THE MEIOTIC EFFECTS OF A DEFICIENCY IN DROSOPHILA MELANOGASTER:

IDENTIFICATION O F TWO DOSAGE-SENSITIVE SITES1

LEONARD G. ROBBINSZ

Department of Zoology und Genetics Program, Colleges of Natural Science and Osteopath'c Medicine, Michigan State University, East Lansing, Michigan 48824

Manuscript received June 13, 1979

ABSTRACT

Heterozygosity for a deficiency for the entire zeste-white region of the X chromosome of Drosophila melanogaster females causes both reduced recombi- nation and increased nondisjunction. The location of the dosage-sensitive sites responsible for these two meiotic defects has been studied by use of a set of deficiencies that subdivide the region. Recombination is reduced when the zw7- zwl l region is present in one dose, while nondisjunction is increased only if the doses of both the zw8-zwl0 and zwb-zwll segments are reduced. Examination of truns heterozygotes of two deficiencies explicitly demonstrates the compound nature of the meiotic dose effect and further delimits the location of the prox- imal disjunctional site to the zwl2-zwll interval. In inversion/deficiency heterozygotes, reduced dose of the zw8-zwl0 region alone, without reduced dose of the proximal site, yields increased nondisjunction, suggesting that the proximal element that affects disjunction is the same as that which affects recombination. Thus, the zeste-white region contains a t least two dosage- sensitive loci that affect meiosis: reduced dosage of one locus, in the zw7-zwfl interval, causes reduced recombination; reduced dose of another, in the zw8- zw10 region, increases the probability that nonexchange chromosomes will nondisjoin. A slight effect on the regional distribution of exchange may also be a property of the zw8-zwlO region locus, but could be an effect of yet another locus or of structural heterozygosity. The implications of these results for understanding meiotic control and the prospects for further analysis of the structure of the zeste-white interval are considered.

zeste-white region of the X chromosome of Drosophila melanogaster has T::en the subject of intensive cytogenetic investigation. All of the essential loci in that region have probably been identified (JUDD, SHEN and KAUFMAN 1972; LIU and LIM 1975) and the zygotic defects of homozygous or hemizygous lethal mutants (SHANNON et al. 1972) and deficiencies (KAUFMAN P t al. 1975) have been described. Most strikingly, there is a one-to-one correspondence of essential loci and chromomeres. Recently. YOUNG and JUDD (1978) identified the locations of a few nonvital loci, as well as breakpoints in nonessential regions, within the zeste-white segment.

Both meiotic recombination and disjunction are abnormal in females hetero-

Research supported by National Science Foundation grant PCMi5-13492 A01 bfailing address Biology Research Center, Mich~gan State Unlverslty, East Lansing, Mlchlgan 48824

Genetics 94: 361-381 February, 1980

362 L. G . ROBBINS

zygous for a deficiency spaniiing the zeste-white region. It was demonstrated (ROBBINS 1977) that the meiotic anomaly is complemented by the addition of a corresponding duplication attached to chromosome 4 and is, therefore, a result of reduced gene dose. Furthermore, the properties of the meiotic defect, partic- ularly the manner in which recombination is reduced, have been shown to be consonant with the effects expected from reduced levels of an enzyme involved in exchange. It has therefore been suggested that zeste-white deficiency females are hemizygous for the structural gene for such an enzyme.

The abnormal meiotic phenotype of the deficiency heterozygotes identifies yet another function in the zeste-white region. Whether that function (or functions, as the case turns out) is determined by any of the already identified loci is a question of some interest. Should the meiotic defect be caused by hemizygosity for previously described loci, some support would be provided for the notion that all functions have been identified in the zeste-white region. Should the meiotic defects prove to be due to hemizygosity for previously undetected loci, one might suspect that the apparent saturation of the functional portions of the zeste-white region is a result of the inability of conventional mutant screens to detect unconventional functions.

As a prelude to attempts to locate the dosage sensitive site(s), a series of overlapping deficiencies has been examined to identify which part(s) of the zeste-white region are involved. The results of those experiments have not yielded the expected identification of a single site, but rather point to the pres- ence of at least two loci in the zeste-white interval with dosage-sensitive effects on meiosis. The experiments that have led to this conclusion and an examination of the interactions between the two loci are presented here.

CROSSES AND ANALYSIS

The deficiencies used in these experiments were supplied by B. JUDD and descriptions may be found in LINDSLEY and GRELL (1968), JUDD, SHEN and KAUFMAN (1972) or KAUFMAN et al. (1975). Except as noted, descriptions of other markers and chromosomes used may be found in LINDSLEY and GRELL. For the sake of readability, Dp(I;I)scV1, which is an essentially normal X chromosome carrying a duplication of the tip attached to the centromere, will be referred to in the text by its marker constitution only. Thus, Dp(I;I)scv", y pn cu m f y+ will be identified as y p n cu m f y + .

Several of the deficiencies used for mapping carried extraneous mutants that interfered with this analysis. Df (2)64j4 and Df( l )64f j were both sterile in deficiency/FM7b,lz (MERRIAM 1969) females, though not as deficiency/FM7a (MERRIAM 1968) or deficiency/FM7c, snce (MERRIAM and DUFFY 1972) females. D f ( I ) w258--45 yielded single crossover X O male offspring that were phenotypically bb, while Of (I)64c4, gave few surviving, obviously abnormal looking X O male single-crosser products, though these did not have a typical bb phenotype. In each instance, recombination between the deficiency and a multiply marked X

MEIOTIC EFFECTS OF A DEFICIENCY 363

chromosome, followed by recombination with wild type (Oregon-R), was used to remove the interfering mutations.

