Copyright 0 1989 by the Genetics Society of America

The Genetic Analysis of Distributive Segregation inDrosophila melanogaster. I. Isolation and Characterization ofAberrant X segregation (Axs),a Mutation Defective in Chromosome Partner Choice

Anne E. Zitron and R. Scott Hawley Department of Molecular Genetics, Albert Einstein Collegeof Medicine, Bronx, New York 10464 Manuscript received December 14, 1988 Accepted for publication March 30, 1989

ABSTRACT We describe the isolation and characterizationof Aberrant X segregation (Axs), a dominant female- specific meiotic mutation. Although Axshas little or no effect on the frequency or distribution of exchange, or on thedisjunction of exchangebivalents, nonexchange X chromosomesundergo nondisjunction at high frequencies in Am/+ and Axs/Axs females. This increased X chromosome nondisjunction is shown to be a consequence of an Axs-induced defectin distributive segregation. In Axs-bearing females, fourth chromosome nondisjunction is observed only in the presence of nonex- change X chromosomes and is argued to be the result of improper X and fourth chromosome associations within the distributive system. In XX females bearing a compound fourth chromosome, the frequency of nonhomologous disjunction of the X chromosomes from the compound fourth chromosome is shown to account forat least 80% of the total X nondisjunction observed.In addition, Axs diminishes or ablates the capacity of nonexchange X chromosomes to form trivalents in females bearing either a Y chromosome or a small free duplication for the X. Axs also impairs compound X from Y segregation. The effect of Axs onthese segregations parallels the defects observed for homologous nonexchange X chromosome disjunction inAxs females. In addition to its dramatic effects on theX chromosome, Axs exerts a similar effect on the segregation ofa major autosome.We conclude that Axs defines a locus required for proper homolog disjunction within the distributive system.

HE general mechanism of disjunctionin most with great efficiency; the frequency of fourth chro- T eukaryotes consists of homologous pairing, ex- mosomenondisjunction is approximately 0.1% (cJ change, and segregation of exchange partners. The BAKERand CARPENTER 1972).These results demon- basic features of the exchange-mediated segregation stratethat an alternative mechanism of exchange- system and our current understanding of the mecha- independent segregation must exist. nisms by which exchangechromosomes disjoin has BRIDGES(1 9 16) obtained evidence that such ex- recently been reviewed by HAWLEY(1988). change-independent segregationsfollowed a different Althoughexchange itself is sufficient toensure set of rules than did segregations mediated by ex- proper chromosome segregation in Drosophila mela- change. When examining sex chromosome disjunc- nogaster females, several lines of evidence suggest the tion in XXY females, he observed frequencies of X existence of another exchange-independentsystem of nondisjunction much higher than those observed in segregation.In females with structurallynormal X XX females. These X nondisjunctional events involved chromosomes, the frequency of nonexchange biva- primarily nonexchangeX chromosomes that appeared lents is approximately 6.0% while the frequency of to segregate from the Y chromosome. When the X spontaneous X nondisjunction is approximately 0.5% chromosomes underwent exchange, they segregated (BRIDGES1916; WEINSTEIN1936). In addition, inver- from each other faithfully with the Y going at random. sion heterozygotes with a reduced frequency of ex- Bridges argued that this nondisjunction resultedfrom change between X chromosomes exhibit spontaneous pairing of an X and Y chromosome while the remain- frequencies of X nondisjunction significantly lower ing X segregated at random; this process was termed than expected if the exchange-mediated system were secondarynondisjunction. COOPER (1 948)later the only disjunction mechanism present in Drosophila showed that secondarynondisjunction can be ac- females (COOPER1945). Finally, although the fourth counted for by the formation of an XXY trivalent in chromosomes in Drosophila are always nonexchange which the Y directs the two nonexchange X chromo-

(STURTEVANT1951), they segregate from each other somes (XX .t, Y). GRELL(1 962a, b) first proposed the existence of a The publication costs of this article were partly defrayed by the payment of page charges. This articlemust therefore by hereby marked“advertisement” novel segregation system, termed distributive segre- in accordance with 18 U.S.C. 61734 solely to indicate this fact. gation, to explain both secondary nondisjunction and

Genetics 122: 801-821 (August, 1989) 802 A.and E. Zitron R. S. Hawley the disjunction of the always nonexchange fourth frequency and distribution of exchange have been chromosomes. Grell also proposed that the segrega- isolated and characterized (reviewed in BAKERand tion of other nonhomologs, of nonexchange X chro- HALL 1976), only three mutations affecting the dis- mosomes (GRELL 1967),and of compound chromo- tributive system have so far been isolated; ald, mei- somes (GRELL1963) was the responsibility of the S51 and nod. The three proposed phases of distribu- distributive system. She argued that this female-spe- tive segregation have been genetically defined by cific distributive system was independent of the ex- these mutations. change-mediated system and does not play a role in The first phase of the model,the selection of the achiasmate meiosis found in Drosophila males nonexchangechromosomes, is defined by the ald (GRELL 1970). (altered disjunction) mutation (O’TOUSA 1982),which GRELLalso presented evidence that the choice of allows exchange chromosomesto enter thesystem and disjunctional partners within the distributive system is participate in nonhomologous disjunctions (such as XX

based on factors such as availability, size, and shape t, Y and XX t, 44).The locus may define a component of chromosomes, and not onhomology (GRELL 1964a, required for proper chiasma maintenance (HAWLEY b; MOORE and GRELL 1972). In other words, if two 1988). The ald mutation also effects the properchoice nonexchange elements are present in a given meio- of disjunctional partners and thus defines the second cyte, they will segregatefrom each other faithfully phase of the model. This defect in partner choice regardless of theiridentity. When morethan two induces high frequencies of nonhomologous disjunc- elements are present however, they will choose dis- tions involving the X and fourth chromosomes (i.e., junctional partners solely on the basis of the size and xx c-) 44). shape of chromosomes. The mei-S51 mutation has also been shown to affect An example of the dependence on size within the partner choice within the distributive system (ROBBINS distributive system is observed in experimentsper- 1971). It is asynthetic mutation composed of two formed by O’TOUSA (1 982).In these studies, a num- recessive genes on thesecond and thirdchromosomes. ber of free X chromosome duplications of varying Females homozygous for mei-S51 exhibit reduced ex- sizes were tested fortheir ability tointerfere with change and high frequencies of nonhomologous dis- fourth chromosome segregation. When the free du- junction, particularly with respect to theX and fourth plication equals the size of the fourth chromosome, chromosomes (i.e, XXw44 segregations are frequent). four nondisjunction rises dramatically, presumably as In addition, mei-S51 decreases the frequency of sec-

