Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Eight eukaryotic promoters have been tested for their activity in vivo in Escherichia coli. The rat beta-actin, rat amylase, rat chymotrypsin B, mouse metallothionein I, rat insulin I, human insulin, Rous sarcoma virus long terminal repeat (RSV LTR) and hepatitis B viral precore promoter activities were measured by using the bacterial chloramphenicol acetyltransferase coding sequences as the reporter function and by primer extension RNA analysis. All eight promoter-chloramphenicol acetyltransferase constructs produce chloramphenicol acetyltransferase activity with the following relative strengths: RSV LTR greater than rat beta-actin greater than rat insulin I greater than rat amylase greater than hepatitis B virus precore greater than human insulin greater than rat chymotrypsin B greater than mouse metallothionein I. A primer extension analysis indicates that transcription from the RSV LTR, rat insulin I, and rat beta-actin promoters initiates at the sites expected for eukaryotic rather than prokaryotic promoters. Thus the site of initiation is determined by the DNA sequence rather than by the RNA polymerase.
...
PMID:Eukaryotic promoters drive gene expression in Escherichia coli. 268 Nov 82

The murine adipocyte lipid binding protein (ALBP) has been cloned into Escherichia coli, purified from expressing cultures, and its ligand binding and phosphorylation properties studied. In the cloning strategy, the recombinant, pT7-5 rALBP, was transformed into E. coli strain K38 harboring plasmid pGP1-2 which directs the synthesis of T7 RNA polymerase. Upon shifting the temperature from 30 to 42 degrees C to induce T7 RNA polymerase expression, the 14.6-kDa recombinant ALBP (rALBP) was expressed for approximately 2 h and accumulated to about 1% of total E. coli protein. The recombinant ALBP was soluble in E. coli extracts and resistant to bacterial proteolysis. A procedure for purifying rALBP was developed utilizing immuno-chemical detection based upon reactivity with anti-murine ALBP antiserum. A combination of acidic ammonium sulfate fractionation, gel permeation chromatography, and carboxymethyl ion-exchange high performance liquid chromatography separation was used to prepare homogeneous rALBP. Sequence analysis of rALBP indicated that the initiating methionine residue had been removed and the amino-terminal cysteine residue was not blocked. Purified rALBP exhibited stoichiometric, saturable binding of oleic acid (n = 1.0, K0.5 approximately 100 microM) and retinoic acid (n = 1.0, K0.5 approximately 170 microM). Incubation of rALBP with wheat germ agglutinin-purified insulin receptor, ATP, and 100 nM insulin resulted in a 5-fold stimulation of rALBP phosphorylation above the basal state. Kinetic analysis of rALBP phosphorylation by the 3T3-L1 insulin receptor kinase yielded a Michaelis constant (Km) of 50 microM and a maximal velocity of 1 mol of rALBP phosphorylated/min/mol insulin binding sites. Phosphoamino acid analysis indicated that phosphorylation occurred upon tyrosine. These results indicate that murine ALBP has been cloned and expressed in E. coli, purified to homogeneity, and is a substrate for the insulin receptor tyrosyl kinase in vitro.
...
PMID:Cloning of murine adipocyte lipid binding protein in Escherichia coli. Its purification, ligand binding properties, and phosphorylation by the adipocyte insulin receptor. 268 57

A cDNA encoding the rat brain glucose transporter was inserted between the 5' and 3' untranslated regions from the Xenopus globin gene and downstream of an SP6 RNA polymerase start site. RNA synthesized from this vector was microinjected into oocytes from Xenopus laevis; this resulted in expression of the glucose transporter, as determined by both immunoblotting and the appearance of transport activity. The properties of the transporter were those expected from previous studies: it was glycosylated, and its activity, measured by 3-O-methylglucose transport, was inhibited by D-glucose and cytochalasin B, but not by L-glucose. The low level of endogenous glucose transport activity found in water-injected oocytes makes this a useful system in which to determine the kinetic parameters of transport. The Km for 3-O-methylglucose was found to be 20 mM under equilibrium exchange conditions. Despite the fact that oocytes exhibit insulin-dependent responses, insulin did not stimulate 3-O-methylglucose transport by injected oocytes.
...
PMID:Expression of a functional glucose transporter in Xenopus oocytes. 269 9

We used a ribonuclease cleavage assay to screen for insulin receptor mRNA sequence alterations in 12 patients with syndromes of severe insulin resistance. Uniformly labeled [32P]antisense RNA probes complementary to insulin receptor mRNA were prepared by an SP6 or T7 RNA polymerase transcription reaction. Four probes ranging in size from 670-1470 bases were used to examine the entire 4.2-kilobase receptor protein-coding region. Patient RNA samples were hybridized to individual probes in solution, and mismatched sequences were detected by susceptibility to cleavage by a mixture of RNAses A and T1. The method was validated with insulin receptor mRNAs from cells transfected with cDNA constructs bearing known point and deletion mutations. Alterations in the insulin receptor mRNA sequence of two patients were detected. A patient with the type A syndrome of severe insulin resistance (A2-Boston) had a mutation in the insulin receptor beta-subunit mRNA sequence that localized to the region coding for amino acid residues 1174-1211 near the tyrosine kinase domain. The second alteration was a sequence polymorphism in the insulin receptor alpha-subunit mRNA in a patient with lipoatropic diabetes (LA-2) that localized to a region within amino acids 268-272. Direct sequence analysis revealed that the ribonuclease cleavage sites in patients A2-Boston and LA-2 were due to distinct single base changes in the insulin receptor gene and mRNA. Additional insulin receptor mRNA sequence polymorphisms were also identified as mismatches between the labeled RNA probes used and mRNA from several cultured human cell types. This study demonstrates that ribonuclease cleavage can rapidly detect and localize insulin receptor mRNA sequence mutations and polymorphic variations as small as single base changes. Further analysis of insulin receptor mRNA sequence alterations identified in this way may elucidate a possible genetic basis for functional insulin receptor defects in patients with severe insulin resistance and can also reveal some insulin receptor sequence polymorphisms that occur in the population at large.
...
PMID:Insulin receptor messenger ribonucleic acid sequence alterations detected by ribonuclease cleavage in patients with syndromes of insulin resistance. 273 94

