DNA in the centromeric heterochromatin of polytene chromosomes is topologically open

Heterochromatin differs from euchromatin by a set of specific features. We suggested earlier that specific features of heterochromatin result from differences in DNA topology of these two chromatin types and provided explanations for the majority of them (Gruzdev 2000). We proposed that, unlike topologically closed euchromatic DNA, the DNA of heterochromatin is topologically open, i.e. it likely contains single- or doublestrand breaks. In this work, we studied the topological state of DNA in a block of centromeric heterochromatin and in a euchromatic banded region of Chironomus melanotus polytene chromosomes by microfluorimetric methods using the fluorescent intercalating dye ethidium bromide (EB). It was demonstrated that the fraction of topologically closed DNA in heterochromatin blocks is five-fold smaller than in the banded region. The data obtained support the hypothesis proposed.     

Analysis of the morphology of short arms of human acrocentric chromosomes by sequential staining for G and C banding

Sequential staining of acrocentric chromosomes from eight individuals for G and C banding showed that a large heterochromatin region is present more often in homologs of chromosome 15 than of 13 and 14, and in the G group it is found more often in pair 22 than in pair 21. As a rule the size of the heterocheomatin region does not correspond to the size of the short arm of the acrocentrics when stained for G banding. The frequency of occurrence of satellites in all eight individuals was approximately the same for all five pairs of acrocentrics. Staining for constitutive heterochromatin revealed heteromorphism for the presence of satellites frequently in the homologs.Academic Group of Academician of the Academy of Medical Sciences of the USSR A. I. Nesterov, Institute of Rheumatism, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. I. Nesterov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 10, pp. 1267–1269, October, 1976.     

Identification of human G–group and Y chromosomes. Demonstration of constitutive heterochromatin by modified DNA/RNA hybridization technique

Treatment of standard human chromosomal preparations with NaOH and standard saline–citrate demonstrated constitutive heterochromatin. It is localized near the centromere or within the centromeric region in all except the Y chromosome. The technique was used for identification of G and Y chromosomes. In karyotypes of individuals with trisomy G1 (Down's syndrome) chromosomes number 21 (in the trisomy stage) possessed heterochromatin in different distribution from number 22 chromosomes; the latter having a distinctly dark stained centromere. The centromere, short arms, and proximal portion of the long arms of the Y chromosome were free of constitutive heterochromatin, which was demonstrated occupying the distal portion of the long arms. The extent of heterochromatin of the Y chromosome was identical with the heterochromatin demonstrated by quinacrine fluorochromes and responsible for the fluorescent "Y body."     

Pattern of Chromosomal Localization of the Hoppel Transposable Element Family in the Drosophila melanogaster Subgroup

We have isolated a Hoppel-like transposon from heterochromatin of the second chromosome of Drosophila melanogaster and used a conserved DNA sequence between the different elements of this family to determine their distribution in both mitotic and polytene chromosomes. The hybridization pattern of polytene chromosomes extends throughout the entire chromocentre, as well as a substantial portion of the fourth chromosome. Analysis of different wild-type strains of D. melanogaster shows variation in euchromatic insertion sites, although most insertions are found near the chromocentre. The positions and the number of heterochromatic clusters of Hoppel on mitotic chromosomes are conserved among the several strains analysed. Accurate mapping of this element was achieved by in situ hybridization on D. melanogaster mitotic chromosomes that had previously been banded with Hoechst 33258. To evaluate the evolutionary stability of this pattern, different species were analysed by in situ hybridization and Southern blotting. We conclude that Hoppel has a conserved distribution in mitotic heterochromatin within the D. melanogaster subgroup, established around 5 million years ago. The overall conservation of heterochormatic organization supports the notion that heterochormatin does perform important structural and functional roles.     

Marker chromosome identification by micro-FISH

Micro-FISH was used to elucidate the chromosomal origin of marker chromosomes in three patients. Ten copies of marker chromosomes were collected with microneedles from GTG banded metaphases, transferred to a collecting drop and amplified by means of DOP-PCR. The PCR products were labeled with biotin-14-dATP and used as FISH probes for hybridization to normal metaphase chromosomes and to metaphase chromosomes of the patients (reverse painting). With the generation of chromosome region-specific painting probes by PCR amplification of microdissected DNA and subsequent FISH it was possible to identify the marker chromosomes in all patients. One marker appeared to be derived from the centromere region of the X-chromosome and the proximal third of the long arm, one from the centromere region of chromosome 17 and one marker chromosome was identified as an isochromosome 18p.     

