Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Corticosteroid-resistant (CR) asthma is not caused by altered bioavailability of the administered drug, altered ligand-binding characteristics, or altered nuclear translocation of the activated human glucocorticoid receptor (hGR) complex. We have tested the hypothesis that CR asthma results from a consistent polymorphism in the functionally diverse hGR cDNA using the sensitive screening technique of polymerase chain reaction (PCR) amplification and chemical mutational analysis. Total RNA was extracted from peripheral blood monocytes derived from six corticosteroid-sensitive (CS) and six CR asthmatic subjects. The RNA was reverse transcribed, and overlapping hGR cDNA fragments were amplified by nested PCR. Double-stranded hGR cDNA fragments were hybridized to corresponding 32P-5'-labeled wild-type fragments, chemically modified with osmium and hydroxylamine, and cleaved with piperidine. The resultant cleaved strands were detected by autoradiography. As controls, single base pair mutated hGR cDNA fragments sensitive to hydroxylamine and osmium modification were used. Using this technique, we did not detect any base pair mismatch between the six CS and six CR patients and the corresponding wild-type hGR, despite a 100% detection of control mutations. We conclude that the defect in CR asthma does not lie in the structure of the hGR.
Am J Respir Cell Mol Biol 1994 Jul
PMID:Chemical mutational analysis of the human glucocorticoid receptor cDNA in glucocorticoid-resistant bronchial asthma. 801 37

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously ts and cold-sensitive (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.
Mol Gen Genet 1994 Mar
PMID:Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae. 812 10

Integration host factor, IHF, is a sequence-specific DNA-binding and DNA-bending protein composed of two related but non-identical subunits. We report the isolation and characterization of hydroxylamine-induced loss-of-function mutations in the genes encoding the IHF subunits. To screen for mutants that preserve proper folding of IHF, clarified extracts were prepared from each mutant and were assayed for production of each subunit by immunoblotting and for formation of heterodimers by chemical cross-linking and subsequent immunoblotting. Extracts from mutants that met these criteria were found to bind a specific IHF site weakly if at all. These alleles therefore identify candidates for residues that may affect the DNA-binding surfaces of IHF. When projected onto the known tertiary structure of the closely related HU protein, these residues are found at the surface; however, with the exception of a single residue, different regions of the protein are implicated in each subunit. This suggests that, despite their homology, each subunit of IHF directs DNA recognition and binding in a distinct manner. To confirm the significance of the differential location of these mutations, we introduced in each subunit alterations that had been isolated as loss-of-function mutations at the corresponding position in the other subunit. In general, the engineered mutants have phenotypes that are strikingly different from those of their hydroxylamine-induced counterparts. In particular, most of the site-directed mutant IHF proteins form or maintain IHF:DNA complexes more readily than mutants that have the same change in the other subunit and were isolated as loss-of-function mutants. We discuss the positions of the mutant amino acid residues as they relate to a proposed molecular model of an IHF:DNA complex.
J Mol Biol 1993 Nov 05
PMID:Characterization of a set of integration host factor mutants deficient for DNA binding. 823 Feb 6

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins the uptake of which through the outer membrane depends on the TonB, ExbB and ExbD proteins. These colicins contain a sequence, called the TonB box, which has been implicated in transport via TonB. Point mutations were introduced by PCR into the TonB box of the structural gene for colicin M, cma, resulting in derivatives that no longer killed cells. Mutations in the tonB gene suppressed, in an allele-specific manner, some of the cma mutations, suggesting that interaction of colicin M with TonB may be required for colicin M uptake. Among the hydroxylamine-generated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This colicin derivative still bound to the FhuA receptor and killed cells when translocated across the outer membrane by osmotic shock treatment. It apparently represents a new type of transport-deficient colicin M. Additional hydroxylamine-generated inactive derivatives of colicin M carried mutations centered on residues 193-197 and 223-252. Since these did not kill osmotically shocked cells the mutations must be located in a region which is important for colicin M activity. It is concluded that the TonB box at the N-terminal end of colicin M must be involved in colicin uptake via TonB across the outer membrane and that the C-terminal portion of the molecule is likely to contain the activity domain.
Mol Gen Genet 1993 Jul
PMID:Domains of colicin M involved in uptake and activity. 834 Dec 56

