That crossing may occur in oogonial cells was suggested by the results of experiments by Whittinghill [1] in which crossing over was induced by x-rays in the X-chromosomes of Drosophila females homozygous for the c3G asynaptic factor. It had previously been demonstrated that induced crossovers recovered from Drosophila males were of spermatogonial origin [2,3]. Cooper's observations [4] of chiasmata in gonial cells of both sexes of Drosophila may provide cytological foundations for the hypothesis of gonial origin of some crossovers, particularly those which are x-ray induced. Somatic crossing over, the basis for the occurranece of twin spots in the hypodermis of Drosophilamales and females [5], closely parallels gonial crossing over in the formation of daughter cells which have become homozygous distally. The possible consequences of oogonial crossing over upon linking data, as recently discussed by Whittinghill [6], illustrate the need for further investigation of this phenomena. This paper reports the results of x-ray induced crossing over in Curly inversion heterozygotes of Drosophila melanogaster females, and these results are interpreted in relation to normal, or random, meiotic events as opposed to oogonial events of recombination

Hinton, Claude W.

Whittinghill, Maurice

English

Caltech Authors - Main

GENETICS: HINTON AND WHITTINGHILL PROC. N. A. S.
THE DISTRIBUTION OF X-RA Y INDUCED CROSSOVERS FROM
CURLY INVERSION HETEROZYGOTES OF DROSOPHILA
MELANOGASTER FEMALES
By CLAUDE W. HINTONt AND MAURICE WHITTINGHILL
DEPARTMENT OF ZOOLOGY, UNIVERSITY OF NORTH CAROLINA
Communicated by J. N. Couch, August 17, 1950
Introduction.-That crossing over may occur in oogonial cells was sug-
gested by the results of experiments by Whittinghill' in which crossing over
was induced by x-rays in the X-chromosomes of Drosophila females homo-
zygous for the c3G asynaptic factor. It had previously been demonstrated
that induced crossovers recovered from Drosophila males were of sperma-
togonial origin.2 3 Cooper's observations4 of chiasmata in gonial cells
of both sexes of Drosophila may provide cytological foundation for the hy-
pothesis of gonial origin of some crossovers, particularly those which are
x-ray induced. Somatic crossing over, the basis for the occurrence of twin
spots in the hypodermis of Drosophila males and females,5 closely parallels
gonial crossing over in the formation of daughter cells which have become
homozygous distally. The possible consequences of oogonial crossing over
upon linkage data, as recently discussed by Whittinghill,6 illustrate the
need for further investigation of this phenomenon. This paper reports
the results of x-ray induced crossing over in Curly inversion heterozygotes
of Drosophila melanogaster females, and these results are interpreted in re-
lation to normal, or random, meiotic events as opposed to o6gonial events
of recombination.
Experimental Methods.-Stocks and experimental prQcedures, with the
exception of the treatment employed in this work, were identical with
those described in the companion paper7 in these PROCEEDINGS. Twenty-
eight adult virgin females between six and twelve hours old were placed
in a 0.7-ml. gelatin capsule and exposed to a total x-ray dose of 2250 r,
which was received at the rate of 350 r per minute from a distance of 20
cm. through a 4-mm. aluminum filter from a tube operating at 140 kv.
These females were allowed to feed for one day before being testcrossed
separately to 4 or 5 It/lt males in consecutive half-pint bottles for life.
Transfers, without etherization, were made every other day for the first
seven cultures and at longer intervals for the final three cultures. As in
the former experiment, all cultures were coded so that classifications were
performed without bias. Uncertain phenotypes were determined by test-
crosses to light homozygotes. Although the spontaneous recombination
data of the preceding report have been employed to some extent as con-
trols, accurate comparison of the frequency of crossing over is not possible
because of the absence of randomly determined recombination offspring.
552
VOL.36,1950 GENETICS: HINTON AND WHITTINGHILL 553
The authors have concentrated their study on the actual distribution of in-
duced recombinants in an attempt to detect the place and manner of their
origin.
