CHROMOSOMA
Chromosoma (Berl) (1989) 98:378 387
9 Springer-Verlag1989
Position-effect variegation and intercalary heterochromatin: A comparative study I.F. Zhimulev, E.S. Belyaeva, V.N. Bolshakov, and N.I. Mal'ceva Institute of Cytology and Genetics, Novosibirsk 630090, USSR
Abstract. The behaviour of IH (intercalary heterochromatin) regions of Drosophila melanogaster polytene chromosomes was compared with that of euchromatin condensed as a result of position-effect variegation. Normally replicating regions, when subject to such an effect, were found to become among the last regions in the genome to replicate. It is shown that the factors which enhance position effect (low temperature, the removal of the Y chromosome, genetic enhancers of position effect) increase the weak point frequency in the IH, i.e. enhance D N A underreplication in these regions. We suggest that the similarity in the properties of IH, CH (centromeric heterochromatin) and the dense blocks induced by position effect is due to strong genetic inactivation and supercondensation caused by specific proteins in early development. The primary D N A structure is not likely to play a key role in this process.
Introduction
It is known that polytene chromosomes contain numerous sites of so-called intercalary heterochromatin (IH). The features displayed by IH in polytene chromosomes include: late replication, frequent breakage due to underreplication, ectopic pairing with centromeric heterochromatin (CH), higher frequency of somatic synapsis and breakpoints of chromosome rearrangements (Zhimulev et al. 1982; Lifschitz 1983; Bolshakov et al. 1985; Lamb and Laird 1987). On the other hand no homology with satellite DNA, the basic component of CH regions, has been found, so that it is not clear if these regions should be regarded as true heterochromatin (Spofford 1976; Hilliker etal. 1980). Many of the IH regions are still likely to comprise repeats of various types: for instance histone genes and 5S rRNA genes in the 39DE and 56EF regions, respectively, a cluster of eight serine t R N A genes (Cribbs et al. 1987) and various types of repetitive sequences in regions 12DE and 16F-17A (Livak 1984; Lamb and Laird 1987; Baumann et al. 1987; Kholodilov et al. 1987, 1988). Thus, similarity between IH and CH does not seem to depend on sequence homology, and, we may suppose, is not dependent on the repetitiveness as such. In polytene chromosomes both types of regions resemble each other cytologically: they are dense and stain deeply. This supercondensation may lead to late replication and to subsequent underreplication with breaks and ectopic pairing. In this
respect, any euchromatic region, when strongly inactivated and condensed, will possess the main properties of heterochromatin. A striking example is the "heterochromatization" of euchromatic regions resulting from position-effect variegation. The genetic repression in thiscase is accompanied by strong condensation of the corresponding regions with fusion of many nearby bands into one compact block (Hartmann-Goldstein 1967; Reuter 1982; Zhimulev et al. 1986, 1988). The removal of the Y chromosome, specific position-effect enhancers and low temperature are known to strengthen genetic inactivation and condensation. If the properties of the IH regions are due to their supercondensed condition rather than their D N A sequence than their behaviour and that of the blocks formed from euchromatin as a result of position effect may be similar. Blocks appearing as a result of position effect should be late replicating with possible underreplication, and show weak points and formation of ectopic threads. It may also be predicted that factors enhancing position effect will strengthen the expression of the properties of IH. As for replication of D N A subject to position effect, a problem first posed by Schultz (1936), this has been discussed frequently since then, but there is still not enough data to allow a conclusion to be drawn (see below). A comparative analysis of the behaviour of the IH regions and blocks arising as a result of position effect has never been made. We have begun such a study and in this paper show that: (i) the euchromatin region, when condensed as a result of position effect, becomes one of the most late-replicating regions in the genome. (ii) The genetic and environmental factors which strengthen condensation resulting from position effect do increase the break frequency in the IH regions of polytene chromosomes. Materials and methods
Larvae were grown in standard medium at 18 ~ and 25 ~ C. The temperature was modified in accordance with the experimental schedule.
Strains. Batumi-L, a wild-type strain, was obtained from the Batumi population by V.A. Lychev in 1963 (Zhimulev 1974). Strain T(1;2)dor~"rT/FM6 (Zhimulev et al. 1986, 1988) will be designated in the text as var7 and the translocated IA-2B7-8 region of the X chromosome by STE (short translocated element).
379
Dp (1 ;f) 1337 contains a fragment of the X chromosome from 1A to 2B7-8 and a non-identified fragment from the heterochromatic part of the Jr" and shows position effect (Lindsley and Grell 1968; Belyaeva et al. 1982). Strain X, y sc ac Pg~w/Dp (1 ; 1)pn2b/C (1) Rm,ywf carries a duplication of region 1A-1EI 2 translocated to CH in the Jf chromosome and shows position-effect variegation. Dp(1;1)pn2b was obtained by crossing over (Tolchkov et al. 1984) from In (1LR)pn2b, induced by X-rays (Ilyina et al. 1980). A strain carrying simultaneously a suppressor (Su-var) and an enhancer (En-var) of variegation was provided by G. Reuter (University of Halle, DDR). The strain is maintained in the cross:
3H]thymidine into polytene chromosome regions was analysed. The banding was interpreted according to the photographic map of Lefevre (1976) with an accuracy of letter subdivisions.