a consequence of duplication t, fourth chromosome ondary nondisjunction in structurally normal XXY fe- segregation while theremaining four segregates at males and increases the frequency of nondisjunction random. The observation that such segregations were in X inversion heterozygotes. ROBBINSproposed that much less frequent when the duplication was larger mei-S51 disrupts a number of aspects of chromosome or smaller than the fourth chromosome provides an pairing and alignment prior to metaphase and there- example of the dependence onsize within the distrib- fore both reduces exchange and prevents proper part- utive system. ner choice within the distributive system. In addition to secondary nondisjunction (described The third andfinal phase of the model is the proper above), the nonrandom assortment of other nonhom- segregation of oriented chromosomal partners to op- ologs (i.e., X and 4, compound autosomes,and various posite poles and is defined by the nod (no distributive other nonhomologous combinations) has also been disjunction) mutation(CARPENTER 1973). The wild- shown to result from nonhomologous disjunction,and type product of this locus is required for the actual has provided evidence that two acrocentric chromo- disjunction of chromosomes within thedistributive somes will generally beoriented by ametacentric system. Females bearing the nod mutation and struc- chromosome when the elements involved are nonex- turally normal X chromosomesproduce approxi- change (COOPER, ZIMMERINC and KRIVSCHENKO mately 90%fourth chromosome loss. Moreover, 1955; SANDLER andNOVITSKI 1956; GRELL 1959a, b, nonexchange X chromosomes disjoin at random in 1962a;OKSALA 1958; FORBES1960; GRELL and females homozygous for nod. The nod mutation also GRELL1960). These experiments demonstrate the disruptssecondary nondisjunction, such thatthe Y dependence of distributive disjunction on shape. chromosome is still capable of committing two nonex- Based on models proposed by CARPENTER (1973) change X chromosomes to segregate to thesame pole, and O’TOUSA(1 982) we outline a three phase model but the disjunction of the Y and XX pair is disrupted for distributive segregation: (1) selection of nonex- (i.e., XXY c, 0 and XX t, Y segregations occurat equal change chromosomes that will enter the distributive frequencies). system; (2) determination and orientation of proper In this report we describe anew dominant mutation (generally homologous) chromosome partners;and (3) Axs (Aberrant X segregation) whose effect is specific to disjunction of chromosomes to opposite poles at the distributive system. Axs induces high frequencies meiosis I. Although many mutationsaffecting the of X nondisjunction in X chromosome inversion het- A Dominant Meiotic Mutation 803 erozygotes, increases frequencies of fourth chromo- In those crosses that involvedsc4sc8/dl-49 females (in some nondisjunction in response to thepresence of which all sc4sc8/0 and dl-49/0 males are inviable) no ad- X justment was attempted. The listed value for total progeny chromosome structural heterozygosity, and strongly reflects only regular and fourth chromosome nondisjunc- decreases the efficiency of XX t* Y trivalent formation tional female progeny and X exceptional offspring. In these in XXY females. Axs also impairs the segregation of crosses the percent X nondisjunction is calculated by divid- nonexchange major autosomes. The primary pheno- ing the total number of X exceptional progeny by the total type of Axs may then be summarized as follows: Axs number of progeny and multiplying by 100. Females bearing a free Y chromosome also pose a com- decreases the frequency of homologous segregations putational difficulty due to the lowered viability of XXYY within the distributive system while increasing the progeny (LINDSLEYand GRELL1968). In these crosses, the frequency of nonhomologous disjunctions. The data number of XXYY zygotes is estimated using the number of suggest that Axs defines a locus required primarily for regular XY male progeny. These males are the reciprocal homologous disjunctions of distributively segregating product of segregations generating these XXYY females. All other corrections or adjustments are listedin the chromosomes, and reveals the existence of a homol- footnotes and adjusted totals are recorded in each table. ogy dependent component of the distributivesystem. Exchange rank distributions are calculated by the method of WEINSTEIN(1936) for regular-X progeny and by the MATERIALS AND METHODS method of MERRIAMand FROST(1964) for exceptional-X All crosses were performed at 23.5" on standardmedium progeny. (LEWIS1960). Both bottles and vials were set up on day 0, Nonhomologous disjunction frequencies are calculated in transferred on day 5 and the parents discarded on day 10. the following manner as described by O'TOUSA(1 982). For Scoring was performed on days 14 and 18. two pairs of chromosomes (AA;BB) being assayed, the non- Mutations and chromosomes used: With the exception homologous disjunction frequency is calculated as: [(AA; 0 of Axs, all mutations and chromosomes referred to in this + 0;BB) - (AA;BB + 0;O)l divided by the total progeny. report are described in LINDSLEYand GRELL(1 968) except The subtraction is carried out in order to account for those for FM7 which is described in LINDSLEYand ZIMM (1987). cases in whichAA and BB disjoined independently and Throughout this report,the balancer X chromosome segregated to opposite poles. When a pair of homologous Znversion(1) Basc (Muller-5) will be abbreviated as Mu-5and chromosomes and a heterologue (AA;E) is being assayed, the fourth chromosome mutation spaPo' will be referred to the nonhomologous disjunction frequency is calculated as: as pol. In addition, Znversion(1) delta-49 will be referred to [(AA + 0;E) - (AA;E + 0;O)ldivided by the total progeny. as dl-49, and Znuersion(1) scute-8 Left scute-4 Right will be For a detailed discussionof the equations see O'TOUSA referred toas sc4sc8. (1982). When two pairs of chromosomes and a third chro- Calculations: An example of the basic experiment for mosome(AA;E;BB) are being assayed, the frequency of monitoring X and fourth chromosome nondisjunction is a AAE c* BB nonhomologous disjunctions is calculated as cross between y/y; pol/pol females and YX-P, Zn(l)EN, v f [(AA;E;O + O;O;BB) - (AA;E;BB + O;O;O)]divided by B/O; C(4)RM, ci eF/O males. This crossallows oneto the total progeny. recognize X chromosome nondisjunctional offspring from The data shown in Figures 2 and 4 were fitted to regres- the mother either as yellow females (diplo-X exceptions) or sionlines using a commercial software plotting program as vermillion, forked, Bar males (nullo-X exceptions). Simi- (Cricket Graph). larly one can recognize fourth chromosome nondisjunc- Isolation of the Axs mutation:Axs (Aberrant X segregation) tional offspring from the mother either as sparkling poliert is an ethyl methanesulfonate-induced X chromosomal mu- flies (diplo-4 exceptions) or as cubitus interuptus eyeless- tation. It was fortuitously recovered in this laboratory in the Russian flies (nullo-4 exceptions). Nondisjunction frequen- course of a screen for mutations on a bb2 X chromosome cies are calculated as the sum of exceptional progeny classes which block rDNA magnification, a process which occurs divided by the sum of all progeny classes. Except where primarily in the male germline and results in reversion of noted, for crosses in which the female test parent contained bbz @e., stable and heritable increases in rDNA redundancy) free X chromosomes, the number of exceptional-Xprogeny (RITOSSA1968; TARTOF1973). During the stock construc- are doubled before computations are made to correct for tion inthis screen it was frequently necessary to make the inviability of triplo-Xand nullo-X progeny. mutagenized X chromosomes (X*) homozygous by crossing Because they cause male inviability, the following two X X*/Mu-5 females to X*/pY brothers.Females heterozygous chromosomes required slightly different methods of calcu- for the Mu-5 balancer chromosome and the Axs-bearing X lating nondisjunction frequencies. The dl-49 Axs chromo- chromosome generated high levels of X nondisjunction. On some used in all experiments carries an unmapped distal this basis, the Axs chromosome was selected forfurther lethal that requires the presence of y+Y in males;X/O males testing. bearing this dl-49 chromosome are therefore inviable. This Construction and use of Axs-bearing and Axs+C(l)RM chromosome originally carried both the f and Rex mutations chromosomes: A compound X chromosome heterozygous and was graciously provided by L. ROBBINS.The proximity for the Axs mutation was constructed by irradiatin y pn cv of Axs to f allowed us to generate a dl-49, Axs car chromo- m f .y+/y cv u Axs car females and crossing to y+bb2/ BHY males. some via recombination between dl-49, f Rex and a y cv v Yellow daughters carrying @Y &e., females arising from Axs car chromosome. Finally, sc4sc8 carries a complete defi- diplo-X exceptions in which the y+ marker had been lost) ciency for the rDNA genes (bb"), and thus, X/O males were selected and backcrossed to y+bb2/PY males to gener- bearing this chromosome are also inviable. Thus, in crosses ate a numberof stocks. We will present evidence below that involving dl-49/y females(i.e., those in which the dl-49 Axs- the Axs mutation is recombinationally inseparable from the bearing X/O males would beinviable), the reported number forked locus. Thus, for the purposes of constructions, the of males represents only the y/O progeny. This number is Axs mutation was followed by the presence or absence off. doubled to estimate the total number of regular male zyg- To maintain heterozygosity of Axs in each stock, phenotyp- otes produced. This adjusted total is used to calculate non- ically heterozygous females were selected each generation disjunction as described above. and their heterozygous daughters were used for the next 804 A. E. Zitron and R. S. Hawley generation only if both f (Axs+) and v car (presumed Axs/ Axs, see below) females were present. A. A single heterozygous Axs stock was chosen and used for all subsequent steps in both constructions and experiments. In order to ensure that all C(I)RM chromosomes used in the experiment were derived from the same irradiation- induced attachment event, a singlef(Axs+) female from this heterozygous stock was used to generate a stock of control females while the putative Axs homozygotes (v car) were selected directly from the heterozygous Axs stock. Cytolog- ical analysis of larval neuroblasts revealed that the com- pound X chromosome constructed in this manner is a C(1)RM chromosome (data not shown). Control and heterozygous Axs crosses were performed in batch matings. For the heterozygous 4xs crosses, we as- B. sumed that (1) if the females tested were wild type with respect to v and car, and (2) each bottle gavefprogeny, then the females used were probably heterozygous for the Axs mutation. All homozygous Axs crosses were performed in vials in the following manner. FI progeny from a single v car mother were scored, and five of the F, daughters were FIGURE1 .-Cytological analysis of the Axs mutant rllromosome. used to generate F2 progeny that were then scored. Ten of (A) The top spread is +/Am. The bottom spread is Am/+ which is the F2 daughters were used to generate FRprogeny. These stretched to reveal theaberration at 15D1. (B) The wild-type were scored and the sum of all generations was used to polytene nap. reprinted from IXFEVRE(1 976). calculate frequencies of nondisjunction. Each generation, and two generations subsequent to F3 were scored for the somes or heterozygous for an inverted, or multiply- presence off: The lines yielding f flies were discarded. Approximately 50 single u car females initiated the experi- inverted, X chromosome. It has been shown previously ment, and five of these lines were finally used to generate that frequencies of X nondisjunction are not greatly the data reported. increased by inversionheterozygosity in otherwise genetically normal females (STURTEVANTand BEADLE RESULTS 1936; COOPER 1945).Indeed Axs+/Axs+ control fe- males in Table 1 exhibit frequencies ofX nondisjunc- Axs (Aberrant X segregation) maps to position on 56 tionthat are low, regardlessof heterozygosity for the standard map andwas not separable from(56.7). f invertedbalancer chromosomes. In females with Among the progeny of +Axs +/v car females, + + f structurally normalX chromosomes, X nondisjunction no crossoversbetween Axs and wererecovered f is elevatedonly slightly in Axs/Axs+ or Axs/Axs fe- among 40 v-frecombinants or 40fcar recombinants males. In the presence ofAxs and a balancer, however, (all recombinants wereAxs+ and all recombinants f f' X chromosome nondisjunction is increased some 10- carried the Axs mutation).Cytological analysis per- to 50-fold. formed by D. WRIGHTin the laboratory revealed that The data in Table 1 also show that Axs increases chromosomes bearing the Axs mutation also carry a the frequency ofX nondisjunction in a semidominant small aberration at band 15D1 on the polytene map fashion. Both the y/dl-49 and thesc4scH/dl-49 females (Figure 1). Thisaberration is notobserved in the parent strain(bb') and is absent in a single spontaneous exhibit an approximately two-fold increase in X non- revertant ofAxs (data not shown). Thisis in agreement disjunction when comparing one versus two doses of with the genetic mapping described above. Experi- the Axs mutation. For normal sequence X chromo- ments are underway to determinethe relationship somes bearing the Axs mutation, the effect is in the between this aberration and theAxs mutation. same direction although much less dramatic. These Axs does not increase either sex or fourth chromo- experiments demonstrate a clear zero, one, two dos- somenondisjunction in Axs/Y males.Control males ageeffect of the Axs mutationon X chromosome exhibitfrequencies of sex and fourth chromosome disjunction in females. nondisjunction of 0.079% and 0.1 19% respectively X nondisjunction in AxslBalancer females appears (N = 1269). These frequencies are similar to those to increase with the degree of structural heterozygos- reported previously (6BAKER and CARPENTER 1972). ity. These data suggest that the frequency of X non- In Axs males, only a single X,4 simultaneous nondis- disjunction is correlated with the frequency ofX chro- junctional offspringwas recovered out of2 131 prog- mosome nonexchange bivalents(Eo tetrads) in Axs- eny scored. ThusAxs has little or no effect on meiotic bearing females. To test this hypothesis it is necessary disjunction in males. to estimate the frequency ofEo tetrads for each inver- Sex chromosome disjunction in Axs females: sioncombination. Because exchange cannot be di- Table 1 presents the results of measuringx and fourth rectly measured in balancer heterozygotes, an indirect chromosome disjunction in females which are either parameter must be used in tetrad analysis. As noted homozygous for two structurally normal X chromo- above, in XXY females, nonexchange X chromosomes A Dominant Meiotic Mutation 805 g; II +r- a s 90? "I- 9 mm m- mmmo m-ar- m- 2 g$ 2 ZG mr- + mm m- mm 2:+ x 2z jgE6 mo vi0 r. r. 8- 2 22 mmom 0-00 ma 22 0 22 m* !Eo -- mm 25% 1 zg e*.. ai5 -- 2 T.2 gJ 8 mz& ; ma r- - h "3 ? y1 ' mm +- mmmr- Lm-mma r-r- 22 2 0 %23 2 mm mz In mm m +m : cgo +a - 2 C&< 3 EZ ++ Y '0% a c sge, 8 .28! ma m 9 r\ SeR 00 -000 22 2 ma- 4 r-mmm no-0 mm z 3 r- I e, IB -2 .g*a 9 22 nn 5 ,& i .-c CVR d ". a y12 8 w 22 2:-m 80 m -0 ';$E 23 n mw + n -nam 3- 3 E+* 3 p ,: 0 m -mmn mm n- z 3% 2z E: -04 nm om5: y11 1 g +w 2 E32 g i 25; Y b aor- a r- x9 0?'9 9 62 g s 3 .u " mr- m mmmm nn r-m m GQ - R 7% - -- r-P- 3 B 22 00 -u3 2 - mm 4h >e*$;z u t E: a$ s O= Q b T\m202 2%". mm m - 3%o\ "u -?X - i 0x2 p .- + mo + m mo-m nn 2 0 s 3-2 't su mm mm 2 n% mm 11 -0$C nn 1 SOC r( 3: 5 -g.Q +m m n "3 8z2 w v3 I4 '94 4 u r-2 mr. mG2r- nm-w m- me =i, e9 $3 mm r-a azcQa 900 m m; g?:$W" e 6 'c,- gz ne0 mr-zi? c1 s v 11 zzg 5 gcc 2 om m fa ;3 45% ? '91 '9 3 mg +m ossa mmP-+ a- mn $ 58 -5 ' 0- r-m o"c5- -n m+ - 2 $2 2 4- am - 3 5 'c, x- +a j $28 - I- 'G x 4- OI- 0 : 3b$ b m p" .3 p 2 " 00-n om 000 0 3 t.g co= -2sn on g 31$$; 2 r-- a0 u=4 2 mw 5 .-F nE? -0 24Yi 5 '5 -52 2 am a 0 i CS$E t 22 "2 b a E 0-on -3 2 u gz :a g+ces nm 2 s Zb4C 0 $ I-+ nn 11 :+EO I " CI 'c, T p) a, w.2 Ia a 1~3m&-bC d c +m m - 8 ;,,e 22 2 z=, ULWm 2 -I. m-nn-mnn +m-n mo 22 3 mm 2 i: mw0- 3, 2s 0- e&bo mm mm .eScggoOC wu"3~~$ ma SSZE E mm -m -mn- 0000 $ a0 ma,on 220 mr- aa 2 wg?.;$ 4 mm ^ao%aS 2 &%; ;I -$ pa 2,; C: C: &: 2

Gamete types Gamete Maternal genotype

dl-491 Mu-5/ FM 71 dl-491 y w' ct6 m f car1 y wa ct6 mf/ Y mfl sc4sc8B+I Mother" Father Mother" Y Y Y Y

Regular (X-XY) ~ X(Y) XY X(Y) 24 1 65 492 1 99 1 X(Y) 0 944311 406 663' X Nondisjunctional (XXoY and XXY-0) XX(Y) 478 224 1156 1274 O(Y) 179 858 33 1 757 Total progeny 955 37861707 3508 56.44 66.58 67.01 70.69 % X67.01 Nondisjunction' 66.58 56.44

