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)

The bacterial tetracycline-resistance determinant from Tn10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/lac operator in a plasmid that provided fusion of TetA to a polyhistidine-carboxy terminal tail. A second plasmid provided a regulated T7 RNA polymerase. The specific activity of the TetA fusion protein was between 10-40% that of the wild-type protein as assayed by tetracycline resistance in cells and by transport in membrane vesicles. The fusion protein, overproduced approximately 3-13-fold, was purified by nickel chelation chromatography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an alpha-helix content of 54-64%, close to the 68% predicted from the amino acid sequence by hydropathy analysis (12 membrane-spanning helices) for the native protein in the membrane bilayer. Fluorescence studies showed binding activity of the purified protein to its substrate, the tetracycline analogue 13-(cyclopentylthio)-5-hydroxy-6-alpha-deoxytetracycline. These findings suggested that the purified protein was in a native state.
...
PMID:Purification of the Tn10-specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein. 882 47

On the basis of position from the transcription start site, the P2 promoter of the gene encoding the major outer membrane protein (ompA) of Chlamydia trachomatis consists of a -35 hexamer region of -42 aaaaaga TATACAaa -28 and an unusual, GC-rich -10 hexamer region of -13 tTATCGCt -6. The P2 promoter was analyzed by in vitro transcription of templates containing deletions and site-specific mutations. The 5' extent of P2 was located at bp -42. Replacement of wild-type sequence with two G's at positions -41 and 40, -35 and 34, and -29 and 28 resulted in severely decreased transcription. Additionally, the spacing between the -35 and -10 hexamers could not be shortened without adversely affecting in vitro activity. Substitution of G at position -13, -10, -7, or -6 had little or no effect on transcription, whereas substitution of G at -11 or -12 significantly decreased promoter strength. Triple point mutations which changed the -10 hexamer from TATCGC to TATTAT,TATATT, or TATAAT had little effect on promoter activity. Unlike the partially purified C. trachomatis sigma66-RNA polymerase used in this study, purified Escherichia coli sigma70-RNA polymerase did not recognize the wild-type P2 promoter. Mutant P2 templates with -10 hexamers that resembled the consensus recognition site were transcribed by E. coli holoenzyme in vitro, suggesting that C. trachomatis sigma66-RNA polymerase has special promoter recognition properties not found in E. coli sigma70-holoenzyme.
...
PMID:Mutagenesis of the P2 promoter of the major outer membrane protein gene of Chlamydia trachomatis. 882 99

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.
...
PMID:Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system. 889 56

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.
...
PMID:Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2). 902 6

Large peptide segments (148-479 amino acids) of the cystic fibrosis transmembrane conductance regulator, which are projected to represent cytoplasmic portions of this large membrane protein, were expressed in Escherichia coli using two T7 RNA polymerase vectors, pET11a and pRSET. Five of the nine peptides were readily expressed at high levels (15-35 mg/liter) and one at an intermediate level (10 mg/liter), but three could not be expressed at >1.5 mg/liter regardless of efforts to further optimize the system. Preinduction testing of these latter plasmids failed to demonstrate any plasmid instability, while bacterial survival was drastically curtailed upon induction, beyond that observed with the other plasmids. Peptides containing the second half of exon 13 (residues 700-830; R domain) appear to be especially toxic to the expressing bacteria. Peptides including this hydrophilic segment may be inhibiting the bacterial permeases which are known to he homologous to other portions of CFTR.
...
PMID:Expression in Escherichia coli of cytoplasmic portions of the cystic fibrosis transmembrane conductance regulator: apparent bacterial toxicity of peptides containing R-domain sequences. 917 82