Results.-Although fertility of the irradiated adults was decidedly lower
than that of controls, recombination values were greatly increased through-
out the egg-laying period (table 1). This persistence of the increase in re-
combination for as long as 31 days after treatment may have been the re-
sult of early as well as late oogonial cells having been affected. Any in-
fluence of age on spontaneous recombination is obscured by the much
larger effect of the x-rays. This effect included a real peak in recombina-
tion between 4 and 8 days after treatment, as shown by the standard
errors for cultures 3 and 4 in table 1.
TABLE 1
COMPARATIVE EFFECTS OF AGE (C) AND X-RAYS (X) ON RECOMBINATION VALUES IN
CONSECUTIVE TESTcRoSs CULTURES OF S Pfd/Cy It L4 FEMALES
CULTURE FERTILE FEMALES TOTAL PROGENY PER CBNT RECOMBINATION
NUMBER (C) (X) (C) (X) (C) (X)
1 18 20 1334 60 0.82 0.24 5.00 2.81
2 18 21 1681 532 0.83 * 0.22 3.95 0.27
3 18 16 1414 250 0.14 i 0.10 10.80 1.96
4 18 14 1511 549 0.07 7.47 1.12
5 18 13 1594 379 0.06 5.01 1.12
6 13 631 3.65 0.75
7 12 467 1.93 0.64
8 9 390 2.82 0.84
9 6 427 4.22 0.97
10 1 34 11.76 5.52
Totals 7534 3719 0.38 l-0.07 4.73 0.35
Three results which are unexpected in random meiotic processes are re-
vealed when the data are recombined into familial groups (table 2). The
first notable feature of the table consists of large deviations from the mean
total recombination value of 4.73 = 0.35 per cent. These values range
from zero to above 12 per cent. Secondly, the usual assumption of linearly
random exchanges is not substantiated due to the wide variations in pro-
portions of region 2 to region 3 crossovers as exhibited chiefly in the families
of Females 5, 11 and 13, as compared with 16. Thirdly, pronounced in-
equalities in complementary crossover classes within a region may be ob-
served in the crossover columns of Females 5, 11, 16 and 23. All of these
three forms of variation from female to female have been shown to be ex-
pected consequences of oogonial crossing over" 6 and may be interdependent
to some extent. The most diagnostic of these consequences, and the one
which lends itself most readily to analysis, is the extent of imbalance be-
tween complementary crossover classes.
GENETICS: HINTON AND WHITTINGHILL PROC. N. A. S.
TABLE 2
FAMLIAL DISTRIBUTION OF TESTCROSS OFFSPRING FROM IRRADIATED ADULT
S Pfd/Cy It L' FEMALES
FEMALE NON-CROSSOVERS SINGLE CROSSOVBRS
NUMBER S Pfd Cy It L4 RBGION 2 RBGION 3
1 96 98 3 0 2 2
2 128 146 5 2 5 2
4 86 95 2 3 3 0
5 274 261 13 7 0 0
8 120 135 1 1 1 0
11 103 85 8 2 0 1
12 33 25 3 3 1 1
13 135 136 9 10 4 2
15 7 8 0 0 1 0
16 225 233 1 1 12 5
18 238 230 5 7 6 5
19 62 59 4 2 2 0
20 36 49 0 1 1 0
22 21 33 3 1 1 3
23 58 62 0 2 5 0
26 85 91 5 2 2 1
12 others 39 52 0 0 0 0
Totals 1746 1798 62 44 46 22
TOTAL PBI;I C]NT
PROGENY RECOMBINATION
201 3.48 * 1.29
288 4.86 * 1.27
189 4.23 *1.46
556 3.96- 0.83
258 1.16 0.67
199 5.53 1.62
66 12.12 4.03
296 8.45 1.62
16 6.25 6.06
477 3.98 *0.90
491 4.68 0.95
129 6.20 2.12
87 2.30 1.67
62 12.90 4.26
127 5.51 2.02
186 5.38 1.65
91 0
3719 4.73 * 0.35
* Includes 1 S It Pfd double crossover.
