Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have constructed a synthetic secretion cassette encoding the alpha-factor prepro leader peptide from Saccharomyces cerevisiae fused to mouse epidermal growth factor (mEGF). This was used to compare the secretion of mEGF, a 53-amino acid polypeptide, in S. cerevisiae and Pichia pastoris. In both yeasts the leader sequence was accurately and efficiently cleaved showing that the S. cerevisiae-derived alpha-factor prepro region is correctly recognised and processed in P. pastoris. Of the total mEGF produced, over 90% was exported to the culture supernatant, although the final level of accumulation was dependent on the composition of the growth medium. With P. pastoris there was instability of the protein in minimal medium (yeast nitrogen base), probably caused by extracellular proteases. This was overcome by adding 1% Casamino acids and buffering the medium to pH 6.0. To increase the level of secreted mEGF we have developed a method for rapidly screening large numbers of P. pastoris transformants for the presence of many copies of a foreign gene. Using this procedure we isolated a strain containing 19 integrated copies of the mEGF gene which secreted 450 micrograms/ml of mEGF in high-density fermentations. Characterisation of the yeast-derived mEGF showed the presence of truncated forms, mEGF1-51 and mEGF1-52, as was found with S. cerevisiae-secreted human EGF [George-Nascimento et al., Biochemistry 27 (1988) 797-802]. In addition, the full-length protein, mEGF1-53, was secreted by P. pastoris.
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PMID:Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. 193 16

The structural gene (glnA) encoding the ammonia-assimulation enzyme glutamine synthetase (GS) has been cloned from the obligate methanotroph Methylococcus capsulatus (Bath). Complementation of Escherichia coli glnA mutants was demonstrated. In vitro expression analysis revealed that the cloned glnA gene coded for a polypeptide of apparent Mr 60,000, as determined by PAGE. Expression of the M. capsulatus (Bath) glnA gene in E. coli was regulated by nitrogen levels in an Ntr+ but not an Ntr- background. The nucleotide sequence of the M. capsulatus (Bath) glnA gene and flanking sequences was determined. This gene, of 1407 bp, encoded a polypeptide of Mr 51717 containing 468 amino acids. The 5' leader region contained three putative promoters. Promoters P1 and P3 resembled the canonical -10 -35 E. coli-type promoter. Promoter P2, which was located between P1 and P3, resembled the NtrA-dependent promoters of enteric organisms. A potential NtrC-binding site was also determined, flanking the Pribnow box at P1. Comparisons of nucleotide-derived amino acid sequences of GS enzymes from prokaryotes and eukaryotes with GS from M. capsulatus are made.
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PMID:Cloning, sequencing and expression of the glutamine synthetase structural gene (glnA) from the obligate methanotroph Methylococcus capsulatus (Bath). 196 23

The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans has been sequenced and its transcript mapped and orientated. A single ORF can encode a protein of 719 amino acids. A 52 amino acid region including a putative 'zinc finger' strongly resembles putative DNA binding regions of the major regulatory protein of erythroid cells. The derived protein sequence also contains a highly acidic region possibly involved in gene activation and 22 copies of the motif S(T)PXX, abundant in DNA binding proteins. Analysis of chromosomal rearrangements and transformation with deletion clones identified 342 N-terminal and 124 C-terminal residues as inessential and localized a C-terminal region required for nitrogen metabolite repressibility. A -1 frameshift eliminating the inessential 122 C-terminal amino acids is a surprising loss-of-function mutation. Extraordinary basicity of the replacement C terminus might explain its phenotype. Mutant sequencing also identified a polypeptide chain termination and several missense mutations, but most interesting are sequence changes associated with specificity mutations. A mutation elevating expression of some structural genes under areA control whilst reducing or not affecting expression of others is a leucine to valine change in the zinc finger loop. It reverts to a partly reciprocal phenotype by replacing the mutant valine by methionine.
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PMID:The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. 197 Feb 93

A spontaneous pleiotropic Nif- mutation in Rhodospirillum rubrum has been partially characterized biochemically and by complementation analysis with recombinant plasmids carrying Azotobacter vinelandii DNA in the vicinity of ORF12 [Jacobson et al. (1989) J. Bacteriol 171: 1017-1027]. In addition to being unable to grow on N2 as a nitrogen source the phenotypic characterization of this and other metronidazole enriched spontaneous mutants showed (a) no nitrogenase activity, (b) the absence of NifHDK polypeptides, (c) a slower growth rate on NH4+, (d) approximately 50% higher glutamine synthetase (GS) activity than the wild-type, which was repressible, (e) an inability to switch-off GS activity in response to an NH4+ up-shift, and (f) an inability to modify (32P-label) the GS polypeptide. The apparent relationship between the absence of nifHDK expression and the absence of GS adenylylation cannot be explained in terms of the current model for nif gene regulation. However, R. rubrum transconjugants receiving A. vinelandii DNA which originated immediately upstream from nifH, restored all aspects of the wild-type phenotype. These data suggest a here-to-fore unrecognized relationship between nif expression and GS switch-off (adenylylation) activity, and the existence of a previously unidentified regulatory locus in Azotobacter that complements this mutation.
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PMID:Complementation of a pleiotropic Nif-Gln regulatory mutant of Rhodospirillum rubrum by a previously unrecognized Azotobacter vinelandii regulatory locus. 198 May 82

