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)

We have shown previously that 48 base pairs (bp) of 5'-flanking sequence are necessary for correct initiation at the major transcriptional start site of the Chinese hamster dihydrofolate reductase (dhfr) gene (Ciudad et al., 1988). As an upstream element, this sequence alone confers 25% of maximum promoter activity. The 5' half of this sequence is particularly well conserved among mammalian species; it contains one Sp1 binding site (GC box) and one CAA element. In the present work, we have analyzed the role of this region by extensive point mutational analysis. Twenty-three dhfr minigene constructs containing 1- or 2-base substitutions in this region of the promoter were tested by measuring their ability to transfect DHFR-deficient Chinese hamster ovary cells to a DHFR+ growth phenotype. Eight mutants, all in or near the GC box, exhibited reduced transfection efficiency. Promoter disfunction in these mutants was confirmed by RNase protection analysis of stable transfectants. Gel retardation experiments showed that mutants affected in the consensus sequence for Sp1 binding were deficient in binding a protein found in nuclear extracts of Chinese hamster ovary cells. Purified human transcription factor Sp1 was also unable to bind a promoter sequence bearing one of these single base substitutions, suggesting that Sp1 itself is involved in dhfr transcription in vivo. We conclude that most single base mutations in the GC box severely cripple or eliminate promoter function by inhibiting binding of transcription factors to this regulatory sequence and that Sp1 is likely to be involved in dhfr transcription in vivo. We also found that the well conserved CAA element is not absolutely necessary for transcription.
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PMID:Point mutational analysis of the hamster dihydrofolate reductase minimum promoter. 174 Apr 17

The carbamoylphosphate synthetase-aspartate transcarbamylase-dihydroorotase (CAD) gene encodes a tri-functional protein catalyzing the first three steps in de novo pyrimidine biosynthesis. Studies correlating CAD gene expression with cellular proliferation indicate the importance of understanding the regulation of the CAD gene. As a first step, the structure of the promoter region of the Syrian hamster CAD gene has been determined. Sequence analysis of 1671 base pairs of DNA revealed that the CAD promoter region is very GC rich. Primer extension analysis indicated that the transcription initiation site of the CAD gene is downstream from two GC boxes (consensus binding sites for the transcription factor Sp1). There is no TATA box appropriately spaced upstream from the transcription initiation site. Using RNase protection mapping, S1 nuclease analysis, and comparison to consensus splice donor/acceptor sites, the 5' end of the CAD gene has been determined to consist of a 241-base pair first exon, a 187-base pair first intron, a 140-base pair second exon, and a second intron that extends at least three kilobase pairs. Using conditions optimized for this GC-rich promoter, accurate transcription can be achieved in vitro. Analysis of CAD promoter deletions indicated that sequences extending only 114 base pairs upstream and 225 base pairs downstream from the transcription initiation site are sufficient for accurate and efficient transcription in vitro. DNase I footprinting reactions using this promoter fragment have identified three regions that bind proteins in a HeLa nuclear extract.
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PMID:Characterization of the 5' end of the growth-regulated Syrian hamster CAD gene. 198 61

The structural organization of the entire nuclear gene (NMDMC) encoding the mitochondrial (mt) NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase enzyme (NMDMC) was determined by analysis of clones obtained from a lambda EMBL3 murine genomic DNA library. The gene is approx. 13 kb in length and contains eight exons and seven introns. All exon/intron splice junctions follow the GT/AG rule. The amino acid presequence, which is essential for transport of the NMDMC enzyme precursor into mt, is encoded almost entirely in the first exon. Two major transcriptional start points (tsp), located 33 and 75 nucleotides upstream from the AUG start codon, were revealed by S1 nuclease mapping and RNase protection analyses. The immediate 5'-flanking region of the first exon contains one CAAT box, a TATA-like box and three sites homologous to the consensus sequence for the binding of transcription factor Sp1.
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PMID:Structural organization of the murine gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase. 199 93

