Two kinds of variation in linkage values in Drosophila experiments are well known but poorly understood. One is the change in crossover values with increasing age of the female as shown by Bridges [1]; the other is excessive variation from female to female as pointed out by Gowen [2]. Studies

reported in the present paper on an age effect in a special situation may contribute to the interpretation of both problems. They also serve as controls for the irradiation paper which follows [3]. 



The chief result of aging is a rapid decrease in recombination values during the first six days of egg-laying, particularly at or near the spindle attachment of the chromosome. After that period smaller changes consist, with variations, of a slight rise and second fall. In recognition of this Bridges [4] has defined as a condition for chromosome mapping the use of data from young females. From such data inferences are made as to the amount of actual crossing over which has occurred between genes which are assumed to be heterozygous at the time when the germ cells enter meiosis. That this procedure may overestimate the amount of crossing over has been elaborated by the senior author both by reasoning back from observable clustering of the data [5] and by deducing certain consequences of crossing over premeiotically, in gonial cells [6]. The present experiment shows an age effect and clustering attributable to oogonial crossing over in the same body of data, where meiotic crossovers have been greatly reduced or eliminated by inversions

Hinton, Claude W.

Whittinghill, M.

Caltech Authors - Main

GENETICS: WHITTINGHILL AND HINTON PROC. N. A. S.
* This investigation was supported in part by a research grant from the National
Institute of Health, U. S. Public Health Service. From the Department of Zoology
and Entomology and the Department of Botany and Plant Pathology (No. 519), Ohio
State University.
1 Beadle, G. W., Chem. Revs., 37, 15-96 (1945).
2 Irwin, M. R., Adv'. Genetics, 1, 133-159 (1947).
* Fox, A. S., Genetics, 34, 647-664 (1949).
4Delbruck, M., in Bonner, D., Cold Spring Harbor Symp. Quant. Biol., 11, 14-22
(1946).
' Hungate, M. V., unpublished M.A. dissertation, Stanford University (1945).
6 Houlahan, M. B., Beadle, G. W., and Calhoun, H. G., Genetics, 34, 493-507 (1949).
7 Fox, A. S., and Gray, W. D. (in preparation).
8 Spiegel-Adolf, M., Biochem. J., 31, 1303-1310 (1937).
9 Baker, M. R., and Andrews, A. C., Genetics, 29, 104-112 (1944).
10 Ginsburg, B. E., Ibid., 29, 176-198 (1944).
11 Nelson, J. M., and Dawson, C. R., Advances in Enzymology, Interscience Pub-
lishers, Inc., New York, Vol. 4, 99-152 (1944).
12 Wagner, R. P., PROC. NATL. ACAD. Sci., 35, 185-189 (1949).
18 Kopac, M. J., Spec. Pubi. N. Y. Acad. Sci., 4, 423-432 (1948).
14 Wright, S., Biological Symposia, 6, 337-355 (1942).
16 Russell, L. B., and Russell, W. L., Genetics, 33, 237-262 (1948).
16 Markert, C. L., Ibid., 35, 60-75 (1950).
17 Wright, S., Ann. Rev. Physiol., 7, 75-106 (1945).
THE NATURE OF LINKAGE VARIATION WITH AGE IN
INVERSION HETEROZYGOTES OF DROSOPHILA
MELANOGASTER
By M. WHITTINGHILL AND CLAUDE W. HINToN*
DBPARTMENT OF ZOOLOGY, UNIVBRSITY OF NORTH CAROLINA
Communicated by J. N. Couch, August 17, 1950
Two kinds of variation in linkage values in Drosophila experiments are
well known but poorly understood. One is the change in crossover values
with increasing age of the female as shown by Bridges'; the other is ex-
cessive variation from female to female as pointed out by Gowen.2 Stud-
ies reported in the present paper on an age effect in a special situation may
contribute to the interpretation of both problems. They also serve as
controls for the irradiation paper which follows.'
The chief result of aging is a rapid decrease in recombination values
during the first six days of egg-laying, particularly at or near the spindle
attachment of the chromosome. After that period smaller changes con-
sist, with variations, of a slight rise and second fall. In recognition of this
Bridges4 has defined as a condition for chromosome mapping the use of
546
GENETICS: WHITT7NCHILL AND HINTON
data from young females. From such data inferences are made as to the
