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)

Using a transient expression assay, we have analysed the effect of novobiocin, DNA topoisomerase II inhibitor, on simian virus 40(SV40) enhancer activities. We used the recombinant clones containing type I or II collagen promoters placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene with or without SV40 enhancer. We observed the expected increase in CAT activities due to the presence of the SV40 enhancer. Interestingly, CAT gene expression of the enhancer-containing constructs were inhibited more sensitively by novobiocin than that of the enhancer-less construct. This findings lead us propose that DNA superhelicity mediated by topoisomeraseII is one of the important factor for the manifestation of SV40 enhancer activity.
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PMID:Novobiocin inhibits the SV40 enhancer activity. 284 73

Two overlapping clones spanning 19 kilobase pairs (kb) of the 5' end of the alpha 1 (IV) collagen gene were isolated and found to contain a single exon which encoded the 5'-untranslated sequence and 84 base pairs of the signal peptide. The 5' end of this exon was determined to be the 5' end of the transcript by S1 nuclease protection and primer extension. The nucleotide sequence of 1 kb of the 5'-flanking DNA was extremely G + C-rich (greater than 70%) and contained two GC boxes and a putative cAMP regulatory sequence. The transcriptional regulation of the alpha 1 (IV) gene was studied with chimeric gene constructs utilizing 2.5 kb of the 5'-flanking sequence coupled to the gene for chloramphenicol acetyltransferase. Transfection of this construct into differentiating F9 cells resulted in low chloramphenicol acetyltransferase activity compared to beta-actin or Rous sarcoma virus long terminal repeat promoters, although these cells produce large amounts of collagen IV. Inclusion of a 2.7-kb sequence 2.3 kb downstream from the first exon in either orientation increased the transcription of the chloramphenicol acetyltransferase construct approximately 10-fold in F9 cells, but was not active in NIH 3T3 cells, which synthesize little collagen IV. These results indicate the presence of an enhancer within the first intron, which increases the expression of this gene.
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PMID:Characterization of the promoter for the alpha 1 (IV) collagen gene. DNA sequences within the first intron enhance transcription. 284 28

The human basement membrane specific collagen type IV is a heterotrimer composed of two alpha 1(IV) chains and one alpha 2(IV) chain. A partial genomic EcoRI library was screened with cDNA clones representing the 5' end regions of the alpha 1(IV) and the alpha 2(IV) mRNA. A 2.2-kb genomic fragment was isolated and sequenced, which contains the 5' terminal exons of both genes located in close vicinity. The two genes were found to be arranged in opposite direction, head-to-head, separated only by a short region of 127 bp, apparently representing promoters of both genes as indicated by the existence of typical sequence motifs (CAT-box, SP1 consensus sequence). These data suggest that the alpha 1(IV) and alpha 2(IV) genes use a common, bidirectional promoter. The striking symmetrical arrangement of sequence elements within the promoter may be of basic importance for the coordination of bidirectional transcription. The promoter region had no detectable transcriptional activity in transient gene expression assays after fusion to the chloramphenicol acetylase (CAT) gene in either direction, indicating the necessity of additional elements for efficient and tissue-specific expression of both genes. Constructs containing different segments of both genes failed to identify regions with enhancing activity for the homologous collagen type IV promoter. When the heterologous HSV thymidine kinase promoter was used, a negatively acting region was identified. This indicates that the alpha 1(IV) and alpha 2(IV) promoter activity is controlled by additional regulatory elements present on distant portions of both genes.
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PMID:The genes for the alpha 1(IV) and alpha 2(IV) chains of human basement membrane collagen type IV are arranged head-to-head and separated by a bidirectional promoter of unique structure. 284 80

We present evidence that the fos oncogene encodes a transcriptional trans-activation function. trans-activation was assayed by cotransfection into NIH 3T3 mouse fibroblasts of v-fos DNA containing plasmids together with a plasmid containing a test promoter. Three v-fos DNAs were used: (i) pFBR-1, a plasmid containing the FBR proviral sequences; (ii) pFBJ-2, a plasmid harboring the FBJ proviral sequences; (iii) pMF-J, a plasmid containing the FBJ fos sequences linked to a mouse metallothionein promoter. Each of the three v-fos DNA plasmids stimulated the expression of a cotransfected chimeric gene consisting of a promoter segment of the mouse alpha 1(III) collagen gene linked to the gene for chloramphenicol transacetylase. In similar experiments the v-fos gene also stimulated the long terminal repeat promoter of Rous sarcoma virus (RSV) but neither the early promoter of simian virus 40 nor the beta-actin promoter. Evidence that the trans-activation function is specified by the v-fos coding sequences comes from the fact that a frameshift mutation in the v-fos coding sequence inhibits the trans-activation. Two mutations that map around nucleotide -100 in the RSV promoter do not respond to cotransfection with v-fos, whereas other mutations respond like the wild-type RSV promoter. These experiments suggest that the v-fos gene either encodes or induces an activator of transcription that recognizes specific sequences in promoters.
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PMID:Transcriptional activation encoded by the v-fos gene. 301 Feb 84

