Homolog pairing and sister chromatid cohesion in heterochromatin in <Emphasis Type="Italic">Drosophila</Emphasis> male meiosis I

Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X–Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.     

Eu-heterochromatic rearrangements induce replication of heterochromatic sequences normally underreplicated in polytene chromosomes of Drosophila melanogaster

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.     

Heterochromatin and interstitial telomeric DNA homology

The purpose of this investigation was twofold. The first objective was to demonstrate that, in most of ten mammalian species commonly used in biomedical research, not all constitutive heterochromatin (C-bands) represents telomeric DNA. For example, the C-bands in human chromosomes, the long arm of the X and the entire Y chromosome of Chinese hamster, and most of the short arms of Peromyscus and Syrian hamster chromosomes are not telomeric DNA. In addition to the usual terminal telomeric DNA in the chromosomes of these mammalian species, the pericentromeric regions of seven or eight Syrian hamster chromosomes and all Chinese hamster chromosomes except pair one have pericentromeric regions that hybridize with telomeric DNA, some in C-bands and some not. The second objective was to describe a simple fluorescence in situ hybridization (FISH) reverse-printing procedure to produce black-and-white microphotographs of metaphase and interphase cells showing locations of telomeric DNA with no loss of resolution. Thus, at least three different types of heterochromatin (telomeric heterochromatin, nontelomeric heterochromatin and a combination of both) are present in these mammalian species, and this simple black-and-white reverse printing of telomeric FISH preparations can depict them economically without sacrificing clarity.     

DNA characterization and karyotypic evolution in the bee genus Melipona (Hymenoptera,Meliponini)

We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini.     

Satellited Y chromosomes: Structure,origin, and clinical significance

Summary Three cases of inherited satellited Y chromosomes (Yqs) were analysed using several cytogenetic techniques. The cytogenetic data of the 14 cases of Yqs chromosomes described to date were reviewed. All Yqs chromosomes carry an active nucleolus organizer region (NOR) in their long arm and must have developed from translocations involving the short arms of the acrocentric autosomes. The structure of the heterochromatic satellite region in the Yqs chromosomes shows conspicuous inter-familial differences; this permits the reconstruction of the translocations from which the various Yqs were derived. Some causal factors leading to the development of Yqs chromosomes are considered: the specific localization of the four satellite DNAs and highly methylated DNA sequences in the karyotype, and some new experimental data on the spatial arrangement of heterochromatic regions in interphase nuclei. These provide distinct evidence for a preferential involvement of the autosomes 15 and 22 in the translocations with the Y heterochromatin. All clinical reports documenting Yqs males born with malformations were reviewed. It appears that the presence of an extra NOR and NOR-associated heterochromatin in the Yqs chromosomes does not cause any phenotypic abnormalities (as long as the Y euchromatin is intact). The possibility that a Yqs chromosome predisposes to non-disjunction and/or to translocations of other chromosomes is discussed.     

Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus

The Y chromosome heterochromatin in Rumex thyrsiflorus has been analyzed. In natural populations the Y chromosome shows a higher morphological variability than the X chromosome. The total duration of replication of Y chromosomes is about 2 hrs longer than that of euchromatin. Autoradiography with tritiated thymidine showed that chromocentres formed by Y chromosomes in interphase nuclei retain their heterochromatic form during DNA replication. — Y chromosome heterochromatin in interphase nuclei is stained pink, while the rest of the nucleus stains green after fast green-eosin staining for histones. — During the premeiotic stage of PMC development Y chromosomes are no longer visible as compact bodies and become more fuzzy in appearance. A diffuse state of Y coincides with intense RNA synthesis. Therefore genetic activity of Y chromosomes or their parts during premeiotic stage of microsporogenesis is postulated.     

Sister chromatid exchange points in the heterochromatin and euchromatin regions of Chinese hedgehog chromosomes

Summary The Chinese hedgehog has a diploid chromosome number of 48 in which there are eleven pairs of telo- or subtelocentric autosomes, twelve pairs of meta- or submetacentric autosomes, a metacentric X chromosome and a telocentric Y chromosome. The heterochromatin is almost completely distributed in five large distal segments of chromosomes nos. 9 to 12 and no. 18. There is no positive C-band in the centromeres of the chromosomes except for the X chromosome which has a small, weakly stained C-band in the centromere. In Chinese hedgehog cells 52.1% of SCEs are found at the junction between the euchromatin and the heterochromatin, 39.5% in the heterochromatin and 8.4% in the auchromatin. The SCE number per unit C-band is double the SCE number per unit euchromatin. The SCE rate in the heterochromatin or euchromatin regions is not proportional to their chromosome length and can be quite different between different pairs of the chromosomes. Our results indicate that there is a non-uniform distribution of the SCEs in the Chinese hedgehog cells.     

