Heterochromatin In Centromeric Regions Research Articles

Oligonucleotide skews enable comprehensive and insightful characterization of GC- and TA-skew properties observed throughout the human genome with support of unsupervised AI with reference to gene- and Alu-polarity skews

GC and TA skew are fundamental genomic features observed widely from bacteria to humans. They have been described as replication- and transcription-coupled skews because they are mainly caused by differences in mutation and repair biases between complementary DNA strands in replication and transcription processes. The skews are clearly observed in bacteria, plants and birds, but are more difficult to accurately identify in mammals, including humans. The main reason for this difficulty is that the correlation between GC and TA skew is not high for their variation in the mammalian genomes. In this study, we focused not only on these mononucleotide skews but also on di- and trinucleotide skews, first in 100-kb sequences of two short human chromosomes (chr21 and 22), and by using the conventional distribution map analysis, we clearly observed these skews over these chromosomes, except for centromeric heterochromatin regions where diverse repetitive sequences are densely clustered. Then, by using a batch learning self-organizing map (BLSOM), which can display large amounts of sequence data at once, we detected the oligonucleotide skews across nearly the entire human genome. Next, to understand skews within centromeric heterochromatin regions, we focused on Alu sequences ubiquitously distributed in the human genome and analyzed mono-, di-, and trinucleotide skews by referring to the polarity of Alu sequences. Since this polarity is unevenly distributed in the human genome, a clear TA skew was observed by reflecting the evident A-richness to T of Alu elements. However, due to some degree of their G-richness to C, TA and GC skews showed inversely correlated variations, when analyzing Alu sequences alone. Notably, when we analyzed genome sequences after excluding Alu sequences, the concordant correlation between GC- and TA-skew variations increased, indicating that a large amount of Alu sequences has made the detection of these mononucleotide skews in the human genome more difficult than in other organisms. Analysis of oligonucleotide skews using BLSOM is thought to be suitable for characterizing sequence features in skew transition regions.

Cytogenetic Analysis of Panaqolus tankei Cramer & Sousa, 2016 (Siluriformes, Loricariidae), an Ornamental Fish Endemic to Xingu River, Brazil

Despite conservation of the diploid number, a huge diversity in karyotype formulae is found in the Ancistrini tribe (Loricariidae, Hypostominae). However, the lack of cytogenetic data for many groups impairs a comprehensive understanding of the chromosomal relationships and the impact of chromosomal changes on their evolutionary history. Here, we present for the first time the karyotype of Panaqolus tankei Cramer & Sousa, 2016. We focused on the chromosomal characterization, using conventional and molecular cytogenetic techniques to unravel the evolutionary trends of this tribe. P. tankei, as most species of its sister group Pterygoplichthini, also possessess a conserved diploid number of 52 chromosomes. We observed heterochromatin regions in the centromeres of many chromosomes; pairs 5 and 6 presented interstitial heterochromatin regions, whereas pairs 23 and 24 showed extensive heterochromatin regions in their q arms. In situ localization of 18S rDNA showed hybridization signals correlating with the nucleolus organizer regions, which are located in the q arms of pair 5. However, the 5S rDNA was detected in the centromeric and terminal regions of the q arms of pair 8. (TTAGGG)_n hybridized only in the terminal regions of all chromosomes. Microsatellite in situ localization showed divergent patterns, (GA)₁₅ repeated sequences were restricted to the terminal regions of some chromosomes, whereas (AC)₁₅ and (GT)₁₅ showed a scattered hybridization pattern throughout the genome. Intraspecific comparative genomic hybridization was performed on the chromosomes of P. tankei to verify the existence of sex-specific regions. The results revealed only a limited number of overlapping hybridization signals, coinciding with the heterochromatin in centromeric regions without any sex-specific signals in both males and females. Our study provides a karyotype description of P. tankei, highlighting extensive differences in the karyotype formula, the heterochromatin regions, and sites of 5S and 18S rDNA, as compared with data available for the genus.

Banded karyotype of Nelore cattle (Bos taurus indicus Linnaeus, 1758).

