San Fernando Valley State College
EFFECTS OF THE CENTROMERE AND HETEROCHROMATIN ON
RECOMBINATION IN DROSOPHILA MELANOGASTER
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Joan Lily Bartholomew
August, 1969
The thesis of Joan Lily Bartholomew is approved:
San Fernando Valley State College
August, 1969
ii.
ACKN OWLEDGJ:.:lENT 8
For the quality of his advice
and direction and for his
unfailing forbearance,
grateful to Dr.
I am
George Lefevre,
iii
Jr.
TABLE OF CONTENTS
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'LIST OF TABlES
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ABSTRACT
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Chapter
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INTRODUCTION.
II. REVIEW OF THE LITERATURE
III.
IV.
v.
MATERIALS AND METHODS
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RESULTS
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DISCUSSION.
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Recombination
homozygotes
Recombination
homozygotes
Recombination
zygotes
Recombination
zygotes
BIBLIOGRAPHY
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in inversion sc8
in inversion rstJ
in wVco heteroVco homoin 'tf.__
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LIST OF TABLES
Table
1
2
3
Page
Crossing Over in Homozygous sc8
Inversion
Crossing Over in Homozygous rstJ
Inversion
The Effect of wVco on Recombination
in the Third Chromosome
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('"··--·-----····
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,
I
ABSTRACT.
EFFECTS OF THE CENTROMERE AND HETEROCHROMATIN ON
RECOMBINATION IN DROSOPHILA MELANOGASTER
by
Joan Lily Bartholomew
Master of Science in Biology
August, 1969
Effects of the centric heterochromatin and the
centromere on recombination frequency were investigated
with the use of two paracentric inversions of the
X
chromosome and a heterochromatic, pericentric inversion
of the third chromosome.
The results clearly support
the hypothesis that crossing over is inhibited in regions
immediately adjacent to the centromere; a decrease from
standard values is obtained when regions are placed, by
inversion, nearer the centromere than is normally the
case, and an increase when regions are moved away from
the centromere.
The influence of centric heterochromatin is not so
easily assessed.
Relative to standard values in normal
chromosomes, increased recombination was obtained between
:markers spanning distally inserted centric heterochromatin
with the two X chromosome inversions.
Although these
data suggest that crossing over occurs more readily once
the heterochromatin is removed from centromeric
.... ... .
vi
... ...·-·--·-·-··-----·-··-·--·--..-
. . ..
inhihition,.that these values might. result from. stimula­
tion of euchromatic exchange can not be excluded,
Interpretation of the data obtained with the third
chromosome inversion presents similar problems,
Increased
recombination in the interval that includes the rearranged
centric heterochromatin was observed, but it was not
possible to determine whether the exchanges actually
occurred in the heterochromatin or in the adjacent
euchromatic regions.
i
Further work utilizing heterochro-
matically located loci is required before the problem
can be fully resolved.
vii
CHAPTER I
INTRODUCTION
Early prophase chromosomes characteristically
'possess extensive regions in the vicinity of the centromere having a dense compact configuration, evidenced b�
·their stainability (Heitz, 1928) .
The term heterochromatin
is commonly used to distinguish these regions from the
remaining euchromatin. Cooper (1959) persuasively argued
that this terminology is inappropriate since it implies
a condition of permanent structural difference between
the two regions, rather than an "out-of-phaseness" of
temporal events. Although his argument has merit, the
terms heterochromatin and euchromatin remain useful in
referring to specific chromosomal regions; they will be
so used below, implying, however, only that the regions
are cytologically distinguishable.
In addition to the �ifference in configuration between
distal and proximal areas, comparison of genetic and
cytological maps, necessarily constructed from independent
data, suggests an inhibition of crossing over in the
neighborhood of the centromere (Dobzhansky, 1930, 1930a,
1932). There is little doubt that genes in close
proximity to the centromere exhibit remarkably little
crossing over.
Beadle (1932) w�s among the first to
2.
report on this effect, basing his conclusions on crossover data for genes of the_right arm of chromosome 3
;that had been translocated to the fourth chromosome.