amount of actual crossing over which has occurred between genes which
are assumed to be heterozygous at the time when the germ cells enter meio-
sis. That this procedure may overestimate the amount of crossing over
has been elaborated by the senior author both by reasoning back from ob-
servable clustering of the data5 and by deducing certain consequences of
crossing over premeiotically, in gonial cells.6 The present experiment
shows an age effect and clustering attributable to oogonial crossing over in
the same body of data, where meiotic crossovers have been greatly reduced
or eliminated by inversions.
The age effect in this stock was encountered by accident. In the course
of stockmaking by the senior author it was noticed that the infrequent
crossovers from al2 Cy It' L4 sp2/S Pfd females appeared in early cultures
but not in later transfers. Of 14 crossovers in the Cy-L interval, 12 were
found in the first two cultures and none at all in the last two of a series of
five transfers. It was not clear whether this was an accidental result until
a larger experiment had been carried out by the junior author and a
genuine age effect found.
The experiment was planned with several improvements on the original
mass matings. By testcrossing to light males use was made of the It
locus, which differentiated the crossovers into two regional kinds. The
map order of the loci, the number designations of the crossover regions and
the extent of the inversions may be diagrammed as follows:
S Pfd
-Cy
---
1 -----tL34_
1 2 3 4
The spindle attachment lies to the right of It. In the experiment, females
were mated individually to these light males, so that the contribution of
each family to the totals would be known. Furthermore, the data for each
family came separately from five consecutive coded cultures which were
not decoded and summed as to families until the experiment was over.
Results and Discussion.-This experiment confirmed the age effect which
had been encountered in mass mating. Hence data from both tests have
been combined in table 1. In the first two cultures crossovers comprised
about 1% of the offspring, in the third they were 0.25% and in the last two
transfers practically zero. Almost all of the crossovers came from eggs.
laid during the first five days of adult life. A chi-square test showed that
this was a highly significant departure from the mean of all 11 days. We
were therefore dealing with an age effect exhibited by these crossovers.
The data were next examined family by family to see whether the cross-
overs had appeared at random or in clusters. Random distribution would
be in accord with the usual assumption that crossing-over occurs at meiosis,
,VoL. 36, 195 547
GENETICS: WHITTINGHILL AND HINTON PRoC. N. A. S.
meaning that no more than one crossover chromatid from each tetrad is re-
coverable by breeding. Clustering would indicate that there was some
gonial influence7 either (a) completed crossing-over in a gonial cell, or (b)
weakening of a certain place in a chromosome followed later by crossing-
over in identical regions in numerous related primary oocytes. Table 2
shows agglutination of the data, as mathematicians call it, after adding up
the cases of crossovers in the two regions of the eighteen families. Al-
though a majority of the 36 cases had no crossovers, paradoxically three-
fourths of the crossovers appeared in groups of two or more, as if from the
same event of exchange. Furthermore the cases of solitary crossovers, 8,
seem too low for a Poisson distribution. Some probabilities will be pre-
sented in later paragraphs.
TABLE 1
SUMMARY OF SINGLE CROSSOVERS RECOVERED FROM Star Pufdi/Curly light Lobe4
INVERSION HETEROZYGOTES IN BOTH EXPERIMENTS ACCORDING TO AGE OF FEMALES
IN DAYS
Cultures 1 2 3 4 5
Age of female 0-3 3-5 5-7 7-9 9-11
Crossovers 17 20 4 1 1
Total offspring 1747 2062 1677 1752 1886
TABLE 2
AGGLUTINATION IN THE FINDING OF SINGLE CROSSOVER FLIES, FOR EITHER REGION,
AMONG 18 FAMILIES OF S Pfd/Cy It L4 INVERSION HETEROZYGOTES
CROSSOVERS/FAMILY CASES TOTAL CROSSOVBRS
0 19 0
1 8 8
2 7 14
3 1 3
4 1 4
Totals 36 29
NoTE: Inclusion of seven multiple crossovers would increase the numnbers of pairs and
especially of triplets.
