CROSSING OVER NEAR THE SPINDLE ATTACHMENT OF THE X CHROMOSOMES IN ATTACHED-X TRIPLOIDS OF DROSOPHILA MELANOGASTER THE PROBLEM

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1 CROSSING OVER NEAR THE SPINDLE ATTACHMENT OF THE X CHROMOSOMES IN ATTACHED-X TRIPLOIDS OF DROSOPHILA MELANOGASTER G. W. BEADLE WilJiam G. Kerckof Laboratories of te Biological Scieces, Calijoria Istitute of Tecology, Pasadea, Califwia Received May 14, 1934 THE PROBLEM A study of crossig over i triploid females of Drosopila melaogaster (BEADLE 1934) wit two of te X cromosomes attaced sowed tat crossig over ear te spidle attacmet is iger betwee te two attaced cromosomes ta betwee eiter of te attaced cromosomes ad te free cromosome. I more distal regios (beyod forked) te cromosomes were foud to be ivolved i crossovers at radom. Sice tese facts ave a bearig o te mode of propase pairig of cromosomes, it was tougt wort wile to ceck te experimet isofar as it cocered crossig over i te proximal portios of te cromosomes. Sice oly a relatively small fractio of te geetic legt of te X cromosome was to be studied, te experimet could be muc simplified over te origial. Because of te less complicated geetic set-up i te X cromosomes it was possible to pla te experimet so tat it would yield iformatio o ay relatio wic migt exist betwee autosomal disjuctio ad detected crossig over i te X cromosome. Tat suc a relatio migt be importat was suggested by results obtaied by Doctors J. SCHULTZ ad H. REDPIELD of tis laboratory (upublised) i studies of free X cromosome triploids. Te results cofirm te fidig of te previous experimet tat crossig over ear te spidle attacmet apparetly does ot ivolve cromosomes at radom, but tey go farter i idicatig tat te iterpretatio of tis fact must take ito accout a relatio sow to exist betwee recovered crossovers i te X cromosome ad te type of disjuctio of te autosomes. PLAN OP THE EXPERIMENT I plaig a experimet wic would give te desired iformatio, several cosideratios were of importace. It was desirable to carry out te experimet i suc a way tat te peotypic frequecies of te progey of triploids of te proper costitutio could be directly used as a measure of crossig over i te several types of gametes to be studied. Te alterative, testig for geotypic costitutio of idividuals gettig more ta oe X cromosome strad from teir moters, must be resorted GENETICS 0: 179 Mr 1935

2 180 G. W. BEADLE to if ay cosiderable portio of te X cromosome is to be followed but as te serious disadvatage tat its use is excluded i te case of itersexes, wic are sterile. Itersex classes are relatively weak ad teir viability is terefore strogly iflueced by may of te caracters ordiarily used i geetic studies. Sice it was igly desirable to make use of itersexes, te miimum possible umber of gees cosistet wit efficiecy was used ad tese were selected after due cosideratio ad bee give to teir effects o viability. Te use of te ciiig gee was avoided as a meas of obtaiig triploids of te proper costitutio sice it was kow from previous experiece (BEADLE 1934) tat a ig - - proportio of te triploid daugters of XXY ciiig females are XXXY i costitutio; te Y cromosome is a udesirable complicatio. Triploids of te desired costitutio were built up by first makig up a stock of XXX triploids of te costitutio v f cr/v f B/v f cr. Tis stock could be maitaied ad multiplied wit little dager of cage due to V I o 63 M.0 FIGURE 1.-Geetic costitutio of te triploids used for a study of crossig over. Te tree cromosomes are desigated a, b, ad c. Stadard map positios of te gees used as markers are idicated. Te spidle attacmet ed is idicated by"%" at te rigt. crossig over. Te oly sigle crossover wic could give a peotypically udetectable cage i costitutio is a reciprocal betwee te attaced cromosomes i te forked U)-caratio (cr) iterval. Suc a cage would ot decrease te usefuless of te stock ad te fact of its avig occurred could be detected i te ext geeratio. Suc females were mated to vermilio (v) caratio males. Vermilio forked Bar (B/+/+ wic ca be distiguised from BIB/ +) triploids were selected from tis matig. Tey were of te proper costitutio for te experimet (figure 1). Suc females were mated to forked caratio males ad allowed to lay eggs for a seve day period after wic tey were discarded. At te ed of tis seve day period te cultures were removed from te 5" C icubator to room temperature (about 0") ad allowed to remai util te couts were completed. Te progey were classified at oe or two day itervals for eleve days. er