Chromosome abnormalities involving heterochromatic regions in monocytic leukemia

We report two cases of monocytic leukemia associated with cytogenetic changes involving the juxtacentromeric heterochromatin of different chromosomes. In a patient with chronic myelomonocytic leukemia (CMMoL) we describe a translocation t(1;9)(q12;q13) in which the duplicated derivative chromosome 9q + showed a huge centromeric C-band, derived by fusion of the heterochromatic regions of chromosomes 1 and 9. The constitutional karyotype showed two heterochromatin polymorphisms, 1qh + and inv(9qh). In the second case, an acute monoblastic leukemia was associated with an abnormally elongated juxtacentromeric heterochromatic region of chromosome 4 that was not constitutionally present.     

A Novel Interspersed Type of Organization of Satellite DNAs in Tribolium Madens Heterochromatin

Analysis of arrangement of satellite DNA sequences in Tribolium madens (Insecta, Coleoptera) by Southern analysis of pulsed-field blots and two colour FISH on extended chromosomes and DNA fibres revealed a novel type of heterochromatin organization. Two satellite DNAs, distributed over the whole pericentromeric heterochromatin of all chromosomes form clusters, ranging in size from 150 kb up to several Mb. Within the clusters, both satellites are in the form of highly interspersed, short homogeneous arrays which vary in size with a lowest length limit of only few kb. The longest arrays composed of a single satellite are relatively short, up to 70 kb for satellite I, and up to 45 kb for satellite II. Only a small fraction of about 15% of satellite II is organized in long tandem repeats, while the rest is in the form of only a few repeats intermingled with satellite I. The results indicate that large clusters composed of interspersed arrays of both satellites represent a major component of T. madens heterochromatin, which is mostly devoid of long regions of other sequences. The same organizational pattern probably also includes a region of the functional centromere. We propose that such an organizational pattern of DNA sequences in heterochromatin might be common in genomes characterized by a high rate of interchromosomal exchange. This pattern of organization is different from that in other animal as well as plant species analysed up to now, in which every satellite in heterochromatin is organized in a small number of large separate domains. This revised version was published online in August 2006 with corrections to the Cover Date.     

Constitutive heterochromatin of chromosomes No. 1, 9, and 16 in 90 patients with malignant disease and 91 controls

The variability of constitutive heterochromatin in chromosomes #1, #9, and #16 was investigated in 90 patients with malignant tumors (neoplasms of the head, neck, breast, and genital organs) and in 91 controls. The investigation was carried out on chromosome preparations from peripheral blood cultures. An analysis showed no significant difference in the frequency of localization variants, that is, inversions, in the patients and in the normal controls. Analysis of the variability of the C-segment size was made by measuring the length of heterochromatin blocks and showed that a correlation with the process of malignant transformation can be sought in the consequences of an uneven distribution (chromosome #1) and in quantitative changes in the constitutive heterochromatin (chromosomes #1 and #9).     

New C band markers of human chromosomes: C band position variants.

Although it is well known that variations in size are common in the centric heterochromatin of human chromosomes, it has not been appreciated that centric heterochromatin may be positioned either entirely on the short arm, centrally, or entirely on the long arm. Inspection of C banded karyotypes in which chromosomes were identified by previous Q banding showed C band position variants in all of the chromosomes of groups A, B, C, E, and F. These 'new' C band markers were followed in families and found to be heritable. By scoring chromosomes for both size and position of C bands it should be possible to distinguish the majority of homologous chromosomes in most people. The ease of scoring and high frequency of variation should make C band position variants extremely useful in linkage studies and population surveys.     