Chromosome banding techniques were applied and standardized to obtain karyotype characteristics for the first time in Brazil of Nelore cattle – Bos taurus indicus Linnaeus, 1758 – (bovine subspecies most prominent in Brazilian livestock). Blood samples were collected from the animals of the School of Agrarian and Biological Sciences of the Pontifical Catholic University of Goiás, two males and two females of pure breed. These samples were submitted to the cell culture method to study metaphase chromosomes. Chromosome banding techniques (C, G and NOR) revealed the karyotype architecture of Nelore cattle common with that of other breeds of zebu cattle formerly karyotyped. The diploid chromosome number was invariably normal, 2n = 60. C-banding revealed C-positive heterochromatin in centromeric regions almost in all chromosomes. G-banding presented the expected band pattern in the respective chromosome pairs in correspondence with the established chromosomal patterns for the species. Ag-staining for nucleolus organizer regions (AgNOR) was identified on the telomeric end of the long arm in 7 autosomal chromosomes. In this study we found more regions in chromosomes with staining than presented in the literature for the Bos indicus group (BIN). These NOR regions were repeated on the same chromosomes for the 4 animals studied.

Karyotype of mitotic metaphase and meiotic diakinesis in non-heading Chinese cabbage

Non-heading Chinese cabbage [Brassica rapa L. ssp. chinensis (L.) Hanelt] is one of the most popular leafy vegetables. Despite the economic importance of non-heading Chinese cabbage, little attention has been given to its cytogenetic profile. This study reveals the karyotype of non-heading Chinese cabbage. Fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes was performed on mitotic metaphase complementary regions. We located 45S rDNA on the centromeric or adjacent region of chromosomes A1 and A2, with the largest on the satellite of chromosome A5. Meanwhile, 5S rDNA co-localized with 45S rDNA on chromosomes A2 and A5, and on the telomeric region of chromosome A10. We performed DAPI fluorescence banding on the same metaphase chromosomes to identify homologous chromosomes. The DAPI fluorescence pattern was observed mainly on the centromeric heterochromatin regions of each chromosome. However, the lengths of chromosomes A2 and A6 were completely stained, except for their telomeric regions. Meiotic diakinesis chromosomes as new substrates in FISH-developed karyotype were revealed for the first time. The karyotype of non-heading Chinese cabbage reveals that it contains eight submetacentric chromosomes, one subtelocentric chromosome (bearing satellite), and one telocentric chromosome. Diakinetic chromosome pairing can overcome the difficulty of unlabeled chromosome identification. This study provided valuable information for cytogenetic research and molecular breeding of non-heading Chinese cabbage by using the combination of FISH and DAPI fluorescence patterns on mitotic and meiotic chromosomes.

C-heterochromatin variation and NOR distribution in the karyotype of water vole, Arvicola terrestris (Mammalia, Rodentia)

A chromosomal study of populations of Arvicola terrestris from Anatolia in Turkey and from Cen- tral Europe was performed. The diploid number of 36 chromosomes was found in all the specimens examined. The autosomal complement consisted of 12 meta- and submetacentric pairs, two large or medium-sized subtelo- centric pairs, and three small acrocentric (Turkey) or subtelocentric (Central Europe) pairs (FNa = 62-68). The X chromosome was medium-sized submetacentric, the Y chromosome was small acrocentric or subtelocentric. All the chromosomes could be reliably identifi ed by their unique G-banding patterns. The C-banding analysis revealed variation in the amount of constitutive heterochromatin in centromeric regions and in short arms of certain autosomes. A unique feature of the C-banded karyotype of individuals from Anatolia was the absence of dark positive regions in most chromosomes. Populations of water vole from Anatolia resemble in their C-band pattern those studied previously in Azerbaijan, and possibly also in the Balkan peninsula, and they are different in this respect from populations in Central Europe and the other parts of the species range. The X chromosome was stained uniformly and C-negatively in populations from Anatolia, whereas a faint dark centromeric C-band was observed in individuals from Central Europe. The Y chromosome was stained C-positively. The active nu- cleolar organizer regions (NORs) were localized in one pair of small metacentric and two acrocentric autosome pairs in the karyotype of individuals from Anatolia.

Karyotype, chromosomal characteristics of multiple rDNA clusters and intragenomic variability of ribosomal ITS2 in Caryophyllaeides fennica (Cestoda)

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A chromosome study and localization of 18S rDNA in Khawia saurogobii (Cestoda: Caryophyllidea)

This paper reports results of the first cytogenetic study carried out on a recently described monozoic tapeworm, Khawia saurogobii Xi et al., 2009, from the Chinese lizard gudgeon (Saurogobio dabryi). The karyotype of this species is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16; n = 3m + 5t), metacentric chromosomes representing the first, sixth, and eight pairs. All chromosomes except the largest pair displayed 4',6-diamidino-2-phenylidole (DAPI) positive heterochromatin in centromeric regions. In mitotic preparations stained with Giemsa, one of the homologues of a smaller metacentric chromosome pair (No. 7) showed a distinct secondary constriction, whereas the other did not. Fluorescent in situ hybridization (FISH) with 18S ribosomal DNA (rDNA) probe revealed that the chromosomes No. 7 carry each a cluster of ribosomal genes associated with the centromeric heterochromatin and confirmed that this chromosome pair contains a nucleolar organizer region (NOR). The rDNA-FISH also confirmed heteromorphism in the size of NOR (i.e., secondary constriction) observed after Giemsa staining. The present cytogenetic analysis revealed species-specific characters of K. saurogobii and showed that FISH may represent a new valuable cytogenetic tool suitable for comparative taxonomic or phylogenetic studies within the order Caryophyllidea in the future.

