MOLECULAR BASIS OF RETROTRANSPOSON MOBILIZATION

逆转录转座子动员的分子基础

• 批准号: 2024565
• 负责人: Victor G. Corces
• 金额: $ 24.73万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2024565
• 关键词: Drosophilidae; Retroviridae; electron microscopy; gene expression; gene mutation; genetic transcription; molecular genetics; polymerase chain reaction; transposon /insertion element; virus envelope; virus protein; viruslike particle

DESCRIPTION: (Adapted from the Investigator's Abstract) Gypsy is a 7kb element with a structure similar to retroviral proviruses featuring LTRs and gag, pol and env genes, features presumably indicative of a transposition mechanism involving reverse transcription and insertion of the cDNA. Retrotransposition has been difficult to study in metazoans because of a low rate of insertion. However, progeny of females homozygous for the X-linked mutation flamenco (flam) undergo high rates of gypsy insertion which can be detected by reversions of the dominant female sterile ovoD mutation (the ovo gene has proved to be a hotspot for retrotransposon insertions) and this has permitted systematic study of the molecular events associated with gypsy insertion. Dr. Corces reports a number of interesting findings using the flam strain. Principally these are: 1) gypsy RNAs are made in follicle cells and some other somatic tissues but are not present in oocytes, suggesting that insertion requires transport or infection from soma to germ line; 2) the insertion events detected in the ovoD assay occur relatively late in the germ line of the progeny (because they are not clustered) and not in somatic cells, indicating a second level of control over processing the RNA into an insertion 3) gypsy mobility is associated with presence of env polypeptides (and a corresponding 2kb RNA) in follicle cells of mothers suggestive of a virus-like stage; 4) virus-like particles can be seen in EM preparations both in fractionated extracts of flam ovaries and in the membranes of stage 10 follicles; moreover the same extract fraction that contains particles also has measurable reverse transcriptase activity; 5) feeding extracts from active strains to flam (but not +) flies lacking endogenous active elements leads to high rates of insertional mutations in their progeny, indicating that the virus particles are infectious and are probably taken in through the gut; 6) the new insertions involve many other retrotransposons besides gypsy, indicating that gypsy infection triggers a general mobilization of retrotransposons, and suggesting that the limiting component may be common to all the retrotransposons. The model favored by Dr. Corces is that infectious particles are generated in follicle cells (in the absence of flam+ protein which probably acts by degrading the RNA); they infect oocytes, losing env protein in the process, then the RNA becomes incorporated into pole cells where it is reverse transcribed and inserted at a later stage. Several viable alternatives are also considered. The proposed experiments focus on 5 aims. The first is to assay for biological activity of the gypsy env protein by determining whether it can substitute for the corresponding protein of a retrovirus, and by engineering a mutation in the env coding region of a solo gypsy element and testing for loss of activity. In addition the env protein will be expressed from a heat-shock promoter at various stages to determine if it can overcome flam+ inhibition of native gypsy elements. If not, constructs expressing other gypsy activities such as integrase, reverse transcriptase, and RNaseH under heat shock control will be added to try to identify the limiting components in flam+ genotypes. The second is to characterize the timing of insertions by PCR and to try to correlate that information with the timing of expression of the various proteins using antibodies. The third is to assess the precise role of virus particles by using antibody staining of EM sections to search for the particles in oocytes, in association with yolk granules (to test the idea that gypsy is transferred from follicles in association with yolk) and in the perivitelline space following fertilization, and whether insertion requires yolk transfer from follicles to oocytes. The fourth is to assess the mechanism of gypsy mobilization of other retrotransposons by testing for associations of gypsy env protein with the RNAs of the other elements which would be indicative of the formation of hybrid particles and of env protein being a generally limiting factor, and to test for stimulation of insertions by overexpression of gypsy integrase which would be consistent with integrase being a common limiting function. Finally purified gypsy particles will be fed to flies of other Drosophila species as a first step toward developing gypsy as a general insect transformation vector.

