Specific Heterochromatic Banding of Metaphase Chromosomes Using Nuclear Yellow

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric hete-rochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.     

The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes

The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A-T and G-C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another. —Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G-C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A-T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A-T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G-C specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.—The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.     

鱼类染色体的荧光显带研究

应用GC碱基特异性荧光染料色霉素A,辅以AT减基特异性荧光染料Hoechst33258,DAPI或喹吖因对鲤鱼,鲫鱼,大鳞副泥鳅和的有丝分裂染色体及黄鳝的有丝分裂和减数分裂染色体进行了荧光显带研究,结果发现,色霉素A3可以特异性地显示鱼类有丝分裂及减数分裂各个时期核仁组织区NORS的存在,Hoechst33258,DAPI或喹吖因则使这些区域（NORs)淡染,大鳞副泥鳞的染色体NORs 分布位置具有性别,根据实验结果,对有关鱼类染色体的荧光染色研究及其应用进行了讨论。     

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.     

QFH banding and DNA distribution in the macrochromosomes of the chicken Gallus domesticus

Chromosomes of Gallus domesticus were stained with rivanolum-SO2 (fluorescence Feulgen reaction) and by Hoechst 33258. Fluorescence photography was performed on a 35 mm film KN-3(KN-3) "Svema". The negatives were analyzed with the microdensitometer. The Feulgen (Fr--) and the Hoechst 3358 (H--) densitometric profiles of chromosomes showed light and dark segments along the metaphase chromosomes. The amount of DNA, as determined by the fluorescence Feulgen reaction, is not constant along the chromosome arms. Consequently, the base composition is not the only factor influencing the fluorescence of Hoechst 33258 along the chromosomes. The comparative analysis of densitometric profiles of the Hoechst 33258 and rivanolum-SO2 stained chromosomes shows that the Iq telomere band consists of GC-rich DNA (about 70% GC). The variation of DNA contents along the metaphase chromosome arms can be realized at both chromoneme and/or subchromoneme levels.     

Use of DNA Fluorochromes for Studying Meiosis in the Woody Species Thryptomene Calycina

The fluorescent DNA probes DAPI and Hoechst 33258 produce superior images to the traditional acetocarmine stain of the small chromosomes of the woody shrub Thryptomene calycina at all stages of microsporocyte meiosis and microspore mitosis. Hoechst 33258 was slightly superior to DAPI because of reduced background fluorescence. Binding with the DNA-specific probes required a fixative containing chloroform to remove autofluorescent materials, a pretreatment with acetic acid and a pH of least 6 during treatment. The nucleoli did not fluoresce after treatment with DAPI or Hoechst 33258. Superior resolution of chromosomes after treatment with the fluorochromes enabled easy determination of the haploid number at metaphase I, metaphase II and at metaphase of the microspore mitosis.     

Banding Human Chromosomes Using a Combined C-Banding-Fluorochrome Staining Technique

Slides pretreated for C-banding and stained with DAPI or CMA3 show different banding patterns in human metaphase chromosomes compared to those obtained with either standard Giemsa C-banding or fluorochrome staining alone. Human chromosomes show C-plus DA-DAPI banding after C-banding plus DAPI and enhanced R-banding after C-banding plus Chromomycin A3 staining. If C-banding preferentially removes certain classes of DNA and proteins from different chromosome domains, C-banding pre-treatment may cause a differential DNA extraction from G- and R-bands in human chromosomes, resulting in a preferential extraction of DNA included in G-bands. This hypothesis is partially supported by the selective cleavage and removal of DNA from R-bands of restriction endonuclease HaeIII with C-banding combined with DAPI or Chromomycin A3 staining. Structural factors relating to regional differences in DNA and/or proteins could also explain these results.     

Bandign isolated metaphase chromosomes by a sequential flurescent G/Q technique.

