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)

Primer extension and RNase protection analyses of the rat beta 2-adrenergic receptor (beta 2AR) gene identify two transcription start points at -64 and -220 nt, respectively. Transient transfections of putative promoter/pCAT constructs into DDT1 MF-2 cells indicate that fragments -36 to -100 (PI) and -186 to -312 (P2) are sufficient to promote transcription, whereas -911 to -1122 contains a negative regulatory element(s). RNase protection analysis of the 3' untranslated region (3'-UTR) indicates the presence of two transcripts with 3'-UTR of 111 and 604 nt exclusive of the poly(A+) tails. Northern blots of beta 2AR mRNA using full-length and partial cDNA probes indicate that a major 2.2 kb and a minor 1.6 kb species arise from the use of alternative promoters as well as different polyadenylation signals. DNase I footprinting and DNA mobility shift assays (DMSA) using rat liver nuclear extracts identify a number of transcription factors binding to sequence elements within or upstream from P1 and P2, including Spl, CRE, CPl, AP-2, NF-1, NF-kappa B, and C/EBP. Supershift assays using antibodies against C/EBP alpha and C/EBP beta and mutational analyses indicate that the protein binding to the C/EBP consensus recognition site at -925 to -933 is C/EBP alpha. The activity of promoter/CAT constructs containing the C/EBP recognition site is significantly decreased by cotransfection of C/EBP alpha but not C/EBP alpha but not C/EBP beta into either DDT1 MF-2 cells or primary rat hepatocytes. Partial hepatectomy causes a transient decrease in C/EBP alpha, as measured by DMSA, and an increase in beta 2 AR mRNA levels and rate of transcription in the remnant liver. Thus, derepression via C/EBP alpha is likely involved in the up-regulation of beta 2AR in the regenerating rat liver.
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PMID:DNA elements and protein factors involved in the transcription of the beta 2-adrenergic receptor gene in rat liver. The negative regulatory role of C/EBP alpha. 885 51

Because the ferric uptake regulator (fur) appears to be an essential gene in Pseudomonas aeruginosa, resistance to manganese was used as an enrichment to isolate strains carrying point mutations in the fur gene in order to assess its role in the co-ordinate expression of siderophores and exotoxin A (ETA). This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, which carry point mutations in the fur gene. Two parental strains were used in this study. Three mutants were isolated from the widely used strain, PAO1. One of these, CS (cold sensitive), has a mutation in the 5' non-coding region of the fur gene while the two other mutants derived from this parent have mutations resulting in the following deduced changes in Fur: mutant A2, H86-->R; mutant A4, H86-->Y. The other mutant (C6) and its revertant (C6Rv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of arginine deiminase and nitrate reduction) that controls anaerobic respiration in P. aeruginosa. Fur from the C6 mutant has an A10-->G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has been changed to Ser at this position. All mutants were examined for alterations in the iron-regulated expression of siderophores and ETA. The A2 and A4 mutants expressed higher levels of siderophores in iron-deficient media and in iron-replete media. The CS mutant constitutively expressed siderophores at 25 degrees C. At 42 degrees C siderophore biosynthesis was iron repressed as in the parental strain PAO1. The deletion of anr in PAO6261 had no apparent effect on the iron-mediated regulation of siderophore synthesis, but the C6 mutant derived from this strain produces siderophores constitutively. The iron-regulated production of siderophores by C6Rv was similar to the parental strain PAO6261 and PAO1. Because one of the parental strains used in this study is an Anr mutant, regulation of ETA production was assessed under aerobic and microaerobic conditions. Iron-dependent repression of ETA synthesis in both parental strains and A2 and A4 mutants was found to be 50-100-fold under aerobic and microaerobic conditions, as assayed by quantitative Western dot-blot assays. By contrast in the CS and C6 mutants, while iron-dependent repression os ETA synthesis was similar to both parental strains under aerobic conditions, ETA production in these mutants was constitutive in a microaerobic environment. RNase protection analysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mutant corroborated the results of quantitative dot-blot assays of ETA. The mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS) that positively regulates the expression of siderophores and ETA. Fur from the A2 and A4 mutants and from the C6Rv revertant was able to bind to the target DNA, but with reduced affinity by comparison to wild-type Fur. Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter region of the pvdS gene, but it retained some weak binding activity in gel mobility shift assays. The data presented in this study not only furnish some additional insights into the structure-function relationships of Fur, but also afford novel perspectives of virulence factors in P. aeruginosa under environmental conditions that have not previously been considered.
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PMID:Ferric uptake regulator mutants of Pseudomonas aeruginosa with distinct alterations in the iron-dependent repression of exotoxin A and siderophores in aerobic and microaerobic environments. 888 70

