7-Amino-actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry

A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10(-5)M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%. The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.     

7-Amino-actinomycin D complexes with deoxynucleotides as models for the binding of the drug to DNA

Fluorescence, CD, absorption, and ¹H-nmr studies are reported for complexes of 7-amino-actinomycin D with deoxydinucleotides, deoxytetranucleotides, and poly(dG-dC)· poly(dG-dC). The optical spectra for the 7-amino-actinomycin D complex with pdG-dC, pdG-dC-dG-dC and pdC-dG-dC-dG are similar in shape to the 7-amino-actinomycin D complex with either DNA or poly(dG-dC). The changes in the ¹H chemical shifts of the 7-amino-actinomycin D and the pdG-dC resonances that accompany complex formation show that 7-amino-actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The magnitudes of the induced chemical shifts for the 7-amino-actinomycin D complex formation with pdG-dC are similar to, but slightly different from, the induced chemical shifts which are obtained when actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The pdN-dG dinucleotides (N = C, A, or T) form stacked complexes with 7-amino-actinomycin D. The presence of the 7-amino-group results in a larger dimerization constant (in aqueous solution) for 7-amino-actinomycin D [K_D(6°C) = 4.4 × 10³M^?1], as compared to actinomycin D [K_D(6°C) = 1.7 × 10³M^?1]; the chemical shifts which accompany dimer formation indicate that the chromophores stack in an inverted manner. Intercalation of 7-amino-actinomycin D into minature double helices, as well as into calf thymus DNA, poly(dG-dC)·poly(dG-dC), and poly(dA-dC)·poly(dG-dT), results in an enhancement of the relative fluorescence intensity and a shift in both the absorbance and corrected emission spectra.     

Spectral properties of chloroplast membranes as a function of physiological temperatures.

The absorption spectrum of chloroplasts changes as a function of temperature. As chloroplasts are cooled from room temperature to 10°C there are increases in absorption at 675, 500 and 436 nm, plus a small decrease at 685 nm. As the chloroplasts are heated to 34°C there are decreases in absorption at 675, 500 and 436 nm plus increases in absorption at 690 and 400 nm. It is concluded that the temperature dependent change in phase of the membrane lipids (solid to liquid crystal state) modifies the state of chlorophyll aggregation.     

7-Azidoactinomycin D: a novel probe for examining actinomycin D-DNA interactions

The technique of photoaffinity labeling is applied to the actinomycin D system to provide a novel probe for the examination of the interactions of actinomycin D with nucleic acids. The capacity for covalent attachment of actinomycin D will aid greatly in the study of target-site specificities and the correlations of biological effects with biophysical DNA interactions. Through chemical modification of the parent actinomycin D molecule with a photoreactive azido substituent, a functional analog of the parent actinomycin D is generated having equilibrium binding properties identical to those of the parent molecule yet with the capacity to form a covalent attachment to DNA upon photolysis. The results presented here describe the noncovalent interactions of this photoreactive probe to DNA (absence of light) and compares the binding properties observed to those of the parent actinomycin D and 7-aminoactinomycin D analog. These studies demonstrate that the DNA binding properties (i.e. binding affinity, binding site size, and sequence specificity) retained by the 7-azidoactinomycin D, thus providing a suitable probe for examining actinomycin D-DNA interactions.     

Deoxyribonuclease II as a probe for chromatin structure. I. Location of cleavage sites

