In polytene chromosomes of D. melanogaster the heterochromatic pericentric regions are underreplicated (underrepresented). In this report, we analyze the effects of eu-heterochromatic rearrangements involving a cluster of the X-linked heterochromatic (Xh) Stellate repeats on the representation of these sequences in salivary gland polytene chromosomes. The discontinuous heterochromatic Stellate cluster contains specific restriction fragments that were mapped along the distal region of Xh. We found that transposition of a fragment of the Stellate cluster into euchromatin resulted in its replication in polytene chromosomes. Interestingly, only the Stellate repeats that remain within the pericentric Xh and are close to a new eu-heterochromatic boundary were replicated, strongly suggesting the existence of a spreading effect exerted by the adjacent euchromatin. Internal rearrangements of the distal Xh did not affect Stellate polytenization. We also demonstrated trans effects exerted by heterochromatic blocks on the replication of the rearranged heterochromatin; replication of transposed Stellate sequences was suppressed by a deletion of Xh and restored by addition of Y heterochromatin. This phenomenon is discussed in light of a possible role of heterochromatic proteins in the process of heterochromatin underrepresentation in polytene chromosomes.

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HETEROCHROMATIN causes inactivation (position-effect variegation, PEV) of euchromatic genes that, due to chromosomal rearrangements, are transposed into its vicinity (SPOFFORD 1976; HENIKOFF 1990; WEILER and WAKIMOTO 1995). At the same time, genes normally located in heterochromatin may be inactivated when they are transposed close to euchromatin (SPOFFORD 1976; WAKIMOTO and HEARN 1990), demonstrating that the integrity of the heterochromatic regions is crucial for their functioning. PEV of euchromatic genes is known to be associated with altered replication of the affected region in polytene chromosomes (ANANIEV and GVOZDEV 1974; SPRADLING 1993; LAVROV et al. 1998). Underreplication (underrepresentation) of heterochromatin in polytene chromosomes is a well-known phenomenon (ZHIMULEV 1999), but the mechanisms of heterochromatin underreplication in the course of polytenization are poorly understood. Underreplication has been interpreted as a prevention or truncation of replication of the heterochromatic DNA (LEACH et al. 2000), which is the last portion of the genome to be duplicated.

Eu-heterochromatic rearrangements may cause abnormal representation of heterochromatin in polytene chromosomes. For example, the heterochromatic right arm of the X chromosome, which is normally contained within the underreplicated chromocenter, can be cytologically visualized in polytene chromosomes when it is relocated to a euchromatic region (KORYAKOV et al. 1999). To get insight into this phenomenon, we used a set of related eu-heterochromatic rearrangements with a breakpoint in the distal heterochromatin of the X chromosome (TOLCHKOV et al. 2000), where a cluster of heterochromatic Stellate repeats is known to be localized (PALUMBO et al. 1994; BOZZETTI et al. 1995; TULIN et al. 1997). Clustered Stellate genes, proposed to encode the ß-subunit of a protein kinase-like protein (LIVAK 1990; PALUMBO et al. 1994), were first localized to the euchromatic 12E region of the X chromosome. These genes were shown to be actively expressed in testes of XO males lacking the Y chromosome. Heterochromatic variants of the Stellate genes were detected in the distal region of the X-linked heterochromatin (SHEVELYOV 1992; NURMINSKY et al. 1994; TULIN et al. 1997) and were also shown to be embedded in amplified, scrambled structures containing middle repetitive DNA (SHEVELYOV 1992; NURMINSKY et al. 1994). These middle repetitive repeats comprise, in addition to the damaged heterochromatic variants of Stellate genes, copia-like elements, LINEs, and rDNA fragments and are thus designated as Stellate-copia-LINE ribosomal DNAs (SCLRs). Distal to the SCLRs is also a 30-kb discontinuous heterochromatic Stellate cluster that contains 20 copies of tandemly repeated Stellate genes (Figure 1). Sequence analysis of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification (TULIN et al. 1997).