1. [125I]-[LTT]SRIF-28 and [125I]-SMS 201-995 were used to identify and characterize somatostatin (SRIF) receptors localized in rat lung tissue. In vitro autoradiography of rat lung tissue sections showed the existence of specific, high affinity binding sites for [125I]-[LTT]SRIF-28 without any significant specific binding of the sst2/sst5-receptor selective ligand [125I]-SMS 201-995. 2. In radioligand binding studies, specific binding of [125I]-[LTT]SRIF-28 to membranes of rat lung was linearly related to the concentration of membrane protein used with only a small portion of nonspecific binding. With [125I]-SMS 201-995 no specific binding could be observed up to a membrane concentration of 0.1 mg of protein/assay tube. 3. [125I]-[LTT]SRIF-28 bound rapidly to rat lung membranes with an apparent association rate constant (kapp) of 1.8 +/- 0.1 h-1 (n = 3). The equilibrium of specific binding was reached after an incubation period of approximately 90 min at room temperature and remained constant for the next 3 h. The association rate constant (k1) was calculated to be 3.7 x 10(10) M-1 h-1. The dissociation reaction followed first order kinetics with a dissociation rate constant (k-1) = 0.44 +/- 0.07 h-1 corresponding to a half-time of 95 +/- 15 min (n = 3). From these kinetic experiments an equilibrium dissociation constant (KD) for the binding of [125I]-[LTT]SRIF-28 was calculated to be 11.9 pM. 4. Saturation binding of [125I]-[LTT]SRIF-28 revealed an equilibrium dissociation constant (KD) of 50.1 pM (pKD = 10.3 +/- 0.1; n = 3) and a receptor density (Bmax) of 78 +/- 3 fmol mg-1 protein. A Hill coefficient not significantly different from 1 indicated saturable binding to a single class of high affinity binding sites. 5. Specific binding of [125I]-[LTT]SRIF-28 to rat lung membranes was inhibited by SRIF-14, SRIF-28 and different SRIF analogues. SRIF and different synthetic short chain SRIF analogues exhibited the following rank order of potency: SRIF-28 > SRIF-14 > CGP 23996 >> RC 160 > BIM 23014 > SMS 201- 995 > BIM 23056 > MK 678. 6. The binding affinities for SRIF and the various SRIF analogues determined using rat lung tissue were in close correlation to those obtained with Chinese hamster ovary (CHO) cells stably expressing sst, (r = 0.92) and sst4 (r = 0.95) receptors, respectively. 7. Reverse transcriptase--polymerase chain reaction (RT-PCR) showed the predominant expression of mRNA specific for sst4 receptors as well as some weak sst1 mRNA expression. 8. The findings suggest that sst4 receptor expression is the predominant form of the somatostatin receptors identified in rat lung tissue. In this study we demonstrated for the first time the existence of sst4 receptors in mammalian tissue.
...
PMID:Identification and pharmacological characterization of somatostatin receptors in rat lung. 922 54

A 3135 bp DNA segment downstream of the spl gene on the Bacillus subtilis chromosome was cloned and its nucleotide sequence determined. An open reading frame capable of encoding a putative protein of 654 amino acids with a calculated molecular mass of 72.1 kDa was identified. The deduced amino acid sequence was similar to the McpA and McpB proteins of B. subtilis. McpA and McpB encode different methyl-accepting chemotaxis proteins (MCPs). A mutant strain containing an antibiotic resistance DNA cassette inserted into the region containing the MCP-like reading frame suffered a complete loss of taxis to the amino acids cysteine, proline, threonine, glycine, serine, lysine, valine and arginine. The open reading frame was designated mcpC. The wild-type and an mcpC mutant strain were analysed for their content of methylated proteins and it was found that mcpC encodes a methylated membrane protein that has previously been designated H3. These results show that mcpC encodes a third MCP in B. subtilis. The transcription start site upstream of the mcpC gene was determined by primer extension analysis and it was found to be preceded by a potential promoter sequence that is recognized by the sigma D form of RNA polymerase. The level of beta-galactosidase expressed from a transcriptional mcpC-lacZ fusion was increased threefold when cells entered the stationary phase. No beta-galactosidase could be detected in a sigD genetic background.
...
PMID:Functional and genetic characterization of mcpC, which encodes a third methyl-accepting chemotaxis protein in Bacillus subtilis. 935 24

