vials at wk 2 and 4? STATISTICAL ANALYSIS SECTION Using th Chi-Squar Tst for Statistical Analysis of Exprimntal Data Exampl 1 Statistics can b usd to dtrmin if diffrncs among groups ar significant, or simply th rsult of prdictabl rror. Th statistical tst most frquntly usd to dtrmin whthr data obtaind xprimntally provid a good fit or approximation to th xpctd or thortical data is th chi-squar tst. This tst can b usd to dtrmin if dviations fro th xpctd valus ar du to chanc alon, or to som othr circumstanc. For xampl, considr corn sdlings rsulting from an F 1 cross btwn parnts that ar htrozygous for color. A Punntt squar of th F 1 cross Gg X Gg would prdict that th xpctd proportion of th grn:albino sdlings would b 3:1. Us this information to fill in th Expctd () column and th (o-) column in Tabl 7.3. Tabl 7.3 Phnotyp Gnotyp # Obsrvd (o) # Expctd () (o-) Grn GG or Gg 72 Albino gg 12 Total 84 Thr is a small diffrnc btwn th obsrvd and xpctd rsults, but ar ths data clos nough that th diffrnc can b xplaind by random chanc or variation in th sampl? To dtrmin if th obsrvd data fall within accptabl limits, a chi-squar analysis prformd to tst th validity of a null hypothsis (that thr is no statistically significant diffrnc btwn th obsrvd and xpctd data). If th chi-squar analysis indicats that th data vary too much from th xpctd 3:1 ratio, an altrnativ hypothsis is accptd. Th formula for chi-squar is:! 2 = "(o-) 2 whr o = obsrvd numbr of individuals = xpctd numbr of individuals " = th sum of th valus (in this cas, th diffrncs, squard, dividd by th numbr xpctd) 1. This statistical tst will xamin th null hypothsis, which prdicts that th data from th xprimntal cross abov will b xpctd to fit th 3:1 ratio. 92

8 2. Us th data from Tabl 7.3 to complt Tabl 7.4. Tabl 7.4 Phnotyp # Obsrvd (o) # Expctd () (o-) (o-) 2 (o-) 2 Grn 72 Albino 12! 2 = "(o-) 2 3. Your calculations should giv you a valu of! 2 = This valu is thn compard to Tabl 7.5. Dgrs of Frdom (df) Probability (p) How To Us th Critical Valus Tabl 1. Dtrmin th dgrs of frdom (df) for your xprimnt. It is th numbr of phnotypic classs minus 1. Sinc thr ar two possibl gnotyps, for this xprimnt df = 1 (2 sampls 1). If th xprimnt has gathrd data for a dihybrid cross, thr would b four possibl phnotyps and thrfor 3 dgrs of frdom. 2. Find th p valu. Undr th 1 df column, find th critical valu in th probability (p) = 0.05 row: it is What dis this man? If th calculatd chi-squar valu is gratr than or qual to th critical valu from th tabl, thn th null hypothsis is rjctd. Sinc for our xampl! 2 = 5.14 and 5.14>3.84, w rjct our null hypothsis that thr is no statistically significant diffrnc btwn th obsrvd and xpctd data. In othr words, chanc alon cannot xplain th dviations w obsrvd and thr is, thrfor, rason to doubt our original hypothsis (or to qustion our data collction accuracy). Th minimum probability for rjcting a null hypothsis in th scincs is gnrally 0.05, so this is th row to us in our chi-squar tabl. 3. Ths rsults ar said to b significant at a probability of p = This mans that only 5 % of th tim would you xpct to s similar data if th null hypothsis was corrct, thus, you ar 95% sur that th data do not fit a 3:1 ratio. 4. Sinc ths data do not fit th xpctd 3:1 ratio, you must considr rasons for this variation. Additional xprimntation would b ncssary. Prhaps th sampl siz is too small, or rrors wr mad in data collction. In this xampl, prhaps th albino sdlings ar undrrprsntd bcaus thy did bfor th counting was prformd. 93

9 Exampl 2 In a study of incomplt dominanc in tobacco sdlings, th counts in Tabl 7.6 wr mad from a cross btwn th two htrozygous (Gg) plants. Tabl 7.6 Phnotyp Gnotyp # Obsrvd (O) Grn GG 22 Yllow Grn Gg 50 Albino gg 12 Total: 84 A Punntt squar for this cross indicats that th xpctd counts should b in a 1 grn:2 yllow grn:1 albino ration (Tabl 7.7). Th xpctd valus for a total count of 84 organisms ar thrfor: 1 grn = 1/4 X 84 = 21 2 yllow grn = 1/2 X 84 = 42 1 yllow = 1/4 X 84 = Tabl 7.7 Phnotyp # Obsrvd (o) # Expctd () (o-) (o-) 2 (o-) 2 Grn Yllow Grn Albino ! 2 = "(o-) Go to th chi-squar tabl, this tim for two dgrs of frdom (thr ar thr phnotyps: 3-1 = 2 df). If th X2 valu wr gratr than or qual to th critical valu of 5.99 w would rjct our hypothsis. Sinc 5.43 is lss than th critical valu at p = 0.05, w accpt th null hypothsis (this scond data st dos fit th xpctd 1 : 2 : 1 ratio). Practic Problm An invstigator obsrvs that whn pur-brding, long wing Drosophila ar matd with pur-brding, shortwing flis, th F 1 offspring hav an intrmdiat wing lngth. Whn svral intrmdiat-wing-lngth flis ar allowd to intrbrd th following rsults ar obtaind: Obsrvd 230 long wings 510 intrmdiat-lngth wings 260 short wings a. What is th gnotyp of th F 1 intrmdiat-wing-lngth flis? b. Writ a hypothsis dscribing th mod of inhritanc of wing lngth in Drosophila (this is your null hypothsis). 94

10 c. Complt Tabl 7.8. Tabl 7.8 Phnotyp # Obsrvd (o) # Expctd () (o-) (o-) 2 (o-) 2! 2 = "(o-) 2 d. Calculat th chi-squar valu for ths data. 1. How many dgrs of frdom (df) ar thr? 2.! 2 (chi-squar) = 3. Rfrring to th critical valus chart, what is th probability valu for ths data?. According to th critical valus of! 2 can you accpt or rjct th null hypothsis? Explain why? 95

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