Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glucosidase I initiates the processing of asparagine (N-) linked glycoproteins by removing the distal alpha1,2-linked glucosyl residue of the tetradecasaccharide Glc(3)Man(9)GlcNAc(2). The gene encoding this enzyme was isolated and its structural organization and promoter activity determined. The major transcript for glucosidase I on northern blot appeared to be 3.1 kb; Southern blotting and DNA sequencing indicated the size of the gene to be 6.8 kb, comprising four exons separated by three introns. The first exon encodes the cytoplasmic tail and transmembrane domain; the fourth encodes the putative catalytic domain of the enzyme. Exon-intron junctions are flanked by consensus splice donor and acceptor sequences. Transcription initiation sites were mapped by primer extension, ribonuclease protection assay and RT-PCR analysis. Primer extension results showed multiple initiation sites at -150, -156, and -272 bp relative to the translation initiation codon ATG. Sequence analysis of 5' flanking region showed no canonical TATA box, a high GC content, Sp1 and ETF binding sites (typical of a housekeeping gene promoter). Also noteworthy, the promoter region contains several generic STAT factor binding sites, one nearly perfect, and two half GR binding elements. Other cis- acting elements recognized by transcription factors such as AP-2, NF-kappaB, estrogen receptor, and progesterone receptor (PR) were also present in the putative promoter region. To determine the promoter activity, a construct encompassing the region between -2114 to -5 bp of the putative promoter was ligated to the chloramphenicol acetyltransferase (CAT) reporter plasmid and transiently transfected into COS 7 cells. CAT assay results clearly show transcriptional activity of the promoter.
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PMID:Genomic organization and promoter activity of glucosidase I gene. 1040 45

Regulation of pituitary vasopressin V1b receptors plays a critical role in regulating pituitary adrenocorticotropic hormone (ACTH) secretion during adaptation to stress. The objective of this study was to isolate the promoter regulatory region of the V1b receptor gene to better understand the molecular mechanisms involved in V1b receptor regulation. Screening of a rat genomic library using probes directed to the coding region and to the 5'UTR of the rat V1b receptor resulted in the isolation of several clones containing the 5'upstream regions of the V1b receptor cDNA. Sequencing of an 11.2 Kb fragment revealed 8.2 Kb upsteam of the reported cDNA sequence, which contains a putative promoter regulatory region. The 3' end of the clone contained 1472 base pairs corresponding to the recognized cDNA sequence, followed by 1506 bp of unknown sequence located at the end of the sixth transmembrane domain, probably corresponding to an intron, characteristic of these family of receptors. An additional 161 bp intron was found in the 5'UTR, similar to that described in the rat oxytocin receptor gene. 5'RACE and RNase protection analysis mapped two major putative transcription start points at -830 and -861 bp from the starting methionine. Analysis of the putative promoter region showed no indication of a proximal TATA box, but the presence of a CACA box, a GAGA box, several AP-1 and AP-2 sites and a cluster of Sp1 sites upstream of the AP-2 sites. A luciferase construct containing a 2.1-kb of putative promoter, and part of the 5'UTR including the first intron, showed promoter activity when transfected into COS-7, CHO and PC12 cell lines but not in AtT-20 cells. A similar construct without the intron and distal 5'UTR sequence has no promoter activity in the same cell lines. In summary, the V1b receptor gene contains at least 3 exons and 2 introns. The 5'flanking sequence contains several potential sites for transcriptional regulation, and induced luciferace activity only in constructs containing intron 1, suggesting that the latter is important for receptor gene activation. The data provide bases for future analysis of the regulatory elements controlling V1b receptor transcription.
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PMID:Isolation and characterization of the promoter region of the rat vasopressin V1b receptor gene. 1079 83