G/Q-banding is a new, rapid, fluorescent technique for banding isolated chromosomes that incorporates characteristics of both G- and Q- banding. G-bands, while easily characterized, are often inconsistent when using isolated chromosomes, and Q-bands, while reliable, fade rapidly under UV exposure, making prolonged observation and photography difficult. G/Q-banding combines these techniques to sequentially utilize quinacrine staining over Giemsa banding to produce slow-fading fluorescent G/Q-bands. The background fluorescence in G/Q preparations fades quickly under continued UV exposure, while the chromosomes remain brightly banded and can be observed and photographed for at least five minutes. G/Q-banding was extended to whole cell chromosome spreads and produced results identical to those obtained with isolated chromosomes. Whole cell karyotypes indicate that G/Q-bands generally correspond to Q-bands. Advantages of G/Q-banding as a unique and universal technique over current double-staining procedures are discussed.     

Use of DNA fluorochromes for studying meiosis in the woody species Thryptomene calycina.

The fluorescent DNA probes DAPI and Hoechst 33258 produce superior images to the traditional acetocarmine stain of the small chromosomes of the woody shrub Thryptomene calycina at all stages of microsporocyte meiosis and microspore mitosis. Hoechst 33258 was slightly superior to DAPI because of reduced background fluorescence. Binding with the DNA-specific probes required a fixative containing chloroform to remove autofluorescent materials, a pretreatment with acetic acid and a pH of least 6 during treatment. The nucleoli did not fluoresce after treatment with DAPI or Hoechst 33258. Superior resolution of chromosomes after treatment with the fluorochromes enabled easy determination of the haploid number at metaphase I, metaphase II and at metaphase of the microspore mitosis.     

Banding isolated metaphase chromosomes by a sequential fluorescent G/Q technique

Summary G/Q-banding is a new, rapid, fluorescent technique for banding isolated chromosomes that incorporates characteristics of both G- and Q-banding. G-bands, while easily characterized, are often inconsistent when using isolated chromosomes, and Q-bands, while reliable, fade rapidly under UV exposure, making prolonged observation and photography difficult. G/Q-banding combines these techniques to sequentially utilize quinacrine staining over Giemsa banding to produce slow-fading fluorescent G/Q-bands. The background fluorescence in G/Q preparations fades quickly under continued UV exposure, while the chromosomes remain brightly banded and can be observed and photographed for at least five minutes. G/Q-banding was extended to whole cell chromosome spreads and produced results identical to those obtained with isolated chromosomes. Whole cell karyotypes indicate that G/Q-bands generally correspond to Q-bands. Advantages of G/Q-banding as a unique and universal technique over current double-staining procedures are discussed.     

Counterstaining human chromosomes for flow karyology

Isolated human metaphase chromosomes stained with the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and chromomycin A3(CA3), and counterstained with nonfluorescent netropsin (NTR), have been analyzed by dual-laser flow cytometry. Counterstaining with NTR reduces DAPI fluorescence except at regions on chromosomes 1,9,15,16, and Y, corresponding to C-band heterochromatin. Bivariate flow karyology of human chromosomes treated with this triple-stain combination resolves chromosomes 1,9, and Y distinctly from the remaining chromosomes and resolves variations between chromosome homologues not detected by staining with propidium iodide (PI) or with the double stain combination Hoechst 33258(HO) and CA3.     

Genetic activity of the G- and R-band DNA of human mitotic chromosomes

The concept of genetic inactivity of G-band DNA had been reinvestigated using the modified approach of Korenberg et al (1978). Coefficients of correlation and partial correlation between the relative gene density (g'), the relative G-band material richness (kH/C) and the relative chromosome size (s') were calculated. The kH/C was calculated as the ratio of brightness of fluorescence of chromosomes stained by Hoechst 33258 (Hi) and by chromomycin A3(Ci). The kH/C is the characteristics of G-band chromosome richness, because G-bands become bright after Hoechst 33258 staining and R-bands are bright after chromomycin A3 staining, while no significant C-bands in chromosomes which may be stained by these fluorochromes are discovered. For the kH/C determination the flow cytometry data of Langlois et al (1982) were used. The relative size of chromosomes was determined, based on the flow cytometry data of Young et al (1979). According to Korenberg, the "gene density" (g') in a chromosome was calculated as a ratio of the number of genes located in the chromosome before 1984 (Human Gene Mapping 7) to the relative size of this chromosome. Correlation between the "gene density" and the G-band richness was rs = -0.65. Out of 107 genes located in either G- or R-bands (Human Gene Mapping 7), 90 were mapped in the R-band and only 17 were ascribed to the G-band in metaphase chromosomes. The data on gene replication time show that all genes of the general cell activity and a portion of tissue-specific genes replicate during the early S-phase, together with R-band materials. These three independent lines of evidence are consistent with the notion that the R-band DNA is more genetically active than G-band DNA. The nature of "junk" DNA of G-bands is discussed.     