DNAase II has been shown to cleave condensed mouse liver chromatin at 100-bp² intervals while chromatin in the extended form is cleaved at 200-bp intervals (Altenburger et al., 1976). Evidence is presented here that DNA digestion patterns of a half-nucleosomal periodicity are also obtained upon DNAase II digestion of chicken erythrocyte nuclei and yeast nuclei, both of which differ in their repeat lengths (210 and 165 bp) from mouse liver chromatin. In the digestion of mouse liver nuclei a shift from the 100-bp to the 200-bp cleavage mode takes place when the concentration of monovalent cations present during digestion is decreased below 1 mM. When soluble chromatin prepared by micrococcal nuclease is digested with DNAase II the same type of shift occurs, albeit at higher ionic strength.In order to map the positions of the DNAase II cleavage sites on the DNA relative to the positions of the nucleosome cores, the susceptibility of DNAase II-derived DNA termini to exonuclease III was investigated. In addition, oligonucleosome fractions from HaeIII and micrococcal nuclease digests were end-labelled with polynucleotide kinase and digested with DNAase II under conditions leading to 100 and 200-bp digestion patterns. Analysis of the chain lengths of the resulting radioactively labelled fragments together with the results of the exonuclease assay allow the following conclusions. In the 200-bp digestion mode, DNAase II cleaves exclusively in the internucleosomal linker region. Also in the 100-bp mode cleavage occurs initially in the linker region. Subsequently, DNAase II cleaves at intranucleosomal locations, which are not, however, in the centre of the nucleosome but instead around positions 20 and 125 of the DNA associated with the nucleosome core. At late stages of digestion intranucleosomal cuts predominate and linkers that are still intact are largely resistant to DNAase II due to interactions between adjacent nucleosomes. These findings offer an explanation for the sensitivity of DNAase II to the higher-order structure of chromatin.     

Spectral properties of phosphopyridoxyl-Lys-7(-41)-ribonuclease A.

We have studied the spectral properties of RNAase A containing a phosphopyridoxyl residue at the epsilon-NH2 group of Lys-7 or Lys-14. The overall conformations of the native and modified enzymes were shown to be rather similar. All three proteins have similar circular dichroism spectra within the 220-300-nm region, and similar thermal transition temperatures. All the changes in the RNAase A molecule modified are located in close proximity to the alkylated lysine residue. The phosphopyridoxyl group of (P-Pxy)-epsilon-Lys-41-RNAase A is situated directly at the enzyme active site and is 25% butied in the protein globule. The P-pyridoxyl group of (P-Pxy)-epsilon-Lys-7-RNAase A was shown to be located in the vicinity of the active site and to be more exposed to the solvent. In the pyridoxyl phosphate absorption band, optical activity is induced in both proteins. Study of the pH dependence of the changes occurring in the circular dichroism and absorption spectra has shown that in the modified proteins, the pyridoxyl phosphate chromophore is rather sensitive to the ionic state of the surrounding medium and serves as a "reporter" group when the relationship between structure and function of the RNAase A active site is being investigated.     

7-Aminoactinomycin as a fluorescent probe for DNA unwinding and denaturation

A fluorescent analogue of antibiotic actinomycin D, 7-aminoactinomycin D (7AAMD), which is widely used in molecular biology, was shown by steady-state, polarization, and phase fluorescent spectroscopy to bind primarily in the unwound regions of DNA with concomitant increase in its emission intensity. The maximum emission intensity of 7AAMD is observed for denatured DNA. Thus, 7AAMD may serve as a good indicator of DNA unwinding, denaturation, and fragmentation.     

Filipin as a cholesterol probe. I. Morphology of filipin-cholesterol interaction in lipid model systems

We report some novel morphological observations on the interaction of the polyene antibiotic filipin (crude complex) with cholesterol, studied in non-cellular systems with replication, freeze-fracture, and negative stain techniques. Cholesterol crystals, lecithin liposomes containing 0 to 20 mole% of cholesterol, and liposomes containing 10 mole% of cholesterol and 5 to 40 mole% of sphingomyelin were incubated for varying lengths of time with filipin at different cholesterol: filipin molar ratios. The resulting filipin-induced lesions (FIL) were pleomorphic in all systems studied. In replicas of crystals, FIL appeared as ridges which were either straight, or curved into C- and S-shaped figures or closed circles. Negatively stained preparations showed FIL as white lines of the same configurations and in addition revealed a delicate veil attached to individual FIL. FIL, fused by their veils into clusters or large sheets ("holey sheets"), were shed from crystals. Incubation of liposomes for 1 h at cholesterol:filipin molar ratios of 4:1, 2:1, 1:1, and 1:5, demonstrated that cholesterol detection (i.e. formation of FIL) depend upon the ratio of cholesterol to filipin. At a 1:1 molar ratio FIL formed on liposomes containing 10 mole% cholesterol or more, but detectability increased to 5 mole% at the 1:5 ratio. Increasing the molar ratio of cholesterol:filipin to 2:1 and 4:1 decreased cholesterol detectability to between 10 and 20 mole%. Increasing concentrations of sphingomyelin decreased cholesterol detectability at the 1:1 cholesterol:filipin ratio; further, FIL in sphingomyelin-containing liposomes tended towards larger diameters. Filipin induced aggregation of liposomes and linked them together by holey sheets, providing evidence for filipin-induced extraction of cholesterol from liposomes. Taken together our morphological observations on filipin-cholesterol interaction in non-cellular systems raise pertinent questions as to the feasibility of filipin as a cholesterol probe in cellular systems.     

