assigned to the pericentromeric heterochromatin or to the euchromatin, This dis- tinction was not made for the entirely heterochromatic Y chromosome or for the.
MUTAGEN SPECIFICITY I N THE INDUCTION OF CHROMOSOMAL ABERRATIONS IN SOMATIC CELLS O F DROSOPHILA MELANOGASTER S. PIMPINELLI, D. PIGNONE, G. SANTINI, M. GATT1 and G. OLIVIERI
Centro d i Genetica Euoluzionistica, Institute d i Genetica, Facoltri d i Scienze, Uniuersitri d i Roma, Italy Manuscript received September 27,1976 Revised copy received November 11, 1976 ABSTRACT
The distribution of chromosomal aberrations between and within chromosoIiies of male D.melanogaster somatic cells after treatment with UV has been analyzed.--Distribution of the breaks between chromosomes was largely nonrandom since we found a higher aberration frequency than that expected on the Y chromosome. Moreover, within the chromosomes the aberrations are clustered in the pericentromeric heterochromatic regions. The above distribution is compared with that of the breaks induced by X rays and methylmethane-sulphonate (MMS) which were distributed in a different pattern.
HE neuroblasts of Drosophila melanogaster constitute a cell system that is highly suitable for the study of induced chromosomal aberrations (GATTI, TANZARELLA and OLIVIERI1974a,b; GATTIet al. 1975; PIMPINELLI et al. 1976). Moreover, various cytochemical techniques have been recently used to show that the heterochromatin of D.melanogaster is heterogeneous, and particularly ATrich heterochromatic regions have been detected (PIMPINELLI, GATTIand DE and SANTINI1976). MARGO 1975; GATTI,PIMPINELLI D. melanogaster neuroblasts can therefore be used advantageously in a systematic study of the distribution of induced aberrations in the genome, in a n attempt to relate the sensitivity of the chromosome to its chemical and structural characteristics. The present study deals with the distribution beween and within chromosomes of the aberrations induced by UV irradiation, a widely studied clastogen (CHU 1965a,b; WOLFF1972). Moreover, we compared the distribution of the breaks induced by UV with those induced by X rays and methyl-methane and OLIVIERI1974b; GATTIet al. sulphonate (MMS) (GATTI,TANZARELLA 1975). MATERIALS A N D METHODS
Three experiments were carried out using the same scheme except for the post-treatment fixation times. Neural ganglia from male third instar larvae of the Oregon-R strain were placed on two layers of blotting paper soaked i n physiological solution in the bottom of plastic Petri dishes. The Petri dishes were then exposed to UV irradiation from a 30-W PHI germicidal lamp giving a n incident intensity at the target level (3 cm away) of 190 ergs/mmz/sec. Immediately Genetics 8 5 : 249-257 February, 1977
s. PIMPINELLI et al.
250
after irradiation the ganglia were transferred to a physiological solution (0.7% NaCl) containing 20% fetal calf serum and were kept in the dark in order to avoid the action of the photoreactivation enzyme in D. melanogaster (TROSKO and WILDER 1973; BOYDand PRESLEY 1974). After 2 h r in the first experiment. 6 h r in the 2nd experiment and 10 hr in the 3rd experiment, the ganglia mere fixed and squashed in acetic orcein according to our usual procedure (GATTI, TANZARELLA and OLIVIERII974a,b). 1.5 h r before each fixation, colchicine was added to the culture solution to a final concentration of 10-5 M. RESULTS
In the first experiment in which only 2 h r elapsed between exposure to UV and fixation, there were no aberrations observed in the 300 metaphases scored. In the second and third experiments in which 6 and 10 hr elapsed between treatment and fixation, aberrations exclusively of the chromatid type were found (Table 1). In Table 1 the aberrations refer to three different groups of chromosomes: autosomes, X chromosome and Y chromosome. It is difficult in fact to discriminate between the two pairs of autosomes and to identify aberrations in chromosome 4 . Within the autosomes and the X chromosome, the breaks were assigned to the pericentromeric heterochromatin or to the euchromatin, This distinction was not made for the entirely heterochromatic Y chromosome or for the chromatid exchanges for which it is difficult to precisely identify the points of breakage. In Table 2 and Figure 1 ., the distribution between and within chromosomes of the aberrations induced by UV is compared with that of the aberrations and OLIVIERI1974b; induced by X rays and by MMS (GATTI,TANZARELLA GATTIet al. 1975). The data presented in Table 1 and 2 show that: ( I ) UV induces aberrations of the chromatid type in cells irradiated in phase 100
UI C 0
-
._ m
L L
m
n m
40
a .
C 0
0 L
al
a
0 EXPECTED
X RAYS
MMS
1
U V I E X P 21
UVIEXPJI
FIGURE 1.-Relative frequencies (%) of chromatid aberrations between different chromosomes after treatment with X rays, MMS or UV. The expected frequencies are derived from relative mitotic lengths of autosomes and sex chromosomes. A = Autosome; X = chromosome; Y = chromosome.