Determination of the temperature-sensitive period of condensation and breaks. To synchronize deposition of eggs a Petri dish with medium was put in a population box for 30 min at 25 ~ or 18 ~ C and larvaewere grown at the above temperatures to the end of the third instar. In other cases embryos or larvae were kept at these temperatures for different developmental periods (Fig. 7).
~-~-wm4h/wm4h; ed dp cl/T(2;3)apXa+In(2L)Cy, apX"Cy, Su-var (3)9/+ x ~J; wm*h/Y; ed dp cl/T (2;3)napXa+In (2L) Cy,apXaCy/En-var (3)201. Half of the males in the offspring should receive En-var. They can be recognized by
Results
the transparent unpigmented cells of the Malpighian tubes (G. Reuter, personal communication). Males without the Y chromosome were derived from the following crosses: (i) Females of Batumi-L (X/X) or var7/FM6 were crossed with XX L. yS, y su (w")wa (the attached X and Y chromosomes) males. All the males in the offspring have X/O. (ii) X/O, Dp (1 ; l)pn2b males were obtained from the cross : ~ xx, y w f x ~2 X/Y/Dp (1;t)pn2b. Off) X/O, Dp (1;f)1337 males were obtained after mating of xx/Dp(1;f)1337, y+ females to yellow males. Among the male progeny those larvae were selected which had the dark mouth hook characteristic of y+, i.e., X,y/O/
In earlier studies of rearrangements showing position-effect variegation it was found that after translocation to CH, euchromatic regions became late replicating (Ananiev and Gvozdev 1974; Wargent and Hartmann-Goldstein 1976). However, the methods used, in particular a comparison of the labelling frequencies of the translocated region and a few nearby regions in the rearranged and normal homologues averaged for a sample of nuclei, and the lack of precise criteria for determining the morphology of the region subject to position effect, did not permit an estimation of the degree of delay in replication and its interrelation with the morphology of the translocated euchromatic region. To avoid the above difficulties we used T(1 ,'2)dorwr7 in which the 1A-2B7 region is translocated to the CH of chromosome 2L. In salivary gland cells the genetic inactivation of this region arising from variegation can be detected by the absence of ecdysone-inducible puffs which always appear in a normal homologue: the defects are due to the inactivation of the ecs locus lying near the 2B3-5 band breakpoint. In females the inactivation of the ecs locus is always accompanied by condensation of the 2B1-7 region that can be easily detected cytologically as the bands in this region fuse into a dense block of chromatin. Our preliminary data have shown that T(1;2)dor v~r7 in females evokes changes in the replication schedule of the chromosome region adjacent to the heterochromatin. When condensed the 2B region becomes late replicating, whereas late replication has never been observed in its normal homologue (Zhimulev et al. 1986, 1988). The replication pattern in the 1A-2B7 region in females and in X/Yand X/O males was studied in the above translocation. After pulse labelling with [3H]thymidine the preparations of salivary gland chromosomes were examined for nuclei with contrasting morphology of the 2B region in the STE (puff block) (see Materials and methods). Then the degree of completion of replication of the 2B region in the STE was analysed on autoradiographs and compared with that of the normal homologue and with late-replicating regions of autosomes. Preliminary cytological analysis of nuclei with distinct morphology of the 2B region in the STE, the X chromosome and segment 75C-80A-C in chromosome 3L yielded from 127 to 432 such nuclei depending on their genotype and morphology. After autoradiography, for varT/FM6 females and var7/O males the fraction of such nuclei showing
Dp(1;f)1337,y + Descriptions of the other mutations marking the strains used can be found in Lindsley and Grell (1968).
Cytological procedures. To determine the morphology of chromosome regions subject to position effect aceto-orceinstained squashed preparations of salivary gland chromosomes were analysed by phase contrast microscopy. The numbers of larvae and chromosomes analysed are indicated in Results. When studying the break frequencies at weak points both whole chromosomes and chromosome fragments were analysed. For each weak point 20-40 regions were studied in each of 8-15 larvae except for the cases specially indicated in Results. The dissection of salivary glands and their labelling with [methyl-3H]thymidine (V/O Izotop, Leningrad, sp.act. 1,700 TBq/mol, 100mCi/ml, incubation time 30min at room temperature), the preparation of slides and autoradiography (M-type liquid emulsion, Gos NII China Photo Project, Moscow, exposure time 7-10 days) were described in Zhimulev et al. (1982). Before being covered with emulsion the preparations were analysed by light microscopy and the coordinates of nuclei (where the morphology of the 2B region not overlapped by the chromocentre, the 75-80A-C segment of chromosome 3L and, if possible, the JF chromosome could be determined) were noted. The morphology of the 2B region in the STE was regarded as normal if the banding in region 2B1~4 was distinct. If the 2B1-4 bands were fused into one dense block as described in Zhimulev et al. (1986, ~988), the morphology was classified as condensed. After autoradiography and development the noted nuclei were examined and incorporation of [methyl-
Changes in replication pattern in the region subject to position effect
380 H
var7/FlVr6
Ioi!
