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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The active forms of all of the matrix metalloproteinases (MMPs) are inhibited by a family of specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Inhibition represents a major level of control of MMP activity. A detailed knowledge of the mechanisms controlling TIMP gene expression is therefore important. We have isolated a genomic clone of the human TIMP-1 gene. A 3 kbp XbaI fragment has been sequenced; this fragment contains 1718 bp 5' flanking sequences, exon 1, a 929 bp intron 1 and part of exon 2. Computer analysis reveals 10 consensus sequences for Sp1, six for activating protein 1 (AP-1), six for polyoma enhancer A3 (PEA3), 12 for AP-2 and five CCAAT boxes. The region hybridizing with a murine TIMP-1 promoter fragment has been subcloned and analysed further. RNase protection identifies six transcription start points, making exon 1 up to 48 bp in length. Transient transfection of promoter-chloramphenicol O-acetyltransferase reporter constructs into primary human connective tissue fibroblasts shows that a 904 bp fragment that hybridizes to a murine TIMP-1 promoter fragment contains a functional promoter. Constructs of -738/+95 to -194/+21 are inducible with serum or phorbol ester to a similar extent to the endogenous TIMP-1 gene. These results and further mapping with 5' deletion mutants from the -738/+95 region have demonstrated that an AP-1 site at -92/-86 is essential for basal expression of the gene. Point mutations within this region have further confirmed the role of this site, along with a more minor role for a neighbouring PEA3 site, in basal expression. Deletions from the 3' end also implicate a region across the exon 1/intron 1 boundary and especially +21 to +58 in basal expression. The +21/+58 region contains a putative binding site for the transcription factor leader-binding protein 1 (LBP-1). Gel-shift analysis shows that protein binds specifically to this region, but competition studies suggest that it is unlikely to be LBP-1.
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PMID:Transcriptional activity of the human tissue inhibitor of metalloproteinases 1 (TIMP-1) gene in fibroblasts involves elements in the promoter, exon 1 and intron 1. 918 25

The locus control region (LCR) regulates transcription of the downstream beta-like globin genes 10 to 50 kb away. Among hypersensitive sites HS4, -3, -2, and -1, which define the LCR in erythroid cells, HS2 possesses prominent enhancer function. The mechanism by which the HS2 enhancer and other functional components of the LCR act over the distance is not clear. We have used reverse transcription-PCR and RNase protection assays to analyze the transcriptional statuses of both the endogenous and the transfected HS2 enhancer in erythroid K562 cells. A novel pattern of HS2 enhancer transcription was observed. The endogenous HS2 enhancer was transcribed predominantly in the direction toward the downstream globin genes. The HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids was also transcribed predominantly toward the CAT gene, regardless of whether the enhancer was placed (i) in the genomic or reverse genomic orientation, (ii) in a position 5' or 3' to the gene, or (iii) at various distances up to 6 kb from the gene. The orientation, position, and distance independence in gene-tropic transcription of the HS2 enhancer correlates with the observed orientation, position, and distance independence of HS2 enhancer function and suggests that enhancer transcription may play a role in enhancer function.
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PMID:Transcription of the HS2 enhancer toward a cis-linked gene is independent of the orientation, position, and distance of the enhancer relative to the gene. 919 30

Cis-acting regions regulating transcription of the alpha1(VI) collagen chain have been investigated in vitro by transfection of promoter-CAT (where CAT is chloramphenicol acetyltransferase) constructs in different types of cultured cells and in vivo in transgenic mice carrying the same CAT constructs or minigenes derived from the fusion of genomic and cDNA sequences in which small deletions of the collagenous domain had been engineered. 215 bp of 5'-flanking sequence showed promoter activity in vitro, yet were not expressed in any tissue of six transgenic lines, indicating that this fragment contains the basal promoter, but not activator sequences. Constructs with 0.6 and 1.4 kb of the 5'-flanking region produced significantly higher CAT activity in transfected cells and were expressed in tissues of about 30% of transgenic lines. Although CAT activity was totally unrelated to the pattern of expression of the alpha1(VI) mRNA, these results suggest the presence of an activator(s) between -0.2 and -0.6 kb from the transcription start site. When the promoter size was increased to 5.4 or 6.5 kb, CAT activity was stimulated severalfold relative to the construct p1.4CAT and p4.0CAT in NIH3T3 fibroblasts and chick embryo chondroblasts. This stimulation was, however, not observed in C2C12 myoblasts. Transgenic mice generated with 6.5CAT construct or minigenes, containing 6.2 kb of promoter, exhibited very high levels of expression, which was similar to the relative amount alpha1(VI) mRNA in the majority of tissues, with the exception of lung, adrenal gland and uterus. CAT activity in tissues was 100-1000-fold higher than that measured in transgenic mice with shorter promoter (0.6 or 1.4 kb). Since expression of minigenes was determined by RNase protection assay, the levels of mRNA per transgene copy were compared to those of the chromosomal gene and found to be always less than one quarter. These data suggest that the region -4.0/-5.4 contains an important activator(s) sequence which induces transcription in several, but not all, type VI collagen-producing tissues. Finally, analysis with the longest promoter fragment (7.5 kb) revealed a complex effect of the region -6.5/-7.5 on alpha1(VI) chain transcription. The sequence was inhibitory in NIH3T3 cells, indifferent in myoblasts and activating in chondroblasts in vitro, whereas transgenic animals generated with 7.5CAT construct produced a pattern of expression comparable to that of 6.5CAT and minigenes. During postnatal development transcription from both the endogenous gene and the transgenes decreased. However, the ratio of transgene/chromosomal gene expression was not constant, but varied in a way dependent on the tissue. This observation suggests that the fragment studied contains key sequences for the age-dependent regulation of the alpha1(VI) gene. No phenotypic alterations were induced by the presence of mutations in the minigenes.
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PMID:Tissue-specific expression of promoter regions of the alpha1(VI) collagen gene in cell cultures and transgenic mice. 924 27

