Patterns of exchange induced by mitomycin C in C-bands of human chromosomes. I. Relationship to C-band size in chromosomes 1, 9, and 16

Summary Frequencies of exchange were determined in C-bands of chromosomes 1, 9 and 16 in six normal males, and related to relative C-band area. Comparing these different chromosomes, more exchanges occurred on average in 9 than in 1 although their mean C-band sizes were similar. Chromosome 16 exchanges were fewer, both overall and relative to C-band area. Comparing the same chromosome between individuals, there was a positive correlation between relative frequency and band size in both 1-1 and 9-9 exchanges. No clear trend was observed for other exchange events.If homology is required for interchange, if cannot be dependent solely on overall C-band size. Perhaps certain DNA sequences, sensitive to mitomycin C damage, are located in part of each C-band, with less per unit area in chromosome 1 than in 9 and still less in chromosome 16.X- and U-type exchanges between chromosome 9s occurred in near equal frequencies in all individuals. If synapsis of specific, affected sequences is a pre-requisite for interchange, this observation suggests that the affected sequence in chromosome 9 is arranged in both orientations relative to the centromere.     

Classification of qh regions in human chromosomes 1, 9, and 16 by C-banding

Summary We present a classification for secondary constriction (qh) regions with C-banding technique in chromosomes 1,9, and 16 by means of comparing them to the short arm of chromosome 16. It is simple and convenient and can be used routinely. It can be incorporated into the modified Paris nomenclature system.     

Human chromosomal heteromorphisms in Delhi newborns. II. Analysis of C-band size heteromorphisms in chromosomes 1, 9 and 16

Quantitative analysis of C-band size heteromorphisms in chromosomes 1,9 and 16 was carried out in 200 Delhi newborns (100 males and 100 females). The percent size heteromorphisms for chromosomes 1,9 and 16 showed nonsignificant differences between the sexes. Homozygous size level combinations showed higher incidence than the heterozygous combinations for all the three chromosome pairs studied in both sexes. Our results are compared with other reported studies and the possible role of these heteromorphisms in ethnic/racial variation and in developmental disturbances is discussed.     

Analysis of the inheritance of heterochromatic regions in human chromosomes 1, 9, 16 and Y

The inheritance of heterochromatic regions of chromosomes 1, 9, 16 and Y was studied in twelve families by means of measuring their C-segments. Maternal and paternal origin of chromosomes 1, 9 and 16 in the child was determined by two methods. The advantages and disadvantages of these methods and possibilities of their application are under discussion.     

Heterochromatic regions of human chromosomes 1, 9, 16 and Y and the phenotype

The relationship between variability of the heterochromatic regions of chromosomes 1, 9, 16, Y and the anthropometric characteristics (the height, the biacromial diameter and weight) was studied in two groups of children; 70 children had embryopathies of unknown etiology and 40 children had the Down syndrome. The positive statistically significant correlation of the C-segments lengths of chromosomes 1, 9, 16, their sum included, and above characteristics was found. The correlation coefficients of Y-chromosome were non-significant. The problems of functional role of the structural heterochromatin and its influence on viability and physical development of the organism are discussed.     

The quantitative analysis of polymorphism on human chromosomes 1, 9, 16 and Y

Summary The generalized characteristic of the C-segment lengths on chromosomes 1, 9, 16, and Y is suggested for a study of population heterogeneity. For this purpose, the concept of the distance D is introduced, taking into account the individual C-segment lengths, the mean lengths and standard deviations of C-segment lengths in a group of subjects, as well as the coefficients of correlation of the C-segment lengths on the said chromosomes.It is demonstrated that distance D may be employed to study the relevance of the given subject to the group studied, the relation to the mean characteristics within the group, and selection of subjects' pairs with almost identical C-segment lengths on respective chromosomes.In the study of such problems as zygosity of twins, family analysis, etc., along with the absolute C-segment lengths, it is recommended to employ the relative C-segment lengths on chromosomes 1, 9, 16, and Y, calculated as a part of the sum total of their absolute lengths.     

The quantitative analysis of polymorphism on human chromosomes 1, 9, 16, and Y

This study was made to establish a stable quantitative characteristic of C segments on chromosomes 1, 9, 16, and Y in an individual karyotype that was reproducible in successive experiments. The C segment of these chromosomes were measured in successive cultures of cells from three males and the C segments of chromosomes 1, 9, and 16 in cells from three pairs of female monozygotic twins were measured. The results show that the absolute lengths of C segments tend to vary considerably with the cell samples analyzed, while the relative length, i.e., the length of a single C segment as a percentage of the total length of all C segments of the chromosomes being studied, is more stable and can be used for individual characteristics.     