TABLE 3
CH SQUARE ANALYSIS OF THE DISTRIBUTION OF COMPLEMENTARY CROSSOVERS WITHIN
FAmiLIES OF IRRADIATED FEMALES
IREGION 2 REGION 3
FEMALB S IsL4: S L4:
NUMIBER Cy Pfd p -LOGOe Cy It Pfd p -LOGS
1 3:0 0.125 2.080 2:2 0.500 0.693
2 5:2 0.227 1.483 5:2 0.227 1.483
4 2:3 0.500 0.693 3:0 0.125 2.080
5 13:7 0.132 2.025 (0:0) ... ....
8 1:1 0.500 0.693 (1:0) ... ....
11 8:2 0.055 2.904 (0:1)
...
....
12 3:3 0.500 0.693 1:1 0.500 0.693
13 9:10 0.500 0.693 4:2 0.344 1.067
16 1:1 0.500 0.693 12:5 0.072 2.632
18 5:7 0.387 0.950 6:5 0.500 0.693
19 4:2 0.344 1.067 2:0 0.250 1.386
22 3:1 0.313 1.161 1:3 0.313 1.161
23 0:2 0.250 1.386 5:0 0.031 3.478
26 5:2 0.227 1.483 2:1 0.500 0.693
Total -log, 18.004 16.059
Chi-square (28 d. f.) 36.008 (22 d. f.) 32.118
Probability 0.154 0.079
Joint Probability = 0.0121
554
VOL. 36, 1950 GENETICS: HINTON AND WHITTINGHILL
In order to answer the question whether the observed deviations from
equality are greater than those allowed by chance, the following analysis
was performed. Probabilities of obtaining the observed distribution of
complementary crossover classes in each family or of any distribution de-
viating more in the same direction were computed (table 3). The -log.,
equal to chi2/2 associated with two degrees of freedom, is additive in char-
acter,8 and this allows an over-all probability to be obtained for either re-
gion for all families. Since the two regions concerned are on opposite
sides of the centromere, one may assume that crossings over occurring
simultaneously in these regions are independent of each other. Distribu-
tions of 1: 0 or 0:1 have been omitted from calculations, for although they
add two degrees of freedom to the total, they can contribute no useful in-
formation here.
While there are no individual probabilities in the above table which are
significant alone, there appears to be a definite excess of distributions of low
likelihood as shown by the probabilities of 0.154 and 0.079 for regions 2
and 3, respectively, all families combined. Furthermore, the joint prob-
ability for both regions is 0.0121, so that a similar over-all distribution
may be expected to occur by chance alone only one time in one hundred
like experiments.
Viability differences in crossover chromosomes may sometimes account
for numerical differences in complementary crossover classes throughout
an experiment; however, this possibility may be reasonably eliminated
here. Six additional testcrosses of males whose genotypes corresponded
to a combination of complementary crossover chromosomes from region 2
exchanges yielded 706 S It L4 and 792 Cy Pfd flies. This inequality is in
the opposite direction from that in the crossover progeny from testcrossed
females which gave a 62:44 majority to the S It L4 class. As there is no
reason to suspect that the relative viabilities of the two chromosomes may
be reversed depending on whether they originate from sperms or eggs,
some cause other than viability differences must be responsible for the
observed inequalities in complementary crossover offspring from irradiated
females. Similar tests yielding 579 SL4: 530 Cy It Pfd indicate that
larval viability differences played little or no part in the 46:22 result
from region 3.
