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)

The correlation between cellular growth and microfilament-dependent morphology was investigated. It was found that the proliferative growth of various nontransformed cell lines does not only depend on cell adhesion to a suitable substratum and cell flattening but also on intact microfilaments. Disintegration of microfilaments by cytochalasin D (CD) as well as lactrunculin (LAT)-A and LAT-B is correlated with a strong decline of the number of DNA-synthesizing cells during a period of 8 to 12 h after application of the drug. RNA and protein synthesis are reduced already after a preincubation time of 2 h. Although microinjected rhodamine-phalloin is colocalized with microfilaments there is no stabilizing effect against CD even at high phaloidin concentrations. Microinjection of DNAse I results in a strong alteration of the microfilament system. The disorganization of microfilaments was correlated with a moderate decrease of protein synthesis 2 h after microinjection, whereas RNA synthesis remained unchanged, if RNase-free DNase I was used. The number of DNA-synthesizing cells was somewhat diminished 24 h after microinjection. Furthermore, the microfilament system is disorganized by microinjection of gelsolin and gelsolin segment 1 + 2, respectively. The severing the microfilaments by gelsolin is correlated with a significant restriction of RNA and protein synthesis during a period of 2 to 4 h after injection, but the labeling index remaining unchanged. Therefore, we assume that inhibition of the G0-G1-S transition is only caused by a disintegration of microfilaments lasting longer than 4 h. The significance of microfilament organization for growth regulation is discussed.
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PMID:Growth regulation by cell shape alteration and organization of the cytoskeleton. 754 9

We have isolated the thermostable 9 kDa SaRD-protein from Sulfolobus acidocaldarius which exhibit RNase activity as well as DNA-binding properties (SaRD). The amino acid composition and the sequence of the 16 N-terminal amino acids show similarities to different RNases as well as to DNA-binding proteins from thermophilic archea. The RNase activity was demonstrated by 5S rRNA degradation, thin layer chromatography and a zymogram. The temperature optimum for the RNase activity is 65 degrees C. The pH optimum ranges from 6.5-7.0. DNA-binding properties were shown by gel-shift assays on agarose gels. In a similar way SaRD mediated protection of DNA against DNase I digestion and Sau3A I restriction could be demonstrated. The melting point (Tm) of genomic DNA was raised from 68 degrees C to 90 degrees C by addition of the SaRD-protein. CD spectroscopy indicated that SaRD is very stable near neutral pH and can neither be unfolded by temperatures up to 85% C nor by addition of 8 M urea.
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PMID:SaRD, a new protein isolated from the extremophile archaeon Sulfolobus acidocaldarius, is a thermostable ribonuclease with DNA-binding properties. 754 5

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre-proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within the trans-Golgi network, where they are finally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellular compartment. We have recently established stably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR-CRH displayed an apparent molecular weight approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule. In this paper, we further characterized the bitopological, i.e. nuclear and cytoplasmic localization of proCRH within transfected CHO-K1 cells. Immunoreactive nuclear CRH was not extractable using detergents (Triton X-100 and CHAPS), 10 mM salt washes or RNase digestion but could be abolished by digestion with DNase I. These results therefore suggest that nuclear proCRH is in close association with DNA/chromatin. Treatment of transfected cells with inhibitors of protein and RNA synthesis for up to 24 h had no effect upon immunoreactive nuclear CRH, indicating that it is very stable with a long half life. Brefeldin A treatment had no effect upon the nuclear translocation of newly synthesized proCRH, suggesting that late stages of the secretory pathway (i.e. post rough endoplasmic reticulum compartments) of the transfected cells do not play a role in proCRH nuclear transport. We also demonstrate that proCRH synthesized within stably transfected CHO-K1 cells is capable of stimulating ACTH release from primary cultures of anterior pituitary cells, therefore showing for the first time that the intact precursor is also biologically active and could act as an ACTH secretagogue in-vivo.
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PMID:Expression of biologically active procorticotrophin-releasing hormone (proCRH) in stably transfected CHO-K1 cells: characterization of nuclear proCRH. 764 68