Glutamine synthetase (GS) converts ammonia and glutamate into glutamine. We assessed the activity of the 5' regulatory region of the GS gene in developing transgenic mice carrying the chloramphenicol acetyltransferase (CAT) gene under the control of 3150 bp of the upstream sequence of the rat GS gene to obtain insight into the spatiotemporal regulation of its pattern of expression. To determine the organ-specific activity of the 5' regulatory region CAT and GS mRNA expression were compared by ribonuclease-protection and semi-quantitative in situ hybridization analyses. Three patterns were observed: the 5' region is active and involved in the regulation of GS expression throughout development (pericentral hepatocytes, intestines and epididymis); the 5' region shows no activity at any of the ages investigated (periportal hepatocytes and white adipose tissue); and the activity of the 5' region becomes repressed during development (stomach, muscle, brown adipose tissue, kidney, lung and testis). In the second group, an additional element must be responsible for the activation of GS expression. The last group included organs in which the 5' regulatory region is active, but not in the cells that express GS. In these organs, the activity of the 5' regulatory region must be repressed by other regulatory regions of the GS gene that are missing from the transgenic construct. These findings indicate that in addition to the 5' regulatory region, at least two unidentified elements are involved in the spatiotemporal pattern of expression of GS.
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PMID:Organ-specific activity of the 5' regulatory region of the glutamine synthetase gene in developing mice. 934 14

Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.
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PMID:Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1. 937 37

Multiple variant alleles of the human arylamine N-acetyltransferase genes, NAT1* and NAT2*, alter the capacity of individuals to metabolize arylamines by N-acetylation. Although biochemical and genetic studies have improved our understanding of the molecular basis of the acetylation polymorphism in humans and other mammals, regulation of NAT* gene expression is not understood. In the present study, a segment of the 5'-untranslated region of mouse Nat2* was sequenced and characterized. Primer extension analysis and RNase protection assays exposed multiple transcription initiation sites located 112 to 151 bases upstream of the translational start site. Computer sequence analysis revealed a promoter-like region located within the region 530 bases upstream of the translational start site consisting of TATA boxes, upstream promoter elements such as a CAAT box and Sp1 binding site, regulatory elements such as a palindromic hormone response element (HRE), and enhancer regions such as an AP-1 transcription factor binding site. Transient expression of CAT reporter constructs of the mouse Nat2*-palindromic HRE demonstrated positive regulation of the HSV-thymidine kinase 1 (tk1) promoter and induced the expression of chloramphenicol acetyltransferase (CAT). This induction was initiated by the addition of hormones such as 5alpha-dihydrotestosterone (DHT) or dexamethasone and was entirely dependent on the presence of androgen or glucocorticoid receptors, respectively. Together with recent discoveries regarding the effects of testosterone on the expression of Nat2* in mouse kidney during development, the findings reported in this article suggest that the HRE found in the promoter region of Nat2* is a potential candidate for the mediation of androgenic regulation of Nat2* in mouse kidney.
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PMID:Characterization of a hormone response element in the mouse N-acetyltransferase 2 (Nat2*) promoter. 957 94

UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) is essential for the normal assembly of complex Asn-linked glycans. Northern analysis showed a major transcript at 2.0 kb and a minor band at approximately 2.9 kb in five different human cell lines. The gene (MGAT2) has three AATAAA polyadenylation sites at 68, 688 and 846 bp downstream of the translation stop codon. 3'-RACE (rapid amplification of cDNA ends) using RNA from the human cell line LS-180 indicated that all three sites were utilized for transcription termination. 5'-RACE and RNase protection analyses showed multiple transcription initiation sites at -440 to -489 bp relative to the ATG translation start codon (+1). The data show that the entire GnT II gene is on a single exon. The gene has a CCAAT box at -587 bp but lacks a TATA box and the 5'-untranslated region is GC-rich and contains consensus sequences suggestive of multiple binding sites for Sp1; these properties are typical for housekeeping genes. A series of chimeric constructs containing different lengths of the 5'-untranslated region fused to the chloramphenicol acetyltransferase (CAT) reporter gene were tested in transient transfection experiments using HeLa cells. The CAT activity of the construct containing the longest insert (-1076 bp relative to the ATG start codon) showed a approximately 38-fold increase as compared to that of the control. Removal of the region between -636 and -553 bp caused a dramatic decrease in CAT activity indicating this to be the main promoter region of the gene.
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PMID:Transcriptional regulation of the human UDP-GlcNAc:alpha-6-D-mannoside beta-1-2-N-acetylglucosaminyltransferase II gene (MGAT2) which controls complex N-glycan synthesis. 957 8

Trophoblast cells are specialized extra-embryonic cells present only in eutherian mammals. They play a major role in the implantation and placentation processes. To understand better the molecular mechanisms that control the development and function of trophoblast cells, we sought to identify the transcription factors that regulate murine adenosine deaminase (ADA) gene expression in the placenta. Here we report a detailed characterization of a placenta-specific footprinting region (FP1) in the Ada placental regulatory element. The sequence of FP1 was mapped by DNase I footprinting and was found to match a consensus AP-2 transcription factor-binding site. Electrophoretic mobility shift assays demonstrated that FP1 interacted with AP-2-like proteins. Further analysis using AP-2 antibody confirmed that AP-2 protein was indeed present in the placenta and bound to FP1. Mutation at the AP-2 site in FP1 abolished the ability of the Ada placental regulatory element to bind AP-2 proteins and failed to target chloramphenicol acetyltransferase reporter gene expression to placentas in transgenic mice, indicating that AP-2 is required for Ada expression in the placenta. In addition, RNase protection assays demonstrated that AP-2gamma was the predominant AP-2 family member expressed in the placenta. In situ hybridization analysis revealed that AP-2gamma expression was enriched in the trophoblast lineage throughout development, suggesting that AP-2gamma may be critical for trophoblast development and differentiation.
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PMID:Transcription factor AP-2gamma regulates murine adenosine deaminase gene expression during placental development. 976 60

Androgen receptor (AR) plays a key role in cell growth both in the normal prostate and in prostate cancer. Androgen ablation and prolonged antiandrogen therapy can give rise to AR-dependent prostate tumors, which nonetheless can grow in the androgen-deprived milieu. Here we describe the ribozyme approach to selectively degrading the AR mRNA and thereby inhibiting AR function. A trans-acting hammerhead ribozyme was designed to cleave the rat AR mRNA at the position +1827/ 1828, a region predicted to be minimally involved in generating stable secondary structures. Using AR mRNA fragments as substrates, it was established that this ribozyme can specifically cleave the RNA target in a sequence-specific manner. Kinetic experiments determined a Km for the substrate of 77 nM and a kcat/Km value of 1.8 x 10(7) M(-1) x min(-1), suggesting a catalytic efficiency similar to that of protein enzymes such as the relatively nonspecific ribonuclease A and a sequence-specific endonuclease EcoRI. Transient cotransfections of prostate-derived PC3 cells with three plasmids, an AR-inducible chloramphenicol acetyltransferase (CAT) reporter, an AR expression vector, and a ribozyme expression vector, showed that the ribozyme was capable of reducing the functional activity of AR. At an equimolar ratio of the AR expression plasmid to ribozyme expression plasmid, androgen-inducible CAT activity was inhibited 70%. Similar extents of inhibition were also observed at the cellular mRNA level using ribonuclease protection assays, indicating that the ribozyme functioned as an AR mRNA cleaving enzyme in cellulo. Immunocytochemical examination revealed a decline of AR immunoreactivity in ribozyme-transfected cells. In addition, no morphologically detectable cellular abnormalities were associated with ribozyme expression, indicating the absence of deleterious side effects. These results offer a new avenue for the control of AR function and cell growth, especially in the case of androgen-resistant, but AR-dependent, prostate cancer cells.
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PMID:Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme. 977 79

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.
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PMID:Transcriptional regulation of the Bmp2 gene. Retinoic acid induction in F9 embryonal carcinoma cells and Saccharomyces cerevisiae. 988 May 12


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