Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Chromosoma (Berl.) 84, 391403 (1981) CHROMOSOMA �9 Springer-Verlag 1981

Cytological Dissection of Sex Chromosome Heterochromatin of Drosophila hydei

Silvia Bonaccorsi, Sergio Pimpinelli, and Maurizio Gatti Centro di Genetica Evoluzionistica del CNR, Istituto di Genetica, Universitfi di Roma, Roma, Italy

Abstract. Prophase chromosomes of Drosophila hydei were stained with 0.5 lag/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. - D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.

Introduction

The autosomal pericentromeric regions, more than half of the X chromosome and the entire Y chromosome of Drosophila hydei (2n = 12) are heterochromatic. Together these three heterochromatic components account for 30 32% of the diploid genome of D. hydei, the X and Y heterochromatin each comprising about 9% of the genome (Hennig, 1972; Pimpinelli et al., 1976b).

Genetic studies have permitted the association of two classes of functions with D. hydei heterochromatin. 1) The bobbed loci are thought to be the repetitive structural genes coding for ribosomal RNA. Cytogenetic studies coupled with

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392 s. Bonaccorsi et al.

in situ hybridization experiments have shown that the X chromosome carries a bobbed locus on the terminal region of its heterochromatic arm, while the Y chromosome accomodates a bobbed locus at each tip of the chromosome (Van Breugel, 1970; Meyer and Hennig, 1974; Hennig et al., 1975). 2) The fertility factors located on the Y chromosome. These factors have been associated with the characteristic lampbrush loops formed by the Y chromosome in primary spermatocytes; 5-6 loop pairs have been observed in D. hydei spermatocytes, each displaying a characteristic morphology and localization along the Y chromosome. These loops, which together account for about 1/12 of the Y chromosome, are very active sites of transcription and their development is required for male fertility (for reviews see Hess, 1973; Hennig, 1978). Although the amount of DNA included in a loop is sufficient to code for hundreds of polypep- tides, recent genetic studies have shown that no more than one complementation group can be detected in each of the lampbrush loop forming sites (Hackstein et al., 1981). These studies have also shown the existence of Y-linked genes located outside the loops whose functioning, although essential for male fertility, is not associated with the appearance of any lampbrush-like structures (Leoncini, 1977; Hackstein et al., 1981).

Cytological and genetic analysis of D. hydei heterochromatin has been paral- leled by a series of studies on its molecular composition and organization. Analytical centrifugation experiments on native and partially renaturated DNA have shown that D. hydei contains a relatively G-C rich satellite DNA banding at 1.714 g/cm 3 and a cryptic satellite DNA included in the light side of the bulk DNA band (Hennig et al., 1970; Hennig, 1972). Moreover, the analysis of karyotypes including different amounts of X heterochromatin has revealed that this region contributes the largest portion of D. hydei highly repetitive DNA. Autosomal heterochromatin contains only a minor portion of this fast reassociating simple sequence DNA, while little or no satellite DNA is located in the Y chromosome (Hennig, 1972). These results have been subsequently corroborated by in situ hybridization experiments on mitotic chromosomes which have shown that highly repetitive DNA-complementary 3H-RNA hybridizes with both the X and autosomal heterochromatin, but not with the entirely heterochromatic Y chromosome which exhibits no hybrid formation at all (Hen- nig, 1973).

These findings have been recently confirmed and extended by Renkawitz (1978b) who isolated two distinct highly repetitive DNA fractions by silver ion and Actinomycin-D binding and centrifugation in isopycnic salt gradients. The 1.696 fraction, which comprises 13% of the genome and corresponds to the cryptic satellite described by Hennig and coworkers, has been shown to be restricted to the X heterochromatin by in situ hybridization (Renkawitz 1978 b). The 1.714 highly repetitive fraction constitutes 4% of the genome and hybridizes in situ with all four acrocentric autosome pairs but neither with the X heterochromatin nor the entirely heterochromatic Y chromosome.

A further advance in the molecular characterization of D. hydei heterochromatin has been provided by the demonstration that also two middle repetitive nuclear fractions (having CsC1 densities of 1.702 and 1.697 respectively) are

Sex Heterochromatin of D. hydei 393

located in specific he terochromat ic port ions (Renkawitz, 1978 a). The 1.702 fraction hybridizes with the he terochromat in of all four acrocentric au tosome pairs, the middle par t of the Y chromosome, but not the X heterochromatin . The 1.697 middle repetitive fraction has a peculiar behaviour hybridizing only with the nucleolus organizer regions o f the X and the Y chromosome.