View larger version (7K): In this window In a new window Download PPT slide   FIGURE 1.— Diagram of the X chromosome carrying heterochromatic (SCLR and hetSte) and euchromatic (euSte) Stellate repeats. Euchromatin is depicted as a line. Distinct segments of the X-linked heterochromatin (Xh) are indicated and numbered according to GATTI et al. (1994). Segment 29 contains the nucleolus organizer (NO). C, centromere; Ste, Stellate cluster; SCLR, centromere proximally located damaged Stellate repeats interrupted by mobile element insertions (NURMINSKY et al. 1994; TULIN et al. 1997).

 
We investigated what happens when parts of the Stellate cluster, normally located inside Xh, are exposed to euchromatin as a consequence of chromosome rearrangements. The heterochromatic Stellate cluster is normally underrepresented in the unrearranged polytene X chromosome (SHEVELYOV 1992; TULIN et al. 1997), but we found that eu-heterochromatic rearrangements can cause Stellate heterochromatin representation (replication) in the course of polytenization. A spreading effect of adjacent euchromatin on Stellate-cluster replication was demonstrated: fragments of the Stellate cluster located farther from the newly formed eu-heterochromatic boundary remain underreplicated whereas those situated closer to euchromatin are replicated. We also demonstrate trans effects exerted by heterochromatic blocks on the replication of the rearranged heterochromatin. This observation allowed us to suggest that heterochromatin-binding proteins participate in the process of underreplication.

Fly stocks and crosses:

The pericentric In(1LR)pn2a rearrangement was isolated after irradiation of the wild-type Batumi line (ILYINA et al. 1980). The structures of the original In(1LR)pn2a rearrangement and its derivatives (m100, m141, r4, r16, and r9) were described earlier (TOLCHKOV et al. 1997, 2000) and are summarized in Figure 2; only the structure of the m141 rearrangement has been sightly corrected as, contrary to what was reported previously, a fragment of the Stellate cluster was shown to be associated with the Xh pericentromeric block. The rearrangements were balanced over FM7, y^31d sc⁸ w^a v^Of B g⁴. The effects of the different rearrangements on the Xh replication in polytene chromosomes were checked in Batumi/R females, where R designates a rearranged X chromosome. Batumi/R females were obtained by crossing R/FM7 females to Batumi males. To discriminate Batumi/R larvae from their Batumi/FM7 sisters, the FM7i chromosome carrying a GFP transgene (FM7i, P{w+mC=ActGFP}JMR/C(1)DX) was used. Females carrying an additional Y chromosome were obtained by crossing recR/FM7 females to y² su(w^a)Y^s.Y^ly⁺ males, where recR designates recombinant chromosome (see RESULTS). All the balancers and markers used are described in LINDSLEY and ZIMM (1992).

View larger version (25K): In this window In a new window Download PPT slide   FIGURE 2.— Diagram of the heterochromatic portions of secondary rearrangements obtained by irradiation of In(1)LR pn2a (pn2a). This chromosome is an inversion with breakpoints in XR (section h34) and euchromatin, carrying a whole intact XL heterochromatin (TOLCHKOV et al. 1997, 2000). The complete structures of complex rearrangements have been presented earlier (TOLCHKOV et al. 2000); here, for simplicity, the translocated centromeric segments of the m100, r16, and r4 rearrangements are omitted. D, C, E, and A indicate the location of the corresponding polymorphic EcoRI Stellate fragments (see Figure 5). C indicates the double B/C band (see text). Solid segments indicate regions of the heterochromatic Stellate cluster that are replicated in polytene chromosomes. Other designations are the same as those in Figure 1.

 

View larger version (74K): In this window In a new window Download PPT slide   FIGURE 5.— Southern blot analysis of genomic Stellate repeats after EcoRI digestion. (a) Underrepresentation of heterochromatic Stellate EcoRI fragments (A, B/C, D, and E) in deficiencies. eu, euchromatic fragments. The sizes of Stellate fragments (in kilobases), including SCLR repeats, are indicated. (b) DNA samples are isolated from females. Recombinant chromosomes carry transposed Stellate repeats. (c) The presence of heterochromatic Stellate fragments in DNA from flies carrying different rearrangements. (d) Diagrams of the X chromosome structures checked by Southern analysis. Dashed lines indicate deleted segments, and other designations are the same as those in Figures 1 and 2.