Previously we isolated and characterized a differentially expressed gene from mouse 3T3-L1 preadipocytes that encodes stearoyl-CoA desaturase (SCD1; Ntambi, J. M., Buhrow, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J., Jr., and Lane, M. D. (1988) J. Biol. Chem. 263, 17291-17300). Genomic Southern blot analysis indicated the existence of another closely related gene. Here we report the isolation and characterization of this gene and the corresponding cDNA which encode a second stearoyl-CoA desaturase, SCD2, 3T3-L1 adipocytes. SCD2 cDNA is 5 kilobase pairs in length and encodes a protein of 358 amino acids with greater than 87% amino acid sequence identity to SCD1. RNase protection analysis reveals a 10-fold increase in the expression of SCD2 mRNA during 3T3-L1 preadipocyte differentiation. SCD2 mRNA is expressed constitutively at a high level in brain, is not expressed in liver, and its expression in kidney, adipose, and lung tissue is increased greatly by shifting mice from a diet containing unsaturated fatty acids to a diet devoid of fat. The tissue distribution and the dietary alteration of SCD1 mRNA expression differs markedly from that of SCD2 mRNA being absent from brain, constitutive in adipose tissue, and subject to negative control in liver by feeding a diet containing unsaturated fatty acids. The SCD2 gene spans approximately 15 kilobase pairs and consists of six exons and five introns, with intron/exon junctions similar to those of SCD1. As determined by primer extension analysis the start site of transcription maps 300 nucleotides upstream of the initiator methionine codon. Unlike the SCD1 gene, SCD2 lacks a typical "TATA" box in the 5'-flanking region, but has two "CCAAT" boxes at positions -90 and -135 relative to the transcription initiation site. The SCD2 promoter contains a 140-base pair sequence (located between nucleotides -54 and -201) which possesses 77% sequence identity to a region (located between nucleotides -472 and -325) in the SCD1 promoter. There is a GC-rich sequence in the SCD2 promoter (at nucleotide -175) similar to the binding site for the nuclear transcription factor Sp1 as well as an element with homology to the core consensus sequence for the glucocorticoid regulatory element position -500 and a potential CCAAT box/enhance binding protein sequence at position -540. The SCD gene family provides a new model system for the study of differentiation-induced as well as tissue-specific metabolite controlled gene expression.
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PMID:Differentiation-induced gene expression in 3T3-L1 preadipocytes. A second differentially expressed gene encoding stearoyl-CoA desaturase. 257 68

Deletion analysis of the 5' flank of the Chinese hamster dihydrofolate reductase (dhfr) gene reveals a promoter region starting 48 base pairs upstream of the major transcriptional start site. A dhfr minigene containing approximately 900 base pairs of 5' flank and one small intron was used as a wild-type standard. Seven deletions were created with BAL-31. Promoter activity was measured in three ways: 1) transient expression of the dhfr gene; 2) frequence of transfection of dhfr- Chinese hamster cells to a dhfr+ phenotype; and 3) RNase protection analysis of dhfr transcripts in pooled populations of permanently transfected cells. The transient expression assay was developed in this work for the rapid analysis of dhfr promoter mutants; this assay could be of general use for analyzing constructs carrying dhfr as a reporter gene. Two of the deletions define a requirement for part or all of the sequence GGGCGT located 48 base pairs upstream of the major transcriptional start site. This site has been shown to bind transcription factor Sp1 in the mouse dhfr gene. The function of the major promoter is independent of the function of the minor promoter. These minigene constructs also contain cryptic promoters located upstream of the natural start sites, probably in the plasmid vector. Transcripts originating from these upstream sites are inefficiently spliced, but do result in messenger RNA molecules that are translated into active dihydrofolate reductase.
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PMID:Deletion analysis of the Chinese hamster dihydrofolate reductase gene promoter. 318 92

