High-resolution idiogram of Giemsa R-banded human prophase chromosomes

The schematic representation of RHG-banded chromosomes (R-banding was produced by heat denaturation followed by Giemsa staining (RHG) in the 850-band range per haploid set, was prepared showing the relative position, the specific size, and the characteristic staining intensity for each band. To this idiogram was adapted the new International Standard Cytogenetic Nomenclature. Our aim was to produce a realistic idiogram which could help in the preparation of R-banded prophase karyotypes and in the localization of chromosomal rearrangements. A comparative analysis of bands at prophase and metaphase revealed certain aspects of the dynamics involved in chromosome condensation and in R-band organization. The effect of chromosome elongation on the appearance of R-bands within heterochromatic regions has also been discussed.     

The ultrastructure of R-banded chromosomes

Electron microscopy has been used to study the fine structural organization of R-banded chromosomes prepared by treatment of the chromosomes with a hot NaH₂PO₄ solution. The results indicate that there is a structural basis for R-banding with this technique. In comparison to untreated control chromosomes, the R-banded chromosomes had a greatly reduced electron density, suggesting that the heat treatment has a general adverse effect on chromosome structure. Chromatin fibers formed a coarse, irregular network throughout the chromosome and were often enlarged, probably as a result of the fusion of two or more native fibers. The chromatin fibers were more aggregated and had an increased electron density in the R-band regions of the chromosome than in the interbands. This indicates that the treatment has a differential effect on the structure of bands and interbands. A comparison of the ultrastructure of R- and G-banded chromosomes demonstrated that the distribution of aggregated chromatin was reversed by these two types of banding techniques; however, the treatments producing R-banding appeared to induce less extreme differences in the degree of chromatin condensation in band and interband regions than those giving rise to G-banding. It is suggested that alterations of DNA-protein interactions may arise from the differential denaturation of proteins and/or DNA in R-band and interband regions during the heat pretreatment. Such differential alterations in DNA-protein interactions may induce localized changes in the organization of chromatin and may account for the subtle morphological differences observed between the band and interband regions.     

Centromeric banding (C) of sequentially Q- and R-banded human chromosomes

Summary A modified C-banding technique is described that produces C bands on human chromosomes after sequential Q and R banding and retains good chromosome morphology. Despite the considerable exposure to UV light during sequential Q and R bandings, clear C bands could still be achieved. Employing the present technique, Q, R, and C polymorphisms can be recorded on a single metaphase.     

Localization of actin-related sequences by in situ hybridization to R-banded human chromosomes

Using a cytoplasmic actin cDNA probe we have localized a number of actin sequences in the human genome using a novel in situ hybridization technique. Metaphase chromosomes treated to produce R-bands were directly annealed with ¹²⁵I-labeled actin probe. Under these conditions many regions of the genome were apparently denatured enough to be capable of hybridizing with the probe. Most of the actin sites detected in prior experiments using chromosome preparations, which had been completely denatured, were recognized in this experiment. The major advantage of this method over standard in situ hybridization techniques is the marked increase in the resolution of subregional localization.     

Autoradiographic studies of human chromosomes

Certain differences in the location of chromosomal material completing DNA synthesis late in the S-period of the cell cycle were demonstrated when a comparison was made between human blood lymphocytes and epithelial cells derived from term amnion grown in vitro for short periods of time. The differences in the patterns of synthesis between these two differentiated diploid cells, each from the same species but of different embryonic origins (mesodermal vs. ectodermal), functions in vivo, and appearances and growth characteristics in vitro, may be reflections of distinctive patterns of condensed interphase chromatin, i.e. a characteristic distribution of heterochromatin, and possibly also of different cellular functions in the organisms.Supported by research grants from the U.S. Public Health Service (HD 04134) and the National Science Foundation (GB 6282). These data were presented at the Fourth Basel Colloquium on Mammalian Sex Chromosomes in Differentiation and Development, March, 1967.     

Establishment of an R-banded rabbit karyotype nomenclature by FISH localization of 23 chromosome-specific genes on both G- and R-banded chromosomes

Direct detection of fluorescent in situ hybridization signals on R-banded chromosomes stained with propidium iodide is a rapid and efficient method for constructing cytogenetic maps for species with R-banded standard karyotypes. In this paper, our aim is to establish an R-banded rabbit karyotype nomenclature that is in total agreement with the 1981 G-banded standard nomenclature. For this purpose, we have produced new GTG- and RBG-banded mid-metaphase karyotypes and an updated version of ideograms of R-banded rabbit chromosomes. In addition, to confirm correlations between G- and R-banded chromosomes, we have defined a set of 23 rabbit BAC clones, each containing a specific gene, one marker gene per rabbit chromosome, and we have localized precisely each BAC clone by FISH on both G- and R-banded chromosomes.     

Autoradiographic studies of human chromosomes

The DNA content of the elongated long arm of an inherited variant No. 16 chromosome was compared with that of the non-elongated long arm of the other No. 16 in the same chromosomal complement. An indirect method of estimation of DNA content was employed, based on the number of autoradiographic grains produced by the segments after they had been labeled with H³-thymidine throughout an S-period. The method proved adequately sensitive to detect a difference in number of grains—and presumably in DNA content—between the short arm and the long arm of a normal chromosome No. 16. The failure to detect an increased number of grains over the elongated long arm of the variant No. 16, in comparison with the other No. 16's long arm in the same cells, favors explanations other than an increase in content of DNA to account for this well-known morphological variation of the human No. 16 chromosome.This research was supported by National Institutes of Health Grant HD 04134-01 and American Cancer Society Grant E-461.     

Autoradiographic studies of human chromosomes

Chromosomal sites of DNA synthesis during the final 30 minutes or less of the S-phase of the cell division cycle of fibroblasts were delinated autoradiographically. Very light labeling was found, indicating that a recognizable but very minor portion of the cell's DNA is synthesized during a few minutes at the extreme end of S. This interval immediately follows those periods near the end of S when prominent synthetic asynchrony exists in different chromosomal regions. A non-random distribution of label, but one different from the more familiar end-of-S pattern, was detected during this final interval. The late-replicating X was less heavily labeled than some autosomes during the final minutes of S, while sites in chromosome No. 3 were somewhat more heavily labeled than those in other chromosomes. The biological significance of these minute, last-to-replicate chromosomal regions is unknown.     

Early studies on human chromosomes

The author describes his introduction to the field of cytogenetics, with his first viewing of himself, cytogenetically, down the microscope, and the progression of human cytogenetics as an area of study up to its modern integration with molecular genetics and computer technology.     

Engineering human chromosomes for gene therapy studies

We now have the capability to engineer human chromosomes that could be used to deliver therapeutic genes in gene therapy studies. These vectors have the advantages of being non-disruptive to the genome, non-immunogenic and are capable of carrying very large genes with all their regulatory sequences. What challenges lie ahead, and what future does this technology hold for gene therapy?     

Immunofluorescent studies of human chromosomes with antibodies against phosphorylated H1 histone

One of the prominent cell cycle-related modifications of histone proteins whose function remains unresolved is the phosphorylation of linker histone H1. In this work we have used indirect immunofluorescence on human cells with antibodies that are specific for phosphorylated histone H1 to examine the cellular distribution and chromosome association patterns of this protein. With confocal microscopy on whole cells, strong immunofluorescence was seen in association with mitotic chromosomes as well as a prominent punctate pattern of labeling throughout the mitotic cell, whereas interphase cells showed very little, if any, specific fluorescence. Multiple patterns of fluorescence distribution were detected with metaphase chromosomes, ranging from apparent tight colocalization with the DNA to expanded ”puffy” mitotic figures to an amorphous network of staining. It was also shown that the ability to label chromosomes could vary drastically with different fixation procedures, adding further complications to interpretation of the potentially complex role of phosphorylated histone H1 in chromatin condensation or decondensation. Received: 8 September 1999; in revised form: 14 September 1999 / Accepted: 17 September 1999     

Cytogenetic studies in human cells exposed in vitro to GSM-900 MHz radiofrequency radiation using R-banded karyotyping

It is important to determine the possible effects of exposure to radiofrequency (RF) radiation on the genetic material of cells since damage to the DNA of somatic cells may be linked to cancer development or cell death and damage to germ cells may lead to genetic damage in next and subsequent generations. The objective of this study was to investigate whether exposure to radiofrequency radiation similar to that emitted by mobile phones of second-generation standard Global System for Mobile Communication (GSM) induces genotoxic effects in cultured human cells. The cytogenetic effects of GSM-900 MHz (GSM-900) RF radiation were investigated using R-banded karyotyping after in vitro exposure of human cells (amniotic cells) for 24 h. The average specific absorption rate (SAR) was 0.25 W/kg. The exposures were carried out in wire-patch cells (WPCs) under strictly controlled conditions of temperature. The genotoxic effect was assessed immediately or 24 h after exposure using four different samples. One hundred metaphase cells were analyzed per assay. Positive controls were provided by using bleomycin. We found no direct cytogenetic effects of GSM-900 either 0 h or 24 h after exposure. To the best of our knowledge, our work is the first to study genotoxicity using complete R-banded karyotyping, which allows visualizing all the chromosomal rearrangements, either numerical or structural.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The association pattern was studied in 1182 mitoses of 21 patients with trisomy 13 and in a control group. In addition, 173 trisomic mitoses were compared with the same number of diploid mitoses in a case of mosaicism.The number of mitoses with associations was no higher in the trisomic cells than in cells with normal karyotypes. Some differences were observed in the frequency of associations per cell and of the types of associations in the patient group and in the trisomic cells of the mosaic case. The number of associations in which more than two acrocentric chromosomes were involved was unexpectedly low in the cells with a supernumerary chromosome 13.The result are interpreted as suggesting the existence of a compensatory mechanism activated by the additional acrocentric chromosome.Parts of this work are included in the doctoral (MD) thesis of DM     

Quantitative studies on the arrangment of human metaphase chromosomes

Summary The pattern of association of acrocentric chromosomes was examined in ten and five carriers of a 15/21 and a 13/14 Robertsonian translocation, respectively, and was compared with that of the same numbers of relatives with normal karyotypes. In the carriers of 15/21 translocation, the number of large associations (involving more than two acrocentrics) and the association frequencies for individual acrocentric chromosomes, were significantly higher than in the control group. The mean number of associations of the single homologs of the translocation chromosomes was much higher than that of the other acrocentrics. In the carriers of 13/14 translocations, only the association frequency for chromosome 13 was higher than in the normal relatives. The uninvolved chromosomes homologous to those involved in translocations showed an insignificant increase in associations in comparison with the other acrocentrics. These results suggest that some mechanism within the cells compensates for the effect of missing acrocentrics or of acrocentrics lacking NORs on the number of associations. The possible relations of this phenomenon to the activity of the nucleolus organizing regions are discussed.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The spatial relationships between the homologous pairs of chromosomes in the normal human colcemid-treated metaphase plate were tested by two different mathematical approaches: (a) determination of the distances between the centromeres of the homologous chromosomes compared to the mean distance of all centromeres of the mitosis in question; (b) measuring the distances of the different chromosomes from the center of the mitosis.The following results were obtained: (1) The arrangement of human metaphase chromosomes does not follow a normal distribution; the distribution is narrower and taller, probably due to an impairment of free chromosome spreading by the cell membrane. We believe that only in membraneless mitotic cells should the chromosome-spread correspond to a normal distribution under the same preparation conditions. (2) There is a positive correlation between decreasing chromosome size and decreasing mean distance between homologous chromosomes. (3) A close positive correlation exists between increasing chromosome size and increasing distance to the barycenter of the mitosis. (4) There is also a close positive correlation between the distance of homologous chromosome pairs and their distance from the center of the mitosis, i.e., with increasing distance from the center of the mitosis, the distance between the homologous partners increases. (5) The following statistically significant deviations from these rules could be established: (a) The large acrocentric chromosomes are closer associated, as one would expect from their size, probably due to their participation in the nucleolus organization; (b) in the female cell one of the two X chromosomes has an extremely peripheral localization; the X chromosomes are furthest apart of all pairs of homologous chromosomes; (c) the chromosome pairs 6 and 8 are relatively close together in spite of their peripheral position, suggesting a truc close association of the homologus partners; (d) the chromosome pair 18 has a more peripheral position than expected, and a relatively large mean distance between the homologous partners; (e) the chromosome pair 1 has a much more central position and a closer association than is expected from its size.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The association pattern was studied in 2715 mitoses of 90 meningiomas with different numbers of acrocentric chromosomes. In cells with monosomy 22, a significant increase of mitoses with associations was observed in comparison to cells with a normal karyotype. The number of associating acrocentric chromosomes was highly significantly increased. This surplus was not only caused by a highly significant increase of associating G chromosomes but also of D chromosomes. The loss of further acrocentric chromosomes had no significant influence on the number of mitoses with associations or the number of associating chromosomes. Based on the well-known correlations between the nucleolus organization and the association pattern, the results seem to indicate a compensation mechanism among the nucleoles organizing regions (NOR's) which keeps the supply of nucleolar material constant and simultaneously causes a higher association tendency between the remaining acrocentric chromosomes. The increase of associations in the 22 monosomic cells was interpreted as a overcompensation after the loss of only one NOR.     

Quantitative studies on the arrangement of human metaphase chromosomes. IX. Arrangement of chromosomes with and without spindle apparatus

Summary The spatial relationships in human male metaphase cells treated with and without colcemide were compared with each other. The following results were obtained: (1) In normal male metaphases the overall distributions of chromosomal distances regardless of chromosome identification numbers did not show normal distribution, neither in the colcemid-free sample nor in the colcemide-treated sample. (2) In both samples larger chromosomes showed a more peripheral position, and smaller chromosomes showed a more central position. This finding was statistically significant. (3) No differences between the two samples could be observed concerning the following parameters: overall distributions of the centromere-centromere distances, distributions of the distances between the homologous chromosomes (except the small acrocentric chromosomes), rank positions of the mean distances between homologous chromosomes, and rank positions of the mean distances of the different chromosomes from the center of the mitosis (except few chromosomes). (4) Visible, but not statistically accessible, differences appeared between the two samples in respect to rank positions of the mean distances of all possible acrocentric pairing groups, rank positions of the mean distances of the homologous acrocentric chromosomes from the center of the mitosis, and distances of the X chromosome from the center of the mitosis. (5) Statistically significant differences appeared between the two samples with respect to distance distributions of the small acrocentric chromosomes and positions of the chromosomes 1, 16, 18, Y, and 21, 22 in relation to the center of the mitosis.