 

In situ hybridization:

The plasmid pSX83.4 (LIVAK 1990) contains the full-length Stellate gene and is thus capable of detecting all the variants of Stellate genes. This plasmid was labeled with biotin (bio-7-dATP; Bethesda Research Laboratories, Gaithersburg, MD) by nick translation. In situ hybridization to salivary gland polytene chromosomes of third instar larvae was performed according to the procedure described in ASHBURNER (1989). Hybridization was detected using the Elite Vectastatin ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine (Sigma, St. Louis).

Southern analysis:

Genomic DNA was isolated as described previously (TULIN et al. 1997). Agarose gel electrophoresis of DNA and blotting to Hybond N membranes (Amersham, Arlington Heights, IL) were performed according to standard techniques (MANIATIS et al. 1989). The Stellate DNA probe was an EcoRI/HindIII fragment from pSX83.4 (LIVAK 1990) labeled with [³²P] by oligo priming (FEINBERG and VOGELSTEIN 1983). EcoRI and HindIII digestions were performed according to the enzyme supplier's instructions.

To define the molecular structure of the Xh Stellate cluster and its pattern of polytenization in rearrangements, we have constructed a number of recombinant chromosomes lacking different portions of the Stellate-containing h26 region.

Recovery of chromosomes Df(1)hSte and Df(1)12:

The Df(1)hSte chromosome lacking all heterochromatic Stellate copies and the SCLRs was selected as a y ac sc male among the progeny of Dp r9/Y males crossed to C(1)/B^SY females. Dp r9 is a derivative of chromosome r9 (TOLCHKOV et al. 2000) and contains a duplication of the euchromatic 1A–2E region carrying the y⁺ ac⁺ and sc⁺ genes (Figure 3). As a result of X-Y recombination in Dp r9/Y males at the nucleolus organizer region, the Dp r9 chromosome lost the h26 segment that contains the Stellate repeats (PALUMBO et al. 1994; TULIN et al. 1997) and acquired the Y chromosome short arm (Y^S) (Figure 3). Males and homozygous females carrying this chromosome are viable and fertile. The absence of Stellate material in the Df(1)hSte chromosome in females was confirmed by Southern analysis of EcoRI restriction fragments, using a Stellate-specific probe (see below).

View larger version (20K): In this window In a new window Download PPT slide   FIGURE 3.— Structure of chromosome Df(1)hSte lacking the heterochromatic Stellate repeats. This chromosome was recovered as a recombinant between a derivative of chromosome r9 carrying the 1A–2E duplication (Dp r9) and the short arm of the Y chromosome. Ys and YL indicate the short and the long arm of the Y chromosome, respectively. Other designations are the same as those in Figure 2.

 
The Df(1)12 chromosome (Figure 2) carries a partial deletion of distal Xh and was selected in the progeny of irradiated (4 krad) In(1LR)pn2a, w males crossed to w^a f su(f) females. F₁ females with yellow rough eyes were expected to carry an irradiated In(1LR)pn2a chromosome with a deletion of su(f), a euchromatic gene located near the eu-heterochromatic boundary (LINDSLEY and ZIMM 1992) and, possibly, a deletion of a portion of the distal h26 region. Df(1)12/FM7 females, carrying a putative deficiency of both su(f) and distal Xh, were then tested for the presence of the nucleolus organizer by crossing to In(1)sc^4L sc^8R/B^SY males, which lack the X-linked nucleolus organizer. Df(1)12/In(1)sc^4L sc^8R females were viable, demonstrating the presence of at least a partial nucleolus organizer in the Df(1)12 chromosome (Figure 2). This chromosome was also confirmed to lack part of the heterochromatic Stellate cluster by Southern analysis (see below).