* Due to the use of an unmarked Y chromosome, the presence or absence of the Y in a given offspring cannot be assessed. This is de- noted by placing the Y in parentheses. The sc'sc8/0 males in this class are inviable. These males represent '/4 of the total number of males in this class and thus, this number is multiplied by 4/3 for use in the calculation of nondisjunction frequency. ' Due to low viability of XXW females, the percent X nondisjunction is calculated as follows: [total X nondisjunctional progeny/regular males + total X nondisjunctional progeny1100.- segregate fromthe Y chromosome at high frequencies. Moreover, the frequencyof secondary nondisjunction has been shown to increase with the extent of struc- tural heterozygosity (COOPER1948), as a consequence of an increased frequency of Eo tetrads. Since the Y has little or no effect on the frequency of exchange (STURTEVANTand BEADLE1936), the frequency of secondary nondisjunction may be used as a valid esti- mate of Eo tetrad frequencies. The useof secondary nondisjunction to estimate the frequency of nonexchange tetrads has been re- cently reviewed by RUTHERFORDand CARPENTER OF 20 40 (1988), however, two points require further mention 0 80 60 % x Eo Tetrads here. First, XX c-, Y segregations have been shown by a number of investigators to account for most, if not FIGURE2,"Percent X EO tetrads (data taken from Tables 2 and 3) us. percent X nondisjunction (data taken from Table 1). all, of the total X nondisjunction observed (BRIDGES 1916; COOPER1948; CARPENTER1973). Second, al- monds) and Axs homozygotes (closed squares). No though secondary nondisjunction represents the pre- such effect is observed in Axs+ control females (open dominant class of segregational events when XXY fe- squares). These data argue that(1) in the presence of males are nonexchange (for a review, see ZIMMERING Axs, nondisjunction increases linearly with thefre- 1976), X t, XY segregational events involving nonex- quency of Eo tetrads and (2) the Axs mutation acts change X chromosomes are also observed. For exam- primarily on nonexchange chromosomes (see below). ple, STURTEVANTand BEADLE(1936) concluded that Four additional features of the X nondisjunction only 90% of the Eo tetrads in In(l)dl-49/+/Y females associated with Axs should be noted.First, unlike most underwent XX f-, Y segregations. Thus, thefrequency previously described meiotic mutants inwhich the of secondary nondisjunction isin fact an underesti- frequency ofnullo-X bearing eggs is substantially mate of the true Eo tetrads. higher than diplo-X bearing eggs (DAVIS1969; BAKER We have measured the frequency of X nondisjunc- and CARPENTER1972; HALL1972; CARPENTER1973; tion in X/X/Y females (secondary nondisjunction) and PARRY1973; O'TOUSA1982), the nondisjunction ob- thus, have derivedan estimated frequency of EO served in Axs females results in nearly equal frequen- tetrads foreach type of inversion-bearing female used cies of nullo- and diplo-X bearing ova. This observa- in these experiments. The data are presentedin Table tion indicates that AXSprimarily induces nondisjunc- 2. tion; chromosome loss, if it occurs at all, is at best a As shown in Figure 2, X chromosome nondisjunc- minor component of the Axs defect. tion increases linearly with the estimated frequency of Second, since in Axs homozygotes the frequency of Eo tetradsfor both Axs heterozygotes (closed dia- X nondisjunction is approximately one half the fre- Dominant Meiotic Mutation Meiotic A Dominant 807 quency of X Eo tetrads, it appears that thedisjunction of one ortwo doses of the Axs mutation (Table 3). of nonexchange X chromosomes is random or nearly The effect of the Axs mutation on exchange fre- random.For example, sc4sc8Axs/dl-49 Axs females, quency and on the interchromosomal effect are pre- which yield anapparent Xchromosome EO tetrad sented in Table 3 and are represented graphically in frequency of approximately 71 % (Table 2), exhibit Figure 3. The data show that recombination levels in 38.5% X nondisjunction. +/Axs and Axs/Axs females are similar to those ob- Third, most, if not all, Axs-induced X nondisjunc- served in +/+ control females either in the presence tion occurs at meiosis I. If a given chromosome has or absence of the SMI chromosome. For both sets of not undergone exchange,nondisjunction at meiosis I1 crosses, however,a 4 X 3 x' test for homogeneity can be observedthrough thehomozygosis of all mark- revealed statistically significant differencesbetween ers contained onthat chromosome. There was no the proportions of noncrossover (NCO), single cross- significant increase inmeiosis 11 nondisjunction of over (SCO), double crossover (DCO) and triple cross- chromosomes in either hetero- or homozygous Axs over (TCO) progeny produced by +/+, +/Axs or Axs/ females. In fact, among all the experiments described, Axs females. (For females bearing normal second chro- the only possible example of meiosisI1 nondisjunction mosomes x* = 27.34, d.f. = 6, P < 0.001 and for was a single yellow crossveinless vermillion carnation SMI-bearing females x' = 14.46, d.f. = 6, P < 0.01.) female among 202 diplo-X female progeny recovered Despite the finding of statistically significant differ- from y cv v Axs carldl-49 mothers. ences in the frequency of crossover recovery, we note Fourth, Axs does not appear to affect mitotic chro- that the observed differencesare small (lessthan 10%) mosome stability or the stability of chromosomes in and that thereis no consistent dosage effect of the Axs early zygotic cleavages that have been transmitted by mutation. Indeed, in females bearing normal second Axs-bearing mothers. Inthe experiments in which chromosomes, +/Axs females display levels of recom- females were heterozygous for the cell-autonomous bination which are virtually identical to those dis- markersf and/or y, no patches of mutant tissue were played in +/+ females; while in the presence of SMI, observed in either hetero- or homozygous Axs moth- the total frequency of X chromosome recombination ers, or their progeny. In addition,only one gynandro- observed in +/Axs exceeds that observed in +/+ con- morphfrom Axs females was observed amongthe trols. Thus, we conclude that Axs has little or no progenyfrom all theexperiments performed. Al- consistent effect on the frequency or distribution of X though these experiments do not rigorously test for chromosome exchange, and that the differences ob- the presence of an effect of the Axs mutationon served most likely reflect other geneticdifferences somatic chromosome disjunction, the results suggest between the three tested genotypes. The observation that Axs does not induce mitotic chromosome loss at that Axs exerts little or no effect on the interchromo- high frequencies. This result is in contrast to those somal effect further suggests that, for most intervals, observed for mutations like nod and candwhich pro- exchange responds to normal controls, even in the duce high frequencies ofzygotic chromosome loss presence of the Axs mutation. (CARPENTER 1973; BAKERand HALL 1976). In the crosses reported in Table 3, control females All of these data strongly suggest that the effect of exhibited a remarkably high frequency of X nondis- Axs is limited to the first meiotic division. junction (Table 4). Indeed, Table 4 also shows that Exchange chromosomes andAxs: The effect of Axs Axs-bearing mothersdid not exhibit increased fre- is much more pronounced in balancer heterozygotes. quencies of X nondisjunctionwhen compared to these This suggests that the Axs mutation only influences controls (approximately 2% in all cases). These obser- the segregation of nonexchange X chromosomes(ie., vations arenot understood. We have repeatedthe X chromosomesin the distributive system). To further experiment utilizing a different set of tester chromo- examine the effects of theAxs mutation on themeiotic somes and, in addition to corroborating the tetrad exchange system, we monitored the frequency and analysis, we have observed substantial increases in X distribution of exchange on the X chromosome in +/ nondisjunction in Axs females relative to frequencies +, +/Axs and Axs/Axs females (Table 3). To test the observed in controls (data not shown). ability of Axs-bearing females to respond to standard In the experiments described in Tables 3 and 4, it modulators of exchange frequency, the effect of Axs was observedthat a significant fraction of diplo-X on the interchromosomal effect was also examined. offspring recovered from all six crosses were homo- The interchromosomal effect has been reviewed by zygous for one or moreX chromosomal markers (data LUCHESSI(1976); briefly, females that are heterozy- not shown). Because such exceptional progeny arise gousfor an autosomal balancer havean increased by nondisjunction of bivalents that have undergone frequency of exchange on other bivalents. To exam- exchange, this observation raised the possibility that ine the effect of Axs in this process, X chromosomal there was a weak effect of Axs on the disjunction of exchange was monitored in females heterozygous for exchange bivalents. To test this hypothesis, diplo-X the second chromosome balancer SMIin the presence exceptionsfrom crosses involving normalsequence 808 A. E. Zitron and R. S. Hawley

TABLE 3

Results of crossing YsX.YL, In(l)EN, vfB/O;C(4)RM, ci ey"/O males to y cv/y wa ct6 m car;pollpol or y cv/y w4Ct6 m car; &W/+; pollpol females

Maternal genotype X Chromosome recombination and tetrad analysis

2nd chromosome: 2nd +I+ SMlf+

X Chromosome: +I+ +/Am AxslAxs +I+ +/Am Aws fAxs

4 4 0 4 0 4 4 0 4 0 NCO 688 19 1580 33 1248 7993 732 510 5 sco 1 (w-cu) 138 412 0 239 7 216 264 10 137 2 (cu-ct) 47 124 4 a5 5 93 a9 2 37 1 3 (ct-m) 1853 432 7 253 229 230 0 148 2 4 (m-car) 29 1 1 56 1 434 a 333 303 2 167 DCO 1, 2 0 1 0 2 0 0 1 0 0 0 1, 3 10 21 0 6 0 28 34 1 13 0 1, 4 43 111 1 53 1 79 107 0 43 0 2, 3 2 5 0 4 0 5 6 0 4 0 2, 4 25 36 0 17 0 37 2a 0 21 0 3, 4 29 55 0 37 0 68 5a 0 31 0 TCO 1,2,3 0 0 0 1 0 0 1 0 2 0 1, 2, 4 0 1 0 0 0 0 0 0 0 0 1, 3, 4 2 5 0 5 0 3 3 0 2 0 2, 3, 4 2 0 0 1 0 1 0 0 1 0 Total males 1462 3372 2446 1891 1864 1127 Map distances for region 1 13.2 16.4 12.8 17.2 22.1 17.6 2 5.2 5.1 4.7 7.2 6.8 5.9 3 15.7 15.5 12.8 17.6 17.9 18.0 4 -26.8 22.9 -22.7 -27.6 -26.9 -23.7 Total 60.9 59.9 53.0 69.6 73.7 65.2 Exchange rank Eo 0.09 0.09 0.15 0.07 0.04 0.1 1 E, 0.62 0.64 0.66 0.45 0.47 0.5 1 J5 0.27 0.26 0.17 0.46 0.47 0.34 E3 0.02 0.01 0.02 0.02 0.02 0.04 second chromosomes were progeny tested and tetrad Both the low frequency of X chromosome nondis- frequencies were calculated according to the method junction in Axs females bearing normal sequence X of MERRIAMand FROST (1964). The resulting data chromosomes and the observed excess of Eo tetrads are presented in Table 4. The tetrad frequencies for amongthe bivalents producing diplo-X exceptions diplo-X exceptional progeny from heterozygous Axs strongly suggest that the Axs mutation has little or no females are Eo, 0.54; El, 0.38;and E2, 0.08. The effect on the disjunction of exchange chromosomes. frequencies for homozygous Axs females are Eo, 0.54; Fourth chromosome disjunction in Axs females: EL,0.29; and EB, 0.17. Tetrad frequencies for diplo- Fourthchromosome nondisjunction remains rela- X exceptional progeny from control females are Eo, tively constant (0.12-0.39%) among Axs+ control fe- 0.26; El, 0.25;and E2, 0.48(taken from MERRIAM males and is not increasedby structural heterozygosity and FROST 1964). Thus, the data reveal a two-fold on the X (Table 1). Although the fourthchromosomes excess of Eo tetrads among theova producing diplo-X are always nonexchange, the Axs mutation does not exceptions in Axs-bearing females. This observed ex- induce high levels of fourth chromosome nondisjunc- cess of nondisjunctional progeny generated from Eo tion in females bearing two structurallynormal X tetrads in Axs-bearing females suggests that Axs does chromosomes. Thus, where the frequency of X Eo not increase the nondisjunction of exchange tetrads, tetrads is low, Axs seems to have no direct effect on but ratherproduces diplo-X exceptional ova primarily the disjunction of chromosome four. as the result of failed disjunction of Eo bivalents. In Axs females heterozygous for anumber of struc- A Dominant Meiotic Mutation 809

TABLE 4 Results of crossing YsX.YL, ln(l)EN, v f BIO C(I)RM,ci eyR/O males toy mly woet6 m car; pollpol females

