Cytosystematics of five North American Dermacentor (Acari: Ixodidae) species.

The karyotypes of five species of Dermacentor ticks found in North America were examined for the presence and location of constitutive heterochromatin (C-bands) and the location of the nucleolar organizing regions (NORs). All specimens examined had 20 autosomes and an XX:XO sex-determining system; the X was the largest element in the karyotype. Except as noted, the autosomes of all species were acrocentric, and heterochromatin was restricted to the centromeric region. The one-host species D. nitens and D. albipictus were distinguished from the three-host species by a lack of additional bands of heterochromatin and by having a subtelocentric X chromosome. However, some individuals of D. albipictus were observed to have acrocentric X chromosomes. D. nitens differed from D. albipictus in having two subtelocentric autosomal pairs (pairs 1 and 8). A single pair of NORs was noted for the one-host species; however, heterochromatin was not associated with the NORs. Among the three-host species, D. occidentalis showed a band of heterochromatin proximal to the centromere in autosomal pairs 1 and 8, and D. andersoni had similar bands in autosomal pairs 1, 3, 7, and 9. D. variabilis displayed heterochromatic bands in autosomal pairs 3, 7, and 9. All three-host species displayed a single pair of NORs, which was associated with the band of heterochromatin on pair 9. Indications of some genetic incompatibility between the two recognized forms of D. albipictus are discussed.

Centromeric heterochromatin instability of chromosomes 1, 9, and 16 in variable immunodeficiency syndrome--a virus-induced phenomenon?

Centromeric instability of chromosomes 1, 9, and 16 has been described in eight patients with variable immunodeficiency. Although the pathogenetic relationship of these cytogenetic abnormalities with the clinical symptoms are unclear, it has nevertheless been proposed that they are a hallmark of this syndrome. Based on the clinical, immunological and cytogenetic data from the literature, a model is presented suggesting that the cytogenetic changes are not causatively involved in the immunodeficiency syndrome, but result from specific virus infections occurring as a consequence of the immunodeficiency in genetically predisposed individuals.

Distribution of chromosome 18 and X centric heterochromatin in the interphase nucleus of cultured human cells.

In situ hybridization of human chromosome 18 and X-specific alphoid DNA-probes was performed in combination with three dimensional (3D) and two dimensional (2D) image analysis to study the interphase distribution of the centric heterochromatin (18c and Xc) of these chromosomes in cultured human cells. 3D analyses of 18c targets using confocal laser scanning microscopy indicated a nonrandom disposition in 73 amniotic fluid cell nuclei. The shape of these nuclei resembled rather flat cylinders or ellipsoids and targets were preferentially arranged in a domain around the nuclear center, but close to or associated with the nuclear envelope. Within this domain, however, positionings of the two targets occurred independently from each other, i.e., the two targets were observed with similar frequencies at the same (upper or lower) side of the nuclear envelope as those on opposite sides. This result strongly argues against any permanent homologous association of 18c. A 2D analytical approach was used for the rapid evaluation of 18c positions in over 4000 interphase nuclei from normal male and female individuals, as well as individuals with trisomy 18 and Bloom's syndrome. In addition to epithelially derived amniotic fluid cells, investigated cell types included in vitro cultivated fibroblastoid cells established from fetal lung tissue and skin-derived fibroblasts. In agreement with the above 3D observations 18c targets were found significantly closer (P less than 0.01) to the center of the 2D nuclear image (CNI) and to each other in all these cultures compared to a random distribution derived from corresponding ellipsoid or cylinder model nuclei. For comparison, a chromosome X-specific alphoid DNA probe was used to investigate the 2D distribution of chromosome X centric heterochromatin in the same cell types. Two dimensional Xc-Xc and Xc-CNI distances fit a random distribution in diploid normal and Bloom's syndrome nuclei, as well as in nuclei with trisomy X. The different distributions of 18c and Xc targets were confirmed by the simultaneous staining of these targets in different colors within individual nuclei using a double in situ hybridization approach.

