Quinacrine fluorescence of specific chromosome regions

The pattern of intense fluorescence of interphase nuclei and metaphase chromosomes after staining with quinacrine is described in Samoaia leonensis. Autoradiographic analysis of interphase nuclei after pulse labeling with tritiated thymidine indicates that there is little or no overlap in the time of replication of the intensely fluorescing and weakly fluorescing regions. Autoradiographic analysis of metaphase figures after continuous labeling with tritiated thymidine shows that the intensely fluorescing regions are late replicating and establishes their order of replication. Autoradiographic analysis of interphase nuclei after pulse labeling with tritiated deoxycytidine and of metaphase figures after continuous labeling with this tracer show that there is little, if any, incorporation of deoxycytidine into those chromosome regions which fluoresce intensely after staining with quinacrine and quinacrine mustard. These results indicate that such chromosome regions are characterized chemically by an extremely high, if not exclusive, content of adenine and thymine.     

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.     

Microfluorometric investigations of chromatin structure. I. Evaluation of nine DNA-specific fluorochromes as probes of chromatin organization

The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes - mithramycin, 7-aminoactinomycin D, Hoechst 33258, DAPI, and propidium iodide - were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes - proflavine, quinacrine mustard, berberine sulfate, and pyronin Y - appeared to be affected significantly by organizational differences of the chromatin. All of the latter "structural probes," except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin D. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.     

Microfluorometric investigations of chromatin structure

Summary The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes—mithramycin, 7-aminoactinomycin d, Hoechst 33258, DAPI, and propidium iodide—were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes—proflavine, quinacrine mustard, berberine sulfate, and pyronin Y—appeared to be affected significantly by organizational differences of the chromatin. All of the latter structural probes, except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin d. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.     

Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increased ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. For L-Pam and UM, increased ionic strength and the cationic DNA affinity binders dose dependently inhibited the alkylation. QM alkylation was less inhibited by salt (100 mM NaCl), ethidium (10 microM), and spermine (10 microM). Distamycin A and netropsin (100 microM) gave an enhancement of overall QM alkylation. More interestingly, the pattern of guanine N7-alkylation was qualitatively altered by ethidium bromide, distamycin A, and netropsin. The result differed with both the nitrogen mustard (L-Pam less than UM less than QM) and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.     

Distamycin A/DAPI bands and the effects of 5-azacytidine on the chromosomes of the chimpanzee, Pan troglodytes

The chromosomes of the chimpanzee were stained with distamycin A/DAPI, which labels specific C-bands. Bright distamycin A/DAPI fluorescence was found in the heterochromatic regions of chromosomes 6, 11, 14 to 16, 18 to 20, and 23 and the Y. Lymphocyte cultures from chimpanzees were treated with low doses of 5-azacytidine during the last hours of culture. This cytosine analog induces highly distinct undercondensations in 28 heterochromatic regions of 19 chromosomes. These 5-azacytidine-sensitive regions are predominantly located in the terminal C-bands of the chromosomes. In vitro treatment with 5-azacytidine also preserves into the metaphase stage somatic pairings between the 5-azacytidine-sensitive heterochromatic regions in interphase nuclei. The homologies and differences regarding the chromosomal localization of distamycin A/DAPI-bright C-bands, 5-azacytidine-sensitive heterochromatin, 5-methylcytosine-rich DNA sequences, and satellite DNAs in the chimpanzee and man are discussed.     

A simple fluorescence staining technique for the differentiation of human tissue transplanted into nude mice.

Human and mouse nuclei can be distinguished by differences in the constitutive heterochromatin when stained with quinacrine dihydrochloride. With the staining method described, mouse heterochromatin during interphase appears as brilliant fluorescent chromocenters. By replacing the commonly used aqueous buffer mounting medium with a xylene-diluted synthetic resin, the haziness of the nuclear fluorescence is eliminated thus allowing identification of the heterochromatin pattern in histological preparations. A requirement for the definite identification of cells of human or murine origin in the nude mouse is the knowledge that the heterochromatin arrangements changes according to the stage of differentiation of the cell of the position of a particular nucleus within the cell cycle.     

