Flow cytometric characterization of a Chinese hamster x man hybrid cell line retaining the human Y chromosome

Summary A Chinese hamster x man hybrid cell line (CH-Y-VII) was established which retains a free human Y chromosome. Exponentially growing CH-Y-VII cells were arrested with colcemid; metaphase chromosomes were isolated and stained with 33258 Hoechst (HO) plus Chromomycin A3 (CA3), or with ethidium bromide (EB). The HO/CA3-stained chromosomes were measured in a dual beam flow cytometer, and bivariate HO/CA3 flow karyotypes and univariate HO and CA3 flow karyotypes were established. EB-stained chromosomes were analyzed in a modified Becton Dickinson FACS-Sorter. For all three stains used, the human Y chromosome forms a separate peak in univariate flow karyotypes; the optimum resolution was obtained for the HO distribution. In the bivariate HO/CA3 flow karyotype, the peak for the human Y chromosome is completely separated from the Chinese hamster chromosomes.Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48 with support from the Deutsche Forschungsgemeinschaft (Cr 60/3-1 and Wo 148/18)     

Cytofluorometric determination of the DNA base content in human chromosomes with quinacrine mustard, Hoechst 33′258, DAPI, and mithramycin

The human chromosomes 1, 9, 16, 21, and Y were analysed cytofluorometrically with the AT-specific DNA ligands quinacrine mustard (QM), Hoechst 33′258, and DAPI, and the GC-specific DNA ligand mithramycin. All three AT dyes give similar results, though QM produces more distinct banding than DAPI or Hoechst. The sum of AT and GC fluorescence is very well correlated to the amount of DNA estimated densitometrically. The AT/GC ratios of chromosomes 16, 22, and Y differ clearly from that of whole nuclei, and accord fairly well with the results obtained by flow cytometry. For the Y a significant difference in calculated base content between donors was found with all three AT dyes even though differences in the karyotypes were not distinguishable by the eye.     

Different Hoechst 33342 and DAPI fluorescence of the human Y chromosome in bivariate flow karyotypes

Summary The staining properties of AT-specific dyes Hoechst 33342 and DAPI as revealed by Hoechst 33342/mithramycin and mithramycin/DAPI bivariate human flow karyotype patterns are different for chromosomes rich in heterochromatin. The peak corresponding to chromosome Y of a given cell line is higher on the A/T axis with mithramycin/DAPI staining than with mithramycin/Hoechst. The chromosome 1 was found slightly more fluorescent in mithramycin/DAPI than in mithramycin/Hoechst as judged by the slight displacement of its area on the Hoechst-DAPI axis. The other peaks did not show major differences. On the same flow karyotypes, chromosomal rearrangements were detected.     

限制酶AluI显带和CA_3/DA/DAPI染色表达的家猪染色体结构异染色质

采用限制酶AluI显带、CA_(?)/DA/DAPI荧光染色和常规C带技术研究了家猪染色体着丝粒结构异染色质,结果表明:着丝粒结构异染色质至少可被区分为3类,并且在染色体组内各有其特异的染色体分布。将家猪染色体DA/DAPI荧光带和限制酶AluI显带与人类染色体比较,发现家猪13—18号端着丝粒染色体显带特征与人染色体1,9、16、Y一致。提示家猪13—18号端着丝粒区结构异染色质存在与人类随体DNA相似的DNA组成。     

Homologous chromosomes characteristics by sequential banding procedures in rainbow trout Oncorhynchus mykiss

The development of high resolution methods of chromosome banding helped the finding of homologous chromosomes, detecting chromosomal abnormalities, and assigning the gene loci to particular chromosomes in mammals. Unfortunately, small and numerous fish chromosomes do not show GC rich and GC poor compartments, this preventing the establishment of G banding pattern. The combination of techniques enabling the identification of constitutive heterochromatin (C-banding), heterochromatin resistant to restriction endonucleas, NOR bearing chromosomes (AgNO3 banding), or AT rich regions on chromosomes (DAPI banding) in sequential staining provides a better characteristic of fish chromosomes. In this work sequentially DAPI, DdeI, AgNO3 stained chromosomes of rainbow trout resulted in the characteristic banding pattern of some homologous chromosomes. Procedure of FISH with telomere probe and DAPI as a counterstaining fluorochrome visualized simultaneous hybridization signals and DAPI banding. Possibility of detection both FISH and DAPI signals can help in procedures of gene mapping on chromosomes.     

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.     

Effects of counterstaining with DNA binding drugs on fluorescent banding patterns of human and mammalian chromosomes

Pairs of fluorescent A-T specific dyes and nonfluorescent agents with similar or complementary base pair binding specificity were used to analyse the extent to which banding patterns in human chromosomes obtained by fluorescent staining can be modified by counterstaining. By testing a variety of different combinations of drugs, essentially three types of alterations were observed. Enhanced contrast of specific heterochromatic regions was obtained with pentamidine, or netropsin, in conjunction with the fluorescent stains Hoechst 33258, DAPI or DIPI, the resulting banding patterns being similar to that reported for distamycin A plus DAPI (DA-DAPI banding [21]. Uniform quenching of Hoechst 33258, DAPI or DIPI fluorescence was induced by counterstaining with stilbamidine or berenil. The combination of echinomycin with DAPI resulted in an improved contrast of DAPI banding on chromosome arms and pale fluorescence on major autosomal C band regions. In addition, a subdivision of the heterochromatic part of the Y chromosome may be discerned by this latter technique.     

