[Metabolic and structural properties of extrachromosomal DNA in mammalian cells]. / Metabolicheskie i strukurnye svoistva vnekhromosomnykh DNK v kletkakh mlekopitaiushchikh.

Discrete bands of free DNAs of approximately 25 kbp were detected in human cell cultures. According to electrophoretic shifts induced by single and double strand breaks, they from topological isoforms (supercoiled, open, and linear). Long-term labeling (24 h) of growing and quiescent cultured cells by [3H]thymidine indicates differences of free versus chromosomal DNAs including (i) significantly lower specific radioactivity in growing cells, (ii) higher specific radioactivity in quiescent cells, and (iii) resistance to fluorodeoxyuridine labeling. During apoptosis of cultured Namalwa cells, heterogeneous fragments are formed which differ from free DNAs. Crosslinking of nascent RNA with DNA template by 8-methoxypsoralen indicated slight transcriptional activity of free DNAs.

A procedure for repeated usage of 33P-labeled DNA probes in hybridization experiments.

We describe a technique for repeated use of 33P-labeled DNA probes in Southern hybridization experiments. A nick-translated 33P-labeled DNA probe in a volume of 0.5-1.0 ml of hybridization mixture (final concentration, 10-100 ng/ml) is used to wet a sheet of filter paper (approx 10 microliters/cm2), which covers a nylon membrane with DNA transferred by Southern blotting, and both are set between two washed X-ray films. The "sandwich" is placed in a plastic bag for hybridization for 16-24 h at 42 degrees C. This very simple procedure using 33P-labeled DNA probes has a number of advantages over the standard method using 32P-labeled probes: (a) a significantly lower biohazard (body/arms exposure); (b) a very small volume of hybridization mixture in contact with a DNA-containing membrane and the higher probe concentrations attainable, causing some increase in sensitivity, and, finally, (c) repeated use of the probe-containing filter (over approx 3 days for unique sequences and up to 2 weeks for reiterated sequences) due to a relatively long 33P half-life (25.3 days).

A procedure for DNA and RNA transfer to membrane filters avoiding weight-induced gel flattening.

We describe a technique of rapid (within 1-2 h) transfer of DNA and RNA from agarose gels to nitrocellulose or nylon membrane filters. It is characterized by nearly complete elimination of mechanical action on the gel (a thin layer of liquid is placed over the gel and, filtering through the gel into a stack of paper towels beneath, it transfers nucleic acids onto the filter under the gel). This "descending" transfer, as opposed to the widely used "ascending" Southern transfer, reduces the transfer time (to about 1 h) with equal or higher quality of the hybridization signal. The comparison of transfer kinetics by the both methods shows that (a) the Southern transfer of large size DNA fragments proceeds quicker than it has been thought so far and is almost complete within 4 h; (b) the descending transfer has an advantage over the ascending one in the rate of transfer (1-2 h) and its efficiency; and (c) the time of transfer may become a critical parameter upon using a filter with an apparently low retention capacity (Hybond N, Amersham) that is manifested by a decreased signal at longer than optimal transfer times.

[Changes in gene expression in the brain of rats with Zajdela's ascitic hepatoma]. / Izmeneniia ékspressii nekotorykh genov v golovnom mozge krys s perevivaemoi astsitnoi gepatomoi Zaidela.

Expression of some genes in the brain of ascitic hepatoma of Zajdela bearing rats was compared with that of control animals using Northern blot hybridization technique. The differences revealed were: an increased expression of actin gene and decreased expression of hsp70 gene in the brain of tumor-bearing animals.

Hydroxyapatite-mediated transfer of DNA from diluted solutions to nitrocellulose or nylon membranes.

Transfer of DNA (from 0.1 to 10 micrograms) from diluted solutions of variable volumes (1-10 ml) and various composition (2 M NaCl; 4 M LiCl, 8 M urea; 4 M CsCl; 20% sucrose) to nitrocellulose or nylon membranes was achieved with the use of hydroxyapatite. This absorbent that binds nucleic acids effectively and independently of ionic strength and composition of solution (except for chelators and phosphate ions) easily dissolves in small volumes of acids (for example, in 10% TCA). This phenomenon provides the opportunity to deliver the acid-insoluble precipitates to membrane filters. After alkaline denaturation on the filter followed by a fixation step (baking or UV irradiation for nitrocellulose or nylon filters, respectively), DNA hybridizes effectively with nick-translated DNA probes. The method is simple, reproducible, sensitive, and useful for working with diluted DNA solutions containing interfering substances.