Conveniently situated PstI sites were used to delete a major segment from the C-peptide coding region of a human pre-pro-insulin cDNA. The resultant mutant cDNA encoded a protein with the structure: pre-peptide B chain--Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Lys-Arg-A chain. Normal and mutant human pre-pro-insulin cDNAs were used as templates for the synthesis of mRNA in a reaction catalysed by T7 RNA polymerase. The mRNAs were then microinjected into Xenopus oocytes to determine the effect of the deletion on the secretion of pro-insulin. When normal pre-pro-insulin mRNA was microinjected, pre-pro-insulin was processed to pro-insulin, which in turn was secreted into the media. When the mutant pre-pro-insulin mRNA was microinjected, however, mutant pro-insulin could be detected in the oocytes but at a much lower level than the normal pro-insulin. No mutant pro-insulin could be detected in the media. The stability of the mRNAs in the oocytes was investigated by microinjecting [32P]mRNA. 24 and 48 h after microinjection, the recovery of [33P]mRNA from the oocytes was 95 and 24% and 20 and 16% of that injected, for the normal and mutant mRNAs, respectively. In a cell-free translation system supplemented with dog pancreatic microsomal membranes, the pre-peptide was cleaved from the normal pre-pro-insulin but not from the mutant pre-pro-insulin. These results suggest that C-peptide plays an important role in the segregation of pro-insulin within and transport through the cellular secretory pathway.
...
PMID:Expression of normal and mutant human pre-pro-insulins in Xenopus oocytes. 284 Sep 76

We have previously shown that when H4 hepatoma cells are pretreated with insulin, plant lectins, phorbol esters, or insulin mediator, the steady state concentration of gene 33 mRNA is markedly increased. The increase in gene 33 mRNA concentration with insulin is due to an increase in the transcription rate of this gene. In the present report we demonstrate that nuclear extracts prepared from H4 hepatoma cells pretreated with insulin exhibit enhanced transcription of gene 33 RNA from a DNA template containing the cap site and 1500 bp upstream of the 33 gene. This is a stable effect of insulin on the nuclear RNA polymerase II system since it is observed in frozen and thawed nuclear extracts as well as fresh nuclear extracts.
...
PMID:Selective transcription of an insulin-regulated gene in nuclear extracts of rat hepatoma cells. 328 2

Actinomycin D, at a concentration that inhibits cellular ribonucleic acid (RNA) synthesis, inhibited the production of foot-and-mouth disease virus-induced RNA polymerase in baby hamster kidney cells. Inhibition was proportional to exposure time and reached 85% when actinomycin D was added 90 min before infection. Polymerase production was inhibited to the same extent in growth and minimal media, and the kinetics of its appearance were slightly different than in untreated cells. Enzyme preparations from actinomycin-treated cells having one-third to one-tenth the activity of untreated samples gave products with RNA profiles similar to those of controls. The 37S viral peak, 20S ribonuclease-resistant peak, and 26 to 28S peaks were present in all cases. Actinomycin D did not consistently inhibit virus production in either medium. Insulin did not prevent the actinomycin induced inhibition of polymerase and virus production from occurring.
...
PMID:Effect of actinomycin D on virus-induced ribonucleic acid polymerase formation in foot-and-mouth disease virus-infected baby hamster kidney cells. 431 46

After the addition of insulin to monolayers of chick fibroblasts previously incubated in serum-free medium, the rates of protein and RNA synthesis increase continuously during the first 8-10 h. Little stimulation of DNA synthesis or mitosis results with the addition of insulin alone in contrast to the addition of fresh serum which stimulates both markedly. The stimulation in RNA synthesis does not result from expansion of the nucleotide pool but is correlated with increases in RNA polymerase activity. All major classes of RNA are stimulated; processing of preribosomal RNA to 28S and 18S and the association of this mature RNA with ribosomes appear to occur normally. The kinetics of stimulation of 5S RNA differ from those of the synthesis of 4S and of ribosomal RNA. Insulin and serum appear to affect the synthesis or stability of certain transcripts differentially.
...
PMID:Stimulation by insulin of RNA synthesis in chick fibroblasts. 485 94

The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver.
...
PMID:RNA synthesis in primary cultures of adult rat hepatocytes. 618 78

Insulin and dexamethasone greatly stimulate the incorporation of 3H-orotic acid into RNA. Such a stimulation is associated to an increase in the uptake of the labelled precursor into the acid soluble fraction as well as in the the specific radioactivity of the nucleoside plus nucleotide pool suggesting that hormone supplementation does not affect RNA synthesis by cultured cells. The lack of effect of insulin and dexamethasone on the level of total RNA polymerase activity in nuclei isolated from cultured hepatocytes is in line with this assumption. The hormone stimulated uptake of orotic acid is dependent on protein synthesis since it is completely abolished by cycloheximide.
...
PMID:Hormonal stimulation of 3H-orotic acid incorporation into RNA by serum-free cultured hepatocytes. 619 40


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---