(:amele rvpes Maternal genotype

\lorher Farher +I+ +fAxs AxsIAxs Regular x4 fig 1624 6099 4231 x4 044 x4 11632246 3509 X Nondisjuncti 11 I 04 x4.2) 1 11 32 24 xx4 OA 14 5 0 23 4 Nontlisjunctio 1 I x44 X)x 0 0 32 32 x0 02 0 5 23 23 40 0 23 x0x44 8; 26 4 37 X.4 Nontlisjunction;ll xx44 2 3 3 00 &? 0 1 3 xxo 0 3 3 044 2%; 1 4 3 Total progeny6668 9787 2820 Ad,justed total6727 9880 2848 96 Nondisjunction X 1.97 1.75 1.88 4 0.39 1.09 2.32 x,4 0.2 I 0.22 0.36 Nunlberexceptiotd of 9 38" 39 fenl;lles tested: Esch;mgc rank Map neglons F4r (0.26)' 0.54 0.54 E, (0.25) 0.29 0.38 FIGURE 3,"Exchange in Axs females. (A) The effect of Axs on E2 (0.48) 0.17 0.08 the frequency of exchange. (B) The interchromosomal effect and Axs. a Due to the low numbers of diplo-X exceptional females gener- ated in this cross, we collected additional exceptional females from crosses that were not scored. turallyabnormal balancer X chromosomes,fourth ' The small numhers obtained in these crosses did not permit chromosomenondisjunction increases with thefre- the calculation of tetrad frequencies using data from these females. 'I'he tetrad frequenciespresented here are from MERRIAMand quency of X chromosome nondisjunction. For exam- FROST (1 964). ple, in y Axsly Axs,y Axsldl-49 Axsand sc4scuAxs/dl-49 Axs, frequencies of fourth chromosome nondisjunc- behavior is the w"Axs/Mu-5 heterozygote, where non- tion are 4.52%,7.42%, and 13.4896, respectively homologous disjunction is low. (Table 1). As was shown in the analysis of X misbehav- The anomalous behavior of the Mu-5 chromo- ior, the effect on thedisjunction of chromosome four some: The Mu-5 chromosome is eccentric in at least two aspects of its response to the Axs mutation. First, is also responsiveto the dosage ofAxs the mut? c tlon.' The correlation betweenX and fourth chromosome the overall frequency of X nondisjunction in AxslMu- 5 females is much lower than would be expected based nondisjunction is expressed graphically in Figure 4. on the estimated frequency of 2). The data show that in Axs females, fourth chromo- Eo tetrads (Table For example, the frequency of X nondisjunction in some nondisjunctionis tightly coupled to the presence AxslMu-5 females is half that observed in AxslFM7 of nonexchange X chromosomes and thus,to X chro- females despite the fact that XIMU-5 females display mosomemisbehavior. Indeed, Table 1 shows that anapproximately equal frequency of X Eo tetrads approximately 50% of the fourth chromosome non- (66.58%) when compared toXIFM7 females (67.01%) disjunction can be accounted for by simultaneous X as derived from Table 2. Secondly, in AxslMu-5 fe- and fourth chromosome nondisjunction.The vast ma- males, simultaneous X,4 nondisjunctional events were jority of these simultaneous nondisjunctional events divided equally between XX - 44 and Xx44 c-, 0; 0 result from nonhomologous segregation of Xthe chro- disjunctions. The absence of a preference for XX c-, mosomes from the fourth chromosomes (ie., XX;OO 44 nonhomologousdisjunctional events is in stark and 00;44ova are producedin vast excess compared contrast to the observations made for several otherX to XX;44 and 0O;OO ova). The exception to this chromosomes tested (Table 1). 810 A. E. Zitron and R. S. Hawley

l4 1 control levels. In females heterozygous for the SMl balancer chromosome and a structurally normal sec- ond chromosome, AXS heterozygotes increase second chromosomenondisjunction over control levels by 1.5-fold and Axs homozygotes exhibit a 7-fold effect. The Axs mutation also reduces the fractionof events that were simultaneously nondisjunctional for the X and second chromosomes in Axs/Axs; +ISM1 females. Moreover, those simultaneous events which did occur were randomized such that among the diplo-2 ova simultaneously nondisjunctional forthe X chromo- somes, nullo-X and diplo-X exceptions were recovered at nearly equal frequencies. This is in contrast to the 2: I I I i vast excess of nullo-X ovaobserved among diplo-2ova 10 20 0 10 30 40 in either +/+ or +/Am females. The effect was con- % x Nondisjunction sistently observed in each bottle and has been subse- FIGURE4.-Percent X nondisjunction us. percent 4 nondisjunc- quentlyrepeated. The significance of this result is tion (data taken from Table 1). considered in the DISCUSSION. The 7- to 12-fold effects on second chromosome Although these observations are not understood, nondisjunction observed in Axs homozygotes are sim- we propose two possible explanations. First, the Mu-5 ilar to those observed for the nod mutation which is chromosome may carry an undefined structural ab- defective in distributive disjunction (CARPENTER erration, such as a heterochromatic deficiency, which 1973; and see Introduction). Two lines of evidence, impairs X and fourth chromosome associations. This however, argue that basing our conclusions solely on possibility is supported by the fact that X/O males the fold-effects exerted by the Axs mutation may be bearing our Mu-5 chromosome display good viability, misleading. despite the statement by LINDSLEYand GRELL (1 968) First, although SMl effectively suppresses the re- that such males should be poorly viable due to posi- covery of exchangeproducts (MACINTYRE and tion-effect variegation of an essential locus, l(1 j'J1. We WRIGHT 1966),it is not clear to what extent exchange have confirmed by polytene chromosomeanalysis that our Mu-5 does possess theeuchromatic inversions is reduced in theseheterozygotes (Eo data are not characteristic of a Mu-5 chromosome(data not available for these aberrations). Thus, it may not be shown). Second, it is possible that Mu-5 carriesa reasonable to compare the effects of this autosomal cryptic meiotic mutation that interacts with Axs. Re- balancer to those exerted by a given X chromosome gardless of which hypothesis is correct, the basic ob- balancer in Axs-bearing females. Second, as was ob- servation points out thenecessity ofutilizing a number served for the X chromosomes, second chromosome of different tester chromosomeswhen characterizing nondisjunction may frequently involve nonhomolo- a novel meiotic mutation. gous 44 t, 22 disjunctions. This class of segregational Distributive disjunctions of a majorautosome and events would berecovered less frequently in these Axs: We tested the effect of Axs on the behavior of crosses because the tester males did not carry a com- the second chromosomes in both the presence and poundfourth chromosome. Thus,our calculations absence of balancers for the X and second chromo- may tend to underestimate the frequency of second somes. Because aneuploidy for the major autosomes chromosome nondisjunction in Axs females. is lethal,these experiments require thatthe males In order toobserve the effect of increased numbers carrycompound autosomes (for review see HOLM of chromosomes in the distributive system, we tested 1976). By providing a diploid complement from the the behavior of the X and second chromosomes in Axs father, nullo-bearing eggs can berecovered as eu- heterozygotes in the presence of both Mu-5 and SMl ploid, and conversely, by providing a nullo-bearing (Table 5b). Although the basal levels of second chro- sperm, one recovers the diplo-bearing egg. In these mosome nondisjunction are elevated in Mu-5/+; crosses all eggs produced from normal segregations SMl/+ females, the effect of Axs on the frequency of are lost, and thus, these experiments only allow for second chromosomeexceptions in these females is the recovery of nondisjunctional progeny. similar to that observed when comparing non-Mu-5- The data in Table 5a show that Axs induces adosage bearing females. Thus,although there is a slightly dependent increase in the frequency of second chro- higher frequency of second chromosome misbehavior mosome nondisjunction.Heterozygous or homozy- in response to the addition of the Mu-5 chromosome gous Axs females carrying two structurallynormal in both control and Axs females, the effects of Axs second chromosomes increase secondchromosome parallel the observationsmade for females bearing nondisjunction by 3- and 12-fold respectively over structurally normal X chromosomes. A Dominant Meiotic Mutation 81 1 TABLE 5 Results of crossing X/y+YBs; C(2)EN,bw sp/O males to X/% +/+ or X/% SMI/+females

Maternal genotype Maternal Female gametic genotype

Diplo 2 Nullo 2" Fraction of si- No. of 2ndChromosome multaneous X- X 2 mothers X xx 0 X XX 0 exceptions/female 2 exceptions 4 +I+ 19+/+ 888 0 0301 0.026 0.044 +/yAxsb +I+ 744 40 1 0604 0.069 0.098 yAxs/yAxsb +/+ 888 227 11 26 1 0 0 0.298 0.140 +/+ SMl/+ 480 24 112 1 0 0 0 0.183 0.285 +/Am car SMl/+ 480 14146 5 2 0 0 0.404 0.273 Axs car/Axs car SMI/+ 504 94943 45 0 0 0 2.058 0.085 b) MU-^ SMI/+ 45618 0.521655 3.136 722 3 30 2 AxsSMI/+ carlMu-5 963480 53 1071 0.483 4.404 3 21 3 The tester males used in these crosses transmit the C(2)EN chromosome at low effkiency. Thus, the number of nullo 2 progeny is low. The numbers used for calculations are not adiusted to account for this behavior. The term y AXSdenotes y m v Axs car. "

Based on the data presented in Table 5, a and b, (ie., nondisjunction is not equal to one-half the fre- we conclude that Axs exerts a significant effect on the quency of C(I)RM c, Y segregations which, in this disjunction of the nonexchange second chromosomes. case, is 100%).Thus, thesedata demonstrate that The effect of Axs in C(I)Z?M/Yfemales: In order there may be some effect of a change in X chromo- to further test the effects of the Axs mutation on large some size or shape on the dominant natureof the Axs metacentric chromosomes, disjunction of a metacen- mutation. triccompound X chromosome, C(I)RM, froma Y Disjunction in XX; C(4)RM females: The experi- chromosome was monitored in the presence of either ments described above demonstrate that Axs-induced zero,one, or two doses of the Axs mutation. The increases in X and fourthchromosome nondisjunction construction of this C(1)RM chromosome is described may be explained by a decrease in homologous dis- in MATERIALS AND METHODS. junctions with a concomitant increase in nonhomolo- Although both arms of the C(1)RM chromosome gous XX c, 44 disjunctions. Unfortunately, the analy- are normalsequence and freely recombine, the sis of the crosses presented in Table 1 is complicated C(I)RM segregates from the Y chromosome (C(1)RM by the presence of four elements in the distributive c, Y) in the distributive system almost 100% of the system. It was possible that if the system couldbe time (for a review see GRELL1976). In other words, simplified such that only two X chromosomes and one exchange events do not preclude a compoundX from heterolog (E) were available, most, if not all, X non- entering the distributive system. One might expect, disjunction could be accounted for by XX c, E segre- therefore, that in the presence of the Axs mutation gational events. To test this prediction, thedisjunction the frequency of C(1)RM t* Y disjunctions observed of the X chromosomes and ametacentric C(4)RM would be strongly decreased. chromosome was tested in the presence and absence As can be observed fromthe data reportedin Table of the Axs mutation. 6,the frequency of nondisjunction involving the The results of examining X and C(4)RM disjunction C(1)RM and Y chromosomes (i.e., C(I)RM/Y c* 0) in in sc4sca/dl-49;C(4)RMIO females bearing zero, one Axs+/Axs+ control females is quite low (0.16%). How- or two doses of the Axs mutation are reported in ever, nondisjunction of the C(1)RM and Y chromo- Table 7. As has been observed previously, X chro- somes is observed in Axs heterozygotes at a frequency mosome nondisjunction rises in a dosage dependent of 3.1% (approximately 20-fold higher than control manner when comparing Axs heterozygotes to AXS levels) and in Axs homozygotes at afrequency of homozygotes. Indeed, the frequencies of Axs-induced 20.8% (approximately 130-fold higherthan control X nondisjunction observed in these females are vir- levels). tually identical to those observed in females bearing These data show that Axs exerts a strong effect on two free fourth chromosomes. Yet, in all three classes disjunctions involving a compound X and a Y chro- of C(4)RM-bearing females (+/+, +/Am, and AxslAxs), mosome, however, there is no longer a clear zero, 7545% of the total X nondisjunctional events occur one, two dosage effect as was observedfor free X as the consequence of nonhomologoussegregation chromosomes.In addition, the nondisjunctionob- from the C(4)RM chromosome. Thus, the proportion served is not 50%in Axs homozygotes but rather20% of X nondisjunction associated with XX c, C(4)RM 812 A. E. Zitron and R. S. Hawley TABLE 6

Results of crossing y cu vfcar/y+Ymales to C(Z)RM/gsYfemales

~~ ~~ Maternal genotype G amete types Gamete AxslAxs" vial numbers 1 2 3 4 5

Mother Father +I+ +lAXS FI + F2 FJ F, + FP F3 F, + Fn Fs Fl + FP Fl Fs + FP Fa Regular C(1)RM226 20 Y 2667 4161 1112 340 197251 1432 108 Y 210 28 X2294 4524 10 279 3210 205159 26 94

Nondisjunctional C(I)RM;Y Y 02 6 10 1 4 0 15 1 6 1 14 7 73 9 67 0; 0 9 73X 7 40 4 0 35 75 135 50 Total progeny scored 8692 4640 572 720 448 303 573 % Nondisjunctionb 0.16 3.10 23.90 18.99 22.54 13.95 14.62 Average frequency of nondisjunction: 20.84%

For complete genotypes of the C(I)RM chromosomes used see MATERIALS AND METHODS. Crosses were performed in vials. FI denotes progeny from a single mother. Five nonvirgin F, females were used to generate FP progeny and ten nonvirgin FPfemales were used to generate FJ progeny (see MATERIALS AND METHODS for further discussion). For reasons detailed in text, the percent nondisjunction is calculated as follows: 12 (male nondisjunctional progeny)/[2 (male nondisjunc- tional progeny) + total regular progeny]) X 100.