It is widely accepted that nuclear genes that encode proteins of the oxidative-phosphorylation system are regulated by nuclear factors believed to be specific for such genes. In the present study we show that the promoter for the human cytochrome c1 gene is an exception, in that it involves only conserved Sp1 core elements and an initiator region. Maximal promoter activity within a 1.4-kb 5' flanking region of the cytochrome c1 gene is contained in a fragment (-72 to +18) that lacks TATA and CCAAT elements. The transcriptional start site was mapped to an initiator region by RNase protection of mRNA from human HepG2 cells, and by primer extension of in vitro-generated transcripts, to a sequence that is highly similar to the dihydrofolate reductase family of initiators. Deletion of this region (+1 to +18) severely impairs transcription initiation. Sp1 core elements centered at nucleotides -21 and -39 define the activation domain of the proximal promoter. Only the -39 element is protected from DNase I in the presence of crude nuclear extracts. However, transfection, gel-mobility-shift, supershift and in vitro-transcription experiments show that the -21 element binds Sp1 protein and contributes to transcription activation. No other functional oxidative-phosphorylation-specific response elements have been identified. These data implicate Sp1 as a single activating factor for an oxidative-phosphorylation gene.
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PMID:Expression of the human cytochrome c1 gene is controlled through multiple Sp1-binding sites and an initiator region. 891 68

The rnb gene encodes ribonuclease II (RNase II), one of the two major Escherichia coli exonucleases involved in mRNA degradation. In this paper, the rnb transcript is characterized regarding its promoter and terminator regions. The combined results from S1 nuclease protection analysis, DNase I footprinting and gene fusions with lacZ have shown that rnb is expressed from two promoters. S1 nuclease protection analysis and DNA footprinting have shown that rnb has two promoters, P1 and P2. Transcriptional and translational lacZ reporter fusions, constructed to the rnb gene, revealed that P2, the rnb proximal promoter, is stronger than P1. However, P2 is not transcribed in vitro, suggesting that an additional factor is required in vivo. The 3' end of the rnb transcript mapped to a stem-loop structure immediately after the translated region.
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PMID:Escherichia coli RNase II: characterization of the promoters involved in the transcription of rnb. 893 10

The broad spectrum of vasoactive intestinal polypeptide (VIP) cellular functions are mediated by high-affinity binding sites. To determine regulation of the VIP receptor gene expression, we have isolated and characterized two genomic clones that contain the first three exons and the 5' flanking region of the VIP receptor gene. Using RNase protection assays, receptor gene expression was detected in adult rat lung, liver and intestine, but not in fetal lung, indicating that VIP receptor is expressed in diverse tissues, and its expression is differentially regulated during lung development. The transcription start site of the gene was mapped to a cytosine residue, 76 bp upstream from the ATG initiation codon. Transfection into rat lung cell lines shows that although 126 bp of the VIP receptor 5' DNA sequences are capable of activating VIP receptor gene basal transcription 30-fold over a promoterless control, 488 bp of the 5' sequences further induce this activation to 97-fold over control. However, inclusion of up to 859 bp 5' sequences results in a decrease in basal promoter activity (31-fold over control), indicating that while sequences between -126 and -488 bp contain potential enhancer sequences, sequences between -488 and -859 bp may include a transcriptional repressor sequence. Deletion analysis shows that transcription factor Sp1 plays an important role in activating basal promoter of the VIP receptor gene. DNase I footprinting and gel-mobility-shift assays show that Sp1 binds to its consensus binding sites in the VIP receptor promoter, suggesting that interaction of Sp1 with VIP receptor promoter transactivates this gene.
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PMID:Characterization of the rat vasoactive intestinal polypeptide receptor gene 5' region. 894 24

Research into the use of new genetic markers is difficult and costly, but it is necessary for more accurate criminal individualization and paternity testing as well as for analysis of genetic diseases. Recently, we discovered that human ribonuclease (RNase), deoxyribonuclease I (DNase I) and deoxyribonuclease II (DNase II) are characteristic markers showing genetic polymorphism and useful for forensic investigation. DNase I is particularly well suited to practical use, since it shows a well-balanced gene frequency, a high concentration in several body fluids (blood, sweat, urine, breast milk and semen) and tissues (pancreas, liver and kidney), stability against severe conditions (exposure of test samples to high temperature, high humidity and long-term storage), and easy and accurate detectability.
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PMID:[Discovery of genetic polymorphism of human nucleases]. 895 29