Covalent Superose microspheres-bound C3b was used as a model system to simplify the analysis of antigen-bound C3b modifications during antigen processing. The model was set up using purified C3 and Superose-bound trypsin. C3b was covalently bound to Superose through an ester link, as indicated by lability to hydroxylamine treatment at alkaline pH. C3b-Superose was incubated with L subcellular fraction, enriched in endosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a proteolytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remained bound to Superose through the original ester link; (ii) an esterolytic activity cleaving the ester bond and releasing C3b. Inhibition experiments pointed to the involvement of serine, aspartyl and cysteine proteases. Cathepsin B appeared most probably as one of the major proteases of L fraction catalysing the proteolysis of the C3b-bound. Kinetic studies were in favour of a good stability on the ester bond, supporting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.
Mol Immunol 1993 Jul
PMID:Associated complement C3b. Towards an understanding of its intracellular modifications. 834 Dec 80

A major problem in the application of polymerase chain reaction (PCR) in diagnostic laboratories is contamination with exogenous nucleic acid, especially aerosolized amplicon, from previous PCR. Although several pre- and post-PCR sterilization techniques have been proposed, an optimal sterilization technique is not yet available. Hydroxylamine hydrochloride is a mutagenic agent that binds to and chemically modifies DNA. In the present study PCR was performed on DNA extracted from Herpes simplex virus (HSV) and Borrelia burgdorferi with two sets of primers that amplified a 92 bp sequence unique to HSV DNA polymerase gene and a 156 bp sequence unique to B. burgdorferi Osp-A gene (35 cycles). Following the amplification, PCR products were treated with 0-500 mM hydroxylamine hydrochloride and incubated at room temperature for 30 min. One microlitre of each hydroxylamine treated PCR product was reamplified for an additional 35 cycles. Pre- and post-hydroxylamine treated PCR products were separated by electrophoresis in 3% agarose gel. Hydroxylamine, at a concentration of 250 mM or higher, was found to effectively modify PCR products and prevent their amplification in subsequent PCR.
Mol Cell Probes 1993 Apr
PMID:Application of hydroxylamine hydrochloride for post-PCR sterilization. 839 42

Cells of ice nucleation active bacterial species catalyse ice formation over the temperature range of -2 to -12 degrees C. Current models of ice nucleus structure associate the size of ice nucleation protein aggregates with the temperature at which they catalyse ice formation. To better define the structural features of ice nucleation proteins responsible for the functional heterogeneity of ice nuclei within a genetically homogeneous collection of cells we used in vitro chemical mutagenesis to isolate mutants with reduced ability to nucleate ice at warm assay temperatures but which retain normal or near normal nucleation activity at cold temperatures (WIND, i.e. warm ice nucleus-deficient mutants). Nearly half of the mutants obtained after hydroxylamine mutagenesis of the iceE gene from Erwinia herbicola had this phenotype. The phenotypes and location of lesions on the genetic map of iceE were determined for a number of mutants. All WIND mutations were restricted to the portion of iceE encoding the repetitive region of the polypeptide. DNA sequencing of two WIND mutants revealed single nucleotide substitutions changing a conserved serine or glycine residue to phenylalanine and serine, respectively. The implications of these findings in structure/function models for the ice nucleation protein are discussed.
Mol Microbiol 1993 Jul
PMID:Isolation and characterization of hydroxylamine-induced mutations in the Erwinia herbicola ice nucleation gene that selectively reduce warm temperature ice nucleation activity. 841 88