CENP-B: a major human centromere protein located beneath the kinetochore.

The family of three structurally related autoantigens CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD) are the best characterized components of the human centromere, and they have been widely assumed to be components of the kinetochore. Kinetochore components are currently of great interest since this structure, which has long been known to be the site of microtubule attachment to the chromosome, is now believed to be a site of force production for anaphase chromosome movement. In the present study we have mapped the distribution of CENP-B in mitotic chromosomes by immunoelectron microscopy using two monospecific polyclonal antibodies together with a newly developed series of ultra-small 1-nm colloidal gold probes. We were surprised to find that greater than 95% of CENP-B is distributed throughout the centromeric heterochromatin beneath the kinetochore. This strongly supports other emerging evidence that CENP-B is specifically associated with alpha-satellite heterochromatin. Although in certain instances CENP-B can be seen to be concentrated immediately adjacent to the lower surface of the kinetochore, the outer plate remains virtually unlabeled. Similar analysis with a human autoimmune serum that recognizes all three CENP antigens reveals an additional unsuspected feature of kinetochore structure. In addition to recognizing antigens in the centromeric heterochromatin, the autoantiserum recognizes a concentration of antigens lateral to the kinetochore. This difference in staining pattern may reflect the presence of a "collar" of chromatin rich in CENP-C and/or CENP-A encircling the kinetochore plates.

Characterization of a tandemly repeated DNA from the fleshfly Sarcophaga bullata.

In studies on the highly repetitive DNA sequences of the flesh fly Sarcophaga bullata, a 279 bp tandem repeat was cloned and sequenced. A 17 bp stretch within the clone was identical to a motif repeated five times in the satellite DNA of the Bermuda land crab. Southern DNA blotting showed the tandem repeat had a high degree of conservation of MboI sites, but had divergence for EcoRI sites; thus, all repeat units were not identical. The cloned DNA localized to the quinacrine-bright centromeric heterochromatin of the C and E autosomes and to sites on the chromosomal arms. In cases of asynapsis of homologs, the probe localized to euchromatic sites on both homologs or sometimes only on one homolog. The probe also localized near, to, or at a major developmental puff (B9). We conclude that blocks of this short interspersed repetitive DNA occur throughout the Sarcophaga genome in both heterochromatin and euchromatin, and also that the variable position of these sequences suggests they possess a degree of instability.

Heteropycnosis of an underreplicating chromosome.

Drosophila nasutoides has an extraordinary genome since 62% of its DNA resides in chromosome 4. This element mainly consists of constitutive heterochromatin which does not polytenize. Earlier studies of heterochromatin attributed little attention to the fact that "condensed" chromosomes often vary in condensation. This paper reports that chromosomes of the same complement display different degrees and kinetics of condensation. In D. nasutoides, even sex specific differences can be observed. The results of a comparative microphotometric study on neuroblast metaphases in both sexes revealed the following picture. The process of chromosome condensation is not restricted to mitotic prophase but continues into the metaphase. The mean condensation is not equal for all chromosomes. In the metaphase of the female, Feulgen density increases from the X chromosome, via 3 and 2, to chromosome 4. In the male, the order is X, 2, 3, Y, and 4. During the metaphase of the male, chromosomes condense with similar kinetics. In contrast, chromosomes of the female display asynchrony as monitored by area and length determinations. The X chromosomes of the female probably have enhanced shortening during prophase. This would explain the metaphase of the female where the X chromosomes shorten less than the autosomes, and why each of the X chromosomes is 15% shorter than the X chromosome in the metaphase of the male. Further differences were observed in the longitudinal and lateral compaction of the chromosomes in males and females. The sex chromosomes and chromosome 3 condense by shortening, while chromosome 2 and 4 preferentially reduce their diameter. The large amount of DNA engaged in heteropycnosis and the isochromosome nature allow the identification of chromosome 4 during interphase. At this stage, a new category of extreme DNA packaging was detected. The interphase density of chromosome 4 can exceed that of metaphase by a factor of up to 8. Two events account for this high degree of condensation: (1) the homologues are particularly associated due to somatic pairing and (2) the arms are further tightened as a result of pericentric folding. The features of the isochromosome suggest that the interaction of chromatids during interphase is essentially caused by specific DNA sequences. The data confirm that heteropycnosis not only interferes with gene expression but also strongly inhibits DNA synthesis in endocycles.