3 ~ CROSSING OVER IN TRIPLOID DROSOPHILA 181 Te triploid females used i te experimet were omozygous for te vermilio gee, te males were ot-vermilio; ece ay idividual i te progey wit two X cromosomes (females ad itersexes) wic sowed vermilio got bot of tese cromosomes from te moter. Tus te female ad itersex progey could be classified peotypically as avig eiter attaced or free X cromosomes. Te geetic costitutio of te triploids is suc tat crossig over betwee te spidle attacmet ad forked (or Bar sice f-b crossig over is egligible i frequecy) ca be detected i ay of te tree possible combiatios of two cromosomes. I certai cases te iterval uder cotrol ca be separated ito two, spidle attacmet-caratio (regio 1) ad caratio-forked (regio ). Uless oterwise stated, crossover values i te discussio of te data refer to te total of te two regios, tat is, spidle attacmet-forked. PROGENY OF XXX TRIPLOIDS A summary of te progey of 13 triploid females is give i table 1. Tese data are more useful i idicatig te types of idividuals produced by XXX triploids ta are tose from te previous experimet already referred to bot because te umbers are larger ad because te geetic set-up is suc tat more iformatio is available. TABLE 1 Summary of te progey of 13 XXX females. CL188 NUMBER Regular males Patroclious males XX females XX females 3 females Matroclious 3 females XX itersexes XX itersexes Superfemales Matroclious superfemales Supermales Patroclious supermales Total 7833 It is well kow tat te distributio of cromosomes at meiosis i te free-x triploid of Drosopila is ot radom but deviates i suc a way tat te tedecy is for equal umbers of cromosomes to be distributed to te two poles at te first divisio. Tis is sow clearly by te data publised by BRIDGES ad ANDERSON (195) ad wic are give i table

4 18 G. W. BEADLE of tis paper. Te same deviatio from a radom distributio is sow i r\ XXX triploids but it is muc less extreme (table ). Te proportio of triploid females ad diploid males from XXX triploids is iger ta from XXX triploids (BEADLE, 1.c.) ad is, of course, a expressio of te differece i te deviatio from radom distributio of cromosomes i te TABLE Compariso of frequecies of differet types of gametes from XXX ad XXX triploids. - FREQUENCY RELATIVE PREQUENCY EGQ SPERM ZYGOTE XXX* xxx xxx XXX X, 1A YA female lx, 1A XA female o 1.0 X, A YA Itersex o 1.o lx, A XA Itersex * Data from BRIDGES ad ANDERSON (195). two types of triploids. Te cause of te deviatio is ukow but te differece betwee XXX ad XXX triploids sows tat bot te mass of cromati ad te umber of idepedet uits (cromosomes) ifluece te distributio. Te summary give i table 1 sows tat XXX triploids produce several types i additio to tose regularly foud i te progey of triploids. Tese are : matroclious (for te X) triploids ad matroclious superfemales (from XXX, A ad XXX, 1A eggs), ad patroclious males ad patroclious supermales (from ox, 1A ad ox, A eggs). Te experimet wit XXX triploids referred to above sowed tat patro- clious males are rater frequet amog te progey of XXXY triploids (13.7 percet of all males). Wit tis fact i mid, tests were made for fertility of te patroclious males i tis experimet. Of 3 tested were sterile ad 1 was fertile. Tere is evidece terefore tat at least oe of te 13 triploid parets carried a Y cromosome. Probably few or o oters were of tis costitutio, oterwise tere sould ave bee a appreciable umber of patroclious males. It is iterestig to compare te relative frequecies of XXX, A ad XXX, 1A ad tose of ox, A ad ox, 1A gametes. Te first two types are recovered i matroclious triploids ad i matroclious superfemales. Te ratios of tese to regular triploids ad superfemales must be used to correct for te violet differece i viability of te two sex types. Te relative frequecies, so cor- - rected, are 1 :48 for XXX, A ad XXX, 1A gametes. NoX, 1A ad ox, A

