CHROMOSOME SIZE AT DISTRIBUTIVE PAIRING IN

CHROMOSOME SIZE AT DISTRIBUTIVE PAIRING IN DROSOPHILA MELANOGASTER FEMALES

RHODA F. GRELL

Biology Diuision, Oak Ridge National Laboratory,' Oak Ridge, Tennessee

Received February 27, 1964

DISTRIBUTIVE pairing is that phase of the meiotic cycle in Drosophila melanogaster females during which chromosomes that have failed to undergo

exchange may associate nonhomologously. At this time, segregation ratios indicate that a single nonrecombinant remains unassociated, that two nonrecombinants, whether homologues or nonhomologues, pair with high frequencies and that three nonrecombinants compete for associations and may exhibit marked preferences (R. F. GRELL 1962a). The present work is concerned with the preferential aspect of distributive pairing. The point of departure for these experiments was the observation that among three nonrecombinant chromosomes, the two that possessed no homology in common, segregated regularly from each other while the third, which carried a centromere and proximal heterochromatin similar in origin to one of the two, assorted randomly with both (GRELL and GRELL 1960). This experiment permitted two conclusions. First, the choice of partners at distributive pairing is not a random event depending upon the chance en- counter between any two centromeres. Rather, it appears to be extremely precise and to involve the entire chromosome, euchromatin as well as heterochromatin. Secondly, it becomes clear that homology, at least for the centromere and proximal heterochromatin, is not the basis for choice of partners. In fact, evidence from a variety of sources negates the notion that homology of any kind plays a decisive role in the selection of associates. If such were the case, the availability of two nonrecombinant homologues would preclude the involvement of either with a nonhomologue. Yet, it is well established that two X chromosomes will frequently associate with an autosome when all are noncrossovers (COOPER, ZIMMERING and KRIVSHENKO 1955) and particularly when one member of the autosomal pair is made unavailable for distributive pairing by translocation (ROBERTS 1962).

One correlation that is evident from the experiment of GRELL and GRELL (1960) described above, is that those chromosomes which segregate regularly are comparatively large whereas the chromosome that assorts at random is small. This observation suggested that chromosome size might be an important factor in distributive pairing. A second possibility concerns the existence of a small number of nonspecific pairing sites so arranged as to favor associations between particular nonhomologues. Experiments were designed to test the possible roles

' Operated by Union Carbide Corporation for the United States Atomic Energy Commision

Genetics 50: 151-1GG July, 1964.

152 R. F. GRELL

of chromosome size and of pairing sites in determining both pairing efficiency between two nonrecombinants in a noncompetitive situation and pairing preference among three nonrecombinants in a competive one. The results indicate that for the chromosomes used, efficiency and preference in pairing are strictly correlated with similarity in chromosome size. An unexpected disclosure is that the occurrence and frequency of trivalent association, as opposed to bivalent- univalent association, as well as the manner of disjunction from a trivalent, are also functions of chromosome size. No evidence was found for the existence of pairing sites.

MATERIALS A N D METHODS

Materials: A number of free X-chromosome duplications that had been obtained and cyto- logically characterized by COOPER and KRIVSHENKO (unpublished) were utilized for the present experiments. All of the duplications carry the distal tip of the X chromosome, including the locus of y+ which was used to follow their segregation, plus varying amounts of proximal X- heterochromatin. In respect to length, the duplications form a series which, upon comparison with a normal metaphase fourth chromosome, ranges in size from < 0.3 to 3.3 times the length of chromosome 4, according to the determinations of COOPER and KRIVSHENKO (Figure 1). As indicated in Figure 1, those duplications larger than 2.0 carry the nucleolar organizer region of X. The genetic content of each duplication for the loci acf, sc+, S U - W ~ + , dor+, p n + . su-f+, and bb+ was determined by COOPER and KIUVSHENKO (unpublished) and is shown in Figure 1. Since some of the duplications of approximately the same size were obtained by irradiation of the sc8 X-chromosome and others of a Canton4 X (Figure l ) , they are expected to carry heterochromatin of similar content but partially reversed sequence. The effect of differences in chromatin order constitutes one of the criteria for evaluating the role of pairing sites. The largest duplication, No. 3 (> 4 times chromosome 4), is one obtained by LINDSLEY and SANDLER (1958) and has been studied previously (GRFLL and GRELL, 1960).

