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:3.1.30.1 (S1 nuclease)
3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Long-term memory for sensitization in Aplysia requires new protein and RNA synthesis. Here, we identify a late protein as calreticulin, the major Ca(2+)-binding protein of the lumen of the endoplasmic reticulum. An antiserum against Aplysia calreticulin reveals an enrichment of calreticulin immunoreactivity in presynaptic varicosities. Quantitative S1 nuclease analysis indicates that the steady-state level of calreticulin mRNA in Aplysia sensory neurons increases during the maintenance phase of long-term sensitization. The finding that this mRNA increases in expression late, some time after training, is consistent with the idea that long-term neuromodulatory changes underlying sensitization may depend on a cascade of gene expression in which the induction of early regulatory genes leads to the expression of late effector genes.
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PMID:Long-term sensitization training in Aplysia leads to an increase in calreticulin, a major presynaptic calcium-binding protein. 146 4

Egasyn (esterase-22), a member of the nonspecific carboxylesterase multigene family (E.C. 3.1.1.1), is the endoplasmic reticulum (ER)-targeting protein of beta-glucuronidase. We utilized the polymerase chain reaction (PCR) in the eventual isolation of murine egasyn cDNAs. PCR primers were based upon: (1) partial amino acid sequences derived from egasyn peptides and (2) a conserved active site region shared by carboxylesterases. The amino acid sequence deduced from the PCR product matched that obtained from egasyn protein. This product was utilized as a probe to screen a cDNA library. Two cDNAs whose composite sequence encoded an open reading frame of 562 amino acids were isolated. A message size of 1700-2000 bp was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses detected mRNA in liver, kidney, lung, and submandibular gland, but not in spleen, brain, and testes. Genetic mapping confirmed the location of an egasyn cDNA fragment in cluster 1 of the esterase region on chromosome 8. Transfection of COS cells with the 2022-bp cDNA resulted in the expression of esterase activity, which comigrated on native gels with liver esterase-22. The features of the deduced amino acid sequence of the egasyn cDNA are compared with previously characterized carboxylesterases and with other lumenal ER proteins.
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PMID:Characterization and functional expression of a cDNA encoding egasyn (esterase-22): the endoplasmic reticulum-targeting protein of beta-glucuronidase. 178 3

The biosynthesis of estrogens from androgens is catalyzed by a enzyme of the endoplasmic reticulum termed aromatase cytochrome P-450 (P-450AROM). The gene encoding P-450AROM was isolated in our laboratory utilizing a full-length P-450AROM cDNA and a primer-extended cDNA obtained from human placental libraries as probes. We have found that the P-450AROM gene spans at least 75 kilobases and the region encoding the P-450AROM protein is comprised of nine exons. In addition, there are at least two untranslated exons, I.1 and I.2, upstream of which are found putative promoter sequences thought to be responsible for expression of P-450AROM in placenta. To determine if these promoters are utilized to regulate P-450AROM expression in adipose tissue, we have used polymerase chain reaction technology in an attempt to amplify the untranslated exons out of human adipose total RNA. The untranslated exons could not be amplified out of adipose RNA although they could be amplified out of placental RNA. When oligonucleotides corresponding to these untranslated exons were used in Northern analysis of RNA from human adipose stromal cells, no hybridizable mRNA species was detectable. Putative promoter sequences 326 and 110 base pairs (bp) upstream of the 5' end of exon II were evaluated as adipose P-450AROM promoters by primer extension analysis and S1 nuclease protection assays. Both methods suggest a start site of transcription 26 bp down-stream of the TATAAA sequence located 110 bp from the placental intron-exon II junction. These results indicate that tissue-specific regulation of aromatase activity in the human is achieved in part by the use of alternative transcriptional start sites and tissue-specific promoters.
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PMID:Tissue-specific expression of human P-450AROM. The promoter responsible for expression in adipose tissue is different from that utilized in placenta. 204 Jun 33