TABLE 7 Results of crossing XYs. YL,Zn(l)EN,y E/O; C(4)RM,poll0 males to sc'sc'E'/~~-49;C(4)~,ci eyR/O females

Gamete types Gamete Maternal genotype M other Father Mother sc'scx/dl-4Y sc'scxAxsldl-49 sc4sc8Axsldl-49Axs Regular" x C(4) k30 1780 1609 773 x0 B C(4) 1434 1288 640 X Nondisjunctional 21 xx C(4) A0 0 12 39 00 XY (44) 5 18 17 xx 0 0534) 53 203 314 0 C(4) XY 0 69 233 425 3353 3372 2208 Total progeny 3372 3353 % X nondisjunctionb 14.09 4.15 36.0 1 (1.49). (1 7.80) (38.51) % XX ++ C(4)nonhomologous disjunction 30.93 11.77 3.13

a Because the dl49 Axs X/0 malesin these crosses are inviable (see MATERIALS AND METHODS) and the Axs+ dl-49 chromosome is not isogenic to the dl49 Ars chromosome, for the purpose of consistency, no dl-49/0 males are recorded. Due to lethality of x/0 males in these crosses (see MATERIALSAND METHODS), the % x nondisjunction is calculated as follows: (total x exceptional progeny/total progeny)100. ' The numbers in parentheses represent frequencies of X nondisjunction for sc4scN/dl-49;pol/pol females (see Table 1). segregations remains thesame in either control or Axs Disjunction in XXY females: To determinethe females, such that the vast majority of X nondisjunc- effect of the Axs mutationon disjunction in XXY tion is associated with XX t, C(4)RM segregation. The females, y/y/y+Y; pollpol and sc4scs/dl-49/y+Y; pol/pol effect of Axs then, is to enhance the frequency of XX females were tested in the presence and absence of e C(4)RM nonhomologous disjunctional events, the Axs mutation (Table 8). Because the frequency of rather than to allow the X chromosomes simply to secondary nondisjunction in sc4scsldl-49/Y females is disjoin at random. lower here than in Table 2, we conclude that a y+Y We conclude from this experiment that most, if not chromosome induces secondary nondisjunction with all, of the X and fourth chromosome nondisjunction observed in Axs females is theconsequence of an reduced efficiency compared to a structurally normal increase in nonhomologous associations. This conclu- Y. Although this difference is unexplained, it may sion is further supported by the following analysis of reflect the altered structureof the y+Y derivative when segregation in females bearing either aY or small free compared to a normal Y (LINDSLEYand GRELL1968). duplication. In order to observe allpossible segregationevents D om inant Meiotic A Dominant Mutation 813 TABLE 8 Results of crossing YsX.YL, Zn(l)EN, y BIO; C(4)RM,ci ef/O males to y/yly+Y; pollpol" or sc'sc8B+ldl-49/y'~ pollpol females

Maternal genotype Gamete types

YlYlY yAxs/yAxs/YyIyA.4Y sc'scn/dl-49/Y sc4scnAxs/dl-49/Y sc4sc8Axs~d1-49AxsjY

MotherFather Oova Yova Oova Yova Oova Yova Yova0 ova Yova0 ova Yova0 ova Regular x4 408 504 k3@1313 169 461 820 273 670144 222 573 521 588 425 1486 1295 394 396 394 1295x4 1486 0425 2 588 600 670 620 X Nondisjunctional 04 k3G 3 30 11 71 11 37825 5 13 355 67 267 xx4 0 4a 57 1 158 11 89 42 5066 806 5 0 641 4 Nondisjunctional x44 GO 3 1 13 2 3 16 3 1 14 3 57 11 3 7 7 9 7 x0 007 0 3 12 (26)(2)' (7) x444 8 (3) 6 2 (2) (2) 12 6 x0 x.Po 44 0 2 0 7 6 3 1 3 12 6 12 33 X,4 Nondisjunctional xx44 00 2 0 4 0 0 1 0 3 1 3 14 4 00 fin3 3 3 1 3 0 0 0 1 0 3 8 xx 0 2 3 1 2 1 228 0 4 0 7 17 0 44 #?5 0 8 1 5 6 0 8 0 3 37 26 1759 4927 1505 2600 2653 3219 2653 2600 1505Total progeny: 4927 1759 5997 1853 2374 2371 2669 Adjusted 2371 total: 16 2374 21 1853 5997 % Nondisjunction X 9.55 9.3 1 10.36 50.25d 44.1 2d 39.94d 4 4.911.65 1.77 0.51 2.83 9.37 % Nonhomologous disjunction xxoY 7.17 7.85 49.75 4.32 41.25 23.68 xx o 44 NA 0.080.27 0.43 0.76 2.96 Yo44 0.28 0.52 1.24 0 0.13 3.07 XXY o 44 NA 0.13 0.32 NA 0.34 1.76

Chromosomes denoted as y are in fact y cvfand those denoted as y Axs are y zu" v Axs car. All sc4sc8 chromosomes used are B+ derivatives. Due to inviability of XXW females, the number of regular y+Y-bearing males in this class replaces the number of XXW females in all calculations. ' The X/yfY,4 nondisjunctional males (in parentheses), are not used to calculate frequencies of nondisjunction due to theinviability of the corresponding classes of X/O 4 nondisjunctional males (see d below). Due to lethality of X/o males in these crosses (see MATERIALS AND METHODS and Table 7 footnote a), % X nondisjunction is calculated as follows: (total X exceptional progeny/total progeny)lOO. NA, not applicable due to theformation of a negative number.

(including XXY t.) 0),it was necessary to use a marked In sc4sca/dl-49/y+Y; pol/pol females there is a sig- y+ Y chromosome in these experiments. nificant decrease in the frequency of X nondisjunction Iny/y/y+Y;pol/pol females, there is little or no effect in Axs females when compared to Axs+ females (from of the Axs mutation on total frequencies of X chro- 50% to 40%). Based on a contingency test, this differ- mosome nondisjunction (approximately 10% in con- ence is highly significant (x2 = 81.6, d.f. = 1, P < trol, heterozygous and homozygous Axs females). The 0.001). In fact, in Axs homozygotes, the presence of frequency ofsecondary nondisjunction (XX * Y), the Y chromosome does not increase the frequency of however, decreases from 82% of the total X nondis- X nondisjunction above that observed in sc4scsAxs/dl- junction in Axs+/Axs+ females to 41% of the total X 49 Axs; pol/pol non-Y-bearing females (the total fre- nondisjunction in Axs/Axs females. Moreover, in Axs quency ofX nondisjunction in these Axs homozygotes, homozygotes, the frequency with which both X chro- 39.9%, is virtually the same as that observed in mosomes and the Y segregate to the same pole (XXY - 0) is increased three-fold over control levels. (In- sc4scaAxs/dl-49 Axs; pol/pol females, 38.5%, Table 1). cluding XXY * 44 segregational events, the frequen- In Axs homozygotes and to a lesser extent in AXS cies for XXY * 0 segregations are as follows: 0.85% heterozygotes, the Y chromosome segregates from two for control females; 1.07% for heterozygous Axs fe- nondisjoining X chromosomes with reduced effi- males; and; 3.07% for homozygous Axs females.) ciency. XX * Y segregation in sc4scaAxs/dl-49Axs These results suggest that Axs/Axs/y+Y females cannot females accounts for 59% of the total X chromosome facilitate XX * Y segregational events as readily as nondisjunction, whereas in sc4scaAxs+/dl-49 Axs+con- Axs+/Axs+/y+ Y control females. trol females, 99.0% of the X chromosome nondisjunc- 814 A.and E. Zitron R. S. Hawley

tion can be accounted for by XX t, Y segregations. tetrads is much lower in the absence of structural The frequency of both the X chromosomes and the Y heterozygosity.) Thus, atleast some component of the segregating to the same pole is increased 29-fold over preferential XX t, Dp segregation is likely to reflect controls in Axs homozygotes (6.74%) with a five-fold factors other than size or shape such as, homology increase over controls in Axs heterozygotes (1.28%). (see DISCUSSION). Similarly, FM7/Axs/y+Y females also exhibit a similar There is little or no effect of Axs on either X or reduction in secondarynondisjunction when com- fourth chromosomenondisjunction (twofold over paredto FM7/+/y+Y control females (35.8% com- control levelsin each case)in y/y; Dp(l;f)l346, y+ pared to 57.5%)(see Table 10). These data,like those females. However, the frequencies of both X and obtained for females with structurally normal X chro- fourth chromosomenondisjunction increase in mosomes, suggest that the ability of a Y chromosome sc4sc8Axs/dl-49 Axs; Dp(1;f )1346, y+ mutant females to direct the segregation of both nonexchange X chro- when compared to sc4sc8/dl-49; Dp(1;f)1346, y+ con- mosomes is strongly reduced in Axs bearing females. trol females. As is observed for sc4sc8Axs/dl-49 Axsly+ The effect of Axs on fourth chromosomes in XXY Y females (Table 8), there is no increase in total X females is more complex. In females with structurally nondisjunction over that observed in sc4scaAxs/dl-49 normal X chromosomes, fourth chromosome nondis- Axs females (Table 1). Rather, these duplication-bear- junction rises only threefold in Axs homozygotes. ing Axs females redistribute X nondisjunctional events

There is little or no effect in Axs heterozygotes. In among XX t, Dp, XXDP t, 44, and XX t, 44 disjunc- contrast, sc4scaAxs/dl-49 Axs/y+Y; pol/pol females ex- tions. This result again indicates that the presence of hibit an 18-fold increase in fourth chromosome non- an additional element in the distributive system does disjunction. This increase is at least partially the result not significantly effect the frequency of X misbehav-

of dramatic increases in the frequency of XX t, 44 ior, just the types of events that contribute to the and XXY t, 44 nonhomologous disjunctional events nondisjunctions themselves. when compared to controls. Indeed, in Axs homozy- The effect of the Axs mutation on the ability of

gotes, XXY t, 44 disjunctional events accountfor 25% Dp(1;f )1346to induce X chromosome nondisjunction

of the XXY or 00 ova (of the 2 17 cases of XXY t, 0 can be assessed by comparing the proportion of X

segregations observed, 156 are theresult of XXY4 t, nondisjunctional eventsdue toXX t, Dp segregations.

