DIPI and DAPI: Fluorescence banding with only negligible fading

Summary DIPI and DAPI produce distinct fluorescent bands in human chromosomes similar to quinacrine banding patterns. Additionally, the AT rich secondary constrictions in the chromosomes Nos. 1, 9 and 16 are brightly fluorescent. On the other hand the brilliantly fluorescent regions after staining with quinacrine mustard in the chromosomes Nos. 3 and 4, satellites and some other regions in the acrocentric chromosomes are less striking. The distal part of the Y, however, is clearly discernible. Thus DIPI and DAPI seem to be strictly AT specific fluorochromes like Hoechst 33258.In interphase nuclei the Y chromosome can be identified. However, quinacrines are superior for Y-body analysis in buccal, hair cell and sperm smears.BrdU labeled chromatids show reduced fluorescence intensity. The difference, however, is less apparent than after staining with Hoechst 33 258.DAPI and especially DIPI are highly resistant to UV-irradiation; there is almost no fading within 30 min when using DIPI. Moreover, fluorescence intensity is stronger than in quinacrines. When photographing, exposure times may be reduced to about one quarter compared to quinacrine mustard.     

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.     

Hoechst 33258 banding of Drosophila nasutoides metaphase chromosomes

Hoechst 33258 banding of D. nasutoides metaphase chromosomes is described and compared with Q and C bands. The C band positive regions of the euchromatic autosomes, the X and the Y fluoresce brightly, as is typical of Drosophila and other species. The fluorescence pattern of the large heterochromatic chromosome is atypical, however. Contrary to the observations on other species, the C negative bands of the large heterochromatic chromosome are brightly fluorescent with both Hoechst 33258 and quinacrine. Based on differences in the various banding patterns, four classes of heterochromatin are described in the large heterochromatic chromosome and it is suggested that each class may correspond to an AT-rich DNA satellite.     

Mithramycin and DIPI: A pair of fluorochromes specific for GC- and AT-rich DNA respectively

Summary The AT specificity of the fluorochromes DIPI and DAPI and the GC specificity of mithramycin are evidenced by observations in human, mouse, and bovine chromosomes. DIPI and DAPI produce a pattern similar to Hoechst 33258 in all three species, whereas mithramycin results in a reverse pattern. The AT-rich centromeric heterochromatin in mouse is brilliantly stained by DIPI or DAPI and remains nearly invisible after mithramycin staining. In the GC-rich centromeric heterochromatin of cattle the opposite behavior is observed.     

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     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

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.     

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.     

A Comparative Study of Dapi, Dipi, and Hoechst 33258 and 33342 as Chromosomal Dna Stains

A comparison of four DNA stains considered to be AT-specific with chromosomes from a clonal Chinese hamster cell line B14F28-C5 have been made. The flow karyotype histograms indicate that DAPI, DIPI, and Hoechst 33258 and 33342 do stain similarly in the same preparation. DAPI staining is specific and highly reproducible in this line. We, therefore, recommend this dye as a single chromosome DNA stain for high-resolution flow cytometric measurements in cytogenetics and mutation research.     

Chromosome CPD(PI/DAPI)- and CMA/DAPI-Banding Patterns in Allium cepa L.

Chromosome banding patterns of Allium cepa L. were obtained by using fluorescent dye combinations chromomycin A₃ (CMA) + 4",6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa,telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After joint staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (2 × SSC, 95°C for 1–3 min) followed by renaturation (2 × SSC, 37°C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of bright DAPI fluorescence in GC-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.     

Characterization of the canine karyotype by counterstain-enhanced chromosome banding

The application of the counterstain-contrasted fluorescent banding technique to canine chromosomes provided an improved capability to highlight specific heterochromatic regions and to produce well defined banding patterns both on mitotic and meiotic chromosomes. Triple staining with chromomycin A3 - distamycin A - DAPI revealed the occurrence of DA - DAPI positive heterochromatin in chromosomes 33, 36, 37, and 38. Pachytene nuclei present more favourable material for the detection of very small amounts of DA - DAPI material than mitotic division stages. Counterstain-enhanced chromomycin R-banding greatly facilitated chromosome identification. A standard R-band karyotype of Canis familiaris is proposed and described in some detail. DAPI - actinomycin D staining produced a QFH-type banding pattern and enhanced differentiation of some polymorphic regions.     