:Studies of the recombinational prop,rties of various
,other chromosomal rearrangements hav� corroborated
Beadle's conclusions (Offerman and Mulller, 1932; Gruneberg,
il935; Mather, 1939; Thompson, 1963),
��d
the concept of
an inhibitory "centromere effect" is now widely accepted.
The question of an effect of heterochromatin itself
1on crossing over still arouses controversy: is the absence
iof crossing over in heterochromatin a result of its
i
iproximity to the centromere, or does its configuration at
ithe time of synapsis preclude exchange?
Offerman and
JMuller (1932), after study of the scute-8 (sc8) and
!de1ta-49 (dl-49) inversions, as we11 as several X-4 trans­
:locations, concluded that proximity to the centromere
I
I
.controls recombination frequencies in euchromatin but not
I
1
!
11n the heterochromatin, this material being refractory
)
1to crossing over wherever located. Later, both Grlineberg
•
! (1935) and Mather (1939) presented data, obtained from
;studies of inversion roughest-3 (rst3 ) homozygotes, that
,they felt could best be accounted for by assuming an
'increased level of crossing over in the distally displaced
heterochromatin.
An objection to this interpretation,
however, was raised by Baker (1958) on the basis of his
study of a Drosophila virilis stock having a 3-5 reciprocal
translocation.
He suggested that increased crossing over
3
,in euchromatin adjacent to displaced heterochromatin was
.an alternative and more plausible explanation for the re­
sults of Gruneberg and Mather.
Support for this hypothesis
came from Braver (1957) , who observed a significant in­
crease in recombination in the uninverted yellow (y) to
:white (�) interval in rstJ homozygotes. Schultz and
'Redfield (1951) had earlier speculated on the importance
:of heterochromatic regions on chromosomal exchange.
They
!postulated that factors influencing "the polarized pattern
'of pairing at meiosis" could affect crossing over,
,heterochromatic association being one of these factors.
The availability of a rather different kind of
chromosomal rearrangement from those previously analyzed
!
'
makes appropriate a further investigation of the problem.
This rearrangement, duplication white-variegated-cobbled
(Dp wVco) , consists of a pericentric inversion in the
:third chromosome whose breakpoints are essentially confined to the heterochromatin.
In addition, a short
euchromatic section of the X-chromosome containing the
�white locus is inte�polated between the breaks in chromosome
.J.
·
Despite this complication, the inversion basically
involves only the heterochromatic region of the third
.chromosome.
The euchromatic loci in the third chromosome,
whose crossover intervals were tested, are not moved from
itheir normal locations.
Their linear sequence, physical
distance from one another (except in the case of the two
spanning the centric region) , and distance from chromosome
4
.ends remain unchanged.
Only their relative proximities
to the centromeres have been altered.
This paper reports
on the results of a study of this chromosome, and, in
addition, on a reinvestigation of recombination in the
i rst3
and sc8 inversions, all carried out under identical
conditions to permit valid comparison.
CHAPTER II
REVIEW OF THE LITERATURE
Investigations of the recombinational properties of
centric regions of Drosophila melanogaster have continued
since Dobzhansky, in a series of papers in 19.30 and 19.32,
'demonstrated a discrepancy between the genetic and cyto­
'logical maps of this organism. The cytological maps were
,constructed using a variety of deficiencies and transloca­
i
:tions, the genetic maps from recombination data. Although
'the two methods resulted in the same linear sequences for
the loci studied, the relative distances separating them
:as determined by cytological methods did not correlate
well with crossover data.
Intervals in the vicinity of
the centromere were shorter genetically than intervals
of comparable cytological length in distal regions.
,Earlier studies by Heitz (1928) had shown that prophase
!
chromosomes exhibit extensive centric regions that stain
more deeply than distal areas for which he proposed the
term heterochromatic solely on the basis of its staining
characteristics.
Dobzhansky (19.32) pointed out that
,the discrepancies between genetic and cytological maps
:could be accounted for by assuming "that crossing over
is not equally frequent in the different regions of the
chromosome."