Loss of the Y chromosome PAR2 region and additional rearrangements in two familial cases of satellited Y chromosomes: cytogenetic and molecular analysis

Two cases of rare structural aberrations of the Y chromosome were detected: a del(Y) (q12) chromosome in a child with mild dysmorphic features, obesity and psychomotor delay, and two identical satellited Y chromosomes (Yqs) in a normal twin, which were originally observed during routine prenatal diagnosis. In both cases a Yqs chromosome was detected in the father which had arisen from a reciprocal translocation involving the short arm of chromosome 15 and the heterochromatin of the long arm of the Y chromosome (Yqh). Cytogenetic and molecular studies demonstrated that in the reciprocal product of chromosomes 15 and Y PAR2 could not be detected, showing that PAR2 had been deleted. It is discussed whether the translocation of the short arm of an acrocentric chromosome to the heterochromatin of the long arm of the Y chromosome causes instability of this region which results either in loss of genetic material or interference with the normal mechanism of disjunction.     

C heterochromatin variation in couples with recurrent early abortions.

The possible influence of the high polymorphic C heterochromatic regions of human chromosomes 1, 9, 16, and Y on meiotic chromosome segregation was investigated. Faulty chromosome segregation may be the result of either an abnormal quantity of C heterochromatin on the homologues, or disequilibrium between the homologues. The aim of our study was to determine whether either a variation in the amounts of total C heterochromatin or differences in the amounts of C heterochromatin between homologues could lead to faulty chromosome segregation. The study was performed on C banded metaphases obtained from peripheral lymphocyte cultures of 15 couples with recurrent early abortions and 15 control couples, all Caucasians. Analysis of variance was first performed on separate metaphases to measure intra-individual, inter-individual, and between population variation in a hierarchical model. Since the significant intra-individual differences covered the other parameters we performed, secondly, a one way analysis of variance on the mean values of metaphases per person in order to measure the inter-individual and between population variation. The results did not show a relationship between C heterochromatin lengths and occurrence of recurrent abortions.     

Polymorphic variants in human chromosome 15

We found eight polymorphic variants in human chromosome 15 using Q, C, Q-C and Ag-NOR staining methods. These variants included brightly or dully fluorescent pericentric segments and satellites, giant satellites, increased amounts of short arm heterochromatin (ph+) and darkly (C band-positive) or lightly (C band-negative) Giemsa-stained pericentric Q-negative segments. These staining properties indicated that the entire short arm of 15 contained at least four distinct chromatin segments: Q-negative centrometric heterochromatin, a Q-vriable distal segment, a Q-negative satellite stalk, and Q-variable satellites, in that order, from proximal to distal ends. The Brd U-Hoechst 33258-stained R bands (RBH) and high resolution G subbands were also studied for karyologic characterization of chromosome 15. Most of these variants were reported also in 13, but insufficiently documented in other D and G chromosomes. Together with polymorphic pericentric fluorochromes seen in 3 and 4, Yq, and nonpathogenic t(D;Yq), the pattern of these variants can be used as karyologic fingerprints for identification of each individual and his or her cell explants both in vivo and in vitro.     

Cytomolecular characterization and origin of de novo formed maize B chromosome variants

B chromosomes are dispensable elements that occur in many species, including maize. The maize B chromosome is acrocentric and highly heterochromatic and undergoes nondisjunction during the second pollen mitosis. In this study, we determined the genetic behavior and organization of two naturally occurring B chromosome variants (designated B^ta and B^tb). The morphology and genetic behavior of the B^ta chromosome were similar to those of the typical B chromosome, but the B^ta chromosome contained a deletion in the first heterochromatin region and had higher transmission frequencies through both male and female parents. The B^tb chromosome was reduced in size, consisted primarily of heterochromatin, and had a lower transmission frequency. The B^tb chromosome lacked nondisjunctional behavior, which was restored by the presence of normal B chromosomes in the cell. Furthermore, the B^tb chromosome contained two centromeric regions, only one of which was active. The organization of these two naturally occurring B chromosome variants was also determined using fluorescence in situ hybridization with B-associated sequences and by amplification of B-specific molecular markers to create possible evolutionary models.     