Mapping the recombination defect: In order to identify the site in the zeste- white region whose reduced dose is responsible for reduced recombination in females heterozygous for a deficiency for the entire region, a series of over- lapping deficiencies was tested for heterozygous effects on recombination.

Recombination was monitored using markers that define two regions spanning the entire length of the X chromosome. Deficiency/y pn v.y+ females were crossed to YsX.YL, y B/O males and recombination was scored among surviving sons. These crosses also yield information about X-chromosome disjunction, which will be considered in the next section along with data from other experi- ments bearing on nondisjunction.

The recombination data from these experiments are presented in Table 1, while these results and the arrangement of the deficiencies are summarized in Figure 1.

All of the deficiencies that expose the zw7-zwli' segment yield a reduction in X-chromosome map length and a marked reduction in the frequency of double- exchange tetrads. Tlie significant variation among these deficiencies suggests, however, that deficiency for the zw8-zwl0 segment might cause a slight addi- tional suppression of recombination.

Thus, most, though perhaps not all, of the effects of reduced dose of the zeste-

TABLE 1

X-chromosome recombination in deficiency heterozygotes

Females Non- tested crossover

Single crossover

pn-u U-y+ Double

crossover Map length

pn-u U-U+ sum Exchange rank Eo E, E2

Coefficient of

coincidence

Controls: Y 977 y rstz 3869 Deficiencies: Df( l )wrJ ' 2417 Df(l)w258-42 1594 Df(1)64j4 1552 Df(lJ62g18 754 D f ( l ) ~ $ 5 8 - 1 1 1713 Df(l)wrJ2 3674 Df(l)64c4 1557 Df( l )K95 1803 D f ( l ) 6 4 f l 1519 Df(l)wxI" 1816 D f ( i ) N r l a 1256 Df(l)65j26 1302 Df(l)w258-45 1129

480 463 1763 1851

726 745 520 414 817 768 383 424 384 400

1272 1324 459 495 811 959 859 848 537 601 353 367 685 676 457 390

134 363

14 14

155 134

3 52 9

143 176 13 10

187 21

29.9 29.1 59.0 27.1 28.2 55.3

19.0 19.5 38.5 21.0 16.8 37.8 29.5 28.0 57.5 30.5 32.9 63.4 15.5 16.1 31.6 20.9 21.8 42.7 18.6 20.0 38.6 25.7 29.7 55.4 30.4 30.1 60.5 18.5 20.7 39.2 18.3 19.0 37.3 30.6 30.3 60.9 23.9 20.6 44.5

0.08 0.66 0.26 0.08 0.74 0.19

0.25 0.74 0.01 0.27 0.71 0.02 0.04 0.77 0.19 0.04. 0.64 0.32 0.38 0.62 0.00 0.18 0.79 0.03 0.25 0.74 0.01 0.05 0.80 0.15

0.23 0.75 0.02 0.28 0.70 0.02 0.05 0.69 0.26 0.15 0.81 0.04

-0.01 0.80 0.21

0.75 0.61

0.10 0.16 0.57 0.79 0.05 0.18 0.10 0.50 0.57 0.1 I 0.14 0.71 0.21

Females heterozygous for each of the deficiency or control chromosomes and y pn v.y+ were crossed to YsX.YL, y B/O males. Exchange parameters are calculated in standard fashion.

364 L. G . ROBBINS

b Double exchange

0. I

0.0

0.1

I Df (1164c4 4

I I D f ( l ) w m - 1 1

Of ( 1 ) wx12 I

I Df(l)K95-

FIGURE 1.-Deficiency map of the recombination effect. The recombination effects of defi- ciency heterozygosity are indicated together with the arrangement of the deficiencies with respect to the lethal and visible loci in the zeste-white region. This figure is based on the data in Table 1. (a) Map length: the ratio of X-chromosome map length of each deficiency hetero- zygotr to that of the (summed) controls is indicated. (b) Double exchange: the deficiency heterozygote to control ratios for the frequency of double exchange tetrads are indicated.

MEIOTIC EFFECTS O F A DEFICIENCY 365

white region on recombination may be ascribed to the presence of a dosage sensi- tive site in the zw7-zwll segment.

Mapping the disjunction defect: Mapping of a site in the zeste-white region responsible for increased nondisjunction ir! deficiency heterozygous females was attempted in similar fashion. The frequency of nondisjunction was measured in females heterozygous for each of the series of overlapping deficiencies that subdivide the region. The crosses already described provided some data, and additional data on X-chromosome disjunction were collected by crossing defi- ciencyly rste females to YsX.YL, y B/O males. Normal disjunction of the X chromosomes in either of these series of crosses yields B / + females and B + , X O males, while nondisjunction yields B+ females and y H males. Regular male offspring from each vial yielding nondisjunctional products were checked for sterility to ensure that females being tested were XX in constitution and not X X Y . The data from both series of crosses are given in Table 2.

