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.     

Identification of several chromosome aberrations in man by the fluorescent method using quinacrine yprite]

The applications of the fluorescent staining of chromosomes with quinacrine mustard allowed to identify a dicentric Y-chromosome in two patients with defected external gynaetalies: a boy of 15 years old and a girl of 2 years old. Both the patients had mosaicism of sex chromosomes: 45, x/46, x dic (Y). The dicentric Y-chromosome, resembling chromosome, 16, had bright luminescence of the thelomeric regions characteristic of the normal Y-chromosome. Besides, a balanced autosomic translocation t (1, 14) (q 31, q 3) was found in the girl identified also with quinacrine mustard fluorescent staining.     

Chromosome regions containing DNAs of known base composition,specifically evidenced by 2,7-di-t-butyl proflavine

After staining by a new proflavine derivative (2,7-di-t-butyl proflavine, DBP), which specifically binds to the A-T base pairs of DNA by an external process, the constrictions of the human chromosomes 1, 16 and to a lesser extent 9 and the centromeric regions of the chromosomes (except the Y) of Mus musculus are brightly fluorescent. These chromosome regions are known to contain repetitive DNAs rich in A-T. On the contrary, the centromeric regions of the autosomes of Bos taurus, which contain a G-C rich DNA, are faintly fluorescent. The arms of the chromosomes of the three species display a banding similar to, but fainter than, the Q-banding. These results are discussed in correlation with physico-chemical studies on the binding and fluorescence processes of the dye bound to DNA and to nucleohistone. The staining properties of DBP are compared to those of quinacrine, quinacrine mustard and proflavine, three intercalative dyes which are also supposed to reveal the A-T base pairs along the chromosomes, but are faintly fluorescent on the human and murine A-T rich regions. This comparison leads us to discuss the mechanisms responsible for the chromosomal banding in relation to DNA base composition and repetitiveness, protein distribution and packing of the chromatin fibers, along the chromosomes.     

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.     

Inhibition of the development of Q-bands on human chromosomes by netropsin

Summary Netropsin, an oligopeptide-type basic antibiotic, having exclusively A-T-specific DNA-binding affinity and situating itself into the minor groove of the double helix, represses the development of Q-bands if human chromosome preparations are treated with it before quinacrine mustard staining. The most probable interpretation of this effect is that netropsin interferes with the intercalation of the dye molecules. It is assumed this phenomenon supports the hypothesis that quinacrine mustard binds preferentially to A-T-rich sequences of DNA in the metaphase chromosomes.     

Identification of human chromosomes by DNA-binding fluorescent agents

The distribution of DNA along metaphase chromosomes that are not excessively contracted can be visualized in the fluorescence microscope with the aid of fluorescent DNA-binding agents. Additional, characteristic details in the fluorescence patterns are obtained with fluorochromes that bind preferentially to certain chromosomal regions. The highly fluorescent alkylating agent quinacrine mustard (QM) effects discrete, fluorescent labeling of both plant and mammalian metaphase chromosomes, presumably by selective binding to guanine residues in DNA, and is also capable of intercalation in the DNA double helix. Chromosome regions fluorescing particularly strongly with QM have been demonstrated in human metaphase chromosomes 3, 13–15 and Y.A convenient measuring technique has been developed for the rapid and accurate recording of fluorescence patterns in human metaphase chromosomes. These photoelectric recordings of the fluorescence patterns contain far greater detail than can be seen by the human eye.The fluorescence patterns described are based on measurements of about 1,000 human metaphase chromosomes. This new technique of determining fluorescence patterns in human chromosomes should be particularly valuable for the identification of chromosomes 4–5 and the individual types in the 6–12 group. Individual, typical patterns also occur within the groups 13–15, 17–18, and 21–22.     

Quinacrine fluorescence of metaphase chromosomes : Identical patterns in different tissues

The quinacrine mustard fluorescence patterns of the metaphase chromosomes of different tissues of the same plant species were found to be identical. Similar studies of the chromosome regions on human material gave the same result.     