F r o m these molecular and cytogenetic studies it is quite clear that the estab- lishing of precise and distinct he terochromat ic landmarks would permit a sub- stantial advance in the molecular and cytogenetic analysis of the he terochromat ic material of D. hydei. An array of such heterochromat ic landmarks is provided by fluorescence and Giemsa banding techniques which produce a highly repro- ducible longitudinal differentiation o f the he terochromat in (Gatti et al., 1976; Pimpinelli et al., 1976 b; Beck and Srdid, 1979). These banding techniques have shown that the he terochromat ic material of D. hydei is subdivided into a series of blocks having different cytochemical features. Thus not only these banding procedures provide a tool comparab le to polytene chromosomes for the cytogenetic analysis o f the heterochromat in , but also permit correlat ion of the cytological characteristics o f a given he terochromat ic region with its molecular and functional properties.

In the present paper we describe the high resolution banding pat tern o f prophase ch romosomes of D. hydei stained with Hoechs t 33258. After Hoechst staining the X ch romosome and the au tosomal he terochromat in fluoresce homogeneously, whereas the Y c h r o m o s o m e exhibits an extremely fine cytological differentiation, being subdivided into 18 different regions. Moreover , we describe the pat tern of lateral asymmetry (Lin et al., 1974) observed in ch romosome preparat ions f rom cells exposed to 5-bromodeoxyur id ine (BrdU) for one round of D N A replication and subsequently stained with Hoechst . Both the X and the Y he terochromat in of D. hydei exhibit complex patterns of contralateral asymmetry. These patterns are compared to the Giemsa and Hoechs t banding patterns and to the regional distr ibution of the highly and middle repetitive D N A fractions observed after in situ hybridization.

Materials and Methods

Stocks. A wild type stock of D. hydei was kindly provided by Dr. Leoncini (Ttibingen) and grown in standard medium at 25~ 1 ~ C.

Slide Preparation. Neural ganglia of third instar larvae were dissected in a drop of saline (NaC1 0.7%) and immediately transferred to a hypotonic solution of 0.5% sodium citrate where they were kept for 10rain at 25 ~ C. The ganglia were then fixed for about 10 sec in a mixture of acetic acid, methanol and distilled water (5.5:5.5:1) and squashed in 45% acetic acid under a siliconized coverslip. The coverslip was removed with a razor blade after freezing on dry ice and slides were air dried at room temperature.

Hoechst 33258 Staining. The slides were rehydrated for 5 rain in 0.15 M NaC1, 0.03 M KC1 0.01 M phosphate adjusted to pH 7, stained for 10 rain with 0.5 lag/ml Hoechst 33258 dissolved in the same buffer and, after rapid washing, air dried. They were then mounted in 0.16 M sodium phosphate, 0.04 M sodium citrate (pH 7), and the coverslips sealed with rubber cement. Some slides were destained in distilled H20 then stained for 10 rain with 4% Giemsa (Merck) in phosphate buffer at pH 7 and mounted in Euparal.

394 S. Bonaccorsi et al.

Lateral Asymmetry. Neural ganglia of third instar larvae were incubated in the dark at 25 ~ C for 4.5 h in saline (NaC1 0.7%) containing 20 pg/ml. To collect metaphases, colchicine (final concen- tration I0-5 M) was added for the last hour of incubation. BrdU-treated ganglia were transferred to hyptonic solution where they were kept for 25-30 rain. This prolonged hypotonic treatment destroys the close apposition of sister chromatids in heterochromatic regions yielding a high frequency of C-anaphases where lateral asymmetry can easily be scored. Slides were then prepared with the procedure previously described, stained for 10 min with 5 gg/ml Hoechst 33258, mounted in 0.16 M sodium phosphate, 0.04M sodium citrate (pH 7) and exposed for 10rain either to sunlight or to a fluorescent bulb to improve the differential staining of sister chromatids. To obtain fluorescence plus Giemsa preparations (FPG), after exposure to light, Hoechst-stained slides were stained for I5 rain with 4% Giemsa in phosphate buffer at pH 7 and mounted in EuparaI.

