Selective excitation of mithramycin or DAPI fluorescence on double-stained cell nuclei and chromosomes

Summary Fluorescence spectra of leukocytes stained by both mithramycin and DAPI showed that the fluorescence of the two dyes can be separated efficiently by using different excitation wavelengths, for instance the 435 nm and the 365 nm mercury lines. In human chromosomes the complementary (reverse) banding pattern produced by these dyes may thus be observed on double stained chromosome spreads. In plants, for instance in Anemone blanda, the two dyes may reveal two different banding patterns. The results of absorption and fluorescence measurements suggest the existence of at least two binding sites, or types, for each dye, with different fluorescent yields and binding strengths.     

DAPI fluorescence in nuclei isolated from tumors.

In DNA histograms of some human solid tumors stained with nuclear isolation medium--4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI), the coefficient of variation (CV) of the G0/G1 peak was broad, and in nuclear volume vs DNA scattergrams, a prominent slope was seen. To determine the cause for this, nuclei from frozen breast tumors were stained with NIM-DAPI and analyzed after dilution or resuspension in PBS. In two-color (blue vs red) analysis, most of the slope and broad CV was due to red fluorescence of nuclei stained with NIM-DAPI, which was reduced on dilution or resuspension in PBS, resulting in elimination of the slope and tightening of the CV.     

DAPI fluorescence of plant chromosomes prestained with actinomycin D

Two plants, Ornithogalum caudatum and Scilla siberica, with contrasting types of heterochromatin, were employed to investigate the effect of prestaining with actinomycin D on the differential DAPI fluorescence in nuclei and chromosomes. Actinomycin D-pretreated preparations exhibited in both plants a significantly lower overall fluorescence intensity than DAPI controls. In O. caudatum the fluorescence properties of DAPI-bright heterochromatin remained qualitatively unchanged, but in S. siberica chromosomes the differential fluorescence of DAPI-negative heterochromatin disappeared or occasionally was reversed. It is shown that the observations cannot be interpreted simply as a result of base specific drug-DNA interaction.     

Mechanisms of quinacrine binding and fluorescence in nuclei and chromosomes

The mechanisms has been investigated whereby quinacrine binds to the DNA of nuclei and chromosomes in cytological preparations fixed in methanol-acetic acid. A variety of evidence is consistent with the idea that the quinacrine binds by intercalation. This is supported by a high value for the affinity of quinacrine for DNA, together with a saturation value of 0.2 quinacrine molecules/nucleotide; binding in the presence of strong salt solutions; and inhibition of fluorescence and banding by denaturation or depurination of DNA. At high quinacrine concentrations, weak binding of quinacrine to nuclei and chromosomes also occurs, but this is not relevant to the production of strong fluorescence or Q-banding patterns. A number of factors were tested which might have affected quinacrine fluorescence and banding. These included: pH; blocking protein amino groups by acetylation or benzoylation; introduction of hydrophobic groups by benzoylation; and dephosphorylation. All these treatments were without effect. However, comparison of the quinacrine fluorescence of human and onion nuclei, which differ substantially in the base composition of their DNA, shows that quinacrine fluorescence can be enhanced in cytological preparations by AT-rich DNA.     

Mechanisms of quinacrine binding and fluorescence in nuclei and chromosomes

Summary The mechanism has been investigated whereby quinacrine binds to the DNA of nuclei and chromosomes in cytological preparations fixed in methanol-acetic acid. A variety of evidence is consistent with the idea that the quinacrine binds by intercalation. This is supported by a high value for the affinity of quinacrine for DNA, together with a saturation value of 0.2 quinacrine molecules/nucleotide; binding in the presence of strong salt solutions; and inhibition of fluorescence and banding by denaturation or depurination of DNA. At high quinacrine concentrations, weak binding of quinacrine to nuclei and chromosomes also occurs, but this is not relevant to the production of strong fluorescence or Q-banding patterns.A number of factors were tested which might have affected quinacrine fluorescence and banding. These included: pH; blocking protein amino groups by acetylation or benzoylation; introduction of hydrophobic groups by benzoylation; and dephosphorylation. All these treatments were without effect. However, comparison of the quinacrine fluorescence of human and onion nuclei, which differ substantially in the base composition of their DNa, shows that quinacrine fluorescence can be enhanced in cytological preparations by AT-rich DNA.In honour of Prof. P. van Duijn     

Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization.

The enormous potential of in situ hybridization derives from the unique ability of this approach to directly couple cytological and molecular information. In recent years, there has been a surge of success in powerful new applications, resulting from methodologic advances that bring the practical capabilities of this technology closer to its theoretical potential. A major advance has been improvements that enable, with a high degree of reproducibility and efficiency, precise visualization of single sequences within individual metaphase and interphase cells. This has implications for gene mapping, the analysis of nuclear organization, clinical cytogenetics, virology, and studies of gene expression. This article discusses the current state of the art of fluorescence in situ hybridization, with emphasis on applications to human genetics, but including brief discussions on studies of nuclear DNA and RNA organization, and on applications to clinical genetics and virology. Although a review of all of the literature in this field is not possible here, many of the major contributions are summarized along with recent work from our laboratory.     

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.     

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.     