Interaction of quinacrine mustard with mononucleotides and polynucleotides

1. The interaction between quinacrine mustard and mononucleotides and polynucleotides was investigated by fluorimetry and absorbance spectrophotometry. 2. The fluorescence spectrum of quinacrine mustard is independent of the ionic strength and pH. The dependence of the quinacrine mustard fluorescence intensity on ionic strength, pH and anions is described. 3. The fluorescence intensity of quinacrine mustard was enhanced with the mononucleotide adenylic acid and polynucleotides such as poly(rA), poly(rU) and poly(rA,rU). 4. Quenching of the fluorescence intensity of quinacrine mustard occurred with the mononucleotide guanylic acid and with poly(rG) and poly(rC,rG). 5. The mononucleotide cytidylic acid or poly(rC) showed no effect on the fluorescence intensity of quinacrine mustard. 6. The interaction between the dye and native DNA species was also dependent on the presence of base-specific binding sites in the DNA. The higher the (G+C) content was in the native DNA tested the higher was the quenching effect on the fluorescence intensity of quinacrine mustard. 7. No interaction was found between the dye and methylated DNA. The binding between quinacrine mustard and apurinic DNA was confirmed to be in the phosphate groups of the purines.     

Cell cycle dependent changes of chromosomes in mouse fibroblasts.

Mouse fibroblast interphase nuclei stained with quinacrine dihydrochloride show distinctive differences in their fluorescent characteristics analogous to those which we have already observed in human and Syrian hamster cells. These patterns reflect the position of any given nucleus within the cell cycle. The brightly fluorescent chromocenters in the mouse nuclei were found to be in absolute aggreement with those stained by the C-banding technique, indicating that they represent centromeric heterochromatin. Furthermore, their number and size per nucleus were shown to vary in relation to the progress of the cell cycle.     

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.     

Reassessment of presumed Y/22 and Y/15 translocations in man using a new technique.

A new chromosome banding technique, distamycin A plus DAPI, has been used to reexamine cases of presumed Y/autosome translocations. In contrast with the results obtained with quinacrine fluorescence (Q-banding), the satellites of acrocentric chromosomes do not fluoresce brightly with this new (DA-DAPI) method, making it more specific for the long arm of the Y chromosome. Previous cases with intensely Q-fluorescent and abnormally long short arms on a chromosome 22 were considered as presumptive 22/Y translocations: The new technique clearly shows that, in these cases, the additional material on 22p is not derived from Yq. In contrast, in other cases the Yq nature of additional material on 15p, in conjunction with the presence of an extra Y-body in interphase nuclei and the presence of a male-specific DNA, supports the previous diagnosis of a presumptive 15/Y translocation.     

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.     

Y-Fluorescence of Interphase Nuclei,Especially Circulating Lymphocytes

Application of a fluorescence technique for the detection of the human Y chromosome in interphase nuclei indicates that the best results may be obtained from the study of lymphocytes in blood smears. The most satisfactory Y-fluorescence is obtained with the use of buffered quinacrine mustard dihydrochloride. The method can be complemented by other standard techniques to obtain a clear idea of the sex-chromosome complement of the individual. The application of this technique to clinical and chromosomal diagnosis and to the prenatal detection of the Y chromosome in amniotic cells seems at least as promising as has proved the earlier identification of the Barr body.     

Identifying the fluorescent body (F-body) with quinacrine mustard staining of canine

The fluorescent body (F-body) was identified with quinacrine mustard (Q-M) staining in spermatozoon and lymphocyte of canine. Well washed sperm suspension was treated with protease (125 mg/ml) or dispase (2000p. u./ml) and staining with Q-M (final dilution 50 micrograms/ml) for 15 min to 24 hr at 37 degrees C. The lymphocyte cultures from whole blood were prepared as routine human investigation. The chromosomal preparation made by air dry method was stained with Q-M (final dilution 0.5 to 50 micrograms/ml) after pretreatment of enzyme digestion. The examination using a reflected fluorescent microscope revealed that the same F-body in human was present in both spermatozoon (20.1-39.7%) and interphase of lymphocyte (0.37.2%) of male origin.     