Counterstained enhancement of TaqI resistant sites after distamycin A/diamidinophenylindole treatment

Numerous selective and differential staining techniques have been used to investigate the hierarchical organisation of the human genome. This investigation demonstrates the unique characteristics that are produced on fixed human chromosomes when sequential procedures involving restriction endonuclease TaqI, distamycin A (DA) and 4,6-diamidino-2-phenylindole (DAPI) are employed. TaqI produces extensive gaps in the heterochromatic regions associated with satellite II and III DNAs of human chromosomes 1, 9, 15, 16 and Y. DA/DAPI selectively highlights, as brightly fluorescent C-bands, the heterochromatin associated with the alpha, beta, satellite II and III DNAs of these chromosomes. When DA and DAPI are used on chromosomes before TaqI digestion, and then stained with Giemsa, the centromeric regions appear to be more resistant, producing a distinct C-banding pattern and gaps in the heterochromatin regions. Sequential use of the DA/DAPI technique after TaqI treatment produces a bright fluorescence on the remaining pericentromeric regions of chromosomes 1, 9, 16 and Y, which also displayed a cytochemically unique banding pattern. This approach has produced specific enhanced chromosomal bands, which may serve as tools to characterize genomic heterochromatin at a fundamental level.     

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A

A DNA-binding AT-specific oligopeptide antibiotic, distamycin A, was used as non-fluorescent counterstain in conjunction with the DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) to investigate the effect of the antibiotic on DAPI fluorescent banding of human chromosomes. Distamycin A-pretreated metaphases and interphase nuclei exhibited a significantly lower overall fluorescence intensity than DAPI controls. Chromosome arms were pale and intercalary DAPI bands (Q bands) were obliterated, but some specific regions of constitutive heterochromatin remained brightly fluorescent. These were mainly the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y. The distamycin A/DAPI banding pattern appears to be comparable to that reported for anti-5-methylcytosine binding [11]. The observations are discussed as they relate to the roles of chromosomal DNAs and proteins in chromosome banding.     

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.     

C-Banding/DAPI and in situ hybridization reflect karyotype structure and sex chromosome differentiation in Humulus japonicus Siebold & Zucc

Japanese hop (Humulus japonicus Siebold & Zucc.) was karyotyped by chromosome measurements, fluorescence in situ hybridization with rDNA and telomeric probes, and C-banding/DAPI. The karyotype of this species consists of sex chromosomes (XX in female and XY1Y2 in male plants) and 14 autosomes difficult to distinguish by morphology. The chromosome complement also shows a rather monotonous terminal distribution of telomeric repeats, with the exception of a pair of autosomes possessing an additional cluster of telomeric sequences located within the shorter arm. Using C-banding/DAPI staining and 5S and 45S rDNA probes we constructed a fluorescent karyotype that can be used to distinguish all autosome pairs of this species except for the 2 largest autosome pairs, lacking rDNA signals and having similar size and DAPI-banding patterns. Sex chromosomes of H. japonicus display a unique banding pattern and different DAPI fluorescence intensity. The X chromosome possesses only one brightly stained AT-rich terminal segment, the Y1 has 2 such segments, and the Y2 is completely devoid of DAPI signal. After C-banding/DAPI, both Y chromosomes can be easily distinguished from the rest of the chromosome complement by the increased fluorescence of their arms. We discuss the utility of these methods for studying karyotype and sex chromosome evolution in hops.     

Reverse fluorescent chromosome banding with chromomycin and DAPI

Two DNA binding guanine-specific antibiotics, chromomycin A₃ (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI. — In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI. — Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The CMA-banding pattern appears to be similar to the pattern found by R-banding procedures.     

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.     

Heterochromatin characterization of sex chromosomes in Triturus marmoratus (Urodela, Salamandridae).

The sex chromosomes of the Iberian marbled newt, Triturus marmoratus, were studied using various banding techniques, including restriction enzyme/nick translation (RE/NT) procedures. Four types of heterochromatin on the sex chromosomes could be distinguished: (1) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT negative, and HaeIII/NT and HinfI/NT positive; (2) distamycin A/DAPI and chromomycin A3/distamycin A positive, but RE/NT negative; (3) AT rich, but RE/NT negative; and (4) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT and HinfI/NT negative, but HaeIII/NT positive. These data suggest a common origin for the terminal heterochromatic domains of both the X and Y chromosomes in this species.     

Semi-automated detection of aberrant chromosomes in bivariate flow karyotypes.