Differences in the organization and chromosomal allocation of satellite DNA between the European long tailed house mice Mus domesticus and Mus musculus.

We compared the organization of satellite DNA (stDNA) and its chromosomal allocation in Mus domesticus and in Mus musculus. The two stDNAs show similar restriction fragment profiles after digestion (probed with M. domesticus stDNA) with some endonucleases of which restriction sequences are present in the 230-240 bp repetitive unit of the M. domesticus stDNA. In contrast, EcoRI digestion reveals that M. musculus stDNA lacks most of the GAATTC restriction sites, particularly at the level of the half-monomer. The chromosome distribution of stDNA (revealed by an M. domesticus stDNA probe) shows different patterns in the M. domesticus and M. musculus karyotypes, with about 60% of M. domesticus stDNA retained in the M. musculus genome. It is particularly noteworthy that the pericentromeric regions of M. musculus chromosomes 1 and X are totally devoid of M. domesticus stDNA sequences. In both groups, the differences in energy transfer between the stDNA-bound fluorochromes Hoechst 33258 and propidium iodide suggest that AT-rich repeated sequences have a much more clustered array in the M. domesticus stDNA, as if they are organized in tandem repeats longer than those of M. musculus. Considering the data as a whole, it seems likely that the evolutionary paths of the two stDNAs diverged after the generation of the ancestral 230-240 bp stDNA repetitive unit through the amplification, in the M. domesticus genome, of a family repeat which included the EcoRI GAATTC restriction sequence.

Heterochromatin heterogeneity in Drosophila chromosomes detected by AluI-banding.

In situ selective digestion of mitotic chromosomes from three Drosophila species (virilis, hydei, and funebris), having a chromosome number 2n = 12, was achieved with AluI restriction endonuclease. The distribution of AluI-bands, revealing a heterogeneity within heterochromatin, was compared with that of quinacrine-bands observed in standard chromosomes. The results confirmed a species-specific AluI-banding pattern, heterochromatin being selectively digested in D. virilis and D. funebris, but unaffected by the enzyme in D. hydei. The overlapping of AluI-bands and Q-bands in D. virilis and in D. funebris is discussed, since these data seem to favour the hypothesis that the induction of AluI-bands does not depend only on the presence of enzyme targets, but also on a suitable conformation of the chromatin.

Cytochemical evaluation of C-heterochromatic-DNA in metaphase chromosomes.

A method is proposed to evaluate the amount of DNA resistant to the C-banding pretreatments (C-heterochromatic-DNA) in metaphase chromosomes. Measurements were performed by microfluorometry on propidium iodide stained metaphases of man, gorilla and mouse; in these species, C-heterochromatin exhibits significant differences of both base composition and distribution along the chromosomes. The amount of C-heterochromatic-DNA was found to be about 16%, 28% and 58% of the total DNA content (genome size) in man, gorilla and mouse, respectively. The areas of C-bands after Giemsa staining were also assessed by microdensitometry, and corresponded to about 8%, 15% and 14% of the total karyotype area of man, gorilla and mouse respectively. No direct relation thus exists between C-band areas and the amount of DNA resistant to the C-banding pretreatments. In man and gorilla, the amount of C-heterochromatic-DNA accounts for the differences observed in genome size.

[Monoclonal antibodies IGR to nuclear antigens of neutrophilic granulocytes of human blood]. / Monoklonal'nye antitela IGR k iadernym antigenam neitrofil'nykh granulotsitov krovi cheloveka.

