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首页> 外文期刊>Molecular and Cellular Biology >An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators.
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An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators.

机译：1型人类免疫缺陷病毒长末端重复序列对不同转录激活因子早期反应的体外转录分析。

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In this report we introduce a simple, fast, and reliable method to prepare whole cell or nuclear extracts from small numbers of cells. These extracts were used to study transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in vitro. Our results revealed that the time courses of activation of extracts derived from cells stimulated with the mitogenic lectin phytohemagglutinin (PHA) or with the tumor promoter phorbol 12-myristate 13-acetate (PMA) are different. PMA induces a rapid onset of increased in vitro transcription from the HIV-1 LTR, while PHA causes a slow and sustained response. The biochemical relevance of protein synthesis inhibition by cycloheximide treatment of cells was investigated. In these studies, PMA induction of a change in in vitro transcriptional activity is not dependent on protein synthesis. Cycloheximide alone is insufficient to induce activation. Oligonucleotide-mediated site-directed mutagenesis demonstrated that mutation of the TATA box in the LTR ablated initiation of both basal-level transcription and activation by extracts from cells stimulated with PMA. Surprisingly, mutation of both kappa B sites in the LTR reduced but did not eliminate the in vitro response to extracts prepared at early time points after PHA or PMA stimulation of Jurkat cells. The reduction was greater in extracts derived from cells treated with PMA. Deletion analysis of the HIV-1 LTR revealed at least one region (-464 to -252) capable of suppressing in vitro transcription in extracts from Jurkat cells stimulated by PMA. This result is consistent with early studies of the HIV-1 LTR in transient transfection assays. We therefore have been able to observe distinct regulatory events at early time points after cells are exposed to agents known to induce transcription of both the HIV-1 LTR reporter gene constructs and the HIV-1 provirus itself.
机译：在本报告中，我们介绍了一种简单，快速且可靠的方法，可从少量细胞中制备全细胞或核提取物。这些提取物用于研究人类免疫缺陷病毒1型（HIV-1）长末端重复序列（LTR）的体外转录激活。我们的结果表明，用促有丝凝集素植物血凝素（PHA）或肿瘤启动子佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）刺激的细胞提取物的活化时间过程是不同的。 PMA引起从HIV-1 LTR体外转录增加的快速发作，而PHA引起缓慢而持续的应答。研究了环己酰亚胺处理细胞对蛋白质合成的抑制作用的生化相关性。在这些研究中，PMA体外转录活性变化的诱导不依赖于蛋白质合成。单独的环己酰亚胺不足以诱导活化。寡核苷酸介导的定点诱变表明，LTR中TATA盒的突变通过用PMA刺激的细胞提取物消除了基础水平转录和激活的启动。出人意料的是，LTR中两个κB位点的突变都减少了，但并没有消除对PHA或PMA刺激Jurkat细胞后早期制备的提取物的体外反应。用PMA处理过的细胞提取物的减少更大。 HIV-1 LTR的缺失分析显示，至少一个区域（-464至-252）能够抑制PMA刺激的Jurkat细胞提取物中的体外转录。该结果与瞬时转染试验中HIV-1 LTR的早期研究一致。因此，我们已经能够在细胞暴露于已知诱导HIV-1 LTR报告基因构建体和HIV-1前病毒本身转录的物质后的早期时间点观察到不同的调控事件。

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