Nucleolar organizing regions of human chromosomes

Summary Silver-Stained cells from 49 parents with a history of several abortions were compared with cells from 35 parents with normal liveborn children. The modal and mean number of silver-stained NORs (Ag-NORs) observed on D- or G-group chromosomes was similar in both groups and between males and females. Ag-NORs were randomly distributed on all five acrocentric pairs. The distribution and size of Ag-NORs within an individual was not random and was fairly consistent from cell to cell.The mean number of associations per cell was similar in both males and females of the abortion group and was less than the number of associations in controls. The probability of D- or G-group chromosomes being associated was near the expected probability of 0.6 for D-association and 0.4 for G-association. The frequency of association of any chromosome combination did not differ statistically from the expected values, though the number of associations, 15/22, was higher than expected.     

The pseudoautosomal regions of the human sex chromosomes

In human females, both X chromosomes are equivalent in size and genetic content, and pairing and recombination can theoretically occur anywhere along their entire length. In human males, however, only small regions of sequence identity exist between the sex chromosomes. Recombination and genetic exchange is restricted to these regions of identity, which cover 2.6 and 0.4 Mbp, respectively, and are located at the tips of the short and the long arm of the X and Y chromosome. The unique biology of these regions has attracted considerable interest, and complete long-range restriction maps as well as comprehensive physical maps of overlapping YAC clones are already available. A dense genetic linkage map has disclosed a high rate of recombination at the short arm telomere. A consequence of the obligatory recombination within the pseudoautosomal region is that genes show only partial sex linkage. Pseudoautosomal genes are also predicted to escape X-inactivation, thus guaranteeing an equal dosage of expressed sequences between the X and Y chromosomes. Gene pairs that are active on the X and Y chromosomes are suggested as candidates for the phenotypes seen in numerical X chromosome disorders, such as Klinefelter's (47,XXY) and Turner's syndrome (45,X). Several new genes have been assigned to the Xp/Yp pseudoautosomal region. Potential associations with clinical disorders such as short stature, one of the Turner features, and psychiatric diseases are discussed. Genes in the Xq/Yq pseudoautosomal region have not been identified to date.     

Preferential breakage of sensitive regions of human chromosomes

Summary Whilst studying the chromosomes of the peripheral blood lymphocytes of normal controls and patients with lymphoproliferative disorders, two examples of preferential breakage of a sensitive chromosomal region were found. A patient with lymphocytic lymphoma had a sensitive region in a C9 chromosome coinciding with the secondary constriction. A healthy woman had one A2 chromosome showing an unusually located secondary constriction in which breakage sometimes occurred.     

Discontinuities and unsynapsed regions in meiotic chromosomes have a trans effect on meiotic recombination of some chromosomes in human males

During meiosis, homologous chromosome pairing and synapsis are essential for subsequent meiotic recombination (crossing-over). Discontinuous regions (gaps) and unsynapsed regions (splits) were most frequently observed in the heterochromatic regions of bivalent synaptonemal complex (SC) 9, and we have previously demonstrated that gaps and splits significantly altered the distribution of MLH1 recombination foci on SC 9. Here, immunofluorescence techniques (using antibodies against SC proteins and the crossover-associated MLH1 protein) were combined with a centromere-specific fluorescence in situ hybridization technique that allows identification of every individual chromosome. The effect of gaps/splits on meiotic recombination patterns in autosomes other than chromosome 9 during the pachytene stage of meiotic prophase was then examined in 6,026 bivalents from 262 pachytene cells from three human males. In 64 analyzed cells with a gapped SC 9, the frequency of MLH1 foci in SCs 5 and 10 and in SC arms 10q, 11p and 16q was decreased compared to 168 analyzed cells with a normally-synapsed SC 9 (controls). In 24 analyzed cells with splits in SC 9, there was a significant reduction in MLH1 focus frequency for SC 5q and the whole SC5 bivalent. The positioning of MLH1 foci on other SCs in cells with gapped/split SC 9 was not altered. These studies suggest that gaps and splits not only have a cis effect, but may also have a trans effect on meiotic recombination in humans.     

Compositional properties of telomeric regions from human chromosomes.