The terminase holoenzyme of bacteriophage lambda is a multifunctional protein composed of two subunits, gpNu1 and gpA. In vitro, under certain conditions, terminase can render DNAs from various sources, of varying lengths and termini, resistant to degradation by high concentrations of DNase I. This reaction is completely dependent on the presence of terminase, proheads, a hydrolyzable triphosphate, and a divalent metal ion, and we propose that it is the result of translocation of DNA into proheads by terminase. This reaction is stoichiometric with respect to terminase, DNA, and proheads and can be supported by all deoxyribo- and ribonucleoside triphosphates, but not by the corresponding diphosphates or nonhydrolyzable ATP analogs. Mg2+ and Ca2+ promote the reaction, but Mn2+ and Zn2+ do not. In the absence of spermidine, translocase activity is low, but addition of the Escherichia coli protein integration host factor (IHF) promotes specific translocation of only those DNA fragments containing the terminase-binding site, cosB. When spermidine is present, nonspecific translocation of DNA from any source is stimulated. Under these conditions IHF no longer promotes specificity, but translocation of only cosB-containing DNA fragments can be restored by addition of small amounts of a dialyzed and RNase-treated E. coli extract, suggesting that additional host factor(s) may be involved in determination of packaging specificity. To a limited extent, gpA alone can promote translocation, but gpNu1, which has no translocase activity on its own, must be added to approach the holoenzyme-like activity levels. Formation of viable phage cannot be accomplished by gpA in the absence of gpNu1.
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PMID:The in vitro translocase activity of lambda terminase and its subunits. Kinetic and biochemical analysis. 765 23

A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/ml), and RNase could not be inhibited at all (MIC, > 7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. 769 Feb 21

Diazepam binding inhibitor (DBI) is a 10-kDa polypeptide that regulates mitochondrial steroidogenesis, glucose-induced insulin secretion, metabolism of acyl-CoA esters, and the action of gamma-aminobutyrate on GABAA receptors. To investigate the regulation of DBI gene expression, three positive clones were isolated from a rat genomic library. One of them contained a DBI genomic DNA fragment encompassing 4 kb of the 5' untranslated region, the first two exons, and part of the second intron of the DBI gene. Two other overlapping clones contained a processed DBI pseudogene. Several transcription initiation sites were detected by RNase protection and primer extension assays. Different tissues exhibited clear differences in the efficiencies of transcription startpoint usage. Transient expression experiments using DNA fragments of different length from the 5' untranslated region of the DBI gene showed that basal promoter activity required 146 bp of the proximal DBI sequence, whereas full activation was achieved with 423 bp of the 5' untranslated region. DNase I protection experiments with liver nuclear proteins demonstrated three protected regions at nt -387 to -333, -295 to -271, and -176 to -139 relative to the ATG initiation codon; in other tissues the pattern of protection was different. In gel shift assays the most proximal region (-176 to -139) was found to bind several general transcription factors as well as cell type-restricted nuclear proteins which may be related to specific regulatory patterns in different tissues. Thus, the DBI gene possesses some features of a housekeeping gene but also includes a variable regulation which appears to change with the function that it subserves in different cell types.
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PMID:Cloning and tissue-specific functional characterization of the promoter of the rat diazepam binding inhibitor, a peptide with multiple biological actions. 769 Sep 62

Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the human vacuolar H(+)-ATPase (V-ATPase). We examined mRNA levels of various V-ATPase subunits during differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell V-ATPase content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5'-flanking region of this gene reveal a TATA-less promoter with a high G + C content. Primer extension and ribonuclease protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. DNase I footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions.
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PMID:Transcriptional regulation of the vacuolar H(+)-ATPase B2 subunit gene in differentiating THP-1 cells. 770 73