A chimeric gene was constructed by fusing the DNA sequences containing the 5' flanking region of the mouse alpha 1(III) collagen gene to the coding sequence of the bacterial chloramphenicol acetyltransferase (CAT) gene. Transient transfection experiments indicated that the alpha 1(III) promoter is active in NIH 3T3 fibroblasts and BC3H1 smooth muscle cells. The activity of the alpha 1(III) collagen promoter-CAT plasmid is stimulated approximately ten fold by the presence of the SV40 enhancer element. Removing sequences upstream of -200 stimulates the activity of the chimeric gene eight fold. Further deletion analysis identified sequences located between -350 and -300 that were instrumental in repressing the activity of the promoter. This 50 bp region contains a direct repeat sequence that may be involved in the regulation of the mouse alpha 1(III) collagen gene. Truncating the alpha 1(III) promoter to -80 further stimulated expression. We propose that the positive regulatory elements of this gene appear to be located within the first 80 bp of the promoter, whereas elements located further upstream exert a negative effect on the expression of the gene. Regulation of the alpha 1(III) gene contrasts with that of the alpha 2(I) collagen gene, which appears to be regulated by several positive elements located in various regions of the promoter.
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PMID:Deletion analysis of the mouse alpha 1(III) collagen promoter. 341 93

Eight transgenic mice were generated in which the promoter of the mouse alpha 2(I) collagen gene (nucleotides -2000 to +54), linked to the bacterial gene for chloramphenicol acetyltransferase (CAT), is stably integrated in the germ line. These strains contain from 1 to 20 copies of the alpha 2(I) collagen-CAT chimeric gene per haploid genome. In seven of the eight strains, the CAT gene is expressed, although the levels of CAT enzyme activity vary considerably from one strain to the other. In six of these strains, the expression of the CAT gene follows the expected tissue distribution pattern of expression of the alpha 2(I) collagen gene. In these six strains, the level of CAT activity is much higher in extracts of tail, a tissue that is very rich in tendons, than in any other tissue that was tested. This distribution parallels the much higher levels of alpha 2(I) collagen RNA that are found in the tail as compared to other tissues. Expression of the chimeric gene is detected in the embryo after 8.5 days of gestation, at approximately the same time that the endogenous type I genes become active. We conclude that the alpha 2(I) collagen promoter sequences present in the recombinant plasmid used for our experiments contain sufficient information to ensure stage- and tissue-specific activity of this promoter.
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PMID:Developmental and tissue-specific expression directed by the alpha 2 type I collagen promoter in transgenic mice. 345 66

The regulation of the collagen II gene was investigated by transfecting plasmids containing potential regulatory sequences of this gene coupled to the gene for chloramphenicol acetyltransferase (CAT) into various cells. The 5' flanking region of this gene functioned as a weak promoter when transfected into chicken chondrocytes or fibroblasts. Inclusion of an 800-base fragment from the first intron, however, increased transcription of CAT approximately 18-fold in chicken chondrocytes and in differentiating limb bud cells but not in fibroblasts, myoblasts, muscle-derived fibroblasts, or teratocarcinoma cells. Furthermore, this fragment was active when placed either upstream or downstream of the CAT gene and when present in either orientation. The activity of this enhancer was examined in relation to differentiation by using "micromass" culture of limb bud mesenchyme cells undergoing chondrogenesis. In this system, no response to the enhancer was observed in undifferentiated limb bud mesenchyme cells. Following differentiation into chondrocytes, a 13-fold enhancer effect was observed in these cells. Finally, all-trans-retinoic acid, a known teratogen, dramatically suppressed enhancer activity in chondrocytes and differentiating limb bud mesenchyme cells. These results suggest that the collagen II gene contains an enhancer element in the first intron that is involved in cell-specific expression.
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PMID:Identification of a phenotype-specific enhancer in the first intron of the rat collagen II gene. 348 May 15