o [~ ~ rD
co
a
b
n---8
r~=16
cO
N
varT/Y
N R
1 r <
cO
c
d
n=22
n=27
var7/O $$
** ~.- ,~-- ,e- ,v-
t--- t ~ t . - ~-- t - - p,- ~C~
,e-- ~.- ~ - ,r-- , r -
e
n=9
t~- t-,- t - - t--- ~O cO
cO
f
~=16
late replication was only t0% 21%, but was 48%-70% for varT/Y males. Moreover, the chromosome morphology became worse after autoradiography in many of these nuclei and could not be analysed. In total the 2B region in STE could be analysed in 2% 13% of the initially selected nuclei. Then the data on the labelling pattern of these nuclei were plotted on a histogram for every variant (Fig. 1). The abscissa shows the labelled regions of the X and the segment of chromosome 3L; the presence/absence of label in these regions is designated by conventional signs. The nuclei are ranked according to decreasing number of labelled regions in segment 75C-80A-C of chromosome 3L, so that at the very top of the histogram there are nuclei with the least number of such regions, i.e. nuclei at the latest stage of replication. Figure 2 shows photographs of some nuclei which are at the stage of discontinuous labelling after a pulse of [methyl-3H]thymidine. At this stage late-replicating X chromosome regions 1A, llE, 12E, 19E and regions 75C, 76B, 77E, 79E and 8 0 ~ C in the segment of chromosome 3L are labelled (Arcos-Teran 1972; Zhimulev et al. 1982). Figure 1 presents data on replication of the 2B region relating
Fig. 1a-f. Replication of the 2B region in the short translocated element (STE) with normal (a, e, e) and condensed (b, d, f) morphology in var7/FM6 females (a, b) and var7/ Y (e, d) and varT/O (e, f) males. The replicating regions of the X chromosome and the 75C-80A-C segment of chromosome 3L are indicated. Every line represents a nucleus analysed, x, labelling; blank, the absence of labelling; *, no data about labelling of this region in a given nucleus, n, number of nuclei analysed
morphology and late-replicating chromosome regions in samples of nuclei. In the normal situation the 2B region is known to complete replication simultaneously with termination of the continuous labelling stage, when almost all X chromosome regions are replicated, both in males and in females (ArcosTeran 1972; Kalish and H/igele 1976). In late-replicating nuclei of var7/FM6 females the 2B region in the STE when morphologically normal does not incorporate label (Fig. 2). The converse is true for the condensed state, although the normal 2B region of the balancer chromosome FM6 (conjugated with STE in region 1A-E) is not labelled (Fig. 2b). The same is found by analysis of samples of nuclei: when normal, the 2B region in the STE does not incorporate label during replication of the 77E region which completes replication a little earlier than other IH regions of the segment of chromosome 3L (Fig. 1 a). But when condensed, the region does incorporate label for much longer (Fig. 1 b). In the case of var7/FM6 females we could not make such a comparison with late-replicating IH regions in the Jr"chromosome, since the rearranged chromosomes vat7 and FM6 had a complex synapsis pattern that could not be analysed.
38]
Fig. 2a-f. Replication of the 2B region (indicated by bracket and arrow) in the STE in late-replicating nuclei of var7/FM6 females (a, b), and var7/Y (e, d) and var7/O (e, f) males. When normal the region is not labelled (a, e, e), but when condensed it is (b, d, f). Late-replicating regions in the X chromosome and segment 75C-80A-C of chromosome 3L are indicated. The 2B region of balancer chromosome FM6 is shown in b. Bar represents 5 mkm
382
Fig. 3a-g. Morphological types of bands of intercalary heterochromatin (IH) in regions 11A6-9 (a-d) and 19E1-4 (e-g) with (c, d, f, g), or without (a, b, e) breaks. 18~ C, Batumi-L strain. Bar represents 5 mkm In late-replicating nuclei of var7/Y and var7/O males the condensed 2B region also incorporates label (Fig. 2d, f) unlike the instances when its morphology is normal (Fig. 2c, e). In var7/Y males replication of the normal 2B region terminates long before its completion in the 4D, 13B, 16F regions of the X chromosome and the 76B, 77A, 77E regions of the chromosome 3L segment (Fig. 1 c), but when condensed, the 2B region completes replication significantly later than these sites (Fig. I d). The same phenomenon was observed in var7/O males when the replication time of the normal and condensed 2B region was compared with that of the 13B region of the X chromosome and the 76B, 77E regions of the chromosome 3L segment (Fig. 1 e, f). Although in our sample we did not have enough nuclei in which the X chromosome had completed its replication and the autosomes were still replicating (Berendes 1966), we observed a few instances in which the male X chromosome was unlabelled while the condensed 2B region in the STE continued incorporating label at the same time as the latereplicating 75C, 79E and 80-C regions of chromosome 3L (upper histogram lines in Fig. 1 d, f). Thus, when condensed as a result of position effect, the 2B region in the STE becomes late replicating and incorporates label simultaneously with the most late-replicating regions of the X chromosome and segment 75C-80A-C of 3L, and in males, after termination of replication in the remainder of the X chromosome. Unfortunately, neither
in var7/FM6 females nor in var7/O males could we observe nuclei at the latest stage of replication when only the chromocentre was still labelling. Therefore, it is not clear whether the condensed 2B region in the STE completes replication at the same time as the most late-replicating regions in the chromosome arms or simultaneously with the CH.