Observation by Fluorescence Microscopy. Both Hoechst banding and lateral asymmetry were examined under a Zeiss fluorescence microscope equipped with a 200 W mercury light source for incident illumination and a combination of filters transmitting light between 380 and 440 nm, i.e., the excitation filters UG5+BG3; the dichroic mirror FT 460 and the barrier filter LP 475. Under these experimental conditions chromosome fluorescence is very stable and the absence of a sensitive fading permits both direct cytological analysis of chromosomes and photography with relatively short exposure times. All microphotographs were taken with Kodak Pan X film.

Results

Hoechst 33258 Banding Pattern

In a previous p a p e r we showed that the f luorescence pa t t e rn of Hoechs t 33258 varies with the concen t ra t ion o f the dye (Ga t t i et al., 1976). Af te r s ta ining with 0.05 gg /ml H. 33258 the euchromat ic a rms o f D. hydei f luoresce homoge ne ous ly while the he t e roch roma t i c regions exhibi t di f ferent degrees o f f luorescence. The he t e roch roma t i c shor t a rms of the four pairs of m a j o r au tosomes are less f luores- cent than euchromat in , whereas the shor t p rox ima l he t e roch roma t i c segments o f their long arms are as b r igh t as euchromat in . The dis ta l tip of the entirely he t e roch roma t i c r ight a rm of the X c h r o m o s o m e and the p rox ima l he te rochro- ma t in of its ma in ly euchromat i c left a rm f luoresce like the euchromat i c ma te r i a l while the rest of the X he t e roch roma t in is dul ler than euchromat in . F ina l ly , the ent i rely he t e roch roma t i c Y c h r o m o s o m e exhibi ts a charac ter i s t ic a r ray o f very bright , b r igh t and more or less dul l bands.

Af te r s ta ining with 0.5 gg/ml , the Hoechs t dul l areas of au tosomes acquire the same f luorescence as euchromat in , while all X he t e roch roma t in becomes un i fo rmly br ighter than euchromat i c arms. The ba nd ing pa t t e rn of the Y chro- m o s o m e does not change by increas ing the Hoechs t concent ra t ion , but the ind iv idua l bands a p p e a r more dis t inct than at 0.05 gg/ml so tha t a very clear long i tud ina l d i f ferent ia t ion is observed. As shown in F igure 1 some long prophase Y c h r o m o s o m e s can be subdiv ided into more than 12 dif ferent regions def ined by the degree of f luorescence and the presence of const r ic t ions (Fig. 2).

Af te r sequent ia l s ta ining with H. 33258 and G iemsa three main const r ic t ions are usual ly seen a long the Y c h r o m o s o m e (Fig. l d - f ) . The shor tes t loca ted within a very Hoechs t -b r igh t region a b o u t 2/3 a long of the Y c h r o m o s o m e co r re sponds to the p r ima ry cons t r ic t ion as shown by the examina t ion of ana- phase c h r o m o s o m e s (Fig. 3). The cons t r ic t ion in the p rox ima l third of the long arm, whose func t iona l s ignif icance is unknown, varies m a r k e d l y in extension


Fig. 1 a-L 1 ExamPles of prophase Y chromosomes, a-c Chromosomes stained with 0.5 gg/ml Hoechst 33258; note the fine banding pattern of the terminal region of Y short shown in a. d and e Chromosomes stained with 0.5 gg/ml Hoechst 33258 and subsequently stained with Giemsa; note that the centromeric constriction (c) subdivides an apparently homogeneous Hoechst bright block. f A prophase Y chromosome stained with Giemsa without prior staining with Hoechst

1 2 3 4 5 6 7 8 9 10 11 12

C a b c d e ?

Fig. 2. Diagrammatic representation of the Y chromosome constructed by coupling the Hoechst banding pattern with the longitudinal distribution of the major constrictions (thin areas). Only very seldom is a short constriction observed involving the distal third of region 1. m Very bright regions, N bright regions; [] dull regions; n very dull regions

Fig. 3. Anaphases of larval neuroblasts. The arrows point to the centromere of the Y chromosome