It had previously been shown, and was confirmed here in the deficiencyly p n v y + crosses, that the nondisjunction occurs at the first meiotic division. Given that this is so, the number of exceptional offspring in all crosses and the iiumber of surviving X O male off spring in the crosses of deficiency heterozygotes were doubled in order to calculate gametic frequencies of nondisjunction.

Although the frequencies of nondisjunction observed in the deficiencyly pn zi.y+ series of crosses are consistently higher than in the deficiency/y rstg series, the pattern is the same in both. That pattern is depicted in Figure 2a and 2b, where the locations of the deficiences and their relative frequencies of nondis- junction are indicated. Unlike the effect of deficiency heterozygosity on recom- bination, the effect on disjunction clearly does not map to a single locus. All of the deficiencies that are deficient for the zw8-zwll region, which contains eleven I ethal complementation groups, yield nondisjunction frequencies markedly above control levels, suggesting that a site responsible for elevated nondisjunction resides in this interval. However, none of the smaller overlapping deficiencies that dissect the zw8-zwll interval exhibit similarly elevated frequencies of non- disjunction. The most straightforward interpretation of this failure to find a single site in the zw8-zwll interval responsible for increased nondisjunction is that there are two loci in the z w 8 - z ~ l l interval, both of which must be deleted in order to increase the frequency of nondisjunction. An alternative hypothesis, that the effect on disjunction of the deficiencies that are deleted for all of the zw8-zwll region is due to stmctural heterozygosity rather than reduced gene dosage, has been ruled out previously ( ROBBINS 1977).

If there are two loci in the zw8-zwll region, both of which must be in one dose in order to cause nondisjunction, then these data delimit their locations. Thus, Df(l)wrJ2, which is deficient for eight of the eleven essential loci located between zw8 and zwl l , fails to increase nondisjunction in heterozygous females. This implies that at least one site having a dose-dependent effect on disjunction must lie outside the region exposed by Df(l)wrJ2; it must therefore be within the zw8-zwl0 segmeiit distal to that deficiency. Similarly, the failure of D f (1) K95 heterozygosity to increase nondisjunction eliminates the zwl3-zw3

366 L. G . ROBBINS

TABLE 2

X-chromosome disjunction in deficiency heterozygotes

Females tested

Controls: + y rst2

Deficiencies:

Df(l)wrJI

Df(I)64j4

D f ( I ) 6Zg18

D f (1)w'JZ

Df(1)64c4

Df(l)wX12

D f (I) 65 j26

Regular females

a 2533 a 9635

b 5372

a 9910

b 5386

a 2888

b 1906

a 9290

b 4021

a 16424

b 2056

a 7163

b 3260

a 8571

b 8033

a 13137

b 3628

a 17565

b 4719

a 8341

b 4208

a 6221

b 3350

a 2261

b 1592

a 2403

b 3826

a 10583

b 2407

Regular males

3573 11 788

7846

8253

3902

2886

2543

7050

3292

10644

1695

7067

2500

5732

6322

8064

2520

10649

3718

5594

3402

5203

2967

2459

1986

1430

2850

7460

1998

Exceptional females

0 4

2

49

37

5

31

1

3

0

0

23

72

6

3

58

36

4

6

2

1

15

37

4

14

2

0

11

5

Exceptional Nondisjunction/ males 103 gametes

1 0.3 7 1 .o 0 0.3

52 7.6

45 12.3

18 5.3

69 27.8

3 0.3

4 1.3

8 0.4

0 0

9 3.0

38 25.9

2 0.8

10 1.3

81 9.4

27 14.3

16 1 .o 8 2.3

1 0.3

0 0.4

12 3.2

27 13.5

16 5.5

41 19.4

0 0.8

0 0

5 1.3

7 3.7

Females heterozygous for each of the indicated X chromosomes and either (a) y rste or (b) y pn v y + were crossed to YSX-YL, y B/O males. The frequency of nondisjunction is calculated as indicated in the text.

MEIOTIC EFFECTS OF A DEFICIENCY 367

...

I Df(iIK95-

FIGURE 2.-Deficiency map of the disjunction effect. The ratio of the frequency of X-chro- mosome nondisjunction for each deficiency heterozygote to that of the summed (and homo- geneous) controls is indicated together with the arrangement of the deficiencies with respect to the lethal and visible loci in the zeste-white region. The results from two series of crosses are shown: (a) deficiency/y rstg females crossed to YsX.YL, y B/O males, and (b) deficiency/ y pn v y f females crossed to YsX.YL, y B/O males. The data from which this figure was compiled are in Table 2.

368 L. G. KOBBINS

segment as the site of the proximal locus and places it in the zwb-zwll region. The relationship of the dosage-sensitive loci: If the meiotic effects of deficiency

heterozygosity of the zeste-white region are indeed the result of the interacting effects of reduced dose of multiple loci, it should be possible to mimic the effects of a larger deficiency by constructing trans heterozygotes of two smaller ones. To test this, control females, Df(l)K95 heterozygotes, D f ( l ) ~ ~ ~ ~ - 4 ~ heterozygotes and Df ( I ) K95/Df ( I ) ~ ” ~ - 4 ~ heterozygotes were compared.