The cellular glucose-regulated protein GRP78-BiP is a member of the HSP70 stress family of gene products, and the protein is a resident component of the endoplasmic reticulum, where it is thought to play a role in the folding and oligomerization of secretory and membrane-bound proteins. GRP78-BiP also binds to malfolded proteins, and this may be one mechanism for preventing their intracellular transport. An induction in synthesis of the GRP78-BiP protein occurs in cells infected with paramyxoviruses (R. W. Peluso, R. A. Lamb, and P. W. Choppin, Proc. Natl. Acad. Sci. USA 75:6120-6124, 1978). We have studied the expression and activity of the GRP78-BiP gene and synthesis of the GRP78-BiP protein during infection with the paramyxovirus simian virus 5 (SV5). We wished to identify the viral component capable of causing activation of GRP78-BiP since GRP78-BiP interacts specifically and transiently with the SV5 hemagglutinin-neuraminidase (HN) glycoprotein during HN folding (D. T. W. Ng, R. E. Randall, and R. A. Lamb, J. Cell Biol. 109:3273-3289, 1989). Expression of cDNAs of the SV5 wild-type HN glycoprotein and a mutant form of HN that is malfolded but not the SV5 F glycoprotein or SV5 cytoplasmic proteins P, V, and M caused increased amounts of GRP78-BiP mRNA to accumulate, as detected by nuclease S1 protection assays. As unfolded or malfolded forms of HN cannot be detected to accumulate during SV5 infection, the data suggest that the flux of HN through the ER in SV5-infected cells can cause activation of GRP78-BiP transcription.
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PMID:Flux of the paramyxovirus hemagglutinin-neuraminidase glycoprotein through the endoplasmic reticulum activates transcription of the GRP78-BiP gene. 204 Oct 85

Based upon the deduced amino acid sequence of a cDNA (cDNA-H4) that had been proposed to encode the peptide core of an eosinophil and a HL-60 cell secretory granule proteoglycan, a 16-amino acid peptide was synthesized. This peptide was then used to elicit rabbit antibodies for study of the translation and post-translational modification of this gene product in hematopoietic cells. When HL-60 cells were radiolabeled for 2 min with [35S]methionine, a protein that migrated in a sodium dodecyl sulfate-polyacrylamide electrophoresis gel with a Mr of 20,000 was immunoprecipitated with the IgG fraction of the anti-peptide serum. Kinetic experiments revealed that within 10 min this radiolabeled precursor protein was converted in HL-60 cells into an Mr approximately 150,000 chondroitin sulfate proteoglycan intermediate. After a 20-min to 1-h chase, this [35S]methionine- or [35S]sulfate-labeled proteoglycan intermediate lost its antigenicity, presumably due to proteolysis of its N terminus. A human genomic library was probed under conditions of high stringency with cDNA-H4 to isolate genomic clones that contain the gene that encodes this proteoglycan peptide core. This gene spans approximately 15 kilobases and consists of three exons. The first exon encodes the 5'-untranslated region of the mRNA transcript, as well as the entire 27-amino acid signal peptide of the translated molecule. The second exon encodes a 49-amino acid region of the peptide core, predicted to be the N terminus of the molecule after its proteolytic processing in the endoplasmic reticulum. The third exon encodes the remainder of the molecule, including its glycosaminoglycan attachment, serine-glycine repeat region. As assessed by S1 nuclease mapping and primer extension analysis, the transcription-initiation site in HL-60 cells for this gene resides 53 base pairs upstream from the translation-initiation site.
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PMID:Characterization of the human gene that encodes the peptide core of secretory granule proteoglycans in promyelocytic leukemia HL-60 cells and analysis of the translated product. 218 Sep 35

We have isolated and sequenced full-length cDNA clones from a rabbit uterine library which encode the smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. These cDNAs resulted from an alternative splice of the cardiac/slow-twitch Ca2+-ATPase gene transcript, and encoded a protein identical to rabbit cardiac/slow-twitch Ca2+-ATPase except for the replacement of the carboxyl-terminal four amino acids with an extended and relatively hydrophobic sequence of 49 amino acids. This cDNA was virtually identical to the alternatively spliced product of the cardiac/slow-twitch Ca2+-ATPase gene recently identified in human kidney (Lytton, J., and MacLennan, D. H. (1988) J. Biol. Chem. 263, 15024-15031) and rat non-muscle tissues (Gunteski-Hamblin, A.-M., Greeb, J., and Shull, G. (1988) J. Biol. Chem. 263, 15032-15040). S1 nuclease mapping of total cellular RNA from a variety of tissues demonstrated that cardiac muscle expressed the cardiac/slow-twitch isoform almost exclusively, most smooth muscle and non-muscle tissues expressed the alternatively spliced smooth/non-muscle isoform almost exclusively, and a few tissues expressed both isoforms in varying amounts. Thus, regulation of alternative splicing of the cardiac/slow-twitch Ca2+-ATPase gene transcript is tissue-specific. The expression of the smooth/non-muscle isoform in every tissue tested supports the hypothesis that this molecule represents the "housekeeping" endoplasmic reticulum Ca2+-ATPase.
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PMID:Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. 252 89