4 segregations, 54 are the result of XXY t, 44 segre- Only 33% of the X nondisjunction can be accounted

gations, and only 7 cases are the result ofall five for by XX t, Dp segregational events in sc4scsAxs/dl- chromosomes proceeding to the same pole). These 49 Axs homozygotes as opposed to 73% in controls.

data suggest that, in the presence of Axs, XX t, 44 Thus, in a manner similar to that observed for the Y nonhomologous disjunctions may prevent or interfere chromosome, Axs impairs the ability of the, duplication

with proper XX c-) Y segregational events in XXY44 to facilitate XX t, Dp disjunctions. females. A comparison of the disjunctional effects of the Y Finally, the frequency of Yt,44 disjunctional events and Dp(1; f)1346,y+ chromosomes allows two further is greatly enhanced in sc4scaAxs/dl-49 Axs/y+Yfemales inferences to be made. when compared with sc4scs/dl-49/y+Ycontrol females. First, in sc4sc8Axs/dl-49 Axs females the frequency A much smaller enhancement is observed in females of XXt, Y segregations (23.7%)is only twofold greater

bearing structurally normal X chromosomes. This cu- than that of XX t, Dp segregations (10.8%). These rious dependence on the frequencyof X chromosome data are in stark contrast to theobservations in sc4sc8/ Eo tetrads is shown below to be a general property of dl-49 control females, in which the Y chromosome and Axs-induced mutant behavior. These datasuggest that the duplication differ by almost 15-fold in their capac- the Axs mutation also increases the ability of a Y ity to direct the segregation of nonexchange X chro- chromosome to direct fourth chromosomedisjunction mosomes. Thus, in Axs females, the segregational

(Y t, 44). behaviors of a large metacentric Y and a small acro- Disjunction in XX; D#(I;f)1346 females: X and centric X duplication are more similar in terms of fourth chromosome disjunction was also examined in their effects on X segregation than in Axs+ control the presence of Dp(l;f)l346, y+, a free X chromosome females. duplication which is approximately two times the size Second, in sc4scaAxs/dl-49Axs females thatcarry of the fourth chromosome (Table 9). either the duplication or the Y chromosome, the pro- In sc4scs/dl-49; Dp(l; f)1346,y+ control females, XX portion of the X nondisjunctional events due to sec-

t, Dp segregations are observed twice as frequently ondary nondisjunction (either XX t, Dp or XX t, Y) as 44 t, Dp segregations despite the small size of this is reduced significantly. These data suggest that the duplication.(Although females bearingstructurally Axs mutation interferes with those associations involv-

normal X chromosomes exhibit frequencies of XX t, ing the X chromosomeand either the Y or Dp(1; Dp segregations lower than 44 f-* Dp segregations, it f)1346 chromosomes that normally result in second- must bekept in mind that the frequency of X Eo ary nondisjunctional events. A Dominant Meiotic Mutation 815 The effect of Dp(1; f)1346 on fourth chromosome this duplication is presumed to include the Axs+ region segregation in Axs-bearing females is more complex. which has been cytologically mapped to 15D (Figure There is a fivefold increase in the total frequency of 1). The effect of this duplication on both X and fourth fourth chromosome nondisjunction in sc4scaAxs/dl-49 chromosome segregation has been monitored in fe- Axs over control levels, but most of this increase can males bearing this duplication chromosome and the be accounted for by XX t) 44, XXDP c-* 44, or XX t, Axs mutation. It should be noted that males bearing Dp44 segregations. Approximately 80% of all fourth this duplication chromosome have extremely low via- chromosome nondisjunction in control females is due bility (Table l l, a and b) and thus, only female prog- to nonhomologous Dp t) 44 segregation and this eny will be presented. proportion remains relatively unchanged inAxs fe- The data in Table 12 indicate that, in Dp(l;4)r+f +- males, regardless of the frequency of X chromosome bearing AX~females, the effects of the AX~mutation Eo tetrads. These experiments again indicate that the are substantially rescued by the duplication. In the effect of Axs on the fourth chromosomes results from absence of the duplication, the number of diplo-X, an increase in the frequency of nonhomologous dis- haplo-4 ovaproduced by Axs females is 58-fold greater junctional events involving the fours and other chro- than that observed for Axs+ controls. In duplication- mosomes present in the distributive system. bearing females, however, the effects of the Axs mu- Although there is no change in the overall propor- tation are less than two-fold over control levels for tion of fourth chromosome nondisjunction due to FM7females and less than three-fold over control nonhomologous Dp t, 44 segregational events, there levels for Mu-5 females.The effects on the production is an increase in the absolute frequency of Dp t) 44 of nullo4 ova parallelthose for haplo 4 ova production disjunctionswhen comparing y Axsly AXS; Dp to described above. These data suggest that Axs is a hypo- sc4sc8Axs/dl-49Axs; Dp females. This observation par- or nullomorphic mutation which can be substantially allels that observed forthe Y t, 44 segregational rescued by a duplication of the wild-type locus. events described above. Thus, as was observed for It is evident that theDp(l;4)r+f + chromosome alone fourth chromosome disjunction (Table l), the total exhibits dramatic effects on the segregation of chro- frequency of nonhomologous Dp t, 44 or Y t) 44 mosomes in control females, the strongest of which is disjunctions responds to an increase in X misbehavior on theX chromosome. The Dp(l;4)r+f + chromosome dueto the presence of high frequencies of X Eo is a fourth chromosome that contains two numbered tetrads. units of X euchromatin and these results may indicate Axs is not a &acting site on the X chromosome: that homologous interactions between this X DNA Since Axs maps to the X chromosome and is associated and themultiply inverted X chromosomes in the meio- with a small aberration, it might be suggestedthat the cyte may disrupt homolog segregation in the distrib- mutation defines a cis-acting chromosomal siteon the utive system. In addition, when comparing the segre- X required for distributive disjunctions. To test this gational behavior of the X chromosomes in females possibility, we examined disjunction in Axs/FM7/y+Y bearing either Mu-5 or FM7, the FM7 chromosome females (Table 10). If the Axs mutation did in fact seems to be affected to a greater degree. This could delete a cis-acting siteand thus, render theAxs-bearing be due either to the higher frequency of Eo tetrads chromosome incapable of nonhomologously associat- generated by the FM7 chromosome and/or to some ing with the Y chromosome the following prediction structural differences between the two chromosomes. Regardless of the basallevels of nondisjunction could have been made: FM7 c) Y segregations, with is clear that the dupli- the Axs chromosome going at random, would be much observed in control females, it cation substantially rescuesthe chromosomes from the more frequent than Axs t* Y segregations with the FM7 chromosome going at random (Axs;Y ova would Axs mutation when compared to these controls. Thus, the analysis of these data, coupled with the semidom- be recovered at much higher frequencies than FM7; inant behavior of the Axs phenotypes described pre- Y ova). As shown in Table 10,in FM7-bearing females, viously, suggeststhat Axs is a loss-of-function mutation progeny were derived from both classes of ova and at a dosage sensitive locus.Unfortunately, the regions were recovered at approximately equal frequencies in both proximal abd distal to the Axs mutation carry both Axs+ and Axs heterozygous females. We conclude haplo-insufficientMinute mutations and thus, there from these data that Axs does not define a cis-acting are no available deficiencies for the region. Efforts X chromosome site necessary for proper chromosome are now underway in the laboratory to generatesmall disjunction. deficiencies containing the Axs region. The nature of the Axs mutation: The ability of a duplication to rescue the Axs mutation has been tested DISCUSSION in the presence of Dp(l;4)r+F which carries the nor- mal X euchromatic DNA from rudimentary to forked The preceding analysis ofthe Axs mutation leads to (bands 14A1- 16A1 on the polytene map) appended six basic conclusions, namely: (1) Axs induces nondis- to the right arm of the fourth chromosome. Thus, junction at meiosis I in Drosophila females, and its 816 A. E. Zitron and R. S. Hawley

TABLE 9 Results of crossing YsX.Y', Zn(I)EN,y B/Q C(4)RM, el ey"/O males to y/y:pol/pol: Dp(I:f)1346,y+" or sc's~~B+/dl-49;PoIIpol; Dp(I.f)1346, y+females