Although bile salts are toxic to the liver at high plasma concentrations, the effects of physiological concentrations of bile salts on normal hepatic function are poorly understood. We examined the effect of taurocholate (TC) on the basolateral uptake of [3H]TC in WIF-B cells, a hybrid cell line stably exhibiting in vitro the structural and functional polarity of hepatocytes. Cells were grown in the absence or presence of TC (50 micromol/L) over 12 days, and then incubated with [3H]TC concentrations ranging from 1 to 250 micromol/L. For both control and TC-grown cells, uptake of [3H]TC was linear over 2 minutes. In control cells, the Km for [3H]TC Na+-dependent uptake over 1 minute was 6 +/- 5 micromol/L, and the Vmax was 45 +/- 6 pmol TC/mg protein/min (+/- SEM). TC-grown cells exhibited no significant change in Km but showed a doubling of Vmax to 87 +/- 6 pmol TC/mg protein/min (P < .005). In both control and TC-grown cells, maximal uptake of [3H]TC occurred following 10 to 12 days in culture, with TC-grown cells consistently showing greater rates of [3H]TC uptake from 4 to 14 days in culture. Western blots immunostained for the basolateral Na+-dependent plasma membrane protein, ntcp, revealed the appropriate approximately 50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na+-dependent plasma membrane transport capacity.
...
PMID:Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt. 942 37

Insulin-like growth factor-I (IGF-I) is implicated in the development, survival and maintenance of function of sympathetic and sensory neurons. These neurons are affected at an early stage during the course of diabetes. Reverse transcriptase polymerase chain reaction (RT-PCR) based assay revealed that rat superior cervical ganglia (SCG) express mRNA transcripts for IGF-I and its receptor. Moreover, specific membrane protein binding sites for IGF-I within the SCG have also been demonstrated using competition-inhibition and affinity cross-linking techniques. An induction of diabetes with streptozotocin (STZ, 55 mg/kg, i.v.) produced a marked decrease in the SCG levels of mRNA transcripts for IGF-I and its receptor. Concentrations of circulating IGF-I and its receptor protein within the SCG were also reduced in this disease state. Insulin treatment partially prevented diabetes-related alterations in circulating IGF-I and the SCG-IGF-I system. Overall, the data described in this study may be of value in understanding the pathogenetic mechanism(s) responsible for the development of diabetic sympathetic neuropathy.
...
PMID:Diabetes-induced suppression of IGF-1 and its receptor mRNA levels in rat superior cervical ganglia. 948 70

Galacturonic acid (GalUA) is a major component of pectin and polygalacturonic acid in the plant cell wall. In the phytopathogen Erwinia chrysanthemi, the uptake of molecules derived from degradation of these polymers is an important early step in the events preceding induction of pectinases, ultimately leading to plant tissue maceration. Uptake systems for GalUA and dimers of GalUA have been described and shown to be inducible in E. chrysanthemi. The GalUA uptake gene (exuT) was cloned and sequenced. Nucleotide sequence analysis identified an open reading frame encoding a 345-amino-acid polypeptide with a calculated mass of 37,825 Da. This polypeptide is predicted to be an integral membrane protein based on its high nonpolar amino acid content and hydropathic profile. Localization studies with the labeled polypeptide in the T7-RNA polymerase system also suggest that ExuT is a membrane protein. This evidence is further supported by the observation of hybrid ExuT-PhoA proteins in the bacterial cytoplasmic membrane following immunoblot analysis. Northern (RNA) analysis indicated that the gene is inducible in the presence of the monomer, GalUA. A targeted mutation in the exuT gene affected the utilization of GalUA as a role carbon source for growth. Maceration of potato tuber tissue by this mutant was delayed and reduced, when compared with the parental strain EC16.
...
PMID:The exuT gene of Erwinia chrysanthemi EC16: nucleotide sequence, expression, localization, and relevance of the gene product. 953 Aug 68


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

---