6
Dobzhansky's findings raised the following question:
was the reduced frequency of recombinations near the
centromere due to the heterochromatic material
.or to an inhibitory effect of the centromere?
� �
Beadle
·(1932) investigated recombination frequencies in a stock
'having a reciprocal translocation between the third and
fourth chromosomes.
Because of the extremely small size
of the fourth chromosome, this rearrangement necessarily
positioned the genes of the translocated right arm of
chromosome 3 much closer to a centromere than had been
'the case in its normal configuration.
A significant
reduction in recombination frequency in the curlec to
stripe and stripe to ebony-sooty intervals led Beadle to
conclude that crossing over was inhibited as a result of
proximity to the centromere.
Offerman and Muller (1932),
after investigating recombination in several translocations and two X-chromosome inversions, also concluded
;that euchromatic crossing over was inhibited by proximity
to the centromere.
In addition, they reported that low
crossing over was characteristic of heterochromatic
.regions regardless of their positions relative to the
centromere.
A more recent consideration of spindle fibre
inhibition by Thompson (1963). using a 3-4 translocation
heterozygote, led him to suggest that following synapsis,
mutual repulsion of homologous centromeres just prior to
·the time of exchange results in low recombination
. frequencies in adjacent regions.
7
These investigations, together with similar work by
Grtmeberg (1935) and Mather (1939), served to establish
:the concept of an inhibitory effect of the centromere.
However, the possibility of an additional heterochromatic
,effect remained a source of controversy. Both Mather
:and Gr8neberg had utilized the roughest-3 inversion of
the X-chromosome in their recombination studies.
In this
inversion the right break occurs in the centric hetero­
:chromatic material. This results in the intercalation of
icentric heterochromatin between euchromatic material at
:a distal location just to the right of the white locus.
!Increased crossing over between markers spanning th is
now distally located heterochromatin was observed by
oth workers, who attributed the result to exchange
taking place in the heterochromatin at a much higher
,frequency than occurs in the uninverted chromosome.
!
This interpretation, however, was challenged by
:Baker (1958). A stock of Drosophila virilis having a
1reciprocal translocation between chromosomes 3 and 5
allowed him to investigate crossing over in heterochromatin
:far removed from the centromere.
·
Observing little or no
lexchange in this material, he suggested that the increased
recombination noted by both Gruneberg and Mather could
have been due to a stimulation of crossing over in the
euchromatic regions adjacent to the inverted heterochromatin.
Earlier work by Braver (1956, 1957) provided data which
supported Baker's proposal.
Using inversion roughest-3
8
homozygotes, Braver obtained recombination frequencies
as high as 6.6% in the distal uninverted yellow-white
interval adjacent to the inverted heterochromatin.
standard value is 1. 5%. )
(The
These findings, however, did
not result in a final settlement of the problem since
Schalet (1967) , using homozygotes for the scute-8
'inversion in which the heterochromatically located bobbed
:
locus is inverted, reported the occurrence of crossing
�ver within the bobbed locus.
CHAPTER III
MATERIALS AND METHODS
The stocks used in the course of this investigation
are described below, together with the markers used in
conjunction with each. More complete descriptions of the
'markers will be found in both Bridges and Brehme (1944)
;and Lindsley and Grell (1968).
Inversion (1) roughest-) (rst3):
an X-ray induced
inversion of the X chromosome discovered by Gruneberg
(1935).
Cytological analysis by Emmens (1937)
placed the left break after salivary chromosome band
JCJ (Bridges� 1938 revised map) and the right break
in the centric heterochromatin, probably in 20B.
The bobbed (bb, 1-66.0) locus was orginally thought
to be included in the inversion, but genetic analysis
by Lindsley (1954) showed this not to be the case.
One rstJ stock used carried the markers yellow (�,
1-0.0); white60i29(w60j.29, 1-1. 5) and carnation
(�, 1-62.5); the other was unmarked.
Inversion (1) scute-8 (sc8):
an X-ray induced
inversion of the X chromosome found by Noujdin
(Bridges and Brehme, 1944). Genetic analysis showed
the left break to be between achaete (�, 1-0.0+)
9
10
and sc, and the right break between bb and the
spindle attachment.
