RESUMO
Transforming growth factor alpha (TGF-alpha) is a transformation-responsive mitogenic polypeptide that is expressed in the brain, epithelial cells, and activated macrophages. We isolated and characterized the TGF-alpha promoter and localized the 5' end of the TGF-alpha transcript to a unique position. Surprisingly, no apparent TATA box was present in the promoter sequence, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.
Assuntos
Genes Homeobox , Genes , Regiões Promotoras Genéticas , Transcrição Gênica , Fatores de Crescimento Transformadores/genética , Sequência de Aminoácidos , Sequência de Bases , Quimera , Humanos , Dados de Sequência Molecular , Plasmídeos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Mapeamento por RestriçãoRESUMO
An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramatically increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either mRNA accumulation, translational efficiency, or both. Our previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using an in vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between +19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.
Assuntos
Regulação da Expressão Gênica , HIV/genética , RNA Mensageiro/biossíntese , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Bases , Linhagem Celular , DNA/metabolismo , Produtos do Gene tat , Humanos , Dados de Sequência Molecular , Família Multigênica , Sequências Reguladoras de Ácido Nucleico , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Short RNA chains initiating at the major promoter sites for simian virus 40 (SV40) late transcription are elongated to approximately 450 nucleotides in a molar ammount greater than that from any other region of the viral DNA. This conclusion is based on the following observations: (i) Transcriptional complexes isolated by Sarkosyl and by hypotonic leaching (minichromosomes) from nuclei of cells infected with SV40 as well as intact nuclei were pulse labeled in vitro with [alpha-32P]TUP and were observed to synthesize short RNA transcripts that hybridized predominantly to a SV40 DNA fragment spanning between 0.67 and 0.76 map units. (ii) In the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a drug known to accentuate premature transcriptional termination, accumulation of these short SV40 RNA chains was enhanced. When SV40-infected cells were pretreated with DRB and then labeled in vivo or in vitro, they synthesized short labeled viral RNAs that hydridized almost exclusively with the DNA fragment spanning between 0.67 and 0.76 map units. These observations suggest a mechanism in the regulation of SV40 late transcription.
Assuntos
DNA Viral/metabolismo , Diclororribofuranosilbenzimidazol/farmacologia , Ribonucleosídeos/farmacologia , Vírus 40 dos Símios/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Células Cultivadas , Haplorrinos , Óperon , RNA Viral/biossíntese , Vírus 40 dos Símios/metabolismo , Fatores de TempoRESUMO
We have used a low-salt detergent-free extraction procedure on cells infected with simian virus 40 to obtain viral nucleoprotein late after infection. Addition of EScherichia coli RNA polymerase and ribonucleotide triphosphates to the viral minichromosomes permitted transcription of RNA from viral templates. This synthesis was initiated predominantly within a fragment of DNA spanning 0.67 to 0.76 map unit on the genome. The synthesis from this region proceeded primarily along the "late" strand in a clockwise direction. These results were in contrast to the synthesis obtained with naked viral DNA in which initiation occurred on other regions of the genome and from which transcription proceeded counterclockwise along the early strand. These findings indicate that the nucleoprotein template or factors tightly associated with it may be responsible for site(s) and strand selection in transcription of simian virus 40.
Assuntos
DNA Viral/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleoproteínas/genética , Escherichia coli/enzimologia , Nucleoproteínas/genética , Vírus 40 dos Símios/genética , Transcrição Gênica , Genes Virais , Óperon , Moldes GenéticosRESUMO
We have derived rat cell lines producing different and regulatable amounts of pp60v-src by introducing the src gene of Rous sarcoma virus (RSV) under the control of the glucocorticoid-responsive transcriptional promoter from the mouse mammary tumor virus (MMTV). We find that the cellular phenotype is strictly dependent upon the dose of pp60v-src with a distinct threshold for changes indicative of neoplastic potential. Cells with low constitutive levels of pp60v-src are not phenotypically distinguishable from cells without v-src, but as little as a 4-fold increment in pp60v-src produces morphological transformation and anchorage-independent growth. These properties of the transformed state are achieved at levels of pp60v-src far below levels found in an RSV-transformed cell line, without detectable increase in phosphorylation of the major cellular target for tyrosine phosphorylation.
Assuntos
Vírus do Sarcoma Aviário/genética , Transformação Celular Neoplásica , Dexametasona/farmacologia , Genes Virais/efeitos dos fármacos , Genes/efeitos dos fármacos , Proteínas Quinases/genética , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais/genética , Animais , Vírus do Sarcoma Aviário/enzimologia , Linhagem Celular , Enzimas de Restrição do DNA , Vetores Genéticos , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Proteína Oncogênica pp60(v-src) , Fosforilação , Plasmídeos , Ratos , Transfecção , Proteínas Virais/metabolismoRESUMO
Nuclear extracts and viral transcribing minichromosomes were prepared from SV40-infected cells and incubated in vitro with [alpha-32P]UTP under conditions which allow the elongation of preinitiated RNA chains. Sucrose gradient lysis of the transcription mixtures revealed two populations of SV40-specific RNA: elongating chains that remain associated with the viral minichromosomes, and, at the top of the gradient, small free RNA detached from the template and hybridizing exclusively to the promoter-proximal region of SV40 DNA. This free RNA was shown by polyacrylamide gel electrophoresis to comprise essentially a 94 nucleotide species, which could, however, at high UTP concentration, be elongated a further few nucleotides before terminating. These results thus show that the actively transcribing minichromosomes provide a sytem in which the attenuated RNA can be released from the template. Moreover, this is the first demonstration of specific in vitro termination of polymerase B transcription. The conditions which lead to transcription termination are discussed.
Assuntos
Vírus 40 dos Símios/genética , Transcrição Gênica , Animais , Sítios de Ligação , Extratos Celulares , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA/biossíntese , RNA Viral/química , Moldes Genéticos , Uridina MonofosfatoRESUMO
The EJ-ras gene was placed under the transcriptional control of the steroid-inducible mouse mammary tumor virus promoter/enhancer and introduced into Rat-1 fibroblasts, yielding the 14C cell line. When these cells were exposed to dexamethasone in vitro, EJ-ras mRNA was induced 15- to 20-fold, the cells grew in agar, and, after injection of cells into syngenic Fischer 344 rats, they produced lethal fibrosarcomas. Inhibitors of poly(ADP ribose) polymerase, which prevent the activation of the purified enzyme by a synthetic octadeoxyribonucleotide duplex, inhibited both in vivo tumorigenicity and in vitro growth in soft agar. The enzyme inhibitor 1,2-benzopyrone, which was studied in detail, and other polymerase inhibitors had no effect on EJ-ras mRNA or p21 protein expression. Poly(ADP ribose) polymerase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, EC 2.4.2.30] was inhibited by the drug in both untreated and dexamethasone-treated cells both in vitro and in vivo to the same extent, but biological consequences of enzyme inhibition were manifest only when the cells were in the transformed tumorigenic state.