'Glowing' chromosomes in cells undergoing rapid division

Previous investigations in which metaphase plates of cells in rapid division were incubated in phosphate buffer at high temperature revealed numerous heterochromatic dots in chromosomes after Giemsa staining. In contrast, chromosomes from cells with a reduced capacity for reproduction were devoid of such dots, or the dots were sloughed-off into rings and patches of heterochromatin. In two types of cells which were rapidly dividing, namely HeLa cells (cervical cancer) and cells from regenerating planaria, phosphate incubation followed by Giemsa staining revealed an 'aura' or 'glowing' effect on the chromosomes, consisting of a densely staining core surrounded by a lightly stained periphery. This finding might be developed into a diagnostic test for certain malignancies, for cells undergoing dedifferentiation, or for tissues undergoing regeneration.     

Further studies on chromocentres and their implications in regeneration

Previous investigations using a number of invertebrates, as well as regenerative tissues/organs of various vertebrates, have promulgated the hypothesis that heterochromatin, in the form of nuclear chromocentres, is correlated with the ability to regenerate. In order to test the universality of this hypothesis, cells from a variety of additional animals were examined for the presence of nuclear chromocentres. In accordance with the hypothesis, cells from these organisms contained numerous chromocentres. Evidence indicates that chromocentres, double minute chromosomes, chromosome 'dots', and telomeres may be different forms of the same heterochromatin entity.     

Translocation of unstable heterochromatin as the mechanism of sister chromatid exchange formation: a proposed hypothesis

The phenomenon of sister chromatid exchange has remained an enigma in that the actual mechanism for its formation has never been elucidated. It has long been suspected that the process involves some form of breakage and rejoining of DNA, but that hypothesis has never been proved. Recent work in this laboratory using cells from a premature aging disorder (Werner's syndrome) has promulgated the hypothesis that heterochromatin may not be an integral structure of chromosomes, but rather serves as a surface feature or covering. Furthermore, heterochromatin in Werner's syndrome chromosomes was found to be unstable and easily sloughed-off the chromosome surface. In this investigation evidence is presented which shows that incorporation of bromodeoxyuridine into DNA causes instability in the purported heterochromatin covering, resulting in translocation of segments of heterochromatin from the unifilarly-substituted chromatid to the bifilarly-substituted sister chromatid. Such translocation may represent the long-elusive mechanism of sister chromatid exchange formation.     

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.     

The quinacrine-fluorescence patterns of the chromosomes of Allium carinatum

Quinacrine staining of somatic chromosomes in Allium carinatum shows intense fluorescence patterns which allow their recognition and the study of their degree of heterozygosity. This makes possible the study of chromosome polymorphism at a level until now impossible to achieve. The intense fluorescence patterns correspond to heterochromatic segments visible as darkly stained regions in prophase chromosomes. Interphase nuclei show fluorescent chromocentres of the same size and distribution as in conventionally stained preparations, and there is a good correlation between intense fluorescence patterns, late replicating DNA and heterochromatin.     

Critical electrolyte concentration of the heterochromatin and euchromatin of Triatoma infestans

The binding of toluidine blue molecules under Mg2+ competitive staining conditions was investigated in chromocentres and the euchromatin of single- and multi-chromocentred nuclei of Triatoma infestans Malpighian tubule cells. It was demonstrated that the chromocentre of single-chromocentred nuclei exhibited the largest critical electrolyte concentration (CEC) value (0.4 M), followed by the chromocentres of multi-chromocentred nuclei (0.3 M) and the euchromatin (0.2 M). The differences in CEC values were assumed to be due to differences in availability of free DNA phosphates and in packing states of the DNA-protein complexes of these chromatin types. Differences in chromatin supra-organization were evident for the chromocentral heterochromatin of single vs multi-chromocentred nuclei. This was also valid for the chromocentral heterochromatin in some multi-chromocentred nuclei, when one of the heterochromatic bodies was especially larger than the others.     

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.     

Cytogenetic Analysis of the Second Chromosome Heterochromatin of Drosophila Melanogaster

This paper reports the cytogenetic characterization of the second chromosome heterochromatin of Drosophila melanogaster. High resolution cytological analysis of a sample of translocations, inversions, deficiencies and free duplications involving the pericentric regions of the second chromosome was achieved by applying sequential Hoechst 33258 and N-chromosome banding techniques to larval neuroblast prometaphase chromosomes. Heterochromatic rearrangements were employed in a series of complementation assays and the genetic elements previously reported to be within or near the second chromosome heterochromatin were thus precisely assigned to specific heterochromatic bands. The results of this analysis reveal a nonhomogeneous distribution of loci along the second chromosome heterochromatin. The l(2)41Aa, l(2)41Ab, rolled (l(2)41Ac) and l(2)41Ad loci are located within the proximal heterochromatin of 2R, while the nine remaining loci in the left arm and two (l(2)41Ae and l(2)41Ah) in the right arm map to h35 and to h46, respectively, the most distal heterochromatic regions. In addition, a common feature of these loci revealed by the cytogenetic analysis is that they map to specific heterochromatic blocks but do not correspond to the blocks themselves, suggesting that they are not as large as the Y fertility factors or the Rsp locus. Mutations of the proximal most heterochromatic loci, l(2)41Aa and rolled, were also examined for their phenotypic effects. Extensive cell death during imaginal disc development was observed in individuals hemizygous for either the EMS 31 and rolled mutations, leading to a pattern of phenotypic defects of adult structures.     