On the Chemical Basis of Chromosome Banding Patterns

A comparative study of the staining characteristics of four reagents for human chromosomes has been carried out. The four reagents are: (I) quinacrine mustard, as an alkylating agent, (II) the dihydrory derivative of quinacrine mustard, (III) quinacrine, and (IV) 9-amino-6-chloro-2-methoryacridine. The last reagent does not possess the amino substituted side chain even though it has the same intercalating nucleus. Comparison of the first three compounds in their staining and banding behavior suggested the initial step leading to banding may be the displacement of the nucleoprotein sites in chromosomes. The Q and G banding could he blocked experimentally by treating the chromosome preparation with dimethylamine solution. This result may suggest that these sites have weaker basic proteins (nonhistone proteins?). The use of compound IV, which does not have the side chain in the molecuk but docs have the same intercalating chromophore, did not lead to handing and gives indirect support to this hypothesis. A combined use of compound IV and quinacrine may be useful for the determination of total DNA vs. banding DNA.     

In situ localization and characterization of different classes of chromosomal DNA: acridine orange and quinacrine mustard fluorescence

In situ denaturation of metaphase chromosomes with alkali results in a shift from green to yellow, orange, brown and red fluorescence with acridine orange, indicating increasing denaturation of chromosomal DNA. The kinetics and characteristics of denaturation are described. Mouse and Microtus agrestis chromosomes denature uniformly but human cells show sequential denaturation. With increasing concentrations of alkali, the secondary constrictions in chromosomes 1, 9 and 16 are the first, and the distal half of the Y chromosome the last, to become denatured. — Reassociation of chromosomal DNA occurs within seconds after the start of incubation in salt solution. Areas containing repetitious DNA, e.g. mouse centromeres, fluoresce much more strongly than other regions with acridine orange after prolonged reassociation. Since human and Microtus centromeric regions behave similarly, it is proposed that they, too, contain repetitious DNA. — Reassociation treatment leads to enhancement of bright quinacrine mustard fluorescence in regions already bright before treatment. Furthermore, regions containing repetitious DNA, e.g. the secondary constrictions in human chromosomes 1, 9 and 16, whose fluorescence is dull before treatment, turn bright after reassociation. — The methods of fluorescence analysis of mammalian chromosomes with acridine orange and quinacrine mustard permit the localization and study of different classes of chromosomal DNA.     

Spectral luminescence studies of cells stained with acrichine derivatives

Excitation and fluorescence spectra are given of quinacrine derivative solutions, of buccal epithelium cell nuclei, of peripheral blood cells, and of isolated chromosomes treated with propyl-quinacrine mustard. It is confirmed that the differential cell treatment with quinacrine derivates may be observed in aqueous solutions only. Data obtained allow us to give some recommendations for employment of optimal filters and dichroic beam-splitters in the fluorescence microscopy of chromosomes treated with quinacrine derivatives.     

The induction of aneuploidy by nitrogen mustard in a human lymphoblastoid cell line

We report that the presence of an extra Y chromosome can be used as a marker for the induction of aneuploidy (mitotic non-disjunction) in a human lymphoblastoid cell line. This endpoint is easily visualized in metaphase chromosome preparations after staining with quinacrine mustard. The induction of cells with two Y chromosomes by nitrogen mustard (NM) was examined. Exposure to 150 ng/ml nitrogen mustard induced a 6-fold increase in aneuploid frequency relative to untreated control levels; maximal induction of aneuploidy was observed 2 days after treatment. Lower concentrations of nitrogen mustard (36 and 75 ng/ml) induced smaller increases in aneuploid frequency, with maximal induction observed 1 day after treatment. This system has the potential to be used as an assay for the induction of aneuploidy in cultured human cells.     

Ethidium bromide counterstain for differentiation of quinacrine stained interphase bodies and brilliant metaphase bands.

Visual perception of quinacrine mustard stained brilliant bands and interphase fluorescent bodies is enhanced by a staining procedure employing ethidium bromide as a counterstain.     