Influence of position-effect modifiers on the frequency of weak points in IH regions We chiefly examined weak point frequency in the X chromosome, because the IH regions in this chromosome exhibit unusual behaviour. The diffuse structure of the male X chromosome, thought to provide dosage compensation, results in earlier completion of replication (Mulder et al. 1968), and breaks in this chromosome are very rare (Zhimul e v e t al. 1982). The X chromosome of females replicates simultaneously with the autosomes and shows numerous weak points in IH regions. Low temperature evidently contributes to the increased break frequencies in the IH regions of Batumi-L larvae (Figs. 3, 4). In 16 out of 25 regions of females studied the frequencies were higher at 18~ C. The breaks observed in the remaining 9 regions were very rare (no more than 1%). Therefore it cannot be ruled out that they were caused fortuitously. As anticipated, at 25 ~ C the break frequency in males was almost zero; it increased at 18~ C, especially in
383 the typical IH regions - 11A6-9, 12E8-9, and 19E1-4 (Figs. 3, 4). Further more detailed analysis was performed in males of the strain carrying a duplication of I A - 2 E I - 2 in the X pericentric heterochromatin - Dp (1;1)pn2b with position effect spreading as far as the sc and y loci (Tolchkov et al. 1984; E.S. Belyaeva, unpublished). In salivary gland cells the morphology of the duplication varies from entirely normal, with all bands from I A to 2E1 2 visible, to completely changed, when only the most distal bands can be basely identified and the remainder of the region is fused into one dense block (Fig. 5 a, f). The most typical intermediate pictures are represented in Figure 5 b-e (more complex instances of morphological changes require further identification and will be described separately). In X/O and X/Y females the degree of condensation and break frequencies in the typical IH regions of the X chromosome and in some IH regions of autosomes (42B, 64C, 70C, 71C, 75C, 84D, 86D) were analysed on the same preparations. When corn-
100-
~ 2o [ [ _l _[~~l'~l
_1 I / [ [ f-
I rgo~
Fig. 4. Frequency of breaks in X chromosome regions of Batumi-L females '(above the chromosome axis) and males (below the axis) at 18~ C (black columns) and 25~ C (whitecolumns). 133-288 nuclei from 9-14 larvae (~/~) and 181-281 nuclei from 12-13 larvae (d~) were studied
paring the intensity of condensation (the frequency and extent of the blocks) in J(/O and X/Y males grown at 18~ and 25~ the enhancement of position effect was found to be correlated with an increase in breaks in IH (Fig. 6). Both values appear more dependent on the presence of the Y chromosome than on temperature, but the maximum condensation and susceptibility to breakage is apparent at 18~ in X/O males (at 14~ C quantitative analysis of the morphology of the duplication is impossible). The same is true for the IH regions in the autosomes (Table 1): the removal of Y and lowering of the temperature results in higher susceptibility to breakage. Thus, the above data show that the break frequencies in IH depend on the presence of the Y and temperature which are the typical modifiers of position effect. Earlier it has been shown that the action of low temperature on genetic inactivation and condensation resulting from position effect is limited chiefly to the early period of development (Hartmann-Goldstein 1967; Zhimulev et al. 1988). To answer the question of whether or not there is also a temperature-sensitive period for break we compared the intensity of condensation and frequency of break of X chromosome of X/O males treated with low temperature during different periods of ontogenesis. The males carried an X chromosome marked with yellow and Dp (1;f)1337 which exhibits position effect. This duplication contains the 1A-2B7-8 region and a non-identified CH region of the J( chromosome. Position effect is cytologically detected by condensation of bands from region 2B to lB. The data presented in Figure 7 show that the action of low temperature on condensation in Dp (1 ;f) 1337 is limited to the initial hours of embryogenesis (not more than 6 h). Later shifting of embryos from 25 ~ to 18 ~ C does not affect the frequency and extent of compact blocks. These data therefore support the view that the early hours of development are crucial for position effect. As for breaks, the action of low temperature on their frequency is apparently not limited to early embryogenesis. The initial 6 h are no more important for temperature sensitivity than the remainder of the polytenization period. Under low temperature conditions the main increase in frequency of breaks was observed throughout the final hours of the first and the whole second instar, when most polytenization cycles occur.