Construction of m100 and m141 derivatives lacking a part of the Xh block:

To produce derivatives of m100 and m141 rearrangements (R) lacking a part of the Xh block, R/FM7 females were crossed to sc^S1 males to generate R/sc^S1 females. Recombinant chromosomes lacking y⁺ ac⁺ sc⁺ and the adjacent Xh block, but carrying the centromeric Xh block of chromosome sc^S1, were selected in the progeny of these females crossed to Df(1)hSte, y ac sc w/Y males (Figure 4). The indicated region of recombination was deduced from Southern analysis of DNA from rec/Df(1)hSte females (Figure 4, inset). The euchromatic Stellate array of In(1)sc^S1 contains many more copies of the 0.95-kb CfoI variant than does Batumi (not shown), and the recombinant chromosomes carry the sc^S1 euchromatic array as expected from an exchange in the region between the euchromatic Stellate cluster at 12E and the f gene. Southern analysis of rec/Df(1)hSte, y ac sc w⁺ females, where rec designates recombinant derivates of either m100 or m141 lacking the proximal Xh segment, was then used to study Stellate repeat organization and replication.

View larger version (24K): In this window In a new window Download PPT slide   FIGURE 4.— Generation of recombinant chromosomes lacking the distal Xh. Heterochromatic blocks are depicted as in Figure 1. Positions of genes are indicated according to the cytological map. Ste, location of euchromatic Stellate repeats. Ste* and Ste are polymorphic euchromatic clusters in the scS1 and pn2a rearranged X chromosomes, respectively. (Inset) Southern analysis of CfoI Stellate restriction fragments in recombinant chromosomes.

 

Mapping of the Stellate cluster restriction fragments:

Heterochromatic Stellate repeats are known to be underreplicated in polytene chromosomes (SHEVELYOV 1992; TULIN et al. 1997), and thus both eu- and heterochromatic Stellate fragments are detected in DNA samples from whole flies, whereas normally only euchromatic Stellate fragments are visible in salivary gland DNA. We have ordered the restriction fragments derived from EcoRI digestion of heterochromatic Stellate repeats on the basis of their sizes and designated them from the largest (>23 kb) to the smallest (<3 kb) as A, B/C, D, and E, respectively. The double B/C band is indicated in Figure 2 as C. Fragments of 1.3 and 0.8 kb derive from SCLR tandem repeats containing damaged Stellate fragments interrupted by inserted transposable elements (NURMINSKY et al. 1994). CfoI digestion of fragment A yielded a 1.15-kb repetitive unit (Figure 4, inset), revealing its regular repeated structure. EcoRI digestion of both salivary gland and adult fly DNA from Df(1)hSte flies yields fragments of 10.5 and 3.5 kb, which correspond to the structural variants of euchromatic Stellate repeats located at 12E. No heterochromatic fragments were evident (Figure 5a), demonstrating that viable homozygous Df(1)hSte females carry a complete deletion of the Xh Stellate cluster. No B/C, D, and SCLR fragments are detected in DNA from whole Df(1)hSte homozygous females, whereas euchromatic bands are easily detected. Df(1)12/Df(1)hSte females carry no A and E fragments, while fragments B/C and D, as well as the proximally located SCLR repeats (TULIN et al. 1997), were visible (Figure 5a). Thus, in Df(1)12 only the most distal region of the Stellate cluster appears to be deleted (Figure 2), which, on the basis of our Southern analysis, seems to contain regular tandem repeats (TULIN et al. 1997) of the A and E fragments.

Previous cytological analysis revealed that in the m100 and m141 rearrangements the Stellate-containing h26 region is transposed to the middle of X euchromatin and to the XL telomere, respectively (TOLCHKOV et al. 2000). To identify the transposed EcoRI Stellate fragments in these rearrangements, we recovered the recombinant chromosomes (rec m100 and rec m141) carrying the transposed h26 region but lacking most of proximal Xh (regions h27–h30; Figure 4). Southern analysis of total DNA from Df(1)hSte/rec100 and Df(1)hSte/rec141 females revealed the presence of the A and E Stellate fragments in the h26 segment transposed into euchromatin of chromosome rec100 while only the A fragment contained within the h26 segment is transposed to the telomere in the rec141 chromosome (Figure 5b). These results confirm that the A and E fragments are located within the most distal portion of the h26 Stellate cluster, while fragments B/C and D are restricted to a more proximal position (Figure 2).

