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

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A DNA fragment from Bacillus natto IFO3936 has been cloned which enhances the production of both extracellular alkaline and neutral proteases in Bacillus subtilis. The DNA sequence analysis around the gene responsible for the hyperproduction, prtR, revealed one open reading frame (comprising 60 amino acid residues) which was bounded by potential transcriptional and translational regulatory signals in its preceding and following regions. This open reading frame was not homologous to the published sequences of the structural genes of the two proteases. The calculated molecular weight (7,109) of the polypeptide predicted from the DNA sequence is much smaller than those of the two proteases, indicating that the gene product is distinct from those enzymes. In-frame fusion between the N-terminal region of the coding sequence and the lacZ gene of Escherichia coli demonstrated that the coding region was indeed translated in vivo. By deletion analysis it was suggested that prtR was the structural gene for the 60-amino-acid polypeptide. Cells carrying a prtR plasmid secreted both proteases 40 to 400 times more than the cells carrying the vector alone. Furthermore, it was found that prtR also enhanced the production of levansucrase by 1 or 2 orders of magnitude. There was no difference, however, in the amount of the other extracellular enzymes such as alpha-amylase, RNase, and alkaline phosphatase. These results indicate that prtR is specific for the hyperproduction of the proteases and levansucrase.
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PMID:Molecular cloning and nucleotide sequence of a DNA fragment from Bacillus natto that enhances production of extracellular proteases and levansucrase in Bacillus subtilis. 308 53

Studies were performed on the prtR gene which enhances the production of the Bacillus subtilis extracellular proteases and levansucrase, but not the alpha-amylase, RNase, and alkaline phosphatase. To investigate the mode of action of prtR, the Escherichia coli bla gene was placed under the control of two promoters. One was the promoter of the alkaline protease gene (aprE), and the other was the promoter of B. subtilis dihydrofolate reductase gene (dfrA). Expression of the bla gene was enhanced by prtR only when the apr promoter was used. From these results, it was concluded that the apr promoter or its vicinity was the target of prtR and that prtR does not affect the process after transcription. The mRNA levels of aprE and nprE (the neutral protease gene) were significantly increased by prtR, but the half-life of the aprE mRNA was not affected. These results show that the prtR gene product enhances protease production by increasing the rate of transcription initiation.
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PMID:prtR enhances the mRNA level of the Bacillus subtilis extracellular proteases. 311 Jan 32

Testosterone-treated calf thymocytes produce increased amounts of proteins, termed lipokinins, that stimulate phospholipase A2 from snake venom and mammalian tissue. The induction of these proteins by testosterone is blocked by cycloheximide and, thus, requires new protein synthesis. These proteins activate phospholipase A2 stoichiometrically. They are inactivated by boiling, trypsin or alkaline phosphatase but not by deoxyribonuclease or ribonuclease. Lipokinins significantly repair the failure of masculinization in the Tfm mouse with an X-linked deficiency of androgen-receptor. Thus, the post-receptor effects of testosterone on embryonic genitalia may be mediated through stimulation of phospholipase A2 by lipokinins. Moreover, lipokinins may be involved as stimulators of the arachidonic acid cascade, as lipocortins are inhibitors.
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PMID:John Lattimer lecture. Lipokinins: novel phospholipase A2 activators mediate testosterone effects on embryonic genitalia. 318 94

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins. 324 13

Escherichia coli strain N100 has been mutagenized by transposon mutagenesis and mutants with a cell surface leaky phenotype have been isolated. The mutant designated as E. coli N100::Tn5 excreted periplasmic proteins like ribonuclease and alkaline phosphatase. When this mutant strain was transformed with plasmids containing cloned cholera toxin genes, the toxin protein synthesized in the cells were excreted. The potentiality of this strain as a live oral vaccine for cholera has been discussed.
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PMID:Excretion of cholera toxin from Escherichia coli: a potential oral vaccine for cholera. 329 72