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation of sn-glycerol 3-phosphate to form 1-acyl-sn-glycerol 3-phosphate, a committed step in triacylglycerol and phospholipid biosynthesis. We have previously reported the cDNA cloning and transcriptional regulation of the murine mitochondrial GPAT (mGPAT). We now report the cloning of the 5'-flanking region of the murine mitochondrial GPAT. The transcription start site was identified by primer extension and RNase protection assays. A TATA box-like motif (TTATTAT) was located between -34 and -29 and a reverse CCAAT box (ATTGG) was located between -78 and -74, relative to the transcription start site. To begin studying mechanisms underlying transcriptional regulation of the mGPAT gene, chimeric luciferase (LUC) plasmids containing serial deletions, from -1447 to -38, of the 5'-flanking region of the murine mGPAT gene were prepared and transfected into 3T3-L1 cells. The fusion construct -1447 GPAT.LUC showed high promoter activity and deletions to -1353, -747, -322, and -86 did not markedly change the promoter activity. With all constructs, luciferase activity was 2-fold higher when plasmids were transfected into 3T3-L1 adipocytes. However, deletion of sequences between -86 and -55 resulted in a 9-fold decrease in LUC activity in both preadipocytes and adipocytes. Deletion of sequences between -55 and -38 did not alter promoter activity. DNase I footprint analysis revealed a protected region between -95 and -65 which included the putative CTF/NF1 binding site. Electrophoretic mobility shift assays demonstrated a single protein-DNA complex formation. Oligonucleotides synthesized according to the CTF/NF1 consensus sequence or the adenovirus NF-1 site showed a different and more complex pattern of protein-DNA interaction and were not able to compete away the mGPAT promoter-protein complex, indicating that a distinct protein was bound to -86/-55, a region important for the basal promoter activity in 3T3-L1 cells. Luciferase activity was increased 2.8- and 8-fold when adipocytes stably transfected with -322 GPAT.LUC were treated with 5 and 25 mM glucose, respectively, in the presence of 10 nM insulin. These results indicate that carbohydrate-responsive sequences are located within -322 base pairs of the mGPAT promoter.
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PMID:Characterization of the murine mitochondrial glycerol-3-phosphate acyltransferase promoter. 783 9

Simian virus 40 (SV40) chromatin extracted from nuclei of infected monkey cells (CV1) was sedimented in neutral sucrose gradients, before and after digestion with bovine pancreatic RNase I-A or DNase I. DNA topoisomerase (TI) activity was found associated with RNase-resistant, DNase-sensitive SV40 nucleoprotein complexes. After polyacrylamide gel electrophoresis, a number of proteins with a molecular mass between 40 and 70 kDa were seen at the level of viral DNA peaks, some of which may represent catalytically active breakdown products of the TI enzyme. Large protein complexes were observed under the electron microscope in association with the viral chromosomes and appear to correspond to the SV40 DNA replication complex, including TI. Our results suggest that TI activity is indeed associated with the viral minichromosomes undergoing replication in vivo.
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PMID:DNA topoisomerase activity associated with simian virus 40 nucleoprotein complexes. 784 Sep 39

The quaternary structure of the adenovirus early region 1B 54K protein has been examined under denaturing and nondenaturing conditions. In the presence of SDS the protein has a strong tendency to form disulfide-linked high-molecular-weight polymers. Under nondenaturing, but reducing, conditions the in vitro-translated 54K polypeptide forms oligomers (probably tetramers) of molecular weight approximately 2 x 10(5). After subcellular fractionation of Ad12 early region 1-transformed cells, the 54K E1B protein present in the cytoplasm had a molecular weight similar to that determined for the in vitro-translated material. However, two populations of the viral protein could be distinguished in the nucleus-one of a size similar to that seen in the cytoplasm and the other of appreciably higher molecular weight (approximately 2 x 10(6)). No difference in migration pattern was observed after treatment of the nuclear extract with DNase I or RNase. A proportion of the Ad12 E1B 54K protein in both the high- and the low-molecular-weight populations in the nucleus was found to form a complex with p53, and it is therefore concluded that the very high molecular weight derives from interaction with an, as yet, unidentified component.
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PMID:The quaternary structure of the adenovirus 12 early region 1B 54K protein. 787 36


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