Muller and Prokofyeva
(Bridges and Brehme, 1944) placed the left salivary·
chromosome break between 1B2 and 1B3 and the right
break to the right of 20B. One sc8 stock used
carried yellow3ld (y3ld) and white-apricot (wa);
the other �' vermilion (�, 1-33.0), and cross-­
veinless (QYJ 1-13.7).
White-Variegated cobbled (w Vco):
a pericentric
inversion in the third chromosome with breaks at
77D4 and 81A, found by Clausen and analyzed by
Schultz (Bridges and Brehme, 1944).
In addition,
a short piece of the X chromosome (2Cl-3C4) is
inserted at 81A. Two marked wVco stocks were
prepared.
One carried the left arm markers hairy
(h, 3-26. 5), thread (th, 3-43.2), scarlet (st,
3-44.0); the other the right arm markers pink­
peach (�, 3-48.0), curled (cu, 3-50. 0), stripe
(g, 3-62. 0).
. .
. 258-45 (w258-45):
Whlte
Deflclency
an X-ray
induced deficiency of the X-chromosome originally
described as deficient solely for 3Cl (Sutton,
cited by Bridges and Brehme, 1944), but more
recently described by Lefevre (1968) as deficient
for 3B3 through 3C2.
In addition, the stock
maintained in this laboratory contains a second
-
�·
7
11
deficiency of unknown origin which extends approximately from 3A7 up to and including 3Bl (Lefevre,
unpublished) .
This doubly deficient chromosome
was illustrated by Lefevre and Wilkins (1966) .
This stock made it possible to obtain fertile females
homozygous for wVco. Initial studies revealed that
females (and males) having nondeficient X chromosomes
Yeo ln homozygous cond·t·
b ut carrylng �
l lOn, though
·
·
·
viable, are not fertile.
The following procedure was used in all experiments
from which quantitative data on recombination were obtained.
Virgin females were collected shortly after emergence and
aged for 3 days. They were then mass-cultured with an
excess of appropriate males for 48 hours, after which the
males were discarded,
The females were then placed
individually in vials· and subcultured three times at two­
day intervals, being discarded on the 6th day.
Counting
and scoring of the progeny in each vial was discontinued
after 17 days in order to avoid including F2 progeny in
the counts. Except for short periods during which flies
were subcultured or progeny examined, the culture vials,
including those from which parental females were obtained,
were maintained in incubators at 25°C.
The breakpoints of the three inversions utilized in
the course of this work are diagrammatically represented
:below at
Figure 1.
sc8
rst3
X
'<'
l_
-�
SC
H
--�----4i==------�---- -.
_______}
j
�-----bb
car
v
cv
III R
-- ----·------r--sr
III
----
Figure l.
sc8, I n(l) rst3 and In(J) vJVco
Diagrammatic representation of In(l) __,
-
��
CHAPTER. IV
RESULTS
Recombination in homozygous
X
chromosome inversions.
Recombination between markers spanning distally
inverted centric heterochromatin, and in adjacent
euchromatic regions, was measured in two X chromosome
inversion homozygotes, sc8 and rstJ.
As shown in Table 1, In (l)sc8 exhibited reduced
crossing over in the cv-wa interval, which occupies a
much more proximal position than it does in the normal
chromosome. Conversely, recombination between y and �
was higher than estimates based on the behavior of the
uninverted chromosome would predict; the y-�interval,
however, includes the distally inverted heterochromatin.
The other two intervals tested (�-y; y-£Y) did not
differ significantly from standard values. None of the
values differed noticeably from those reported by Mather
(1939).