5 CROSSING OVER IN TRIPLOID DROSOPHILA gametes are recovered i patroclious males ad i patroclious supermales. Te ratio of tese two types (excludig te oe culture te moter of wic was sow to be XXXY), corrected for viability differeces, is 1 :4. Tese ratios are based o relatively small umbers but everteless idicate extreme deviatios from te 1 :1 relatio expected wit idepedet assortmet of te cromosomes. Tey sow tat te frequecy of large deviatios from a approximately equal distributio of te cromosomes to te two poles of te first meiotic spidle is very muc lower ta tat expected wit radom distributio. CROSSING OVER Sice itersex classes are to be used i measurig crossig over i certai types of gametes produced by triploids, it is ecessary to examie te data to see weter or ot viability complicatios migt ivalidate comparisos betwee values based o ormal ad tose based o itersex classes. Te experimet is so arraged tat crossovers recovered i XX, A gametes ca be directly compared i te two sex classes, triploids ad XX itersexes. Tese two classes are differetiated by te sperm ad sould, aside from viability differeces, give similar results. Te frequecies of differet crossover types i tese classes are give i table 3. Te percetages of detected crossovers betwee te cromosomes of te tree combiatios are summarized as follows: CBROYOSOMES TBIPLOIDB INTEWEXEB DIFmRENCE B.B. a-b b-c a-c Te values for a-b crossovers represet oly oe alf te frequecy of potetially detectable crossovers. Tey are preseted i tis way because of ucertai classificatio of oe type of crossover (v f BB) i te XX itersexes. It ca be see tat te values from te two sex classes agree very well; all of te differeces are cosiderably less ta teir stadard errors. Tis agreemet meas tat tere are o appreciable differetial iviability effects i te differet peotypes used as measures of crossover frequecies, ad it is terefore legitimate, i tis experimet, to compare crossover values based o itersex classes wit tose based o ormal sex classes. Crossover Values i Digeret Types of Gametes Tere are tree combiatios of two cromosomes i wic crossovers ca be detected i te experimet as set up (a-b, a-c, ad b-c i figure 1).

6 ~~ 184 G. W. BEADLE Te relative frequecies of tese i differet classes of idividuals are give i table 3. Crossovers betwee te two attaced cromosomes TABLE 3 Progey of XXX trilpoids (v f crjv j Bjv cr) mated to j cr males. PRWEM CLAS6 CBROMOBOMES BEQION PHENOTYPa NUMBER PERCENT C 0 c-b 1 c-a lor c-b 1 ad v cr VfB vfbcr vfcr V XX females (1186) C 0 c-b 1 c-a 1 or c-b 1 ad cr * 0.08 Males ad XX females C 0 (18) c-b 1 c-a lor c-b 1 ad c-b-a lad * 0.05 XX itersexes (1854) C 0 c-b 1 c-a 1 or c-b 1 ad Triploids (871) 0 a-b 1 lor b-c 1 a-c 1 or oo * 0.3 It 0.11 c.\ XX ibrsexes (700) 0 a-b 1 1 or b-c 1 a-c 1 or vf B v jcr vf vfbb v cr V VB