Sex Chromosome Meiotic Drive in DROSOPHILA MELANOGASTER Males

In Drosophila melanogaster males, deficiency for X heterochromatin causes high X-Y nondisjunction and skewed sex chromosome segregation ratios (meiotic drive). Y and XY classes are recovered poorly because of sperm dysfunction. In this study it was found that X heterochromatic deficiencies disrupt recovery not only of the Y chromosome but also of the X and autosomes, that both heterochromatic and euchromatic regions of chromosomes are affected and that the "sensitivity" of a chromosome to meiotic drive is a function of its length. Two models to explain these results are considered. One is a competitive model that proposes that all chromosomes must compete for a scarce chromosome-binding material in Xh(-) males. The failure to observe competitive interactions among chromosome recovery probabilities rules out this model. The second is a pairing model which holds that normal spermiogenesis requires X-Y pairing at special heterochromatic pairing sites. Unsaturated pairing sites become gametic lethals. This model fails to account for autosomal sensitivity to meiotic drive. It is also contradicted by evidence that saturation of Y-pairing sites fails to suppress meiotic drive in Xh(- ) males and that extra X-pairing sites in an otherwise normal male do not induce drive. It is argued that meiotic drive results from separation of X euchromatin from X heterochromatin.     

Studying Trans-Effects of Modifiers on the Position-Effect Variegation in a Set of Euchromatin–Heterochromatin Rearrangements in Drosophila melanogaster

The effects of suppressors of position-effect variegation were studied in a set of euchromatin–heterochromatin rearrangements of the X chromosome accompanied by inactivation of the gene wapl.The rearrangements differed from one another in the size of the heterochromatic block adjacent to euchromatin, with the euchromatin–heterochromatin border remaining unchanged. In one rearrangement (r20), the position effect caused by a small block of adjacent heterochromatin may be determined by its interaction with the neighboring main heterochromatic region of the X chromosome. Chromosome 3 (the RT chromosome) was found to have a strong suppressing effect on all rearrangements, irrespective of the amount of heterochromatin adjacent to euchromatin. Su-var(3)9, a known suppressor of the position-effect variegation, had a considerably weaker suppressing effect. The RT chromosome had the strongest suppressing effect on the rearrangement r20.     

MU2 and HP1a regulate the recognition of double strand breaks in Drosophila melanogaster

Chromatin structure regulates the dynamics of the recognition and repair of DNA double strand breaks; open chromatin enhances the recruitment of DNA damage response factors, while compact chromatin is refractory to the assembly of radiation-induced repair foci. MU2, an orthologue of human MDC1, a scaffold for ionizing radiation-induced repair foci, is a widely distributed chromosomal protein in Drosophila melanogaster that moves to DNA repair foci after irradiation. Here we show using yeast 2 hybrid screens and co-immunoprecipitation that MU2 binds the chromoshadow domain of the heterochromatin protein HP1 in untreated cells. We asked what role HP1 plays in the formation of repair foci and cell cycle control in response to DNA damage. After irradiation repair foci form in heterochromatin but are shunted to the edge of heterochromatic regions an HP1-dependent manner, suggesting compartmentalized repair. Hydroxyurea-induced repair foci that form at collapsed replication forks, however, remain in the heterochromatic compartment. HP1a depletion in irradiated imaginal disc cells increases apoptosis and disrupts G2/M arrest. Further, cells irradiated in mitosis produced more and brighter repair foci than to cells irradiated during interphase. Thus, the interplay between MU2 and HP1a is dynamic and may be different in euchromatin and heterochromatin during DNA break recognition and repair.     

Scanning Electron Microscopy of Heterochromatin in Chromosome Spreads of Male Germ Cells in Schistocerca gregaria (Acrididae, Orthoptera) after Trypsinization

