C-band length variability and reproductive wastage

Summary The possible influence of total Y chromosome length and the C-band size variability of chromosomes 1, 9, 16, and Y, on reproductive wastage was investigated. One hundred couples with recurrent reproductive wastage and 106 control couples with at least two healthy children and no miscarriages were cytogenetically studied. Total Y chromosome length was evaluated as the Y/F index and the C-band size was analyzed quantitatively according to the linear measurement method of Baliek et al. (1977). The different degrees of mitotic contraction were corrected on the basis of the linear correlation found between heterochromatin and euchromatin length. Statistical comparison between results of Y chromosome from both samples demonstrated, in the test group, an increase in the mean value of the Y/F index, but the increase of Y C-band length did not reach significance. In addition mean values of C-band length on chromosomes 1, 9, and 16 in couples from the test group and especially those who had had two or more abortions, were lower than those in the controls. Among the latter the frequency of chromosomes included in the category of very large heterochromatin size is higher. However these length differences have been demonstrated only in specific subgroups, and in each one for a different chromosome. Our results indicated that Y chromosome length as well as C-band size variabilities are not directly related to reproductive wastage.     

Equivalence of the total constitutive heterochromatin content by an interchromosomal compensation in the C band sizes of chromosomes 1, 9, 16, and Y in Caucasian and Japanese individuals

A quantitative analysis of C bands by densitometric measurements in chromosomes 1, 9, 16, and Y was conducted in Caucasians and Japanese living in Brazil. Sixty normal unrelated subjects (30 males and 30 females) were studied in each racial group. Caucasians presented C bands of chromosomes 1, 9, and 16 larger than Japanese, but, on average, only the difference for C bands of chromosome 9 was statistically significant. In the Japanese, the C band sizes of chromosomes Y were, on average, significantly larger than in the Caucasians. The mean C band size of chromosome 9 and the sum of the three pairs were significantly larger in Caucasian than in Japanese males. The total values of constitutive heterochromatin, sigma (1qh,9qh,16qh,Yq12), did not show significant difference between Caucasian and Japanese males. The relative C band sizes of chromosomes 1, 9, and 16 were, on average, similar in Caucasians and Japanese. No sex difference was found in both racial groups. As regards the heteromorphism, only the values of C bands of chromosome 9 were, on average, significantly larger in Caucasians than in Japanese. Partial inversions were detected only among the Caucasians.     

Variability and familial transmission of constitutive heterochromatin of human chromosomes evaluated by the method of linear measurement

Summary Using the method of linear measurement, the lengths of constitutive heterochromatin of chromosomes 1, 9, 16, and Y were determined in 125 unrelated individuals, and in 30 members of ten families. The method used eliminates the variations in the C-band length due to different degrees of contraction of chromosomes in different mitoses, and enables the size of heterochromatin blocks to be expressed. It was found that the distribution of C-band lengths in the group of 125 individuals was normal, i.e., Gaussian, for all four classes of chromosomes measured. On the basis of length distribution and by computing the P₁, P₁₀, P₉₀ and P₉₉ percentiles, the actual numerical limits could be proposed for the five-step evaluation of heterochromatin length according to the Paris Conference (1971), Supplement (1975), for chromosomes 1, 9, 16, and in a preliminary way also for Y. When applying the proposed limits to data obtained in the present study, 165 C-band variants could be identified among the 125 individuals.In ten families, C-block lengths of the chromosomes transmitted from parents to progeny could be determined in 63 cases. The mean difference in C-band length of transmitted chromosomes, as measured in parents and in children, was 0.46×10^-7 m. An analysis was carried out to detect the factors upon which the magnitude of this difference depends, and to define what differences are attributable to methodological errors. The results revealed that the difference rises slightly with the increasing length of the measured C block. Three degrees, defined by concrete ranges of difference in C-block length, were proposed for expressing the probability that the compared chromosomes had been transmitted.The study further attests to the effectiveness of the method of constitutive heterochromatin measurement for paternity testing. In our set of ten families, the comparison of C-band lengths of chromosomes 1, 9, 16, and Y led to rejection of paternity in 64% of unrelated individuals; excluding the Y chromosome, the percentage decreased to 61. As many as 47% of the individuals were rejected by a difference higher than two units (i.e., transmission of the compared chromosome highly improbable).     