The quantitative analysis of polymorphism on human chromosomes 1, 9, 16, and Y

Summary Comparative analysis of the polymorphism of C segments on chromosomes 1, 9, 16, and Y was conducted in 50 normal boys and 50 normal girls. Quantitative methods revealed that the mean lengths of C segments, their variability, and their distribution on the chromosomes mentioned are quite identical in the two groups. Methodological problems related to the study of chromosome polymorphism are discussed.     

The quantitative analysis of polymorphism on human chromosomes 1, 9, 16, and Y

Summary A paracentric inversion of chromosome 5 was detected after RHG banding in a subject affected by Klinefelter's syndrome. The inversion was also observed in the patient's mother, and was confirmed by QFQ-and RBA-banding techniques.A second paracentric inversion affecting chromosome 7 was detected in a woman with Turner's syndrome. The same structural anomaly was found in her father and her half-brother.The possible relationship between sex chromosome nondisjunction and paracentric inversion is discussed.Furthermore, the inversion of chromosome 7 reproduces exactly the chromosome 7 of the gorilla, which is presumed to be ancestral to the human 7. This therefore appears to be the first reported case of reverse chromosomal mutation.     

The segregation of C-band polymorphisms on chromosomes 1, 9, and 16.

Eleven normal families with at least four children were studied cytogenetically using the C-band technique to identify polymorphisms in the constitutive heterochromatin of chromosomes 1, 9 and 16. Thirteen individuals showed one or more variants in such chromosomes. The analysis of the segregation ratios in the 35 offspring of these 13 individuals showed that these marker chromosomes generally segregated according to the expected 50:50. However, one of these variants, chromosome no. 9 with an increased heterochromatin block in the secondary constriction, has an apparently preferential segregation, when the findings from this study are combined with those of other authors.     

Relation between the size of C-segments and corresponding euchromatic regions of human chromosomes 1, 9 and 16 during their mitotic condensation

The nature of associations between the length of C-segments and the corresponding euchromatic regions of chromosomes 1, 9, and 16 in the process of their mitotic condensation has been studied. Their statistically significant linear nature in the range of chromosome 2 condensation from 11 to 4 micron has been established. Within the interval of 6.5-8.5 micron the above association is less significant, at the same time minimal variability of C-segment length is observed as compared to other stages of mitotic condensation. It is recommended to define the absolute size of C-segments in chromosomes 1, 9 and 16 by measuring their dimensions in metaphase plates with chromosome 2 length from 6.5 to 8.5 micron. The regressional correction of the results of C-segment measurements or approximation of values depending on the statistical significance of linear regression equation coefficient has been demonstrated.     

Polymorphism of human chromosomes 1, 9, 16, Y: Variations,segregation and mosaicism

Summary A study was carried out on C-banded chromosomes 1, 9, 16, Y from an unselected population and from 30 normal families. We found: a) great variability in length and position of the C-bands; b) somatic mosaicism involving C-bands; c) variants in children that were not present in parental patterns. The possible role of crossing-over in generating the last two phenomena is discussed.     

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.     

Dynamic G- and R-banding of human chromosomes for electron microscopy

Synchronized human lymphocytes were exposed to 5-bromo-2-deoxyuridine (BrdUrd) for incorporation in either G-or R-bands. The substituted bands were revealed by monoclonal anti-BrdUrd antibodies disclosed with either gold-labeled antibodies or with the protein A-gold complex. Sharp G-or R-banding, specific for electron microscopy (EM), was obtained. These banding patterns, referred to as GB-AAu (G-bands by BrdUrd using Antibodies and gold [Au]) and RB-AAu (R-bands by BrdUrd using Antibodies and gold [Au]), resemble dynamic band patterns (GBG and RBG) much more than they do morphologic band patterns (GTG and RHG). The G- and R- band patterns allow accurate chromosome identification and karyotyping. An actual karyotype of human GB-AAu-banded chromosomes at the 750 band level, photographed in the EM, is presented. The method produces excellent band separation and band contrast. Variations in band staining intensities were noted and correlated with BrdUrd enrichment. The C-band regions were positively stained after GB-AAu banding while they were negatively stained after RB-AAu banding. Telomeres appeared heterogeneous after GB-AAu banding suggesting that part of the telomeric bands might be late replicating.     

Detection of small, single-copy genes on protein-G-banded chromosomes by electron microscopy.

