Distribution of induced chromosome rearrangement breakpoints along the chromosome length and the problem of intercalary heterochromatin in Drosophila melanogaster

Historically, the term “intercalary heterochromatin” was based on the finding that induced chromosome rearrangements occur at a higher frequency in the corresponding regions. The available molecular genetic data and, in particular, the results of the Drosophila Genome Project made it possible to decide between two possible explanations of the preferential location of chromosome rearrangement breakpoints in intercalary heterochromatin regions. Namely, a higher frequency of radiation-induced rearrangements in these regions correlates with the DNA content and probably lacks an association with the features of chromatin organization.     

Changes in the properties of intercalary heterochromatin in Drosophila melanogaster induced by position effect modifiers

Compaction of chromosome regions translocated to the pericentric heterochromatin and the break frequency in intercalary heterochromatin (IH) regions as affected by position effect modifiers (low temperature, removing the Y chromosome, genetic enhancers and suppressors) were studied. It has been shown that the factors enhancing compaction also increase the break frequency in IH regions. Data obtained are discussed from the point of view of proteins affecting the compaction of intercalary and pericentric heterochromatin regions translocated to heterochromatin under position effect.     

Transcription of intercalary heterochromatin regions in Drosophila melanogaster cell culture

Labelled RNA preparations (total newly synthesized RNA, as well as stable cytoplasmic RNA) isolated from a cell culture of D. melanogaster were hybridized in situ with polytene chromosomes. Apart from the nucleolus, in all cases the regions adjacent to the chromocentre in the polytene chromosomes and the intercalary heterochromatin regions in the X chromosome and the autosomes are the most intensively labelled. In the case of asynapsis of polytene chromosomes in heterozygotes the label is detected in a number of intercalary heterochromatin sites in one homologue only ("the asymmetrical label"). The same kind of radioactivity distribution in intercalary heterochromatin regions was observed after a hybridization of polytene chromosomes with cloned DNA fragments (Ananiev et al., 1978, 1979) coding for the abundant classes of messenger RNA (Ilyin et al., 1978) in a cultured D. melanogaster cells. In some regions of intercalary heterochromatin which do not contain these fragments the "'asymmetrical" type of label distribution is observed after hybridization with cell RNA. - These results lead one to regard the intercalary heterochromatin regions as "nests" comprising different types of actively transcribable genes, the composition of each nest varying in different stocks of D. melanogaster.     

Intercalary heterochromatin in Drosophila

The relative amount of DNA in defined segments of salivary gland chromosomes of Drosophila melangogaster from the Oregon R stock was determined by autoradiography. The data obtained were then used to estimate the possible correlation between DNA content and the degree of manifestation of charcters such as weak-point behavior, late replication, strong synapsis, breaks of chromosome rearrangements, hybridization with cRNA, and localization of mobile elements. Of 380 regions investigated 274 have showed deviations in the degree of manifestation of these features from that predicted on the basis of the DNA content of these regions. Regions, previously shown to consist of intercalary heterochromatin (IH, Zhimulev et al. 1982), were found to have a significantly higher frequency of the simultaneous manifestation of several of the above-mentioned features, with the exception of localization of mobile elements. These findings support the earlier suggestion that a high frequency and a simultaneous manifestation of IH features depend on some peculiarities of the molecular organization of IH regions, but not on a high DNA content.     

Cytogenetic analysis of the third chromosome heterochromatin of Drosophila melanogaster

Previous cytological analysis of heterochromatic rearrangements has yielded significant insight into the location and genetic organization of genes mapping to the heterochromatin of chromosomes X, Y, and 2 of Drosophila melanogaster. These studies have greatly facilitated our understanding of the genetic organization of heterochromatic genes. In contrast, the 12 essential genes known to exist within the mitotic heterochromatin of chromosome 3 have remained only imprecisely mapped. As a further step toward establishing a complete map of the heterochomatic genetic functions in Drosophila, we have characterized several rearrangements of chromosome 3 by using banding techniques at the level of mitotic chromosome. Most of the rearrangement breakpoints were located in the dull fluorescent regions h49, h51, and h58, suggesting that these regions correspond to heterochromatic hotspots for rearrangements. We were able to construct a detailed cytogenetic map of chromosome 3 heterochromatin that includes all of the known vital genes. At least 7 genes of the left arm (from l(3)80Fd to l(3)80Fj) map to segment h49-h51, while the most distal genes (from l(3)80Fa to l(3)80Fc) lie within the h47-h49 portion. The two right arm essential genes, l(3)81Fa and l(3)81Fb, are both located within the distal h58 segment. Intriguingly, a major part of chromosome 3 heterochromatin was found to be "empty," in that it did not contain either known genes or known satellite DNAs.     