As seen in Table 2, In (l)rstJ exhibited higher
recombination frequencies in both the y-� and �-£££
(which includes the inverted heterochromatin) intervals
:than occur in the normal chromosomes. Although the
present results are comparable with those obtained by
Mather, crossing over between y and car was significantly
lJ
Table 1
Crossing Over in Homozygous sc8 Inversion
y-ear
3.5+*
2
X =19,45
P<<.Ol
5. 8 (188)
�·
2 =0,14
.7<P<. 8
v-cv
19. 3
2
X ::::2,41
.l<P<.2
cv-wa
12. 2
2
X ::::26.20
P<<.Ol
28.9 (937)
20, 2 (653)
6.9 (107)
n=l559'H'"
car-v
2 8.5
----- --------------------·----------------------------------------------------�----------
Standard values
X
X
2=4. 2
3
,02<P<.05
X
4.99
n=l424
18.24
n=4046
7.09
n::.4046
2:::1,30
.2<P<o3
Inversion homozygotes
- Mather (1939)
*
**
X
/
"'--
;/
2=0,09
o7<P<. 8
This value is used as a standard since it represents the ----car-bb interval in the
normal chromosome.
Since an appropr Aately marked backcross chromosome was not available, recombination
between cv and w could be detected only in male progeny.
-
-
x2 and P values lie between entries the significance of whose difference they test.
f-1
·�
15
Table 2
Crossing Over in Homozygous rst3 Inversion
y-w
Standard values
2=38.3
p <<. 01
w-ear
3.7+*
.
2
X =288. 3
P<<. Ol
y-ear
5.2
(by addition)
2
X =28. 56
P<<O. Ol
2. 8 (270)
n=9699
12.3 (707)
n=5759**
14.6
(by addition)
2
X =1. 60
0. 3>P> 0.2
1.5
X
y w6o:r"" 29rst3car
+ +
rst) +
Inversion
homozygotes
- Mather (1939)
Inversion
homozygotes
'- Braver (1957)
.
15.6
(n=l999)
x2=12.6
P<<O.Ol
5.1
(averaged)
13.4
(averaged)
Inversion
homozygotes
- Gruneberg
(1935)
*
This estimated value includes the 0.2%
between � and rst plus 3. 5% between � and
the right break is to the left of bb.
18.5
(by addition)
18.6
(n=7296)
recombination
bb, although
** Since the presence of -w masks the expression of car,
recombination between w and car could be detected only-in w+ progeny.
x2 and P values lie between entries the significance of
whose difference they test.
16
.lower in �his �xperiment thari in those reported by Braver
(1957) and Gruneberg (1935) . It is perhaps appropriate
;to note that cytological examination of the salivary
'gland chromosomes of both rst3 stocks used in this cross
revealed no autosomal inversions which might have produced ;
an interchromosomal stimulati6n of crossing over.
Recombination in wVco.
The effects of the presence of the wvco inversion on
recombination in the third chromosome are recorded in
Table 3.
Control values are reported for two separate
crosses, one having all the markers in coupling, the
second with the left arm markers in repulsion to those
of the right arm. In all cases the values obtained were
lower than standard values. Nevertheless, the presence
of wVco appears to stimulate recombination in both the
st-� and �-£Q intervals; whereas, the intervals tested
in the left arm are unaffected or, possibly, slightly
depressed. The stimulatory effect is noticeable in the
inversion heterozygo.tes, but is quite pronounced in the
homozygotes.
Table 3
The Effect of wVco on Recombination in the Third Chromosome
th-st
h-th
Standard values
Controls
!Lth �t J.2P C!:-!
+ + + + +
n=4877
h th st + +
�u
n=3782
Average
rrr;vers-ion ·heterozygotes
i y_ w258 -45 h th_.§J 12p__£Q
+ + +WVc o+ +
+ +
n=2
883
i
.: Inversion homoz.ygotes
+
. w258-4·5 -h th st wVco
v- + ­
p
co
�758:::-r
r:s- + + + w
p cu
y w
n=l627
-
�
�
st-pP
------
-pP-cu
16.7
0. 8
4.0
2, 0
14. 2 (694)
0.3 (17)
0.8 (37)
l. 1 (54)
15. 8 (598)
0.5 (18)
1. 6 (59)
1.0 (37)
14. 9
0. 4
1.1
1.1
0. 03 (1)
l. 6 (46)
2,4 (69)
0
9. 0 (146)
7.8 (127)
·-- ·
··
9. 8 (281)
-
-
�
�
10,0 (163)
}-J
"-'1
CHAPTER V
DISCUSSION
Recombination in Inversion sc8 Homozygotes
Support for the hypothesis that recombination is
'inhibited by the centromere, first proposed by Beadle in
19.32, is provided by the results obtained for the £:;L-Wa
,. terva1 1n the
,1n
.