The crosses were also designed to determine, with greater precision than was possible in crosses in which the X chromosome was divided into only two regions: (1) whether the effect of reduced dose of the zw7-zwll region alone on recom- bination is analogous to the effect of reduced dose of the entire zeste-white region, (2) whether reduced dose of the distal region has a slight effect on recombina- tion, as suggested by the mapping experiment. and (3) whether there is any interaction of the two regions with respect to recombination.

The meiotic effects of Df(I)K95 and of D ~ ( I ) w ~ ~ ~ - ~ ~ were separately examined by comparing X-chromosome recombination and disjunction in Df ( I ) K95, y 2 / y pn cu m f y+ and Df(I)w258-45, y z / y pn cv m f y + females to that of y / y p n cu m f.y+ controls. Interactions between the deficiencies were explored by comparing D f ( l ) ~ ” ~ - 4 ~ , y z cu m f y + / y and D f ( l ) ~ 9 ~ ~ - 4 ~ , y9 cu m fy+/Df(I)K95, ye lemales. In each instance the females were crossed to y pn cu m f/BSY males. Because there was substantial variation in the preceding experiments, the execu- lion of this experiment was arranged to minimize the effects of extraneous sources of variation. Small numbers of each of the five matings were made at the same time and replicates were mated on several successive days. All females were three to five days post-eclosion at the time of mating. In addition, to reduce background differences among the genotypes, both deficiencies were first made heterozygous with y pn cu m f y+. crossovers between the deficiencies and cu were recovered, and these chromosomes were, in turn, made heterozygous with y to remove the proximal markers. Within each cross, the results were homo- geneous among replicates.

Nondisjunction of the X chromosomes is signalled by the production of BSY/ X / X females and B+ males, while normal disjunction yields B+ females and BS males. Although nondisjunction of the X and Y chromosomes of the male parent also yields B S Y / X / X females and B+ males. these products are, in most cases, phenotypically distinrt because of the other markers. There are only two pos- sible cases of ccnfusion. Equational, diplo-X ova that are crossover in the short f-centromere region could be misclassified as paternally derived exceptions. Instances of regular ova bearing crossovers in that same region, fertilized by nondisjunctional nullo-X, nullo-Y, sperm could be misclassified as maternally derived exceptions. Neither of these possibilities represents a serious source of error. All exceptional male offspring were tested for fertility to ensure that the parental females were not X X Y ; all were, as expected, sterile.

The results of these crosses are classified in Table 3 according to the disjunc- tional origin of the offspring. They are unequivocal. Df(l)K95 and Df(I )wZs8-J5 separately have little effect on disjunction, while the trans heterozygote of the

MEIOTIC EFFECTS OF A DEFICIEhTCY

5 a

369

3 70 L. G . ROBBINS

two deficiencies produces nondisjunctional offspring at a rate comparable to that observed for deficiencies spanning both regions. Progeny tests of the exceptional females also indicate that the nondisjunction occurs at the first division and involves only nonexchange chromosomes, as had been found to be the case for the larger deficiencies.

Crossovers in four regions can be detected in the y / y p n cu m f y + , Df(l)K95, y*/y pn cu m f y + , and D f ( l ) ~ P ~ - 4 ~ , y % / y p n cu m f.y+ crosses. While all prod- ucts are recoverable as female offspring, only one of each reciprocal pair of products is recoverable among the sons of deficiency females. Only the daughters are used in the following analysis. The few crossovers between y and p n that were detected in the deficiency crosses are included among the noncrossovers because they are undetectable in the control cross.

Crossovers in only the three regions proximal to cu can be detected in the cross of D f ( l ) z ~ 9 ~ ~ - 4 ~ , y# cu m f y f / D f ( l ) K 9 5 , y" females, and no viable male products of normal disjunction are produced. While one of the two reciprocal crossover products of exchange distal to cu can be detected in crosses of D f ( l ) ~ " ~ - ~ ~ , y" cu m f y + / y females, they are counted as noncrossovers for this comparison with the Of ( I ) W ~ " - ~ ~ / D ~ ( I ) K95 results.

The results of these crosses with respect to recombination are given in Table 4, and the various parameters of recombination calculated from them are listed in Table 5. Df (I)uP58-45 heterozygosity has an obvious effect on recombination, and the pattern produced is similar to that produced by deficiency heterozygosity for the entire zeste-white region (ROBBINS 1977) : (1) overall recombination is reduced, (2) almost no triple exchanges are detected, the frequency of double- exchange tetrads is reduced about 60%, and the frequency of single-exchange tetrads is slightly increased, ( 3 ) interference is increased, and (4) recombina- tion is most affected in the tip and basal regions where exchange is normally less frequent per unit physical length. (Those reductions are 29% in the pn-cv interval, 19% in the cv-m interval, 20% in the m-f interval, and 38% in the f-y+ interval.)