1135
1056
6 (Exp.2)
10 (Exp.3)
152
91
Number of cells with aberrations
X
56 6 (36) (3) 50 11 (31) (11)
A
__---
-
-
1 (1)
Y
Chromatid deletions
8 (6) 39 (31)
A
Y
1 21 (1) (21) 7 52 ( 7 ) (52)
X
Isochromatid deletions*
1
A-X
-
A-Y
1 9 - - -
4
A-A
-
X-Y
Chromatid exchanges
18
3
17
X
21
A
61
5
-
24
9 1 2 4 5
-
Y
A
X
______
Isochromatid gaps
Y
Chromatid gaps
* These aberrations can also result from the replication of a chromxome break (chromosome deletion). Within brackets the aberrations involving the centromeric heterochromatic region or the intermediate areas between euchromatin and heterochromatin; the Y chromosome is considered entirely heterochromatic. A = Autosome; X = X chromosome; Y = Y chromosome.
Number of cells scored
Post-irradia tion time of fixation ( h )
Distribution of chromaiid aberraiions between and within chromosomes in larual ganglia cells of Drosophila melanogaster ( 8 8 ) after treatment with UV
TABLE 1
E 3
3
P
2!
20
H U
Exp. 3
64.5
70.9
103
197
88.1
83.1
A
354
1478
-
9.1
7.8
8.2
9.7
X
Percentage of aberrations involving
26.4
21.3
3.7
7.2
Y
7.1
9.1
10.8
8.5
A/ X
2.4
3.3
24.0
11.6
A/Y
Aberration ratios
107
71
217
1150
Aberrations Total+
74.8
64.8
68.2
48.9
25.2
35.2
31.8
51.1
Heterochromatin Euchromatin
Percentage of aberrations involving
______
Chromatid and isochromatid gaps have not been included i n this analysis. * Chromatid and isochromatid deletions scored as a single event; chromatid exchanges as two events. -f This total is lower then the total in column 1, since Y chromosome aberrations and exchanges have not been included. $ Data from GATTI,TANZARELLA and OLIVIERI1974b. Data from GATTIet al. 1975. A = Autosome; X = X chromosome; Y = Y chromosome.
uv
time of fixation 2.4.6h.;$) MMS (post-treatment time of fixation 4.8.12h.iS) Exp. 2
X rays (1250 R ; post-treatment
Treatnient
Aberratims Total*
Sensitiuity of different chromosomes and of differentchromosome regions after treatment wiih X ray, M M S , or UV
TABLE 2
g
E 2.
2r
HE!
?
CHROMOSOMAL ABERRATIONS IN
D.melanogaster SOMATIC
CELLS
253
G1 or S, but not in cells irradiated in G2. Previous experiments have in fact shown that 6-10 hr before mitosis most of the neuroblasts of D.mezanogaster are in G1 or S (PIMPINELLI et al., 1976). The present data therefore confirm that UV irradiation has an S-dependent action in inducing chromosomal aberrations (BENDER,GRIGGS and WALKER 1973; GRIGGS and BENDER1973; WOLFF,BODYCOTE and PAINTER 1974). (2) The distribution of the breaks between chromosomes after UV treatment is nonrandom. If it is assumed that X and Y chromosomes offer a target that is about 1/8 that of the autosomal one, there is a greater frequency of breaks on the Y chromosome than expected. Moreover, the breaks induced on this chromosome are only of the isochromatid type. (3) The distribution of the aberrations within the chromosomes is nonrandom. The heterochromatin near the centromere occupies a length of from 30% to 50% of the X chromosome and about 20% of the second and third chromosomes (for and SANTINI1976). The frequency of breaks review see GATTI,PIMPINELLI in the heterochromatin of these chromosomes is much higher than expected (see Table 2). Moreover many breaks are localized in distal heterochromatic regions and appear to involve the junctions between eu- and heterochromatin. (4) Like X rays and MMS, UV induces aberrations mainly in the pericentromeric heterochromatic regions of the autosomes and the X chromosome. Unlike X rays, and above all MMS, UV specifically induces aberrations on the Y chromosome in which the proportion of breaks produced go respectively from 3.7% (MMS) and 7.2% (X rays) to 21.3% (UV experiment 2) and 26.4% (UV experiment 3 ) . As far as the sensitivity of heterochromatin with respect to euchromatin is concerned, the situation can be schematically represented as follows:
Mutagen treatment
X rays MMS UV rays
Heterochromatin of chromosome X , 2 and 3
+ ++
++
Heterochromatin of Y chromosome
-
++
DISCUSSION
The three mutagen treatments examined (X rays, MMS and UV rays) did not induce terminal deletions on the Y chromosome. We feel, however, that this is a trivial phenomenon due merely to the impossibility of microscopically scoring the deletions induced on this chromosome. Indeed, in the Y chromosome, the sister chromatids are so close to each other that the broken chromatid fragments remain in situ. This interpretation is borne out by the sporadic observation of terminal deletions on the Y chromosome in some colchicine-induced C-anaphases. As far as the isochromatid breaks are concerned, it was noted that after X ray treatment, and especially after treatment with MMS, these aberrations were
254
s. PIMPINELLI et at.