7 CROSSING OVER IN TRIPLOID DROSOPHILA 185 TABLE 3 (Cotiwed) PBOQENX CLASS CaaOPOBOME8 REQION PHENOTYPE NUMBER PER" XX females (1968) 0 VfB a-b 1 VfCT vf lor vfbb b-c 1 v cr V a-c 1 or vb vfbcr v B cr * 0.05 * From XXX paret of te costitutio v f/v f B CT/V cr-sould be added to c-a sigle crossovers. t a-b-c- double crossover wit te b-c crossover betwee f ad B-treated as a-b sigle. 1 b-a-b or b-c-b doubles i regios 1 ad -because a-b crossig over is more frequet i tis type of gamete ad because b-a-b doubles are detected wit twice te frequecy of b-c-b doubles, tese are treated as b-a-b crossover. (a-b) will be cosidered first. Te detected crossover frequecies (oe alf te total excage frequecy betwee te two desigated cromosomes) for differet types of gametes are summarized as follows: QAYETE SPERM ZYQOTE PERCENT CBOSBOVERS 8.1. x^x, A XA 3 female YA XX itersex - XX, 1A YA XX female Te value for XX itersexes, 7.16, is obtaied by multiplyig te omozygosis value for cromosome a by two; omozygosis for Bar (cromosome b) caot be measured because of difficulties of classificatio for tis gee i itersexes. Te values are i fair agreemet. Te average of te two values for XX, A gametes differs from te value for XX, 1A gametes by 0.81 percet, a value less ta its stadard error. Tus tere is o idicatio tat crossig over betwee te two attaced cromosomes is ay differet i te two types of gametes i wic it ca be measured. Te frequecies of detected crossovers betwee oe of te attaced cromosomes ad te free cromosome (a-c or b-c) are:

8 ~ ~~ ~ ~~ 186 G. W. BEADLE lx, 1A YA or XA male or female a-c 7.19 b-c 7.7 Total lx, A XA XX itersex a-c 3.45 b-c 3.56 Total XX, 1A YA Xx female a-c 4.73 b-c 3.68 Total XX, A XA 3 female a-c 7.3 b-c 7.01 Total YA xx itersex a-c 7.30 b-c 7.9 Total I cotrast to tose for crossig over betwee te two attaced cromosomes, it is clear tat tese values sow strikig differeces depedig o weter oe or two sets of autosomes were preset i te egg. Te gamete types lx, 1A ad XX, A give values about twice te magitude of te comparable values for lx, A ad XX, 1A gametes. Te differece betwee lx, 1A ad lx, A values is 7.6 times its stadard error; tat be- twee XX, la, ad XX, A is 5.5 times its stadard error. Tere ca be o questio as to te statistical sigificace of tese differeces. It is clear, te, tat crossig over betwee attaced ad free cromosomes i regios ear te spidle attacmet is i some way related to autosomal disjuctio. A ispectio of te separate values for regios 1 ad wic are based o smaller umbers ad terefore less reliable ta te combied values, suggests tat te differeces are greater i regio 1 (earest te spidle attacmet) ta i regio ; te data are iadequate to establis clearly tis relatio. Crossover values betwee diferet omologs For compariso of crossover values betwee te two attaced cromosomes wit tose betwee attaced ad free cromosomes te data ca be summarized as follows: PERCENT CROWOVER8 WHITE SPERM ZYQOTE A-E A-0 OR R-C DIIFEBENCE B.E. r? x^x,la YA XX female XX,A XA 3 female r? &,A YA XX itersex

9 ~ ~~ ~ ~ ~ CROSSING OVER IN TRIPLOID DROSOPHILA 187 Tese values are comparable ad represet detected crossig over (oe alf total excage) for oe combiatio of two cromosomes; te a-c or b-c values are te average of te two. It ca be see tat i XX, 1A gametes crossig over betwee te attaced cromosomes (a-b) is sigificatly iger ta betwee attaced ad free cromosomes (a-c or b-c); te differece is 4.8 times its stadard error. It is a oteworty fact tat suc a differece does ot exist i XX, A gametes. I fact te observed differeces are i te opposite directio but are clearly ot statistically sigificat; i oe case te differece is equal to its stadard error, i te oter it is muc less. Compariso of triploid ad diploid excage values A compariso of crossig over ear te spidle attacmet of te X cromosome i triploids wit tat of diploids is complicated by te relatio betwee crossig over i tis regio i te triploid ad te type of disjuctio of te autosomes. It is evidet tat te triploid values will vary TABLE 4 Homozygosis values i XX daugters of triploids used i tis experimet. M)NSTITUTION PROGENY CROSWVER REGION NUMBER PERCENT vfcr - vfb vfb vfcr Of vfbb lor vfcr - v cr v cr vfcr lor widely depedig o te type of gamete i wic tey are measured. A questio also arises as to te coice of a diploid cotrol. Crossig over i diploid XX daugters of te triploids used i te experimet as bee measured. Tis was doe i o-crossover XX daugters ad also i XX daugters i wic te b cromosome ad bee replaced, by crossig over i regio 1, wit te c cromosome. Te data are give i table 4. Te two values agree but bot are lower ta stadard values (see BEADLE ad EMERSON 1935, for discussio of tis differece). Te total excage i te triploid calculated i various ways, togeter wit te stadard diploid value (twice stadard map distace) ad te value obtaied i te XX diploid cotrol (twice or four times omozygosis values, depedig o weter omozygosis for bot or oe cromosome could be detected) are :