.
8 1nvers
lOne
.§.2_
.
The reduction in exchange
1frequency between the two markers, as compared with
standard values, is highly significant and is most reasoni
ably explained by the assumption of a centromere effect.
1As can be seen in Table 1, the present results are in
good agreement with those of Mather (19.39) , who also
[ascribed thi� diminution to a centromere effect.
The results obtained for the �-�and y-£:;L intervals
also compare well with those reported by Mather. The
region between� and�' in the inverted chromosome,
includes virtually all of the proximal heterochromatin,
:and shows significantly more recombination than do the
comparable chromosomal regions in the normal configuration.
In view of the inhibitory influence of the centromere on
crossing over between £::!.. and wa in their new proximal
,Positions, it seems reasonable to attribute increased
recombination in the�-� interval to a release from
centromere inhibition and to conclude that the euchromatic
18
19
portion of this interval, at least, crosses over more
freely than it do.es in the normal chromosome.
Recombination in Inversion rstJ Homozygotes
Since only distal intervals were considered in the
;investigation of crossing over in rstJ, inhibition in the
1vicinity of the centromere was not detectable.
However,
.an increase over standard values was observed in both of
.the distal intervals (�-� and �-�) investigated.
Although no real difference exists between the present
results and those of Mather, they are significantly lower
than those obtained by Braver (1957) and Gruneberg (1935) .
The reason for this difference can not be ascertained
beyond doubt, but several possible explanations can be
suggested. A difference in the environmental conditions
is one of these; temperature, for example, has been
shown by several investigators, including Mather, to be
a factor governing recombination frequency, particularly
in heterochromatic regions. Also, Schultz and Redfield
1 (1951) have shown that the age of the mother is a factor
influencing exchange frequency. Finally, there is the
possibility of undetected autosomal rearrangements
stimulating crossing over in the X chromosome. With
this latter possibility in mind, the salivary gland
chromosomes of the stocks used in this experiment were
examined, but no abnormalities were found.
The high frequency of exchange observed between
�
20
and w in the uninverted euchromatic tip provides support
for the proposal of Baker (1958) that stimulation of
crossing over in euchromatin adjacent to inverted heterochromatin was a plausible interpretation of the results
.obtained by Mather and Gruneberg.
Nevertheless, the
surprisingly high crossover values observed for the �-�
�nterval, which includes the inverted proximal hetero­
�hromatin, is difficult to explain on the basis of
euchromatic exchange alone.
Bridges (19J7) introduced a method for quantifying
regional differences in recombination along a chromosome
by finding the ratio of crossover frequency to salivary
hromosome length.
Lefevre and Moore (1968) utilized a
modification of this scheme, calculating coefficients of
�r.ossing over on the basis of crossover units per salivary
gland chromosome band.
This latter method is useful in
�etermining the extent of effects resulting from. chromo­
s' omal rearrangements such as the inversions used in this
work.
When the normal X chromosome is considered as a
�hole, a value of 0.07 crossover units per band is obtained,
The euchromatic region present between
£§£
and the centric
heterochromatin gives a slightly lower value of 0. 06,
possibly as a result of centromeric influence.
12. J% recombination between � and
�
If
the
in the rst 3 inver­
sion is attributed solely to euchromatic crossing over,
a
coefficient of 0.23 crossover units per band would be
21
'require?; this value would be four times higher than
normal for the interval. Thus, it seems probable that
.some crossing over is occurring within the inverted
heterochromatin itself, even though virtually none occurs
.in the proximal heterochromatin of normal chromosomes.