Quantitative analysis of C bands in chromosomes 1, 9, 16 and Y in Caucasian and Japanese males

A comparative analysis of the C bands of chromosomes 1, 9, 16 and Y of 27 Caucasian and 27 Japanese males is reported. The mean of the total centromeric heterochromatin of the three pairs (sigma h1, 9, 16) is larger in Caucasian than in Japanese subjects, but Caucasians showed a lower mean of C band size of chromosome Y. Heritability of the C band of the Y chromosome was studied in 26 families.     

Relationship of the variability of the heterochromatic regions of chromosomes 1, 9, 16, and Y to some anthropometric characteristics in children with embryopathies of unknown etiology and in children with Down syndrome

Summary The relationship between variability of the heterochromatic regions of chromosomes 1, 9, 16, and Y, and anthropometric characteristics (length and mass of body, shoulder diameter) in 70 children with embryopathies of unknown etiology and in 40 children with Down syndrome was studied. The positive statistically significant correlations of the C segment lenghts of chromosomes 1, 9, 16, their sum included, and the above characteristics were found. The correlation coefficients of Y chromosome were not significant. The questions of the functional role of the structural heterochromatin and its influence on the viability and physical development of the organism are discussed.     

Heterochromatin is not an adequate explanation for close proximity of interphase chromosomes 1--Y, 9--Y, and 16--Y in human spermatozoa

Analysis of human spermatozoa and lymphocytes using C-banding techniques and in situ hybridization has shown a higher order packaging of the human genome. Chromosomes are not distributed entirely at random within the nucleus. In particular, chromosomes 1, 9, and 16, carrying large blocks of pericentromeric heterochromatin, and the Y chromosome, carrying heterochromatin in Yq12, are in close proximity to each other within the nucleus and are involved in somatic pairing with nonhomologous chromosomes. In order to determine whether the close proximity of these chromosomes in any way is attributable to the distribution of heterochromatin, double in situ hybridization was performed on chromosomes 1--Y, 9--Y, and 16--Y as well as on 1--X, 9--X, and 16--X-with chromosome X as the other gonosome carrying less heterochromatin-in human spermatozoa. Each pair was found to have a nonrandom spatial distribution. However, comparison of the arrangement of chromosomes 1--Y versus 1--X and 9--Y versus 9--X revealed that heterochromatin cannot be the only cause for the tendency of chromosome fusion, because only the results of the chromosome pair 1--Y/1--X could support this proposition. In conclusion, the heterochromatin effect cannot be, in itself, an adequate explanation for chromosome association, implicating as well other mechanisms.     

Analysis of centromere size in human chromosomes 1, 9, 15, and 16 by electron microscopy.

Human chromosomes were treated with 5-azacytidine and analyzed by whole-mount electron microscopy. This base analogue produces undercondensation of heterochromatin and separation of the centromere from the bulk of pericentromeric heterochromatin in chromosomes 1, 9, 15, and 16, which allows clear delimitation of the centromere regions. A quantitative analysis of centromeres showed that chromosomes 1, 9, and 16 have centromeres of different size. The centromere of chromosome 15 is similar in size to that of chromosome 9 and different from those of chromosomes 1 and 16. No interindividual variation for centromere size was found. A positive correlation between centromere and chromosome size was found for the chromosomes analyzed.     