1 Bars represent 5 lam


Fig. 4. Partial neuroblast prometaphase sequentially stained with Hoechst 33258 and Giemsa. The arrows indicate the nucleolar constrictions of the X and the Y chromosomes

from cell to cell, being very long in some prophase chromosomes and almost undetectable in condensed metaphase chromosomes. The long constriction on the tip of the short arm often exhibits a complex cytological organization after Hoechst staining, showing a characteristic sequence of six bands (Fig. la , b). Most likely this constriction corresponds to one of the nucleolus organizer regions (NOR) which in D. hydei are located at both ends of the Y chromosome and on the tip of the right arm of the X chromosome (Hennig et al., 1975). Interestingly, while the constrictions on the tips of XR and Y short are con- sistently observed in our material (Figs. 1 and 4), only very seldom is the constriction on the tip of Y long clearly apparent. If one assumes that the presence of nucleolar constrictions reflects gene activity in these regions, our observation suggests the possibility that in most neuroblast cells of D. hydei only two nucleolus Organizers are active. Unfortunately this hypothesis is very difficult to test since the silver staining technique for active NORs (Miller et al., 1976) does not specifically stain the NORs of D. hydei (Beck and Srdi6, 1979).

Lateral Asymmetry

About 25% metaphases treated for 4.5 h with BrdU exhibit a characteristic pattern of differential staining of sister chromatids in heterochromatic regions.


Fig. 5. C-anaphases treated for 4.5 h with 20 gg/ml BrdU and subsequently stained with 5 gg/ml Hoechst 33258. Note the clear lateral asymmetry of many regions of sex chromosome and autosomal heterochromatin. The arrows indicate the autosome pair which shows a very limited asymmetric staining of the short arm

Several considerations indicate that the asymmetric regions are not generated by incorporation of BrdU for two cell cycles: 1) the cell cycle of D. melanogaster lasts for about 8 h (Pimpinelli et al., 1976a) and consequently D. hydei, which has a longer generation time than D. melanogaster, is expected to have a compar- ably longer cell cycle; 2) typical second mitotis sister chromatid differentiation has never been observed in our preparations; 3) the complex patterns of lateral asymmetry shown by the X and Y chromosomes are very consistent, thus they cannot be generated by sister chromatid exchanges.

To clearly score lateral asymmetry in the heterochromatin we destroyed the close chromatid apposition present in these regions by prolonged hypotonic treatment. However this procedure produces an excessive chromosome spreading so that complete C-anaphases are only occasionally found. Figure 5 shows examples of complete C-anaphases which exhibit a clear lateral asymmetry involving the short arms of the four major autosome pairs, the heterochromatic arm of the X chromosome and the Y chromosome. One autosome pair shows an


Fig. 6a-d. Examples of X chromosomes showing a clear contralateral asymmetry in the heterochromatic regions, a and b Partial C-anaphases sequentially stained with Hoechst and Giemsa; (b is taken from Fig. 5b), e and d Partial C-anaphases stained with Hoechst and the FPG technique respectively. Due to the higher rate of BrdU substitution, the weakly stained chromatid segments often appear more elongated than the corresponding heavily stained segments

Fig. 7. Diagrammatic representation of the pattern of contralateral asymmetry shown by the X chromosome. The heterochromatic region is depicted with the sister chromatids closely apposed

asymmetr ic spot which is smaller and less differentiated than those present in the other major autosomes and the dot chromosomes (chromosomes 6) never show lateral asymmetry.

While the autosomes exhibit a simple pat tern of lateral asymmetry : i.e., one chromat id fluorescent and its sister dull, the X and the Y chromosomes have complex patterns o f contralateral asymmetry. Three and five contralateral asymmetr ic regions are respectively observed in the X heterochromat in and the Y ch romosome (Figs. 6-9). However, while the asymmetr ic regions o f the X ch romosome show a high degree of differential fluorescence between sisters, one chromat id being brighter and the other duller than euchromatin, in the


Fig. 8a and b. Examples of BrdU treated Y chromosomes sequentially stained with Hoechst and Giemsa a is a part of Fig. 5a). The weakly stained regions of the Y chromosome with a high rate of BrdU substitution are more elongated than the corresponding heavily stained regions. C Centromere

Fig. 9. Diagrammatic representation of the pattern of controlateral asymmetry shown by the Y chromosome. The diagram which is representative of the Giemsa stained chromosomes has been normalized for the differential elongation of sister chromatids in the asymmetric regions

Y chromosome the differential fluorescence between sisters is less pronounced and varies slightly f rom region to region.