Previous studies of induced crossing over in Drosophila have revealed
a tendency of some recombinants to appear simultaneously among the test-
cross progeny, and several instances of this irregularity occurred in this
experiment. A cluster of 6 region 2 recombinants distributed equally
was recovered from the third culture of Female 13 among a small brood of
19 flies which included only one crossover from the third region. By
comparison, the mean brood size per fertile culture was found to be 33.50
of which 1.58 flies would be expected to be recombinants, assuming a ran-
555
GENETICS: HINTON AND WHITTINGHILL PROC. N. A. S.
dom distribution. The fourth culture of Female 4 contained only 3 re-
combinants, identical region 2 triplets, among a total brood of 42. At
this same age, Females 11 and 18 each produced identical quadruplets
from region 2, and each set was accompanied by twins of the complemen-
tary class; in addition, the latter female had twins from region 3. While
these examples are from the two cultures which exhibited the maximum re-
combination values, clustering was not limited to this period. It is very
unusual that although Female 5 produced 7 region 2 recombinants out of 182
offspring in Culture 9 (20-26 days), region 3 recombinants were not repre-
sented; the Culture 10 (27-31 days) brood of 34 from this same female in-
cluded 4 crossovers, all from region 2. Other clusters of lesser magnitude
might be cited, and it is possible that some clusters were missed due to
splitting them when transferring the cluster-producing female into a new
culture.
Two light homozygotes, classified with the broods of Females 18 and 23,
have not been included in the data because of the possibility of their being
contaminants. Yet, they are not too improbable representatives of quad-
ruple crossing over when compared with similar multiple crossovers which
appeared in controls. Furthermore, in order to recover any recombinants
from a region included in an inversion, at least double crossing over must
occur since single exchanges alone lead to the production of lethal dicentrics
and acentric chromosome fragments.9 One is also reminded that although
the Curly inversions do severely limit the frequency of single exchanges in
adjacent non-inverted regions, the non-random occurrence of recombinants
resulting from such exchanges is not explained by the presence of these in-
versions.
Discussion.-If an exchange of homologous segments of paired bipartite
prophase chromosomes should occur in only a relatively few oogonial cells,
and the products of such an exchange be erratically reproduced before the
initiation of meiosis, one may well expect the resulting recombination off-
spring to be distributed non-randomly as recorded in this experiment.
Or, as suggested by the distribution of spontaneous recombinants from
Curly inversion heterozygotes, oogonial exchange of homologous segments
may not be necessary for agreement with the results if only a localized
weakening of one chromatid at this time may predispose meiotic crossing
over at this point after several mitotic divisions have intervened. It
seems plausible that these alternatives could be tested by a cytological
examination of the first meiotic division, for if exchange occurred prior to
this division, the regions distal to the break and including the inversion
would frequently become homozygous, allowing normal synapsis in this
arm and free chiasma formation.
On either hypothesis, it is necessary to assume unequal multiplication of
the affected chromosomes, and the agencies responsible for this may be
556
VOL. 36, 1950 GENETICS: HINTON AND WHITTINGHILL
several. For example, different rates of division might be conditioned
by the exchange products themselves, or could be imposed by chance ac-
cording to the relative positions the daughter oogonia occupy in the
ovary; some of them may be crowded aside from the main line of division
either to be lost entirely or relegated to later appearance. However,
some causal factor other than larval viability differences must operate pref-
erentially in one direction in order to explain the total inequalities in conm-
plementary crossover adults. Cell lethals could presumably explain this
if some oogonia become homozygous due to prior exchange.
Applying this idea to the present experiment, we may assume that Star
and Curly may become homozygous in adjacent cells after region 2 ex-
changes, and that Pufdi and Lobe can become homozygous after region 3
exchanges. If any of these mutants are homozygous cell lethals (all are
homozygous lethal in larvae), they could not cause more than a 50 per cent
reduction of any crossover chromosome, since such chromosomes may seg-
regate into heterozygous situations as frequently as into the homozygous
condition. We would then infer from the 46 S L4 :22 Cy It3 PJd ratio of
recombinants that Pufdi is completely lethal to cells homozygous for this
factor; i.e., in S Pfd/Cy It Pfd gonia. In the case of region 2 exchanges,
Curly would appear to be detrimental, although not completely lethal, to
o6gonial cells homozygous for it as judged from the ratio of 62 S It3 L4
to 44 Cy Pfd recombinants from this region. While this hypothesis is de-
pendent on oogonial exchange, other possibilities may be found which
agree equally well with o6gonial weakening and subsequent meiotic cross-
ing over.