Chromosome banding in amphibia

The chromosomes of 26 species of Anura from variously highly evolved groups were analysed with the fluorescent GC-specific antibiotics mithramycin and chromomycin A₃ as well as with the AT-specific quinacrine. The mithramycin- and chromomycin A₃-stainings generally resulted in a pattern of the constitutive heterochromatin opposite to the one obtained with quinacrine stain. The weaker a heterochromatic region fluoresces with quinacrine, the stronger is the intensity of the fluorescence achieved with mithramycin and chromomycin A₃. Some of the telomeric and interstitial heterochromatic regions, however, exhibit no enhanced fluorescence with any of the fluorochromes. The nucleolar constrictions of the nucleolus organizer regions (NORs) displayed the brightest mithramycin- and chromomycin A₃-fluorescence in the karyotypes and interphase nuclei of all species examined. The contrast of the brightly fluorescing GC-rich heterochromatin and of the NORs is considerably enhanced, when the non-fluorescent AT-specific oligopeptide distamycin A is employed as a counterstain. No banding patterns were observed with the fluorochromes in the euchromatic regions of the metaphase chromosomes; this is attributed to the strong spiralization of the anuran chromosomes. A cytochemical classification of the various chromatin types in the anuran chromosomes is discussed on the basis of the differential labelings found on the constitutive heterochromatin by means of the fluorochromes.This paper is dedicated to Professor Dr. Hans Bauer on the occasion of his 75th birthday     

Optical studies of complexes of quinacrine with DNA and chromatin: implications for the fluorescence of cytological chromosome preparations

The fluorescence and circular dichroism of quinacrine complexed with nucleic acids and chromatin were measured to estimate the relative magnitudes of factors influencing the fluorescence banding patterns of chromosomes stained with quinacrine or quinacrine mustard. DNA base composition can influence quinacrine fluorescence in at least two ways. The major effect, evident at low ratios of quinacrine to DNA, is a quenching of dye fluorescence, correlating with G-C composition. This may occur largely prior to relaxation of excited dye molecules. At higher dye/DNA saturations, which might exist in cytological chromosome preparations stained with high concentrations of quinacrine, energy transfer between dye molecules converts dyes bound near G-C base pairs into energy sinks. In contrast to its influence on quinacrine fluorescence, DNA base composition has very little effect on either quinacrine binding affinity or the circular dichroism of bound quinacrine molecules. The synthetic polynucleotides poly(dA-dT) and poly(dA)-poly(dT) have a similar effect on quinacrine fluorescence, but differ markedly in their affinity for quinacrine and in the circular dichroism changes associated with quinacrine binding. Quinacrine fluorescence intensity and lifetime are slightly less when bound to calf thymus chromatin than when bound to calf thymus DNA, and minor differences in circular dichroism between these complexes are observed. Chromosomal proteins probably affect the fluorescence of chromosomes stained with quinacrine, although this effect appears to be much less than that due to variations in DNA base composition. The fluorescence of cytological chromosome preparations may also be influenced by fixation effects and macroscopic variations in chromosome coiling.     

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.     

A new differential technique for staining the heteropycnotic X-chromosome in female mice

The technique described here is the result of a successful attempt to identify the heteropycnotic X-chromosome at metaphase in female mice. A new modification of the commonly used air-drying technique for spreading chromosomes was employed using bone marrow cells. After treatment with hypotonic solution of 0.5% KCl at 50 °C for 30 min, all the chromosomes except one of the two X-chromosomes were only palely stained with Giemsa solution (pH 6.9) or quinacrine mustard. This X-chromosome with high stainability was not observed in males.     

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.     

Non-random position of homologous chromosomes (no. 9 and YY) in interphase nuclei of human fibroblasts (author's transl)]

The position of chromosomes No. 9 and of the Y chromosomes in interphase nuclei was observed by means of the Giemsa-11 staining and the quinacrine mustard fluorescence staining respectively. 3 fibroblast cultures from normal female persons and 1 culture from a person with the karyotype 47/XYY were used. The distance between the two homologous chromosomes was compared with the theoretical expected distance between two points which are randomly positioned in a circular area. The distance between the chromosomes No. 9 as well as between the two Y chromosomes is significantly smaller than expected with a random position. This tendency for a somatic association is stronger in the case of the two Y chromosomes than in the two chromosomes No. 9.