[Identification of the stable DNA-matrix complex (type II) as a site of replication fork]. / Identifikatsiia prochnogo kompleksa DNK-matriks (tip II) kak mesta nakhozhdeniia replikativnoi vilki.

The two types of DNA-matrix complexes (the weak and tight ones, or type I and type II, respectively) identified in our previous work were studied with respect to their involvement in DNA replication. Nuclei isolated from human fibrosarcoma HT1080 cell line were treated with either restriction endonucleases or ultrasonic desintegrator and afterwards subjected to the triple-gradient Nucleoprotein--Celite chromatography. This permitted fractionation of nuclear DNA into fragments not attached, weakly attached, and tightly attached to the nuclear matrix (DNA 0, DNA I, and DNA II, respectively). It was shown that pulse labelled RNA migrates from DNA II fraction where it resides initially to DNA 0 and further to DNA I during the 2 h chase period. This finding allowed us to consider the tight DNA-matrix complex as the replicative one. The experiments aiming to follow the movements of specific DNA sequences (histone genes) in relation to the DNA-matrix attachment sites were conducted on synchronous HT1080 cells progressing through S phase. The histone sequences appeared to undergo similar movements during the first 30 min of S phase. They reside initially in DNA 0 and DNA I fractions, but as soon as DNA synthesis was restored they migrate consequently to DNA II and DNA 0 fractions. This approach can appear to be a useful tool for studying the schedule of replication of specific genes during S phase.

[A new method of DNA and RNA transfer onto nitrocellulose filters during blot hybridization]. / Novyi sposob perenosa DNK i RNK na nitrotselliuloznye fil'try v metode blot-gibridizatsii.

A quick (1-2 hour) method of DNA and RNA transfer onto nitrocellulose filters for subsequent blot-hybridization was elaborated. The main features of the method proposed are, firstly, almost complete exclusion of the mechanical impact on the gel and, secondly, addition to the transfer medium (20 X SSC) of a chaotropic agent, 0.5 M NaClO4. The latter results in a slight dissolution of the gel matrix and, on the other hand, somewhat increases the binding of the nucleic acid to the nitrocellulose. The method shortens significantly the time of DNA or RNA transfer at equal, or even higher, quality of hybridization.

[Glucocorticoid-mediated degradation of DNA in thymocytes of rats with transplantable Zajdela ascites hepatoma]. / Oposredovannyi gliukokortikoidami raspad DNK v timotsitakh krys s perevivaemoi astsitnoi gepatomoi Zaidela.

Tumour growth was shown to be associated with DNA breakdown in thymocytes of rats bearing Zajdela ascites hepatomas. The tumour action on the thymus is mediated through adrenal glands since bilateral adrenalectomy completely prevents DNA breakdown in thymocytes. Using Southern hybridization of DNA genome with probes for histone, ribosomal and heat shock gene (hsp 70), it was shown that the degradation products of specific DNA sequences are as heterogenous as those of total DNA, although marked differences in appearance of nucleosomal ladder were seen. These data were interpreted to indicate different patterns of DNA breakdown in dying thymocytes. DNA breakdown in thymocytes in vivo and in isolated rat liver nuclei in vitro seems to proceed by similar mechanisms.

[Radioactive labeling of DNA with 3H-thymidine: effect of the internal irradiation on cell growth, chromatin structure and gene activity]. / Radioaktivnoe mechenie DNK 3H-timidinom: vliianie vnutrennego oblucheniia na rost kletok, strukturu khromatina i gennuiu aktivnost'.

