[TATA box polymorphisms in genes of commercial and laboratory animals and plants associated with selectively valuable traits].

Most of more than 11 million experimentally established polymorphisms, accumulated in dbSNP, were identified in the intergenic spacers or coding DNA regions. This fact enables interpretation of the former polymorphisms as neutral, while the latter make clear the biological sense of the associated mutant phenotypes, "the defect of certain proteins". The association of polymorphisms in regulatory DNA regions with mutant phenotypes is poorly studied. Specifically, the defects in certain DNA/protein binding sites were identified in less than 500 cases. In TATA-containing genes of eukaryotes the TATA box, the TBP (TATA-binding protein) binding site, is located about 30 bp upstream from the transcription start site. Interaction between DNA and TBP triggers assemblage of the preinitiation complex. For 37 TATA box polymorphisms in the genes of commercial and laboratory animals and plants, the effect on TBP-binding activity was evaluated using the equilibrium equation for the four subsequent steps of TBP/TATA box binding (nonspecific binding <----> sliding <----> recognition <----> stabilization). According to the GenBank data, these 37 polymorphisms were associated with the changes in a number of selectively valuable traits. Statistically significant congruence of in silico analysis performed with mutant phenotypes (a < 0.05, binomial law) provides suggestion of the mechanism of phenotypic manifestation of these polymorphisms (changing of the TBP-binding activity), as well a validates the possibility of developing the universal test system for experimental-computer prediction of the effects of TATA box mutations in specified genes on selectively valuable traits of the species, varieties, and breeds.

TATA box polymorphisms in human gene promoters and associated hereditary pathologies.

TATA-binding protein (TBP) is the first basal factor that recognizes and binds a TATA box on TATA-containing gene promoters transcribed by RNA polymerase II. Data available in the literature are indicative of admissible variability of the TATA box. The TATA box flanking sequences can influence TBP affinity as well as the level of basal and activated transcription. The possibility of mediated involvement in in vivo gene expression regulation of the TBP interactions with variant TATA boxes is supported by data on TATA box polymorphisms and associated human hereditary pathologies. A table containing data on TATA element polymorphisms in human gene promoters (about 40 mutations have been described), associated with particular pathologies, their short functional characteristics, and manifestation mechanisms of TATA-box SNPs is presented. Four classes of polymorphisms are considered: TATA box polymorphisms that weaken and enhance promoter, polymorphisms causing TATA box emergence and disappearance, and human virus TATA box polymorphisms. The described examples are indicative of the polymorphism-associated severe pathologies like thalassemia, the increased risk of hepatocellular carcinoma, sensitivity to H. pylori infection, oral cavity and lung cancers, arterial hypertension, etc.

[Prognosis of affinity change of the TATA-binding protein to TATA-boxes upon polymorphisms of the human gene promoter TATA boxes].

TATA-binding protein (TBP) is a subunit of basal transcription factor TFIID that recognizes and binds to the TATA-box on TATA-containing promoters of class II genes, and starts assembling RNA polymerase II basal transcription complex. It is shown in many works that the sequence of TATA-box with its flanking regions affects the level of basal and activated transcription. TATA-box polymorphisms and human hereditary diseases associated with them show that TBP/TATA interaction may indirectly affect gene regulation in vivo. The object of this work is to determine changes in the TBP/TATA affinity upon polymorphisms in TATA-boxes of human gene promoters. We assess changes in TBP/TATA affinities in silico by using our formula of equilibrium TBP/TATA binding upon four consecutive steps: nonspecific binding <--> sliding <--> braking (stopping) <--> stabilization. Our prognoses agree with known examples of TATA-box polymorphisms and human hereditary diseases associated with them.

[Interaction of proteins from general transcription complex RNA polymerase II with oligoribonucleotides].

We have analyzed an interaction of the general transcription complex RNA polymerase II proteins (RNA polymerase II, factors TBP, TFIIB, TFIIF, TFIIE and TFIIH) S. cerevisiae with the oligoribonucleotides. With the help of method EMSA was shown that labeled 32P labeled oligoribonucleotide 5'-ACUCUCUUCCGCAUCGC-3' (r-17) binds with the proteins and generates three species of the complexes with the three major shifts. All the three species of the complexes are RNA specific because a total RNA S. cerevisiae was a competitor for all three species but the TATA-containing oligodeoxyribonucleotide (500-fold molar excess) was not a competitor for its. Complexes 32P-r-17 with the proteins belonging to the middle shift are the sequence specific because unlabeled r-17 was a competitor for its binding (100-fold molar excess) but unlabeled UA-rich oligoribonucleotide (5'-AUAUUAUGUUCAAAA-3) was not a competitor for this shift (500-fold molar excess). Complexes belonging to the upper shift are RNA specific probably. We think 32P-r-17 interaction with the proteins belonging to the under shift is nonspecific corresponding to a sorbtion of 32P-r-17 on a protein. The data presented demonstrate that oligoribonucleotide and oligodeoxyribonucleotide don't compete for the binding sites on a basal transcription complex proteins.

[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.

[Interaction of Escherichia coli RNA polymerase with eukaryotic TATA-binding protein]. / Vzaimodeistvie RNK-polimerazy Escherichia coli s TATA-sviazyvaiushchim belkom éukariot.