D i s c u s s i o n

Hoechst 33258 Banding Pattern

After staining with 0.05 ~tg/ml Hoechst 33258 the heterochromatic regions of the major autosomes and the X chromosome of D. hydei show a limited longitudinal differentiation and moreover with 0.5 gg/ml they fluorescence homogeneously. By contrast at both concentrations of Hoechst 33258, and especially at 0.5 lag/ml, the Y chromosome exhibits a characteristic array of bands having different degrees of fluorescence. The presence of very fluorescent material on the Y chromosome is rather surprising since Hoechst 33258 is known to bind specifically A T rich D N A (Comings, 1975; Latt and Wohlleb, 1975) and the Y chromosome of D. hydei does not contain any particularly A - T rich D N A fraction (Hennig, 1973; Renkawitz, 1978a). However, it has been reported that poly(dA)-poly(dT) polymers produce a stronger enhancement of Hoechst 33258 fluorescence than poly(dAdT)-poly(dAdT) polymers (Latt and Wohlleb, 1975). According to Latt and Wohlleb this may determine the very bright fluorescence


of mouse centromeres which contain the A-T rich mouse satellite DNA having an asymmetric distribution of thymine residues between complementary polynu- cleotide chains (Flamm et al., 1967). The intensely fluorescent regions of the Y chromosome ofD. hydei show a clear lateral asymmetry after BrdU incorporation and probably contain a DNA with an asymmetric distribution of thymine residues (see next section). We suggest therefore that these regions, even without being particularly A-T rich, bind Hoechst 33258 selectively, thereby showing a very bright fluorescence.

Hoechst stained high resolution prophase Y chromosomes can be subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus the cytological entities visualized by Hoechst banding provide suitable chromosome landmarks for a systematic cytogenetic analysis of the Y chromosome of D. hydei. Following an approach we already used for the Y chromosome of D. melanogaster (Gatti and Pimpinelli, in manuscript 1981), the different cytological entities of the Y chromosome of D. hydei could be associated with specific genetic functions. For example, a careful cytological characterization of the numerous chromosome rearrangements involving the Y chromosome of D. hydei (cf. Hackstein et al., 1981) would permit the loop- forming and the non-loop-forming fertility factors to be precisely localized. This may also permit the estimation of the relative physical dimensions of these two types of loci and give some insight into their genetic organization. On the other hand, once cytologically characterized, these rearrangements could be successfully employed for a precise localization of the molecular components of the Y chromosome, and this would permit the integration of molecular, cytological and functional data for a better understanding of the organization and expression of the fertility factors.

Lateral Asymmetry

Lateral asymmetry was first observed in mouse centromeric heterochromatin by staining with Hoechst 33258 chromosomes treated with BrdU for only one round of DNA replication (Lin et al., 1974). Since the mouse satellite DNA present in centromeric heterochromatin has a disproportionate distribution of thymine residues between the two DNA strands (Flamm et al., 1967), and since Hoechst fluorescence is quenched by BrdU incorporated into the DNA instead of thymine, it has been suggested that the differential staining of sister chromatids in mouse heterochromatin is a consequence of the interstrand asymmetry in thymine content (Lin et al., 1974). Subsequently lateral asymmetry was demonstrated in human chromosomes (Angell and Jacobs, 1975, 1978; Emanuel, 1978), in Dipodomys ordii (Bostock and Christie, 1976), in Drosophila nasuta (Lakhotia et al., 1979) and in Vicia faba (Schubert and Rieger, 1979). In all cases but one (Emanuel, 1978), lateral asymmetry is found only in heterochromatic C- banded material. The general interpretation of these results is that lateral asymmetry can be demonstrated in chromosome areas which contain sufficiently long segments of highly repetitive simple sequence DNA with an interstrand


bias of thymine content. However the observation of lateral asymmetry in the q12 C-band negative region of human chromosome 6 (Emanuel, 1978) has suggested that even moderately repetitive DNA can be organized in such a way as to produce cytologically detectable lateral asymmetry.

Studies on lateral asymmetry have also shown that the thymine rich strand of mouse satellite DNA has uniform polarity in the genome in relation to the centromeres of all autosomes (Lin and Davidson, 1974; Holmquist and Comings, 1975). However, the discovery of contralateral asymmetry in human chromosomes (Angell and Jacobs, 1975; 1978) has raised some doubts as to the generalization of this situation. In fact the contralateral asymmetry observed in the C-bands of human chromosomes 1 and 9 could either mean that satellite polarity is not always uniform or that switches of lateral asymmetry are due to the presence of two or more satellites in these chromosome regions.