The mutants used in this study with their symbols and genetic locations are: yellowl

NATURE OF X-DUPLICATIONS

CHROMOSOME

Chromosome 4 X-Dp # 1187 X-Dp # 1162 X-Up # 816 X-Dp # 1204 X-Dp # 1193 X-Dp # 1339 X-Dp # 1144 X-Dp # 1337 X-Dp # 1186 X-Dp # 1346 X-Dp# 1328 x-Dp # 1488 X-Dp # 1343 X-Dp# 856 X-Dp # 1498 X-Dp# 3

ORIGIN

SCE

sc 8

c- s S C E

sc 8

c- s c-s c- s

c- s c- s c-s c-s c-s c- s c- s

S C E

LENGTH X-LOCI PRESENT AT MITOTIC METAPHASE Y + OC+ 9C+ S U - W a + dor+ pn+ su- f+ bb+

1.0 + + - - - - - - g 0.3

0.5 + + - - - - - - + + + - - - - - 0.7

0.9 + + - - - - - - + + - - - - - - 1.0

4.1 + + + + - - - - + + - - - - - - 1.1

i.4 + + + + + f.6 + + - - - 2.0 + + + - - 2.1 + + + + - 2.5 + + + + - 2.6 + + + + - 3.0 + + + - - 3.3 + + + + - 4 + + + + - -

FIGURE 1 .-Nature of X-duplications, showing their mitotic lengths with chromosome 4 taken as unity. The small circle in the heterochromatin (Dps 1328-3) symbolizes the nucleolar organizer region. (Tests for the presence of ci+, guI+, ey+, and su+ in the duplications were uniformly negative.)

DISTRIBUTIVE PAIRING 153

(y , 1-0.0), yellow2 (ye , 1-O.O), achaete (ac, 1-O.O), scute (sc, 1-O.O), suppressor of apricot ( s u - ~ , 1-0.05), deep orange (dor, 1-0.3), prune (pn, 1-0.8), Bar (B, 1-57.0), red (red, 3-55.0), cubitus interruptus (ci, 40.0), cubitus-interruptus-Dominant ( 9 , 4-O.O), grooveless ( g d , 4-O.O) , eyeless (ey, 4-O.O), and shaven (su, 4-0.0). The rearrangements that were used include Duplication (l;f)3 [Dp(l;f)3] (LINDSLEY and SANDLER, 1958), Transposition (3) Vein off, Vein off (Tp(3)Vno, Vno) (GRELL, E. H. 1959; NICOLETTI and LEWIS 1960), Translocation (3;4) 86D [T(3;4)86D] (GRELL, R. F. 1959) and an attached XY (LINDSLEY and NOVITSKI 1950). Fuller descriptions of the mutants and rearrangements may be found in BRIDGES and BREHME (1944.) or in the references cited.

Method: Two sets of experiments were performed utilizing the following kinds of females: Type 1. For the noncompetitive studies, each duplication was introduced into females of the genotype yz/y*; T(3;4) 86D,red/T(3;4)86D,r; c i D . The X-duplication and the dominantly marked free 4 are practically always nonrecombinant chromosomes and, in the absence of competitors, are expected to pair distributively and segregate from one another. Thus the progeny from such females should consist predominantly of two reciprocal classes, those that carry the free 4 and lack the duplication and are y2;ciD in phenotype and those that carry the duplication but lack the free 4 and are yf in phenotype. Failure of distributive pairing between the X- duplication and the free 4 is expected, half of the time, to lead to products which either carry both chromosomes and are yf;ciD or carry neither and are yz. The other half of the time, the products will be identical to those arising from distributive pairing between the duplication and the 4. Thus nondisjunction occurs only one half of the time between unassociated chromosomes so that n = % (1 - a ) where n = nondisjunction and Q = association. The frequency of association between the X-duplication and the 4 at distributive pairing may be calculated from the observed frequency of nondisjunction and is negatively correlated with it. Nondisjunction frequency is used, therefore, as a measure of the efficiency of pairing between the two nonhomologues such that the lowest nondisjunction frequency indicates the greatest pairing efficiency and the highest nondisjunction frequency, the least.

Type 2. For the competitive studies each duplication was introduced into females of the genotype y2/y2; T(3;4) 86D,red/Tp (3R)Vno,Vm; ciD. T(3;4) 86D is a reciprocal translocation in which 3R is broken about one third of the distance from its centromere at 86D and chromosome 4 is broken close to the heterochromatic-euchromatic boundary at 101F. The results are one large, J-shaped chromosome designated T, which bears 3L, the three centromere and the proximal third of 3R which is capped with the euchromatic portion of chromosome 4; and one smaller acrocentric chromosome, designated T,, which bears the centromere and proximal heterochromatin of 4 plus most of the right arm of 3. At metaphase T, appears approximately five to six times the length of chromosome 4 from which it may be concluded that T, is a predominantly euchromatic element with heterochromatin constituting less than % of its content. The presence of the dominantly marked transposition, Vno, in the distal half of the right arm of the nontranslocated third chromosome serves to reduce exchange between T, and its homologue to less than 2 percent. Therefore females of this genotype carry, in over 98 percent of their primary oocytes, three nonrecombinant chromosomes, namely an X-duplication marked with y f , a free 4 marked with 9, and T, whose presence is recognized among the progeny by the absence of Vno. Nondisjunction frequencies are measured for the three nonrecombinants taken two at a time. It should be noted that for viability a zygote must carry both T, and T, or neither chromosome.

