[Expression of the pro-opiomelanocortin gene in rats with inherited arterial hypertension]. / Osobennosti ékspressii gena proopiomelanokortina u krys s nasledstvenno obuslovlennoi arterial'noi gipertenziei.

Expression of pro-opiomelanocortin (POMC) gene in pituitary of rats from newly developed hypertensive strain (ISIAH strain) was studied by dot hybridization. The pUC8 plasmid containing 900 base pair (bp) segment or the human POMC gene corresponding to the major portion of the 3'-nontranslated mRNA region and 60 bp coding for the signal peptide, was used as a probe for hybridization. It was found that the expression of the POMC in pituitary of the hypertensive JSJAH rats was more than 3-fold gene lower as compared to normotensive Wistar strain. The latter is the original strain from which the ISIAH rats were bred. The mechanism of this phenomenon and its possible relation to the arterial hypertension are discussed.

[Increased content of nucleotide sequences in transcriptionally active DNA and poly(A)+-mRNA of the rat liver and a rise in its translation activity as affected by inducers]. / Vozrastanie soderzhaniia nukleotidnykh povtorov v transkriptsionno aktivnoi DNK, v poliA+-mRNK pecheni krys i uvelichenie ee transliatsionnoi aktivnosti pod deistviem induktorov.

Cortisol and 3'-methyl-4-dimethyl-amino-azobenzene induce an increase in the content of repeated sequences (RS) in transcriptionally active (TA) DNA, while the content of respective RS in potentially active DNA fractions enriched with regulatory regions of the genome decreases. RS content in induced poly A+-mRNA also rises, as determined by the nature of hybridization of respective c DNA with total DNA. The translation of induced poly A+-mRNA rises essentially, with the qualitative distinctions in in vitro synthesized protein product spectrum being absent. Inducible RS with unstable chromatine conformation are thought to provide a universal system of rapid response of the genetic apparatus to extreme situations, serving as transcription intensifiers in TA DNA and as translation intensifiers in induced poly A+-mRNA.

[Detection of DNA sequences sensitive to glucocorticoids in the thymus of AKR mice and their inactivation in thymoma]. / Obnaruzhenie chuvstvitel'nykh i gliukortikoidam posledovatel'nostei DNK v timuse myshei linii AKR i ikh inaktivatsiia v timome.

A high increase in transcriptionally active DNA fraction (TA DNA) has been detected in the thymus of young AKR mice and in thymoma. Hybridization of TA DNA with [32P] cDNA synthesized on poly A+-mRNA of cortisol-induced animals has shown that TA DNA of young AKR mice contains 40 times as much cortisol-activated repeated sequences (RS) as that of thymoma. The decrease of cortisol-activated (RS) in AKR thymoma TA DNA was found to be the result of their transition into transcriptionally inactive (TI) DNA. It is concluded that chromatin conformation changes take place in cortisol-activated RS DNA region of AKR mouse thymus before transformation. Unstable conformation of RS DNA in the thymoma can possibly promote their transition into TI DNA.

Purified glucocorticoid-receptor complexes from rat liver cytosol preferentially bind in vitro to a homologous DNA fraction whose transcription is activated by cortisol.

The in vitro binding of 2000-fold purified rat liver glucocorticoid-receptor complexes to functionally differing homologous DNA fractions has been studied. The effectiveness of receptor binding to the transcriptionally active DNA, isolated from rat liver 4 h after cortisol administration, increased by 1.6-1.8-fold with simultaneous reduction in receptor affinity for the hardly extractable DNA fraction. The analogous DNA fractions from control animals did not differ in ability to bind the receptor. This suggests that sites of high affinity for the receptor are directly involved in the in vivo process of hormonal transcription activation.

[Cortisol-induced changes in methylation of repeating sequences of transcriptionally active DNA from rat liver]. / Izmenenie metilirovaniia povtoriaiushchikhsia posledovatel'nostei transkriptsionno aktivnoi DNK pecheni krys pod deistviem kortizola.

The m5C content was determined in three functionally different fractions of rat liver DNA isolated by a modified phenol fractionation. The transcriptionally active DNA-I contains up to 12-14% of hybrid RNA not hydrolyzed by RNAase, is enriched with unique sequences and makes up to about 20% of total DNA. In its total nucleotide content (43 mol.% of GC-pairs) DNA-I does not differ from the major fraction of DNA-II making up to about 70% of the genome, and from hardly extractable DNA-III, whose content in total DNA does not exceed 10%. However, the degree of methylation of the transcriptionally active DNA-I (1.28 mol.% of m5C) is higher than that of DNA-II and DNA-III (1.0 mol.% of m5C). Under cortisol-induced activation of transcription the number of repeating sequences in DNA-I is increased by about 10% concomitant with a decrease of these sequences in DNA-III. The hormone-induced changes in the transcriptionally active fraction of DNA are reversible. 4 hrs after cortisol injection a demethylation of highly repeating and supermethylation of normally repeating sequences in DNA-I are observed. The level of methylation of the unique sequences in DNA-I (Cot greater than 200) remains unchanged. The degree of methylation of various subfractions of nucleotide sequences of DNA-II and DNA-III afer cortisol injection does not differ from normal. Presumably the hormone-induced supermethylation of normally repeating sequences may control the transcription.