Heterochromatic Stellate fragments are replicated in polytene chromosomes of specific rearrangements:

In(1LR)pn2a contains the entire Stellate X-heterochromatic block (Figure 2) but no heterochromatic Stellate fragments were detected after EcoRI digestion of polytene chromosome DNA from In(LR)pn2a/Batumi females (Figure 5c). By contrast, we found that heterochromatic Stellate repeats are represented in salivary gland DNA from flies carrying secondary rearrangements. In m100/Batumi females, the A, E, and B/C fragments, which are relocated adjacent to euchromatin in this rearrangement, are represented in salivary gland DNA, while the D fragment, which remains embedded within heterochromatin, is almost not detected (Figure 5c). The D fragment is much more abundant than the euchromatic 3.5-kb fragment in DNA from whole flies, but this band is negligible compared to the 3.5-kb fragment in salivary gland DNA. Southern analysis of DNA from polytene chromosomes of Batumi/m141 females demonstrates substantial replication of the E fragment while only a minor amount of the A fragment is detected (Figure 5c). Interestingly, cytogenetic analysis of m141 revealed the presence of additional heterochromatic material at the telomere region of this chromosome (TOLCHKOV et al. 2000). Thus, it is conceivable that replication of the E fragment is caused by its proximity to euchromatin whereas the more distal fragment A (Figure 2) remains subsantially underrepresented due to its association with telomeric heterochromatin. Only a vestige of the A band is detected in salivary gland DNA from r16/Batumi females (Figure 5, c and d). No polytenization of heterochromatic Stellate material was observed for the r4 and r9 rearrangements (900 nuclei, 60 larvae scored). The r4 rearrangement contains an undamaged distal Xh region, comprising the h26-h29 sections and an intact eu-heterochromatic transition zone (Figures 2 and 5d). r9 is characterized by shuffling of internal segments of the Xh block (Figure 3 and TOLCHKOV et al. 2000), with no alteration of the eu-heterochromatic boundaries.

Supplementary evidence of heterochromatic Stellate overrepresentation was obtained by in situ hybridization of Stellate sequences to polytene chromosomes. No labeling was detected on the chromocenter of pn2a/Batumi females (Figure 6a) (800 nuclei, 50 larvae scored), while a strong signal is detected at the 12E region, where the euchromatic variants of Stellate repeats are located (PALUMBO et al. 1994). By contrast, highly reproducible and strong staining of the chromocenter region is observed on polytene chromosomes from both m141/Batumi and r16/Batumi females (Figure 6, b and c), consistent with the results obtained by Southern analysis of these genotypes. Due to the presence of extensive ectopic pairing in polytene chromosomes from m100/Batumi females, analysis of Stellate sequence representation was performed by in situ hybridization on m100/Y males. The transposed A and E fragments were clearly detected adjacent to polytene region 5D, as transposition of a heterochromatic block into euchromatin generates a "weak point" (Figure 6d). Ninety percent of nuclei (900 nuclei, 50 larvae scored) from m100/Y salivary glands exhibit a Stellate signal in the chromocenter. This result is consistent with the observed abnormal replication of the EcoRI C fragment that in m100 is still associated with the main Xh block but is adjacent to a new eu-heterochromatic boundary (Figures 2 and 5, c and d). The signal in the chromocenter observed in m100/Y males is not from hybridization to the Y chromosome-linked Supressors of Stellate repeats (LIVAK 1990) that are homologous to Stellate, because the Y chromosome is completely underreplicated in the course of polytenization. In experimental confirmation of this, no Stellate signal was detected in the chromocenter of In(LR) pn2a/Y males (500 nuclei, 30 larvae scored).