Mapping of the juxtacentromeric heterochromatin-euchromatin frontier of human chromosome 21

Euchromatin and heterochromatin are functional compartments of the genome. However, little is known about the structure and the precise location of the heterochromatin-euchromatin boundaries in higher eukaryotes. Constitutive heterochromatin in centromeric regions is associated with (1) specific histone methylation patterns, (2) high levels of DNA methylation, (3) low recombination frequency, and (4) the repression of transcription. All of this contrasts with the permissive structure of euchromatin found along chromosome arms. On the sequence level, the transition between these two domains consists most often of patchworks of segmental duplications. We present here a comprehensive analysis of gene expression, DNA methylation in CpG islands, distribution of histone isoforms, and recombination activity for the juxtacentromeric (or pericentromeric) region of the long arm of human chromosome 21. We demonstrate that most HapMap data are reliable within this region. We show that high linkage disequilibrium between pairs of SNPs extends 719-737 kb from the centromeric alpha-satellite. In the same region we find a peak of histone isoforms H3K9Me3 and H3K27Me (715-822 kb distal to the alpha-satellite). In normal somatic cells, CpG islands proximal to this peak are highly methylated, whereas distal CpG islands are not or very little methylated. This methylation profile undergoes dramatic changes in cancer cells and during spermatogenesis. As a consequence, transcription from heterochromatic genes is activated in the testis, and aberrant gene activation can occur during neoplastic transformation. Our data indicate that the frontier between the juxtacentromeric heterochromatic domain and euchromatic domain of the long arm of chromosome 21 is marked by a heterochromatic peak located approximately 750 kb distal to the alpha-satellite.

Chromosomal distribution and organization of three cervid satellite DNAs in Chinese water deer (Hydropotes inermis)

The species-specific profile and centromeric heterochromatin localization of satellite DNA in mammalian genomes imply that satellite DNA may play an important role in mammalian karyotype evolution and speciation. A satellite III DNA family, CCsatIII was thought to be specific to roe deer (Capreolus capreolus). In this study, however, this satellite DNA family was found also to exist in Chinese water deer (Hydropotes inermis) by PCR-Southern screening. A satellite III DNA element of this species was then generated from PCR-cloning by amplifying this satellite element using primer sequences from the roe deer satellite III clone (CCsatIII). The newly generated satellite III DNA along with previously obtained satellite I and II DNA clones were used as probes for FISH studies to investigate the genomic distribution and organization of these three satellite DNA families in centromeric heterochromatin regions of Chinese water deer chromosomes. Satellite I and II DNA were observed in the pericentric/centric regions of all chromosomes, whereas satellite III was distributed on 38 out of 70 chromosomes. The distribution and orientation of satellite DNAs I, II and III in the centromeric heterochromatin regions of the genome were further classified into four different types. The existence of a Capreolus-like satellite III in Chinese water deer implies that satellite III is not specific to the genus Capreolus (Buntjer et al., 1998) and supports the molecular phylogeny classification of Randi et al. (1998) which suggests that Chinese water deer and roe deer are closely related.

Monitoring dynamic protein interactions with photoquenching FRET

The mammalian cell nucleus is a dynamic and highly organized structure. Most proteins are mobile within the nuclear compartment, and this mobility reflects transient interactions with chromatin, as well as network interactions with a variety of protein partners. To study these dynamic processes in living cells, we developed an imaging method that combines the photoactivated green fluorescent protein (PA-GFP) and fluorescence resonance energy transfer (FRET) microscopy. We used this new method, photoquenching FRET (PQ-FRET), to define the dynamic interactions of the heterochromatin protein-1 alpha (HP1alpha) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) in regions of centromeric heterochromatin in mouse pituitary cells. The advantage of the PQ-FRET assay is that it provides simultaneous measurement of a protein's mobility, its exchange within macromolecular complexes and its interactions with other proteins in the living cell without the need for corrections based on reference images acquired from control cells.

Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome.

Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries approximately 500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed. --The nucleotide sequence data were uploaded to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AG020727 to AG025611 and AG205093 to AG215049.