The cathepsin D (cath-D) gene, coding for a ubiquitous lysosomal aspartyl protease, is overexpressed in aggressive human breast cancers, and its transcription is induced by estrogens in hormone-responsive breast cancer cells. We have determined the structure and function of the proximal 5' upstream region of the human cath-D gene from MCF7 cells. We show that the promoter has a compound structure with features of both housekeeping genes (high G+C content and potential transcription factor Sp1 sites) and regulated genes (TATAA sequence). By RNase protection assay, we show that transcription is initiated at five major transcription sites (TSSI to -V) spanning 52 base pairs. In hormone-responsive breast cancer cells, estradiol increased by 6- to 10-fold the level of RNAs initiated at TSSI, which is located about 28 base pairs downstream from the TATA box. The specific regulation by estradiol of transcription starting at site I exclusively was confirmed by primer extension. Moreover, the same estradiol effect was observed in the ZR75-1 cell line and in MDA-MB231 estrogen-resistant breast cancer cells stably transfected with the estrogen receptor. Site-directed mutagenesis indicated that the TATA box is essential for initiation of cath-D gene transcription at TSSI. In breast cancer biopsy samples, high levels of TATA-dependent transcription were correlated with overexpression of cath-D mRNA. We conclude that cath-D behaves, depending on the conditions, as a housekeeping gene with multiple start sites or as a hormone-regulated gene that can be controlled from its TATA box.
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PMID:Cathepsin D gene is controlled by a mixed promoter, and estrogens stimulate only TATA-dependent transcription in breast cancer cells. 841 24

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

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains three binding sites for the viral origin binding protein (OBP) flanked by transcriptional regulatory elements of the immediate-early genes encoding ICP4 and ICP22/47. To assess the role of flanking sequences in oriS function, plasmids containing oriS and either wild-type or mutant flanking sequences were tested in transient DNA replication assays. Although the ICP4 and ICP22/47 regulatory regions were shown to enhance oriS function, most individual elements in these regions, including the VP16-responsive TAATGARAT elements, were found to be dispensable for oriS function. In contrast, two oriS core-adjacent regulatory (Oscar) elements, OscarL and OscarR, at the base of the oriS palindrome were shown to enhance oriS function significantly and additively. Specifically, mutational disruption of either element reduced oriS-dependent DNA replication by 60 to 70%, and disruption of both elements reduced replication by 90%. The properties of protein-DNA complexes formed in gel mobility shift assays using uninfected and HSV-1-infected Vero cell nuclear extracts demonstrated that both OscarL and OscarR are binding sites for cellular proteins. Whereas OscarR does not correspond to the consensus binding site of any known transcription factor, OscarL contains a consensus binding site for the transcription factor Sp1. Gel mobility shift and supershift experiments using antibodies directed against members of the Sp1 family of transcription factors demonstrated the presence of Sp1 and Sp3, but not Sp2 or Sp4, in the protein-DNA complexes formed at OscarL. The abilities of OscarL and OscarR to bind their respective cellular proteins correlated directly with the efficiency of oriS-dependent DNA replication. Cooperative interactions between the Oscar-binding factors and proteins binding to adjacent OBP binding sites were not observed. Notably, Oscar element mutations that impaired oriS-dependent DNA replication had no detectable effect on either basal or induced levels of transcription from the ICP4 and ICP22/47 promoters, as determined by RNase protection assays. The Oscar elements thus appear to provide binding sites for cellular proteins that facilitate oriS-dependent DNA replication but have no effect on transcription of oriS-flanking genes.
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PMID:Cellular transcription factors enhance herpes simplex virus type 1 oriS-dependent DNA replication. 955 44

In this report, we examined the role of activin in the regulation of cell growth inhibition of human hepatocarcinoma cells. Using RNase protection assay for various cell cycle regulators and Western blotting experiments, we show that activin treatment of HepG2 cells leads to increased gene expression of the cyclin-dependent kinase inhibitor (CDKI) p15INK4B. Furthermore, transient co-transfection studies of the p15INK4B promoter/luciferase construct performed in HepG2 cells demonstrates that activin induction of the p15INK4B promoter is mediated through the Smad pathway. p15INK4B gene promoter mapping analysis revealed a 66-bp region within the proximal domain of the promoter, which contains a consensus site for the transcription factor Sp1, as critical for mediating the activin effect on p15INK4B gene expression. Finally, gel mobility shift experiments, using the Sp1 consensus site, revealed increased DNA binding of Sp1 in response to activin treatment of HepG2 cells, further confirming the involvement of Sp1 in activin-mediated p15INK4B gene promoter activation. Together, our data indicates an important role for the cyclin-dependent kinase inhibitor p15INK4B in activin-induced cell cycle arrest in liver cells.
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PMID:Activin induces hepatocyte cell growth arrest through induction of the cyclin-dependent kinase inhibitor p15INK4B and Sp1. 1509 10