The growth of djungarian hamster fibroblasts 4/21 is inhibited by 3H-thymidine present in a culture medium in concentrations from 18.5 to 740 KBq/ml. As judged from the gradient elution of DNA from isolated nuclei (the nucleoprotein-celite chromatography), DNA fragmentation increases together with the increase in 3H-thymidine concentration and the decrease in the cell growth rate. DNA fragmentation does not activate the family of heat shock genes (hsp70). On the contrary, the hsp70 gene transcription is somewhat inhibited in both heat shock and non-heat shock conditions even at a concentration of 3H-thymidine of as low as 37 KBq/ml. Hence the 3H labelling of radiosensitive cultured cells can lead to some deviations in cellular processes under study.

[Hydroxyapatite-mediated transfer of DNA on nitrocellulose filters in dot-hybridization experiments]. / Oposredovannyi oksiapatitom perenos DNK na nitrotselliuloznye fil'try v metode gibridizatsii v piatnakh.

The method of hydroxylapatite-mediated rapid and effective transfer of DNA onto nitrocellulose filters for following dot-hybridization was elaborated. The analysed DNA occurred initially in diluted and large volume solutions (from 1 to 10 ml) with various composition (2 M NaCl; 4 M LiCl--8 M urea; 4 M CsCl; 5 and 20% sucrose) was adsorbed on hydroxylapatite and quantitatively transferred onto nitrocellulose after hydroxylapatite solubilization in a small volume of acid (usually, 200 microliters of 10% TCA). As exemplified by the hybridization of total rat liver DNA with the plasmid ph22 DNA containing a cluster of sea urchin histone genes, the method presented appears to be not only simple and useful for handling multiple probes of diluted DNA solutions with high concentrations of salts, sucrose and urea but also more sensitive than some convenient DNA dot-hybridization methods.

[Turnover of nuclear RNA. III. Detection and characterization of superstable molecules]. / Obmen iadernykh RNK. III. Vyiavlenie i kharakteristika sverkhstabil'nykh molekul.

In prolonged experiments in which rats were injected with 14C-orotic acid for 7-10 days with subsequent measurements (during 1-2 months) of the label in various rat liver RNA fractions and in the acid-soluble pool, a superstable nuclear RNA fraction was found (T1/2-4.2 days in quiescent rat liver and 4.3 days in regenerating rat liver). A small fraction of this RNA (5-10% of total) is polyadenylated and heterogeneously distributed when electrophoresed in 1% agarose under partially denaturing conditions. Using the conveyer model of synthesis and processing of nuclear RNA advanced earlier the spectrum of turnover rates of superstable transcripts was determined. The data obtained are discussed in light of the hypothesis on posttranscriptional regulation of gene expression.

[Nuclear and cytoplasmic ribonucleoprotein complexes of normal mouse liver cells, Guelstein hepatomas 22A and 48 and of liver of tumor-bearing animals]. / Iadernye i tsitoplazmaticheskie ribonukleoproteidnye kompleksy normal'nykh kletok pecheni myshi, gepatom 22A i 48 Gel'shtein i pecheni zhivotnykhopukholenostelei.

Nuclear and cytoplasmic RNP complexes obtained from normal mouse liver cells, Guelstein hepatomas of different degrees of malignancy (22A and 48) as well as from liver of tumor-bearing mice were subjected to chromatography on a celite column (NPC--chromatography). In addition cytoplasmic RNP complexes were centrifuged in sucrose and CsCl density gradients. The results of the NPC-chromatography indicate that nuclear rapidly labelled RNA species of all tissues under study are constituents of the two main types of RNP particles differing from each other by the tightness of RNA-protein bonds. No precursor-product relationship could be revealed between the above types of RNP-particles of nucleus, labelled under conditions of a partial Actinomycin D block. Rapidly labelled nonribosomal cytoplasmic RNAs represent constituents of RNP-particles resembling nuclear ones in their degree of heterogeneity and chromatographic position. Sedimentation analysis of cytoplasmic RNP-particles from tumours showed an increase in relative proportion of monoribosomes and informosomes (free non-ribosomal cytosol RNP-complexes) at the expense of polyribosomes and mRNP complexes. Thus, the liver cells of experimental tumour-bearing animals undergo changes (although not very well-defined), typical for tumour cells.