In both sets of experiments, single females were mated within 10 hours after eclosion to three males of the genotype attached-XY, y B/Y. The presence of y and B in the male parent permits recognition of matroclinous females as B+ and patroclinous males as y B.

RESULTS A N D ANALYSES

Noncompetitive situation: The frequencies of nondisjunction between each of the X-duplications and chromosome 4 are given in Table 1. The highest nondis-

154 R. F. GRELL

TABLE 1

Noncompeiiiiue system. Nondisjunction frequencies between chromosome 4 and X-duplications from the cross:

y2/yz/Dp(l;f),y+; T(3;4)86D,red / T(3;4)86D,red; ciD 0 x attached-XY,yB / Y 8

Duplication Mitotic No. length

1187 Q 0.3 1162 0.5 816 0.7

1204 0.9 1193 1 .o 1339 1.1 114.1. 1.1 1337 1.4 1186 1.6 1346 2.0 1328 2.1 1488 2.5 1343 2.6 856 3.0

1498 3.3 3 4 f

Totals

12,113 8,000 9,324 7,391 5,371 7,126 8,476 5,635 5,991 8,011 2,789 2,665

3,732 2,767

34.4

3,343

Dp,4 nondisjunction among X-regulars

(percent 1

X-nondis- Dp,4 nondisjunction junction among X-exceptions (percent) (percent)

~

4.49 f 0.19 0.39 f 0.07 0.22 f 0.05 0.08 k 0.03 0.13 i- 0.05 0.08 f 0.03 0.02 f 0.02 0.38 f 0.08 0.39 f 0.08 0.42 f 0.07 0.43 & 0.12 1.94 f 0.27 2.20 ir 0.25 1.90 k 0.24 1.63 f 0.24 2.00 f 0.75

0.46 f 0.06 0.32 f 0.06 0.86 f 0.10 0.62 k 0.10 0.37 f 0.08 0.76 f 0.10 0.88 f 0.10 0.75 f 0.12 0.70 k 0.11 0.97 f 0.11 1.14 i- 0.20 1.20 f 0.21 1.79 t 0.23 1.39 f 0.19 1.01 t 0.19

29.6 7.7 0 0 0 3.7 2.7 0 4.8 0 6.6

12.5 16.7 23.1 14.3

junction value occurs with the smallest duplication (1 187, < 0.3) and is approximately 4.5 percent or in terms of association approximately 91 percent. As the duplications become larger and approach chromosome 4 in size, nondisjunction frequencies progressively decrease which means that association frequencies increase. The lowest nondisjunction values, and hence the highest association values, are found with those duplications closest to the 4 in size (0.9 to 1.1) and average approximately 0.1 percent for the former or approximately 99.8 percent for the latter. Nondisjunction rises again as the duplications become larger than the 4 (sizes 1.4 to 2.1). A sharp increase occurs between sizes 2.1 and 2.5 from which point the values plateau at approximately 2 percent regardless of increase in duplication size. Since the highest association frequencies occur with duplications closest to the 4 in length and the lowest with duplications most dissimilar to the 4, it may be concluded that pairing efficiency between the two nonhomologues is closely correlated with their size similarity.

Changes in duplication size are not always associated with corresponding changes in nondisjunction frequency as evidenced by the plateaus between sizes 1.4 to 2.1 and between 2.5 to 4+. If the plateaus occurred as the duplication size was increasing and approaching 1 .O, it might be assumed that the number of sites are small and that the end of a plateau indicated the addition of a pairing site. There is, however, no apparent reason why a site addition should lead to less efficient pairing. Furthermore, as was noted above, differences in chromosome origin lead to differences in the sequence of heterochromatin within the duplication and could be expected to change the position of sites. Yet, duplication 816,


of Canton-S origin, displays a nondisjunction value consistent with its size position in the series, despite the fact that the two duplications preceding it as well as the two duplications following it are of sc8 origin. These results lend no support to the notion that pairing sites are a factor in distributive pairing.

X-chromosome nondisjunction values (Table 1, column 5 ) are, in general, higher for larger duplications than for smaller ones. Females carrying duplications of C-S origin tend to show more X-exceptions than those of sc8 origin. Very small duplications (< 0.3 and 0.5) and those longer than 2.0 are probably involved in X-nondisjunction as shown by the markedly higher frequencies of Dp, 4 nondisjunction among the X-exceptions than among the progeny regular for X segregation (Table 1, columns 6, 4). With duplications of intermediate length (0.7 to 2.0), Dp, 4 nondisjunction is little, if any, higher among the X-exceptions than among the X-regulars, suggesting that these duplications are much less frequently involved.

Competitiue situation: The addition of a third element, i.e. T,, to the distributive pool increases the possibilities for association from one type of bivalent (Dp-4) to three types of bivalents (Dp-4; T,-4; T,-Dp) and a trivalent (Dp-4-T4). The raw data are shown in Table 2 and nondisjunction frequencies for the three pairs of chromosomes are given in Table 3. An examination of Dp, 4 nondisjunction (Table 3, column 3) shows that, as in the noncompetitive case, the highest values occur when the two chromosomes are most dissimilar in size and the lowest values when they are most similar. The effect of the competitor, T,, on Dp, 4 nondisjunction (Figure 2) is apparent when the difference in size between the duplication and 4 roughly exceeds one third. Thus, within the range 0.7 to 1.6, Dp, 4 nondisjunction is similar. Outside of this range, Dp, 4 nondisjunction is significantly greater in the presence of T,. It increases as the size discrepancy between the duplication and 4 increases and the plateaus observed in the noncompetitive situation are eliminated.

A more comprehensive interpretation of the data can be made from Figure 3, in which nondisjunction frequencies for each pair of the three competitors are

301 25 i

WPLICATION LENGTH

FIGURE 2.-Comparison of Dp, 4 nondisjunction in competitive and noncompetitive systems.

156 R. F. GRELL

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

h

. . . h 8 v

a o o o o - o o o o o o + a o o


158 R. F. GRELL

plotted and can be examined simultaneously. For the smallest duplication (< 0.3), Dp, 4 nondisjunction is equal to T,, 4 nondisjunction. As duplication size approaches 4 size (i.e. 1.0), Dp, 4 nondisjunction decreases and T,, 4 nondisjunction increases. At apparent equality between the duplication and the 4, their nondisjunction is minimal and at this point T, shows about 50 percent nondisjunction with both elements. It should be noted that equality of size at mitotic metaphase may not completely reflect equality of size at the time distributive pairing occurs. Perhaps for this reason, the midpoint of the curve is at approximately 1.3 instead of 1.0. A shift of the curve to the right, however, might con- ceivably be attributed to slight viability differences between the major classes of progeny such that the triplo-4 class, T; ciD, is less viable than the diplo-4 class, Vno;ciD. Beyond the point of equality, as the duplication begins to approach T, in size, Dp, T, nondisjunction decreases and Dp, 4 nondisjunction increases. The two curves cross at about 3.0 and with the largest duplication (4+), Dp, 4 nondisjunction greatly exceeds Dp, T, nondisjunction. (The nondisjunction values for Dp 1498 from both experiments suggest that this duplication has undergone a change in size and is now smaller than 3.3). If the data for the BEY, which is larger than any duplication, are included (GRELL and GRELL 1960), BsY,T, nondisjunction falls to 2 percent and chromosome 4 shows independent assortment with both the BsY and T, (Figure 3, dotted lines). The relationship among the three chromosomes at this point is closely analogous to that existing with duplication 1337. In both cases the two chromosomes of similar size segregate regularly and the dissimilar chromosome assorts randomly. Thus, with the BSY, it is the Y and T, which disjoin regularly and the small fourth chromosome which assorts independently, while with duplication 1337 it is the duplication and 4 which segregate regularly and the large T, element which now shows independent assortment.

As indicated above, the competitive situation is complicated by the possible

4 YBs T. t r -

WPLICATION LENGTH

FIGURE 3.-Relation of nondisjunction frequencies to duplication length.


occurrence of trivalents. Perhaps the simplest analysis would be to assume that trivalents do not exist. As can be seen from the graph (Figure 3 ) in almost every case, one of the painvise combinations shows nondisjunction frequencies greater than 50 percent. This means that trivalents are indeed occurring and furthermore that in the trivalent, chromosome segregation is nonrandom. The type of nonrandomness which must be assumed to explain the data is such that the element of intermediate length directs the small and large chromosomes to the same pole. Thus, for duplications smaller than 1.4 the directing element is the fourth chromosome, and for those greater than 1.4 it is the duplication. In other words, it would appear as if the element intermediate in length pairs with the other two simultaneously and directs both of them to the same pole. The point at which the shift in directing elements occurs (between 1.1 and 1.4) corresponds to the point at which the requirement for trivalents is eliminated and is probably the most sensitive indicator of similarity in size between the duplication and chromosome 4 at distributive pairing.

While it is necessary to postulate that trivalents must exist, quantitative estimates of their frequency are impossible from genetic data alone. At one extreme, invariable trivalent formation might be imagined in which case size-determined disjunction rather than size-determined association would be responsible for the segregation results. Since the present data show that invariable bivalent association does not occur in the noncompetitive situation, there is no reason for assuming that invariable trivalent association occurs in the competitive one. Presuming, then, that both bivalents and trivalents are formed, since there is compelling evidence that bivalent association is predicated on size similarity of chromosomes, perhaps the most reasonable assumption, to carry the analysis further, is that association between the two most unlike elements is negligible. With nonrandom disjunction from the trivalent, such that the intermediate element directs the other two to the same pole, it can be shown that the above assumptions lead to a minimal estimate for the frequency of trivalents, hence maximal estimates for the frequency of the other two types of bivalents. Such estimates are tabulated in Table 4. Other estimates, compatible with the data, but assuming bivalent formation between the two most unlike elements, may be obtained by the method indicated under Table 4.