Energy transfer and binding competition between dyes used to enhance staining differentiation in metaphase chromosomes

The ability of electronic energy transfer and direct binding competition between pairs of dyes to enhance contrast in human or bovine metaphase chromosome staining patterns is illustrated, and the relative effectiveness of these two mechanisms compared. The existence of energy transfer between quinacrine or 33258 Hoechst and 7-amino-actinomycin D in doubly stained chromosomes is demonstrated directly by microfluorometry. The ability of the dyes 7-amino-actinomycin D, methyl green, or netropsin, acting as counterstains, to displace quinacrine, 33258 Hoechst, or chromomycin A₃ from chromosomes, is estimated by quantitative analysis of energy transfer data, by photobleaching of the counterstains, or by selective removal of counter-stains by appropriate synthetic polynucleotides. Effects on the fluorescence of soluble 33258 Hoechst-DNA complexes due to energy transfer or binding displacement, by actinomycin D or netropsin, respectively, are further differentiated by nanosecond fluorescence decay measurements. Examples are presented of dye combinations for which (a) energy transfer is the primary mechanism operative, (b) binding competition exists, with consequences reinforcing those due to energy transfer, or (c) binding competition is the most important interaction. These analyses of mechanisms responsible for contrast enhancement in doubly stained chromosomes are used to derive information about the relationship between chromosome composition and banding patterns.     

High resolution chromosome analysis: one and two parameter flow cytometry

Isolated mammalian chromosomes have been quantitatively classified by high resolution flow cytometry. Chinese hamster chromosomes stained with 33258 Hoechst and excited in the UV showed a fluorescence distribution in which the 14 types of Chinese hamster chromosomes were resolved into 16 groups seen as distinct peaks in the distributions. Chinese hamster chromosomes were also stained with both 33258 Hoechst (HO) and chromomycin A3 (CA3); the two dye contents were measured by selective excitation in the UV and at 458 nm in a dual beam flow cytometer. The resulting two parameter distribution (HO versus CA3) showed 10 chromosome groups¹. Human strain LLL 761 chromosomes stained with HO and excited in the UV showed a fluorescence distribution in which the 23 types of human chromosomes were resolved into 12 groups. Human chromosomes stained with both HO and CA3 and measured in the dual beam flow cytometer produced two parameter fluorescence distributions which showed 20 groups. The chromosomes associated with each group were determined by quinacrine banding analysis of sorted chromosomes and by DNA cytophotometry of preidentified metaphase chromosomes. The relative HO and CA3 stain content and frequency of occurrence of chromosomes in each group were determined from the fluorescence distributions and compared to the results from DNA cytophotometry. The chromosome to chromosome variations in HO and CA3 staining are attributed to variations in chromosomal base composition.     