Evidence that heterochromatin is actually capable of
recombination was provided by Schalet (1967), who reported
on the occurrence of crossing over within the bb locus
. sc8 homozygotes.
ln
A second problem arises, however, when one compares
the rst3 results with those with sc8• Essentially the
.same ammount of euchromatin is present in the y-�
interval of sc8 as in the �-£§£ interval of rst3. In the
latter case, appreciably less proximal heterochromatin
is intercalated qetween the two markers, yet exchange
occurs much more frequently, 12. 3% versus 5. 8%.
Various explanations for this apparent inconsistency
might be suggested. One feature differentiating the two
.inversions is the proximity of the left break to the
:chromosome tip. If the telomere acts as a suppressor
:of crossing over, in a manner analogous to that of the
centromere, there should be less recombination in the
�-£§£region of sc8 than in the �-� interval of rst3.
The ';L-� results in rst3 support this argument since,
even with the increase obtained, the crossover coefficient
for this region remains at a low value of 0.02 units per
:band.
Nevertheless, much of this is mere speculation and
a model_might equally well be proposed in which all of
the increased recombination is due solely to exchange in
the euchromatic region, with the amount of stimulation
being inversely proportional to the amount of inter­
.calated heterochromatin. Some support for this latter
,idea may be obtained from a consideration of recombination
:in the standard chromosome in regions such as JC, where
ithere is evidence for a relatively small amount of
intercalary heterochromatin (Kaufmann, 1946) and the
'coefficient of crossing over is exceptionally high.
Recombination in wVco Heterozygotes
Analysis of the results obtained with the wVco inversion is complicated by the fact that the control values
are low as compared with standard map distances.
Probably,
this is a consequence of the experimental conditions,
since cytological examination revealed no chromosomal
abnormalities. However, all crosses were carried out
under the same conditions and, thus, comparison of
control and experimental data should be valid.
In the wvco heterozygotes, recombination in the left
1arm appears to be inhibited. There are two possible
1explanations for this effect. Reduced recombination
within, and adjacent to, heterozygous euchromatic inver­
·Sions is an expected and frequently observed effect.
Thus the decrease for the h-th and th-st intervals may
:be due to the wVco inversion extending somewhat into the
23
euchromatin of the left arm at ??D. However, this in',
version also results in bringing the centromere into
.closer proximity to the left arm markers than is true in
the normal configuration, so that the reduced crossing
:over might be attributed to an inhibitory centromere
effect.
The inversion also affected recombination between
·� and
£.Q
in the right arm, where the heterozygote showed
a doubling in exchange frequency as compared with the
,controls.
An increase was also observed for the st-�
'interval, which spans the centromere and centric heterochromatin.
Interpretation of these latter two results
is difficult.
Thompson (1963) has proposed that pairing
rof the centromeres at synapsis, and repulsion prior
!
:to the time of exchange, could be responsible for the
inhibitory effect of these structures.
His model
suggests a possible explanation for the increased recom1bination in the centric region and right arm of wvco
!heterozygotes, since synapsis of homologous centromeres
could conceivably be interrupted as a consequence of
the inversion. In this case, however, one could not
'attribute the decrease in the left arm to centromeric
inhibition. The data for all four of the tested intervals
rcan be correlated by assuming an intrachromosomal effect:
inhibition of crossing over in the left arm resulting in
a stimulation of exchange in the right arm.
24
Vc o
Rec ombination in Y:!_ Homozygotes
The dec rease in rec ombination in the left arm
'intervals of the homozygote was no greater than that
;observed in the heterozygote.
However�
the dec rease in
the homozygote c an not be a c onsequenc e of struc tural
dissimilarity between the two c hromosomes.
The c entro-
mere effec t is now a muc h more probable explanation.
The
results for the right arm intervals are extremely inter:esting.
A c onsiderable inc rease over both c ontrol and
standard values was observed in both the
�-�
and st-�
:intervals.
Sinc e the c hromatic material between
not been altered in any way,
�
and £Q has
the inc rease must result
from the influenc e of other c hromosomal regions;
but is
it due to a reduc tion in c entromeric inhibition or to a
stimulation of euc hromatic exc hange by inverted hetero­
'c hromatin?