10 188 G. W. BEADLE Stadard diploid?x diploid XX, A gametes i 3 daugters XX, A gametes i itersexes XX, 1A gametes i XX daugters XX, A ad X, A gametes Average of all gametes Percet Excage It is clear tat tere is a marked regioal icrease ear te spidle attacmet i te triploid as compared wit te diploid. Tese results corroborate tose previously obtaied (BEADLE 1934). From te experimet it is possible to determie weter or ot te two marked regios are icreased proportioately i te triploid. Tis ca coveietly be doe by comparig te ratios of crossover values for te two regios i te triploid ad diploid. Te ratios (regios 1 : ) are: Stadard 1:1.7 X^x diploid 1 : 1.3 Triploid 1:0.6 Te ratio for te triploid represets te uweigted average of te ratios for te four types of gametes. I all types of gametes te triploid sows a iger proportio of crossovers i regio 1 ta does te diploid. It is terefore quite clear tat te icrease i crossig over i te triploid falls off progressively wit distace from te spidle attacmet (te distal regios of te cromosome are of course ot cosidered ere). DISCUSSION Relatio of autosomal disjuctio ad X cromosome crossig over Te evidece preseted above demostrates tat tere is a relatio i attaced-x triploids betwee disjuctio of te autosomes ad crossig over ear te spidle attacmet betwee attaced- ad free-x cromosomes. Tis relatio is suc tat attaced ad free cromosomes sow low crossig over i gametes wic result from disjuctio wic gives as ear equal umbers of cromosomes to te two poles of te first meiotic spidle as is cosistet wit te formatio of gametes wic ca give viable zygotes wit ormal spermatozoa, ad ig crossig over i gametes from disjuctio wic distributes two members of eac of te tree major

11 CROSSING OVER IN TRIPLOID DROSOPHILA 189 groups of omologues to oe pole ad oe member of eac to te oter pole of te spidle. Tese two types of disjuctio are illustrated i figure. From te fact tat te cromosomes of triploids are ot distributed to te two poles of te first meiotic spidle at radom it is kow tat tere must be a correlatio i te orietatio of differet potetial trivalets o te equatorial plate. Te evidece preseted ere sows tat suc selective A orietatio of te XXX trivalet wit respect to te autosome trivalets is iflueced by crossig over ear te spidle attacmet, betwee attaced ad free cromosomes, i suc a way tat if crossovers occur i tis regio, te XXX trivalet is less likely to be selectively orieted ta if suc crossovers are abset. A possible explaatio of te facts ca be made o te basis of two assumptios, (1) a geeral ad o-specific force A B FIGURE.-Represetatio of te two types of disjuctio differetiated i te preset study. A-lX, PA:=, 1A type wic gives low crossig over betwee attaced ad free cromosomes. B-XX, A: lx, 1A type wic gives ig crossig over betwee attaced ad free cromosomes. of repulsio actig at ad before metapase (LILLIE 1905, KUWADA 199, DARLINGTON 193) ad () a relatio betwee excages ad metapase associatio of omologous cromosomes (DARLINGTON 1931). If excages are assumed to old omologs togeter at te poit of crossig over durig teir orietatio o te equatorial plate, te, followig a excage ear te spidle attacmet betwee oe attaced ad te free-x cromo- some, te members of te XXX trivalet will be so associated tat tey will bear approximately te same relatio to te geeral mass of autosomes regardless of teir orietatio. If, o te oter ad, te free-x is distally associated wit oe attaced cromosome, a cage i orietatio of te XXX trivalet will cosiderably cage te relatio of its mass to tat of te autosomes; ece suc a type of XXX trivalet ca easily be imagied to be selectively orieted wit respect to te autosomes. I a similar way, te differece betwee X cromosome disjuctio i triploids wit free-x s ad tose wit two of te X s attaced ca be accouted for. It is evidet tat te relatio discussed above as a bearig o te problem of measurig crossig over i spidle attacmet regios i triploids. Te reciprocal relatio to tat sow ere, amely, tat betwee auto-