G amete types Maternal types Gamete genotype YAXSIYAXS~sc~sca/dl-491Dp sc'scaA~/dl-49/ sc4Sc8A~~/dl- YlYlDP ylyAxslDp DP DP DP 49AxslDp

~~~~~ 0 Dp 0 Dp 0 Dp 0 Dp 0 Dp 0 Dp Mother Father ova ova ova ova ova ova ova ova ova ova ova ova ova ova ova ova ova ova ova ova ova ova ova Father Mother Regular x4 1511 1399 1476 1515 751 548 1997 1764 18651795 12121096 x4 1984 1705 1670 1491 700 435 X Nondisjunctional 04 a4'4 2 8 10 5 36 5 35 72 166 107 251 xx4 02 4 17 23 3 13 3 123 4 349 57 521 188 4 Nondisjunctional x44 X94 55 1 94 2 6 47 2 49 67 132 170 x0 04 3 37 3 43 1 17 x44 63 0 79 5 37 0 x0 #X 42 4 50 3 36 6 1 29 4 82 20 136 X,4 Nondisiunctional xx44 1 2 2 3 116 4 2 4 5 26 00 84% 3 2 0 1 10 4 3 0 3 6 11 xxo 1 1 0 2 02 0 1 2 13 22 55 0 44 K2 0 0 0 1 012 6 13 2 66 22 Total progeny 6845 6480 261 1 4032 4501 3927 Adjusted total 6886 6530 2642 4032 450 1 3927 % Nondisjunction R 1.19 1.53 2.35 4.71' 15.24' 32.60b 4 3.21 4.67 5.98 2.58 4.38 14.06 % Nonhomologous disjunction XX c, Dp 0.55 0.70 0.83 3.42 8.13 10.85 xx c* 44 NA NA 0 NA 0.40 2.98 Dp c, 44 2.69 3.84 4.84 1.84 3.71 9.58 XXDp c, 44 NA NA 0 NA 0.47 2.80

a In Axs heterozygotes, y Axs denotes y cv v Axs cur and y Axsly Axs homozygotes are y w" v Axs curly cv v Axs cur. * Due to the lethality of XI0 males (see MATERIALS AND METHODS and Table 7 footnote a) and the lowered viability of X/O; Dp(I:f)1346 (which are not reported here), the % X nondisjunction is calculated as follows: (total X exceptional progeny/total progeny)100. NA, not applicable due tothe formation of a negative number.

TABLE 10 Results of crossing X/Ymales to FM7/X/Yfemales

A) y N u f carlY X FM7Iy w" cP B) y uf cP f carlY x FM7/y cv u f car/y+Y" Axs carly+Y' Female Male Female Male progeny progeny progeny progenySegregation inprogeny mother progeny progeny

479 470 224 178 XIY 224c, Y 470 479 X/X/Y c, Y 2 4 13b 30 x c, X/Y FM 7c* XIY 480 329 327 378 X c, FM 7/Y 237 324 292 324 Total progeny 2339 1768 Adjusted2247 total 3290 % X41.1 nondisjunction 57.9

% Nonhomologous XlX c, Y disiunction 57.5 35.8

The FM7 chromosome carries the fld, w", v, and B markers and thus, segregation of all chromosomes can be followed in these crosses. ' Due to lowered viability of XXW females, the number of males in this class replaces this number in all calculations. A Dominant Meiotic Mutation 817 TABLE 11 nondisjunction (see below). Similar results have been Results of crossea involving Dfi (1;4)r*f obtained for the second chromosome, when compar- ing the effect of Axs in females bearing two structur- Gamete types ally normal second chromosomes to those heterozy- Mother Father No. gous forthe autosomal balancer SMl. These data a) Results of crossing YsX.YL,Zn(l)EN, v f B/O; C(4)RM, ci qR/0males to argue strongly that the province of the Axs defect is C(l)Dx,y f/L?'Y; Dfi(1;4) r+f/pol females limited to the distributive system. Y4 fi C(4) 59 Y Dp(1;4) PY C(4) 7 The relationship between the frequency of nonex- No. of mothers 20 change X chromosomal bivalents andX chromosome b) Results of crossing y w" ct6 m f/y+Y; pol/pol males to C(I)Dx,y f/ nondisjunction: As shown inFigure 2, X chromosome EY; Dp( 1.4) r+f+/polfemales nondisjunction increases in an apparently linear fash- Y4 98 ion with the frequency of X chromosome EO tetrads fi&1;4) Y4 152 in both hetero- andhomozygous Axs females. Indeed, Y 4 x4 Y4 49 in the presence of two doses of Axs, X chromosome ~ p(1;4) x4 Y ~p(1;4) 8 No. of mothers 40 disjunction withinthe distributive system isapparently random. There are more than twenty mutations that result effect is limited to chromosomes in the distributive in increased frequencies of Eo tetrads for all chromo- segregation system; (2) there is a simple and appar- somes in the genome by decreasing the frequency of ently linear relationship between the frequency of X exchange (BAKERand HALL1976; see also HAWLEY nondisjunction and the frequency of X chromosome 1988). Unlike the direct relationship between X non- nonexchange tetrads, such that in Axs homozygotes X disjunction and X Eo tetrads observed in Axs females, nondisjunction is apparently equal to one-half the in these exchange deficient mutations X nondisjunc- frequency of Eo tetrads; (3) nondisjunction of the tion rises in a nonlinear fashion (X nondisjunction is fourth chromosomes results primarily from nonho- however, linear with respect to Eo cubed, BAKERand mologous XX t, 44 disjunctions and occurs at high HALL1976). frequency onlyin the presence of nonexchange X BAKERand HALL(1976) have argued that the non- chromosomes; nonhomologous disjunctions in- (4) linear relationship between X nondisjunction and X volving the X and other chromosomes account for a Eo tetrads observed in exchange-defective mutations large fraction of the total X chromosome nondisjunc- may reflect a requirement for theX chromosome and tion observed; (5) Axs exerts a qualitatively similar both arms of a major autosome (A) to nonexchange effect onthe disjunction of a nonexchange major be in order forX nondisjunction to occur. The resulting autosome (second chromosome); (6) Axs is not a cis- acting site on the X chromosome, but rather is appar- X nondisjunctional events would presumably be the ently a loss-of-function mutation at a dosage sensitive product of XX t, AA nonhomologous segregations locus. This characterization of the Axs phenotype is (for reviews,see BAKERand HALL 1976; HAWLEY based solely on the analysis of the one existing allele. 1988). However, as shown in Table 5, Axs has little Before we can conclude that these phenotypes are a or no effect on simultaneous X and second chromo- general consequence of the loss of Axs+ function, it some nondisjunction except in Axs/Axs; SMI/+ fe- will be necessary to characterize a number of alleles. males where it acts to reduce the frequency of these The data obtained so far, however, do allow us to simultaneous events. Thus, in femaleshomozygous present below a simple model in which the Axs+ gene for Axs, X nondisjunction does not appear to require product is required for homolog recognition within the presence of nonexchange autosomes, but rather, the distributive system. Wepropose that theAxs defect the absence of an X chromosome exchange event is may be understood by the failure ofhomologous itself sufficientto randomize X disjunction. chromosomes to properly identify their partners fol- In addition, the relationship of X chromosome non- lowed by the concomitant occurrence of nonhomolo- disjunction to the frequency of Eo tetrads in the pres- gous segregations. ence of Axs is unchanged by the addition of a Y Axs specifically affects the distributive segrega- chromosome, a free X duplication, or a compound tion system: The data in Tables 3 and 4 show that fourth chromosome to the distributive system. The Axs has little or no affect on exchange events along ability of Axs to induce X nondisjunction is therefore the length of the X chromosome. Moreover, the dis- unaffected by the number, structure, or identity of junction of X chromosomes which have undergone other chromosomes undergoing distributive segrega- exchange seems to be unaffected by the presence of tions. This observation, in addition to the apparently either oneor two doses ofthe Axs mutation. Axs does, random disjunction of distributively segregating X however, affect the disjunction of nonexchange X chromosomes inAxs homozygotes, might suggest that chromosomes, such that, as the frequency of X chro- the nonexchange X chromosomes simply segregate at mosome Eo tetrads rises, so does the frequency of X random in the absence of Am+. Yet, paradoxically, 818 A. E. Zitron and R. S. Hawley

TABLE 12 Results of crossing YsX.Y', Zn(l)EN,v f B/O; C(4)RM,ei eyR/Omales to X/& pol/pol or X/& Dp(l;4)r'r/pol females

G amete types Maternal genotype Maternal types Gamete FM7/y/ FM7/yAxs/FM7/y/ Mother Father FM7/yAxsFM7/yDp(1;4)Dp(1;4) Mu-5/w"MuJ/w" Axs Mu-5/wa/DP(1;4)Mu-5/w"Axs/Dp(l;4) Regular" x4 fi.0888 8115 1566 1442 1275 2452 58 1 704 X Nondisjunctional xx 4 042 10 71 50 83 0 101 2 7 4 Nondisjunction I x0 B 9/46 12 20 4 6 0 27 0 6 X,4 Nondisjunctional xx 0 0'0 0 26 3 12 1 9 0 0 Total progeny 8137 1005 1623 1543 1276 2589 583 717 % Progeny from XX;4 ova 0.12 7.07 3.08 5.38 0 3.90 0.34 0.98 X, 0 ova 0.15 1.99 0.25 0.39 0 1.04 0 0.84 XX;0 ova 0 2.59 0.19 0.78 0.08 0.35 0 0 Data are taken from Table 1 and treated in a manner similar to those obtained for duplication-bearing females. Chromosomes denoted as yAxs are in fact y N u Axs car, and those denoted as w"Axs are y w" u Axs cur. They chromosome used in the FM7 control is y N vfcar and chromosomes referred to as w' also carry y, ct6J and cur. a Due to low viability of male progeny from duplication bearing females (see Table 1 l), only female progeny are reported here. ' Because the Dp(l;4)r+f*chromosome is heterozygous with a normal pol fourth chromosome, the only fourth chromosome nondisjunctional products able to be scored are from nullo-4-bearing ova.

nonhomologous disjunctional events involving the X nondisjunction hasalso been demonstrated fora num- chromosome, specifically XX c* 44 and XX * C(4)RM, ber of exchange defective meiotic mutants (BAKER are frequently observed in Axs females. and HALL 1976). As more chromosomes enter the The effectof Axs on fourth chromosome disjunc- distributive system in females bearing one of these tion: In females with structurally normal X chromo- mutations, fourthchromosome nondisjunction in- somes, Axs exhibits a weak effect on the disjunction creases. Although this is similar to the trend seen in of thenonexchange fourth chromosomes, while Axs-bearing females, the high frequency of nonho- fourth chromosome nondisjunction dramatically in- mologous X and fourth chromosomesegregation ob- creases in Axs females heterozygous for X chromo- served in Axs-bearing females is in stark contrast to some balancers. Thus,fourth chromosome nondis- observationsmade for exchange-defective meiotic junctional events inducedby the Axs mutation require mutants. In these mutants, simultaneous nondisjunc- the presence of nonexchange X chromosomes. Two tion of the X and fourth chromosomes is apparently lines of evidencesuggest that theAxs-induced increase random, such that approximately equal frequencies in fourthchromosome nondisjunctionresults from of XXOO, 00;44,XX,44 and 0O;OO ova are pro- the failure of nonexchange X and fourth chromo- duced (for review see BAKERand HALL1976). somes to properlyrecognize their homologs, followed Although the mechanism by which the X chromo- by the formation of improper X-4 associations. some can interfere with proper fourth chromosome First, approximately one-half of the fourth chro- disjunction remains unclear, BAKERand HALL(1976) mosome nondisjunction observed in Axs females car- have speculated that "nonexchange X chromosomes rying an X chromosome balancer occurs concurrently could associate with fourth chromosomes, and thus with X nondisjunction, and these simultaneous X,4 disrupt fourth chromosomedisjunction, but thatthese nondisjunctional events are almost always the conse- associations were not sufficiently stable to allow non- quence of nonhomologous XX c-* 44 segregations. homologous segregations of the X and fourth chro- Second, as shown in Figure 4, fourth chromosome mosomes.'' Both cytological andgenetic evidence nondisjunction is linear with respect to X nondisjunc- suggest that the X and fourth chromosomes are evo- tion in both Axs hetero-and homozygotes. These lutionarily related (for review see HOCHMAN1976). observations suggest that the primaryeffect of Axs on Moreover, MIKLOSet al. (1988), have recently shown fourth chromosome disjunction may be aresult of that the fourth chromosome and the proximaleu- improper associations with anonexchange X chro- chromatin of the X are highly homologous with re-

mosome. This would lead to frequentX t, 44 disjunc- spect to the number,composition, and distribution of tions, leaving the otherX to segregate at random. certain classes ofrepetitive sequences. Thus, it is A relationship between increased X chromosome possible that homologous sequences present on both nondisjunction and increased fourthchromosome the X and fourthchromosomes provide opportunities D ominant Meiotic A Dominant Mutation 819 for frequent associations that can lead to improper suppresses exchange, small X chromosomal free du- segregational events. plications were capable of inducing high levels of X Finally, we note that the frequent X-4 associations nondisjunction. Indeed, as shown in Table 9, in Ax$+ and improper disjunctions observed in females bear- females heterozygous for two X inversions, a free X ing structurally aberrant X chromosomes are much duplication approximately two times the size of the less common in either Am+ or Axs females bearing fourth chromosome induces greater than twiceas structurally normal X chromosomes @e., a much many XX t, Dp disjunctions as it does 44 t, Dp smaller fraction of fourth chromosome nondisjunc- disjunctions. This is so despite a greater similarity in tion is associated withX nondisjunction). This suggests sizeof the duplication to the fourth chromosome. the possibility that naturally occurring nonexchange Thus, in competitive situations, homology may well X chromosomes are in some sense different from, or play somerole in partner choice withinthe distributive unique, compared to those nonexchange X chromo- system. somes produced by inversion-bearing females. We propose that one function of the Axs+ locus is Nonhomologousdisjunctions involving the X to mediate homolog recognition within the distribu- chromosome in Axs-bearing females: The random tive systemby promoting tight pairings between hom- disjunction of nonexchange X chromosomes in Axs/ ologs, thus preventing pairings (such as X-4) which Axs females is not due to a failure of the X chromo- are based on more limited homologies. In this sense, somes to participate in distributive disjunctions per se, the Axs mutation might be likened to the Ph mutation because nonhomologous disjunctions involving the X on chromosome 5B in wheat which allows homeolo- and, in particular, the fourth chromosomes, are ob- gous pairing at the expense of proper meiotic pairing served at high frequencies in Axs females. Indeed, in of homologs (for review see SEARS1976). AXShomozygous