Our data on the pattern of lateral asymmetry in D. hydei have a number of interesting implications concerning the chromosomal organization of highly and moderately repetitive heterochromatic DNA.

The X heterochromatin of D. hydei contains a single simple sequence satellite DNA (Hennig, 1973; Renkawitz, 1978b) and exhibits a complex pattern of controlateral asymmetry. The simplest interpretation of these results is that: 1) this satellite DNA has disproportionate numbers of thymine residues between strands and 2) it is arranged in long contiguous segments where the thymine rich strands have opposite polarity with respect to the centromere. Thus D. hydei provides a clear example of an organism where a single satellite DNA does not have uniform polarity.

In addition to the X heterochromatin the heterochromatic short arms of the major autosomes of D. hydei also exhibit a clear lateral asymmetry. Accord- ing to Renkawitz (1978b) these heterochromatic regions accomodate the 1.714 satellite DNA containing 55% G-C base pairs. Thus one interpretation of our results is that also the relatively G-C rich 1.714 satellite DNA has an interstrand bias in its thymine content reflected by a cytological asymmetry. The autosome pair which exhibits short and poorly differentiated asymmetric spots, may contain only a small amount of 1.714 satellite DNA. Alternatively it may contain a DNA having a partial homology with the 1.714 satellite DNA and a limited interstrand bias in thymine content. In this respect it should be noted that in Fig. 8A of Hennig (1973), which shows the in situ hybridization pattern of highly repetitive DNA of D. hydei, only three pairs of the major autosomes exhibit autoradiographic grains over their heterochromatic short arms.

The most interesting aspect of the lateral asymmetry pattern of D. hydei is the presence of five contralateral asymmetric regions in the Y chromosome, which does not contain any highly repetitive DNA (Hennig, 1973; Renkawitz, 1978b). This finding suggests that the moderately repetitive DNA present in the Y chromosome is organized in long segments having a disproportionate content of thymines in one strand and a corresponding deficiency in the other. Moreover the thymine rich strands of these segments appear to have different polarities with respect to the centromere of the Y chromosome.


Besides in D. hydei, a s y m m e t r i c h e t e r o c h r o m a t i c reg ions have been observed in D. nasuta ( L a k h o t i a et al., 1979) a n d f o u n d in D. melanogaster, D. virilis, D. texana, D. americana a n d D. novamexicana (Bonaccors i , P imp ine l l i a n d Gat t i , u n p u b l i s h e d ) . M o r e o v e r in m o s t o f these species com p lex con t r a l a t e r a l pa t t e rns of a s y m m e t r y have been f o u n d even in c h r o m o s o m e areas tha t are n o t d i f feren- t ia ted by b a n d i n g techniques . T h u s la te ra l a s y m m e t r y coup l ed wi th c h r o m o s o m e b a n d i n g has revealed tha t an ex t remely f ine r eg iona l d i f f e ren t i a t ion of the he te ro- c h r o m a t i c m a t e r i a l is a c o m m o n fea ture of m a n y Drosophila species. However , this pecu l i a r s t ruc tu ra l o r g a n i z a t i o n o f Drosophila h e t e r o c h r o m a t i n is still to be ass igned a f u n c t i o n a l s ignif icance.

Acknowledgments. The authors thank Dr. W. Hennig for providing information prior to its publica- tion and Dr. G. Prantera for a critical reading of the manuscript. This work has been in part supported by grants from EURATOM (contract N. B10-E-400-I) and from Progetto Finalizzato Biologia della Riproduzione.

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Renkawitz, R.: Characterization of two moderately repetitive DNA components localized within the fl-heterochromatin of Drosophila hydei, Chromosoma (Bed.) 66, 225--236 (1978a)

Renkawitz, R. : Two highly repetitive DNA satellites of Drosophila hydei localized within the c~-heterochromatin of specific chromosomes. Chromosoma (Berl.) 66, 23%248 (1978b)

Schubert, I., Rieger, R, : Asymmetric banding of Vicia faba. Chromosomes after BrdU incorporation. Chromosoma (Bed.) 70, 385 381 (1979)

Received July 30, 1981 / Accepted by W. Beermann Ready for press August 30, 1981

Documents

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei