The ring chromosome 13 syndrome

Summary A study of the ring chromosome 13 syndrome is presented with detailed clinical and cytogenetic features of three new unrelated cases. The clinical limits of this syndrome can now be defined. An analysis of these cases together with those in the literature indicates that the syndrome forms a continuous spectrum, and no further taxonomic subdivision is possible at this stage of knowledge. The chromosome breakpoints in the first two cases are 13p11 and 13q32 and in the third case 13p11 and 13q33 or 13q34. All described cases of the ring 13 syndrome have breakpoints within the region bounded by bands 13q21 to 13q34. All rings are negative for silver banding. Peripheral blood cultures showed an average of 88% of metaphases to be 46.XX,r(13), with the remaining 12% manifesting either random loss or ring duplication. The rings vary in size and show a variable number of centromeres. An estimate of the birth incidence of this condition in the Anglo-Saxon population is 1 in 58,000. Parents of affected children are clincally and cytogenetically normal, the rings in affected offspring being meiotic in origin.     

Synchronization of human leukemic cells: Relevance for high-resolution chromosome banding

Summary The cell-cycle kinetics of synchronized K562 human leukemic cells and bone marrow cells from adults with acute leukemia were studied in order to develop more reliable methods for producing increased numbers of mitoses, particularly those with elongated chromosomes suitable for high-resolution banding. Parameters examined included DNA content, mitotic index (MI), and chromosome preparations. K562 cells synchronized with methotrexate (MTX), thymidine (Tdr), or hydroxyurea (HU) showed two-fold increases in peak MI. Optimal harvesting times after release from block were approximately 10.5, 12.5, and 14.5 h for MTX, HU, and Tdr, respectively. MTX was selected for studies with cells from patients. Cells from 7 of the 10 patients studied showed 4.4-fold increases in peak MI. The optimal harvesting time was 9.5 to 11.5 h after release from block, considerably later than the 6 h time previously assumed in studies using stimulated lymphocytes. Cells from the three remaining patients showed no increase in MI after synchronization: and the lack of response may have been related to the high proportion of cells in G₀+G₁ prior to MTX exposure. For both the K562 cell line and most patient specimens, the combination of synchronization with appropriate release times and short Colcemid exposure (10 min) resulted in substantially improved chromosome preparations.     

Zebrafish chromosome banding.

Banding techniques were carried out on metaphase chromosomes of zebrafish (Danio rerio) embryos. The karyotypes with the longest chromosomes consist of 12 metacentrics, 26 submetacentrics, and 12 subtelocentrics (2n = 50). All centromeres are C-band positive. Eight chromosomes have a pericentric C-band in each arm and 22 chromosomes have one in the longest arm. Two chromosomes have a slightly heterochromatic long arm and five chromosomes have an Ag-NOR at the terminal end of the long arm. Other banding patterns and sex chromosomes could not be revealed.     

Mechanisms of chromosome banding. V. Quinacrine banding.

A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quanacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound be intercalation with relatively little sid binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nuclei acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2 times 10-6 to 2 times 10-5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescenc. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCL AND Cs-2SO-4-Ag+ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding.We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins.     

A case of D13 ring chromosome

Summary A case of D ring chromosome identified with trypsin banding as a 13 with loss of the bands p12 and q34 is reported. The clinical features characteristically associated with the loss of these specific segments were present.     

Improved definition of chromosomal breakpoints using high-resolution multicolour banding

Characterisation of chromosome rearrangements using conventional banding techniques often fails to determine the localisation of breakpoints precisely. In order to improve the definition of chromosomal breakpoints, the high-resolution multicolour banding (MCB) technique was applied to identify human chromosome 5 breakpoints from 40 clinical cases previously assessed by conventional banding techniques. In 30 cases (75%), at least one breakpoint was redefined, indicating that MCB markedly improves chromosomal breakpoint localisation. The MCB pattern is highly reproducible and, in contrast to conventional banding pattern, is consistent in both short and elongated chromosomes. This might be of fundamental interest for the detection of chromosomal abnormalities, especially in tumour cells. Moreover, MCB even allows the detection of abnormalities that remain cryptic in GTG-banding analysis.     

Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high-resolution chromosome banding

Ethidium bromide (EB) is known to intercalate between stacked base pairs without specific base-pair preference. Its use in cultured human lymphocytes and Burkitt's lymphoma cells resulted in the accumulation of cells in prophase and prometaphase stages. Inhibition of mitotic chromosome condensation as a possible mechanism involved in this phenomenon is discussed. A simple method for obtaining high-resolution banding patterns on elongated chromosomes was devised as follows: Human lymphocytes cultured for 3 days with phytohemagglutinin were exposed to EB (5-10 micrograms/ml) and Colcemid (0.02 micrograms/ml) simultaneously for 2 h and then routinely harvested for chromosome preparation. High-resolution G-bands were obtained by Giemsa staining following mild trypsin treatment.     

Mechanisms of ring chromosome formation in 11 cases of human ring chromosome 21

We studied the mechanism of ring chromosome 21 (r(21)) formation in 13 patients (11 unique r(21)s), consisting of 7 from five families with familial r(21) and 6 with de novo r(21). The copy number of chromosome 21 sequences in the rings of these patients was determined by quantitative dosage analyses for 13 loci on 21q. Nine of 11 r(21)s, including the 5 familial r(21)s, showed no evidence for duplication of 21q sequences but did show molecular evidence of partial deletion of 21q. These data were consistent with the breakage and reunion of short- and long-arm regions to form the r(21), resulting in deletion of varying amounts of 21q22.1 to 21qter. The data from one individual who had a Down syndrome phenotype were consistent with asymmetric breakage and reunion of 21q sequences from an intermediate isochromosome or Robertsonian translocation chromosome as reported by Wong et al. Another patient, who also exhibited Down syndrome, showed evidence of a third mechanism of ring formation. The likely initial event was breakage and reunion of the short and long arms, resulting in a small r(21), followed by a sister-chromatid exchange resulting in a double-sized and symmetrically dicentric r(21). The phenotype of patients correlated well with the extent of deletion or duplication of chromosome 21 sequences. These data demonstrate three mechanisms of r(21) formation and show that the phenotype of r(21) patients varies with the extent of chromosome 21 monosomy or trisomy.     

Functional implications of differential chromosome banding.

Information on DNA content and banding patterns has been utilized in the construction of a novel ideogram of the human complement. Correspondence between certain features of this ideogram and the clinical record of human autosomal imbalance supports the idea that banding patterns reflect structural as well as functional heterogeneity of chromosomes.     

Mechanisms of chromosome banding

A thorough understanding of the mechanisms of R-, C-and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral adsorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. — Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not a necessary component for banding. — The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their adsorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.     

Mechanisms of chromosome banding

The interaction of Hoechst 33258 with DNA has been examined to help clarify the mechanisms of banding. 1. In agreement with previous studies Hoechst fluorescence is enhanced to a greater degree in AT-rich compared to GC-rich DNA. 2. Hoechst causes an increase in the DNA Tm which is greater at the higher AT content of the DNA. 3. There is a decrease in extinction coefficient and shift in the adsorption spectra to a higher wavelength when Hoechst binds to DNA. 4. DNA is completely precipitated at a ratio of one dye molecular per base pair, and this precipitation is not affected by salt. 5. There is no increase in viscosity or change in the circular dichroism of DNA when bound to Hoechst. These findings suggest Hoechst does not bind to DNA by intercalation or by ionic interaction with the phosphate groups, but rather binds by an attachment to the outside of the double DNA helix by interacting with the base pairs. This type of binding allows greater sensitivity to the base composition than occurs with intercalating agents. In this respect its binding is similar to that of dibutyl proflavine (Muller et al., 1973).     

Mechanisms of chromosome banding

The G-band patterns of mitotic metaphase chromosomes No. 1 and 2 of the Chinese hamster cells correlate closely to the chromomere patterns of the meiotic pachytene bivalents. This is interpreted to indicate that the regions of centromeric and intercalary heterochromatin, which are more tightly condensed or more tightly packaged during interphase, tend to remain so during meiosis and mitosis.     

