ANALYSIS OF THE CHROMOSOME ABERRATIONS INDUCED BY X-RAYS IN SOMATIC CELLS OF DROSOPHILA MELANOGASTER GATT1 M., C. TANZARELLA AND G. OLIVIERI Istituto di Genetien, Facolt6 di Seienz?, Universitci di Roma, Rome, Italy Manuscript received February 28, 1974 ABSTRACT A technique has been perfected for enabling good microscope preparations to be obtained from the larval ganglia of Drosophila melanogaster. This system was then tested with X-rays and an extensive series of data was obtained on the chromosome aberrations induced in the various stages of the cell cycle.- The analysis of the results obtained offers the following points of interest: (1) There exists a difference in radio-sensitivity between the two sexes. The females constantly display a greater frequency of both chromosome and chro- matid aberrations. They also display a greater frequency of spontaneous aber- rations. (2) In both sexes the overall chromosome damage is greater in cells irradiated in stages G, and GI. These two peaks of greater radiosensitivity are produced by a high frequency of terminal deletions and chromatid exchanges and by a high frequency of dicentrics, respectively. (3) The aberrations are not distributed at random among the various chromosomes. On the average, the Y chromosome is found to be more resistant and the breaks are preferen- tially localized in the pericentromeric heterochromatin of the X chromosome and of the autosomes. (4) Somatic pairing influences the frequency and type of the chromosome aberrations induced. In this system, such an arrangement of the chromosomes results in a high frequency of exchanges and dicentrics between homologous chromosomes and a low frequency of scorable translo- cations. Moreover, somatic pairing, probably by preventing the formation of looped regions in the interphase chromosomes, results in the almost total absence of intrachanges at both chromosome and chromatid level. ROSOPHZLA melanogaster lias been an organism of fundamental import- ance for studies on mutagenesis; in fact it has enabled a vast quantity of information to be collected regarding every type of genetic damage (SOBELS 1971). However, genetic damage in this organism has been analyzed only in the generations subsequent to the one that has been irradiated. This procedure has a considerable limitation, since especially for certain types of damage (e.g., chro- mosome aberrations), it does not allow examination of those mutations that do not go beyond either gametogenesis or the development of the zygote. Also in research on somatic crossing over, the chromosome damage has been studied after its segregation and selection by various cell divisions (STERN 1936). We have therefore thought it useful to make a direct study in somatic cells of Dro- sophila of the type and relative frequency of spontaneous and induced chromo- some aberrations. We feel that these sttidies are interesting also because the presence of somatic pairing in the cells of Drosophila melanogaster makes it possible to assess the Genetics 77: 701-719 August, 1974.
Transcript
ANALYSIS OF THE CHROMOSOME ABERRATIONS INDUCED BY X-RAYS IN SOMATIC CELLS OF DROSOPHILA MELANOGASTER
GATT1 M., C. TANZARELLA AND G. OLIVIERI
Istituto di Genetien, Facolt6 di Seienz?, Universitci di Roma, Rome, Italy
Manuscript received February 28, 1974
ABSTRACT
A technique has been perfected for enabling good microscope preparations to be obtained from the larval ganglia of Drosophila melanogaster. This system was then tested with X-rays and an extensive series of data was obtained on the chromosome aberrations induced in the various stages of the cell cycle.- The analysis of the results obtained offers the following points of interest: (1) There exists a difference in radio-sensitivity between the two sexes. The females constantly display a greater frequency of both chromosome and chro- matid aberrations. They also display a greater frequency of spontaneous aber- rations. (2) In both sexes the overall chromosome damage is greater in cells irradiated in stages G, and GI. These two peaks of greater radiosensitivity are produced by a high frequency of terminal deletions and chromatid exchanges and by a high frequency of dicentrics, respectively. (3) The aberrations are not distributed at random among the various chromosomes. On the average, the Y chromosome is found to be more resistant and the breaks are preferen- tially localized in the pericentromeric heterochromatin of the X chromosome and of the autosomes. (4) Somatic pairing influences the frequency and type of the chromosome aberrations induced. In this system, such an arrangement of the chromosomes results in a high frequency of exchanges and dicentrics between homologous chromosomes and a low frequency of scorable translo- cations. Moreover, somatic pairing, probably by preventing the formation of looped regions in the interphase chromosomes, results in the almost total absence of intrachanges at both chromosome and chromatid level.
ROSOPHZLA melanogaster lias been an organism of fundamental import- ance for studies on mutagenesis; in fact it has enabled a vast quantity of
information to be collected regarding every type of genetic damage (SOBELS 1971). However, genetic damage in this organism has been analyzed only in the generations subsequent to the one that has been irradiated. This procedure has a considerable limitation, since especially for certain types of damage (e.g., chro- mosome aberrations), it does not allow examination of those mutations that do not go beyond either gametogenesis or the development of the zygote. Also in research on somatic crossing over, the chromosome damage has been studied after its segregation and selection by various cell divisions (STERN 1936). We have therefore thought it useful to make a direct study in somatic cells of Dro- sophila of the type and relative frequency of spontaneous and induced chromo- some aberrations.
