ANALYSIS OF HIV MESSENGER RNA STRUCTURE

HIV信使RNA结构分析

• 批准号: 3916915
• 负责人: J A LAUTENBERGER
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3916915
• 关键词: AIDS; complementary DNA; gene expression; genetic library; genetic manipulation; human immunodeficiency virus 1; messenger RNA; molecular cloning; nucleic acid hybridization; oligonucleotides; transposon /insertion element; virus RNA; virus genetics

Human immunodeficiency virus (HIV) infection has been shown by several laboratories to use a complex set of RNA synthesis and processing events in order to control viral expression. In order to determine if messages exist that have not been previously characterized, we have begun to directly analyze cDNA copies of HIV messages by Southern hybridization and DNA sequence analysis. A cDNA library in lambda gt10 has been prepared from RNA isolated from HIV-infected H9 cells. A large number of phages from this library were found to hybridize to viral sequences. A total of 19 such phages were plaque-purified an' analyzed by Southern hybridization using labeled oligonucleotides or restriction fragments derived from various locations on the HIV genome. The specific region include: (A) 5'-end of tat: (B) 5'-end of trs; (C) 5'-portion of env (excluding tat and trs region); (D) pol/sor region. Clones with the following hybridization pattern have been isolated: class I, A+B-C-D+; class II, A-B-C+D+; class III, A-B-C- D+. We have purified several of these phages and have subcloned their insert DNA into pIBI vectors. Several of these phages have been characterized by Southern blot hybridization. We are currently sequencing these DNA segments to determine if they represent novel HIV messages and, thus, might reveal new exons or splice sites.

人类免疫缺陷病毒（HIV）感染已被证明， 几个实验室使用一套复杂的RNA合成， 处理事件以控制病毒表达。 为了 以确定是否存在先前未 我们已经开始直接分析HIV的cDNA拷贝， 通过Southern杂交和DNA序列分析， 从分离的RNA制备了λ gt 10的cDNA文库， 来自感染艾滋病毒的H9细胞。 大量的大麻从这个 发现库与病毒序列杂交。 共有19 将这些噬菌斑纯化并通过Southern杂交分析 使用标记的寡核苷酸或限制性内切酶的杂交 来自HIV基因组上不同位置的片段。 的 特异性区域包括：（A）达特5 ′-末端;（B）trs 5 ′-末端;（C） env的5 '-部分（不包括达特和trs区域）;（D）pol/sor 地区 具有以下杂交模式的克隆已被 分离：I类，A+B-C-D+; II类，A-B-C+D+; III类，A-B-C- D+。 我们已经纯化了其中的几种， 将其插入pIBI载体中。 其中几个人 通过Southern印迹杂交进行了表征。 我们 目前正在对这些DNA片段进行测序， 代表新的HIV信息，因此可能揭示新的外显子或 剪接位点