Mechanisms of chromosome banding

Photo-oxidation of mitotic human chromosomes has been used in conjunction with anti-cytosine and anti-adenosine antibodies to produce R-banding. To elucidate the mechanism of this banding procedure we have examined the effect of photo-oxidation alone on chromosomes and nuclei. With short exposures to light in the presence of dilute methylene blue, C-band areas on chromosomes 1, 9, 16 and the terminal segment of the Y stain poorly. We call this phenomena reverse C-banding. After 18 h of exposure to light the chromosomes are swollen and show very little staining with quinacrine or Giemsa. Quantitative autoradiography shows that their DNA is almost completely extracted. Cytophotometric measurements also confirm that nuclear DNA is progressively extracted according to the length of exposure to light. When chromosomes are exposed to dilute methylene blue alone, without light, G-banded chromosomes result. We suggest the following explanation for these observations. In dilute methylene blue, C-band regions take up the greatest amount of dye and after short periods of photo-oxidation the DNA of these regions is preferentially destroyed resulting in reverse C-banding. Autoradiography in photo-oxidized chromosomes suggested that this preferential destruction of C-segments occurred in our experiments. With more prolonged exposure the DNA of the G-bands regions is preferentially destroyed and staining the remaining DNA with sensitive fluorescent labeled anti-C antibodies results in R-banding.     

Mechanisms of chromosome banding

Prior studies on subfractions of mouse and Kangaroo rat DNA have suggested that variations in base concentration within a given genome may not be great enough to account for Q-banding. To examine this with another species, calf DNA was subfractionated by CsCl ultracentrifugation into GC-rich satellites and the main band DNA was further fractionated into AT-rich, intermediate and GC-rich portions. The effect of varying concentrations of these DNAs on quinacrine and Hoechst 33258 fluorescence was examined. Although with both compounds there was less fluorescence in the presence of the GC-rich satellites than main band fractions, these results per se did not answer the question of whether the variation in base composition alone was adequate to account for chromosome banding. To answer this the fluorescence observed in the presence of DNA of a given base composition was related to the fluorescence observed in the presence of DNA of 40% GC content (F/F40). This allowed the derivation of a term B which indicated the relative change in fluorescence per 1% change in base composition of DNA. To determine the percent change in fluorescence observed in Q-banding, the photoelectric recordings of Caspersson et al. (1971) were used. From these data we conclude: 1. Quinacrine is twice as sensitive to changes in base composition as Hoechst 33258. 2. Variation in the base content of DNA along the chromosome is sufficient to account for most Q-banding, except possibly for some of the extremes of quinacrine fluorescence. This was further examined with daunomycin. Even though daunomycin gives good fluorescent banding, DNAs varying in base composition from 100 to 40% GC content all resulted in the same relative fluorescence of 0.03. However, in the presence of poly (dA-dT) the relative fluorescence was 0.85, indicating a great sensitivity to very AT-rich DNA. This suggests that with daunomycin and possibly other fluorochromes, stretches of very AT-rich DNA may be more important in fluorescent banding than simple variation in mean base composition.     

Mechanisms of chromosome banding

A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quinacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound by intercalation with relatively little side binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nucleic acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2×10^?6 to 2×10^?5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescence. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCl and Cs₂SO₄-Ag⁺ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding. — We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins.     

Mechanisms of chromosome banding

The binding of methylene blue to DNA and chromatin treated in various ways was examined by equilibrium dialysis. The maximum r value (moles of bound dye/mole of nucleotide) was 1.0 for DNA, 0.6 for unfixed chromatin, and 0.83 for chromatin fixed in methanol-acetic acid. When fixed chromatin was treated with saline-citrate at 60° C for 3 hours, as used for G-banding chromosomes, the r value decreased from 0.83 to 0.55. When unfixed chromatin was treated as for R-banding the r values also dropped. Equilibrium dialysis indicated there was no disproportionate increase of dye binding as the concentration of DNA increased. — These results, and others, suggest that some of the Giemsa negative regions of G- and R-banded chromosomes are due to the denaturation of non-histone proteins so that they more effectively cover the DNA and prevent side binding of the thiazin dyes.     

Analysis of banding patterns and mosaic configurations in a case of ring chromosome 15

Summary Cytogenetic studies on lymphocytes from a 14-year-old mentally retarded girl with somatic anomalies suggestive of a chromosomal abnormality revealed a ring chromosome 15. The long arm of the defective chromosome is broken at band q24 or q25. The silver staining technique for nucleolus organizer regions showed that the ring had lost the achromatic stalk and the satellite. The chromosomal mosaicism resulting from the structural instability of the ring chromosome was analyzed and compared with 6 cases reported in the literature. It is proposed that the clinical manifestations in the different patients with ring chromosome 15 result from both the deficiency in the long arm and the mosaic configurations.