The nucleotide sequence of the Aspergillus nidulans crnA gene for the transport of the anion nitrate has been determined. The crnA gene specifies a predicted polypeptide of 483 amino acids (molecular weight 51,769). A hydropathy plot suggests that this polypeptide has 10 membrane-spanning helices with an extensive hydrophilic region between helices six and seven. No striking homology was observed between the crnA protein and other reported membrane proteins of either prokaryotic or eukaryotic organisms, indicating that the crnA transporter may represent another class of membrane protein. Northern blotting results with wild-type cells show that (i) control of crnA expression is subject to nitrate (and nitrite) induction as well as nitrogen metabolite repression and (ii) regulation of the crnA gene is exerted at the level of mRNA accumulation, most likely at transcription, in response to the nitrogen source in the growth medium. Furthermore, similar studies with mutants of nirA and areA control genes and the niaD nitrate reductase structural gene show that crnA expression is mediated by the products of nirA (nitrate induction control gene), areA (nitrogen metabolite repression control gene), and niaD (involved in autoregulation of nitrate reductase).
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PMID:crnA encodes a nitrate transporter in Aspergillus nidulans. 198 67

The URE2 gene of Saccharomyces cerevisiae has been cloned and sequenced. It encodes a predicted polypeptide of 354 amino acids with a molecular weight of 40,226. Deletion of the first 63 amino acids does not have any effect on the function of the protein. Studies with disruption alleles of the URE2 and GLN3 genes showed that both genes regulate GLN1 and GDH2, the structural genes for glutamine synthetase and NAD-linked glutamate dehydrogenase, respectively, at the transcriptional level, but expression of the regulatory genes does not appear to be regulated. Active URE2 gene product was required for the inactivation of glutamine synthetase upon addition of glutamine to cells growing with glutamate as the source of nitrogen. The predicted URE2 gene product has homology to glutathione S-transferases. The gene has been mapped to chromosome XIV, 5.9 map units from petX and 3.4 map units from kex2.
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PMID:The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to the nitrogen source and has homology to glutathione s-transferases. 199 Feb 86

A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source.
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PMID:Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa. 212 8

The tonoplast mediates the transport of various ions and metabolites between the vacuole and cytosol by mechanisms that remain to be elucidated at the molecular level. The primary structure of only one tonoplast protein, the H(+)-ATPase, has been reported to date. Here we report the primary structure of tonoplast intrinsic protein (TIP), a 27-kilodalton intrinsic membrane protein that occurs widely in the tonoplasts of the protein storage vacuoles (protein bodies) of seeds [Johnson, K.D., et al. (1989). Plant Physiol. 91, 1006-1013]. Hydropathy plots and secondary structure analysis of the polypeptide predict six membrane-spanning domains connected by short loops and hydrophilic, cytoplasmically oriented N- and C-terminal regions. TIP displays significant homology with several other membrane proteins from diverse sources: major intrinsic polypeptide from bovine lens fiber plasma membrane; NOD 26, a peribacteroid membrane protein in the nitrogen-fixing root nodules of soybean; and interestingly, GIpF, the glycerol facilitator transport protein in the cytoplasmic membrane of Escherichia coli. Based on the homology between TIP and GIpF and the knowledge that the protein storage vacuolar membrane and the peribacteroid membrane are active in solute transport, we propose that TIP transports small metabolites between the storage vacuoles and cytoplasm of seed storage tissues.
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PMID:An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GIpF). 215 74

Eighty-three percent of polyphosphoinositide-specific phospholipase C activity was recovered in a cytosolic fraction after nitrogen cavitation of turkey erythrocytes. This activity has been purified approximately 50,000-fold when compared to the starting cytosol with a yield of 1.7-5.0%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phospholipase C preparation revealed a major polypeptide of 150 kDa. The specific activity of the purified enzyme was 6.7-14.0 mumol/min/mg of protein with phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 4-phosphate as substrate. Phospholipase C activity was markedly dependent on the presence of Ca2+. The phospholipase C showed an acidic pH optimum (pH 4.0). At neutral pH, noncyclic inositol phosphates were the major products formed by the phospholipase C, while at pH 4.0, substantial formation of inositol 1:2-cyclic phosphate derivatives occurred. Properties of the purified 150-kDa turkey erythrocyte phospholipase C were compared with the approximately 150-kDa phospholipase C-beta and -gamma isoenzymes previously purified from bovine brain (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1987) J. Biol. Chem. 262, 12511-12518). The turkey erythrocyte phospholipase C differed from the two mammalian phospholipases with respect to the effect of sodium cholate on the rate of polyphosphoinositide hydrolysis observed. Moreover, when presented with dispersions of pure inositol lipids, phospholipases C-beta and -gamma displayed comparable maximal rates of polyphosphoinositide and phosphatidylinositol hydrolysis. By contrast, the turkey erythrocyte phospholipase C displays a marked preference for polyphosphoinositide substrates.
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PMID:A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. I. Purification and properties. 216 32

15N-1H correlation spectroscopy with detection at the 1H frequency has been used at natural abundance to detect nitrogen nuclei bonded to protons in the ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429). Side-chain aromatic nitrogens, main-chain amides, and side-chain amides have been assigned to specific residues by comparison to previous proton assignments. Assignment ambiguities arising from overlap in the proton dimension have been resolved by examining spectra as a function of temperature and pH. Nitrogen chemical shifts are reported at pH 4.6 and 9.4 and three temperatures, 32, 50, and 60 degrees C. Significant differences arise from the observed protein shifts and expected shifts in the random coil polypeptide.
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PMID:15N NMR spectroscopy of Pseudomonas cytochrome c-551. 217 26


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