We feel that these sttidies are interesting also because the presence of somatic pairing in the cells of Drosophila melanogaster makes it possible to assess the Genetics 77: 701-719 August, 1974.
702 M. GATTI. C. TAIVZARE1,I.A A N D G . OLIVIERI
influencc that this arrangement of the chromosomes has on the type and fre- quency o l induced aberrations. lye consider that a study of the relationships between chromosome pairing am1 induced aberrations is important because it allows a mort cbffectivc asws4ment o f the genetic damage that can be induced in the meiocytcs.
A sniall part of the data given in ihc present work has been summarized in a previous short note (GATTI, TANZAREILA and Or.IvIERI 1974).
MATERIALS A N D METHODS
In all the cxprrimrnts pcrformrd, third instar larvae of the Oregon R stock, raised at !Tk0 2 1 were ur;ctl. IJlirs were matmi in hottlrs (ahout 50 pairs per hottle) and allowcd to deposit rggs for -1.8 hours. Third instilr liir-var wrrr collrctrtl ilt 7-8 days n f t r r the Iwginning of culture. Larvae grown under these conditions show Icnv variahility in size and weight, the averagr weight being ahout 2 mg. Larvae of discrqmnt sizr wrrr rrjrctrd.
The following technique W A S usrd for the prrparation of the slides: 2 hours before every fixation a suficirnt numhrr of liin.ac were dissected and the ganglia wrre placed in a 0.7% solu- tion of SaCl in watcr coritairiing 1 0 - 1 M colchicine; they wrre thrn traiisferrcd for ten minutes into a 1 % citrate hypotonic solution and, after 3 minutes’ fixing in a mixture of acrtic acid and mrthyl alcohol (I: l ) , they wrre squashed in acetic orcein.
It \vas ohserrcd that krrping thr ganglia in physiological solution containing colchicine does not apprrciahly nffrct the mitotic cycle of the gangliar cells. In fact, preliminary experiments had shown us that ganglia trentrd for 2 hours with physiological solution and colchicine hare mitotic indices very similar to those of ganglia obtained from larvae fed on colcemid for an cqual prriocl of tirnr.
In the colchicinized metaphases, the pairing of the chromosomes is mostly lost, but it is clearly presrnt up to late prophase and is also evident in cells in which the chromosomes are lrss contractrd. Figure 1 shows examples of crlls in which somatic pairing is clearly present and of othrrs in which it is beginning to he lost.
containing a similar number of males and females were placed in plastic Petri dishes, on the y, rirroiis . rxp!.rimrnts wrre prrformd, following the same general plan: groups of Inn-ae
FIGURE 1 .-Colchicinizcd metaphases showing that the pairing of the chromosomes is present up to late prophase and then progressively lost.
CHROMOSOME ABERRATIONS I N DROSOPHILA 703
bottom of which had first been placed two or three layers of blotting paper soaked in physiologi- cal solution. The Petri dishes were then irradiated with 1250 R of X-rays (180 kV, 6 mA, 3 mm A1 and 125 R/min) and a t various times after irradiation (2,4,6,8, 10, 12,14 hours) the ganglia obtained from the dissection were fired by the method described above. The overall data con- cerning the frequency and type of the chromosome aberrations present a t the various times of fixing were obtained by adding together partial data obtained in a series of similar experiments. In each separate experiment and for each time, very similar numbers of male and female cells were scored and nJt more tliilri 100 crlls were rrnd in each preparation. For each time and for each sex a total of IO00 metaphases were scored; for the control, 5000 metaphases for each sex were scored.
RESULTS
Figures 2 and 3 show examples of the aberrations scorable in the cells of ganglia of Drosophila melanogaster. In these figures it may be noted that the aber- rations are clearly detectable and that the autosomes and the allosomes (hetero- chromosomes) are easily distinguishable from one another. The overall results
: * . ' e
b 4 . ... h
FIGURE 2.-Deletions induced in chromosomes of larval ganglion cells of Drosophila melano- gaster. (a) ( 9 ) euchromatic chromatid deletion of an aubsame; (b) ( 0 ) heterochromatic chro- matid deletion of an autosome; (c) ( 0 ) heterochromatic chromatid deletion of an X chromosome; (d) ( a ) two euchromatic isochromatid deletions of two autosomes; (e) ( 0 ) heterochromatic isochromatid deletion of an autosome; ( I ) ( 0 ) two heterochromatic isochromatid deletions of two autosom-ne with distal union; (g) ( 8 ) heterochromatic isochromatid deletion of an X chromosome; (h) ( 9 ) heterochromatic isochromatid deletion with distal union of an X chromo- some; (i) ( 8 ) euchromatic isochromatid deletion of an X chromosome.