As can be seen from Figure 3, the data suggest that the highest trivalent frequencies, as indicated by maximal nondisjunction values, occur when the length of the middle element is roughly midway between the other two on the graph. Since the length scale is logarithmic, this implies that maximum nondisjunction, hence maximum trivalents, occurs when the length of the middle element is the geometric mean of the lengths of the other two (i.e. when the ratio of a:b equals the ratio of b:c where a, b and c symbolize the lengths of the small, intermediate and large chromosomes, respectively) and would mean that it is the ratio of the lengths of chromosomes rather than their absolute sizes which is important in distributive pairing.

If it is true that the requirements for maximal trivalents is given by the expression b = d;, then, for the duplications studied, two maxima are expected,

160 R. F. GRELL

TABLE 4

Patterns of some possible pairing configurations in the competitive system

Percent bivalents Percent trivalents

Dp-4.T, Dp-T,,S T4-4,Dp Dp-4-T, 4-Dp-T4 4-T4-Dp Duplication No. I m g t h Amax Bmin ‘ma, “mi“ Emin F m i n S,.X

1187 G0.3 29.6 0 30.8 39.6 0 0 29.6 1162 0.5 53.6 0 6.6 39.8 0 0 6.6 816 0.7 64.0 0 0.7 35.3 0 0 0.7

12Q4 0.9 73.4 0 0.2 26.4 0 0 0.2 1193 1 .o 86.9 0 0.1 13.0 0 0 0.1 1339 1.1 88.1 0 0.7 11.2 0 0 0.7 1 I44 1.1 91.5 0 0.5 8.0 0 0 0.5

Duplication No. Length Amax B,,,

1337 1.4 98.4 1.7 1186 1.6 89.4 1.0 1346 2.0 72.3 5.4 1328 2.1 53.4 13.4 1488 2.5 55.0 16.8 1343 2.6 37.4 32.0 856 3.0 27.8 37.8

1498 3.3 36.4 31.6 3 4+ 9.4 55.6

0 0 9.6 0

22.3 0 33.2 0 28.2 0 30.6 0 34.4 0 32.0 0 35.0 0

1.7 1 .o 5.4

13.4 16.8 32.0 27.8 31.6 9.4

T h e z l u e s in this table are obtained from>e nondisjunction frequencies in Table 3 i* following way: If Dp+=duplication 4 nondisjunction. T 4 =translocation, 4 nondisjunction. and DpT= duplication, translocation

A B C D E F

nondisjunction, and if the three types of hicalents and the three types of trivalents de designated,

DP-4 bivalent Dp-T, bivalent 4-T, bivalent Dp-4-T4 4-DpT4 4-T;Dp T+ univalent 4 univalent Dp univalent trivalent trivalent trivalent

then the following equations describe the origin of the three nondisjunctional types: - - D p 4 = 0 . A + ‘ / z ’ B + ‘/ . C + O . D + O . E + l . F - T4 = % . A + % . B + O . C + O , D + l . E + O . B DpT = ’/ . A 4- 0 . B + $4 . C + 1 . D + 0.22 + 0 . F

We assume: (1) Segregation from the trivalent is nonrandom such that the intermediate element directs the other two to the same pole. (2) Bivalents between the tw-o most dissimilarelements are negligible. I t follows, when chromosome 4 is intermediate (Case I) B = E = F = 0, and 1 %C, T4 ‘ / A , and F T = %A + %C + D, which leads to

- Cmaz = 2 Dp4

A,,, 2 T4 D,*, = DpT - Dp4 -E Bmtn = 0

- - -

- BmaZ = 2 EW -

The values obtained from Table 3 in this way are given in the upper half of the table. For the case where the dupli- F = 0 and analogous calculation leads to cation is intermediate (Case 11), the above assumptions mean that C = D

Amas 2 IIpT E,<,

. . - - T4 - Dp4 - DpT { c,<, = 0

and the values obtained from Table 3 are given in the lower half of the table. (T, is never intermediate, hence A = D = F = o ’ 1s not applicable here).

The justification for the “maximum” and “minimum” labels is as follows: for Case I, B = 0 is clearly the Bmin; we drop this assumption and assume B = 6 and as above that E = F = 0.


