Staining of human metaphase chromosomes with fluorescent conjugates of polylysine

The interaction of polylysine and partially substituted dansyl, fluorescein, and quinacrine conjugates of polylysine with cytological preparations of human metaphase chromosomes has been studied by fluorescence microscopy. The fluorescence intensity along chromosomes stained with the dansyl and fluorescein conjugates exhibits little variation, suggesting that regions capable of binding these polycations are nearly evenly distributed. In contrast, the quinacrine derivatives of polylysine stain the chromosomes in a banded fluorescence pattern resembling that observed following quinacrine or quinacrine mustard treatment.     

Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry

In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific ‘paints’ from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10–12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.     

Banding of Allium chromosomes protected against DNase digestion by DNA-binding drugs

Metaphase chromosome bands were induced in Allium flavum (Liliaceae) by protecting the chromosomal DNA with DNA-binding compounds of different base specificities against DNase digestion. The bands obtained represented different subsets of C-band heterochromatin.     

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.     

Identification of human and mouse chromosomes in human-mouse hybrids by centromere fluorescence

Differences between the centromeric regions of mouse and human chromosomes have been revealed with a staining technique which is based on the quenching by 5-bromodeoxyuridine of the fluorescence of the dye 33258 Hoechst. The differences can be used to distinguish between human and mouse chromosomes in human-mouse hybrids.     

Identification of cells with lowered sensitivity to cytostatic agents by the use of fluorescent dyes

With a help of stepwise increase of vincristine concentrations in culture medium several lines of mouse myeloma X63 Ag 8.863 cells resistant to low concentrations of vincristine (6-35-fold) were selected. Rhodamine 123 stained resistant cells and wild-type cells with an equal intensity. However, resistant cells differ significantly from the sensitive ones by the rate of rhodamine efflux. The rate of the efflux was in proportion to the degree of resistance. The efflux of the dye could be blocked by the addition to reserpine, the inhibitor of multidrug resistance. Thus, fluorescent dyes can be used for the detection of cells with low levels of multidrug resistance.     

Sister chromatid exchanges--a sensitive assay of agents damaging human chromosomes.

Sister chromatid exchange frequencies in human lymphocyte chromosomes are greatly increased by alkylating agents, but ionizing radiation has little if any such effect. Scoring these exchanges may provide a useful technique for exploring the mechanisms of chromosome breakage and repair.     

Mapping homology between human and black and white colobine monkey chromosomes by fluorescent in situ hybridization

We used in situ hybridization of chromosome specific DNA probes (“chromosome painting”) of all human chromosomes to establish homologies between the human and the white and black colobus (Colobus guereza 2n = 44). The 24 human paints gave 31 signals on the autosomes (haploid male chromosome set). Robertsonian translocations between chromosomes homologus to human 14 and 15, 21 and 22, form colobine chromosomes 6 and 16, respectively. Reciprocal translocations were found between human chromosomes 1 and 10, 1 and 17, as well as 3 and 19. The alternating hybridization signals between human 3 and 19 on Colobus chromosome 12 show that in this case a reciprocal translocation was followed by a pericentric inversion. The hybridization data show that in spite of the same diploid number and similar Fundamental Numbers, the black and white colobine monkey differs from Presbytis cristata, an Asian colobine, by 6 reciprocal translocations. Comparisons with the hybridization patterns in other primates show that some Asian colobines have a more derived karyotype with respect to African colobines, macaques, great apes, and humans. Chromosome painting also clearly shows that similarities in diploid number and chromosome morphology both between colobines and gibbons are due to convergence. Am. J. Primatol. 42:289–298, 1997. © 1997 Wiley-Liss, Inc.     

In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure

In situ hybridization and immunocytochemical procedures are described which allow identification and localization of specific DNA sequences in human chromosomes by fluorescence microscopy. With this method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum. Visualization of the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno-fluorescence. The antiserum against RNA-DNA hybrid molecules was raised in a rabbit injected with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of Sephadex beads to which various nucleic acids had been coupled. To obtain optimal specific fluorescence and very low aspecific background staining, several modifications of the in situ hybridization and the immunocytochemical procedures were investigated. The use of aminoalkylsilane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochromelabelled antibody solutions and application of a proteinase K treatment during the hybridization procedure and the immunocytochemical procedure proved to be essential for optimal results.     

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.     

Fluorescent staining of human chromosomes: Identification of some common aberrations

With the development of fluorescent staining techniques and their application to human chromosomes, identification of the entire human karyotype is now possible. Of considerable value are the elimination of the guesswork in the pairing of the C group and sex chromosomes and the recognition and characterization of the translocation chromosomes in mongolism.