I;IGL.RK 3.--Eschangrc iri:litcrd in chromosomrs of l a 1 ~ a 1 ganglion crlls of Drrophiln mrlm- ogacter. (a) ( 9 ) symmrtrical chromatid exchange hrtween autosomes; (h) ( 9 ) asymmetrical chromatid cscliangr hetwren autosomrs; (c) ( 9 ) symmrtricnl chromatid excliange between autosomrs plus asymmrtrical chromatid exchange hct\vcrn autosome and X chromosome; (d) ( 9 ) asymmrtrical chromatid exchange hetwecn nutosnme and X chromosome; ( e ) ( E ) sym- nirtrical rxchange hetwcrn autosomc and Y chroniosomc: ( f ) ( 9 ) triradial hetwecn autosomes; (g) ( 9 ) dicrntric hetwrrn autosomes; (h) ( 0 ) dicentric hetwrcn autosome and X chromosome; ( i ) ( 6 ) dicrntric between nutoaomrs plus dicmtric hctwren X and Y chromosome; (j) ( 9 ) di- crntric hct\vcrn X chromosomrs; (k) ( 9 ) translocation hrtwcrn aiitosomrs: (1) ( 9 ) transloca- tion Iwtwrcn autosome ancl X chromosome.
TA
BL
E 1
Fre
quen
cies
of
chro
mat
id a
nd c
hrom
osom
e ab
erra
tions
in l
arva
l ga
nglia
cel
ls of
Dro
soph
ila
mel
anog
aste
r aft
er t
reat
mn
t wit
h X
-ray
s (1
250
R) 1000 m
etap
hase
s of
at le
ast t
en
larv
ae sc
ored
for
each
poi
nt;
cont
rol:
5000
met
apha
ses s
core
d fo
r eac
h po
int
Isoc
hrom
atid
Po
st-i
rrad
iatio
n N
umbe
r of
de
letio
ns a
nd
Tot
al
time
of f
ixat
ion
cells
with
C
hrom
atid
ch
rom
osom
e C
hrom
atid
Sc
orab
le
Scor
able
C
hrom
atid
Is
ochr
omat
id
__-
Q
(h)
Sex
aber
ratio
ns
dele
hons
de
letio
x#
exch
ange
s D
icen
tric
s tr
ansl
ocat
ions
in
vers
lons
gaps
gaps
7 8
AB
-
-
8 12
9 i
-
31
17
E 8
8
448*
* 14
8 30
6 72
-
526*
* 59
8**
-
-
28
12
8 k 88
362*
* 72
35
7 16
8
453*
* 47
7**
-
-
11
10
g
Con
trol
99
33
5
28
-
-
33
33
-
-
17*
18*
-
88
17
* 6
10
1
2 P
P
540
21 9
33
8 13
0 -
687
817
-
4 9
9
505
115
477
54
16
662
732
-
-
16
18
6 P
P
439
70
376
47
51
544
61.2
8 -
7 6
3
88
35
8* *
56
299
34
ffi
435*
* 51
5**
3 -
10
50
li z
8 P
P
436
19
394
4 12
5 54
2 67
1 25
5
5 4
v,
Y
2 3
4z
8
8
314*
* 25
26
1 3
77
366*
* 44
6**
10
10
PO
435
10
296
-
238
544
782
26
10
4 2
88
29
4* *
6 23
3 3
110
352*
* %
**
17
5 3
2
12
99
24
4 10
15
6 3
87
256
3ffi
18
4
3 4
v
-
1 *
14
9P
19
4 9
133
1 75
21
8 29
4 12
1
88
131*
* 6
92
-
44
142*
* I%
**
7
U
id 8
88
17
3**
4 12
3 3
59
189*
* %
I**
3 -
2 1
8
1 -
-
* * S
igni
fican
t in
x2
test
with
P <
0.01.
* S
igni
fican
t in x2 te
st w
ith
P <
0.05
. t A
n is
ochr
omat
id d
elet
ion
can
resu
lt both f
rom
the
repl
icat
ion
of a
chr
omos
ome
brea
kage
(ch
rom
osom
e de
letio
n) a
nd f
rom
an
isoc
hrom
atid
br
eaka
ge.
Con
sequ
ently
thi
s type o
f ab
erra
tions
is p
rese
nt both
in t
he e
arly
and
the
lat
e fi
xatio
n tim
es p
ost-
irra
diat
ion.
T
otal
A:
Eac
h ab
erra
tion
scor
ed a
s a s
ingl
e eve
nt.
Tot
al B
: C
hrom
atid
and
isoc
hrom
atid
del
etio
ns sc
ored
as
sing
le e
vent
; ch
rom
atid
exc
hang
es a
nd d
icen
tric
s as
two
even
ts.
U
0
m
706 M. GATTI, C. TANZARELLA AND G . OLIVIERI
regarding the frequency of the aberrations observed are shown in Table 1 and in the graph of Figure 4. For every type of aberration, and for the various post- irradiation times of fixation, an analysis was also made of the distribution within and between chromosomes. The results are given in Table 2 (terminal deletions), Table 3 (isochromatid and chromosome deletions), Table 4 (chromatid ex- changes) and Table 5 (dicentrics).
% 100-
80 -
60-
40-
20 -
Variations in radiosensitivity with sex:
The results given in Table 1 and in Figure 4 clearly show that the sexes have
% 100.
80.