Characterization of human heterochromatin by in situ hybridization with satellite DNA clones

Biotinylated DNA from two satellite-related, repetitive DNA clones, pHuR 98 and pHuR 195 (specific for chromosomes 9 and 16, respectively), and from a Y-specific clone, pY-3.4A, were hybridized to human metaphase chromosomes using fluoresceinated avidin to detect binding. The chromosomes were simultaneously counterstained with distamycin-DAPI to identify the AT-rich heterochromatin of chromosomes 1, 9, 15, 16, and the Y chromosome. With this method, clear results were obtained under both normal and low stringency conditions, allowing hybridization between molecules sharing 80-85% and 60-65% identity, respectively. Thus, additional sites related to the probes could be identified. A close relationship was shown between the heterochromatin of chromosomes 1 and 16, both hybridizing with clone pHuR 195 under low stringency. Hybridization with clone pHuR 98 was highly specific for chromosome 9, even under low stringency. A relationship between chromosomes 9, 15, and the Y chromosome, however, was shown by hybridization with clone pY-3.4A. The chromosomal distribution of the three repetitive DNA clones used in this study, and data from the literature, are in accordance with the distribution of the heterochromatin types characterized by staining with different fluorescent dyes and dye combinations. Furthermore, our sequence data for clones pHuR 98 and pHuR 195 may explain the fluorescent properties on which the cytogenetic classification of the heterochromatin is based.     

Attraction between centric heterochromatin of human chromosomes.

An analysis of the pattern of association of acrocentric chromosomes with nonacrocentric chromosomes in human lymphocyte metaphases was performed. This pattern in nonrandom with respect to chromosome length and intrachromosomal distribution. There is a general preference for the centric regions, most pronounced at the proximal segments of the long arms of chromosomes 1, 9, and 16, which is interpreted to reflect heterochromatin attraction during interphase. Comparison of the association patterns of homologous chromosome 1's differing with regard to the size of their heterochromatic regions corroborates this interpretation. The possible significance of heterochromatin attraction for the formation of spontaneous and induced chromosome anomalies is discused.     

Variability of the C-segment sizes of chromosomes 1, 9, 16 and Y in the human chromosome set]

The investigation of chromosome polymorphism by quantitative methods is a rather hard task. The manual method for measuring C-segments of chromosomes 1, 9, 16 and Y in man is suggested, which is not difficult, being reasonably precise for the population research. Metaphases of the average level of chromosome condensation were taken for analysis. Only the C-segments were measured without measuring chromosomes. The negative chromosome image was 4000-fold magnified, compared to the chromosome natural size, and the boundaries of C-segments of each chromosome were five-fold dotted on a sheet of paper specially printed for this purpose. C-segments were measured by magnifying glass with 0.025 mcm scale unit. For every individuum, C-segments were measured in 5-7 cells only. The data are presented on the estimation of measurement errors and on individual (intercellular) and population (interindividual) variations of C-segments of chromosomes.     

Quantitative analysis of C-segments of chromosomes 1, 9, 16 and Y in couples with reproductive disorders

A comparative analysis of structural variability of C-bands on chromosomes 1, 9, 16 and Y was conducted in 50 phenotypically normal adults and 25 couples with repeated spontaneous abortions. Reduction of both the total amount of heterochromatin in the cell and the lengths of these regions on chromosomes 1, 9, and 16 is revealed in the group of pathology. No differences were found in the lengths of C-bands on Y chromosome.     

Cathepsin L stabilizes the histone modification landscape on the Y chromosome and pericentromeric heterochromatin