Genomic rearrangements involving rDNA and centromeric heterochromatin in vulvar epidermoid carcinoma cell line A-431

The cytogenetic and molecular cytogenetic characterization of the human cell line A-431 derived from a vulvar epidermoid carcinoma is presented. A combination of karyotyping, fluorescence in situ hybridization (FISH) with chromosome- and/or region-specific probes, M-FISH, RxFISH, and comparative genomic hybridization (CGH) analysis was used. Six marker chromosomes with rearrangements involving insertions of single or double nucleolar organizing regions (NORs) and/or homogeneously staining regions containing active and overexpressed NORs and regions of centromeric heterochromatin were found: der(6), der(7), der(17), der(21), dic(13;14), and dic(14;18). The chromosomal origin of 14 other marker chromosomes was elucidated. Amplification of the C-MYC oncogene at 8q24 was revealed in two marker chromosomes: dup(8)(q24) and der(15)t(8;15)(q22;p11). Confirming previous reports, amplification of the cyclin D1 gene within an abnormal chromosome 11, that is, der(11)t(7;11)(p15;q21), was also detected. Loss of the TP53 tumor suppressor gene was evidenced over two der(17). Good concordance was found among karyotyping, FISH analysis, and CGH. Although reasons for NOR amplification or ectopic location in the epidermal carcinoma A-431 cell line are not clear yet, our data suggest that these phenomena play a supporting role with regard to other amplified genes. Thus, the A-431 cell line would be an appropriate model to study the different mechanisms involved in human tumorigenesis.

Replication banding patterns in the spined loach, Cobitis taenia L. (Pisces, Cobitidae).

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.

Imaging the Localized Protein Interactions Between Pit-1 and the CCAAT/Enhancer Binding Protein α in the Living Pituitary Cell Nucleus

The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPalpha) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBPalpha was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that coexpressed Pit-1 redistributed C/EBPalpha to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBPalpha was recruited by Pit-1, the average distance separating the fluorophores labeling the proteins was less than 7 nm. A mutation in the Pit-1 homeodomain, or truncation of the C/EBPalpha transactivation domain disrupted the redistribution of C/EBPalpha by Pit-1. Fluorescence resonance energy transfer analysis revealed that the mutant Pit-1 still associated with C/EBPalpha, and the truncated C/EBPalpha still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBPalpha that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBPalpha to DNA is a critical first step in specifying its association with Pit-1. These findings indicated that the protein domains that specify the interaction of Pit-1 and C/EBPalpha are separable from the protein domains that direct the positioning of the associated proteins within the nucleus. The intimate association of Pit-1 and C/EBPalpha at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression.

Functional redundancies, distinct localizations and interactions among three fission yeast homologs of centromere protein-B.

Several members of protein families that are conserved in higher eukaryotes are known to play a role in centromere function in the fission yeast Schizosaccharomyces pombe, including two homologs of the mammalian centromere protein CENP-B, Abp1p and Cbh1p. Here we characterize a third S. pombe CENP-B homolog, Cbh2p (CENP-B homolog 2). cbh2Delta strains exhibited a modest elevation in minichromosome loss, similar to cbh1Delta or abp1Delta strains. cbh2Delta cbh1Delta strains showed little difference in growth or minichromosome loss rate when compared to single deletion strains. In contrast, cbh2Delta abp1Delta strains displayed dramatic morphological and chromosome segregation defects, as well as enhancement of the slow-growth phenotype of abp1Delta strains, indicating partial functional redundancy between these proteins. Both cbh2Delta abp1Delta and cbh1Delta abp1Delta strains also showed strongly enhanced sensitivity to a microtubule-destabilizing drug, consistent with a mitotic function for these proteins. Cbh2p was localized to the central core and core-associated repeat regions of centromeric heterochromatin, but not at several other centromeric and arm locations tested. Thus, like its mammalian counterpart, Cbh2p appeared to be localized exclusively to a portion of centromeric heterochromatin. In contrast, Abp1p was detected in both centromeric heterochromatin and in chromatin at two of three replication origins tested. Cbh2p and Abp1p homodimerized in the budding yeast two-hybrid assay, but did not interact with each other. These results suggest that indirect cooperation between different CENP-B-like DNA binding proteins with partially overlapping chromatin distributions helps to establish a functional centromere.

