[Site-specific scission of DNA of polytene chromosomes of Chironomus thummi in situ by the oligoadenylate alkylating derivatives]. / Induktsiia napravlennykh razryvov v DNK politennykh khromosom Chironomus thummi in situ pri deistvii alkiliruiushchikh proizvodnykh oligoadenilatov.

The possibility of site-specific scission of DNA in polytene chromosomes in situ by means of the method of complementarily addressed fragmentation is demonstrated. The fragmentation of polytene chromosomes of Chironomus thummi resulting from the alkylation of the denatured chromosomes with oligoadenylate derivatives, followed by scission of the DNA in specific sites and enlargement of nicks with exonucleases was investigated. Single-stranded regions were registered by means of luminescence microscopy after staining the chromosomes with acridine orange. The pattern of degradation of the chromosomes depends on the length of the oligoadenylate part of the reagents and is different from that obtained with uridine-5'-methylphosphate alkylating derivative which does not form any complexes with DNA. Oligoadenylates inhibit the action of the alkylating derivatives on the chromosomes.

[Preparation and properties of stretched polytene chromosomes. 2. Mechanism of stretching]. / Poluchenie i svoitsva rasianutykh politennykh khromosom. 2. Mechanizm rastiazheniia.

Isolated polytene chromosomes were stretched in a 0.125 M NaCl solution with constant speed, by constant force and by cyclically changing force. For each regime, the dependence of chromosome length on the time and force magnitude were recorded. From this it may be concluded that three processes are involved in chromosome stretching: viscoelastic deformation, viscous flow of DNP segments, and cristallization, i.e. intermolecular cross-linking of neighbour segments. At a high rate stretching (V greater than Vo) chromosome may be torn like at small deformation; when rate is V greater than Vo chromosome deformation is mostly viscoelastic; at rates V approximately Vo viscous flow of DNP segments if predominant. We estimate Vo approximately less than 3--6 mum/s. Electron microscopy shows that during chromosome stretching its DNP fibers are oriented along chromosome axis without detectable breaks.

[Preparation and properties of elongated polytene chromosomes. I. The method]. / Poluchenie i svoistva rastianutykh politennykh khromosom. I. Metod.

Compactness of eukaryotic, particularly polytene chromosomes pose difficulties for investigation of their functional organization. For example, 0.1 --1.0 micrometer thick bands of polytene chromosomes contain some dozens microns pieces of DNA molecules. Therefore the useful resolving power of autoradigraphical methods is reduced by 10--100 times of its upper limit. To overcome the mentioned difficulty, a new method has been developed which permits to attain the upper limit of resolution. An isolated polytene chromosome from salivary gland nucleus of Chironomus thummi larva is stretched by microneedles to obtain a bundle of oriented DNP-fibers. A previously chosen small region of the chromosome (band or puff) is stretched simultaneously in a transverse direction by a stick frame made of another chromosome. Electron microscopy of the preparation reveals a meshwork of DNP fibers as presented on fig. 6.

[Stretched polytene chromosomes--model for studying the functional organization of eukaryotic chromosomes]. / Rastianutye politennye khromosomy--model' dlia izucheniia funktsional'noi organizatsii khromosom éukariot

To localize functional loci on cytological maps of polytene chromosomes we propose to use 10-100 times stretched chromosomes. Three different ways of stretchening are briefly considered: the squash tissue preparation, when chromosomes are stretched by hydrodynamical forces; the treatment of isolated polytene chromosomes in 10-minus 4M EDTA OR 0.8M NaCL with subsequent change of these solution for saline when abrupt structural changes occur in chromosomes and they become morphologically homogeneous threads (Gruzdev and Belaya, 1973); and, finally, the use of microneedles of the micromanipulator. After an intense (ca. 100 times) stretchening, the autoradiography is sufficient to localize the loci within one micron length of double helical DNA molecule.