Fluorescent labeling of fragments of high molecular weight RNA.
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Acriflavine↗
Biomedical subjects
J W Levinson
Publications and source records attributed to J W Levinson.
Correlation among the rates of dimer excision, DNA repair replication, and recovery of human cells from potentially lethal damage induced by ultraviolet radiation.
The kinetics of excision repair in confluent cultures of diploid human fibroblasts after ultraviolet irradiation at varying doses was measured by three different methods: (a) removal of thymine-containing dimers, (b) DNA excision repair synthesis, and (c) biological recovery of cells from the potentially lethal effects of the irradiation. Each method gave similar results and indicated that the excision rate was dependent upon the number of thymine-containing dimers induced (substrate concentration). For example, at a dose of 40 J/m2 (0.2% dimerization), the repair rate was 1.6 J/m2 per h as determined by a modified method to measure the number of thymine-containing dimers remaining in DNA and 1.65 J/m2 as measured by excision repair synthesis. At a dose of 7.5 J/m2, the repair rate was 0.5 J/m2 per h as measured by biological recovery, and at a dose of 7 J/m2, the repair rate was 0.46 J/m2 per h as measured by excision repair synthesis.
Cell Cycle↗
An improved acriflavine-Feulgen reagent for quantitative DNA cytofluorometry.
We have modified the acriflavine-Feulgen histochemical method for the quantitative determination of DNA by performing the staining of hydrolyzed cells affixed to cover slips with acriflavine dissolved in 90% ethanol. Compared to conventional techniques, in which the acriflavine is dissolved in aqueous HCl plus potassium metabisulfite, this method not only decreased non-Feulgen (background) staining in both the nucleus and the cytoplasm, but also increased the fluorescent intensity of Feulgen stained nuclei more than four-fold. Cells stained by our modified method exhibited a fluorescence maximum of 507 nm, which is similar to the 500 nm fluorescence maximum obtained with acriflavine bound to apurinic acid solution by a modification of the acriflavine-Feulgen method. These fluorescence maxima are in contrast to the 510 nm to 625 nm fluorescence maxima which are obtained when cells are stained according to conventional protocols. The fluorescence intensities of nuclei of synchronized cells stained by the modified method were proportional to DNA content. Thus, by the criteria of staining specificity, in situ and in solution fluorescence spectra agreement and quantitative staining, we conclude that our modified acriflavine-Feulgen method is more satisfactory for quantitatively measuring DNA in situ by cytofluorometry than the usual acriflavine-Feulgen method.
Acriflavine↗
Purification of commercial acriflavine by Sephadex LH-20 column chromatography.
Acriflavine supplies commercially was purified by removing contaminating proflavine by chromatography on a column of Sephadex LH-20 eluded with 65% methanol. The purified acriflavine, recovered in a yield that was 52% of the theoretical maximum, had a visible absorption maximum of 452 nm with a band width of 47 nm. These spectral properties agreed with those of acriflavine purified by precipitation, repeated recrystallization and chromatography on aluminum oxide, which results in a yield of 13% at best.
Acridines↗
An improved acriflavine-Feulgen method.
The acriflavine-Feulgen method for the histochemical demonstration of deoxyribonucleic acid was modified by staining hydrolyzed cells with 0.01% acriflavine dissolved in 90% ethanol. This method offered the following advantages: (a) it simplified the preparation of the acriflavine-Feulgen reagent; (b) it left the cytoplasm essentially unstained while staining the nuclei bright green in hydrolyzed cells and left the cytoplasm and nuclei essentially unstained in unhydrolyzed cells; (c) it eliminated poorly defined reagents from the staining solutions. Because of these staining properties, this technique may be especially useful in the quantitative determination of deoxyribonucleic acid by cytofluorometry.
Acridines↗
A moving spot high resolution scanning microspectrometer using focused laser illumination: illustrations with acriflavin stained sephadex beads and human fibroblast nuclei.
We describe a high resolution moving spot scanning microspectrometer, capable of absorption or fluorescence detection, using focused laser illumination which is moved over the sample by rotating the laser beam direction prior to focusing. This rotation is achieved by reflecting the beam from mirrors mounted on bending mode piezoelectric transducers which, when bent by an applied voltage, cause the mirrors to rotate. The images of optically thin samples are analyzed by considering the convolution of the focused spot intensity distribution with the absorbance of a uniformly stained spherical particle. This analysis is verified experimentally with data from acriflavin stained Sephadex beads. Data from acriflavin-Feulgen stained human fibroblasts indicate that the efficiency of this type of nuclear staining is about 2 to 3 dye molecules incorporated per 100 nucleotide pairs. Quantitative data on fading of acriflavin fluorescence in stained fibroblasts indicate that fading is negligible in the time required to record the microscope images.
Acridines↗
Fluorescent labeling of DNA in solution with covalently bound acriflavin.
We have prepared a fluorescent derivative of DNA based on the acriflavin-Feulgen histological procedure for staining DNA. Our procedure involved binding acriflavin to DNA in solution by reacting the acriflavin with aldehydes formed on the deoxyribose of DNA by controlled removal of a few percent of the purine bases of the DNA. Partially depurinated DNA was reacted with the acriflavin reagent, and unbound acriflavin was removed by chromatography on Sephadex G-25 eluted with phosphate buffered guanidine -HCl. Such single-stranded depurinated DNA bound 0.36 acriflavin molecules per 100 purine bases per h of depurination. DNA containing one bound acriflavin per 200 bases reassociated at 85% of the value of control DNA. The acriflavin - DNA complex showed new absorption maxima at 466 and 370 nm. The fluorescent product had excitation maxima at 304 and 465 nm and an emission maximum at 502 nm. This labeling procedure should be useful in place of or in addition to radioactive labeling for DNA.
Acridines↗