60 - 40 -
20-
50 -
40-
30 -
A
I 2 4 6 8 10 12 14
t i m r (h)
C
2 4 6 8 10 12 14 t i m e (h)
B
I , . ; b 10 12 14 t imr ( h )
D
t i m e ( h )
FIGURE 4.-Relationship between fixation time and frequency of the various types of aberra-
A) Total number: each aberration scored as a single event ( .---a 0 0 ; 0-0 8 8 ). B) Total number: chromatid and isochromatid deletions scored as single event; chromatid
exchanges and dicentrics as two events ( 0- 0 0 ; 0-0 8 8 ) ; C) Chromatid deletions (m-m 0 0 ; D---n 8 8 ) ; chromatid exchanges ( .---a
Q 0 ; 0-0 8 8 ); i s o c h a t i d deletio~ns and chromosome deletions (A-A 0 0 ; A-A 8 8 ) ;
tion induced.
D)Dicentrics(.---. 00;O-0 8 8 ) .
CHROMOSOME ABERRATIONS IN DROSOPHILA 707
TABLE 2
Distribution of chromatid deletions between and within chromosomes after treatment with X-rays
Postirradiation Autosomes X chromosomes Y chromosomes time of ____ fixation Hetero- Euchro- Hetero- Euchro-
( h) Sex chromatin matin Total chromatin matin Total Total Totals
Dishbution of isochromatid deletions and chromosome deletions between and within chromosomes after treatment with X-rays
Post-irradiation Autosomes X chromosomes Y chromosomes time of -
fixation Hetero- Euchro- Hetero- Euchro- (h) Sex chromatin matin Total chromatin matin Total Total Totals
2 Q 0 130 135 0" 8 127 114
4 0 0 199 178 8 8 131 153
6 0 Q 161 138 8 0" 132 117
2 f 4 $ 6 9 9 490 451 6 6 390 384
8 0 0 154 169 8 8 122 91
10 ? 9 113 102 $8 IC4 82
8f10 9 9 267 266 8 0" 226 173
265 2 41
377 284
299 243
941 7 74
318 213
21 5 186
533 399
51 22 73 19 12 31
74 26 100 25 14 39
52 25 77 19 12 31
177 73 250 63 38 101
M 32 76 19 14 33
46 35 81 12 12 24
90 67 157 31 26 57
- 34
- 34
- 19
- 87
- 15
- 23
- 38
338 3016
477 357
3 76 299
1191 962
394 261
296 233
6901 494
different radiosensitivity. For all the different fixing times the males show fewer aberrations than the females. This difference in radiosensitivity is sigEificant for every type of aberration and is greater for the chromatid exchanges and the di- centrics. The distribution of aberrations among the various chromosomes and within chromosomes is similar in the two sexes (see below) and therefore does not interfere with the phenomenon described above. Also, in the controls the two sexes show a significant difference in the total number of spontaneous aberrations.
TAB
LE 4
Dis
trib
utio
n of
chro
mat
id e
xcha
nges
bet
wee
n ch
rom
osom
es a
fter
trea
tmen
t w
ith X
-ray
s &-
+I
"2
2 P
P
28
57
a5
20
14
34
53
8 -
-_
-
--
3
130
3 G ? 1
47
2 i4
Chr
omat
id e
xcha
nges
invo
lvin
g:
P
iixat
ion
(h)
Sex
SA
T
SA
T
S A
T
S
AT
S
AT
T
rira
dial
s T
otal
s &-
Aut
osom
es a
nd
X an
d A
utos
omes
and
Po
st-ir
radi
atio
n A
utos
omes
X
-chr
omos
omes
X
chro
mos
omes
Y
chro
mos
omes
Y
chro
mos
omes
4
_-
-
time
of
M 8
8
21
37
58
--
-
11
2
61
7 -
33
2
72
2 54
8
8
6 7
13
--
-
3-
3
34
8 21
29
-
-_
-
--
2
13
1
12
-
$8
6
231
2++
k6
0 O
$8
35
65
100
--
-
11
2
11
2 13
1
45
2
1 22
--
-
--
_
4 PO
15
18
33
10
2 12
4
37
6 P
O
7 26
33
3
7 10
-
33
50
101
151
33
23
56
9 9
18
&-
--
_
-
--
-
__I -
__
I" r 21 0
-_
-
-_
_
I+
S =
sym
met
ric;
A =
asym
met
ric;
T =
tota
l.
