Electrofusion of fibroblasts on the porous membrane.

Electric fusion of cells is usually performed in two steps: the first is the creation of tight intercellular contact, the second is an application of electric pulses which induce membrane fusion proper. In the present work a new technique of cell electrofusion on the porous film is described. It consists of preliminary cultivation of cell monolayer on the porous film (protein-coated cellophane). Then cells of the same or any other type are added from above to form a second cell layer upon the first one. The pulses of the electric field are applied normally to the plane of the double cell layer to induce cell fusion. After pulse application a picture of mass polynucleation was observed. At the same time we did not obtain fusion of L cells by means of dielectrophoretic electrofusion technique. This difference in efficiency could be explained by the formation of broad zones of membrane contact between the cells adherent to the film, while during intensive dielectrophoresis only the point contacts were revealed. The high-conducting medium for electric treatment providing an efficient fusion on the film and high cell viability was composed. Neither cytochalasin B nor colcemid affected cell fusion noticeably; however the sodium azide (added with 2-deoxyglucose) inhibited fusion completely. The short hypotonic shock after electric treatment enhanced the rate of polycaryon formation.

Structural rearrangements and insertions of dispersed elements in pericentromeric alpha satellites occur preferably at kinkable DNA sites.

Centromeric region of human chromosome 21 comprises two long alphoid DNA arrays: the well homogenized and CENP-B box-rich alpha21-I and the alpha21-II, containing a set of less homogenized and CENP-B box-poor subfamilies located closer to the short arm of the chromosome. Continuous alphoid fragment of 100 monomers bordering the non-satellite sequences in human chromosome 21 was mapped to the pericentromeric short arm region by fluorescence in situ hybridization (alpha21-II locus). The alphoid sequence contained several rearrangements including five large deletions within monomers and insertions of three truncated L1 elements. No binding sites for centromeric protein CENP-B were found. We analyzed sequences with alphoid/non-alphoid junctions selectively screened from current databases and revealed various rearrangements disrupting the regular tandem alphoid structure, namely, deletions, duplications, inversions, expansions of short oligonucleotide motifs and insertions of different dispersed elements. The detailed analysis of more than 1100 alphoid monomers from junction regions showed that the vast majority of structural alterations and joinings with non-alphoid DNAs occur in alpha satellite families lacking CENP-B boxes. Most analyzed events were found in sequences located toward the edges of the centromeric alphoid arrays. Different dispersed elements were inserted into alphoid DNA at kinkable dinucleotides (TG, CA or TA) situated between pyrimidine/purine tracks. DNA rearrangements resulting from different processes such as recombination and replication occur at kinkable DNA sites alike insertions but irrespectively of the occurrence of pyrimidine/purine tracks. It seems that kinkable dinucleotides TG, CA and TA are part of recognition signals for many proteins involved in recombination, replication, and insertional events. Alphoid DNA is a good model for studying these processes.

[Subregional localization of recombinant cosmids, containing microsatellite sequences, on human chromosome 13]. / Subregional'naia lokalizatsiia na khromosome 13 cheloveka rekombinantnykh kosmid, soderzhashchikh mikrosatellitnye posledovatel'nosti.

Twenty four recombinant cosmids were subregionally localized by fluorescent in situ hybridization on human chromosomes. Fifteen of the clones were found to belong to only one chromosome: 13 clones located on chromosome 13, one located on chromosome 1, and one on chromosome 11. Nine cosmids were located in nuclear organizer regions. The clones gave signals from NOR regions of chromosome 13 and all other chromosomes containing the NOR region. The cosmid probes were selected from the chromosome 13 cosmid library as ones containing microsatellite repeats with motifs GACA, GACT, GATG, TCC, and CA. Each of the 9 clones located in the NOR region contains microsatellites GACA and TCC. Among the 15 clones giving unique signals, we found 9 clones with the GACT microsatellite, and three clones containing one of the microsatellites GATG, TCC, and CA. These microsatellite-containing clones can be used to make polymorphic genetic markers for fine genetic mapping of chromosome 13.

[A beta-casein gene fragment is located in region 1p3 of human chromosome 1]. / Fragment beta-kazeinovogo gena lokalizovan v oblasti 1p3 pervoi khromosomy cheloveka.

Analysis of the nucleotide sequence of a cloned fragment of the human beta-casein gene was performed. A conserved DNA locus, present with a varying degree of degeneracy in introns of [beta]-casein genes of several species and in the intron of an oncogene of the sarc family, was revealed. It was located in region 1p31-32 by means of in situ hybridization. A clearly visible additional hybridization site was observed in region 1p36. The data obtained are discussed in the light of available information on several oncogenes that are located in region 1p3. A relation was found between this region and the occurrence of some cancers, including breast cancer.

[Use of photo-anchoring of DNA probes for fluorescent in situ hybridization]. / Ispol'zovanie fotoprishivaemykh DNK-zondov dlia fluorestsentnoi gibridizatsii in situ.

A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization.

[The composition of the blood in pups of the Baikal seal Phoca sibirica during forced diving]. / Sostav krovi shchenko baikal'skoi nerpy Phoca sibirica pri prinuditel'nom nyrianii.

A set of parameters, such as pO2, pCO2, the content of glucose, glycogen, lactate, pyruvate, cholesterol, insulin, triglycerides, cAMP in the blood from the venous sinus has been investigated in puppies of Baikal seals during forced diving and rehabilitation. The dynamics of these parameters is presented.

[RNA and nuclear protein synthesis in regenerating and uninjured rabbit tissues]. / Sintez RNK i iadernykh belkov v regeneriruiushchei i netravmirovannykh tkaniakh krolikov.

Synthesis of RNA and nuclear proteins was activated in regenerating rabbit muscles within 2-8 days after trauma; within the subsequent periods (13-31 days) it was comparable with control values in most cases. The trauma affected the RNA synthesis in several other nontraumatized tissues of animals.