Posttranslational histone modifications and histone variants form a unique epigenetic landscape on mammalian chromosomes where the principal epigenetic heterochromatin markers, trimethylated histone H3(K9) and the histone H2A.Z, are inversely localized in relation to each other. Trimethylated H3(K9) marks pericentromeric constitutive heterochromatin and the male Y chromosome, while H2A.Z is dramatically reduced at these chromosomal locations. Inactivation of a lysosomal and nuclear protease, cathepsin L, causes a global redistribution of epigenetic markers. In cathepsin L knockout cells, the levels of trimethylated H3(K9) decrease dramatically, concomitant with its relocation away from heterochromatin, and H2A.Z becomes enriched at pericentromeric heterochromatin and the Y chromosome. This change is also associated with global relocation of heterochromatin protein HP1 and histone H3 methyltransferase Suv39h1 away from constitutive heterochromatin; however, it does not affect DNA methylation or chromosome segregation, phenotypes commonly associated with impaired histone H3(K9) methylation. Therefore, the key constitutive heterochromatin determinants can dynamically redistribute depending on physiological context but still maintain the essential function(s) of chromosomes. Thus, our data show that cathepsin L stabilizes epigenetic heterochromatin markers on pericentromeric heterochromatin and the Y chromosome through a novel mechanism that does not involve DNA methylation or affect heterochromatin structure and operates on both somatic and sex chromosomes.     

Variation of constitutive heterochromatin in the sex chromosomes of the rodent Bandicota bengalensis bengalensis (Gray)

Bandicota bengalensis bengalensis (Gray) trapped from different localities of India and Nepal exhibited a marked variation in the size and morphology of sex chromosomes. Three types of X's were found; A) simple acrocentric, B) composite subtelocentric and C) composite submetacentric X with their relative sizes 5.9%, 7.5% and 9.6% of the genome respectively. The autosomes remained unaltered. It was shown that this variation in the size of sex chromosomes was caused by deletion of constitutive heterochromatin. The Y chromosome was also found to be variable. Usually a large X was combined with a large Y. The preponderance of homozygotes for each type of X chromosome in populations, suggested the probable role of sex chromosomes heterochromatin in speciation.     

Lateral asymmetry in human constitutive heterochromatin

Human lymphocytes were grown for one replication cycle in BrdU, stained with 33258 Hoechst, exposed to UV light and subsequently treated with 2 x SSC and stained with Giemsa. This technique differentially stains the constitutive heterochromatin of chromosomes 1, 9, 15, 16, and the Y. In the heterochromatin of chromosome 9 both sister chromatids stained darkly and symmetrically but in the other four chromosomes the heterochromatin showed lateral asymmetry, one chromatid being darkly stained while its sister chromatid was as pale or paler than the rest of the chromosome. The lateral asymmetry is presumed to reflect an underlying asymmetry in distribution of thymine between the two strands of the DNA duplex in the satellite DNA component of the chromosomes. In some number 1 chromosomes compound lateral asymmetry was seen; darkly staining material was present on both sister chromatids although at any given point lateral asymmetry was maintained so that if one chromatid stained darkly the corresponding point on the sister chromatid was very pale. The pattern of compound lateral asymmetry varied among the number 1 chromosomes studied but was constant for any one homologue from one individual. This technique reveals a previously unsuspected type of polymorphism within the constitutive heterochromatin of man.     

白颊长臂猿染色体的研究

本文对我国云南南部的白须长臂猿（Ｈ．ｌｅｕｃｏｇｅｎｙｓ）染色体的Ｇ带、Ｃ带、晚复制带及Ａｇ－ＮＯＲｓ进行了较为详细的研究。它的２ｎ＝５２,核型公式为４４（Ｍ或ＳＭ）＋６（Ａ）,ＸＹ（Ｍ,Ａ）。Ｃ带表明一些染色体着丝点Ｃ带弱化;有的染色体出现插入的和端位的Ｃ带;Ｘ染色体两臂有端位Ｃ带,Ｙ染色体是Ｃ带阳性和晚复制的。Ａｇ－ＮＯＲｓ的数目,雌体有４个,雄体有５个,Ｙ染色体上具ＮＯＲ。本文对白颊长臂猿与其它长臂猿间的亲缘关系、核型进化的可能途径进行了讨论。     