View larger version (90K): In this window In a new window Download PPT slide   FIGURE 6.— Detection of Stellate repeats in polytene chromosomes by in situ hybridization. Designations of rearrangement structures are the same as those in Figure 2. C indicates the double B/C band (see text). The unpaired 1A–2E euchromatic region of the Batumi chromosome is indicated. Region 2E either is visualized near the chromocenter (b and c) or may be detached from it. (a) pn2a/Batumi females. Detection of the euchromatic Stellate cluster 12E (arrow), with no staining of Stellate repeats in the chromocenter, is shown; the thick arrow indicates the 2F telomere region (T) of pn2a and the base of the unpaired 1A–2F region of the Batumi chromosome. (b) Detection of Stellate repeats in the chromocenter and in the 12E region of m141/Batumi females (the internal X chromosome fragment distal to the 12E region is not shown). (c) Similar pattern of staining in r16/Batumi females. (d) Stellate hybridization on polytene chromosomes from m100/Y males. Note the broken X chromosome at the newly formed weak point at 5D; the chromocenter, as well as the 12E and 5D regions, is clearly stained.

 

An Xh block as well as a Y chromosome exert trans effects supporting replication of the rearranged Stellate material:

We found that the Stellate signal observed at the 5D region in polytene chromosomes of m100-carrying flies disappeared in polytene chromosomes (500 nuclei, 35 larvae scored) from rec m100/Df(1)hSte females, which lack most of the Xh present in the original m100 chromosome (Figure 7a). Underreplication of this region in polytene chromosomes was maintained in successive generations of rec m100/Df(1)hSte females (400 nuclei, 30 larvae scored). In line with this observation, Southern analysis of salivary gland DNA from rec m100/Df(1)hSte females failed to detect a significant representation of the A and E Stellate fragments, which are nevertheless detected in adult DNA from the same flies (Figure 5b). Analogous results were obtained with rec m141/Df(1)hSte females, as the E fragment that is clearly detected in the salivary gland DNA of m141/Df(1)hSte flies (Figure 5c) completely disappeared in polytene DNA from rec m141/Df(1)hSte females, which carry a substantial deletion of the Xh present in the original m141 chromosome (Figure 5b). However, in situ hybridization revealed that replication of the AE fragment is restored in rec m100/ XY females, carrying an additional Y chromosome (Figure 7b) (40% of nuclei; 600 nuclei, 50 larvae scored). Together, these results indicate that addition and deficiency of heterochromatin can exert a suppression and an enhancer effect, respectively, on the underreplication of rearranged Stellate heterochromatic repeats in polytene chromosomes.

View larger version (73K): In this window In a new window Download PPT slide   FIGURE 7.— Deficiency of the X heterochromatin causes underreplication of relocated Stellate fragments, while addition of the Y chromosome restores replication. (a) Absence of Stellate signal at the 5D region in rec m100/Df(1)hSte larvae. (b) Restoration of relocated Stellate replication. (c) Diagram of polytene chromosomes in rec m100/Df(1)hSte females. Chromosome rec m100 carries the terminal duplicated 2F–5D region depicted by a thick dashed line, which is paired with Df(1)hSte (thin solid line); the rest and main part of the rec m100 chromosome (dashed line) is represented by the paired and unpaired region. Ectopic pairing is observed between the 5D and 20F regions. (d) Representation of the X chromosome inherited from XY males (solid line) paired with rec m100 (thin dashed line) and the 2F–5D fragment of this chromosome (thick dashed line).