Immune function in aged mice. I. T-cell responsiveness using phytohaemagglutinin as a functional probe.

The loss of cell-mediated immunity with age was assessed by a detailed analysis of the in vitro response of murine lymphocytes to the well-defined probe of T-cell function, PHA (phytohaemagglutinin). The reduced mitogenic activity of lymphoid cells from old mice compared with young mice could not be explained in terms of a shift in kinetics of the responding cells. Removal of macrophages, which are known to exert a regulatory effect on T-cell function, failed to reverse the poor response of old lymphoid cells. Furthermore, no evidence was found for a role of soluble inhibitors released by either lymphocytes or macrophages in the decreased response of old cells. Not only were old cells less efficient in producing such factors, but in addition, they responded less well to them than did young cells. Taken together, these observations implied that the defect in PHA responsiveness of old cells is due to a disturbance in the T cells themselves rather than to any extracellular influences. The total number of T cells, assessed by labelling with anti-Thy-1 serum was comparable in old and young animals. Selective depletion of a subpopulation of PHA-reactive cells was excluded by direct quantitation of PHA-binding cells. Thus, 25% of small lymphocytes from the spleens of old mice bound ¹²⁵I-labelled PHA ([¹²⁵I]PHA) compared with 15% in the case of young mice. To show that the cells binding PHA were those reacting to it, a suicide technique was used. Spleen cells pretreated with [¹²⁵I]PHA failed to respond to subsequent challenge with the specific mitogen, but could mount a normal response to a control (B-cell), mitogen, LPS (lipopolysaccharide). When PHA cultures were carried out in the presence of colchicine, fewer cells from old mice were found to react to the mitogenic signal. In the absence of evidence for depletion of precursor cells, the conclusion was reached that the T-cell defect in old mice is more likely to be qualitative than quantitative, perhaps due to metabolic or structural abnormalities preventing lymphocyte transformation and/or proliferation.     

An investigation of the conformational properties of ribosomes using N-ethylmaleimide as a probe.

The reactivity of ribosomal proteins towards N-ethylmaleimide has been examined in a variety of ribosome and ribosomal subunit preparations from Escherichia coli. The data show that samples which would be regarded as equivalent operationally can differ significantly in conformation, as judged by reactivity, depending on the method of preparation. The washing of ribosomes with high concentrations of salt has a particularly dramatic effect on protein reactivity. The implications of these results for our understanding of ribosome conformation and for the further study of conformation by chemical reactivity are discussed.     

7-Azido-actinomycin D: a photoaffinity probe of the sequence specificity of DNA binding by actinomycin D

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5'-dGpdC-3' sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5'-flanking base was a pyrimidine and the 3'-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5'-TGGG-3'.     

Photographic colorimetry as a quantitative cytochemical method

Summary A method for quantitative cytophotometry based on the photographic method of Ornstein is described. Taking special precautions, photographic negatives are made of microscopic objects with light from the appropriate part of the spectrum. Enlarged prints, developed in a blue colour, are made from these negatives. Stirring with a stream of nitrogen was applied in this procedure.The images of the objects in the blue print are cut out, and the amount of dye proportional to the integrated extinction of the objects is measured in a colorimeter. This method, for which the name photographic colorimetry is proposed, was tested on Feulgen-stained nuclei from various sources. The reproducibility of the method was found to range from 3 to 5%.Submitted in partial fulfillment of the requirements for Ph. D. degree (Den Tonkelaar 1963).