C-band polymorphism: Comparison between trisomy 21 cases and mentally retarded controls

C-banding was done prospectively on 50 Down syndrome (trisomy 21) cases and 50 mentally retarded controls. Heterochromatin was quantitated by measuring the lengths of heterochromatin blocks and comparing these segments to the length of the short arm of chromosome 16 for 1, 9 and 16 heterochromatin, and to the total length of the Y chromosome for the Y heterochromatin in the distal long arm. For the first 30 individuals in each group, there was no difference in the mean lengths of C-band blocks of the 1, 9, 16 and Y chromosomes. For the total sample, there also was no difference between the trisomy 21 cases and controls in the number or size of pericentric inversions involving the heterochromatin blocks of chromosomes 1 and 9. Assuming random segregation of the parental C-band polymorphisms, this study gives no evidence for an association between such polymorphisms of the 1, 9, 16 and Y chromosomes and nondisjunction of chromosome 21.     

Histone H3 phosphorylation of mammalian chromosomes

Inferences about the role and location of phosphorylated histone H3 are derived primarily from biochemical studies. A few direct observations at chromosome level have shown that phosphorylation begins at the site of heterochromatin and spreads throughout the chromosome. However, a comparative study of chromosomes of mouse (L929 cells), Chinese hamster (CHO 9 cells) and the Indian muntjac (male cells) reveals some distinguishable details among mammalian species. Whereas the L929 cells exhibit the typical pattern of phosphorylation at the region of centromeric heterochromatin associated with the active centromere, the heterochromatin blocks associated with the inactive centromeres also show label of about equivalent intensity. Throughout the cell cycle, heterochromatin exhibits sharper (denser) and better defined label than does euchromatin which expresses somewhat diffuse label. The centromere constriction on biarmed chromosomes, originating in Robertsonian translocations, appears phosphorylated in some, if not all chromosomes. A similar situation was found for the CHO 9 cells indicating that phosphorylation does include the region in which H3 is supposedly replaced by CENP-A. An interesting feature of the CHO cell line was the dense label at and near the telomeres; this feature was not observed in either the mouse or the Indian muntjac. The centromere regions of the Indian muntjac chromosomes showed three sites of label in the multicentric X chromosome and two each on chromosome pair number 1 and Y₂; the sites coinciding with the reaction sites of antikinetochore antibodies. Also, the X and Y₁ chromosomes of Indian muntjac show intense phosphorylation at the sites of secondary constrictions.The chromosomes of all three species were phosphorylated throughout the cell cycle. As the chromosomes started to decondense during anaphase, heavy phosphorylation was observed in the form of discontinuous beaded structures indicating partial despiralization of the chromosome. Interestingly, when cells had completed karyokinesis and resolved into two independent nuclei, the phosphorylation was observed at the midbody. At this stage, the cytoplasm appeared to be again phosphorylated.     

Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH)

Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes; (c) absence of hybridization to the ribosomal gene probes; (d) C-band-positive centromeric regions; and (e) euchromatic arms. They differ as follows: (a) the larger B element hybridizes to the centromeric heterochromatin (pMeg-1) probe whereas the smaller B element does not; (b) the amount of C-band-positive material is reduced in the smaller B chromosome relative to that present on the larger B chromosome; and (c) the smaller element is reduced in size by about a third. It is concluded that the larger B chromosome arose as a leftover centromere from centric fusion, whereas the smaller element has a different origin — perhaps as an intact fragment or as an amplified region from the A chromosomes. The presence of euchromatic regions on B chromosomes may account for their survival in the karyotype.This revised version was published online in November 2005 with corrections to the Cover Date.     