A comparative study of DAPI, DIPI, and Hoechst 33258 and 33342 as chromosomal DNA stains

A comparison of four DNA stains considered to be AT-specific with chromosomes from a clonal Chinese hamster cell line B14F28-C5 have been made. The flow karyotype histograms indicate that DAPI, DIPI, and Hoechst 33258 and 33342 do stain similarly in the same preparation. DAPI staining is specific and highly reproducible in this line. We, therefore, recommend this dye as a single chromosome DNA stain for high-resolution flow cytometric measurements in cytogenetics and mutation research.     

Effects of DAPI on human leukocytes in vitro.

DAPI (4'-6-diamidino-2-phenylindole), a fluorochrome specific for AT-rich DNA, was supplied for 24 h at various concentrations to human leukocytes in culture. This treatment caused the appearance on the chromosomes of specific areas lacking spiralization. In particular, the centromeric regions of chromosomes 1,9, and 16, a short region on the long arm of chromosomes 1 and 2, and the distal heterochromatic part of the long arm of the Y chromosome were despiralized. The despiralization pattern of DAPI is compared with those previously obtained with Hoechst 33258 and Distamycin A.     

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.     

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.     

Cytofluorometric determination of DNA base content in plant nuclei and chromosomes by the fluorochromes DAPI and Chromomycin A3

The fluorescence of DAPI (AT-dye) and Chromomycin A3 (GMA; GC-dye) was measured in mitoses and interphase nuclei of nine species of plants having moderate or strong fluorescent bands—or none at all. In Scilla sibirica chromosomes, band and non-band regions were analysed. The results are compatible with a linear base-dependent fluorescence of the two dyes; their fluorescence can thus be utilized for cytofluorometric base content determination. The measurement of fluorescence fading of DAPI gave identical curves in band and non-band regions, whereas a different fading pattern could be observed with another AT-dye (Hoechst 33258). CMA also yielded different fading curves in band and non-band regions, which indicates a structural difference of the chromatin-dye complex.     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

Chromosome CPD(PI/DAPI)- and CMA/DAPI-banding patterns in Allium cepa L

Chromosome banding patterns of Allium cepa L. were obtained by using fluorochrome combinations chromomycin A3 (CMA) + 4',6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa, telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (95 degrees C for 1-3 min) followed by renaturation in the 2 x SSC buffer (37 degrees C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of the NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of DAPI fluorescence in GR-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.     

Evolutionary diversity of reverse (R) fluorescent chromosome bands in vertebrates

Mitotic chromosomes, interphase cell nuclei, and male meiosis of 41 species representing all vertebrate classes were analyzed with distamycin A/mithramycin counterstaining. The purpose of the study was to recognize differences and common characteristics in the reverse (R) fluorescent banding patterns in the chromosomes of vertebrate species at various stages of evolution. In contrast to the warm-blooded mammals and birds, the euchromatic segments in the chromosomes of most reptiles, amphibians, and fishes contain no multiple fluorescent R-bands. This is thought to be due to the absence of the long homogeneous regions (isochores) in the DNA of the cold-blooded vertebrates. Distamycin A/mithramycin banding specifically reveals the GC-rich constitutive heterochromatin in all vertebrates. In most of the vertebrate chromosomes examined, the heterochromatic regions have opposite staining properties with mithramycin and quinacrine. Mithramycin labels the nucleolus organizer regions very brightly in the karyotypes of fishes, amphibians, reptiles and birds, but not of mammals. The lack of mithramycin fluorescence at the nucleolus organizer regions of mammals is attributed to the relatively low level of redundancy of the GC-rich ribosomal DNA in their genomes. Studies on the various meiotic stages of the cold-blooded vertebrates show that the mithramycin labeling of the nucleolus organizers is independent of their state of activity. This can be confirmed by mithramycin fluorescence at the nucleoli of actinomycintreated cells.Dedicated to the memory of Professor Dr. Hans Bauer