石貂的染色体研究

本文对分布在我国的石貂北方亚种染色体进行了较详细的研究。结果表明２ｎ＝３８,核型为１４（Ｍ）＋４（ＳＭ）＋１８（ＳＴ）,ＸＹ（Ｍ,Ａ）。Ｃ－带显示该亚种的一些染色体着丝粒区域结构异染色质弱化或消失。Ｎｏ,９染色体的短臂完全异染色质化;Ｘ染色体长臂丰出现插入杂色质带;Ｙ为完全结构异染色质组成。     

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

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

Further evidence of intraspecific heterochromatin variants in Drosophila melanogaster

The analysis of inter-strain heterochromatin polymorphism in mitotic chromosomes of Drosophila melanogaster was extended to some stocks characterized by chromosomal mutations. In particular, the present investigation aims to compare, in the same cell, the quinacrine banding of two different Y chromosomes of male hybrids derived from crosses using special stocks. A direct comparison of homologous heteromorphic chromosomes in F₁ hybrids provided additional evidence of differences in the fluorescence pattern of the Y chromosome, as well as in the length of the heterochromatin segment of the X chromosome.     

Comparison of areas of quinacrine mustard fluorescence and modified Giemsa staining in human metaphase chromosomes

The methods of quinacrine mustard fluorescence and modified Giemsa staining were compared in view of the structural details revealed in human mitotic chromosomes derived from the peripheral blood of normal healthy humans. Over the chromatids both techniques produced a crossbanding pattern where larger segments of heavy staining in the latter technique and the fluorescing bands in the former occurred at similar locations. The centromeric heterochromatin, intensely stained with Giemsa was, however, negative in fluorescence, except for chromosome no. 3 and less often no. 6. The regularly occurring secondary constrictions in chromosomes 1, 9, and 16 behaved generally like areas of centromeric heterochromatin. The area of secondary constriction in the Y chromosome as also that of chromosome 9 in the ASG modification of the Giemsa technique was both non-fluorescent and non-staining.     

Experimental condensation inhibition in constitutive and facultative heterochromatin of mammalian chromosomes

What drives the dramatic changes in chromosome structure during the cell cycle is one of the oldest questions in genetics. During mitosis, all chromosomes become highly condensed and, as the cell completes mitosis, most of the chromatin decondenses again. Only chromosome regions containing constitutive or facultative heterochromatin remain in a more condensed state throughout interphase. One approach to understanding chromosome condensation is to experimentally induce condensation defects. 5-Azacytidine (5-aza-C) and 5-azadeoxycytidine (5-aza-dC) drastically inhibit condensation in mammalian constitutive heterochromatin, in particular in human chromosomes 1, 9, 15, 16, and Y, as well as in facultative heterochromatin (inactive X chromosome), when incorporated into late-replicating DNA during the last hours of cell culture. The decondensing effects of 5-aza-C analogs, which do not interfere with normal base pairing in substituted duplex DNA, have been correlated with global DNA hypomethylation. In contrast, decondensation of constitutive heterochromatin by incorporation of 5-iododeoxyuridine (IdU) or other non-demethylating base analogs, or binding of AT-specific DNA ligands, such as berenil and Hoechst 33258, may reflect an altered steric configuration of substituted or minor-groove-bound duplex DNA. Consequently, these compounds exert relatively specific effects on certain subsets of AT-rich constitutive heterochromatin, i.e. IdU on human chromosome 9, berenil on human Y, and Hoechst 33258 on mouse chromosomes, which provide high local concentrations of IdU incorporation sites or DNA-ligand-binding sites. None of these non-demethylating compounds affect the inactive X chromosome condensation. Structural features of chromosomes are largely determined by chromosome-associated proteins. In this light, we propose that both DNA hypomethylation and steric alterations in chromosomal DNA may interfere with the binding of specific proteins or multi-protein complexes that are required for chromosome condensation. The association between chromosome condensation defects, genomic instability, and epigenetic reprogramming is discussed. Chromosome condensation may represent a key ancestral mechanism for modulating chromatin structure that has since been realloted to other nuclear processes.