The 14.1 surrogate light chain protein is expressed on human pre-B lymphocytes in association with Vpre-B and the mu Ig heavy chain to form the pre-B receptor. The 14.1 chain has also been called the lambda (lambda)-like (LL) protein and is homologous to murine lambda5. The 14.1(IGLL1) gene is expressed in a lineage- and stage-restricted manner. To understand the molecular mechanism of the 14.1 gene tissue- and stage-specific expression, we analyzed the 5'-flanking region and characterized the promoter for this gene. In this report, we identify two DNase I-hypersensitive sites located at 2.6 kilobases (HSS 1) and 0.2 kilobases (HSS 1) upstream of 14.1 exon 1. These hypersensitive sites are present in pre-B lymphocyte cell lines, but absent in mature B and T cell lines. We have used RNase protection analysis to localize the 5' major transcriptional start site and rapid amplification of 5' cDNA ends to identify multiple start sites within the TATA-less, GC-rich 14.1 promoter. The region encompassing HSS 2 was analyzed for promoter activity. Transfection of cell lines with a series of truncated segments of the 5' flanking region linked to the luciferase reporter gene revealed that the 14.1 promoter is lineage- and stage-specific, and we localized this activity to positions +150 to +227 relative to the 5' major transcriptional start site.
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PMID:The 14.1 surrogate light chain promoter has lineage- and stage-restricted activity. 902 4

POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.
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PMID:Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression? 903 92

After androgen ablation by castration, the epithelial cells of the rat ventral prostate are eliminated by apoptosis. The number of cells showing apoptotic chromatin degradation increases with time up to day 3 after castration as verified by in situ end labeling of fragmented DNA. Apoptotic chromatin degradation is catalyzed by a Ca2+, Mg2+-dependent endonuclease. Recently, evidence has been presented that suggests deoxyribonuclease I (DNase I) is identical or very closely related to the apoptotic endonuclease (Peitsch, M.C., B. Polzar, H. Stephan, T. Crompton, H.R. MacDonald, H.G. Mannherz, and J. Tschopp. 1993. EMBO [Eur. Mol. Biol. Organ.] J. 12:371-377). Therefore, the expression of DNase I in the ventral prostate of the rat was analyzed before and after androgen ablation at the level of protein, enzymatic activity, and gene transcripts using immunohistochemical and biochemical techniques. DNase I immunoreactivity was detected only in a few single epithelial cells before androgen ablation. After castration, a time-dependent increase in DNase I immunoreactivity was observed within the epithelial cells. It first appeared after about 12 h in the apical region of a large number of epithelial cells. Up to day 3 after castration, the intracellular DNase I antigenicity continuously increased, and the cell nuclei gradually became DNase I positive. At day 5, almost all nuclei of the epithelium were stained by anti-DNase I. DNase I immunoreactivity was particularly concentrated in cells showing morphological signs of apoptosis, like nuclear fragmentation, and in many cases was found to persist in apoptotic bodies. DNase I gene transcripts were detected in control animals using dot and Northern blotting as well as RNase protection assay. After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5. Their exclusive localization within the epithelial cells was verified by in situ hybridization. Before castration, the DNase I gene transcripts were homogeneously distributed in all epithelial cells. At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology. Using the zymogram technique, a single endonucleolytic activity of about 32 kD was detected in tissue homogenates before castration. After androgen ablation, the endonucleolytic activity increased about four- to sevenfold up to day 3. At day 5, however, it had dropped to its original level. At day 1, three new endonucleolytic variants of higher molecular mass were expressed. At day 3, the predominant endonucleolytic activity exhibited an apparent molecular mass of 32 kD. Enzymatic analysis of the endonucleases present in prostate homogenates before and after castration demonstrated properties identical to DNase I. They were inhibited by chelators of divalent cations, Zn2+ ions and monomeric actin. Immunodepletion was achieved by immobilized antibodies specific for rat parotid DNase I. A polyclonal antibody raised against denatured DNase I was shown by Western blotting to stain a 32-kD band after enrichment of the endonuclease from day 0 and 3 homogenates by preparative gel electrophoresis. The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.
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PMID:Androgen ablation leads to an upregulation and intranuclear accumulation of deoxyribonuclease I in rat prostate epithelial cells paralleling their apoptotic elimination. 915 93

The precise quantification of rare mRNA copies from intronless genes by reverse transcription polymerase chain reaction (RT-PCR) requires the complete removal of genomic DNA because discrimination of cDNA and DNA amplification products by differing sizes of PCR products is not possible. Elimination of DNA is achieved by treating the RNA sample with RNase-free DNase I before RT-PCR. The lack of a PCR product from DNase-treated RNA samples before RT is usually accepted as a proof of efficient DNA destruction. However, this may vary depending on the metal cofactor used in the DNase I cleavage. Treating DNA-contaminated RNA samples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after additional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor, RT-created DNA fragments do not appear. This is because in the presence of manganese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended or that have protruding termini of only one or two nucleotides in length. However, overlapping fragments with the potential to recombine are created by DNase digestion with magnesium as cofactor. Because one cannot differentiate between a PCR signal produced by RNA and one produced by recombined DNA after DNase I digestion and RT, all such DNase I assays should be performed with manganese instead of magnesium.
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PMID:Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase I digestion. 918 63


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