CHROMOSOME ABERRATIONS IN DROSOPHILA
TABLE 5
Distribution of dicentrics between chromosomes after treatment with X-rays
709
Dicentrics involving Post-irradiation
time of Autosomes and X chro- Autosomes and X and Y fixation (h) Sex Autosomes X chromosomes mosomes Y chromosomes chromosomes Totals
13 7
42 43
95 63
181 85
331 198
2 1
9 1
20 6
46 5
77 13
1 -
10 -
11 -
22 -
2
-
4
-
18
-
24
- 4
-
2
-
6
16 8
51 46
125 77
238 110
430 241
The relation between cell stage and aberration yield and type:
The total aberration yield can be calculated in two ways, according to whether or not distinction is made between aberrations due to one break or to two breaks. With both criteria of assessment, a clear drop in aberration yield is noted at the 12-hour and 14-hour fixation times. Taking into account the times at which chro- matid and chromosome aberrations are present, it is probable that thsmetaphases scored at 12 hours and 14 hours derive from cells that have already completed a cell cycle after irradiation; that is to say, they would be at the second mitosis after irradiation. Consequently, most of the cells with aberrations would have been arrested or eliminated (WOLFF 1972). On the basis of these considerations we maintain that at the 2,4, and 6 hour times, the metaphases scored derive from cells irradiated in the S-G, stage, and at the 8 and 10 hour times from cells ir- radiated in the GI stage. Following this criterion, we consider as chromatid aber- ratiorzs the isochromatids that we find at the first three sampling times, and as chromosome terminal deletions those that we find at the 8 and 10 hour times. At 6 hours, we find both chromatid aberrations and a number of dicentrics; this fact might be due both to a mixing of irradiated cells in different stages (S and GI) and to the induction of dicentrics also at the beginning of the S stage (see DISCUSSION). If the radiosensitivity of the various stages of the cell cycle is esti- mated from the total number of aberrations produced, by assessing for the GI stage not only the asymmetrical aberrations scored but also an equal number of symmetrical ones (HEDDLE 1965), it may be concluded that there are two peaks of greater sensitivity at the 2 and 10 hour times.
Regarding the frequency of the various types of chromatid aberration scored at the first three times, it may be observed that, as mitosis is approached, there is a considerable increase in chromatid exchanges and terminal deletions, whereas the isochromatid deletions are fairly constant in S and G,.
710 M. GATTI, C. TANZARELLA AND G. OLIVIERI
Difference in radiosensitivity between chromosomes:
Assuming that the X and Y chromosomes offer a target that is about % of the autosomic one in the females and l/s in the males, it may be stated that there are no great differences in radiosensitivity between autosomes and X chromosomes with regard to either the chromatid and chromosome terminal deletions or the isochromatid deletions (Table 6 ) . On the other hand, the Y chromosome shows a characteristic radioresistance for the chromatid terminal deletions, which, in practice, are not induced on this chromosome. The Y chromosome also seems more resistant with regard to isochromatid deletions and chromosome deletions. As regards chromatid exchanges and dicentrics it is very difficult to make com- parisons of sensitivity differences between the various chromosomes as these aberrations are largely conditioned by other factors, such as somatic pairing.
As may be noted in Tables 4, 5 and 7, a large majority of the chromatid ex- changes and the dicentrics involve the autosomes. With regard to the sex chromo- somes, there is a different situation according to whether chromatid exchanges or dicentrics are considered. In the first case the exchanges between the two X chromosomes or between the X and Y are more frequent than exchanges between autosomes and sex chromosomes; in the second case they are less frequent. How- ever the frequency, of both chromatid exchanges and dicentrics involving at the same time autosomes and sex chromosomes, is lower than that expected for random involvement of chromosomes in interchanges (see Table 7) . This result suggests that in somatic cells of Drosophila melanogaster the interchanges in- volving homologous chromosomes are strongly favored. It is also interesting to note that the chromatid exchanges and the dicentrics between sex chromosomes are less frequent in percentage in the male than in the female (24.9 versus 10.8; 5.1 uersus 2.5.
Localization of aberrations:
Assuming that in Drosophila melanogaster the heterochromatin near the cen- tromere occupies a length of from 30% to 50% of the X chromosome and about 20% of the second and third chromosomes (HEITZ 1934a, b; KAUFMAN 1934; COOPER 1959; GALL, COHEN and POLAN 1971), the data regarding terminal and
TABLE 6
Radiosensitivity of different chromosomes for different types of aberration
Aberration ratios
Fixation Type of Autosome/X Autosome/Y time (h) aberration Sex chrommome chromosome
Theoretical expectation for random involvement 58.6 39.0 2.4
Theoretical expectation for interchanges involving homologous chromosomes at non identical arms 88.9 0 11.1
Theoretical expectation for interchanges involving homologous chromosomes at identical arms 80.0 0 20.0
* The theoretical expectation is based upon the assumption that the X and Y chromosomes are involved with the same frequency in an exchange and that, according to RIEGER et al. (1973), the probability for the occurrence of an interchange between two chromosomes is proportional to the product of their metaphase length.
A = Autosome; X = x-chromosome; Y = Y-chromoeone.
isochromatid deletions indicate that the heterochromatic centromeric region in these chromosomes is more sensitive to the induction of aberrations. In fact, as may be noted in Table 8, these aberrations occur in heterochromatic area of the autosomes and the X chromosome with frequencies of greater than 20% and 50%, respectively. Moreover, many of these breaks (see Figure 2 e,f,g,h) seem to occur at the point of junction between the two areas. It is also interesting to note that the aberrations induced in the heterochromatin region more oiften involve both chromatids (isochromatid deletions) .