Fig. 5a-f. Morphology of the duplicated fragment of the X chromosome in a strain with the duplication Dp(1;1)pn2b in a normal chromosome (a) and those with various degrees of condensation (b-f). Bar represents 5 mkm
384
.o a X/Dp pn2bl)'~
'~
I
I
I
40
X/Oppn2b/O
8oi X/Op pn2b/Y~i 18~
2~I
g
II
i
l
i
i
i
i
i
i
--
i
I
60t
d
X/Dp pn2b/Y4o 25oc
-
h
20-
N 2C
2B 2A
1F
IE
ICD IB
IA
3C 11A 19E
To investigate the action on position effect o f both the enhancer and suppressor on the breakage at weak points of the I H regions the b r e a k frequencies in two samples of males were compared. Samples o f larvae were obtained from the same cross (see Materials and methods), one with an enhancer and another with a suppressor (Table 2). Breaks were extremely rare in the X c h r o m o s o m e o f males with a suppressor. F o r example, the only instance o f a break was observed in the 19E region; in other regions breaks did not occur. The break frequency in larvae with an enhancer was much higher. F e w breaks were detected in 4D, l l D , 13B, 16F and 19F, and this was never observed in the corresponding sample o f larvae with a suppressor. Moreover, in the enhancer strain changes in the m o r p h o l o gy of the X c h r o m o s o m e t o o k place: bends, helical turns and ectopic threads between some regions appeared. As shown previously (Zhimulev et al. 1982), the ectopic threads were found to connect regions with breaks. Therefore invisible breaks are thought to cause ectopic pairing and, as a consequence, c h r o m o s o m e bends. W e assume that the break frequency in this case should be higher than is shown in Table 1. It is difficult however to ascertain the effect of every factor without d a t a on the frequency o f breaks in the same strain lacking an enhancer or suppressor. A t the same low temperature (18 ~ C) the suppressor is not likely to change the frequency o f breaks dramatically, and the enhancer, in its turn, increases it, but the opposite situation cannot be excluded. It is more likely that b o t h the enhancer and suppressor display entirely different weak effects - the suppressor decreasing and the enhancer increasing the frequency of breaks. In any case, however, it was shown that susceptibility to breakage of the I H regions could be affected by genetic modifiers o f position effect.
Fig. 6a-h.
Influence of the Y chromosome and temperature on break frequency in X chromosome regions (e-h) and on the extent of position-effect condensation (a~l) in chromosome rearrangement Dp (1; 1)pn2b. Abscissa a-d state of condensation of the chromosome fragment from 2EI-2 to the region indicated. The most distal region with normal morphology is shown, e--h Breaks in the 3C, 11A6-9 and 19E regions. Ordinate percentage frequency. 120-200 nuclei studied (20 chromosomes from 6-10 larvae) Nnormal chromosome
Discussion Our d a t a show that condensation o f the 2B region in the rearranged X c h r o m o s o m e in T(1;2)dor va'7 leads to a significant delay in its replication in comparison with its normal homologue. The region becomes late replicating to the same extent as late-replicating regions 1A, 3C, l l A , 12E,
Table 1. Dependence of break frequencies in autosomes on temperature and the Expt. Genotype
Y chromosome
Temperature Number Break frequencies in regions (%)a (~ of 42B 64C 70C larvae
1
X/Dp(1;1)pn2b/Y 25
13
2
X/Dp(1;1)pn2b/Y 18
14
3
X/Dp(1;1)pn2b/O 25
9
4
X/Dp(1;1)pn2b/O 18
7
5
X/Dp(1;1)pn2b/O 14
10
70.8_+16.2 (236) 75.8-+15.3 (245) 80.5_+16.2 (157) 86.2_+ 9.5 (174) 96.7+ 3.5 (180)
64.9_+14.0 (231) 68.9_+15.0 (237) 90.6_+11.5 (160) 96.0_+ 4.6 (175) 97.2_+ 3.6 (180)
76.5_+ 18.7 (246) 83.7__ 12.0 (211) 85.6_+ 18.0 (160) 88.5_+ 13.9 (174) 97.0-+ 3.8 (175)
71C
75C
84D
86D
77.6_+12.4 (258) 79.3+13.1 (215) 82.5--+14.6 (160) 88.5_+13.9 (174) 94.8-+ 3.6 (175)
86.9_+6.6 (260) 86.4__9.0 (220) 89.2_+4.9 (120) 94.9_+5.5 (175) 98.9_+2.2 (177)
48.3_+17.8 (240) 44.6+22.8 (252) 78.6+10.3 (140) 82.1+ 9.5 (140) 96.1+ 4.2 (178)
67.6__.13.0 (238) 69.5+16.4 (252) 78.6+12.1 (140) 95.7_+ 3.5 (139) 97.8+ 3.6 (178)
Break frequencies in all regions in experiments 3 and 5 differ significantly with 0.95 < P < 0.999 a Number of regions studied in parentheses
385
8
8 0 ~
20 0
40
101 []
b
zo p
40
10
.I C
40
10
40
10
eo, I
2o S
d
'o l 2o t
40
g 2s~
10
n
80~ J ~ 40
20 10
18~
0 embryo, 3 6 21larval 123 h instars
1
2
11A 1 9 E
Fig. 7a-u. Dependence of position-effect condensation (h-n) and break frequencies (o-u) on temperature during development of X/ O/Dp(1;f)1337 males, a - g Changes in temperature. Abscissa h - n first column, duplications with any degree of condensation; second column, extensive condensation, i.e. from the breakpoint in 2B7 8 to 2A1-2 and further, o-u Breaks in llA and 19E. Ordinate percentage frequency. 182427 chromosomes studied from 11-15 larvae (h-n), and 312-354 from 11-15 larvae (o-u)