Chromosome spreads, prepared from testes of the desert locust Schistocerca gregaria, were analyzed using scanning electron microscopy (SEM) after varying periods of preincubation in trypsin. The emphasis of the study was on the appearance of heterochromatin. A trypsin pretreatment of 5 sec resulted in a smooth surface on the chromatin throughout and the heterochromatin was highly electron-emissive. The facultatively heterochromatic X chromosome was clearly visible in interphase spermatogonia and in pachytene and late prophase I spermatocytes. Chromomeres of autosomal bivalents could be recognized in pachytene cells. Centromeric heterochromatin segments were very prominent in autosomes of late prophase I spermatocytes and some chromosomes showed interstitial and telomeric bands. Longer trypsin treatment (10 sec) resulted in a fine globular surface on the chromatin; however, the electron emission of heterochromatic chromosome segments was lower under these conditions. The result of trypsin pretreatment of euchromatin differed only slightly from that of the heterochromatin. Extensive trypsin treatment (20 sec) did not alter further the relative electron emission of heterochromatin and euchromatin, but the regular globular appearance was lost, apparently owing to damage on the chromatin surface. The loss of electron emission from the centromeric heterochromatin of the autosomes and the facultatively heterochromatic X chromosome after extended trypsin treatment suggests a central role of proteins in mediating the heterochromatic status in meiotic chromo somes of the locust. Information obtained using scanning electron microscopy of chromosome spreads is complementary to that obtained by C-banding in that facultative heterochromatin is visualized with particular clarity.     

Alpha and beta heterochromatin in polytene chromosome 2 ofDrosophila melanogaster

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization with Rsp sequences (H _o clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed. Received: 17 November 1995; in revised form: 10 April 1996 / Accepted: 18 September 1996     

Trans-effect of modifiers on position-effect variegation in a set of euchromatin-heterochromatin rearrangements in Drosophila melanogaster]

The effects of suppressors of position-effect variegation were studied in a set of euchromatin-heterochromatin rearrangements of the X chromosome accompanied by inactivation of the gene wapl. The rearrangements differed from one another in the size of the heterochromatic block adjacent to euchromatin, with the euchromatin-heterochromatin border remaining unchanged. In one rearrangement (r20), the position effect caused by a small block of adjacent heterochromatin may be determined by its interaction with the neighboring main heterochromatic region of the X chromosome. Chromosome 3 (the RT chromosome) was found to have a strong suppressing effect on all rearrangements, irrespective of the amount of heterochromatin adjacent to euchromatin. Su-var(3)9, a known suppressor of the position-effect variegation, had a considerably weaker suppressing effect. The RT chromosome had the strongest suppressing effect on the rearrangement r20.     

Equal induction and persistence of chromosome aberrations involving chromosomes 1, 4 and 10 in thyroid cancer patients treated with radioactive iodine

A number of in vitro studies have questioned the assumption of random distribution of breaks in radiation-induced chromosome aberrations. The therapeutic application of radioactive 131I in thyroid cancer patients offers a good opportunity to study the induction and persistence of cytogenetic damage involving different chromosomes in vivo. Using whole-chromosome painting probes and triple colour painting by fluorescence in situ hybridization (FISH), we have analysed the frequency of chromosomal aberrations (CAs) involving chromosomes 1, 4 and 10 in peripheral blood lymphocytes of 10 thyroid cancer patients sampled before and 1 week, 1 year and 3.5 years after therapeutic application of radioactive iodine in a self-controlled, longitudinal study. A highly significant 3.4-fold increase in the frequency of chromosome breaks was observed 1 week after treatment with a similar representation of all chromosomes analysed. Although a significant decrease in dicentrics was observed during the first year after treatment, the frequency of chromosome aberrations remained over control levels until the last sampling time, 41-47 months post-treatment. The same behaviour, in terms of induction and persistence, was observed for all three chromosomes, confirming our previous results in vitro and rejecting the reported suggestion that chromosome 10 is radiosensitive in vivo. Our finding that the dynamics of radiation-induced CA in vivo is independent on the chromosome of choice suggests that this variable is not important in retrospective studies.     

Different DNA-PKcs functions in the repair of radiation-induced and spontaneous DSBs within interstitial telomeric sequences

Interstitial telomeric sequences (ITSs) in hamster cells are hot spots for spontaneous and induced chromosome aberrations (CAs). Most data on ITS instability to date have been obtained in DNA repair-proficient cells. The classical non-homologous end joining repair pathway (C-NHEJ), which is the principal double strand break (DSB) repair mechanism in mammalian cells, is thought to restore the morphologically correct chromosome structure. The production of CAs thus involves DNA-PKcs-independent repair pathways. In our current study, we investigated the participation of DNA-PKcs from the C-NHEJ pathway in the repair of spontaneous or radiation-induced DSBs in ITSs using wild-type and DNA-PKcs mutant Chinese hamster ovary cells. Our data demonstrate that DNA-PKcs stabilizes spontaneous DSBs within ITSs from the chromosome 9 long arm, leading to the formation of terminal deletions. In addition, we show that DNA-PKcs-dependent C-NHEJ is employed following radiation-induced DSBs in other ITSs and restores morphologically correct chromosomes, whereas DNA-PKcs independent mechanisms co-exist in DNA-PKcs proficient cells leading to an excess of CAs within ITSs.     