A method for the detection by electron microscopy of chromosome banding after in situ hybridization of small, nonradioactive DNA sequences is described. Typical high-resolution G-banding is produced by adding 5-bromodeoxyuridine (BrdU) during the last part of the S-phase and by applying a monoclonal antibody against the BrdU-substituted chromosome segments, followed by the addition of protein G, but no further treatment. A protocol for in situ hybridization of small, single-copy biotinylated DNA sequences and their detection by immunogold tagging on banded chromosomes is also described. This combined approach permits high-resolution mapping of small DNA sequences and should be useful in discriminating between neighboring DNA fragments.     

Analysis of deoxyribonucleic acid replication in human X chromosomes by fluorescence microscopy.

The genetically inactive, late-replicating human female X chromosome can be effectively distinguished from its more active, earlier-replicating homologue, when cells are grown according to the appropriate BrdU-33258 Hoechst protocol. Results obtained from a fluorescence analysis of DNA replication in X chromosomes are consistent with those from previous autoradiographic studies, but reflect additional sensitivity and resolution offered by the BrdU-Hoechst methodology. Both qualitative and quantitative differences in 33258 Hoechst fluorescence intensity, reflecting alterations in replication kinetics, can be detected between the two X chromosomes in female cells. The pattern of replication in the single X chromosome in male cells is indistinguishable from that of the early female X. Intercellular fluctuations in the distribution of regions replicating early or late in S phase, particularly with reference to the late female X, can be localized to structural bands, suggesting multifocal control of DNA synthesis in X chromosomes.     

Mitomycin C induced structural changes in heterochromatic regions of human chromosomes 1, 9, 16 and Y

Aberrations and variations in the heterochromatic blocks of chromosomes 1, 9, 16 and Y were found under the influence of mitomycin C in cultured lymphocytes of peripheral human blood. Lymphocytes were cultured during 96 hours, mitomycin C in final concentration of 0.3 mkg/ml was present in the culture during the latest 24 hours of culturing. Different changes in the heterochromatic regions of chromosomes were found in approximately 30% of cells: in 6.3% of cells mitotic chiasmata were indicated. In 9.5% of cells isolocus breaks were observed in heterochromatic region of chromosome 1 in segment 1q11. In the latter case this may be a fragile site detected under the influence of mitomycin C on the lymphocytes.     

Differential digestion of the centromeric heterochromatic regions of the 5-azacytidine-decondensed human chromosomes 1, 9, 15, and 16 by NdeII and Sau3AI restriction endonucleases

A study on the factors involved in chromosome digestion by restriction endonuclease was carried out on 5-azacytidine treated and untreated human chromosomes 1, 9, 15 and 16 by using NdeII and Sau3AI isoschizomers. After treatment with 5-azacytidine, chromosomes 1, 9, 15, and 16 showed two differentiated areas at the centromeric regions: the centromere, fully condensed, and the pericentromeric heterochromatin, decondensed. Chromosomes not treated with 5-azacytidine after digestion with Sau3AI and NdeII showed all the centromeric regions undigested, except pair number 1, digested at the pericentromeric area. Digestion of the 5-azacytidine decondensed chromosomes with Sau3AI and NdeII showed the centromeres undigested in the four chromosome pairs while the pericentromeric heterochromatin appeared largely digested. Other factors, different to target distribution, are necessary to explain the pattern of restriction endonuclease digestion observed in this communication.     

Scanning electron microscopy of giemsa-stained chromosomes and surface-spread chromosomes

Giemsa-stained chromosomes as prepared for light microscopy, and including G-banded, C-banded, and FPG-stained chromosomes, were examined by scanning electron microscopy. Although suitable for light microscopy, these chromosomes were too flat for a close examination of their fine structure by scanning electron microscopy. The surface of Giemsa-positive regions was rough and bright, whereas that of unstained or poorly stained regions was smoother and less bright. Giemsa-staining, therefore, seems to produce the bulkiness of the chromosomes. On topographical examination by scanning electron microscopy, the transparent chromosomes as observed with the light microscope proved to be footprints. Stereographical examinations of surface-spread chromosomes showed that minimally stretched chromosomes were composed of a mass of nodular and twisted looping fibers with an average diameter of about 300 Å. The substructure of these chromosome fibers was not determined. The kinetochore region was discernible as a constriction in the mass of the chromosome fibers, and was distinguishable from gaps by the presence of several chromosome fibers parallel to the axis of the chromatid. The organization of the chromosome fibers, however, was disordered rather than regular.