Four monoclonal antibodies (Mabs) of series IGR-1 and IGR-2 to nuclear antigens of neutrophilic granulocytes of human peripheral blood were obtained. Mabs IGR-1 2B8 and IGR-1 6B5 are bound to their specific antigens in the nuclei of all the investigated human cell lines. These Mabs were also specific for metaphase chromosomes of cell lines HL-60 and U-937. Investigations on the ultrastructural level showed that Mabs IGR-1 6B5 reacted with the HL-60 nuclear heterochromatin region. Mabs IGR-1 3D3 and IGR-2 2F1 manifested high specificity only for the nuclei of mature neutrophils and of plasma cells.

Characterization of the heterochromatin in moose (Alces alces) chromosomes.

The karyotype of moose (2n = 68) is characterized by very large C-bands close to the centromeres of most chromosomes. The C-banded material represents 40% of the genome. For further characterization of the heterochromatin chromosome spreads were treated with restriction endonucleases and the restriction enzyme (Re) banding pattern was analyzed. HaeIII, AluI, MboI, RsaI and HinfI produced informative Re-bands. DdeI induced an even digestion with no banding. Staining with chromomycin A3 produced bright fluorescence in regions corresponding to C-bands. Labeling with BrdUrd during late S phase differentiates four regions in the C banded area. The sequence of these regions from centromere to telomere are: late, early, late and early replicating. The authors propose the existence of five satellite DNA families with distinctive characteristics of G-C and A-Trichness and different replication timing, and point out the different clusters for the endonucleases detailed above and their varying location in the chromosomes examined.

Variability of euchromatic and heterochromatic regions of Y chromosome in two types of cancer patients.

Heteromorphism of Y chromosome was studied in head and neck cancer patients and leukemia patients. The results were compared with similar data obtained for healthy men. It was observed that, compared to the controls, mean lengths of Y chromosome were nonsignificantly higher for leukemia patients and lower for head and neck cancer patients. The euchromatic region of Y chromosome (Y-eu) remained comparable in the controls and the leukemia patients, whereas it was smaller in patients with head and neck malignancies. The heterochromatic region (Y-het) was more or less analogous in controls and head and neck cancer patients, however, it was significantly larger in patients with leukemia (P less than 0.02).

Mapping the Drosophila genome with yeast artificial chromosomes.

The ability to clone large fragments of DNA in yeast artificial chromosomes (YAC's) has created the possibility of obtaining global physical maps of complex genomes. For this application to be feasible, most sequences in complex genomes must be able to be cloned in YAC's, and most clones must be genetically stable and colinear with the genomic sequences from which they originated (that is, not liable to undergo rearrangement). These requirements have been met with a YAC library containing DNA fragments from Drosophila melanogaster ranging in size up to several hundred kilobase pairs. Preliminary characterization of the Drosophila YAC library was carried out by in situ hybridization of random clones and analysis of clones containing known sequences. The results suggest that most euchromatic sequences can be cloned. The library also contains clones in which the inserted DNA is derived from the centromeric heterochromatin. The locations of 58 clones collectively representing about 8 percent of the euchromatic genome are presented.

Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila.

We have previously reported the identification of a nonhistone chromosomal protein (nhcp-19; now called HP1) preferentially associated with the heterochromatin of Drosophila melanogaster. A detailed study of the HP1 distribution pattern on polytene chromosomes by immunofluorescent staining, using monoclonal antibody C1A9, has been carried out. The results indicate that this protein is found within the centric beta-heterochromatin, in cytological regions 31, 41 and 80, and throughout polytene chromosome 4. Staining of telomeres is frequently observed, those of chromosome arms 2R and 3R and the X chromosome being the most conspicuous. Analysis of a fourth chromosome insertional translocation T(3;4)f/In(3L)P confirms an autonomous interaction with chromosome 4 material. Similarly, the beta-heterochromatin distal to light on chromosome arm 2L, moved to position 97D2 on chromosome arm 3R in the rearrangement ltx13, is prominently stained using the C1A9 antibody. Staining of intact salivary glands indicates that this rearranged segment of beta-heterochromatin is not associated with the polytene chromocenter, but provides an independent structural reference point. HP1 is not observed in the nuclei of the early syncytial embryo, but becomes concentrated in the nuclei at the syncytial blastoderm stage (ca. nuclear division cycle 10). This suggests that heterochromatin formation occurs at approximately the same stage at which nuclei first become transcriptionally competent. Thus, the C1A9 antibody may serve as a useful marker for both structural and functional studies of the Drosophila nucleus.