Cot-1 banding of human chromosomes using fluorescencein situ hybridization with Cy3 labeling

We developed a new chromosome banding method by in situ hybridization of human Cot-1 DNA as a probe. Clear banding was produced on metaphase chromosomes of lymphoblastoid cells after probe detection with a fluorescent dye Cy3. Comparison with the known banding patterns revealed a similarity to the R-banding with some significant differences: some centromeric heterochromatin regions show Cot-1 positive bands. This suggests that some repetitive sequences from the heterochromatin regions constitute a major component of Cot-1 DNA. This unique chromosome banding method, Cot-1 banding, may be used as a supplement to the conventional karyotype analysis. Scanning analysis of the fluorescence intensities of Cot-1 banding and Q-banding are useful for objectively analyzing the banding pattern including a detection of chromosome aberrations. The Cot-1 banding with Cy3 is particularly powerful when applied for the gene mapping by fluorescence in situ hybridization (FISH) because red fluorescence of Cy3 for chromosome staining can be readily distinguished from green fluorescence of fluorescein isothiocyanate (FITC) for probe labeling. Using this novel method, we mapped a 4 kb-DNA fragment from myelin protein zero (MPZ) gene on the chromosome 1q22 to q23.

Characterization of two abundant satellite DNAs from the mealworm Tenebrio obscurus

Two highly abundant satellite DNAs comprise 36% of the Tenebrio obscurus (Tenebrionidae, Coleoptera) genome. They are designated as satellite I and satellite II with the monomer length of 344 and 142 base pairs (bp), respectively. Both satellites differ in their nucleotide (nt) sequences, but the frequency of point mutations, well-conserved length of monomer variants, stretches of shared mutations characteristic for the process of gene conversion, and distribution of both satellites in regions of centromeric heterochromatin of all chromosomes indicate that the same evolutionary processes act on both of them with the same, or similar, rate. While satellite I shares no sequence similarity with any other known nt sequence, satellite II is 79.7% homologous with the highly abundant satellite from closely related Tenebrio molitor. Difference in the frequency of point mutations and absence of shared mutations indicating gene conversion strongly suggest that in these two closely related species mutational processes affecting satellite DNAs seem to be changed. Retarded electrophoretic mobility, due to sequence-induced curvature of DNA helix axis, was observed for T. obscurus satellite II, but not for satellite I. Although evolutionary processes act with different rates in T. obscurus and T. molitor satellites the monomer length and sequence-induced curvature are well preserved in both 142-bp satellites, as well as in, at the nt sequence level completely divergent, Palorus ratzeburgii (Tenebrionidae) satellite, indicating potential importance of these parameters in their evolution.

New distinctions between regions of centromeric heterochromatin in human chromosomes by treatments with the isoschizomers NdeII–Sau3AI

The isoschizomers NdeII-Sau3AI (decreases GATC) have been used to characterize heterochromatic regions in human chromosomes. The findings with NdeII are identical with those previously published with MboI, but the results with Sau3AI provide evidence for new distinctions of centromeric heterochromatin in chromosomes 5 and 6. The results are discussed in relation to the chromatin organization at these regions an the mechanisms of the action of restriction endonucleases.

Differential sensitivity of constitutive and facultative heterochromatin in orthopteran chromosomes to digestion by DNaseI

In situ pancreatic DNaseI digestions were used as probes to study the structural organization of facultative and constitutive heterochromatin during both mitotic and meiotic divisions. Three different types of heterochromatic regions from three insect species were chosen for this study. These regions had been previously characterized by in situ treatments with restriction endonucleases (AT and GC rich DNA sequences). Progressive increase in DNaseI concentration (from 10 to 200 ng/ml) or in incubation time (from 5 to 30 min) revealed a specific pattern of sequential digestion of the constitutive heterochromatic regions, the centromeric ones (AT-rich DNA) being the most resistant to DNaseI action. The interstitial C-bands (with AT or GC-rich DNA) were more sensitive to DNaseI, and the band 4.4 from Baetica ustalata was the most resistant of the non-centromeric bands. Similar results were obtained during meiosis, but increased accessibility to DNAseI was observed compared to mitosis. DNA methylation in the non-centromeric band 4.4 of B. ustulata could be responsible for its differential digestion with respect to the remaining intercalar heterochromatin. Facultatively heterochromatic regions (X chromosomes) were found to exhibit a differential response to DNaseI attack from mitosis to meiosis. While they behaved as cuchromatin during mitosis, they were the most resistant together with centromeric heterochromatin regions, during metaphase I and II. The different responses to digestion of the X chromosome and X-derived regions between somatic and meiotic divisions are probably a consequence of the changes in the organization of this chromosome during the facultative heterochromatinization process.

Inhibition of condensation of human Y chromosome by the fluorochrome Hoechst 33258 in a mouse-human cell hybrid.

The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.