Relationships between the various types of aberration:
Our data clearly show that, in the formation of chromosome aberrations, cer- tain types of rejoining are not observed. In particular, ring chromosomes or in- terstitial deletions have never been observed. It is also interesting to note the high frequency of isochromatid and chromosome deletions compared to the other types of aberration. Lastly, mention should be made of the higher frequency of dicentrics compared with the chromatid exchanges and the low frequency of translocations. However, this last type of aberration was scored only in the more obvious cases and it is probable that many translocations representing pseudo
712 M. GATTI, C. TANZARELLA A N D G . OLIVIERI
TABLE 8
Radiosensitivity of the centromeric region for different types of aberration
Type of aberration and fixation timo (h)
Percentage of aberrations involving the centromeric (heterochromatic region)
Chromosome deletions (S+10) P P 50.1 57.3 88 56.1 54.4
crossing over between homologous chromosomes ( OLIVIERI and OLIVIERI 1964) may not be clearly identifiable under the microscope.
I t would also have been interesting to study how the relationships between symmetrical and asymmetrical exchanges vary during the cell cycle. Unfortu- nately not enough information has been collected to provide us with precise facts in this area. The data that we possess would only indicate that the frequency of the asymmetrical exchanges is higher the further the cells are removed from mitosis, in the exchanges between autosomes, and that the contrary occurs in the exchanges between the two X chromosomes.
DISCUSSION
Differences in radiosensitivity between the two sexes:
The clear difference that we found between the males and the females of Drosophila melanogaster, with regard to radiosensitivity and the spontaneous rate of chromosome aberrations, is surprising both because it is not found in other species and because a fact of this type cannot be deduced from the (extensive) literature on Drosophila melanogaster (see GRAF 1972 and WURGLER 1972 for review). The absence of data in literature in favor of a higher rate of spontaneous or induced chromosome mutations in the females might be explained in two ways: ( I ) either the difference in radiosensitivity that we observed concerns only the chromosome damage produced in the somatic cells of the ganglia, or (2) the type of observation so far carried out, regarding the cells of the gameto- genesis, has not allowed any comparison between cells of the same type in the two sexes.
At the present stage of our studies it is difficult to explain what are the mechanisms underlying the greater radiosensitivity of the chromosomes of the female cells. This phenomenon might be due to greater resistance to breakage among the chromosomes of the males o r to a different efficiency of the repair mechanisms in the two sexes. This difference in radiosensitivity might therefore be associated with the presence or greater activity in the fcmales of the enzyme system that allows the crossing over; these enzymes, which are inactive or only slightly active in the somatic cells of the male, might interact with the damaged
CHROMOSOME ABERRATIONS I N DROSOPHILA 713
DNA and, according to the misrepair hypothesis (EVANS 1966, 1967; KIHLMAN and HARTLEY 1967; DUBININ and SOYFER 1969; EVANS and SCOTT 1969), would turn potential damage into real damage. This would indicate that the mechanism of X-ray-induced aberration formation may involve a process similar to meiotic crossing over (REVELL 1953; WHITEHOUSE 1965). This interpretation would also be in agreement with the idea that “chromosome exchanges, whether induced by physical o r chemical agents or occurring spontaneously at mitosis giving mitotic crossing over, or at meiosis giving meiotic crossing over, are all basically similar events ultimately utilizing the same pathways in the cell” (EVANS 1966). A series of studies carried out on bacteria and fungi (see NEWCOMBE 1971 for review) seem to indicate effectively that UV repair, X-ray repair and recombi- nation are processes controlled by different metabolic paths which have, never- theless, one o r more steps in common. WATSON (1969) has shown that in the oocytes of the desynaptic Drosophila mutant c3G, an effect of fractionation of the dose is absent. According to this author, this would indicate that the same enzymes are involved both in the repair and in the recombination. HOW- ewer, no direct relationship has yet been proved between chromosome aberrations and absence of unscheduled DNA synthesis ( WOLFF and SCOTT 1969) or recom- bination (SWIETLINSKA and EVANS 1970). However, we feel that the absence of this correlation is inconclusive, since it is not known what enzyme activity is absent in the mutants studied. In fact, it is possible that only certain enzymatic steps are common to the different metabolic paths involved in the recombination, in the repair and in the formation of the chromosome aberrations. In this case, for mutations in enzymatic steps that are not common to all three processes, one would logically not expect to find any correlation. The male of Drosophila melanogaster which can be considered a desynaptic individual, might be a case in which one or more eniymes involved in recombination and in the formation of chromosome aberrations are absent or show reduced activity. In this respect also it would be interesting to chock whether other species, in which crossing over does not occur in one of the two sexes, confirm what we have found in Drosophila melanogaster.
A second factor with which to explain our data might be the presence of a different metabolism in the two sexes which results in a different intracellular 0, tension. In fact, this might have a marked influence both on the breakability of the chromosomes and on the repair mechanism (OSTER 1958; SOBELS 1969).
Lastly, it should be borne in mind that certain gangliar cells-though having a diploid numbor of chromosomes-would contain a quantity of DNA equal to 8C (GAY 1967). If these cells were present in different numbers in the two sexes and had a different radiosensitivity, this would constitute a further factor to be taken into account for explaining our data.