[Specificity of the location of human chromosomes in the nucleus of a moving cell]. / Spetsifichnost' raspolozheniia khromosom cheloveka v iadre dvizhushcheisia kletki.

Data are presented on the distribution of centromeric heterochromatin of the human X-chromosome in the interphase nucleus of a moving cell. The in situ hybridization made it possible to obtain some results leading to the following conclusions: in moving fibroblasts centromeric heterochromatin of the X-chromosome is located in end regions of the interphase nucleus; there was no preferential localization noted of the centromeric region of the X-chromosome in the front or back areas of the nucleus as to the direction of the movement.

Histone variants in mouse centromeric chromatin.

Highly purified centromeric heterochromatin was isolated from mouse liver nuclei and the pattern of core histone variants was analyzed. In comparison with total chromatin, the centromeric heterochromatin of young animals was characterized by (1) enrichment in the replication-dependent variants H2A1, H2B2 and H3(2), (2) reduced amount of the minor variant H2Az and (3) absence of ubiquitinated molecules of H2A. This specific variant pattern changed upon ageing as a result of accumulation of replacement variants so that in adult animals both chromatin preparations exhibited similar pattern for H2A and H2B, while the difference in the profile of H3 variants was preserved.

[Changes in the properties of intercalary heterochromatin in Drosophila melanogaster induced by position effect modifiers]. / Izmenenie proiavleniia svoistv interkaliarnogo geterokhromatina u Drosophila melanogaster pod vliianiem modifikatorov éffekta polozheniia.

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.

Position effect variegation in Drosophila melanogaster: relationship between suppression effect and the amount of Y chromosome.

Position effect variegation results from chromosome rearrangements which translocate euchromatic genes close to the heterochromatin. The euchromatin-heterochromatin association is responsible for the inactivation of these genes in some cell clones. In Drosophila melanogaster the Y chromosome, which is entirely heterochromatic, is known to suppress variegation of euchromatic genes. In the present work we have investigated the genetic nature of the variegation suppressing property of the D. melanogaster Y chromosome. We have determined the extent to which different cytologically characterized Y chromosome deficiencies and Y fragments suppress three V-type position effects: the Y-suppressed lethality, the white mottled and the brown dominant variegated phenotypes. We find that: (1) chromosomes which are cytologically different and yet retain similar amounts of heterochromatin are equally effective suppressors, and (2) suppression effect is positively related to the size of the Y chromosome deficiencies and fragments that we tested. It increases with increasing amounts of Y heterochromatin up to 60-80% of the entire Y, after which the effect reaches a plateau. These findings suggest suppression is a function of the amount of Y heterochromatin present in the genome and is not attributable to any discrete Y region.

Histone H1 in the centromeric heterochromatin of Glyptotendipes barbipes larval polytene chromosomes.

Immunofluorescent analysis with antibodies against histone H1 failed to detect H1 in the centromeric heterochromatin blocks of the polytene chromosomes of Glyptotendipes barbipes larvae. Centromeric regions were dissected microsurgically and acid-extracted. Electrophoresis in SDS and acid-urea gels revealed a band comigrating with H1 of calf thymus and of Gl. barbipes salivary gland nuclei. ELISA dot assay of the extracted material gave a positive reaction with anti-H1 monoclonal antibodies and with anti-H1 affinity-purified polyclonal antibodies. This shows that the centromeric heterochromatin contains histone H1 but packed in a way which prevents the H1 antigenic determinants from reacting in situ with the specific antibodies.

Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography.

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.