clustered on the heterochromatin of the X chromosome and of the autosomes, but not on that of the Y chromosome. In fact, after MMS treatment the Y chromosome had a frequency Qf isochromatid breaks which was lower than that for euchromatic regions of the same length. It was suggested that this discrepancy was due to the different base composition of the Y chromosome or to the absence in this entirely heterochromatic chromosome of those intermediate areas between euchromatin and heterochromatin which seem to be the sites of a great number of breaks (GATTI,TANZARELLA and OLIVIERI1974b; CARRANO and WOLFF1975). Some recent studies have made it possible to establish that the Y chromosome has the following characteristics: (1 ) It contains a high proportion of the d (A-T) rich satellite DNA (BLUMENFELD and FORREST 1971). (2) It is entirely decondensed by the benzimidazole derivative Hoechst 33258 which binds (COMINGS 1975; LATTand WOHLLEB 1975) and specifically decondenses AT-rich DNA (PIMPINELLI, GATTIand DE MARCO 1975; GATTI.PIMPINELLI and SANTINI1976), while the heterochromatin of the autosomes and of the X chromosome show limited areas of decondensation. (3) Tritiated actinomycin which interacts specifically with the GC bases (REICHand GOLDBERG 1964) is bound to the Y chromosome to a lesser extent than to the other heterochromatic regions (PIMPINELLI and GATTI,unpublished results). (4) This chromosome. with the exception of few regions. fluoresces brightly after staining with quinacrine or 33258 Hoechst, which are both fluorescent and probes specific for DNA rich in AT (for review see GATTI,PTMPINELLI SANTINI1976). It appears therefore that the Y chromosome of D.melanogaster is on the average richer ill AT than are the other heterochromatic regions. We suggest that the base composition of the Y chromosome of D.melanogaster plays an important part in determining its response to the various mutagen treatments. Thus, it would be highly sensitive to UV rays. which induce chromatid-type aberrations mainly through the formation of thymine dimers (BENDER.GRIGGSand WALKER 1973; GRIGGSand BENDER1973), but would, however, be more resistant than the rest of the genome to MMS which, as is known, interacts mainly with GC-rich DNA (cf. LAWLEY 1966). Numerous authors have described a nonrandom distribution between and within chromosomes of the aberrations induced by various mutagens (see for review, NATARAJAN and SCHMID1971; RIEGERet at. 1975). Recent banding techniques have also made possible a more careful analysis of the location of both spontaneous and induced aberrations and sister chromatid exchanges (JACOBS et al. 1974; COOKE, SEABRIGHT and WHEELER 1975; SCHUBERT and RIEGER1976; SMYTHand EVANS 1976). Taken as a whole, the above data point to a nonrandom distribution of the aberrations due to a nonrandom distribution in the genome of areas having heterochromatic properties. However, the differences in the base composition of different parts of the genome are not great enough to reflect a nomaEdom distribution of aberrations (EVANS and SCOTT1969). We feel, how-
CHROMOSOMAL ABERRATIONS IN
D.melanogaster SOMATIC CELLS
255
ever, that in this context the case of the Y chromosome of D. mezanogaster is exceptional. In fact, as mentioned, there is evidence that the Y chromosome is richer at AT than the rest of the genome, and it is therefore likely that it is, to a large extent, made up of the 3 AT-rich satellite DNAs of Drosophila melanogaster. These DNAs constitute about 18% of the genome, and the two analyzed to date (PEACOCK et al. 1973) have a great enough AT content (93% and 78% of AT respectively) to explain the resistance of the Y chromosome to MMS. In addition, the high proportion of adjacent thymidines present in both these DNAs (BRUTLAGand PEACOCK 1975; ENDOW, POLAN and GALL1975) makes them especially susceptible to UV lesions and explains the specific response of the Y chromosome to this mutagen. In conclusion, the distribution of aberrations after the three mutagen treatments examined has shown that there are two classes of heterochromatin in D. melarnogaster. The first class, which includes the heterochromatin of the X chromosome and of the autosomes, has a consistently greater sensitivity than euchromatin, independent of the mutagen used. This class probably has a base composition on the average not very different from that of euchromatin, and its “sensitivity” could be related to factors such as its greater compaction in interphase (SMYTHand EVANS1976) or to its peripheral location in the nucleus (Hsu 1975) or possibly to different repair mechanisms (FALK1961). The Y chromosome constitutes the second class of heterochromatin, which differs from the first in its exceptional AT richness which is related to its different sensitivity with respect to euchromatin and the other heterochromatic regions, depending on the type of mutagen used. The authors wish to thank MISSM. P. BELLONIfor her excellent technical assistance. This work was supported i n part by the Association between Euratom and C.N.R., contract NO. 136-71-7 BIOI. L I T E R A T U R E CITED
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