A method is described that is designed to compare, in a standardized procedure, bivariate flow karyotypes of Hoechst 33258 (HO)/Chromomycin A3 (CA) stained human chromosomes from cells with aberrations with a reference flow karyotype of normal chromosomes. In addition to uniform normalization of normal and abnormal flow karyotypes, the main purpose is detection of structurally abnormal chromosomes in often complex karyotypes of tumor cells. The method, which has been implemented in a computer program, consists of a comparison of individual chromosome peaks with the positions of peaks in the flow karyotype constituted by normal chromosomes and takes into account the natural variability in base composition of normal chromosomes among healthy individuals. Flow-karyotypes are normalized using an iterative fitting procedure, using corrections for (1) amplification of HO and CA fluorescence, (2) cross-talk between the fluorescence signals of HO and CA, and (3) offset of the HO and CA origin. Flow karyotypes of two cell lines, one with a simple deletion and the other with more complex karyotypic changes, were analyzed. The results of flow analysis were found to be in general agreement with the cytogenetic analysis of quinacrine banded karyotypes.     

Patterns of chromosome banding in four nabid species (Heteroptera, Cimicomorpha, Nabidae) with high chromosome number karyotypes

Male Nabis (Aspilaspis) indicus (St?l), N. (A.) viridulus Spinola, Himacerus (Himacerus) mirmicoides (O. Costa) (2n=32+XY) and Prostemma guttula (Fabricius) (2n=26+XY) were studied using C-banding, silver nitrate staining and base-specific fluorochrome (DAPI and CMA(3)) staining. N. indicus differed from N. viridulus in distribution pattern of C-bands, which were telomeric in the former while interstitial in the latter. H. mirmicoides showed interstitial C-bands in the majority of autosomes. P. guttula had no conspicuous C-bands in other chromosomes, but only in the Y, which was totally heterochromatic. C-heterochromatin was labelled with DAPI, indicating that it was AT-rich. In every species, both X and Y chromosomes were NOR-bearing, and the NOR regions were GC-rich. In H.mirmicoides and P. guttula, NORs showed sub-median location in the X and distal in the Y, such a pattern being probably common in Nabidae. The present paper provides new information on the genome organization and new cytological markers useful for a better insight into karyotype evolution of nabid species.     

Karyotype analysis of Nicotiana kawakamii Y. Ohashi using DAPI banding and rDNA FISH

Prometaphase cells were used to analyze the karyotype of Nicotiana kawakamii Y. Ohashi by means of sequential Giemsa/CMA/DAPI staining and multicolor fluorescence in situ hybridization with 5S and 18S rDNA. Observation of the DAPI-stained prometaphase spreads indicated that N. kawakamii had six pairs of large chromosomes, one pair of medium-sized chromosomes and five pairs of small chromosomes. The six pairs of large chromosomes possessed remarkable DAPI bands, and each could be identified from both the DAPI banding pattern and the length of the short arm. The DAPI banding pattern was approximately identical to the CMA and Giemsa banding patterns. Hybridization signals of the 18S rDNA probe were detected on two pairs of large chromosomes. In addition, two pairs of small chromosomes were identified based on the position of the 5S rDNA signals. An idiogram of N. kawakamii chromosomes was produced based on DAPI bands and rDNA loci. Received: 17 July 2000 / Accepted: 4 September 2000     

Correlation of pachytene chromomeres and metaphase bands of human chromosomes, and distinctive properties of telomeric regions

By means of double staining with DAPI and chromomycin A3, we show that the chromomeres of human pachytene chromosomes are generally DAPI positive and chromomycin negative, like the G- or Q-bands of mitotic chromosomes. Thus we have demonstrated, using an objective technique not based on morphological comparisons, that chromomeres and G-bands are equivalent. However, terminal chromomeres and the ends of mitotic chromosomes, as well as a few other sites, are chromomycin positive and DAPI negative. The ends of human chromosomes appear, therefore, to contain a distinctive class of GC-rich DNA.     

Optimising human chromosome separation for the production of chromosome-specific DNA libraries by flow sorting

Summary A number of cell lines, some containing chromosomes with distinctive heteromorphisms, have been flow karyotyped using a single laser flow sorter in an attempt to select those suitable for sorting all human chromosomes individually. Using the non-base-specific DNA stain ethidium bromide, chromosomes 3,4,5, and 6 form individual peaks in practically all normal subjects, while the right combination of heteromorphisms enables chromosomes 1, 2, 8, 9, 13, 16, 17, 18, 19, 20, 21, 22, and Y to be sorted separately. Two male cell lines, one containing a duplication and one a deletion of the X, produce flow karyotypes suitable for sorting chromosomes 7 and 8. The use of numerical chromosome abnormalities to enrich the sex chromosomes and the autosomes 18 and 21 is also illustrated. The DNA stain Hoechst 33258 binds preferentially to AT base pairs. Flow karyotypes produced with this fluorochrome separate some chromosomes not well separated with ethidium bromide. Chromosomes 5, 6, 8, 13, 14, 15, 17, and 20, and Y can be sorted individually with Hoechst 33258 with the right combination of heteromorphisms. Using these techniques, all human chromosomes apart from 10, 11, and 12 have been found as individual flow karyotype peaks, suitable for sorting with a high degree of purity.