Two hypotheses of the mode of action of x-rays in inducing crossing
over have appeared in the literature, and these may be briefly considered.
In attempting to demonstrate a single mechanism of crossing over for both
male and female Drosophila, Friesen10 found that the Curly inversions did
not limit x-ray induced crossing over between black and cinnabar in males
as much as in females. On the basis of this evidence, the lack of sponta-
neous male crossing over was ascribed to an incomplete conjugation of
homologous chromosomes (the greater the normal degree of synapsis, the
greater the depressing influence of the inversions). Friesen further ex-
plained the action of x-rays while increasing recombination to result in "a
closer intercourse of the long autosomes (first of all in the central regions)"
for both sexes. This hypothesis obviously fails to account for the non-
random occurrence of recombinants in our experiments, unless it is applied
to the chromosomes of gonial cells. Friesen's omission of this application
apparently indicates abandonment of his own earlier proposal that cross-
overs induced in males were of spermatogonial origin.
Another possible mode of action of x-rays in inducing crossing over which
more nearly conforms to our experimental results has recently been ad-
557
GENETICS: HINTON AND WHITTINGHILL PROC. N. A. S.
vanced by Cooper4 as a result of observations of chiasma-like configura-
tions in Drosophila gonial cells of both sexes. While it is clear that such
chiasmata do not regularly lead to spontaneous recombination, at least in
the male, Cooper supposes that these configurations may be competent
targets for ionizing particles, and he concludes that x-ray induced cross-
overs are probably of gonial origin. This would be the place of origin of
the crossovers found in this experiment in view of the tendency for com-
plementary crossovers to vary in either direction from the expected.
Summary.-The induced crossover offspring from testcrossed S Pfd/Cy
It' L4 females x-rayed with 2250 r as adults were so distributed that their
meiotic origin is unlikely. The effects of the x-rays were evident for as long
as 31 days, with a peak in recombination between 4 and 8 days after treat-
ment. Wide variations from female to female were observed in total per
cent recombination, in the relative numbers of recombinants from adja-
cent regions, and in the distribution of complementary crossover classes
within each region. Some tendency toward clustering of the recombinants
among the testcross offspring was revealed. These results are indicative
of some oogonial influence upon recombinants. This influence may be
merely a weakening of oogonial chromosomes followed much later by
meiotic crossing over at the weak point, or it may be completion of cross-
ing over in the o6gonia.. The data on the variable balance of complemen-
tary classes favors the latter hypothesis.
Acknowledgments.-The authors wish to express their appreciation to Dr.
Robert James Reeves, Head of the Department of Roentgenology of Duke
University, for making x-ray facilities available; and to Mr. H. C. Yeat-
man of this Department for coding the testcross cultures.
t Present address: Biology Division, Calif. Inst. Tech., Pasadena.
1 Whittinghill, M., Genetics, 23, 300 (1938).
2 Whittinghill, M., Ibid., 22, 114 (1937).
Friesen, H., Z. i. A. V., 71, 501 (1936).
4 Cooper, K. W., J. Morph., 84, 81 (1949).
cStern, C., Genetics, 21, 625 (1936).
o Whittinghill, M., Ibid., 35, 38 (1950).
7 Whittinghill, M., and Hinton, C. W., these PROCEnDINGS, 36, 546-551 (1950).
8 Fisher, R. A., Statistical Methods for Research Workers, 10th ed., 99 (1943).
' Sturtevant, A. H., and Beadle, G. W., Genetics, 21, 554 (1936).
Friesen, H., J. Genetics, 35, 141 (1937).
558


The Distribution of X-Ray Induced Crossovers from Curly Inversion Heterozygotes of Drosophila melanogaster Females

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The Distribution of X-Ray Induced Crossovers from Curly Inversion Heterozygotes of Drosophila melanogaster Females

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