We have investigated the GC levels of third codon position of genes localized in G- (Giemsa), R-(reverse) and T-(telomeric) bands of human metaphase chromosomes, as well as the hybridization of telomeric probes on fractionated human DNA. The first set of results shows much higher GC levels for genes localized in T-bands than in G- or R-bands (the latter being higher than the former). The second set of data shows that telomeric probes corresponding to T-bands hybridize on the GC-richest family (H3) of isochores, whereas telomeric probes corresponding to R-bands hybridize on GC-rich families H1 and H2; in agreement with these findings, the telomeric repeat common to all chromosomes hybridized on isochore families H1, H2 and H3.     

Polymorphism of nucleolar organizer regions of chromosomes in human embryos

NOR activity in metaphase chromosomes from extraembryonic and embryonic tissues of 9-12 week human fetuses was studied after standard silver staining. Significant interidividual variations in the average cummulative NOR activity was assessed by means of one-factor dispersion analysis. No significant intertissue fluctuation of NOR activity was found. Total number of NOR+ chromosomes demonstrated no correlation with the embryonic age. Steady growth of an average cummulative NOR activity respective of progressive embryonic age was proven by correlation analysis method. Unequal participation of NOR-bearing chromosomes of D- and G-groups during early embryonic development in human was shown.     

Quantitative assessment of the size of Ag-stained nucleolus-organizing regions in human chromosomes

The squares of Ag-stained nucleolar organizing regions of metaphase chromosomes have been estimated by scanning their negative images on the film and computer data processing. The intercellular variation of the sum of squares of nucleolar organizing regiones of five individuals was studied. The coefficient of variation for these individuals varied from 11.2 to 24.6%. The analysis of the mean sum of squares of nucleolar organizing regiones per metaphase has revealed reliable differences for all the individuals. This value, therefore, can be taken as individual characteristics in the population research.     

Preferential somatic pairing between homologous heterochromatic regions of human chromosomes.

The cytidine analog 5-azacytidine (5-azaC) induces an undercondensation of the heterochromatin in human chromosomes 1, 9, 15, 16, and Y when it is added in low concentrations to the late S-phase of growing lymphocyte cultures. In interphase nuclei, these heterochromatic regions are frequently somatically paired. The somatic pairing configurations are preserved up to metaphase stage in the 5-azaC-treated cultures and are thus susceptible to a direct microscopical examination. The statistical analysis of 1,000 somatic pairing configurations from 5-azaC-treated cells showed that the somatic pairing between the heterochromatic regions of homologous chromosomes is preferred over that between nonhomologous chromosomes.     

Co-occurrences of polymorphic heterochromatin regions of chromosomes and effect on reproductive failure

Although the polymorphic heterochromatin regions of chromosomes (heteromorphisms) have been extensively studied for their phenotypic effects on humans, co-occurrences of chromosome 1, 9, 16 and Y heteromorphisms and of acrocentric variants have never been studied on humans with an objective scoring system. Here we compared the frequencies of individual heteromorphisms on a total of 602, 768 and 224 patients with the indications of infertility, recurrent miscarriage and in vitro fertilization (IVF) failure, respectively and on 272 controls. Then we examined whether there were significant co-occurrences between heteromorphisms within and between the groups. There were no statistically significant differences in the frequencies of heteromorphisms between the groups. Both statistically significant and non-significant correlations were observed within the non-acrocentric and certain acrocentric heteromorphisms in each group. When these co-occurrences were examined between the groups, a 2.2 fold increased risk of IVF failure in males in the presence of either chromosome 13 or chromosome 21 variants was observed (95 %CI:1.1–4.2). We conclude that the simultaneous manifestations of heteromorphisms have no effect on reproductive failure. There seems to be a correlation between the non-acrocentric heteromorphisms (1qh+, 9qh+, 16qh + and Yqh+/-), which might be the result of complex interactions of formation of these heterochromatin regions. The correlations observed between certain acrocentric chromosomes might be related to satellite association and nucleolus formation. The increased risk observed in males with IVF failure in the presence of either chromosome 13 or 21 variants should be interpreted cautiously due to the heterogeneity of the group.     