One of the minor features of the effect of deficiency heterozygosity of the entire zeste-white region is not matched by the effect of reduced dose of just the zw7-zwll region. The data for deficiency heterozygosity for the entire region (ROBBINS 1977) had indicated that the effect on single exchange, though slight, was not regionally uniform in either the X or second chromosomes. The data for Of heterozygotes do, however, fit the expectations from regionally uni- €orm effects on the frequencies of both single- and double-exchange tetrads. To examine this, maximum likelihood estimates of the frequencies of single exchange for each region, of double exchange for each pair of regions and of regionally uniform effects on the frequencies of single and double exchange, were obtained by numerical approximation using the control and Df ( I ) w~5s8-4s, y" / y p n cu m f y + data of Table 4. Expected numbers of non, single and double crossovers were calculated using these estimates, and these expectations were compared to the observations by x2. The few triple crossovers were included in estimating the exchange parameters, but were not included in testing goodness-of-fit because

MEIOTIC EFFECTS OF A DEFICIENCY

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3 72 L. G. ROBBINS

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MEIOTIC EFFECTS O F A DEFICIElVCY 373

of their rarity. Regionally uniform, though quite different, effects on the fre- quencies of single and double exchange yield x2 = 15.1 with 8 degrees of freedom, a satisfactory fit. Thus, the regionally nonuniform effect of Df ( l )w258-45 on map length is a reflection of the normal, underlying, nonuniform distribution of single and double exchanges.

Heterozygosity for Df( l )K95 causes but a slight reduction in map length and has no discernable effect on the tetrad distribution or on interference. The most prominent feature of its effect is its regionally uneven nature. Recombination is reduced in the medial cu to m region, but is much less affected, and is even increased, in the tip and basal regions. Maximum-likelihood estimates of ex- change frequencies for control and Df( l )K95, y z / y p n cu m f.y+ crosses indicate that the frequencies of single and dmble exchanges are not affected in a region- ally uniform fashion (x2 = 24.5, 8 d.f.. P < 0.005). The nonuniformity can be accounted for by nonuniformity of effects on either single exchange (x2 = 3.2, 5 d.f., P > 0.6) or double exchange (x2 = 4.2, 3 d.f., P > 0.2). Interference is not altered, but the effect on exchange is so slight that it seems unlikely that an effect on interference would have been detectable.

The effect of Df( l )K95 on recombination could be an effect of either the same zwS-zwl0 locus that affects disjunction or a different locus in the zw8-zwlO region. Alternatively, this slight effect could result from structural heterozygos- ity. Though addition of a duplication complements the meiotic effects of zeste- white region deficiency heterozygosity ( ROBBINS 1977), that experiment would not have been sufficiently sensitive to preclude a slight, residual, structural com- ponent of the meiotic anomaly.

Simultaneous heterozygosity for both Df ( 1 ) K95 and Df (l)zoP58--C5 results in a iurther reduction in recombination that is qualitatively similar to what might be expected from the combined effects of the two deficiencies. In particular, the cv-m region. which is the only region in which Df(l)K95 heterozygosity evinces a substantial effect, is also the only region in which the double-deficiency hetero- zygotes differ substantially from Df

Furthermore, the effects of simultaneous heterozygosity for Df ( l )K95 and Df ( 1 ) wz58-45 on exchange are indistinguishable from those reported previously for deficiency heterozygosity for the entire zeste-white region. The frequency of double exchange is markedly reduced, the frequency of single exchange is only slightly affected, map distance is most affected at the tip and base and the regional variation. in effect, can not be accounted for by regionally uniform (but different) effects on single and double exchange (x2 =31.8, 4 d.f., P < 0.0001). The regional variation can be accounted for by regional variation in the frequency of single exchange (x2 = 0.8, 2 d.f., P > 0.6), though not by regional variation in the occurrence of double exchange (x2 = 30.5, 2 d.f., P < 0.0001).

Although the qualitative differences between Df ( 1 ) w258-45 heterozygotes and Df (1) w258-45 /D f ( l )K95 females are as expected from the effect of reduced dose of the distal region, the interaction is neither proportionate nor additive. Ex- change is more seriously affected in the double heterozygote than would be

heterozygotes.

3 74 L. G. ROBBINS

expected from simple combination of the effects of the two deficiencies. This may be examined by inquiring whether identical changes in exchange fre- quencies resulting from Df (I)K95 heterozygosity could account for both the dif- ference between the Df (I)R95 heterozygotes and controls and the difference between the D ~ ( Z ) W ~ ~ ~ - ~ ~ , y L cv m fy+/Df(I)K95, y2 and Df(I)f158-45, y 2 cu m f y + / y crosses. The data for the Df(I)K95 and control crosses were regrouped into the classes that would have been observed had only three regions been marked, as was the case in the other two crosses. Six parameters were assigned to the frequencies of single and double exchange in the control cross, another six were assigned io the exchange frequencies in Df ( I ) w258-b5 heterozygotes and six parameters were used to represent the change in frequency of each event occasioned by the presence of Df(l)K95 in the other two crosses. Maximum likelihood estimates of these parameters were obtained by numerical iteration, expected numbers of the various offspring classes were obtained for the four crosses and the difference between expectations and observations was evaluated by x2. Neither an equal, proportionate change produced by Df(I)K95 hetero- zygosity (for which x2 = 64.4, 6 d.f., P < 0.0001) nor an equal but additive change produced by Df(l)K95 heterozygosity (x2 = 18.9, 6 d.f., P < 0.005) provides an adequate fit of the observed effect of Df(I)K95 in the presence and absence of heterozygosity for the other deficiency.