On the chemical basis of chromosome banding patterns.

A comparative study of the staining characteristics of four reagents for human chromosomes has been carried out. The four reagents are: (I) quinacrine mustard, as an alkylating agent, (II) the dihydroxy derivative of quinacrine mustard, (III) quinacrine, and (IV) 9-amino-6-chloro-2-methoxyacridine. The last reagent does not possess the amino substituted side chain even though it has the same intercalating nucleus. Comparison of the first three compounds in their staining and banding behavior suggested the initial step leading to banding may be the displacement of the nucleoprotein sites in hcromosomes. The Q and G banding could be blocked experimentally by treating the chromosome preparation with dimethylamine solution. This result may suggest that these sites have weaker basic proteins (nonhistone proteins?). The use of compound IV, which does not have the side chain in the molecule but does have the same intercalating chromophore, did not lead to banding and gives indirect support to this hypothesis. A combined use of compound IV and quinacrine may be useful for the determination of total DNA vs. banding DNA.     

Acetylcholine receptor. Binding properties and ion permeability response after covalent attachment of the local anaesthetic quinacrine.

Membrane vesicles rich in nicotinic acetylcholine receptor prepared from Torpedo californica electric tissue have been irreversibly modified with quinacrine mustard, an alkylating derivative of the local anaesthetic quinacrine. The reaction blocked the ion channel regulated by the acetylcholine receptor. Acetylcholine still bound to the modified membrane vesicles with KD approx. 10(-8). The number of binding sites was reduced by up to 50%. Stopped-flow experiments showed that in contrast to what had been found with the reversibly binding quinacrine no fluorescence changes caused by energy transfer from the irradiated protein to the fluorescent local anaesthetic occurred after addition of agonist. This indicates that the conformational changes associated with the activation of the ion channel are blocked by the covalent reaction with quinacrine mustard. Analysis of the membrane vesicles by SDS-polyacrylamide gel electrophoresis showed that all polypeptide chains assumed to be part of the receptor complex had reacted with the mustard. Even small components, probably lipids, migrating with the dye front, showed fluorescence.     

A comparison of the fluorescent karyotypes of the chimpanzee (Pan troglodytes) and man

Individual chimpanzee chromosomes have been identified by their characteristic banding revealed by quinacrine fluorescent staining. A fluorescent karyotype of this species was set up to be compared with the standard human fluorescent karyotype. It was found that chromosomes 1, 3, 11, 12, 14 and X-chromosome of the chimpanzee appear to have banding patterns similar to the equivalent human chromosomes. Chromosomes 6, 7, 8, 10 and 13 also had a fluorescent pattern corresponding to the human chromosomes of the same number, particularly in the long arm. Remarkable variation in intensity and/or size of fluorescent regions was frequently found in the short arm of satellited acrocentric chromosomes 13, 14, 15, 22 and 23. Variations occurred between homologues and between individuals. Such variable fluorescence in a specific chromosomal region of an individual animal is a reproducible characteristic. Unlike its human counterpart, the distal segment in the long arm of the chimp's Y-chromosome is not brightly fluorescent. An earlier report is thus confirmed.     

Fluorescence of quinacrine mustard conjugated to proteins.

Proteins are readily labeled by quinacrine mustard to yield conjugates whose spectral properties are well-suited for fluorescence studies. Data on these conjugates and on the parent compound, quinacrine, are presented including lifetimes, quantum yields, and corrected excitation and emission spectra. Polarization studies using the Perrin-Weber equation show that rotational relaxation times can be obtained with quinacrine mustard conjugates. Such conjugates had lifetimes ranging from 4 to 13 ns and quantum yields from about 0.1 to 0.3. Quinacrine mustard is a useful reporter group, as shown by the changes in fluorescence parameters of conjugates undergoing conformational changes induced by denaturants. An excited state $\text{p}\text{K}{}_{\mathrm{a}}{}^1$ of 4.9 was identified for quinacrine, but the protonation was suppressed in the mustard conjugate of serum albumin until the N-F transition took place. The properties of the mustard conjugates are discussed in terms of potential uses and compared with properties of other types of fluorescent conjugates.     