 

Eu-heterochromatic rearrangements are known to cause underrepresentation of euchromatic regions transposed to heterochromatin. Spreading of chromatin compaction and underrepresentation from the eu-heterochromatic boundary into euchromatin has been extensively documented on polytene chromosomes (ANANIEV and GVOZDEV 1974; SPRADLING 1993; LAVROV et al. 1998; ZHIMULEV 1999). Here we report evidence of a "euchromatization" phenomenon, which is the spreading of euchromatic replication toward adjacent heterochromatin in polytene chromosomes carrying eu-heterochromatic rearrangements. This phenomenon may be considered as a peculiar type of position effect, resulting in the violation of the normal process of heterochromatin underrepresentation in polytene chromosomes. We determined the relative positions of the EcoRI fragments corresponding to the heterochromatic Stellate cluster along the distal Xh region (region h26). A series of rearrangements involving h26 were induced and their heterochromatic breakpoints were cytologically and molecularly defined. This allowed us to demonstrate a spreading effect exerted by the adjacent euchromatin on the replication of heterochromatic regions that, as a consequence of chromosomal rearrangements, are relocated at the eu-heterochromatic boundary. The regions of the Stellate cluster that, due to the rearrangements, are positioned closer to euchromatin were shown to be overrepresented in polytene chromosomes, whereas those located more distally from euchromatin showed normal underreplication. To our knowledge, this is the first demonstration that a spreading effect can be exerted by euchromatin on the replication of adjacent heterochromatin. This spreading effect might be explained by the involvement of euchromatic origins in the replication of Stellate repeats. It was proposed that "instructions" for underreplication may be emanated from satellite blocks that are located inside the X-linked heterochromatin, proximally to the Stellate cluster (LEACH et al. 2000). However, our observations that the spreading effect starts from the adjacent euchromatin allow us to propose that the eu-heterochromatic border normally acts as a boundary between the underreplicated heterochromatin and polytenized euchromatin.

The recently described Su(UR) (Supressor of underreplication) gene modifies the manifestation of the cytological characteristics of intercalary and, in many cases, pericentric heterochromatin (ZHIMULEV and BELYAEVA 2003). In particular, the Su(UR) mutation strongly suppresses underreplication of some regions of pericentric heterochromatin. However, we observed no effects of the Su(UR) mutation on the replication of Stellate repeats. Thus, we suggest a peculiarity of the regulatory cues affecting heterochromatin underreplication, taking into account that distal X heterochromatin is represented by the distinct arrays of repeats possibly involved in performing different functions.

The abnormal replication of heterochromatic Stellate sequences observed in salivary glands of flies carrying eu-heterochromatic rearrangements is stably suppressed by a deficiency of heterochromatin, while addition of a Y chromosome restores Stellate polytenization in Xh-deleted flies. This trans effect implies that abnormal heterochromatic replication in polytene chromosomes may be considered as an epigenetic phenomenon, like the classical type of position-effect variegation of euchromatic genes. We also demonstrated that heterochromatic blocks can exert trans effects on the replication of a fragment of the Stellate cluster transposed into euchromatin: underreplication of an Xh fragment stably prevented Stellate polytenization while addition of a Y chromosome suppressed this effect. These observations strongly suggest that specific heterochromatic proteins could be involved in the phenomenon of underreplication. The enhancer effects of heterochromatic deficiencies on the inactivation of euchromatic genes caused by position effect are considered to be the result of a titration of the repressor heterochromatic proteins by an excess of heterochromatic DNA (KHESIN and LEIBOVITCH 1978; WEILER and WAKIMOTO 1995; ZHIMULEV 1999). Here we observed an opposite effect, as a deficiency of heterochromatin restored the normal underreplication of rearranged heterochromatic Stellate sequences in polytene chromosomes. Our results agree with the observation that in salivary glands of XY males there is an increase in the amount of heterochromatic minichromosomes that are substantially eliminated in XO males (LEACH et al. 2000). We propose that heterochromatic proteins may play an active role in the underreplication of heterochromatin during polytenization. Studies of the effects of suppressors of classical position-effect variegation on the abnormal heterochromatin polytenization might clarify the mechanisms of heterochromatin underreplication and its dynamic functions in gene expression.

We thank E. G. Pasyukova for help in the studies of polytene chromosome structures. We thank anonymous reviewers for improving this manuscript. This work was supported by the Russian Academy of Sciences program for Molecular and Cell Biology and by grants from the Russian Foundation for Science School (2074.2003.4) and the Russian Foundation for Basic Researches (05-04-48034).

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Communicating editor: M. J. SIMMONS

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