19E in the X chromosome and segment 75C-80A-C in chromosome 3L; moreover, in males it turns out to be the latest replicating region of the X chromosome. However, we could not ascertain whether its replication terminated simultaneously with the late-replicating regions in the chromosome arms or still later, together with the CH. In our investigation of males without a Y we did not find a delay in replication of the IH regions of the X chromosome as reported by Ananiev and Gvozdev (1975). Data from earlier communications by different authors reporting a replication delay in euchromatic regions juxtaposed to pericentric heterochromatin in rearrangements displaying position effect do not allow a realistic estimate of the magnitude of this delay. For example, in the study of [3H]thymidine pulse labelling of polytene chromosomes of salivary glands of Dp (1;J)R males Ananiev and Gvozdev (1974) found an increase in the labelling frequency of the 1D-E5 region in the duplicated segment 1 ~ 3 A 1 - 2 at low temperature and in the absence of the Y chromosome, when compared with this region in the normal chromosome. This frequency was comparable to that of the late-replicating region 1A1-B4 located in the X chromosome segment investigated. However, the morphology of the late-replicating 1D-E5 region was not determined so there was no evidence for its condensation in that case. Wargent and Hartmann-Gotdstein (1976) studied T (1 ; 4) w"258- 21, a rearrangement showing position effect, and found that the 3E region, lying near the breakpoint of the rearranged X chromosome, had a higher labelling frequency if it was heterochromatized than when it appeared euchromatic as in the normal homologue. This frequency was close to that of the late-replicating 3CI 7 region in the X chromosome segment studied. Yet, due to the experimental procedure (larvae were grown at 15 ~ C), the 3CI-7 region itself was very often involved in heterochromatization (Hartmann-Goldstein 1967) and it was not clear whether late replication in that case was caused by the condensation of the 3E region itself or by its fusion with latereplicating region 3C1-7. The T(1;2)dor ~ar7 translocation used in the present study has some advantages in this respect: (i) the 2B region does not contain large dense bands, so that the normal decondensed state can be easily distinguished from the condensed one (with dense block formation). (ii) The 2B1-2 bands comprising this region are early replicating, and there are no late-replicating bands nearby. (iii) Only nuclei with the 2B region in the STE not overlapped by the late-replicating chromocentre and lying apart from it were analysed. (iv) The replication time of the 2B region was determined relative to the late-replicating chromosome regions in the same nucleus. All this allows the relationship between the condensation and replication delay of the 2B region to be ascertained precisely. Thus, the variegated region undergoes condensation along with replication delay. The late replication of the condensed region may presumably lead to underreplication and this, in its turn, to breaks and ectopic pairing. The data in support of this view (G.H. Umbetova et al. in preparation; E.S. Belyaeva et al. in preparation) suggest that the variegated euchromatin region should develop morphological and other properties similar to the so-called IH regions. On the other hand, susceptibility to breakage, the most characteristic feature of IH, is strengthened by the same factors that enhance position effect. Our data show a signif-
386 Table 2. Influence of a genetic suppressor and an enhancer of position effect on break frequencies in male X chromosome regions (at 18~ C) Expt.
1 2
Genotype
Jg/Y,En-var(3)2 X/Y, Su-var(3) 9
Number of larvae 18 24
Number of nuclei 110 t 18
Break frequencies in X chromosome regions (%) 7B
9A
llA
12E
19E
0.9 0.0
0.9 0.0
4.5 0.0
3.7 0.0
15.5 0.8
Total breakability is higher in experiment I than in 2 (P > 0.999)
icant difference in the frequency of weak points in the IH regions of males with an XO and X Y constitution in all genotypes investigated (Figs. 6, 7); these data confirm that the genetic factors affect susceptibility to breakage in the IH regions. In the absence of the Y chromosome the break frequency in the IH regions increases and is higher in individuals with an enhancer than in those with a suppressor. A complete explanation of this phenomenon cannot be given because the compared samples differed not only in modifiers present in the genome but in genetic background. However, we noticed regularly that the factors acting as position effect enhancers simultaneously increased the susceptibility to breakage in the IH regions. The most reliable results were obtained for males carrying Dp (1; 1)pn2b. In this case a direct correlation between the strengthening of position effect and susceptibility to breakage in the IH regions in the absence of the Y chromosome was observed. It is important to stress that both features were analysed in the same sample of cells in every variation of the experiment. It is obvious that low temperature as a well-known modifiez of position effect affects susceptibility to breakage too. It is more complicated to explain the action of low temperature. While for condensation the temperature-sensitive period is restricted to the initial 3-6 h of embryogenesis, for increase in susceptibility to breakage in IH it covers the whole period of polytenization. It is also possible that temperature affects susceptibility to breakage through condensation (the more condensed the region, the stronger its underreplication appears). But we have to bear in mind that in salivary gland of larvae grown at low temperature there are more nuclei with higher polyteny (Rodman 1967; Hartmann-Goldstein and Goldstein 1979). Underreplication in chromosomes with higher polyteny might be expressed to a greater extent. Moreover, breaks might be more easily detected for purely technical reasons (Zhimulev et al. 1982). Position-effect modifiers seem to cause not only an increase in break frequencies in the regions known as weak points but also the appearance of breaks in regions where they have never been detected. Therefore the combination of modifiers (low temperature and removing the Y chromosome) suggests the appearance of some additional regions with breaks. The mechanism of break formation has been interpreted in the following way: the bands in the IH regions are more compact than usual D N A bands, making replication difficult and resulting in late replication and, as a consequence, underreplication in extreme cases. So, the formation of a break could be the result of local underreplication. Under-
replication in weak points has been shown (Zhimulev et al. 1982; Lifschytz 1983; Lamb and Laird 1987). If the above assumption is correct we can expect that factors increasing susceptibility to breakage primarily affect DNA condensation in the IH regions. Following from the above analysis of the results, blocks arising as a result of position effect acquire properties traditionally ascribed to IH and CH. However, in the case of position effect it is an euchromatin region that becomes supercondensed. Hence, the similar behaviour of the three types of regions is probably not due to the primary D N A structure, as anticipated earlier (Zhimulev et al. 1982; Kholodilov et al. 1988), but to the supercondensed state characteristic of all of them. A similar conclusion has been drawn by Lamb and Laird (1987) and Healy et al. (1988) who suppose that features of IH do not depend on the sequence organization. We can assume that there are some D N A sequences which should be strongly repressed from early in development. The mechanism of such repression may be through condensation controlled by specific histone or nonhistone proteins. Specific interaction of such proteins with D N A of heterochromatin is presumed to play a key role in packaging of centromeric heterochromatin and variegated euchromatin (Zuckerkandl 1974; Khesin and Leibovitch 1976). Some authors have already reported finding specific proteins in heterochromatin (Levinger 1985; James and Elgin 1986). With the removal of the Y chromosome (that is a considerable portion of heterochromatin) "condensation proteins", because of a lack of an adequate amount of target heterochromatin for binding, start packing the retained heterochromatin which becomes denser, involving the nearby euchromatin region in the same changes. From this point of view the role ascribed to enhancers and suppressors as modifiers of position effect and break formation can be easily interpreted if we regard them as the structural genes of chromatin proteins (Dorn et al. 1986; Eissenberg et al. 1987; Sinclair et al. 1989). Taking all the results together we are inclined to explain the similar behaviour of IH, CH and blocks resulting from position effect by the likeness of their functional state a strong and irreversible repression during early ontogenesis caused by specific condensation proteins. The primary D N A structure of sucti regions may be very different.
Acknowledgements. We are very indebted to Olga Kharlamova for translation of this paper into English and to Dr. G. Reuter for providing us with Drosophila stocks with an enhancer and a suppressor.
387
References Ananiev EV, Gvozdev VA (1974) Changed pattern of transcription and replication in polytene chromosomes of Drosophila melanogaster resulting from eu-heterochromatin rearrangement. Chromosoma 45:173-191 Ananiev EV, Gvozdev VA (1975) Differences in D N A replication pattern in the X chromosome of males, females and intersexes of Drosophila melanogaster. Chromosoma 49 : 233 244 Arcos-Teran L (1972) DNS replication und die Natur der sprit replizierenden Orte im X-Chromosom yon Drosophila melanogaster. Chromosoma 37:233-296 Baumann A, Krah-Jentgens I, Muller R, Muller-Holtkamp F, Seidel R, Kecshemethy N, Casal J, Ferrus A, Pongs O (1987) Molecular organization of the maternal effect region of the Shaker complex of Drosophila: characterization of an IA channel transcript with homology to vertebrate Na + channel. E M B O J 6 : 2 9 49 Belyaeva ES, Aizenzon MG, Kiss I, Gorelova TD, Pak SD, Umbetova GH, Kramers PGN, Zhimulev IF (1982) Reports on new mutants. Dros Inf Serv 58:184-190 Berendes H D (1966) Differential replication of male and female X chromosome in Drosophila. Chromosoma 20 : 32-43 Bolshakov VN, Zharkikh AA, Zhimulev IF (1985) Intercalary heterochromatin in Drosophila. II. Heterochromatin features in relation to local D N A content along the polytene chromosomes of Drosophila melanogaster. Chromosoma 92 : 200-208 Cribbs DL, Leung J, Newton CH, Hayashi S, Miller RC Jr, Terner GM (1987) Extensive microheterogeneity of serine t R N A genes from Drosophila melanogaster. J Mol Biol 197:397-404 Dorn R, Heymann S, Lindigkeit R, Reuter G (1986) Suppressor mutation of position-effect variegation in Drosophila melanogaster affecting chromatin properties. Chromosoma 93 : 398-403 Eissenberg JC, Elgin SCR, James TC (1987) A heterochromatic specific chromosomal protein gene in Drosophila melanogaster. Genetics 116:(suppl) 4 Hartmann-Goldstein IJ (1967) On the relationship between heterochromatization and variegation in Drosophila, with special reference to temperature-sensitive periods. Genet Res 10:143 159 Hartmann-Goldstein I, Goldstein DJ (1979) Effect of temperature on morphology and DNA-content of polytene chromosomes in Drosophila. Chromosoma 71 : 333-346 Healy MJ, Russel RJ, Miklos GLG (1988) Molecular studies on interspersed repetitive and unique sequences in the region of the completion group uncoordinated on the X chromosome of Drosophila melanogaster. Mol Gen Genet 213:63-71 Hilliker AJ, Appels R, Schalet A (1980) The genetic analysis of Drosophila melanogaster heterochromatin. Cell 21:607-619 Ilyina OV, Sorokin AV, Belyaeva ES, Zhimulev IF (1980) Report on Drosophila new mutants. Dros Int Serv 55:205 James TS, Elgin SCR (1986) Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene. Mol Cell Biol 6 : 3262-3282 Kalish WE, Hrigele K (1976) Correspondence of banding patterns to 3H-thymidine labelling patterns in polytene chromosomes. Chromosoma 57:19-23 Khesin RB, Leibovitch BA (1976) Chromosome structure, histones and gene activity in Drosophila. Mol Biol (USSR) 10:3 33 Kholodilov NG, Bolshakov VN, Blinov VM, Soloviev VV, Zhimulev IF (1987) Molecular and genetic characteristic of Drosophila melanogaster new genomic element with varying location. D A N USSR 295:984-989 Kholodilov NG, Bolshakov VN, Blinov VM, Solovyov VV, Zhimulev IF (1988) Intercalary heterochromatin in Drosophila. III. Homology between D N A sequences from the Y chromosome, bases of polytene chromosome limbs, and chromosome 4 of Drosophila. Chromosoma 97 : 247-253
Lamb MM, Laird CD (1987) Three euchromatic D N A sequences underreplicated in polytene chromosomes of Drosophila and localized in constrictions and ectopic fibers. Chromosoma 95 : 227-235 Lefevre G (1976) A photographic representation and interpretation of the polytene chromosomes of Drosophila melanogaster salivary glands. In: Ashburner M, Novitski E (eds) The genetics and biology of Drosophila, vol la. Academic Press, London New York San Francisco, pp 461-480 Levinger L (1985) Nucleosomal structure of two Drosophila metanogaster simple satellites. J Biol Chem 260:11799 11804 Lifschitz E (1983) Sequence replication and banding organization in the polytene chromosomes of Drosophila melanogaster. J Mol Biol 164:17-34 Lindsley DL, Grell EH (1968) Genetic variations of Drosophila melanogaster. Carnegie Inst Wash Publ No 627, p 472 Livak KJ (1984) Organization and mapping of a sequence on the Drosophila melanogaster X and Y chromosomes that is trancribed during spermatogenesis. Genetics 107 : 611-634 Mulder MP, Duijn P, van Gloor HJ (1968) The replication organization of D N A in polytene chromosomes of Drosophila hydei. Genetica 39 : 385-428 Reuter G, Werner W, Hoffmann H (1982) Mutants affecting position-effect heterochromatinization in Drosophila melanogaster. Chromosoma 85 : 53%551 Rodman TC (1967) Control of polytenic replication in Dipterian larvae. II. Effect of growth temperature. J Cell Physiol 70:187-190 Schultz J (1936) Variegation in Drosophila and the inert chromosome regions. Proc Natl Acad Sci USA 2 2 : 2 ~ 3 3 Sinclair DAR, Lloyd VK, Grigliatti TA (1989) Characterization of mutations that enhance position-effect variegation in Drosophila melanogaster. Mol Gen Genet 216:328-333 Spofford JB (1976) Position effect variegation in Drosophila. In Ashburner M, Novitski E (eds) The genetics and biology of Drosophila, vol 1C. Academic Press, London New York San Francisco, pp 955-1018 Tolchkov EV, Balakireva MD, Alatortsev VE (1984) Inactivation of the X chromosome region with a known fine structure as a result of the variegated position effect in Drosophila melanogaster. Genetica (USSR) 20 : 1846~1856 Wargent JM, Hartmann-Goldstein IJ (1976) Replication behaviour and morphology of a rearranged chromosome region in Drosophila. In: Chromosomes today, vol 5. Pearson PL, Lewis K R (eds). John Wiley and Sons, New York, pp 109-116 Zhimulev IF (1974) Comparative study of the function of polytene chromosomes in laboratory stocks of Drosophila melanogaster and the l(3)tl mutant (lethal tumoraes larvae). I. Analysis of puffing patterns in autosomes of the laboratory stock BatumiL. Chromosoma 46 : 59-76 Zhimulev IF, Semeshin VF, Kutichkov VA, Belyaeva ES (1982) Intercalary heterochromatin in Drosophila. I.Location and general characteristics. Chromosoma 87 : 197-228 Zhimulev IF, Belyaeva ES, Fomina OV, Protopopov MO, Bolshakov VN (1986) Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster. I. Morphology and genetic activity of the 2AB region in chromosome rearrangement T (1 ; 2)dor varT. Chromosoma 94: 492-504 Zhimulev IF, Belyaeva ES, Bgatov AV, Baricheva EM, Vlassova IE (1988) Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster. II. Peculiarities of morphology and genetic activity of the 2B region in the T (1 ; 2)dor vary chromosome in males. Chromosoma 96 : 255561 Zukerkandl E (1974) Recherches sur les propri6t6s et l'activit6 biologique de la chromatine. Biochimie 56:937-954 Received January, 1989 / Revised form July 19, 1989 Accepted by W. Beeremain