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its sigma subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of 32P-labeled phosphamide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme-oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither minimal enzyme nor the sigma subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the sigma subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.

[The thermoregulatory response to noradrenaline in rats tested for stress or pharmacological exposures in the early postnatal period]. / Termoreguliatornyi otvet na noradrenalin u krys, ispytavshikh stressovye ili farmakologicheskie vozdeistviia v rannem postnatal'nom periode.

Norepinephrine-induced increase of the thermoconductivity of the body surface was depressed in rats exposed to cold, handling or treatment with thyroid hormone or alpha-adrenomimetic (mezatone) during the first week after birth. This effect was enhanced by prolonged cold exposure of adult animals. On the contrary, the norepinephrine-induced increase of heat losses from the tail surface was higher in new-born rats, exposed to cold or treated with beta-adrenomimetic (novodrine) than in control or mezatone-treated groups. The effects of neonatal stress or pharmacological treatment on oxygen consumption and rectal temperature responses to norepinephrine were unsignificant.

[Changes in noradrenaline metabolism in the rat subjected to cold in the early postnatal period]. / Izmeneniia metabolizma noradrenalina u krys, podvergnutykh kholodovomu vozdeistviiu v rannii postnatal'nyi period.

The adrenal gland norepinephrine content is elevated and 3H-norepinephrine lowered in adult rats exposed to cold during early postnatal period. 7-week test cooling of cold-imprinted animals induced a more obvious adrenal norepinephrine exchange than in control rats, phenylethanolamine-N-methyltransferase activity falling by a factor of two. The brown adipose tissue 3H-norepinephrine exchange was first intensified and then returned to normal level in control rats in cooling. The 3H-norepinephrine exchange was reduced in cold-imprinted animals after 7-week cooling.

[Carbohydrate and lipid metabolism in rats during adaptation to cold]. / Uglevodno-zhirovoi metabolizm u krys v usloviiakh adaptatsii k kholodu.

Cold adapted rats are shown to have glucose and fatty acids concentration in blood inchanged, lactate concentration increased and triglyceride concentration decreased against the control level. Glucose utilization rate in the tolerance test grows. Glycogen content falls, hexokinase and succinate dehydrogenase activity increases, glucose-6-phosphatase and NAD+-isocytratedehydrogenase activity decreases in the liver of experimental animals. The results indicate that utilization of carbohydrate and lipid substrates for thermogenesis is intensified in cold-adapted rats. The hypothesis is supported by the data of tests dealing with IPNA injection or with bringing the animals back under thermocomfortable conditions.

[Causes of decreased enzyme induction after long-term hormone administration: cortisol receptors in the rat liver cells]. / Issledovanie prichin snizheniia induktsii fermentov pri dlitel'nom vvedenii gormona: retseptsiia kortizola v kletkakh pecheni krys.

The results are presented of experimental studies of hepatic cytosol, nucleus and chromatin hormone-receptor activity of animals with decreased enzyme induction of gluconeogenesis, resultant from prolonged cortisol injection. In such animals in vivo 3H-cortisol incorporation into hepatocyte subcellular fractions was changed, being reduced in cytosol and enhanced in microsomes, nuclei and chromatin. 3H-Cortisol incorporation into chromatin was the highest in combination of the experimental rat hepatic nuclei with cytosol of intact animals after in vitro incubation with reconstructed homogenate labelled hormone from hepatocyte nuclei and cytosol. After incubation of experimental and intact rat nuclei with cytosol of the animals under study 3H-cortisol binding in chromatin was 30% lower than after incubating the nuclei with cytosol from intact rats.

[Thermoregulation characteristics of rats exposed to cold in the early postnatal period]. / Osobennosti termoreguliatsii u krys, ispytavshikh vozdeistvie kholodom v rannii postnatal'nyi period.

Cold adaptation of adult rats (at 4-5 degrees C for 7 weeks) increased their ability to respond to noradrenaline by the rise of body temperature and heat radiation, led to an almost 2-fold increase in the relative brown fat mass (BFM). Adult rats which experienced "cold imprinting" (from the first to the seventh day after birth, 15 min at 4-5 degrees C) showed a far less increment of the BFM on cold adaptation, no additional rise of body temperature and heat radiation in response to noradrenaline. In cold-imprinted rats, the relative surface of the tail and the body surface heat radiation transfer conefficient were found to be reduced. This attests to stable adaptive changes in physical thermoregulation, directed toward increase in animals' heat insulation.

Affinity labeling of RNA-polymerase II in the transcriptionally active complex by a phosphorylating analog of the initiation substrate.

Affinity modification of RNA-polymerase II by a phosphorylating analog of the initiation substrate carrying a zwitterionic 5;-terminal phosphate group with a 4-N,N-dimethylaminopyridine residue (DMAP-pA) was studied during specific transcription initiation controlled by the late adenoviral promotor. Super-selective affinity labeling and standard conditions of affinity modification resulted in labeling a polypeptide with molecular weight corresponding to that of the third subunit of the enzyme, RPB3 (45 kD). The initiation substrate (ATP) protects RNA-polymerase II from modification. The third subunit may be involved in the formation of the substrate-binding site of the enzyme.