females bearing a compound fourth According to this model, the Axs mutation impairs chromosome, XX t* C(4)RM nonhomologous disjunc- the mechanism by which homologous chromosomes tions account for more than 80% of the X nondisjunc- recognize each other. In this way, Axs allows for the tion observed. formation of less homologous pairings and/or com- The relationship of Axs to nonhomologous distrib- plex multivalents in which improper nonhomologous utive disjunctions involving the X chromosome may associations occur as a secondary effect. Our model be summarized asfollows; the frequency of “im- also requires that not all secondary associations are proper” segregations (such as XX t* 44 and XX t* equallylikely to occur or to cause nondisjunction. C(4)RM), are dramatically increased in females with This assumption is necessary to explain the decreased high X EO tetrads, while the frequency of “proper” ability ofthe Y or Dp(l;f)1346 chromosomes to impair distributive disjunctions (such asXX t* Y)are strongly 4 t, 4 segregation in Axs females bearing structurally decreased. The effect of Axs on secondary nondis- normal X chromosomes where X Eo tetrads are low. junction in fact parallels the Axs defect in X t* X For example, we propose that in sc4scsAxs/dl-49Axs; distributive disjunctions, in that failed secondary non- C(4)RM females, the nonexchange X chromosomes disjunction (such as, XXY c, 0)frequently occurs as are incapable of pairing with each other and conse- the result of XX t* 44 segregational events (ie., XXY quently one X pairs with the C(4)RM chromosome t-, 44). Perhaps the effect of Axs on both proper and while the other segregates at random. Thus, these X- improper distributive segregations is a consequence C(4)RM nonhomologous disjunctions would result in of an Axs-induced defect preventing homolog recog- X nondisjunction in approximately one-half of the X nition which allows for a more promiscuous choice of Eo tetrads. We would further propose that, as more partners for theX and fourth chromosomes. elements are added to the distributive system, non- A simple modelof Axs’ function: The observations homologousassociations involving the X chromo- described above are most easily understood in light of somes and other heterologs continue to occur in the a model in which the Axs+ gene product is required form of more complicated multivalents. for homolog recognition within the distributive sys- This model explains the following observations:(1) tem. Such an assertion contradicts the widely held allof the sc4scaAxs/dl-49Axs females tested exhibit belief that distributive disjunctions are entirely inde- frequencies of X chromosome nondisjunction approx- pendent of homology (for reviews see GRELL1976; imately equal to one-half the EOtetrads, regardless of HAWLEY1988). the presence or absence of other potential disjunc- Severalstudies have demonstrated however, that tional partners; (2) XX t* Y distributive disjunctions the parameters of size and shape alone may be insuf- are disrupted by the Axs mutation in a manner similar ficient to explain partner choice and have suggested to X t, X distributive disjunctions and thus, the fre- a possible role of homology in distributive disjunc- quency of secondary nondisjunction in XXY females tions. For example, GERSHENSON(1940) and Lm- homozygous for the Axs mutation is reduced; (3) as is DSLEY and SANDLER(1958) observed that in females strikingly apparent in the C(4)RM experiment (where heterozygous for an X chromosome inversion which only three elements are present in the distributive 820 A. E. Zitron and R. S. Hawley system), X chromosomes in Axs females are free to C. NEW for valuable technical assistance and B. BRODEUR,M. disjoin from a nonhomolog with high efficiency; and FEANY,W. WHYTE,and P. ZHANC for valuable discussion of this fourth chromosomenondisjunction rises in re- work and manuscript. In addition, we thank A. CARPENTERand P. (4) SZAUTERfor critically reading the manuscript. This manuscript is sponse to increases in X EOtetrad frequency regardless dedicated to the memory of LARRY SANDLER,whose energy and of the presence of other elements in the system. guidance was felt throughout thecourse of this work. It should be noted that in X/X/y+Y or X/X/Dp(l; f)1346 females bearing two structurallynormal X LITERATURECITED chromosomes, Y t) 44 or Dp c-) 44 disjunctions are not increased in Axs heterozygotes and areonly mod- BAKER,B. S., and A. T. C. CARPENTER,1972 Genetic analysis of sex chromosomal meiotic mutants in Drosophila melanogaster. erately increased in Axs homozygotes. Dramatic in- Genetics 71: 255-286. creases, however, in these disjunctional events, as well BAKER,B. S., and J. C. HALL,1976 Meiotic mutants: genic control as XXY c* 44 or XXDP t) 44 disjunctions, are observed of meiotic recombination and chromosome segregation, pp. in the presence of heterozygosity for structural aber- 351-434 in Genetics and Biology of Drosophila la, edited by E. NOVITSKIand M. ASHBURNER.Academic Press, New York. rations that suppress X chromosome exchange. To BRIDGES,C. B., 191 6 Nondisjunction as proof of the chromosome explain these results, we propose that all four types of theory of heredity. Genetics 1: 1-52, 107-163. nonhomologous segregation result from the forma- CARPENTER,A. T. C., 1973 Amutant defective in distributive tion of complex multivalents involving both nonex- disjunction in Drosophila melanogaster. Genetics 73: 393-428. COOPER,K. W., 1945 Normal segregation without chiasmata in change X chromosomes, both fourth chromosomes, female Drosophila melanogaster. Genetics 30 472-484. and either theY or the duplication. We further argue COOPER,K. W., 1948 A new theory of secondary nondisjunction that such multivalents result from the same relaxation in female Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 34: 179-187. of homology dependent pairing postulated to account COOPER, K. W., S. ZIMMERINC and J. KRIVSHENKO, 1955 for XX t-) 44 disjunction in X/X females. Interchromosomal effects and segregation. Proc. Natl. Acad. Whether or not our proposed mechanism for the Sci. USA 41: 91 1-914. precise action of the Axs mutation is correct, it is clear DAVIS,D. G., 1969 Chromosome behavior under the influence of claret-nondisjunctional in Drosophila melanogaster. Genetics 61: that Axs defines a locus required for the proper dis- 577-594. junction of homologs within the distributive system. FORBES,C., 1960 Nonrandom assortment in primary nondisjunc- In light of previously described schemes of meiosis, tion in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 46: we believe that the Axs+ locus controls a crucial step 222-225. GERSHENSON,S., 1940 Thenature of the so-called genetically in the differentiation of homologous vs. nonhomolo- inert parts of chromosomes. Vidensk. Akad. Nank. URRS 3: gous elements prior to disjunction. In addition, the 3-1 16. effect on X chromosome disjunction revealed by the GRELL,E. H.,1963 Distributive pairing of compoundchromo- Axs mutation, taken together with the high degree of somes infemales of Drosophila melanogaster. Genetics 48: 1217- 1229. fourth chromosomeinterference that the mutation GRELL,R. F., 1959a Nonrandom assortment of nonhomologous promotes, indicates atight coupling of the X and chromosomes in Drosophila melanogaster. Genetics 44: 42 1- fourth chromosomes within the distributive system 435. that was also indicated by the ald and mei-S51 muta- GRELL,R. F., 1959b Effect of chromosome four on segregation of sex chromosomes in Drosophila melanogaster. Genetics 44: tions. 514. Perhaps the wild-type functions defined by these GRELL,R. F., 1962a A new hypothesis on the nature andsequence three mutations aid in the suppression of X,4 associa- of meiotic events in the female of Drosophila melanogaster.Proc. Natl. Acad. Sci. USA 48 165-172. tions which arise as a consequence of limited homol- GRELL,R. F., 1962b A new model for secondary nondisjunction: ogy between the chromosomes. Thus, a mutation in the role of distributive pairing. Genetics 47: 1737-1 754. one of these loci leads to high frequencies of nonho- GRELL,R. F., 1964a Chromosome size at distributive pairing in mologous disjunctions. If such a model is correct, then Drosophila melanogaster females. Genetics 50 150-1 66. GRELL,R. F., 1964b Distributive pairing: the size-dependent the analysis of the Axs mutation, along with the ald mechanism for regular segregation of the fourth chromosomes and mei-S51 mutations, has revealed the existence of in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 52: 226- a novel component of distributive pairingin Drosoph- 232. ila which allows for the recognition of limited homol- GRELL,R. F., 1967 Pairing at the chromosomal level. J. Cell. Physiol. 70 (Supp. 1): 119-145. ogies between heterologous chromosomes. GRELL,R. F., 1970 The time of initiation of segregational pairing between nonhomologues in Drosophilamelanogaster: a reex- This work was supported in part by grants to R.S.H. from the amination of wm4. Genetics 64: 337-365. National Science Foundation (DCB-8815749), the American Can- GRELL,R. F., 1976 Distributive pairing, pp. 435-486 in Genetics cer Society (JFRA-98and FRA-324), the Irma T. Hirschl Monique- and Biology of Drosophila, Val. la, edited by E. NOVITSKIand M. Weill Caulier Trust, and the Searle Scholars Foundation (84-E- ASHBURNER.Academic Press, New York. 102). A.E.Z.was supported by National Institutes of Health GRELL,R. F., and E. H. GRELL,1960 The behavior of nonho- (5T32-GM07491). The data in this paper are from a thesis submit- mologous chromosomal elements involved in nonrandom as- ted by A.E.Z.in partial fulfillment of the requirements for the sortment in Drosophila melanogaster. Proc. Natl. Acad. Sci.USA Degree of Doctor of Philosophy in the Sue Golding Graduate 46: 5 1-57. Division of Medical Sciences, Albert Einstein College of Medicine, HALL,J. C., 1972 Chromosome segregation influenced bytwo Yeshiva University. We wish to acknowledge L. MARZULLOand D. allelesof the mutation c(3)G in Drosophilamelanogaster. Ge- WRIGHTfor their contributions to this analysis. We wish to thank netics 71: 367-400. A Dominant Meiotic Mutation 82 1

HAWLEY,R. S., 1988 Exchange and chromosomal segregation in MOORE,C. M., and R. H. GRELL,1972 Factors affecting recog- eukaryotes, pp. 497-525 in Genetic Recombination, edited by R. nition and disjunction of chromosomes at distributive pairing KUCHERLAPATIand G. SMITH.American Society for Microbi- in female Drosophila melanogaster. Genetics 70: 567-58 1. ology, Washington, D.C. OKSALA,T., 1958 Chromosome pairing, crossing over and seg- HOCHMAN,B., 1976 The fourth chromosome of Drosophila mela- regation inmeiosis in Drosophilamelanogaster females. Cold nogaster, pp. 903-928 in Genetics and Biology of Drosophila, Vol. Spring Harb. Symp. Quant. Biol. 23: 197-210. Ib, edited by M. ASHBURNERand E. NOVITSKI.Academic Press, O’TOUSA,J., 1982 Meiotic chromosome behavior influenced by New York. mutation-altered disjunction in Drosophilamelanogaster fe- HOLM,D. G., 1976 Compound autosomes, pp. 529-561 in Ge- males. Genetics 102: 503-524. netics and Biology of Drosophila, Vol.Ib, edited by M. ASHBURNER PARRY,D. M., 1973 A meiotic mutant affecting recombination in and E. NOVITSKI.Academic Press, New York. female Drosophila melanogaster. Genetics 73: 465-486. LEFEVRE,G., 1976 A Photographic Representation and Interpre- RITOSSA,F. W., 1968 Unstable redundancy of genes for ribosomal tation of the Polytene Chromosomes of Drosophila melanogaster RNA. Proc. Natl. Acad. Sci. USA 60 509-576. Salivary Glands, pp. 32-66 in Genetics and Biology of Drosophila, ROBBINS,L. G., 1971 Nonexchange alignment: a meiotic process Vol. la, edited by M. ASHBURNERand E. NOVITSKI.Academic revealed by a synthetic meiotic mutant of Drosophila melano- Press, New York. gaster. Mol. Gen. Genet. 110 144-166. RUTHERFORD,S. L., and A. T. C. CARPENTER,1988 The effect LEWIS,E. B., 1960 A new standard food medium. Drosophila of sequence homozygosity on thefrequency of X-chromosomal Inform. Serv. 117-1 18. 34 exchange in Drosophilamelanogaster females. Genetics 120 LINDSLEY,D. L., and E. H. GRELL, 1968 GeneticVariations of 725-732. Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627. SANDLER, L.,and E. NOVITSKI,1956 Evidence for genetic homol- LINDSLEY,D. L., and L. SANDLER,1958 The meiotic behavior of ogy between chromosomes I and IV in Drosophila melanogaster, grossly deleted X chromosomes in Drosophila melanogaster. Ge- with a proposed explanation for thecrowding effect in triploids. netics 43: 547-563. Genetics 41: 189-193. LINDSLEY,D. L., and G. ZIMM,1987 The genome of Drosophila SEARS,E. R., 1976 Genetic control of chromosome pairing in melanogaster; Part 3, edited by P. W. HEDRICK.Drosophila wheat. Annu. Rev. Genet. 1031-53. Inform. Serv. 65 89. STURTEVANT,A. H., 1951 A map of the fourth chromosome of LUCCHESI,J. C., 1976 Inter-chromosomal effects, pp. 315-329 in Drosophila melanogaster based on crossing-over in triploid fe- Genetics and Biology of Drosophila, Vol. Ib, edited by M. ASHBUR- males. Proc. Natl. Acad. Sci. USA 37: 405-407. NER and E. NOVITSKI.Academic Press, New York. STURTEVANT,A. H., and G.W. BEADLE,1936 The relations of MAC~NTYRE,R. T., and T. R.F. WRIGHT,1966 Recombination inversions in the X chromosomes of Drosophila melanogaster to in FM4/; SMI/;Ubx’”/heterozygotes. Drosophila Inform. crossing-over and disjunction. Genetics 28: 554-604. Serv. 41: 141-142. TARTOF,K. D., 1973 Regulation of ribosomal gene multiplicity MERRIAM,J. R., and J. N. FROST,1964 Exchange and nondisjunc- in Drosophila melanogaster. Genetics 73: 57-77. tion of the X-chromosomes in female Drosophila melanogaster. WEINSTEIN,A,, 1936 The theory of multiple-strand crossing over. Genetics 49 109-1 22. Genetics 21: 155-199. MIKLOS,G. L. G., M. T. YAMAMOTO,J. DAVIES and V. PIRROTTA, ZIMMERING,S., 1976 Genetic and cytogenetic aspects of altered 1988 Microcloning reveals a high frequency of repetitive segregation phenomena in Drosophila, pp. 569-61 3 in Genetics sequences characteristic of chromosome 4 and the beta-heter- and Biology of Drosophila, Vol. lb, edited by M. ASHRURNERand ochromatin of Drosophila melanogaster. Proc. Natl. Acad. Sci. E. NOVITSKI.Academic Press, New York. USA 85 2051-2055. Communicating editor: W. M. GELBART