[Increase in the number of repeating sequences of DNA in transcriptionally active sites of rat genome induced by cortisol and phenobarbital]. / Uvelichenie doli povtoriaiushchikhsia posledovatel'nostei DNK v transkriptsionno aktivnykh uchastkakh genoma krysy pri induktsii kortizolom i fenobarbitalom.

The effects of transcription inducers, e. g. cortisol, phenobarbital and 3-acetate-16 alpha (beta)-isothiocyano-pregnenolone (AIP), which, similar to phenobarbital, activates the multifunctional oxidase system, on the nucleotide sequence arrangement in the DNA fractions corresponding to functionally different sites of the genome, were investigated. Three DNA fractions were isolated from rat liver cells, using different concentrations of NaCl and different pH values of aqueous solutions of phenol used for deproteination. The DNA fraction I extracted with 1 M NaCl and phenol (pH 6.4) down to the water phase contains 12-14% of non-hydrolyzed by hybrid RNA RNAase, is rich in unique sequences, makes up to about 20% of the genome and probably corresponds to the transcriptionally active sites of chromatin DNA. The DNA fraction II extracted by 0.14 M NaCl and "alkaline" phenol (pH 8.5) makes up to 70% of total cellular DNA, in which the hybrid RNA content does not exceed 2%. The amount of weakly extractable and, possibly, membrane-bound DNA III does not exceed 10% of total DNA content. During induction of transcription the number of repeating sequences is increased by about 10% in DNA I and is simultaneously decreased in DNA III. This can both be due to the arrangement of sequences between chromatin fractions corresponding to membrane DNA and the transcriptionally active sites of the genome. Hybridization of [3H]rRNA demonstrated that the contribution of ribosomal genes to the above changes in the arrangement of sequences in DNA fractions under study is insignificant.

[Changes in the composition of nucleotide sequences in the transcriptionally active fraction of rat liver DNA during cortisol induction]. / Izmenenie sostava nukleotidnykh posledovatel'nostei v transkriptsionno-aktivnoi fraktsii DNK pecheni krys pri induktsii kortizolom.

The renaturation kinetics of functionally different DNA fractions of rat liver isolated by modified phenol fractionation were studied. It was found that the transcriptionally active fraction of DNA (DNA-I), making up to approximately 20% of total DNA, is enriched with unique sequences. Under cortisol-induced DNA-dependent synthesis of RNA in the liver the amount of repeated sequences in this DNA fraction is increased (approximately by 10-11%), whereas the type of renaturation of a transcriptionally inactive major DNA fraction (DNA-II), making up to approximately 70% of total DNA, remains unchanged. The renaturation kinetics of a weakly extractable DNA fraction (DNA-III) making up to 5-10% of total DNA are similar to those of the major DNA fraction. Under cortisol-induced transcription the number of repeated sequences in this fraction is decreased as compared to DNA-III of intact liver (by approximately 10-12%). The increase of the number of repeated sequences observed after 4-8 hrs (i. e. peak of induction) is decreased after 16 hrs, resulting in a complete reconstitution of the original type of DNA-I renaturation on the 24th post-induction hour.

[Properties of replicating DNA in the regenerating rat liver isolated during phenol fractionation]. / Izuchenie svoistv replitsiruiushcheisia DNK regeneriruiushchei pecheni krys, vydelennoi pri fenolovom fraktsionirovanii.

Using phenol fractionation in the absence of detergents three DNA fractions differing by the incorporation of radioactive thymidine after pulse label are obtained from regenerating rat liver cells. Two fractions extracted under variety of conditions represent the main bulk of cell DNA (85--90%). DNA non-extractable under conditions used (DNA III) incorporates labelled thymidine 10--15 times faster than the first two DNA fractions. DNA III purified from the interphase layer by pronase, sodium dodecylsulfate and phenol sediments at 26S and has a hyperchromic effect about 40% after alkaline denaturation. Alkaline sucrose density gradient centrifugation of pulse-labelled DNA III revealed that nascent DNA consisted of heterogeneous fragments similar in size to the replication fragments in bacterial cells (9--10S). It was shown by CsCl equilibrium centrifugation that buoyant density of heat denatured DNA III labelled for 5 min with [3H]thymidine is heavier than the bulk of DNA prelabelled for 2 h with [14C]thymidine. After hydrolysis with RNase or alkali, buoyant densities of both DNAs became the same. These results support the idea of initiating role of RNA in the synthesis of discontinuous replicating fragments in regenerating rat liver cells. Specific radioactivity of RNA associated with replication fragments which are labelled for 5 min with [14C] orotic acid is 20 times more than of the same RNA labelled for 30 min. These data demonstrate high metabolic activity of initiating RNA.