An inactivated gene for Bacillus amyloliquefaciens extracellular ribonuclease (barnase) has previously been cloned and sequenced following transposon mutagenesis. The intact gene could not be assembled in Escherichia coli and is presumed to be lethal. Therefore, we introduced specific mutations into the barnase gene to prevent its lethal effect. A Gln-73 mutant gene was stable in E. coli but only produced low amounts of barnase antigen. Mutants containing Asp, Gln or Arg, instead of His-102, at the active site were identified by immunological screening for barnase antigen. None of the mutant proteins with alterations at aa residue 102 possessed RNase activity. The level of barnase (Asp-102) was higher in E. coli than in B. subtilis but the protein was not processed to the correct size in E. coli. To obtain correct processing, the barnase (Asp-102) structural gene was fused to the E. coli alkaline phosphatase promoter and signal sequence (phoA). Cells containing this construct secreted correctly processed barnase (Asp-102) into the periplasmic space and culture supernatant at a level of 20 mg/l. Barnase (Asp-102) was purified and found to have an identical N-terminus and a thermal unfolding curve that was nearly identical to that of active barnase (His-102). The cloning and expression of barnase in E. coli will allow detailed analysis of barnase protein folding by molecular genetic approaches.
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PMID:Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation. 329 26

A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (alpha MSH/ACTH[4-11]), was synthesized and labelled in the 3'-end by use of terminal transferase. Probes tailed with either [3H]- or biotin-labelled nucleotides could be used for in situ hybridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidin-alkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridization (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.
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PMID:Detection of proopiomelanocortin mRNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry. 339 4

A factor, termed neutrophil alkaline phosphatase-inducing factor (NAP-IF), that has the capacity to increase the NAP activity of granulocytes was characterized by using two samples: cystic fluid (CF) and conditioned medium of a tumor cell line (T3M5). The molecular weight of NAP-IF was shown to be between 13,000 and 45,000, and its isoelectric point was between 5.5 and 6.2. It was sensitive to heat and proteolytic enzymes, but was resistant to DNase and RNase, suggesting that NAP-IF is an acidic protein or glycoprotein. These characteristics of NAP-IF seem to be similar to those of granulocyte-macrophage colony-stimulating factor (GM-CSF) that is also present in the CF. NAP-IF rich fractions obtained by isoelectric focusing from CF were also found to be rich in a subclass of GM-CSF: granulocyte-CSF (G-CSF). Furthermore, a high correlation was noted between the activities of G-CSF and NAP-IF (gamma = 0.798, P less than 0.005). These results suggest that the two activities, i.e., G-CSF and NAP-IF, may be attributable to an identical macromolecule.
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PMID:Characterization of neutrophil alkaline phosphatase-inducing factor (NAP-IF). 387 40

An enzyme that converts [3H, 32P]ATP, with a 3H:32P ratio of 1:1, to oligoadenylates with the same 3H:32P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of 3H:32P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities. 399 78

A 125-kilodalton (kDa) phosphoprotein was isolated from nucleoli of Novikoff hepatoma cells in the presence of various inhibitors of proteases, alkaline phosphatase, and RNase. This protein was the most highly phosphorylated protein found thus far in the nucleolus. The half-life of [32P]phosphate in the 125-kDa phosphoprotein was approximately 60 min. Amino acid analysis of the protein showed it had a high serine content (15.5 mol %), a high glutamine plus glutamic acid content (15.5 mol %), and a high lysine content (10.3 mol %). Phosphoserine was the only phosphorylated amino acid identified. After alkaline hydrolysis of the 32P-labeled protein, ribonucleotides were found which accounted for approximately 8.5% of the [32P]phosphate. After cytidine 3',5'-[32P]diphosphate ([32P]pCp) labeling by RNA ligase, several oligoribonucleotide sequences were purified including GGGCOH and GGGGCOH. The binding of oligonucleotides to peptides was stable under denaturing fractionation conditions including 6 M urea treatment and incubation at 100 degrees C for 10 min in sodium dodecyl sulfate and beta-mercaptoethanol. Furthermore, when nucleotide-peptide complex was treated with ribonuclease T2 followed by snake venom phosphodiesterase, the junctional nucleotide pCp was released. These results suggest that one or more ribonucleotides are covalently bound to the 125-kDa phosphoprotein.
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PMID:Isolation and characterization of a 125-kilodalton rapidly labeled nucleolar phosphoprotein. 408 83


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