Several lines of evidence have suggested that the regulation of type I collagen gene transcription is complex and that important regulatory elements reside 5' to, and within, the first intron of the alpha 1(I) gene. We therefore sequenced a 2.3-kilobase HindIII fragment that encompasses 804 base pairs of 5' flanking sequence, the first exon, and most of the first intron of the alpha 1(I) human collagen gene. A 274-base-pair intronic sequence, flanked by Ava I sites (A274), contained a sequence identical to a high-affinity decanucleotide binding site for transcription factor Sp1 and a viral core enhancer sequence. DNase I protection experiments indicated zones of protection that corresponded to these motifs. When A274 was cloned 5' to the chloramphenicol acetyltransferase (CAT) gene, driven by an alpha 1(I) collagen promoter sequence, and expression was assessed by transfection, significant orientation-specific inhibition of CAT activity was observed. This effect was most apparent in chicken tendon fibroblasts, which modulate their level of collagen synthesis in culture. We propose that normal regulation of alpha 1(I) collagen gene transcription results from an interplay of positive and negative elements present in the promoter region and within the first intron.
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PMID:Regulatory elements in the first intron contribute to transcriptional control of the human alpha 1(I) collagen gene. 348 May 16

Glucocorticoids have been shown to be useful in the treatment of certain types of chronic liver disease both by inhibiting fibrosis and by improving liver function. We have previously demonstrated in an in vivo model of hepatic fibrogenesis that dexamethasone inhibits the synthesis of types I and IV collagen. In the present study we have evaluated the level of regulation responsible for the dexamethasone-induced changes in collagen gene expression in a defined in vitro system. Primary cultures of adult rat hepatocytes treated with and without dexamethasone under classical cell culture conditions or using defined media were evaluated for synthesis and abundance of procollagen and beta-actin mRNAs. Cells treated with dexamethasone had decreased types I and IV procollagen mRNA steady state levels due in part to diminished transcription rates of the genes. On the other hand, beta-actin mRNA levels were unaffected by dexamethasone. Transient expression experiments were performed to more precisely define the mechanism whereby dexamethasone affects type I procollagen gene transcription. The recombinant plasmid, pAZ1009, containing the mouse alpha 2(I) procollagen gene promoter linked to the chloramphenicol acetyltransferase gene, was transfected into mouse fibroblast cell lines. Cells transfected with the pAZ1009 plasmid in the presence of dexamethasone had a significant decrease in chloramphenicol acetyltransferase activity when compared to cells not exposed to dexamethasone. These data suggest that dexamethasone inhibits collagen synthesis through a direct effect on the collagen gene promoter and appears also to have a post-transcriptional effect on procollagen mRNA content.
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PMID:The effects of dexamethasone on in vitro collagen gene expression. 358 2

A chimeric gene was constructed in which sequences between 2,000 base pairs upstream of the start of transcription of the mouse alpha 2(I) collagen gene and 54 base pairs downstream of this site were fused to the chloramphenicol acetyltransferase (CAT) gene. We present evidence suggesting that this collagen gene segment is sufficient for cell-specific expression of the chimeric gene. Indeed, the levels of CAT activity in transient expression experiments were at least 10 times higher after transfection of NIH 3T3 cells than after transfection of a mouse myeloma cell line, whereas much less difference was found after transfection of these two cell types with pSV2-CAT, a plasmid in which the early simian virus 40 promoter is fused to the CAT gene. Several deletions were introduced in the same 5'-flanking segment of the alpha 2(I) collagen gene, and the effects of these deletions were examined after DNA transfection of the chimeric collagen-CAT gene into NIH 3T3 cells. At least two segments broadly located between -979 and -502 and between -346 and -104 are needed for optimal expression of the chimeric gene. These results were obtained both in transient expression experiments and by analysis of pools of NIH 3T3 cells that were stably transfected with the different mutants. In general, the effects of the deletions on the activity of the alpha 2(I) collagen promoter were analogous, whether the plasmids harbored the simian virus 40 enhancer sequence or not, although the overall levels of expression of the chimeric gene were increased when the recombinant plasmids contained this sequence.
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PMID:Transcriptional control of the mouse alpha 2(I) collagen gene: functional deletion analysis of the promoter and evidence for cell-specific expression. 378 51


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