These results are in accord with those obtained in the earlier deficiency map- ping of disjunctional effects. The effect of zeste-white region deficiency hetero- zygosity on disjunction requires reduced doses of two sites, even though reduced dose of either region alone has only a slight effect on disjunction. The effects on recombination are also separable into effects of two regions. The major recom- bination effects of zeste-white region deficiency heterozygosity are exposed by the proximal deficiency, Df ( I ) w2558-45, while a slight effect on recombination is produced by heterozygosity for the distal deficiency, DfCI) K95. A synergistic interaction between the two deficiencies produces an effect identical to that produced by deficiency heterozygosity for the entire zeste-white region.

The dependence of the disjunction effect on the frequency of exchange: BAKER and HALL (1976) have noted that, for a series of mutants that reduce exchange to varying degrees and cause nondisjunction of nonexchange chromosomes, the pattern of nondisjunctional products is nearly identical. To explain this similar- ity, they suggested that the occurrence of nondisjunction is simply, and sec- ondarily, related to the increased frequency of nonexchange tetrads. Because the frequency of X-chromosome nondisjunction is proportionate to the cube of the frequency of X-chromosome nonexchange tetrads, they have suggested a linear relationship between the frequency of X-chromosome nondisjunction and the frequency of cells simultaneously nonexchange for the X chromosome and both arms of a major autosome.

Reduced dose of the zw7-zwIZ region causes reduced recombination but, by itself, has little effect on the frequency of nondisjunction. Is this observation inconsistent with the notion that the frequency of nondisjunction is directly, though nonlinearly, related to the frequency of nonexchange tetrads? Probably

MEIOTIC EFFECTS O F A DEFICIENCY 3 75

not. The frequency of nonexchange tetrads in zw7-zwll region deficiency het- erozygotes is low enough that only the barest increase in the frequency of non- disjunction could be expected in any case. D f ( l ) ~ " ~ - 4 ~ heterozygotes yield an estimated frequency of X-chromosome nonexchange tetrads of 0.14 when recom- bination is measured over the entire length of a well-marked chromosome (Table 5 ) . For this frequency of nonexchange tetrads, the expected frequency of X-chromosome nondisjunction from the relationship of the frequency of non- disjunction to the cube of the frequency of X-chromosome nonexchange tetrads (BAKER and HALL 1976) is one to two exceptions per thousand gametes. The observed rate was 1.6 exceptions per thousand gametes (Table 3).

Thus, the effect of reduced dose of the zw7-zwll' region on recombination is consistent with its minimal effect on disjunction. In concert with zw8-zwlO deficiency heterozygosity, however, one observes a marked increase in the fre- quency of nondisjunction. Does reduced dose of the zw7-zwll region produce this interaction because it affects exchange, or is it necessary to invoke a more direct effect on disjunctional processes?

Reduced dose of the zw8-zwlO region alone is nearly undetectable when either disjunction or exchange is examined. The slight effect cm exchange does, how- ever, become more prominent when the dose OP the zw7-zwll' region is also reduced. Is this minimal effect on exchange, and the synergistic interaction, sufficient to account for the markedly increased nondisjunction observed when females are heterozygous for deficiencies of both regions?

Suppose that the effect of a deficiency on disjunction were solely a consequence of its effect on recombination. If recombination were eliminated by some other means, the deficiency would then have no effect on disjunction. If, on the other hand, a deficiency directly affected the disjunction of nonexchange chromosomes, reducing recombination by any means would expose the disjunctional effects of the deficiency. To examine this, heterozygosity for the multiply inverted bal- ancer chromosome, FM7, was used to reduce exchange, and X-chromosome non- disjunction was measured in females heterozygous for FM7 and for each of the various overlapping deficiencies.

FM7/deficiency females were crossed either to wild-type males, when the deficiency chromosome carried y or ya, or to y rsta males, when the deficiency chromosome was y+. In the first case, normal disjunction yields y+ B / + and y f B + females and FM7/Y ( ya wa B ) males, while nondisjunction is detected by the production of ya B/+ females and +/0 males. In the second case, the regular progeny are + and yz B/+ females and FM7/Y males, while the nondisjunc- tional offspring are y+ B/+ females and y rst*/O males. Control crosses of both y rst"FM7 and +/FM7 females were also done. In each case, the deficiency was introduced by use of deficiency/w+Y males, and the sterility of the nondis- junctional male progeny was confirmed to eiisure that the parental females were deficiency/FM7 and not deficiency/FM7/Y.

The results of these crosses are given in Table 6 and are diagrammed in Figure 3. As in the preceding experiments, there is variation among those deficiencies that affect disjunction. There is, however, no apparent correlation between the

3 76 L. G . ROBBINS

TABLE 6

X-chromosome disjunction in deficiency/FM7 heterozygotes

Females tested

Regular Regular Exceptional Exceptional Nondisjunction/ female$ males females males lo3 gametes

Controls:

y rst2 + 4902 4408 12 12 5.1

1561 1173 3 3 4.4

Deficiencies: Df (I)wrJ1, y2 2285 803 27 54 40.0 D f ( 1 ) w 2 5 8 - 4 2 , y 3298 1501 53 99 46.0 Df(1)641'4, Y 2999 1210 3 10 4.8

Df ( I ) wZ58-11 , Y 3609 1642 29 54 23.5 Df(I )wrJ2 , yf 5492 2460 11 15 5.0 Df(1)64c4, yf 6218 2812 34 56 15.0 D f ( l ) K 9 5 , y2 4955 1930 42 62 23.1

