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)

Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme and ion transport systems in eukaryotic cells. We have studied G-protein-coupled processes that appear to be developmentally regulated in polarized pig kidney cells (LLC-PK1). Following trypsinization, LLC-PK1 cells differentiate from a rounded cell type to a fully polarized epithelium by 7 days of culture. During this differentiation, the expression of G-protein alpha i-2 subunit mRNA was not detected until day 4 of culture, it peaked at day 6, and declined thereafter. In contrast, G-protein alpha s subunit mRNA which peaked on day 4 was easily detected on all culture days. The presence of the alpha i-2 protein on epithelial cell basolateral membranes followed the same pattern of mRNA expression during culture. To understand the developmental expression of the alpha i-2 subunit in non-polarized cells and its potential regulation by hormones and second messengers in polarized cells at the transcriptional level, genomic DNA segments encoding the alpha i-2 gene promoter were isolated from an EMBL-3 porcine genomic library. S1 nuclease analysis of LLC-PK1 mRNA with cRNA probes derived from these DNA segments revealed major and a minor transcriptional start sites 131 and 171 base pairs upstream of the translation initiation site. The porcine and human alpha i-2 subunit genes shared a 78% sequence identity in their 5' flanks which suggested an evolutionary conservation of cis elements required to influence their transcription. The porcine alpha i-2 gene promoter was identified by fusing DNA segments encoding putative 5'-flanking areas of the gene to a plasmid that contained a firefly luciferase reporter gene but lacked a promoter. The minimal promoter was found between -130 and -60 base pairs from the major transcription start site. No typical "TATA-like" sequences were found. However, a "GC" box and a "TGTGG" sequence were two potential cis elements required for basal transcription of the porcine gene promoter which shared a 76% sequence identity to the promoter of another GTP-binding protein, the human c-Ha-ras proto-oncogene. Transcription of the gene was inhibited following treatment of renal cells with 10(-8) M dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of the G-protein alpha i-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter. 189 94

A bovine cDNA probe for the type I regulatory subunit of the cAMP-dependent protein kinase [Lee et al. (1983) Proc. Natl Acad. Sci. USA 80, 3608-3612] was used to screen two lambda gt11 libraries constructed from poly(A)-rich RNA from the porcine kidney cell line, LLC-PK1. A series of overlapping clones were isolated and characterized. The largest clone, lambda RI15, of 1426 bp was found to code for the entire RI protein but was apparently missing the 3' end of the mRNA. The porcine cDNA codes for a protein of 389 amino acids that shows 99% homology to bovine RI and hybridizes to two major mRNA transcripts of approximately 2.0 kb and 4.5 kb from LLC-PK1 cells. The porcine cDNA for RI was used to screen a genomic library of LLC-PK1 DNA constructed in the EMBL-3 vector and several clones were isolated and characterized. By using a probe from the 5' end of the RI cDNA we isolated the 5' end of the gene and 700 bp of the promoter region of the gene were sequenced. The promoter region lacks a characteristic TATA box but contains two inverted CAAT boxes and is rich in G + C residues. Several sequence motifs were identified in the 5' promoter region which could be responsible for the regulation of synthesis of this gene. Multiple transcription initiation sites were identified by S1 nuclease mapping.
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PMID:Isolation of a cDNA and characterization of the 5' flanking region of the gene encoding the type I regulatory subunit of the cAMP-dependent protein kinase. 304 Apr

The gene encoding the calcitonin receptor (CTR) was isolated from a porcine kidney epithelial cell line (LLC-PK1) genomic library and found to span approximately 70 kilobases. Analysis of the gene sequence revealed that the CTR mRNA encompasses 14 exons with 12 exons encoding the protein. Two splicing acceptor sites separated by 48 nucleotides were found in intron 7. The expression of two mRNA species in LLC-PK1 cells was subsequently confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing. In LLC-PK1 cells the mRNA encoding the shorter CTR (CTR-1a) is approximately 1,000 times more abundant than the longer variant (CTR-1b), as estimated by the competitive RT-PCR. The transcription initiation site of the CTR gene was mapped by primer extension, S1 nuclease, and RT-PCR analysis. The proximal promoter region of 500 base pair is GC-rich (66%) and CpG-rich (CpG/GpC ratio 0.71). Transient transfection of CTR gene promoter-luciferase chimeras in LLC-PK1 cells led to the expression of luciferase activity. The CTR gene was mapped to chromosome band 9q11-q12.
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PMID:Isolation, characterization, and chromosomal localization of the porcine calcitonin receptor gene. Identification of two variants of the receptor generated by alternative splicing. 803 23

The activity of the apical membrane Na+/H+ exchanger NHE3 isoform of renal or intestinal epithelial cells is chronically regulated by a wide variety of stimuli, including acidosis, cAMP, glucocorticoids, and thyroid hormone. To understand the molecular mechanisms responsible for long term regulation of this cation transporter, we have isolated and determined the structure of this gene from a rat genomic library. The Nh3 gene spans > 40 kilobases and contains 17 exons that are flanked by typical splice donor and acceptor sequences at the exon-intron boundaries. The transcription initiation site was mapped by S1 nuclease protection analyses of mRNA from rat kidney and intestine. Multiple start sites were clustered between nucleotides -100 and -96 relative to the translation initiation codon. An atypical TATA-box and CCAAT-box are centered 30 and 147 nucleotides, respectively, upstream of the predominant transcription initiation site. Sequence analysis of approximately 1.4 kilobases of the 5'-flanking promoter region also revealed the presence of other putative cis-acting elements recognized by various transcription factors (e.g. AP-1, AP-2, C/EBP, NF-I, OCT-1/OTF-1, PEA3, Sp1, glucocorticoid, and thyroid hormone receptors), some of which may participate in the chronic regulation of this gene. The glucocorticoid responsiveness of the Nhe3 gene was assessed by fusing its 5' regulatory region to the firefly luciferase reporter gene and then by measuring the expression of the chimeric gene in transiently transfected renal epithelial OK and LLC-PK1 cells. Glucocorticoid treatment significantly increased the luciferase activity of the chimeric gene in both cell lines, thereby indicating that glucocorticoid regulation of Nhe3 is mediated primarily by a transcriptional mechanism.
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PMID:Genomic organization and glucocorticoid transcriptional activation of the rat Na+/H+ exchanger Nhe3 gene. 863 55