Then,

whirh leads to

Since S is always positive, the assumption 6 = 0 leads to maximum values for A and C and minimal values for B and D,

hence

For Case 11, a similar ralrulation leads to

one when Dp = d 4 x T , and one when 4 = dDpxT, . Assuming that the two instances of random assortment of one of the three chromosomes represent the points at which the remaining two chromosomes are closest in size at distributive pairing, values of about 1.3 for chromosome 4 and by extrapolattion, about 5.5 for T, are obtained. Substitution of these values in the two expressions gives duplication lengths of 0.3 and 2.7 for the two maxima. While the two maxima are plateaus (Figure 3 ) , these values clearly fall within the plateaus. I t is of interest that the values 0.3 and 3.0 coincide with the points where in one instance the curves representing Dp, 4 nondisjunction and T,, 4 nondisjunction cross and in the other instance those representing Dp, 4 nondisjunction and T,, Dp nondisjunction cross. Thus, it appears that when the intermediate chromosome associates equally well with the large and small chromosomes and engages them in the greatest amount of trivalent formation, its length is the geometric mean of theirs.

The X-exceptions are classified with respect to their X-duplication and their chromosome 3 and 4 content (Table 3 ) . The duplications and chromosome 4 are approximately equally distributed among the male and female exceptions after a correction is made for the inviability of haplo-4 females. On the other hand, all of the female exceptions carry the nontranslocated third chromosome marked with Vno whereas 98 percent of the male exceptions carry the translocation. This evidence, thait the T, is highly involved in X-nondisjunction whereas the X-duplications and chromosome 4 are not, is consistent with the preceding findings that pairing preference is correlated with similarity in chromosome length.

DISCUSSION

The present studies of distributive pairing indicate that for the two situations that have been investigated, pairing efficiency and pairing preference are

1 62 R. F. GRELL

functions of the similarity in length of the chromosomes involved. Since lengths are determined by mitotic metaphase measurements, it may be assumed that mitotic and meiotic metaphase lengths are closely correlated. Furthermore, it may be assumed that either distributive pairing occurs at or close to metaphase or the size relationships of the chromosomes at metaphase are indicative of their size relationships at an earlier meiotic stage. It should be noted that the chromosomes used in this work include X-duplications which are predominantly heterochromatic, the T, which is predominantly euchromatic and chromosome 4 which probably occupies an intermediate position. In spite of these differences, pairing behavior is closely related to metaphase length. Therefore, behavior at distributive pairing is clearly not a property of the euchromatin or heterochromatin per se, or of the ratio between the two, but rather of the entire chromosome.

Are there any grounds for assuming that size-dependent pairing at this time may be characteristic of all of the chromosomes of D. melanogaster? Analysis of the X-exceptions (Table 3 ) has shown that the matroclinous females never carry the translocation whereas the patroclinous males almost invariably do. Yet, the assortment of the X-duplication and chromosome 4 to the male and female exceptions is apparently random. Of the three elements, other than the X’s, available in the distributive pool, it is only the one closest in size to the X, namely the T,, which segregates nonrandomly from it. Similar results have been observed when the system is sensitized through the introduction of In( l )dl-49 into one X (GRELL and GRELL 1959). In this experiment, the X-duplication is absent so that the mother’s genotype is dl-49/+; T(3;4)86D / Vno;+. Here, X-nondisjunction reaches about 20 percent and again the T, is found almost exclusively in the male exceptions, although in the absence of the X-duplication it is frequently accompanied by the fourth chromosome. Studies of competitive pairing among compound chromosomes (E. H. GRELL, unpublished data) show that when the three chromosomes in the distributive pool are a compound X, a Y and a compound 4, in decreasing order of size, the X and Y pair preferentially and the compound 4, which is minimally involved, prefers the Y. Total Y association significantly exceeds 100 percent so that the trivalent association must be inferred with the Y directing the compound X and compound 4 to the same pole. Similarly, studies of a Y, a compound 4 and a free 4 (E. H. GRELL, unpublished) reveal the same behavior pattern. In this case, preferential pairing occurs between the free and compound 4 and the Y is minimally involved. Again trivalent association is indicated with the compound 4, which is now intermediate in size, directing the Y and free 4 to one pole. The experiments of DOBZHANSKY (1934) and STURTEVANT (1936) with a compound X, a Y, and a free X-duplication have shown a tendency for the small X-duplication and the large compound X to be recovered more frequently at one pole while the Y, which is intermediate in size, is recovered more frequently at the other. Finally ROBERTS’ (1962) investigations into crossing over and disjunction of a variety of heterozygous X-inversions demonstrate that X-nondisjunction, which is increased in the presence of a heterozygous inversion in either autosome, falls to control levels when such inversions are present in both autosomes. ROBERTS has suggested that either crossing over is so increased in X, through the interchromosomal effect, as to practi-


cally eliminate the simultaneous occurrence of noncrossover-X and autosomal tetrads, or alternatively, nonhomologous pairing occurs preferentially between chromosomes 2 and 3. Although an overall reduction in Eo’s undoubtedly occurs, preferential 2,3 pairing may be a contributory factor in the elimination of X-exceptions. If so, preference based on size similarity would again be indicated.