Genome and chromosome structure: Twelve dynamic and evolving genomes

Chromosomes are not inert structures that haul the genome through cell division. The dynamic properties of chromosomes, during the cell cycle, the lifetime of the organism and across evolutionary time, featured prominently at the 49(th) Annual Drosophila Research Conference. Platform presentations, workshops and posters focused on many aspects of chromosome structure and function including chromosome interactions such as trans-silencing and pairing between homologous and non-homologous chromosomes, specialized portions of the chromosome including the centromere and telomeres, the structure, function and evolution of the large heterochromatic domains such as the Y and 4(th) chromosomes, centric heterochromatin and subtelomeric heterochromatin. The speed of evolutionary changes in these regions, and the consequences for speciation and hybrid-incompatibility, were recurring themes. Finally, there was considerable new insight offered into the mechanics by which chromosomes are rearranged and changes in the types of alterations occurring over the lifetime of the organism, which can result in novel genes and gene flow between chromosomes. The availability of the twelve sequenced Drosophila genomes has allowed new insights into the structure, function and evolutionary transformation of chromosomes and genomes that will continue to transform our view of the chromosome as a dynamic and flexible entity that houses and regulates the genome.     

Genome and chromosome structure: Twelve dynamic and evolving genomes

Chromosomes are not inert structures that haul the genome through cell division. The dynamic properties of chromosomes, during the cell cycle, the lifetime of the organism and across evolutionary time, featured prominently at the 49th Annual Drosophila Research Conference. Platform presentations, workshops and posters focused on many aspects of chromosome structure and function including chromosome interactions such as trans-silencing and pairing between homologous and non-homologous chromosomes, specialized portions of the chromosome including the centromere and telomeres, the structure, function and evolution of the large heterochromatic domains such as the Y and 4th chromosomes, centric heterochromatin and subtelomeric heterochromatin. The speed of evolutionary changes in these regions, and the consequences for speciation and hybrid-incompatibility were recurring themes. Finally, there was considerable new insight offered into the mechanics by which chromosomes are rearranged and changes in the types of alterations occurring over the lifetime of the organism, which can result in novel genes and gene flow between chromosomes. The availability of the twelve sequenced Drosophila genomes has allowed new insights into the structure, function and evolutionary transformation of chromosomes and genomes that will continue to transform our view of the chromosome as a dynamic and flexible entity that houses and regulates the genome.     

A distal heterochromatic block displays centromeric activity when detached from a natural centromere

We repeatedly released a distal block of heterochromatin lacking a natural centromere in mitotic cells and assayed its segregation. At anaphase, control acentric fragments typically remained unoriented between daughter nuclei and were subsequently lost. Fragments containing the brownDominant (bWD) heterochromatic element displayed regular anaphase movement upon release. These fragments were found to segregate and function based on both cytological and phenotypic criteria. We also found that intact bWD-containing chromosomes normally display occasional dicentric behavior, suggesting that bWD has centromeric activity on the intact chromosome as well. Our findings suggest that centromere competence is innate to satellite-containing blocks of heterochromatin, challenging models for centromere identity in which competence is an acquired characteristic.     

The structure of heterochromatic DNA is altered in polyploid cells of Drosophila melanogaster.

DNA sequences within heterochromatin are often selectively underrepresented during development of polyploid chromosomes, and DNA molecules of altered structure are predicted to form as a consequence of the underrepresentation process. We have identified heterochromatic DNAs of altered structure within sequences that are underrepresented in polyploid cells of Drosophila melanogaster. Specifically, restriction fragments that extend into centric heterochromatin of the minichromosome Dp(1;f)1187 are shortened in polyploid cells of both the ovary and salivary gland but not in the predominantly diploid cells of the embryo or larval imaginal discs and brains. Shortened DNA molecules were also identified within heterochromatic sequences of chromosome III. These results suggest that the structure of heterochromatic DNA is altered as a general consequence of polyploid chromosome formation and that the shortened molecules identified form as a consequence of heterochromatic underrepresentation. Finally, alteration of heterochromatic DNA structure on Dp(1;f)1187 was not correlated with changes in the variegated expression of the yellow gene located on the minichromosome.     