12 190 G. W. BEADLE soma1 crossig over ad X cromosome disjuctio as ot bee ivestigated but sould be take ito accout i ay attempt to measure accurately crossig over i spidle attacmet regios. Propase pairig of cromosomes Te fact tat i XX, 1A gametes te two attaced cromosomes cross over more frequetly wit eac oter ta does eiter wit te free cromosome was previously iterpreted (BEADLE 1934) as idicatig tat propase pairig is by twos ad tat te two attaced cromosomes ave a pairig advatage. Te fact tat tis relatio does ot old i all types of gametes ecessitates a recosideratio of te iterpretatio. Cosiderig all types of gametes te et differece i crossig over is i favor of te attaced cromosomes, tat is, te average of te ig ad low values for attaced ad free cromosome crossig over is less ta te average value for crossig over somes. Tese values are: a-b a-c or b-c Differece betwee te two attaced cromo Stadard error 0.6 Te differece is ot large but is probably statistically sigificat. However te evidece for te iterpretatio previously give is cosiderably weakeed by te additioal iformatio ow available ad te questio as to te ature of propase pairig i triploids of Drosopila must, as far as evidece i tis orgaism goes, remai ope. SUMMARY Crossig over was studied ear te spidle attacmet of te X cromosomes i triploids of Drosopila melaogaster wit two attaced cromosomes. Te experimet was so arraged tat crossovers could be detected i four types of gametes, amely, lx, 1A; lx, A; XX, 1A; ad XX, A. Te results suggest tat crossig over betwee te two attaced cromosomes is costat i te two types of gametes i wic it ca be studied. O te oter ad, crossig over betwee eiter of te attaced cromosomes ad te free cromosome is markedly differet i differet types of gametes. It is low i tose gametes wic ave as ear alf te total um- ber of major cromosomes as is possible (lx, A ad XX, 1A) ad is ig i tose wose cromosome umbers are lower or iger ta tis (lx, 1A ad FX, A).

13 CROSSING OVER IN TRIPLOID DROSOPHILA 191 No-radom distributio of cromosomes at meiosis i triploids of Drosopila must mea tat tere is a correlated orietatio of cromosomes o te equatorial plate. Te evidece summarized above sows tat tis selective orietatio is iflueced by crossig over. Te relatio of te results to te problem of measurig crossig over i triploids ad to te questio of te ature of propase pairig of cromosomes is discussed. LITERATURE CITED BEADLE, G. W., 1934 Crossig over i attaced-x triploids of Drosopila melaogaster. J. Geet, 9: BEADLE, G. W. ad EMERSON, S., 1935 Furter studies of crossig over i attaced-x cromosomes of Drosopila melaogaster. Geetics 0: DARLINGTON, C. D., 1931 Meiosis. Biol. Rev. 6: Recet advaces i cytology. Lodo: Curcill. KUWADA, Y., 199 Cromosome arragemet. I. Model experimets wit floatig magets ad some teoretical cosideratios o te problem. Mem. Coll. Sci. Kyoto 4: LILLIE, R. S., 1905 Te pysiology of cell divisios.-i. Experimets o te coditios determiig mitosis. Amer. J. Pysiol. 15: