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In vitro methylation inhibits the promotor activity of a cloned intracisternal A-particle LTR. 总被引:9,自引:2,他引:7
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We studied the relation between LTR methylation and expression of the family of endogenous retrovirus-like elements related to mouse intracisternal A-particles (IAP). Comparative HpaII/MspI and HhaI restriction analysis of genomic DNA's showed that in cells and tissues with a low level of IAP gene expression, HpaII and HhaI sites within the 5' LTR were heavily methylated, while in cells abundantly expressing IAP's 20 to 30% of the 5' LTRs were demethylated at these sites. The effects of methylation on the promoter activity of a cloned IAP 5' LTR was studied directly, using the plasmid pMIA5' L-cat in which this LTR was linked to the chloramphenicol acetyl transferase (CAT) gene. In vitro methylation of three HhaI sites located between -137 and -205 bp from the RNA start site of this LTR completely inactivated the promoter activity of pMIA5' L-cat transfected into COS7 cells. Methylation of a HpaII site located 94 bp downstream from the RNA start site reduced the promoter activity by 75%. The results show that methylation at sites both upstream and downstream from the RNA start site profoundly effects the promoter activity of this LTR and suggest that methylation within the 5' LTR can serve to regulate IAP gene expression in vivo. 相似文献
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A single species of pX mRNA of human T-cell leukemia virus type I encodes trans-activator p40x and two other phosphoproteins. 总被引:26,自引:14,他引:12
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Human T-cell leukemia virus type I (HTLV-I) contains the pX sequence which codes for the trans-activator of the long terminal repeat (LTR) and is thus postulated to be associated with leukemogenesis in adult T-cell leukemia. Overlapping open reading frames (ORF) in the pX sequence were recently found to code for p27x-III and p21x-III by ORF III, in addition to p40x coded for by ORF IV. The mechanism of expression of these newly identified proteins and their possible association with trans-activation were studied. On transfection of an expression plasmid that contains a cDNA sequence of the pX mRNA, products from both ORFs III and IV were detected in the cells. The RNA was synthesized in vitro from the cDNA clone by SP6 RNA polymerase and translated in a rabbit reticulocyte lysate. As translation products, two proteins, p27x-III and p21x-III, were detected in addition to p40x. Elimination of the first and second ATG codons in ORF III resulted in loss of the ability to code for p27x-III and p21x-III, respectively, which indicated that the translations from these two ATG codons were independent. A mutant that lacked both ATG codons was fully active in trans-activation of chloramphenicol acetyltransferase gene expression directed by the LTR. These results indicate that a 2.1-kilobase pX mRNA of HTLV-I independently encodes three proteins, p40x, p27x-III, and p21x-III, by different ORFs and that the last two proteins are not involved in trans-activation of the unintegrated LTR. 相似文献
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Inhibition of HIV-LTR gene expression by oligonucleotides targeted to the TAR element. 总被引:15,自引:4,他引:11
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T Vickers B F Baker P D Cook M Zounes R W Buckheit Jr J Germany D J Ecker 《Nucleic acids research》1991,19(12):3359-3368
All human immunodeficiency virus mRNAs contain a sequence known as TAR (trans-activating responsive sequence). The TAR element forms a stable RNA stem-loop structure which binds the HIV tat (trans-activator) protein and mediates increased viral gene expression. In principle, molecules which bind to the TAR RNA structure would inhibit trans-activation by perturbing the native RNA secondary structure. We have constructed a series of phosphodiester and phosphorothioate antisense oligonucleotides which specifically bind to the HIV TAR element. Specific binding to the TAR element was demonstrated in vitro with enzymatically synthesized TAR RNA. The TAR-directed phosphorothioates inhibited trans-activation in a sequence-dependent fashion in a cell culture model using an HIV LTR/human placental alkaline phosphatase gene fusion and tat protein supplied in trans. The molecules also inhibited HIV replication in both acute and chronically infected viral assays, but without sequence specificity. We have constructed a series of vectors consisting of the MMTV promoter and 5'-untranslated region of four different mRNAs, including the TAR region, to study the effect of TAR on gene expression in heterologous systems. The results suggest that, in the absence of the HIV LTR, the TAR element has a repressive effect on gene expression, which is relieved by tat. 相似文献
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B R Cullen 《Cell》1986,46(7):973-982
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Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific. 总被引:36,自引:16,他引:20
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The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream. 相似文献