Df( l )WX12, Y 463 1 1601 19 22 10.4 DfCI)N7la, Y 1693 839 17 22 22.6 Df(1)65j26, yf 5156 1976 4 4 1.8 D j ( 1 ) w258--45, y2 4709 1773 6 6 2.9

Df(l)6Zg18, Yf 4798 2107 3 13 3.5

D f ( 1 ) 64f1, Y + 2785 1156 1 4 2.0

Females heterozygous for FM7 and for each of the chromosomes listed were crossed to either wild-type males (where the deficiency carried y or y2) or to y rst2 males (where the deficiency was y + ) . The classes recovered and the calculation of the frequency of X-chromosome nondis- junction are described in the text.

t Df ( 1 ) w 2 ~ ~ - ~ ~ 9.2 I

I Df (1164~4 3.0 I

I 4'6 Df(llK95-

FIGURE 3.-Nondisjunction in deficiency/FA47 heterozygotes. The ratio of the frequency of nondisjunction of each deficiency/FM7 heterozygote to that of the control is indicated together with the arrangement of the deficiencies with respect to the lethal and visible loci in the zeste-white region. The data from which this figure was compiled are in Table 6.

MEIOTIC EFFECTS O F A DEFICIENCY 377

variation of this and the preceding mapping experiment, suggesting that the variation is not an intrinsic property of the deficiencies themselves. While FM7 heterozygosity itself causes increased nondisjunction, it also exposes the effect of reduced dose of the zw8-zwl0 region. Df( l )K95, as well as the other defi- ciencies that are deficient for the zw8-zwl0 segment, now yields a clearly ele- vated frequency of nondisjunction. In addition, there is no longer any indication in these data of an interaction between the ZW~-ZW?O and proximal regions, sug- gesting that the effect of FM7 is, in this sense, equivalent to that of hemizygosity for the zw6-zwl? region.

The disjunctional effect of the zw8-zwl0 segment can not, therefore, be viewed as a consequence of its minimal effect on exchange. Reduced exchange, whether as a result of inversion heterozygosity or because of reduced dose of the zw7-zwll region, exposes a disjunctional effect of the zw8-zwlO region. I t is likely. then, that reduced dose of the zw8-zw20 region directly affects the processes that normally ensure disjunction of nonexchange chromosomes and increases the probability of nondisjunction of nonexchange chromosomes.

In contrast to this, heterozygosity for FM7, rather than exposing a disjunc- tional effect of the zw7-zwll region, obscures the interaction of that region with reduced dose of the zw8-zwl0 segment. Thus, the disjunctional effect of reduced dose of the xw7-zwll region can be accounted for entjrely as an indirect conse- quence of its effect on exchange.

The relationship between exchange and disjunction in Drosophila females has been explored by a number of investigators in normal, chromosomally aber- rant and mutant situations. (See GRELL 1976; BAKER and HALL 1976; and BAKER et al. 1976 for reviews.) Though there has been serious division over the timing and nature of events governing disjunction (see GRELL 1976 review, also NOVITSKI 1964, 1978), it is apparent that processes distinct from exchange exist to ensure disjunction and that these processes are usually of consequence only for nonexchange chromosomes. Comparison of the array of nondisjunctional events occurring in zeste-white deficiency heterozygotes with those occurring in recombination defective meiotic mutants provides some suggestion of how the disjunction of nonexchange chromosomes is disrupted by reduced dose of the zw8-zwlO region.

The pattern of nondisjumtion produced by zeste white region deficiency het- erozygotes has been previously described (ROBBINS 1977). As for recombination- defective mutants, but unlike what is observed for the disjunction-defective mutant nod (CARPENTER 1973), nondisjunctional X and third chromosomes often disjoin from each other. Furthermore, as for recombination-defective mutants, but unlike what is observed for the disjunction defective mutant mei-SSI ( ROBBINS 1971 ) . X and fourth chromosome nondisjunction are cor- related, but the X and fourth chromosomes do not disjoin from one another. Thus, both the rules of distributive recognition and the ability of nonhomologs to disjoin are apparently unaffected in the deficiency heterozygotes. If zeste- white deficiency heterozygotes are normal with respect to both distributive recog-

378 L. G . ROEBINS

nition and distributive disjunction, perhaps their abnormality lies in reduced strength of the distributive association.

Descriptions of the disjunctional patterns of the various meiotic mutants gen- erally refer to the errors of distributive disjunction-nondisjunctional events- and not to the normal distributive outcome, i.e., the regular separation of non- exchange homologs to opposite poles. Association of nonhomologs would yield disjunction of the nonhomologs to opposite poles, but failure of association of an homologous pair would yield nondisjunction of only that pair. If zeste-white deficiency heterozygosity produces weakened associations, a larger fraction of cells exceptional for one chromosome should be regular for others than is the case for strictly recombination-defective mutants. Extant data that may be used for this comparison are summarized in Table 7. Though the types of disjunctional products produced by zeste-white deficiency heterozygotes are the same as those recovered from recombination-defective mutants, the frequencies of the products differ. A substantially larger fraction of cells exceptional for the second chromo- some are regular for the X chromosome in zeste-white deficiency heterozygotes than is the case for the recombination-defective mutants (mei-251, mei-195, mei-SS1, mei-41, mei-9 and mei-218), while 4 3 ) G , which may have some effect on disjunction in addition to its effect on exchange (HALL 1972), is intermediate.