描述：（改编自研究者的摘要）Gypsy 是一个 7kb 的 具有类似于逆转录病毒原病毒结构的元素，具有 LTR 和 gag、pol 和 env 基因，可能表明转座的特征 涉及逆转录和cDNA插入的机制。 由于逆转录转座率低，因此在后生动物中进行逆转录转座研究很困难。 插入率。 然而，X连锁纯合雌性的后代 突变弗拉门戈（flam）经历了很高的吉普赛插入率，这可以 通过显性雌性不育 ovoD 突变（ovo 基因已被证明是逆转录转座子插入的热点），这已经 允许对与吉普赛人相关的分子事件进行系统研究 插入。 Corces 博士报告了一些有趣的发现 火焰应变。 主要是：1) gypsy RNA 在卵泡中产生 细胞和一些其他体细胞组织，但不存在于卵母细胞中， 表明插入需要从体细胞到细菌的运输或感染 线; 2) ovoD检测中检测到的插入事件相对发生 处于后代种系晚期（因为它们不聚集）并且 不在体细胞中，表明对加工的第二级控制 RNA 插入插入 3) 吉普赛人的流动性与 母亲卵泡细胞中的 env 多肽（和相应的 2kb RNA） 暗示病毒样阶段； 4) 电镜中可见病毒样颗粒 火焰卵巢分馏提取物和 10期卵泡膜；此外，相同的提取部分 含有的颗粒还具有可测量的逆转录酶活性； 5） 将活性菌株的提取物喂给缺乏的flam（但不是+）果蝇 内源活性元件导致高插入突变率 它们的后代，表明病毒颗粒具有传染性并且 可能是通过肠道摄入的； 6）新的插入涉及许多其他内容 除吉普赛人外还存在反转录转座子，表明吉普赛人感染引发了 逆转录转座子的普遍动员，并表明限制 成分可能是所有反转录转座子所共有的。 深受青睐的车型 Corces 博士认为，传染性颗粒是在毛囊细胞中产生的（在 缺少 flam+ 蛋白，该蛋白可能通过降解 RNA 发挥作用）；他们 感染卵母细胞，在此过程中丢失env蛋白，然后RNA变成 并入极细胞，在那里它被逆转录并插入 稍后的阶段。 还考虑了几种可行的替代方案。 拟议的实验集中于 5 个目标。 首先是测定 吉普赛环境蛋白的生物活性，通过确定它是否可以 替代逆转录病毒的相应蛋白质，并通过工程改造 单独吉普赛元素的 env 编码区的突变并进行测试 失去活动能力。 此外，env 蛋白将由 在各个阶段进行热激启动子以确定是否可以克服flam+ 抑制本土吉普赛元素。 如果不是，则构造表达其他 吉普赛活动，例如整合酶、逆转录酶和 RNaseH 将添加热冲击控制以尝试识别限制组件 在 flam+ 基因型中。 第二个是表征插入的时机 通过 PCR 并尝试将该信息与时间相关联 使用抗体表达各种蛋白质。 第三是评估 通过使用 EM 抗体染色来确定病毒颗粒的精确作用 用于搜索卵母细胞中与卵黄相关的颗粒的切片 颗粒（测试吉普赛人是从毛囊转移的想法 与蛋黄的关联）和随后的卵周空间 受精，以及插入是否需要从卵泡转移卵黄 到卵母细胞。 四是评估吉普赛人动员机制 通过测试吉普赛环境蛋白与其他逆转录转座子的关联 其他元素的 RNA 可以指示形成 杂化颗粒和 env 蛋白通常是限制因素，并且 测试吉普赛整合酶过度表达对插入的刺激 这与整合酶是一种常见的限制功能是一致的。 最后纯化的吉普赛颗粒将被喂给其他果蝇的苍蝇 物种作为将吉普赛人发展为一般昆虫的第一步 变换向量。