Chromosome damage and lethality:
It is becoming increasingly obvious that chromosome aberrations can cause cell death (see WOLFF 1972 fo r review). In Drosophila melanogaster it has been shown that the death of larvae subjected to irradiation is mainly due to chromo-
714 M. GATTI, C. TANZARELLA A N D G. OLIVIERI
some breaks followed by loss of the chromosome (OSTERTAG 1963). This author also finds that larval mortality arter irradiation is higher among the males than among the females. It will be seen that this fact is not in accordance with what we have reported. However, we maintain that a possible agreement may be found between our data and those reported by OSTERRAG if the €act that in Dro- sophila cell death is chiefly due to the complete absence of the X chromosome or of a part of it is taken into account. Males are therefore more exposed to this danger since they possess only a single X chromosome. For this reason, for an equal radioisensitivity of the chromosome aberrations, the males should show a far higher mortality compared with the females than that described by OSTERTAG. It is therefore possible that what has been observed by OSTERTAG is the resultant effect of at least two components: the greater radiosensitivity of the males, due to the hemizygous condition of the X chromosome, and the greater radiosensi- tivity of the females to the induction of chromosome aberrations.
Aberrations and cell cycle:
The sensitivity of the various stages of the cell cycle to the different types of aberration confirms results found by other authors in studies on other materials (BREWEN 1964, 1965; CHU 1964; Fox 1967; SCOTT and EVANS 1967). As the cycle proceeds towards mitosis there is a considerable increase in terminal dele- tions and chromatid exchanges; the iswhromatid deletions on the other hand, do not change much in their frequency. The overall radiosensitivity of the various stages of the cell cycle i s higher at two fixation times (2 and 10 hours). The first peak is produced mainly by the high frequency olf terminal deletions and chrom- atid exchanges, the second by the dicenixics. This would seem to indicate a greater resistance by stage S, compared with stages G, and GI, and would agree with what has been reported by SINCLAIR and MORTON (1966) for Chinese hamster cells and by ADAMS and EVANS (1970) for human leukocytes. These findings, however, do not agree with the more generalized knowledge that there is an increase in radiosensitivity from G, through S to G, (WOLFF 1968).
The high sensitivity in G, observed in our system is related to the frequency of exchanges which is higher in GI, than in S or G,. These results are in contrast with the majority of data obtained on different animal and plant cells (WOLFF 1967), although similar results were obtained on human leukocytes by BREWEN (1965) and by PREMPREE and MERZ (1969). A possible explanation for such a discrepancy is, according to BREWEN (1965), that the chromosomes of G, nuclei show a closer spatial relationship than do those of S and G,. However, it is diffi- cult to correlate the sensitivity of the various stages of the cell cycle with regard to the chromosome aberrations since different components, which can vary inde- pendently, must be taken into account. The breakability of the chromosome, the repair, the arrangement of the chromosomes and hence the number of sites, the volume of the nucleus and that of the chromosomes are factors that can influence the formation of chromosome aberrations through the variations that occur in them throughout the cell cycle (EVANS 1962; WOLFF 1967).
CHROMOSOME ABERRATIONS IN DROSOPHILA 715
Distribution of the breaks:
In order to compare the distribution of aberrations produced within and between different chromosomes, we considered chromatid and chromosome terminal deletions and the isochromatid aberrations. This was because chromatid exchanges and dicentrics are difficult aberrations in which to identify the points at which exchange has occurred and also because their distribution is greatly influenced by the spatial arrangement of the chromosomes (see below).
The distribution of the breaks along the chromosome is chiefly relevant with regard to the controversial question of the different sensitivity of euchromatin and heterochromatin. Drosophila melanogaster provides indirect data on this question. These data, which have been deduced from analysis of the F, stage after treatment of the cell during spermatogenesis, are of a contrasting nature and can fundamentally be related to two different hypotheses: greater sensitivity of the heterochromatin (MULLER 1954) and equal sensitivity (KAUFMAN 1954).
Taken as a whole, our data indicate a greater sensitivity of the heterochromatic region, especially in the autosomes and especially for the isochromatid deletions. The absence of terminal deletions in the Y chromosome and the presence of iso- chromatids in the same chromosome would appear to confirm that the second type of aberration can be induced more easily than the first in the heterochro- matin. We consider that this may be due to the close pairing of the two daughter chromatids in the heterochromatic region. In fact, a closer pairing might favor the breaking of both chromatids instead of only one, or else, according to the misrepair hypothesis, the cross-polymerization of two very close lesions.
The distribution of the aberrations among the various chromosomes has shown that the X chromosome has roughly the same radiosensitivity as the autosomes. On the other hand, as has been seen, the Y chromosome not only shows a marked radioresistance as regards the terminal deletions, but is also more resistant with regard to the isochromatid and chromosome terminal deletions.
This result does not agree with what has been said about the higher radio- sensitivity of the heterochromatin, since, as is known, the Y chromosome is one that is entirely heterochromatic. It is difficult to explain this discrepancy; it can only be thought that the heterochromatin of the Y chromosome is different from that of the X chromosome and the autosomes. In this respect, BLUMENFELD and FORREST (1971) have suggested that poly d(AT) is preferentially, if not exclu- sively, localized o n the Y chrolmosome. FACCIO DOLFINI (1974) has also recently shown that, on staining with quinacrine, the Y chromosome shows a brighter fluorescence than the heterochromatin of the other chromosomes. Since a spe- cificity olf quinacrine for A-T-rich sequences has been reported (ELLISON and BARR 1972), it is very probable that the heterochromatin of the Y chromosome actually has a specific composition. However, it is hard to believe that the greater radioresistance of the Y chromosome may be connected only with the base com- position of its DNA; it is possible that other factors, such as the protein composi- tion or the coiling of the chromosome filament, may play an important role in determining the resistance of the Y chromosome. It should also be considered that a high sensitivity might be a characteristic not of the heterochromatin in itself,
716 M. GATTI, C. TANZARELLA A N D G. OLIVIERI
but of the intermediate areas between heterolchromatin and euchromatin; these intermediate areas would be absent or less numerous in an entirely heterochro- matic chromosome.