Although it would be c onsistent to interpret
this as heteroc hromatic
stimulation c omparable to that
of the �-� interval of inversion rst3,
a few simple
c alc ulations suggest that in this c ase a large part of
!the inc rease is probably due to a reduc tion in the inhi'bitory influenc e of the c entromere.
c hromosome 3 c ontains
:is about
58.4
1178
salivary c hromosome bands and
map units long,
ation c oeffic ient of
0.05
The right arm of
whic h gives a mean rec ombin­
map units per salivary band
,for the right arm as a whole.
While
�(48.0)
and
£..1!(50.0)
25
have no� been precisely located cytologically,
separated by approximately
150
Thus,
0.01
a low coefficient of
they are
salivary chromosome bands.
map units per band is
characteristic for this region in the standard chromosome.
The
recombination obtained for the interval in
?.8%
Vco
homozygous w
brings the coefficient back up to the
normal average of
0.05,
which suggests that a release
'from inhibition by the increased distance from the cen­
:tromere was responsible for the change.
'course,
All of this,
of
depends on the assumption that the calculated
oefficients represent an index of centromeric inhibition.
The st-� interval also showed a marked increase
crossing over.
Included in this interval,
the centromere and centric heterochromatin,
!euchromatic portion of the
X
chromosome,
bute somewhat to the increase.
·however,
only about
It is worth noting,
far from the
this region represents a genetic length of
1.0
but in its new,
less.
is a short
which may contri­
that even in its n6rmal location,
centromere,
in addition
Thus,
crossover units (Lefevre and Moore,
more proximal po�ition,
196a),
probably much
we must explain at least a doubling of
,recombination frequency over standard values in this
;interval,
;
perhaps more if the control figures presented
here are representative of the true exchange frequency
.under the applied experimental conditions.
In the left
:arm of the inverted chromosome there is a short
'
heterochromatic region followed by a euchromatic region
26
of approximately
:the st locus.
280
While this euchromatic region,
.standard conditions,
,between st and
bands between the centromere and
�
contributes
3.7
of the
(Lindsley and Grell,
under
4. 0
1968),
map units
it appears
Vco
most probable that in the w
inversion the closer
proximity of the centromere would act to reduce exchange •
.The observed reduction in the more distal left arm inter­
,vals supports this contention.
This leaves the chromatin
:present between the centromere and the
right arm,
�
locus,
in the
as the most likely area showing increased
crossing over.
Unfortunately it i's not possible to
i
idifferentiate
between euchromatic and heterochromatic
.
;
exchange in the system under discussion,
can not say, with certainty,
and so we
which of the two chromosomal
regions is responsible for the increase.
doubt that
There is no
euchromatic bands between the hetero­
170
chromatic junction and the
�
locus could accommodate
1this amount of recombination without displaying any
unusual characteristics.
Even a full
8%
in this interval alone, would give just
units per band,
which as we have seen,
recombination,
0.05
crossover
is typical of
JR
··as a whole.
In conclusion,
the data presented here do not fully
resolve the problem of whether heterochromatin is in
its�lf refractory to crossing over.
by inversion,
In regions where,
it has been removed from the vicinity of
the centromere, there is an increase in recombination
27
frequency.
At least part of this increase is due to
increased exchange in the adjacent euchromatin:
heterochromatic exchange,
possible.
: in
as well,
increased
remains distinctly
The problem can be finally resolved only when,
systems such as those described abov�recombination
'
'is measured between markers actually located in the
heterochromatin.
28
B IBLI OG·RAPHY
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c. B., 1937
correspondences between li��age maps
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74'5-755.
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The mutants of Droso£hila
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_______
cooper,
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Cytogenetic analysis of major
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Cytological map of the second
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Translocations involving the third
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Genetics 12:
347-399.
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29
Dobzhansky, Th. � .1932
Cytological map of the X chromosome
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Salivary gland cytology of roughcst-3
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Heitz, E., 19 28
Das heterochromatin der moose I. Jb.
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Cytogenetic studies
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, 1968
Tests for deficiencies in the vicinity of
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----
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____
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----
Mather, K., 1939
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1
30
Schultz J., and H. Redfield, 1951
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