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REGULATORY ELEMENTS OF AVIAN SARCOMA VIRUS LTRS

禽肉瘤病毒 LTRS 的调控元件

基本信息

批准号：
2376858
负责人：
RAMAREDDY V GUNTAKA
金额：
$ 14.18万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-05-01 至 1999-02-28
项目状态：
已结题

项目摘要

The overall objective of this research project is to understand the host transcription factors involved in the determination of species-specific and tissue-specific expression of Rous sarcoma virus genome. The long terminal repeats (LTR) of the virus contain powerful cis-acting elements to which host factors bind and activate transcription. For some time we have been studying in detail the molecular reactions between host factors and cis- sequences in the promoter-enhancer region (U3) of the RSV lTR. We, and others, have found that at least three well-defined cis elements are required for maximal expression of LTR-linked genes and three different factors interact with these motifs. We have also succeeded in isolating three different yet related cDNA clones encoding polypeptides that specifically bind to distinct sequence motifs. We have shown that one of these factors, mainly present in the avian fibroblasts, and in muscle tissue, binds to a specific site, E3/E3-1, localized at nucleotides -151 to -171. The importance of this cis-acting motif for viral LTR function has not ben examined. The aims of the current study are; (i) to define the role of the cis-acting sequence E3/E3-1 (-151 to -171) in LTR function and characterize the protein binding to this sequence as it appears that this factor is present only in avian fibroblasts, the target tissue for efficient expression of viral genome; (ii) to determine the functional domains of the cloned factors and their cognate cis-acting sequences; (iii) to establish the role of the cloned cDNA products in RSV gene expression. In-frame deletions and point mutations will be made in the cDNAs to identify the various DNA binding domains of the factors. We will also address the issue of whether phosphorylation and homo- or heterodimerization of various factors are required for their interaction, using antibodies raised against the purified factors. These studies will provide further insights into the regulation of viral gene expression and the contribution of host factors in determining tissue tropism and species- specific expression of viral genes. Preliminary results indicated that the GT-rich sequence of the U5 region of the lTR contributes to transcription termination and/or the 3' end processing of the RNA. We intend to continue these studies with an objective of identifying precisely the nucleotides involved in the 3' end maturation of the mRNA, which will be determined by S1 protection assays, oligonucleotide-directed mutagenesis and by studies using polymerase chain reaction.

该研究项目的总体目标是了解宿主参与确定物种特异性和劳斯肉瘤病毒基因组的组织特异性表达。长终端病毒的重复序列（LTR）含有强大的顺式作用元件，宿主因子结合并激活转录。一段时间以来，我们一直详细研究宿主因子和顺式之间的分子反应 RSV ITR 启动子-增强子区 (U3) 中的序列。我们，和其他人发现至少三个明确定义的顺式元素 LTR相关基因最大表达所需的以及三种不同的因素与这些主题相互作用。我们也成功隔离了三个不同但相关的 cDNA 克隆编码多肽，特异性结合不同的序列基序。我们已经证明其中之一这些因子主要存在于禽类成纤维细胞和肌肉中组织，结合特定位点 E3/E3-1，位于核苷酸 -151 至 -171。这种顺式作用基序对于病毒 LTR 功能的重要性没有经过检查。当前研究的目的是； (i) 定义顺式作用序列 E3/E3-1（-151 至 -171）在 LTR 功能中的作用和表征与该序列结合的蛋白质，因为这似乎是该因子仅存在于禽类成纤维细胞中，这是病毒基因组的高效表达； (ii) 确定功能克隆因子的结构域及其同源顺式作用序列； (三) 确定克隆的 cDNA 产物在 RSV 基因表达中的作用。将在 cDNA 中进行框内删除和点突变，以识别因子的各种 DNA 结合域。我们还将解决磷酸化和同源或同源的问题各种因子的相互作用需要异二聚化，使用针对纯化因子产生的抗体。这些研究将提供对病毒基因表达调控的进一步见解宿主因素在决定组织向性和物种方面的贡献病毒基因的特异性表达。初步结果表明 lTR U5 区域富含 GT 的序列有助于转录 RNA 的终止和/或 3' 末端加工。我们打算继续这些研究的目的是精确识别核苷酸参与 mRNA 3' 端的成熟，这将由下式确定 S1 保护测定、寡核苷酸定向诱变和研究使用聚合酶链式反应。