Human IgG allotypic markers Gm(a)[Glm(1)], Gm(x)[Glm(2)]; Gm(f)[Glm(4)], Gm(b)[G3m(5) and (11)] and Gm(g)[G3m(21)] were studied after chemical modification of IgG histidines by diethylpyrocarbonate, tyrosines by N-acetylimidazole and lysines by formaldehyde and sodium borohydride. Degrees of substitution were estimated by trinitrobenzenesulfonic acid assay. IgG of known Gm phenotype isolated from serum of hyperimmune anti-tetanus toxoid donors was studied. Histidyl modification resulted in virtually complete loss of Gm(a) and Gm(g) antigenicity but preservation of Gm(x), Gm(b) and Gm(f). Reconstitution of the histidyl residues using hydroxylamine resulted in virtually complete restoration of Gm(a) and Gm(g) antigenicity. Histidine modification resulted in no significant decrease in ELISA anti-tetanus antibody activity. Alteration of tyrosyl residues using N-acetylimidazole considerably diminished Gm(a) and Gm(f) expression. This effect was reversed by hydroxylamine treatment. Moreover, chemical alteration of tyrosyl residues produced a complete loss of Gm(g) antigenicity which was only partially restored after deacylation. A urinary H chain fragment containing the VH region directly linked to C gamma 3 which contained the Gm(a) specific and Gm(x) specific amino acid residues was positive for Gm(a) but negative for Gm(x). Another urinary H chain fragment containing only the C gamma 3 domain was negative for both Gm(a) and (x). These findings indicate that Gm allotypic markers may depend on conformational determinants in which strongest expression for Gm(a) and (x) depends on structures expressed by C gamma 3 linked to C gamma 2 domains. Although RFs react with the region encompassing the C gamma 2-C gamma 3 interface, Gm-specificities of such reactions are affected allosterically through single or double amino acid substitutions at a relatively distant site.
Mol Immunol 1993 Mar
PMID:Conformational dependency of human IgG heavy chain-associated Gm allotypes. 845 36

Methoxyamine, N4-methoxycytidine and its 2'-deoxyribo analogue are transition mutagens. The mechanism by which the latter acts after incorporation into or generation within DNA has been ascribed to the ability of the base analogue to pair effectively with both adenine and guanine. To obtain a detailed understanding of these interactions, the solution structures of the self-complementary octanucleotide d(CGGATCCG) and its analogues d(CGGATTCG), d(CGGATMCG) and d(CGGATPCG) (designated 8mer-GC, -GT, -GM and -GP, respectively) were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2] oxazin-7-one. A variable temperature study showed the order of stability as 8mer GC > GP > GT > GM. Nuclear Overhauser spectroscopy permitted the assignment of the base, anomeric and H2'/H2" protons in these 8mers. All had spectra consistent with regular B-DNA duplex structures. Imino proton spectra showed that the 8mers GC, GP and GM involved Watson-Crick base-pairing but that the G.P and to a greater extent G.M base-pairs were in slow exchange on the nuclear magnetic resonance time-scale with the wobble configuration. Indeed, the G.M pair showed an additional exchange process interpreted in terms of the presence of syn and anti conformers of the methoxy group in the wobble pair. This accounts for the destabilization of M compared with the P-containing duplex. The observations are compared with those made earlier on the corresponding AT, AP and AM octamers. It is evident that M and P can form stable base-pairs with both A and G with essentially Watson-Crick geometry. This confirms the earlier, although unsubstantiated explanation for the transition mutational propenstty of methoxyamine which, in turn, was based on the fact that methoxycytosine bases have tautomeric constants (KT) much nearer to unity than the normal bases. The same general explanation for hydroxylamine and hydrazine-induced mutations is correspondingly rendered more certain.
J Mol Biol 1993 Apr 05
PMID:Molecular basis for methoxyamine-initiated mutagenesis: 1H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-modified cytosine residues. 847 18

The Bacillus subtilis gnt operon is negatively regulated by the gnt repressor (GntR, 243 amino acids), which is antagonized by gluconate. The GntR protein belongs to a new family of bacterial regulatory proteins (GntR family). To locate the DNA-binding domain of the GntR protein, we obtained mutations of this protein, by hydroxylamine mutagenesis, which diminish its operator binding ability. Sequence analysis of these mutations indicated that the mutant GntR proteins (GntR43L, GntR66T, GntR74K and GntR75Q) had amino acid substitutions (Ser43 to Leu, Ala66 to Thr, Glu74 to Lys and Arg75 to Gln), respectively. They were all located within the N-terminal conserved region of the GntR family. In vivo and in vitro analysis of these GntR proteins indicated that their relative operator binding abilities became weaker in the order of GntR (wild type), GntR66T, GntR75Q, GntR74K and GntR43L. The equilibrium dissociation constants of GntR (wild type), GntR66T, GntR75Q and GntR74K as to operator binding were determined by gel retardation assays to be 0.43, 2.6, 4.2 and 8.8 M x 10(-10), respectively.
J Mol Biol 1993 May 20
PMID:Missense mutations in the Bacillus subtilis gnt repressor that diminish operator binding ability. 851 Jan 40


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