Thus, it may be suggested (with these results alone only a suggestion is intended) that reduced dose of the zw8-zwl0 region increases the probability of nondisjunction of nonexchange chromosomes by reducing the strength of homol- ogous (and possibly nonhomologous) distributive associations.

DISCUSSION

The data presented here indicate that the meiotic effects of reduced dose of [he zeste-white region are the composite of effects of reduced dose of at least

TABLE 7

Distribution of X chromosomes among second chromosome exceptional gametes

Genotype tested

x . 22 ah x ; o

Eg genotype

a& 0 : 22

Bx.0 xx . 2 2 a& 0 ; o

Df (1)wTJ' mei-251 mzi-195 mei-S51 mei-41 mei-9 mei-218 c(3)G1' c(3)G68

0.69 0.25 0.34 0.37 0.32 0.42 0.36 0.53 0.51

0.30 0.69 0.66 0.57 0.64 0.55 0.59 0.34 0.39

0.01 0.05 0.00 0.06 0.04. 0.03 0.05 0.13 0.1 1

The frequencies of different disjunctional classes are calculated from the data of ROBBINS 1977 (Table 11) for Df(1)w'Jl and from the summary of BAKER and HALL 1976 (Table 11) for the meiotic mutants. The observed numbers are corrected for recoverability of half the X exceptional gametes and, in the case of Df(I)w'J', for the lethality of deficiency/O zygotes.

MFIOTIC EFFECTS O F A DEFICIENCY 3 79

two loci: one located in the ZW7-ZWll region that is involved in recombination and one located in the zw8-zwlO region that is involved in the orderly dis- junction of nonexchange chromosomes. While the latter locus might also have a slight regional exchange effect, it is not possible to rule out a slight structural effect of deficiency heterozygosity, nor can the possibility of a dose effect of yet a third locus be excluded.

Reduced dose of the zw7-zwll region alone accounts for the major features of the recombination effect of hemizygosity for the entire zeste-white region: (1) the frequency of double exchange is reduced severely, while the frequency of single exchange is hardly affected, and (2) exchange is most severely depressed in the centromere and telomere regions. Reduced recombination entirely ac- counts for the disjunctional effect of zw7-zwll deficiency heterozygosity.

It has previously been suggested that the recombination effects of zeste-white region deficiency heterozygosity could be understood as an effect of reduced level of a protein acting at the site of exchange. In reaching that conclusion, a model was developed that attempted to portray (albeit in oversimplified form) the effects expected from that reduction (ROBBINS 1977). The effects of reduced dose of the zw7-zwll region remain consistent with that model. The interaction of deficiency heterozygosity of the zw7-zwll and Z W ~ - Z W I O regions lends some additional support to the model, even though the cause of the slight recombina- tion effect of zw8-zwlO region deficiency heterozygnsity remains ill defined. Under the model, any reduction in the probability of an exchange, given an enzyme contact. would yield a synergistic interaction that would be strongest in regions where the probability of exchange is most reduced. The interaction of Df(l)K95 and D f ( l ) d 5 * - J 5 is synergistic, and the strongest interaction occurs in the cv-m region where the effect of Df( l )K95 is strongest.

While reduced dose of the zw7-zwll site has effects that are compatible with a model of a reduced amount of a recombination enzyme, it is also analogous to a mutant postulated by CARPENTER and SANDLER (1974), but not then repre- sented among the known meiotic mutants. That is, reduced dose of this site mimics a mutant that differentially affects the frequencies of single and multiple ex- changes, but that has no effect on the regional distribution of either. Following their paradigm, the deficiency would alter the frequency, but not the distribu- tion, of recombination nodes.

Deficiency heterozygosity for the zw8 -zwlO region increases the frequency with which nonexchange chromosomes nondisjoin. The array of nondisjunc- tional products produced is identical to that produced by recombination defective mutants, but nonhomologs apparently interact less frequently in the deficiency heterozygote than in the recombination defective mutants. It is possible that this dosage-sensitive disjunction locus is analogous to one of the several dose- sensitive pairing promoters that have been identified in wheat (see SEARS 1976 for review).

While these results demonstrate that at least two loci with dose-dependent meiotic functions are contained in the short, intensively studied zeste-white region, these results do not, of themselves, provide any fuel for debate about the

‘80 L. G . ROBBINS

number of functional units within the region. It is simply impossible to use them to argue that the meiotic functions defined by deficiency heterozygosity do, or do not. represent functions that are essential for survival. Were the meiotic dose effects simply pleiotropic effects of essential loci, the same loci would have also been detected in screens for lethal mutants. These results do, however, suggest which loci might be examined for meiotic effects, and they do provide some caveats about the manner in which that examination should proceed. For exam- ple, mutants at zw8, zw4 or zwl0 might affect disjunction, but the effect might not be evident unless recombination is, by some other means. reduced as well. Whether the dosage-sensitive loci are essential, or are nonessential (though clearly not Eunctionless) remains a question of some interest.

The author is grateful to NANCY VEENSTRA for her assistance and to BRUCE MCKEE, CLAUDE HINTON and LAURENCE SANDLER and his students for their critical reading of a draft of this report.

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MEIOTIC EFFECTS OF A DEFICIENCY 38 1

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