RETROVIRUS REGULATION IN VITRO AND DURING DEVELOPMENT

拟议中的实验的长期目标是了解 逆转录病毒序列在许可条件下对基因表达的调控 条件，如在质粒分子中导入培养细胞 以及这些序列是如何克服发育信号而诱导 大鼠染色质结构变化与基因异常表达 插入基因组后在细胞DNA的侧翼。这些问题 对于理解病毒调控和病毒 致病机制，尤指整合前病毒的能力 调节前病毒相邻细胞癌基因的表达- 诱导肿瘤来源的细胞。 以前的工作主要集中在内源性病毒的表达上。 在发展过程中，以及综合前病毒之间的相互作用 以及邻近的细胞DNA。这项提案的重点是病毒 鸡红细胞生成过程中的基因表达及其顺式效应 关于氯化蛋白结构和转录的综合前体 在这些细胞中的侧翼宿主序列的活性。这将是 使用内源性前病毒EV-6完成，它是由 细胞促进剂。该病毒及其邻近宿主序列的分析 因此将允许调查前病毒的影响 整合在已知启动子元件的活性上。上一首 结果表明，分化的红细胞是一个有趣的 系统来调查序列内容、 染色质的结构和转录是由于这样一个事实，虽然这些 细胞经历了全球凝聚和转录失活 大多数细胞基因，前病毒DNA能够同时逃脱这两种 流程。 第二组实验旨在补充和扩展体内的 通过直接分析上述三个实验的作用 来自致病性、外源性和非致病性病毒的病毒序列， 内源性病毒对连锁LTRs转录调控的影响。这个 这些研究的目的是确定与转录有关的序列 对病毒和邻近宿主特异序列的调节。三位一体 将进行测试的内源性和外源性病毒的区域包括 来自前病毒LTRS的U3区，是env基因中的一个区域，作为 与其他前病毒和细胞转录调节区一起，是 包括在核酸酶敏感部位，并受到特殊保护 来自发育过程中的从头甲基化和3‘端未翻译 内源性病毒的区域，这已被牵连到抑制 连接的LTRs的增强子/启动子活性。这些实验将 通过分析重组人、转染人的RNA表达情况进行 通过瞬时表达分析获得重组表达载体。

The long range goal of the proposed experiments is to understand how retroviral sequences mediate control of gene expression under permissive conditions such as in plasmid molecules introduced into cultured cells and how such sequences overcome developmental signals to induce chromatin structural changes and aberrant expression of genes in flanking cellular DNA after insertion into the genome. These questions are important in understanding both viral regulation and viral pathogenesis, particularly the ability of integrated proviruses to modulate the expression of adjacent cellular oncogenes in provirus- induced tumor-derived cells. Previous work has focused on the expression of avain endogenous viruses during development, and the interactions between integrated proviruses and adjacent cellular DNA. The focus in this proposal is on virus expression during avian erythorpoiesis and the cis-effects induced by integrated proviruses on the chormatin structure and transcriptional activity of flanking host sequences in these cells. This will be accomplished using the endogenous provirus ev-6, which is drived off of a cellular promoter. Analysis of this virus and adjacent host sequences will therefore allow the investigation of the effects of provirus integration on the activity of a known promoter element. Previous results indicate that differentiating erythrocytes are an interesting system to investigate the relationship between sequence content, chromatin structure and transcription due to the fact that, while these cells undergo a global condensation and transcriptional inactivation of most cellular genes, proviral DNA is able to escape both of these processes. The second set of experiments seeks to complement and extend the in vivo experiments described above by directly analyzing the role of three viral sequences from pathogenic, exogenous viruses and non-pathogenic, endogenous viruses in transcriptional regulation of linked LTRs. The aim of these studies is to define sequences involved in transcription regulation of viral and adjacent host-specific sequences. The three regions from endogenous and exogenous viruses that will be tested are teh U3 regions from proviral LTRs, a region in the env gene which, as with other proviral and cellular transcriptional regulatory regions, is included in a nuclease hypersensitive site and is specifically protected from de novo methylation during development and the 3' untranslated region from endogenous viruses, which has been implicated in inhibiting the enhancer/promoter activity of linked LTRs. These experiments will be conducted by analyzing RNA expression of recombinant, transfected plasmids by transient expression assays.