Importance of the spatial arrangement of the chromosomes:
The data presented in this work constitute further support for the theory of sites (see WOLFF 1967 for review) and confirm that the spatial arrangement of the chromosomes influences both the type and the frequency of the chromosome aberrations induced. We showed that in endoreduplicated Chinese hamster cells the exchanges within diplochromosome are far more frequent than the exchanges between diplochromosomes (GATTI et al. 1973). Similarly, in the cells of Dro- sophila, where somatic pairing is present, we find a high frequency of chromatid exchanges and dicentrics between homologous chromosomes. The importance that the distance between the chromosomes has in the genesis of the chromosome aberrations is proved by the fact that when the pairing is of a lesser degree, as occurs between the X and Y chromosomes in the male (MOORE 1971), there is also a lower frequency of exchanges and dicentrics. This is clearly seen from the Table 7, if the percentage of exchanges and dicentrics between the two X chromo- somes in the female is compared with that for the X and Y chromosomes in the male. Table 7 also shows another interesting fact: there is less autosome-sex- chromosome interaction for the chromatid exchanges than for the dicentrics (8.0 compared with 17.9 in the females and 5.9 compared with 15.3 in the males). This fact indicates that the two sex chromosomes, both in the female and in the male, probably draw closer together as the cell cycle proceeds towards mitosis. This could be correlated with the progressive formation of the nucleolus.
In our opinion, somatic pairing also determines the low frequency of scorable translocations. In fact, it is probable that many translocations are not scorable, since they represent, as in the spermatogonia, induced pseudo-crossing over (OLIVIERI and OLIVIERI 1964).
I t has been reported that in Drosophila melanogaster the spermatocytes are the cells of the spermatogenesis that are most sensitive to the partial or total loss of the sex chromosomes and to the deletions of the same; on the other hand, the highest frequency of translocations has been found in the spermatids (See LEIGH 1969 for review). We maintain that in the light of our data this can be explained. In fact, it is very likely that in the spermatocytes, since a close pairing is still present between homologous chromosomes, most of the breaks interact in such a way as to produce exchanges between homologous chromosomes (pseudo cross- ing over). In the spermatids, however, since they are in haploid condition, the breaks would more readily interact with others present on different chromo- somes, giving rise to translocations in the case of symmetrical rejoining. Another phenomenon that can probably be correlated to somatic pairing is the possible induction of dicentrics at the beginning of stage S. In fact, a similar phenomenon has been observed in diplochromosomes of endoreduplicated cells (GATTI et aZ. 1973) and has been interpreted as the result of an asymmetrical double chromatid exchange involving the four chromatids making up the diplochromosome.
C H R O M O S O M E ABERRATIONS IN DROSOPHILA 71 7
The almost total absence of both chromosome and chromatid intrachanges is a fairly surprising fact since it is known that such types of aberrations have been detected in the off spring after irradiation of the sperm (See LINDSLEY and GRELL 1968 for a review). This fact could not even be deduced from our work on endo- reduplicated cells, since in this type of material we have often observed both interstitial deletions and rings. We consider that this fact too can be associated with the presence of somatic pairing in the cells of Drosophila melanogaster. Indeed, it is possible that this type of spatial arrangement of the chromosomes and the bond that holds them together does not allow the formation of looped regions in interphase. If this is true, it would be necessary to consider that the chromosome pairing present in the endoreduplicated cells and in somatic cells of Drosophila are of a different nature and are probably mediated by different cellular structures.
C O N C L U S I O N S
Taken as a whole, our data have shown that the cells of the larval ganglia of Drosophila melanogaster represent a system of cells in uiuo that is highly suitable for a study of induced chromosome aberrations. The small number of chromo- somes per cell and their easy recognition make scoring very quick and allows an accurate analysis of the distribution of the aberrations within and between chromosomes. Furthermore, this system calls for simple techniques and the cost is very low. For these reasons we consider that the larval ganglia of Drosophila can in future constitute a useful test system for assessing the genetic damage produced by chemical substances.
In addition, this system, with the others already available for Drosophila melanogaster (ABRAHAMSON and LEWIS 1971). may enable a model to be estab- lished for correlating the damage induced in the germinal line, estimated by genetic methods, and the damage induced in the somatic cells. Since the only genetic damage easily detectable in man is represented by chromosome aberra- tions in the leukocytes, such model studies, as has been emphasized by SOBELS (1971), may be a valuable aid for a better assessment of the genetic risk in man.
We are grateful to MISS M. P. BELLONI for the excellent technical assistance.
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