Intranuclear destruction of heterochromatin in two species of armored scale insects

Spermatogenesis is described in two species of armored scale insects,Parlatoria proteus andParlatoria ziziphus. In the males of both species, a haploid set of four chromosomes becomes heterochromatic during early embryogeny. The heterochromatic chromosomes are lost later by two different mechanisms during spermatogenesis. Just before meiosis begins one or more heterochromatic chromosomes disappear from each primary spermatocyte as a consequence of a rapid intranuclear chromosome destruction. Meiosis consists of a single achiasmatic division. At prophase four euchromatic and from one to three heterochromatic chromosomes are present in each cell. Although both the euchromatic and remaining heterochromatic chromosomes divide, the heterochromatic chromosomes are later eliminated by posttelophase ejection; the eliminated chromosomes then disintegrate slowly in the cytoplasm. Each of the two species displays a species specific level of heterochromatin retention and both differ in this regard from the previously describedParlatoria oleae. The evolution of a chromosome system involving intranuclear chromosome destruction is discussed.     

Werner's syndrome: A review of recent research with an analysis of connective tissue metabolism,growth control of cultured cells,and chromosomal aberrations

Summary Werner's syndrome is a rare, autosomal recessive condition with multiple progeroid features, but it is an imitation of aging rather than accelerated or premature senescence. Somatic chromosome aberrations occur in multipe tissues in vivo and in vitro, and there is an increased incidence of neoplasia. Thus, Werner's syndrome can be classified in the group of chromosome instability syndromes. Recent findings provide additional support for the concept that there is aberration of connective tissue metabolism in Werner's syndrome, but it is unclear whether this is a primary or secondary manifestation of the underlying genetic defect. Abnormal growth characteristics are observed in cultured skin fibroblast-like cells and this provides another avenue for current research. Identification of the basic genetic defect in Werner's syndrome might clarify our understanding of the normal aging process in general, or might elucidate specific aspects such as the development of neoplasia, atherosclerosis, diabetes, or osteoporosis.     

Nuclear structure and gene activity in human differentiated cells

The nuclear arrangement of the ABL, c-MYC, and RB1 genes was quantitatively investigated in human undifferentiated HL-60 cells and in a terminally differentiated population of human granulocytes. The ABL gene was expressed in both cell types, the c-MYC gene was active in HL-60 cells and down-regulated in granulocytes, and expression of the RB1 gene was undetectable in HL-60 cells but up-regulated in granulocytes. The distances of these genes to the nuclear center (membrane), to the center of the corresponding chromosome territory, and to the nearest centromere were determined. During granulopoesis, the majority of selected genetic structures were repositioned closer to the nuclear periphery. The nuclear reposition of the genes studied did not correlate with the changes of their expression. In both cell types, the c-MYC and RB1 genes were located at the periphery of the chromosome territories regardless of their activity. The centromeres of chromosomes 8 and 13 were always positioned more centrally within the chromosome territory than the studied genes. Close spatial proximity of the c-MYC and RB1 genes with centromeric heterochromatin, forming the chromocenters, correlated with gene activity, although the nearest chromocenter of the silenced RB1 gene did not involve centromeric heterochromatin of chromosome 13 where the given gene is localized. In addition, the role of heterochromatin in gene silencing was studied in retinoblastoma cells. In these differentiated tumor cells, one copy of the RB1 gene was positioned near the heterochromatic chromosome X, and reduced RB1 gene activity was observed. In the experiments presented here, we provide evidence that the regulation of gene activity during important cellular processes such as differentiation or carcinogenesis may be realized through heterochromatin-mediated gene silencing.     

Heterochromatin and rDNA sites distribution in the holocentric chromosomes of Cuscuta approximata Bab. (Convolvulaceae).

Cuscuta is a widely distributed genus of holoparasitic plants. Holocentric chromosomes have been reported only in species of one of its subgenera (Cuscuta subg. Cuscuta). In this work, a representative of this subgenus, Cuscuta approximata, was investigated looking for its mitotic and meiotic chromosome behaviour and the heterochromatin distribution. The mitotic chromosomes showed neither primary constriction nor Rabl orientation whereas the meiotic ones exhibited the typical quadripartite structure characteristic of holocentrics, supporting the assumption of holocentric chromosomes as a synapomorphy of Cuscuta subg. Cuscuta. Chromosomes and interphase nuclei displayed many heterochromatic blocks that stained deeply with hematoxylin, 4',6-diamidino-2-phenylindole (DAPI), or after C banding. The banded karyotype showed terminal or subterminal bands in all chromosomes and central bands in some of them. The single pair of 45S rDNA sites was observed at the end of the largest chromosome pair, close to a DAPI band and a 5S rDNA site. Two other 5S rDNA site pairs were found, both closely associated with DAPI bands. The noteworthy giant nuclei of glandular cells of petals and ovary wall exhibited large chromocentres typical of polytenic nuclei. The chromosomal location of heterochromatin and rDNA sites and the structure of the endoreplicated nuclei of C. approximata seemed to be similar to those known in monocentric nuclei, suggesting that centromeric organization has little or no effect on chromatin organization.     

Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.