Follistatin is a secreted protein, which functions as an antagonist of different members of the TGF-beta superfamily, including activin and bone morphogenetic proteins. Expression of follistatin is tightly regulated during mouse development both spatially and temporally. In order to study the regulation of follistatin expression in the mouse embryo we have cloned and analyzed part of the 5' flanking region of the murine follistatin gene. Primer extension and RNase protection assays demonstrate that the murine follistatin promoter region has at least three distinct transcription initiation sites, which are each preceded by a TATA box. All of the transcription initiation sites are located within the first 500 bp upstream of the translational start site. Sequence analysis of this 500 bp region revealed several consensus binding sites for transcription factors including AP-1, Brachyury-T, CREB, Sp1, AP-2 and Tcf. To test whether the 5' region displays promoter activity, we transfected various 5' flanking region deletion constructs into F9 embryonal carcinoma (EC) cells and into P19 EC cells. In these two cell lines a region of only 262 bp upstream of the translation start site could drivereporter expression in a manner that reflects endogenous mRNA expression.
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PMID:Cloning and analysis of the mouse follistatin promoter. 1125 2

Two cDNA clones (OlGC2 and OlGC7) and their genomic DNA clones encoding medaka fish homologs of mammalian natriuretic peptide receptor/membrane guanylyl cyclase A (GC-A) were isolated, and their complete nucleotide sequences were determined. The open reading frame predicts a protein of 1,063 amino acids for OlGC2 cDNA (4,283 bp), and one of 1,055 amino acids for OlGC7 cDNA (3,721 bp), respectively. Northern blot analyses demonstrated 4.7 kb OlGC2 transcripts in the kidney and gill, and 4.0 kb OlGC7 transcripts in the kidney, brain, and ovary, while RNase protection analyses revealed that both genes are expressed in various adult organs. Both the OlGC2 (about 33.0 kbp) and OlGC7 (about 44.3 kbp) genes consist of 22 exons with an exon/intron organization similar to those of the human GC-A gene (about 16.6 kbp) and medaka fish GC-B homolog gene (OlGC1, about 93 kbp). Intron 4 of OlGC2 contains two repeated sequence (RS) clusters, designated as RS1 (about 1 kbp) and RS2 (about 5 kbp), consisting of nucleotide 5'-AGCCTCTGCTCCTCCTTC-3'. In addition, many identical but variably sized nucleotide sequences were found in introns in OlGC1, OlGC2, OlGC6, and OlGC7. The OlGC2 and OlGC7 genes both have no apparent TATA box in the 5' flanking region upstream of the putative transcription initiation point, but several consensus sequences for cis-regulatory elements, including C/EBP, CREB, NF-IL6, and Sp1 and AP-2, NF-IL6, c-Myb, and Sp1 are present in the 5'-flanking region of OlGC2 and OlGC7, respectively.
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PMID:Expression and exon/intron organization of two medaka fish homologs of the mammalian guanylyl cyclase A. 1143 78

Onconase is an RNase with a very specific property because it is selectively toxic to transformed cells. This toxin is thought to recognize cell surface receptors, and the protection conferred by metabolic poisons against Onconase toxicity indicated that this RNase relies on endocytic uptake to kill cells. Nevertheless, its internalization pathway has yet to be unraveled. We show here that Onconase enters cells using AP-2/clathrin-mediated endocytosis. It is then routed, together with transferrin, to the receptor recycling compartment. Increasing the Onconase concentration in this structure using tetanus toxin light chain expression enhanced Onconase toxicity, indicating that recycling endosomes are a key compartment for Onconase cytosolic delivery. This intracellular destination is specific to Onconase because other (and much less toxic) RNases follow the default pathway to late endosomes/lysosomes. Drugs neutralizing endosomal pH increased Onconase translocation efficiency from purified endosomes during cell-free translocation assays by preventing Onconase dissociation from its receptor at endosomal pH. Consistently, endosome neutralization enhanced Onconase toxicity up to 100-fold. Onconase translocation also required cytosolic ATP hydrolysis. This toxin therefore shows an unusual entry process that relies on clathrin-dependent endocytic uptake and then neutralization of low endosomal pH for efficient translocation from the endosomal lumen to the cytosol.
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PMID:Intracellular pathway of Onconase that enables its delivery to the cytosol. 1737 40


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