Localization and characteristics of DNA underreplication zone in the 75C region of intercalary heterochromatin in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes

In Drosophila polytene chromosomes, regions of intercalary heterochromatin are scattered throughout the euchromatic arms. Here, we present data on the first fine analysis of the individual intercalary heterochromatin region, 75C1-2, located in the 3L chromosome. By using electron microscopy, we demonstrated that this region appears as three closely adjacent condensed bands. Mapping of the region on the physical map by means of the chromosomal rearrangements with known breakpoints showed that the length of the region is about 445 kb. Although it seems that the SUUR protein binds to the whole 75C1-2 region, the proximal part of the region is fully polytenized, so the DNA underreplication zone is asymmetric and located in the distal half of the region. Finally, we speculate that intercalary heterochromatin regions of Drosophila polytene chromosomes are organized into three different types with respect to the localization of the underreplication zone.     

Intercalary heterochromatin in polytene chromosomes of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Intercalary heterochromatin consists of extended chromosomal domains which are interspersed throughout the euchromatin and contain silent genetic material. These domains comprise either clusters of functionally unrelated genes or tandem gene duplications and possibly stretches of noncoding sequences. Strong repression of genetic activity means that intercalary heterochromatin displays properties that are normally attributable to classic pericentric heterochromatin: high compaction, late replication and underreplication in polytene chromosomes, and the presence of heterochromatin-specific proteins. Late replication and underreplication occurs when the suppressor of underreplication protein is present in intercalary heterochromatic regions. Intercalary heterochromatin underreplication in polytene chromosomes results in free double-stranded ends of DNA molecules; ligation of these free ends is the most likely mechanism for ectopic pairing between intercalary heterochromatic and pericentric heterochromatic regions. No support has been found for the view that the frequency of chromosome aberrations is elevated in intercalary heterochromatin.     

Intercalary heterochromatin in Drosophila

Sites of intercalary heterochromatin (IH) in the complete set of Drosophila melanogaster polytene chromosomes were localized and studied according to the following criteria: tendency to break (weak points), ectopic pairing and late replication, the existence of repeats (in X and 2R) including those enriched with A-T bases. Correlation between these features investigated, the highest correlation coefficients found between weak point behavior, late replication, and ectopic pairing. The frequency of breaks in weak points in some IH bands was shown to be different in different tissues, strains and closely related Drosophila species. Sexual differences in morphology and manifestation of IH features were found in bands of the X chromosome: weak point behavior and participation in ectopic pairing of IH bands are an order of magnitude less frequent in male X chromosomes than in female X chromosomes. In autosomes such differences have not been observed. IH bands in male X chromosomes look more massive than the homologous ones in female X chromosomes: the DNA content of the 11A6-9 region is four times less in females than in males. The hypothesis is proposed that the specific features of intercalary heterochromatin bands are determined by tandem repetitiveness and late replication. The latter, if it occurs in a cluster of repetitions, could cause incomplete polytenization of the region and, as a consequence, breaks (or weak points) and the appearance of adhesive ends which may take part either in realization of ectopic contacts or in fixation of those occurring previously. Breaks caused by chromosome aberrations in regions with repeats may not result in a sharp decline of viability, so that break points of chromosome rearrangements in intercalary heterochromatin may be more frequent than in other regions.     

Changes in the duration of replication of the polytene chromosome region during position effect in Drosophila melanogaster

Replication pattern of the X chromosome 2B region translocated to the pericentric heterochromatin in the 2L chromosome within the T(1; 2) dorvar7 rearrangement which causes position effect was studied. It was found that this pattern is affected by the 2B region morphology. When normal, i.e. with decondensed bands in this region, it completes replication early. But after compaction, i.e. fusion of bands in the 2B region into dense block, due to position effect, its late replication was observed, together with the most late replicating intercalary heterochromatin regions in the X chromosome and the 75C-80A-C segment of the 3L chromosome. Possible mechanisms of replication delay and their role in promoting the position effect are discussed.     

A strategy for mapping the heterochromatin of chromosome 2 of Drosophila melanogaster

The heterochromatin of chromosome 2 of Drosophila melanogaster has been among the best characterized models for functional studies of heterochromatin owing to its abundance of genetic markers. To determine whether it might also provide a favorable system for mapping extended regions of heterochromatin, we undertook a project to molecularly map the heterochromatin of the left arm of chromosome 2 (2Lh). In this paper, we describe a strategy that used clones and sequence information available from the Drosophila Genome Project and chromosome rearrangements to construct a map of the distal most portion of 2Lh. We also describe studies that used fluorescent in situ hybridization (FISH) to examine the resolution of this technique for cytologically resolving heterochromatic sequences on mitotic chromosomes. We discuss how these mapping studies can be extended to more proximal regions of the heterochromatin to determine the structural patterns and physical dimensions of 2Lh and the relationship of structure to function.     

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS–GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR ⁻ phenotype and induces specific swellings in heterochromatin.     