Demonstration of two different regions of lateral asymmetry in human Y chromosomes

Summary Two differently stained regions of lateral asymmetry were observed in the long arm of the human Y chromosome, following FPG staining. The first asymmetry was confined to band q12 of the long arm. The second asymmetrically stained region was located at the junction between bands q11 and q12. In the non-fluorescent Y chromosomes only one region of lateral asymmetry was found at the end of the long arm and its staining properties were similar to the region situated at the junction between q11 and q12 bands in the fluorescent Ys. The two morphologically distinguishable regions of lateral asymmetry are presumed to indicate sites containing different satellite DNAs in the human Y chromosome.     

Recent evolution of DNA sequence homology in the pericentromeric regions of human acrocentric chromosomes

A search for genes located on human chromosome 21 resulted in the isolation of a HeLa cDNA clone, pUNC724, which hybridized to 3.7 and 2.5 kilobase (kb) EcoRI fragments on each of the human acrocentric chromosomes. In situ hybridization further localized pUNC724 to the pericentromeric region of the human acrocentrics. Two other EcoRI fragments that hybridized to pUNC724 were assigned to the long arms of chromosomes 1 and 18. The pUNC724 sequence does not appear to be related to ribosomal or satellite DNA sequences. The juxtaposition of DNA sequences homologous to pUNC724 and ribosomal DNA sequences presumably occurred within the past thirty-five million years, following the divergence of the lines leading to man and the New World owl monkey, Aotus trivirgatus--pUNC724 is not syntenic with the single chromosome containing ribosomal DNA sequences in the owl monkey.     

Clonal analysis of the intercellular variability in the functioning of human chromosomal nucleolus organizer regions

Intercellular variability of NOR activity detected with the aid of Ag-staining of human chromosomes was studied in mass and cloned fibroblast cultures obtained from 3 individuals. The intercellular variability was determined by different staining of one of 10 NORs. According to this trait the heterogeneity of the cell population was discovered in all cloned lines, with this heterogeneity being the same as in the parent cultures. That concerned the number of a variable chromosome and the percentage of the cells with Ag-stained and unstained chromosomes. It is suggested that genetic determination in the progenies of the somatic cells concerns the whole spectrum of potential variability observed in cell populations.     

Negative silver staining in A-T and satellite DNA-rich regions of human chromosomes

Human metaphase chromosomes were stained with silver following a pretreatment with a heated alkaline solution. The most conspicuous feature of the stained metaphases was the omission of silver staining in the secondary constrictions of chromosomes 1,9 and 16, and on the distal Yq. Our evidence indicates that the negative silver binding is due to the preferential removal or alteration of non-histone proteins associated with these regions. The cytochemical significance of these findings is discussed.     

Combined silver staining of the nucleolus organizing regions and Giemsa banding in human chromosomes

Summary A combination of the silver-staining method of the nucleolus organizer regions (NORs) with a Giemsa-banding method is deccribed. This double staining allows a rapid identification of the NOR-bearing chromosomes.Supported by the Deutsche Forschungsgemeinschaft (Za 32/14).     

The effect of tritiated thymidine on human chromosomes

Summary The effect of low dosages of³H-thymidine on human chromosomes was investigated. Two established human cell lines, amnion and HeLa cells, were treated for various periods of time 0.125, 0.25, 0.50 or 0.75 μCi/ml of this agent. Chromatid-type aberrations were observed, occurring only if³H-thymidine was incorporated into DNA during the S phase of the cell cycle. The higher the dosage and the longer the³H-thymidine acted on chromatin, the greater the number of aberrations induced. Chromosomes were most sensitive to the effects of³H-thymidine during the early S phase of the cell cycle. This work was supported by the Medical Research Council, Canada Grant MA 1639.     

In human chromosomes telomeric regions are enriched in CpGs relative to R-bands

Human chromosomes were in situ nick-translated using as nicking agents the endonucleases MspI (CCGG), its methyl-sensitive isoschizomer HpaII, HaeIII (GGCC), SacII (CCGCGG), EcoRI (GAATTC) and DNaseI. We show that in metaphase chromosomes R-bands are enriched, as compared with G-bands, in the dinucleotide CpG but no more than what is expected on the basis of their relative G+C content. The telomeric regions, on the contrary, besides having a chromatin conformation that is particularly relaxed and accessible to endonucleases, also show an enrichment in CpGs.