A mouse liver genomic library was probed with a 450-base pair AccI----3' gene-specific fragment of a mouse bone marrow-derived mast cell proteoglycan cDNA to isolate 15-18-kilobase (kb) genomic clones containing the gene that encodes the peptide core of mouse secretory granule proteoglycans. Based on the nucleotide sequences of its 2.0-3.5-kb subcloned fragments, this mouse gene consists of three exons. The first exon contains 41 base pairs of untranslated nucleotides that are present in the 5' region of the transcript and also encodes the hydrophobic 25-amino acid signal peptide. The second exon encodes a 48-amino acid sequence that would be predicted to be the N terminus of the peptide core after the signal peptide has been removed in the endoplasmic reticulum. The third exon encodes a 79-amino acid sequence that includes the 15 amino acids immediately preceding an alternating serine-glycine 21-amino acid sequence for glycosaminoglycan attachment, and the subsequent C-terminal 43 amino acids; this exon also contains the 424 untranslated nucleotides present in the 3' region of the transcript. Primer extension and S1 nuclease protection analyses were performed to determine the transcription initiation site of the mouse gene. Rat-1 fibroblasts were cotransfected with the selectable marker pSV2 neo and a lambda clone (lambda MG-PG1) to obtain two rat-1 fibroblast cell lines that had the mouse secretory granule proteoglycan gene integrated into their genomes. RNA blot analysis of both cell lines revealed the presence of the 1.0-kb secretory granule proteoglycan peptide core mRNA transcript, indicating that lambda MG-PG1 contained the entire mouse secretory granule proteoglycan peptide core gene including some of the regulatory elements in its promoter region. The gene that encodes the peptide core of mouse secretory granule proteoglycans is the first proteoglycan gene to have its complete exon/intron organization determined and to be transfected and expressed in another cell type.
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PMID:Cloning and characterization of the mouse gene that encodes the peptide core of secretory granule proteoglycans and expression of this gene in transfected rat-1 fibroblasts. 277 4

Ehlers-Danlos syndrome type IV (EDS IV) is an autosomal dominant disorder characterized by fragile skin, blood vessels, and internal organs and associated with decreased production, secretion, or thermal stability of type III procollagen. Mutations in the gene for type III procollagen have been identified in patients exhibiting decreased secretion or thermal stability of the protein, but no defect has been elucidated to explain the decreased production of type III procollagen in some patients with EDS IV. We report on a patient with a moderate case of EDS IV who produced decreased amounts of type III procollagen despite normal levels of translatable type III procollagen mRNA. S1 nuclease analysis of the type III procollagen mRNA indicated a defect in the region encoding exon 27. Sequence analysis of cDNA clones and genomic fragments generated by polymerase chain reaction amplification revealed that sequences encoded by exon 27 were absent from 3 out of 5 cDNA clones and that a G at the +5 position of the splice donor site in intron 27 was changed to an A in one allele of the patient's type III procollagen gene. Using a cDNA-genomic DNA hybrid probe in S1 nuclease analysis, fragments consistent with mRNA species containing and lacking exon 27 were detected in a 1:1 ratio. Pulse label and chase experiments in the presence or absence of brefeldin A indicated that most of the type III procollagen molecules synthesized by the patient's fibroblasts were not secreted into the medium but were degraded in the endoplasmic reticulum-Golgi compartment by a nonlysosomal mechanism.
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PMID:Aberrant splicing of the type III procollagen mRNA leads to intracellular degradation of the protein in a patient with Ehlers-Danlos type IV. 758 95

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.
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PMID:A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. 809 58

Cathepsin W is a member of the papain-like family of cysteine proteases. In this report, we have isolated the cDNA for murine CtsW (mCtsW) from a splenocyte library. The deduced 371-amino-acid sequence shares 68% identity with human CtsW and includes the conserved catalytic triad cysteine, histidine, and asparagine found in all members of this family. In addition to the fulllength form of mCtsW, we have isolated an alternatively spliced form of the mRNA that lacks a complete catalytic triad. An S1 nuclease protection assay and a Western blot analysis showed that mCtsW is mainly restricted to the CD8(+) T cell and natural killer cell compartments. In addition, we confirmed that, like its human homologue, mCtsW is localized mainly to the endoplasmic reticulum and its expression is up-regulated upon activation. We also characterized the mCtsW locus using bacterial artificial chromosome clones. The gene consists of 10 coding exons and 9 introns spanning 3.2 kb. To elucidate the physiologic role of this protease, we generated mice deficient in mCtsW. Our data establish that mCtsW is not required for cytotoxic lymphocyte-induced target cell death in vitro. In addition, mCtsW deficiency does not alter the susceptibility of cytotoxic lymphocytes to suicide or fratricide after degranulation. Thus, mCtsW does not have a unique role in target cell apoptosis or cytotoxic cell survival in vitro.
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PMID:Characterization of murine cathepsin W and its role in cell-mediated cytotoxicity. 1508 52


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