A question which arises is whether the critical size factor is the length of the chromosome arm or the length of the entire chromosome. The present experiments provide no answer since the chromosomes which were used are all acro- centrics. The experiments of E. H. GRELL, which tested a Y, a compound 4 and a free 4, suggest it may be chromosome rather than arm length which is the determinant, since here the compound 4 was found to behave as the intermediate element.

A consideration of the pertinent data reveals that the behavior of any two chromosomes in the distributive pool depends not only upon their lengths but also upon the residue of chromosomes in the pool. In the simplest situation, that of the normal genome, there is now convincing evidence that the fourth chromosomes are always members of the pool and that association between the 4’s, initiated at distributive pairing, is responsible for their regular segregation (R. F. GRELL, unpublished). Under normal conditions the X chromosomes are also members of the pool about 5 percent of the time (R. F. GRELL, 1962b). Despite the simultaneous availability of both the X’s and the 4’s, nondisjunction of both pairs is very low indicating that distributive pairing, in this circumstance, is largely confined to homologues. In view of the present results, regular segregation of the X’s and 4’s may be interpreted as a consequence of their difference in size. In fact, if the X’s are made part of the distributive pool over 90 percent of the time, as happens when Ins(l)dZ-49,BM1 are introduced into one X, X-nondisjunction remains less than 1 percent (COOPER, 1945).

On the other hand, if instead of two X and two fourth chromosomes, the distributive pool consists of two X’s, one 4 and the T, (when the genotype of the mother is dl-49/+; T(3;4)86D / Vno;+ (GRELL and GRELL 1959), X-nondisjunction reaches 19.8 percent and the T, is recovered in the nullo-X gametes about 99 percent. Surprisingly, in about 90 percent of the nullo-X gametes, the free 4 is also present. Thus, it appears that when a larger chromosome, the T,, is substituted for one of the 4’s, it will successfully engage one X with a rather high frequency and the other fourth chromosome can, in this situation, become involved with the other X, in spite of their size differences. Similarly, if only two chromosomes are present in the pool, one a large chromosome such as a Y and the other a small one such as the 4, association will occur between them about 84 percent of the time. While this frequency appears high, it is low by contrast with association values found here for an X-duplication and a 4 which is about 96 percent for the largest duplication studied and about 99.9 percent for a duplication very close to the 4 in length.

A different situation is encountered when three chromosomes constitute the distributive pool. If all three chromosomes are different in size, some trivalent formation occurs. A remarkable feature of the trivalents is that the element of intermediate length either invariably or predominantly, depending upon the

164 R. F. GRELL

model postulated, directs the other two elements to one pole while it proceeds to the other. The frequency of trivalents is also size-dependent such that the greatest number is found when the length of the intermediate element b is related to the length of the smaller and larger elements by the expression b = dz. When two of the chromosomes are approximately the same length and the third is different, bivalent, univalent association appears to occur. For the chromosomes used, the length ratio of the similar: dissimilar chromosomes is about 1 : 4. Finally, the remaining situation, in which all three chromosomes are more or less equal in length, has not been studied in these experiments. This category would probably include the XXY case and here trivalent association must, under certain conditions, again be invoked (COOPER 1948).

The competitive system described in these experiments constitutes an independent genetic method for the determination of fragment lengths. If a fragment of unknown size is introduced into a suitably marked Tp(3)Vno,Vno; ciD / T(3;4) 86D female, the disjunctive behavior it exhibits with respect to the free 4 and the T, chromosomes as well as its effect on the disjunction of the 4 and T, should assign i t to a position (Figure 3) which could be read on the abscissa as a specific metaphase length relative to chromosome 4.

Pairing for exchange is defined as occurring exclusively between homologous loci while distributive pairing, which may occur with extraordinarily high frequencies between nonhomologues (99.9 percent), is clearly not predicated on homology. These experiments were undertaken for the purpose of ascertaining what factor or factors are the determinants of distributive pairing. The results make it strikingly clear that it is either chromosome length o r some quality of the chromosome closely correlated with length such as mass, that is responsible for chromosome behavior at distributive pairing. By way of contrast, small free fragments may reduce crossing over 50 percent or more in the regions of duplication (DOBZHANSKY 1934) indicating that for exchange pairing, length is of minor o r negligible importance and homology is the prime determinant. Thus, the present findings disclose that distributive pairing, in its size dependency, possesses an additional characteristic which serves to distinguish it from exchange pairing.

The author is particularly indebted to DRS. KENNETH W. COOPER and JAKOV KRIVSHENKO for the use of their duplications without which this study could not have been made. These duplications were produced to test the pairing properties of heterochromatic regions with the view in mind of finding the location of pairing sites. The many valuable suggestions of DR. CHARLES STEINBERG in the treatment of the data are gratefully acknowledged.