Chromatin preferences of the perichromosomal layer constituent pKi-67

The proliferation-associated nuclear protein pKi-67 relocates from the nucleolus to the chromosome surface during the G2/M transition of the cell cycle and contributes to the formation of the perichromosomal layer. We investigated the in-vivo binding preferences of pKi-67 for various chromatin blocks of the mitotic chromosomes from the human and two mouse species, Mus musculus and M. caroli. All chromosomes were decorated with pKi-67 but displayed a gap of pKi-67 decoration in the centromere and NOR regions. pKi-67 distribution in a rearranged mouse chromosome showed that the formation of the centromeric gap was controlled by the specific chromatin in that region. While most chromatin served as a substrate for direct or indirect binding of pKi-67, we identified three types of chromatin that bound less or no pKi-67. These were: (1) the centromeric heterochromatin defined by the alpha satellite DNA in the human, by the mouse minor satellite in M. musculus and the 60- and 79-bp satellites in M. caroli; (2) the pericentromeric heterochromatin in M. musculus defined by the mouse major satellite, and (3) NORs in the human and in M. musculus defined by rDNA repeats. In contrast, the conspicuous blocks of pericentromeric heterochromatin in human chromosomes 1, 9 and 16 containing the 5-bp satellite showed intense pKi-67 decoration. The centromeric gap may have a biological significance for the proper attachment of the chromosomes to the mitotic spindle. In this context, our results suggest a new role for centromeric heterochromatin: the control of the centromeric gap in the perichromosomal layer.     

Cytogenetics of the genus Leporinus (Pisces, Anostomidae). 1.Karyotype analysis, heterochromatin distribution and sex chromosomes

Cytogenetic analyses (Giemsa staining, C-banding, AgNO3 labelling of nucleolus organizer regions (NORs) and staining with base-specific fluorochromes) were performed on the South American fish species Leporinus friderici, L. obtusidens and L. elongatus. The overall karyotypic structure, position of NORs, as well as the amount,distribution and composition of constitutive heterochromatin were determined. Particular attention was given to the highly differentiated ZZ/ZW sex chromosome system of L. obtusidens and L. elongatus. Sharing the apparently ancient macroscopic karyotype of Anostomidae, all three species have 2n=54 meta- or submetacentric chromosomes. NORs were found exclusively on chromosome pair 2, which may represent the ancestral NOR-bearing chromosome of the anostomid karyotype. Observed differences in the relative position of NORs along chromosome 2 and variations in the amount and distribution of constitutive heterochromatin throughout the karyotype were most probably caused by heterochromatin-mediated chromosome rearrangements. Detailed analysis of the morphologically similar heteromorphic ZZ/ZW sex chromosomes of L. obtusidens and L. elongatus allowed detection of differences in the DNA composition of the largely heterochromatic W chromosomes. However, since these and the W chromosomes of three other Leporinus species exhibit homologies with respect to their relative size, centromere position and amount and distribution of heterochromatin, it is concluded that they evolved from the same ancestral W chromosome. This revised version was published online in August 2006 with corrections to the Cover Date.     

Disclosure of five breakpoints in a complex chromosome rearrangement by microdissection and FISH.

Microdissection and fluorescence in situ hybridisation (FISH) were used to elucidate the nature of a complex chromosome translocation, after GTG banding failed in the complete characterisation of the structural rearrangement between chromosomes 6 and 12. These chromosomes were painted with chromosome specific paints and one of the chromosome regions involved in the translocation was isolated by microdissection. Ten copies of the microdissected region were collected with microneedles from GTG banded metaphases, transferred to a collecting drop, and amplified by means of DOP-PCR. The PCR product was labelled with biotin-14-dATP and used as a FISH probe for hybridisation to normal metaphase chromosomes and metaphase chromosomes of the patients (microFISH). FISH with this chromosome region specific painting probe and with chromosome band specific probes enabled the characterisation of a complex chromosome rearrangement with five breakpoints in two chromosomes. This resulted in the following karyotype: 46,XY,t(6;12)(6pter--> 6q12::12q24.1-->12qter;12qter-->12q13.3:: 6q16.2-->6q26::12q13.3-->12q24.1::6q12--> 6q16.2::6q26-->6qter).     

Constitutional C-band pattern in patients with adenomatosis of the colon and rectum

The pattern of polymorphism in the C-band-positive constitutive heterochromatin of chromosomes #1, #9, and #16 was studied in fibroblasts from 23 unrelated patients with adenomatosis of the colon and rectum and in peripheral lymphocytes from 78 control persons. The parameters of the heterochromatic regions analyzed were relative size, symmetry-asymmetry within homologous chromosome pairs, and frequency of inversions. The polyposis coli patients had a significantly higher frequency (p less than 0.05) of partial and total heterochromatin inversion on chromosome #9 than the control group (37.0% compared with 21.8%). In the other parameters studied, no significant differences were found between patients and controls.