The band patterns of twelve D 98/AH-2 marker chromosomes and their use for identification of intraspecific cell hybrids

The chromosomal complement of the human cell line D98/AH-2 has been studied by quinacrine mustard and trypsin Giemsa banding techniques. The dispersion of chromosome counts has been shown to be due to non-random variation involving mainly a few particular chromosomes. — Twelve different marker chromosomes could be distinguished and the presumptive derivation of most of their chromosomal material from normal human chromosomes has been determined. Most cells in 6 different hybrid clones derived from fusion of D98/AH-2 cells with skin fibroblasts from a cystinotic patient contained a single copy of each marker chromosome.Supported by: united States Public Health Service Grant HD 04608, National Institute of General Medical Sciences Grant GM 17702 and American Heart Association Grant 71-981.Established Investigator of the American Heart Association.     

Irreversible Inhibition of Calcium Uptake in Synaptosomes by Quinacrine Mustard: Relationship to Labeling Sites of Quinacrine Mustard

The sites of interaction of quinacrine with synaptic membranes were labeled with quinacrine mustard. Quinacrine mustard had an inhibitory effect on depolarization-induced calcium uptake by synaptosomes similar to that of quinacrine. The inhibition of depolarization-induced calcium uptake by quinacrine was reduced by 70% after washing, whereas that by quinacrine mustard was not affected. Fluorescence electrophoretograms of the quinacrine mustard-treated synaptic membranes showed that quinacrine mustard specifically labeled two proteins, with corresponding molecular weights of about 37,000 and 32,000.     

Acetylcholine receptor binding properties and ion permeability response after covalent attachment of the local anaesthetic quinacrine

Membrane vesicles rich in nicotinic acetylcholine receptor prepared from Torpedo californica electric tissue have been irreversibly modified with quinacrine mustard, an alkylating derivative of the local anaesthetic quinacrine. The reaction blocked the ion channel regulated by the acetylcholine receptor. Acetylcholine still bound to the modified membrane vesicles with K_D approx. 10^?8. The number of binding sites was reduced by up to 50%. Stopped-flow experiments showed that in contrast to what had been found with reversibly binding quinacrine no fluorescence changes caused by energy transfer from the irradiated protein to the fluorescent local anaesthetic occurred after addition of agonist. This indicates that the conformational changes associated with the activation of the ion channel are blocked by the covalent reaction with quinacrine mustard. Analysis of the membrane vesicles by SDS-polyacrylamide gel electrophoresis showed that all polypeptide chains assumed to be part of the receptor complex had reacted with the mustard. Even small components, probably lipids, migrating with the dye front, showed fluorescence.     

Chromosome heteromorphisms in the gorilla karyotype. Analyses with distamycin A/DAPI, quinacrine and 5-azacytidine

The chromosomes of one male and three female gorillas were extensively studied with various regional banding methods. The chromosomes were stained with the fluorescent dyes quinacrine mustard and distamycin A/DAPI (DA/DAPI), which label different subsets of heterochromatin in the chromosome complement. Furthermore, lymphocyte cultures were treated with the cytidine analog 5-azacytidine (5-azaC). The 5-azaC-induced undercondensations were found in most of the DA/DAPI-bands as well as in many telomeric C-bands. The karyotype of the gorilla exhibits a considerable number of heterochromatin variants. Of the different types of heteromorphisms noted, the most striking is that involving the short arm regions of chromosomes 12 to 16 and 23 (satellite stalk regions) and the paracentromeric heterochromatin of chromosomes 17 and 18. There also are numerous heteromorphic C-bands localized in the telomeric regions of homologous chromosome arms. In comparison, only few heteromorphisms occur between C-bands in the centromeric and pericentromeric regions of homologs. Finally, a variability in the fluorescence intensity of quinacrine-bright satellites in the short arms of chromosomes 12 to 16, 22, and 23 is observed.