SUMMARY

The relation of the size of two nonhomologues to the regularity of their association has been investigated in a noncompetitive situation by measuring the nondisjunction frequencies between a free fourth chromosome and a series of free X-duplications in the progeny of females of the genotype y2/y2/Dp( l ; f ) , yf; T(3;4)86D, red/T(3;4)86D, red; ci” mated to attached-XY, y B/Y males. The relation of the size of three nonhomologues to preferences in association has been


investigated in a competitive situation by measuring the nondisjunction frequencies between the three sets of chromosomes Dp-4, Dp-T, and 4-T, in the progeny of females of the genotype y2/ye/Dp(l;f), yf; T(3;4)86D, red/Tp(3)Vno, Vrzo; ciD mated to attached-XY/Y males.

In the noncompetitive situation, pairing efficiency between two nonrecombinant chromosomes, as measured by their nondisjunction frequency, is closely correlated with their similarity in length. If the metaphase length of chromosome 4 is arbitrarily set at 1 .O, nondisjunction between an X-duplication and chromosome 4 reaches maximal values of 4.5 and approximately 2.0 percent with the smallest ( G 0.3) and largest (2.5 to 4+) duplications, respectively, and minimal values of 0.02 to 0.13 percent with those duplications closest to the 4 in length (0.9 to 1.1).

In the competitive situation, pairing preferences among three nonrecombinant chromosomes, as interpreted from measurements of nondisjunction frequencies, are also strictly correlated with similarity of chromosome lengths. One of the three chromosomes appears to act as a univalent if the remaining two are identical or nearly so in length and the third is markedly different. For all other situations studied, some trivalent association occurs. The data suggest that maximal trivalents obtain when b = q/cwhere b represents the length of the intermediate chromosome and a and c the lengths of the other two. Disjunction from the trivaIent follows a pattern in which the chromosome of intermediate length, invariabIy or predominantly, directs the other two to the same pole.

It is concluded that distributive pairing is dependent on chromosome size in contrast to exchange pairing which is dependent on homology.

LITERATURE CITED

BRIDGES, C. B., and K. S. BREHME, 1944 The mutants of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 552.

COOPER, K. W., 1946 Normal segregation without chiasmata in female Drosophila melanogaster. Genetics 30: 47-4. - 1948 A new theory of secondary nondisjunction in female Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S. 34: 179-187. 1959 Cytogenetic analyses of major heterochromatic elements (especially Xh and Y) in Dro- sophila melanogaster and the theory of “heterochromatin.” Chromosoma 10 : 535-588.

-

COOPER, K. W., S. ZIMMERING, and J. KRIVSHENKO, 1955 Interchromosomal effects and segregation. Proc. Natl. Acad. Sci. US. 41 : 91 1-914.

DOBZHANSKY. TH., 1934 Studies on chromosome conjugation. 111. Behavior of duplicating fragments. Z. Ind. Abst. Vererb. 68: 134-162.

GRELL, E. H., 1959

GRELL, R. F., 1959

Zn(3R)Vno:Inversion(3R) with Vein off. Drosophila Inform. Serv. 33: 94.

The Dubinin effect and the Y chromosome. Genetics 44: 911-922. - 1962a A new hypothesis on the nature and sequence of meiotic events in the female of Drosophila melanogaster. Proc. Natl. Acad. Sci. US. 48: 165-172. - 196213 A new model for secondary nondisjunction: The role of distributive pairing. Genetics 47: 1737- 1754.

GRELL, R. F., and E. H. GRELL, 1959 Correlation of primary nondisjunction of X chromosomes with the segregation of certain autosomes in oocytes of Drosophila melanogaster. Drosophila

166 R . F. GRELL

Inform. Serv. 33: 137-139. __ 1960 The behavior of nonhomologous chromosomal elements involved in nonrandom assortment in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S. 46: 51-57.

LINDSLEY, D. L., and E. NOVITSKI, 1950 sophila Inform. Serv. 21 : 84-85.

The synthesis of an attached XY chromosome. Dro-

LINDSLEY, D. L., and L. SANDLER, 1958 The meiotic behavior of grossly deleted X chromosomes in Drosophila melanogaster. Genetics 43 : 547-563.

NICOLETTI, B., and E. B. LEWIS, 1960

ROBERTS, P., 1962

Tp(3)Vno: Transposition(3) Vein off. Drosophila Inform. Sen . 34: 53.

Interchromosomal effects and the relation between crossing-orer and nondisjunction. Genetics 47: 1691-1 709.

STURTEVANT, A. H., 1936 Preferential segregation in triplo-IV females of Drosophila melanogaster. Genetics 21 : 444-466.

Documents

CHROMOSOME SIZE AT DISTRIBUTIVE PAIRING IN