Detection of retinoblastoma gene copy number in metaphase chromosomes and interphase nuclei by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) was applied to detect the copy number of the retinoblastoma (RB1) tumor suppressor gene in metaphase chromosomes and interphase nuclei. We used 14 lambda phage clones spanning the whole RB1 gene region as a probe and obtained a specific hybridization signal in normal metaphase chromosomes at 13q14. Normal interphase nuclei showed two RB1 signals in about 90% of cases, whereas two cell lines with cytogenetically defined deletions involving the RB1 gene showed only one hybridization signal in about 80% of the nuclei. Analogous changes were detected in metaphase chromosomes. Multicolor FISH with subsets of the phage clones allowed visualization of subregions within the 200-kb gene in interphase nuclei. Analysis of clinical breast cancer samples showed that most of the cells contained two copies of the RB1 gene, even when restriction fragment length polymorphism analysis showed loss of heterozygosity (LOH) at the RB1 locus. This indicates that LOH at the RB1 locus in breast cancer cells probably involves mechanisms other than physical deletion.     

Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae.

Repetitive DNA was cloned from HindIII-digested genomic DNA of Larix leptolepis. The repetitive DNA was about 170 bp long, had an AT content of 67%, and was organized tandemly in the genome. Using fluorescence in situ hybridization and subsequent DAPI banding, the repetitive DNA was localized in DAPI bands at the proximal region of one arm of chromosomes in L. leptolepis and Larix chinensis. Southern blot hybridization to genomic DNA of seven species and five varieties probed with cloned repetitive DNA showed that the repetitive DNA family was present in a tandem organization in genomes of all Larix taxa examined. In addition to the 170-bp sequence, a 220-bp sequence belonging to the same DNA family was also present in 10 taxa. The 220-bp repeat unit was a partial duplication of the 170-bp repeat unit. The 220-bp repeat unit was more abundant in L. chinensis and Larix potaninii var. macrocarpa than in other taxa. The repetitive DNA composed 2.0-3.4% of the genome in most taxa and 0.3 and 0.5% of the genome in L. chinensis and L. potaninii var. macrocarpa, respectively. The unique distribution of the 220-bp repeat unit in Larix indicates the close relationship of these two species. In the family Pinaceae, the LPD (Larix proximal DAPI band specific repeat sequence family) family sequence is widely distributed, but their amount is very small except in the genus Larix. The abundant LPD family in Larix will occur after its speciation.     

Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides

Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides. DAPI decay at pH 7 was decomposed using two exponential components of 2.8 and 0.2 ns of lifetime values, respectively. The double exponential character of the decay was maintained over a large pH range. Phase- and modulation-resolved spectra, collected between 420 and 550 nm, have indicated at least two spectral components associated with the two lifetime values. This, plus the observation of the dependence of the emission spectrum on the excitation wavelength, suggests a lifetime heterogeneity originating from ground-state molecular conformers, partially affected by pH changes. DAPI complexed with natural polydeoxynucleotides retained most of the features of DAPI decay in solution, except for the value of the long lifetime component that was longer (approximately 4 ns) and the relative fractional fluorescence intensities of the two components that were inverted. AT polymers/DAPI complexes show single exponential decay. Solvent shielding when DAPI is bound to DNA changes the indole ring solvation and stabilizes the longer lifetime decay component. For poly(GC)/DAPI complex, the decay was similar to that of free DAPI in solution, proving the dependence on the polydeoxynucleotides sequence the different types of binding and the reliability of the fluorescence method to solve them.     

Unique techniques for cell analysis utilizing mithramycin and flow microfluorometry.

Cell cycle analysis through utilization of the mithramycin/flow microfluorometric technique combines simplicity (one-step cell preparation) with speed (results available within 20 min after removal of a sample from a culture). Furthermore, the technique is useful in many situations in which standard ³H-thymidine autoradiography and mitotic accumulation are unsatisfactory. Examples are provided which demonstrate a continuous monitoring of experiments in progress, including analysis of populations devoid of cells in S or M phases, analysis of arrested or slowly progressing populations resulting from exposure to toxic agents, detection of abnormalities in mitosis such as nondisjunction and polyploidization, and localization within the cell cycle of dying cells in cultures exposed to toxic agents.     

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

 Apoptotic cell nuclei are known to stain hyperchromatically with absorption dyes and dimly with many DNA fluorochromes. We hypothesised that both optical phenomena have the same cause - the ability of apoptotic chromatin to aggregate cationic dyes. This hypothesis was tested using prednisolone-primed rat thymus, which is known to contain apoptotic cells. The apoptotic cells were classified as early and late, based on their morphology, in thin and semithin sections and in thymus imprints on slides. Direct reaction for DNA strand breaks (TUNEL) indicated the presence of breaks in both categories of cells, with more intense labelling in late apoptosis. The chromatin ultrastructure of early apoptotic cells initially retained the supranucleosomal order of packaging which characterises control cells, whereas the dense chromatin of late apoptotic cells possessed the degraded structure. Absorption spectra of the toluidine blue-stained early apoptotic cell chromatin revealed a metachromatic shift, indicating a change of DNA conformation and polymerisation of the dye. When the staining was performed by acridine orange (preceded by a short acid treatment), a paradoxical several-fold increase of fluorescence intensity at a several-fold dilution of the dye was found. The simultaneous reduction of the ratio of red to green components of fluorescence confirmed that the concentration-dependent fluorescence quenching was due to aggregation of the dye. The results suggest that the enhanced affinity of the chromatin of early apoptotic cells for cationic dyes is associated with conformational relaxation rather than degradation of DNA. In late apoptotic cells, the very dense packaging of degraded DNA promotes further aggregation of dyes. The results suggest alternative methods for detection and discrimination of early and late apoptotic cells. Accepted: 12 February 1997