C-band distribution,DNA content and base composition inAdoxa moschatellina (Adoxaceae), a plant with cold-sensitive chromosome segments

The chromosomes ofAdoxa moschatellina (2n = 36, paleo-4x) contain mostly terminal, occasionally intercalary, negatively heteropycnotic cold-induced regions which correspond to all major C-bands including the satellites, as revealed by sequential analysis. Positively C-stained are also centromeres, the dotlike arms of the 7 telocentric chromosome pairs, and some very narrow intercalary bands; their cold-sensitivity is hardly traceable. There exists a fraction of condensed interphase chromatin, at least after chilling, which is virtually not C-banded (possibly condensed euchromatin).The DNA amount is 14.3 pg (1 C). The heterochromatin content is 13.0%. The thermal melting profile (Tm corresponding to 38.6% GC) does not reveal a particular AT- or GC-rich fraction. Significantly, the heterochromatin respond to the Hy-banding procedure is neutral.The distribution of cold-sensitive regions in plants was analysed with the arm-frame method: Intercalary positions, clearly, are not especially favoured regions. The obvious deficiency at centromeric positions may depend on the action of natural selection against mechanically labile centromeric regions.Dedicated to Univ.-Prof. Dr.Lothar Geitler on the occasion of his 80th birthday.     

Identification of chromosome aberrations observed in human cancerous cells by labeling of R bands]

A precise identification of chromosome abnormalities observed in cancerous human cells may be demonstrated by chromosome banding. The types of newly formed chromosomes seem to indicate the possibility of a correlation between chromosome rearrangements and regions of heterochromatin. The evolution of neoplastic cells would be due to a balance between an increase in the level of chromosome mutation and stable changes.     

Gene-rich chromosome regions and autosomal trisomy

Summary Cases of autosomal trisomy and trisomy mosaicism among liveborn infants are reviewed, and a second case of chromosome 3 trisomy mosaicism is described. The occurrence of autosomal trisomy for a particular chromosome is in general negatively correlated with the number of genes which have been localized to that chromosome. It is also positively related to the Q-brightness of the chromosome, which reflects its content of intercalary heterochromatin. Furthermore there are significantly fewer autosomal trisomics for chromosomes which contain hot spots for mitotic chiasmata in Bloom syndrome (chromosomes 1, 3, 6, 11, 12, 17, 19, and 22), compared with similar-sized control chromosomes 2, 4, 7, 9, 10, 18, 20, and 21. This is interpreted as further evidence for the gene richness of the hot spots which, being active, are extended in interphase and are therefore available for mitotic crossing over. The gene richness of these short Q-dark regions is also borne out by the scarcity of trisomic abortions for the chromosomes involved (the embryo dies before the abortion is recognized) and by the higher number of genes localized to these chromosomes compared with the control chromosomes.     

Pericentromeric and intercalating alpha-heterochromatin of polytene chromosomes of the malaria mosquito

The properties of heterochromatin from polytene chromosomes of the malaria mosquito Anopheles messeae Fal. and A. atroparvus V. Tiel. were studied by various methods of differential staining and by hybridization in situ with two repetitive DNA sequences of Drosophila melanogaster. In malaria mosquito, the heterochromatin was heterogeneous. Two forms of alpha-heterochromatin were revealed: pericentromeric and intercalary heterochromatin, which is localized within the internal chromosome regions.     

Three euchromatic DNA sequences under-replicated in polytene chromosomes of Drosophila are localized in constrictions and ectopic fibers

We examined three regions of under-represented euchromatic DNA sequences (histone, Ubx, and 11 A), for their possible correlation with euchromatic constrictions in polytene chromosomes of Drosophila melanogaster. Cloned sequences were hybridized to filters and to chromosomes prepared for light microscopy. Under-represented sequences hybridized to DNA within constrictions and in ectopic fibers. In contrast, adjacent sequences that were fully endoreplicated in the Ubx and 11A regions in polytene cells hybridized to sites just adjacent to their respective constrictions. For one region (Ubx), sequences under-represented in salivary gland cells were fully endoreplicated in fat body cells. For this particular region, the morphology of the polytene chromosomes differs between these two cell types in that the specific constriction is absent at this region in fat body polytene chromosomes, thus strengthening the correlation between under-representation and chromosome constrictions. Although all three sequences are in regions that have been classified by others as intercalary heterochromatin, we detect no common functional or sequence organizational feature for these examples of under-represented DNA. We suggest that the lower efficiencies of the replication origins, or special regions of termination at these sites, are the primary cause of the under-replication, and that this under-replication is sufficient to confer the properties of intercalary heterochromatin.     

Distorted heterochromatin replication in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes as a result of euchromatin-heterochromatin rearrangements

Studies of the position effect resulting from chromosome rearrangements in Drosophila melanogaster have shown that replication distortions in polytene chromosomes correlate with heritable gene silencing in mitotic cells. Earlier studies mostly focused on the effects of euchromatin-heterochromatin rearrangements on replication and silencing of euchromatic regions adjacent to the heterochromatin breakpoint. This review is based on published original data and considers the effect of rearrangements on heterochromatin: heterochromatin blocks that are normally underrepresented or underreplicated in polytene chromosomes are restored. Euchromatin proved to affect heterochromatin, preventing its underreplication. The effect is opposite to the known inactivation effect, which extends from heterochromatin to euchromatin. The trans-action of heterochromatin blocks on replication of heterochromatin placed within euchromatin is discussed. Distortions of heterochromatin replication in polytene chromosomes are considered to be an important characteristic associated with the functional role of the corresponding genome regions.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/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.