Differences in the meiotic pairing behavior of gonosomal heterochromatin between female and male Microtus agrestis: implications for the mechanism of heterochromatin amplification on the X and Y

It is generally thought that pairing and recombination between the X and Y chromosome in eutherian mammals is important for the occurrence of normal meiotic division and the production of functional gametes. Microtus agrestis is one of the examples whose giant and heterochromatin-rich sex chromosomes fail to establish a durable association at any stage of the first meiotic division in males. In contrast, in females, synapsis starts in the euchromatic short arm and pairing progresses unidirectionally and continues until both X chromosomes have paired completely, as can be demonstrated by the use of fluorescence in situ hybridization with a sequence confined to the non-centromeric, gonosomal heterochromatin. However, compared with euchromatin, this association is apparently ephemeral and breaks off precociously in the pachytene and metaphase I stages. We demonstrate that a middle repetitive element is localized interspersed in the noncentromeric heterochromatin of both X and Y, except the telomeric region of the Y. No differences could be detected at the molecular level between male and female DNA, indicating that at least the bulk of these elements are organized in the same manner on the X and Y. Our data imply that the loss of synapsis and recombination between the X and Y might have preceded the process of heterochromatin amplification in the course of Microtinae evolution. Since asynapsed elements are particularly susceptible to DNA strand breaks during prophase I, DNA repair of double-strand breaks involving heterochromatic segments of the X and Y could have resulted in translocations of larger segments from the X to the Y or vice versa during the course of chromosome evolution of the gonosomes, explaining the homology at the molecular level between the heterochromatin of the asynaptic X and Y in M. agrestis.     

Spatial distribution of sperm-derived chromatin in zygotes determined by fluorescence in situ hybridization.

Fluorescence in situ hybridization was used to determine the spatial distribution of chromatin in zygote pronuclei. A hybrid system involving golden hamster eggs and individual human sperm permitted use of DNA probes specific for the entire human chromosome 4, for the heterochromatic region on the long arm of the human Y chromosome and for unique DNA sequences on human chromosome 19. Chromosome 4 occupied a circumscribed domain in the pronuclei, similar to findings in somatic interphases. Unlike the situation in somatic interphases, the Y heterochromatin was extended throughout the first cell cycle. Pronuclear chromatin was extended 3- to 4-fold compared to somatic interphase chromatin. The extended pronuclear chromatin conformation is likely to affect a zygote's susceptibility to environmental hazards.     

C-bands on chromosomes of four beetle species (Coleoptera: Carabidae, Silphidae, Elateridae, Scarabaeidae)

The C-banding pattern of Bembidion geniculatum, Silpha atrata, Prosternon tesselatum, and Epicometis hirta are presented. All analysed species have pracentromeric C-bands on autosomes and chromosome X but the widest ones are visible in the karyotype of B. geniculatum. In S. atrata, P. tesselatum, and E. hirta sex chromosome y is heterochromatic, only B. geniculatum having the Y chromosome wholly euchromatin. The results indicate that on the chromosomes of the investigated species do not have a terminal and an intercalar segments of heterochromatin.     

Intragenomic Distribution and Stability of Transposable Elements in Euchromatin and Heterochromatin of Drosophila melanogaster: Elements with Inverted Repeats Bari 1, hobo, and pogo

The elements of the Bari 1, hobo, and pogo transposon families that are located in euchromatin, heterochromatin, and on the Y chromosome have been identified, and their stability has been assessed by Southern blot analysis. The fraction of heterochromatic elements appears to be distinctive of all transposon families tested, except for Bari 1. Evidence for instability of heterochromatic elements is described. The analysis of unstable elements in different Drosophila stocks suggests that the host genome contributes to the stability/instability of transposon families. Received: 21 August 1996 / Accepted: 24 March 1997     

Differential reaction of condensed and diffuse chromatin to polyamines. II. Effect of putrescine on the chromatin of mitotic chromosomes]

The response of euchromatin and heterochromatin to putrescine was studied using chinese hamster mitotic chromosomes with heterochromatic segments delayed in condensation due to BUdR treatment. These heterochromatic segments did not react to the condensing effect of putrescine looking during metaphase still more elongated. Additional decondensed segments occurred in chromosomes. This is interpreted as the condensing effect of putrescine on euchromatic segments which accelerates transition of the cells into metaphase with preserved BUdR-induced chromosomal decondensation.