More detailed familial data appear in table 3. The smallest family,
No, 1, contained 4 crossovers in region 2, and these were not all from the
same culture, so that they tend to confirm each other. The next smallest
family had 3 crossovers in region 3, all found in the same culture. In
seven other cases 2 crossovers of a kind were found, and more often than
not these were in separate cultures rather than together. Many families,
representative in size, failed to have any crossovers in one or both regions.
Because of this clustering the crossover values showed great variability
among the different families, from about 5 per cent to zero.
One may compute the probability that these are merely chance deviations
548
VOL. 36, 1950 GENETICS: WHlTTINGHILL AND HINTON
from randomness. In region 2 the over-all frequency of crossovers is ap-
proximately 0.002, (15/7534 from table 3). In ten families having some
4700 offspring no such crossovers were found, and the probability of this as
a random result is 0.9984700, or 0.00008. On the same hypothesis, the
probability of drawing a sample of 108 including as many as 4 similar
crossovers (Family 1) is 0.0008. Then the joint probability of occurrence
of these two aspects of the experiment is 6.4 X 10-8. Inclusion of the
fact that pairs of crossovers were found more often than singles would make
it even more improbable that the crossovers in region 2 were independently
determined.
TABLE 3
SumiARY OF TESTCROSS DATA FROM 84 FERTILE, CODED CULTURES AS REASSEMBLED
INTO THE ORIGINAL 18 FAmiLIES AFTER CLASSIFICATION. FAMILIES LISTED IN ORDER
OF INCREASING SIZE
FAMILY SINGLE CROSSOVERS MULTIPLE
NO. TOTAL PROGENY REI:GION 2 REGION 3 CROSSOVERS
1 108 4a 1
2 135 1 3
3 268 1 0
4 387 0 1
5 408 1 2a
6 417 2 0
7-11 5X439.4 0 0
12 445 2a 2
13 481 0 0 S Cy;' It, wild.
14 490 2a 1
15 520 0 2 Cy L
16 533 2' 1
17 533 0 1 S lt Pfd
18 612 0 0 S It Pfd L;6 L.
Totals 7534 15 14 7
aDenotes crossovers obtained from two different coded cultures.
Similar calculations for region'3 show that events there are, very far
from random. The probability that famili; containing aLtotal of 3975 off-
spring would have no crossovers, if the latter were randomly distributed at
a frequency of 0.183%, is 0.998173975, which is less than 0.0006. Turning
to the other end of the distribution we find that the probability of getting
as many as 3 crossovers in a family of only 135 offspring is 0.0157 on the
same assumption. The joint probability for the two extremes is more re-
mote, and the further joint probability for region 2 crossovers and region 3
crossovers is 6 X 10-15.
Up to this point the negative aspects of data of table 3 have been dis-
cussed. The kind of non-randomness pointed out may be explained by a
549.
GENETICS: WHITTINGHILL AND HINTON PROC. N. A. S.
limited amount of gonial crossing over plus gonial multiplication. While
this is perhaps the better explanation, there is another interpretation which
could reserve crossing over until meiosis. Thus a tendency to produce
crossovers near the spindle attachment regardless of region may be pos-
sessed by some heterozygotes more than by others. In table 3 one may
see that pairs and larger clusters in one region are almost always accom-
panied by at least one crossover in the adjacent region. This is true in
6 out of 8 families; only females No. 6 and No. 15 had 2 crossovers in one
region without any single exchanges in the other. Yet a double crossover,
a Curly Lobe fly, appeared in family No. 15 in the same culture with 2
Star Lobe single crossovers. This makes 7 cases of crossing over adjacent
to the regions represented by the 8 clusters.
Further evidence for this alternative may be obtained from the multiple
crossovers as a whole, which have therefore been included in table 3, even
though they are not fully understood. The most plausible multiple cross-
overs are the Curly Lobe fly just mentioned and the Star light Pufdi of
family No. 17. These could be region 2-3 double crossovers, and each was
formed in a female which also had one or two single crossovers in region 3.
The expectation for these double crossovers in an entire experiment of this
size is less than one fly even if figured on the basis of the initial high point
