Application of 7-amino-actinomycin D for the fluorescence microscopical analysis of DNA in cells and polytene chromosomes.

The cytochemical properties of a guanine-specific synthetic fluorescent analogue of actinomycin D, 7-amino-actinomycin D, have been studied in fixed and living preparations of L cells and polytene chromosomes of salivary glands of Chironomus thummi thummi and Drosophila lummei (Hackman). 7-Amino-actinomycin D has been shown to bind to DNA-containing structures, thereby inducing in them a bright red fluorescence. No specific fluorescence has been found in RNA-containing structures treated with this fluorescent probe. The fluorescence pattern of some regions of polytene chromosomes with a known nucleotide composition was analysed. It has been established that 7-amino-actinomycin D induces a very weak fluorescence in GC-poor chromosome regions of the Drosophila lummei toromere structure. Data indicating a nonlinear dependence between the fluorescence intensity of a stained chromosome region and the GC content in its DNA have been obtained. The influence of DNA nucleotide composition in a chromosome region on the fluorescence of 7-amino-actinomycin D is discussed. In combination with quinacrine staining and the Feulgen fluorescence reaction, treatment with 7-amino-actinomycin D provides useful information about the distribution of GC base pairs in the chromosome region under study.

[Fluorescence microscopic and autoradiographic analyses of the differential reaction of various regions of polytene chromosomes in Chironomus to prolonged exposure to 7-amino-actinomycin D and 3H-actinomycin D]. / Fluorestsentno-mikroskopicheskii i avtoradiograficheskii analizy differentsial'noi reaktsii razlichnykh uchastkov politennykh khromosom khironomusa na dlitel'noe vozdeistvie 7-amino-aktinomitsina D i 3H-aktinomitsina D.

Dynamics of binding of a fluorescent analogue of actinomycin D -- 7-amino-actinomycin D -- and 3H-actinomycin D with polytene chromosomes of Ch. thummi was studied. Biological effects of AMD, 7-amino-AMD, and 3H-AMD on polytene chromosomes were found to be similar. These ligands provoke the reduction of the nucleolus and Balbiani rings and the appearance of giant pseudo-puffs in heterochromatic centromere regions of polytene chromosomes. There was no intermediate binding of 7-amino-AMD to DNA in vivo both after a longterm treatment of larvae with fluorochrome and in chase experiments. It was found that a loosening of chromatin in centromere regions accompanied by a weakening of its fluorescence took place in the formation of pseudo-puffs. Possible mechanisms of pseudo-puff formation under the influence of AMD and 7-amino-AMD are discussed. Essential factors may be peculiarities of DNA nucleotide composition in centromere regions, DNA packing, alteration of physico-chemical properties of DNA in the complex with AMD (despiralizations and elongation), and an inhibition of RNA synthesis necessary for the maintenance of normal structure of polytene chromosomes.

[Effect of pilocarpine on the puffing of polytene chromosomes in the salivary glands of Chironomus thummi]. / Vliianie pilokarpina na puffing politennykh khromosom sliunnykh zhelez Chironomus thummi

Under the influence of pylocarpine on polytene chromosomes of the salivary glands of Chironomus thummi at the end of the 4th age, the puff size increased in the region 3-A5cd, 3-C3ghi, 4-KB1 and 4-KB2, in parallel with the increase of the amount of secrete in the central reservoire. In this connection, the above indicated puff may be considered as closely correated in their function with the secretory process of salivary gland cells.