Hoechst 33258 fluorescent staining of Drosophila chromosomes

Metaphase chromosomes of D. melanogaster, D. virilis and D. eopydei were sequentilly stained with quinacrine, 33258 Hoechst and Giemsa and photographed after each step. Hoechst stained chromosomes fluoresced much brighter and with different banding patterns than quinacrine stained ones. In contrast to mammalian chromosomes, Drosophia's quinacrine and Hoechst bright bands are all in centric heterochromatin and the banding patterns seem more taxonomically divergent than external morphological characteristics. Hoechst stained D. melanogaster chromosomes show unprecedented longitudinal differentiation by the heterochromatic regions; each arm of each autosome can be unambiguously identified and the Y shows eleven bright bands. The Hoechst stained Y can also be identified in polytene chromocenters. Centric alpha heterochromatin of each D. virilis autosome is composed of two blocks which can be differtiated by a combination of quinacrine and Hoechst staining. The distal block is always Q-H- while the proximal block is, for the various autosomes, either Q-H-, Q+H- or Q+H+. With these permutations of Hoechst and quinacrine staining, D. virilis autosomes can be unambiguously distinguished. The X and two autosomes have H+ heterochromatin which can easily be seen in polytene and interphase nuclei where it seems to aggregate and exclude H- heterochromatin. This affinity of fluorochrome similar heterochromatin was been seen in colcemide induced multiple somatic non-disjunctions where H+ chromosomes were distributed to one rosette and H- chromosomes were distributed to another. Knowing the base composition and base sequences of Drosophila satellites, we conclude that AT richness may be necessary but is certainly an insufficient requirement for quinacrine bright chromatin while GC richness may be a sufficient requirement for the absence of quinacrine or Hoechst brightness. Condensed euchromatin is almost as bright as Q+ heterochromatin. While chromatin condensation has little effect on Hoechst staining, it appears to be "the most important factor responsible for quinacrine brightness.' All existing data from D. virilis indicate that each fluorochrome distinct block of alpha heterochromatin may contain a single a single DNA molecule which is one heptanucleotide repeated two million times.     

Application of laser scanning cytometry to the analysis of chromosomal aberrations induced by benzo[a]pyrene in CHO-WBLT cells

BACKGROUND: A recently developed laser scanning cytometry technique was applied to cytometric studies to detect rapidly stable chromosomal aberrations induced by a carcinogen in a Chinese hamster fibroblast cell line, CHO-WBLT. METHODS: Individual chromosomes were collected from metaphase cells by a syringe technique and spread on slides. The DNA content of each chromosome stained with propidium iodide was measured with a laser scanning cytometer (LSC). A characteristic DNA histogram, designated as the "laser scanning karyotype (LSK)," was obtained from about 20,000 chromosomes of CHO-WBLT cells. Each chromosome was confirmed morphologically under the microscope by using a "re-location" system built into the LSC. RESULTS: A total of 21 chromosomes, including marker chromosomes specific to the cell line, were assigned to 10 major peaks in the LSK, which was analogous to the karyotype demonstrated with the classical Q-banding technique. In contrast, clonal sublines isolated after exposure to the carcinogen benzo[a]pyrene showed LSKs different from those found in untreated control cells, and seven of 20 clones were found to be abnormal, with a small number of chromosomal translocations and/or deletions, which were confirmed by Q-banding. CONCLUSIONS: The laser scanning cytometry technique was employed to detect stable chromosomal aberrations in CHO-WBLT cells after treatment with benzo[a]pyrene. The results obtained with this technique were comparable to those obtained by Q-banding; therefore, this method may be useful for rapid primary screening to detect stable, abnormal karyotypes induced by environmental chemicals and/or radiation.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-UV irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Summary Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-U.V irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.In honour of Prof. P. van Duijn     

Chromosome banding in the genusPinus

Somatic chromosomes ofPinus nigra var.maritima (2n=24) were sequentially stained with DNA binding base-specific fluorochromes, chromomycin A₃ (CMA) and DAPI. Many CMA- and DAPI-bands appeared at intercalary and/or proximal regions of most chromosomes. These banding patterns reversely related. Individual chromosomes were easily identified using these fluorescent banding patterns.     

Banding analysis of the somatic chromosomes of the domestic dog (Canis familiaris).

The chromosomes of the domestic dog (Beagle) were investigated by several different staining techniques. G-banding, Q-banding, and the bis-benzimidazol derivative Hoechst 33258, make possible the identification of all 39 chromosome pairs. Constitutive heterochromatin (C-bands) was present on a few chromosomes as distinctive, large stained areas; on the other autosomes there was little or no heterochromatin detectable.