of recombination values, 1%, and with no interference. The other multiple
crossovers are far less likely than the preceding. The finding of a wild and
a light fly, the latter verified by breeding, in the same culture of family
No. 13 might be explained as contamination; or these might be triple
crossovers, or even quadruples, in some way related to a Star Curly quad-
ruple crossover independently classified in a previous culture. In the
subsequent experiment in which similar heterozygotes were irradiated
(Hinton and Whittinghill3), two more light phenotypes were found in
different cultures, so it remains a possibility that this It fly was a 1-2-4
triple crossover. It is indeed strange that there were no single crossover
offspring in this family which had two different triples and a quadruple
crossover. In one other family, 18, single crossovers were lacking. The
only crossovers in this the largest familyS of all, were a S It Pfd L4 2-3-4
triple and a veri4d L4 1-3 dbuble. Neither kind was detected in the
next experiment. However the sporadic nature of the multiple crossovers
may be viewed as an extension of the non-randomness already demon-
strated among the single crossover types of regions 2 and 3. No single
crossovers were recovered from regions 1 or 4 due to the inversions there.
Summary.-There is a decrease in the frequency of crossover offspring
from S Pfd/Cy It L4 females as they age. Crossover values drop prac-
tically to zero in seven days, in agreement with the previously known
decrease from normal females. Variability in crossover values from family
to family was greater than one would expect by chance if each crossover
550
VOL. 36, 1950 GENETICS: WHITTINGHILL AND HINTON
had been formed separately and independently of every other crossover,
the usual assumption about meiosis.8 Two better explanations have been
considered. An entire cluster of crossovers may represent only one actual
crossing over in an oogonial cell which was the common ancestor of several
eggs. Such an hypothesis is preferred, but an alternate explanation may
fit the data of this one experiment without recourse to gonial crossing over.
The clustering may begin with a gonial conditioning, such as a permanent
weakening of a chromosome, which may influence the location of several
later meiotic crossings over. Because identical crossovers and adjacent
crossovers tended to be found in the same families in this favorable stock
of flies, either of the new hypotheses is appropriate for the present data.
Great variability of spontaneous crossover values near spindle attachments
would seem to be inescapable in experiments based on the early broods
prescribed by Bridges. Hence the collection of data for maps might well
be done from a later egg-laying period and, particularly, from large numbers
of females to get a measure of the variability.
Acknowledgments.-These experiments were substantially aided by a
grant from the Carnegie Research Fund of the University of North Carolina
during the winter of 1949, for which we are very grateful. For aid in mak-
ing certain calculations we wish to thank Dr. A. S. Householder, Head of
the Mathematics Panel, Oak Ridge National Laboratory. We thank
Miss Margaret Stewart of this laboratory for coding the 90 cultures to hide
their familial identity.
* Present address: Biology Division, Calif. Inst. of Technology, Pasadena.
1 Bridges, C. B., Carnegie Inst. Wash. Pub. 399, 63-89 (1929).
'Gowen, J. W., Genetics, 4, 205-250 (1919).
' Hinton, C. W., and Whittinghill, M., these PROCEEDINGS, 36, 552-558 (1950).
4 Bridges, C. B., and Brehne, K. S., Carnegie Inst. Wash. Pub. 552, 6 (1944).
' Whittinghill, M., Genetics, 32, 608-614 (1947).
' Whittinghill, M., Ibid., 35, 38-43 (1950).
7 Whittinghill, M., Oak Ridge Nat'l. Lab. Report 817. Health & Biology, (1950)
' Gowen, J. W., these PROCEEDINGS, 15, 266-268 (1929).
551


The Nature of Linkage Variation with Age in Inversion Heterozygotes of Drosophila melanogaster

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The Nature of Linkage Variation with Age in Inversion Heterozygotes of Drosophila melanogaster

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