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《调查者摘要》)吉普赛人有7kb 具有与逆转录病毒前病毒类似的结构的元件，具有LTRS和 Gag、pol和env基因，这些特征可能表明转座。 涉及反转录和插入cDNA的机制。 后生动物中的逆转座很难研究，因为 插入率。然而，雌性纯合子后代为X连锁 突变弗拉门戈(Flam)经历了很高的吉普赛插入率，这可能是 通过显性雌性不育卵子突变(卵子)的逆转检测到 基因已被证明是反转录转座子插入的热点)，这 允许对与吉普赛人有关的分子事件进行系统研究 插入。考克斯博士报告了一系列有趣的发现 火焰应变。主要是：1)吉普赛RNA是在卵泡中制造的 细胞和其他一些体细胞组织，但不存在于卵母细胞中， 提示插入需要从胞体到生殖细胞的运输或感染 行；2)在Ovod检测中检测到的插入事件相对发生 在后代的胚系中较晚的(因为它们没有聚集在一起) 不是在体细胞中，这表明对加工的第二级控制 插入吉普赛人的RNA的流动性与存在 母亲卵泡细胞中的环境多肽(及相应的2kb RNA) 提示处于病毒样阶段；4)EM中可见病毒样颗粒 火绒卵巢分级提取物和黄连中的制剂 10级卵泡的膜；此外，相同的提取物组分 含有还具有可测量的逆转录酶活性的颗粒；5) 用活性菌株提取物喂养缺乏的火焰蝇(但不是+) 内源性活性元件导致高插入突变率 它们的后代，表明病毒颗粒是有传染性的，并且 可能是通过肠道被吸收的；6)新的插入涉及到许多其他 除了吉普赛人之外的反转录转座子，这表明吉普赛人感染引发了 反转录转座子的普遍动员，并表明限制 组分可能是所有反转录转座子共有的。最受欢迎的模式 科尔塞斯博士认为，感染颗粒是在毛囊细胞中产生的(在 缺乏可能通过降解RNA起作用的Flam+蛋白)；它们 感染卵母细胞，在这个过程中失去env蛋白，然后rna变成 整合到极细胞中，在那里被反转录并插入到 更晚的阶段。还考虑了几种可行的替代方案。 建议的实验集中在5个目标上。第一个是化验 检测吉普赛病毒包膜蛋白的生物学活性 取代逆转录病毒的相应蛋白质，并通过工程 一个单独的吉普赛元素的env编码区的突变和检测 失去活力。此外，env蛋白将从一个 不同阶段的热休克促进剂，以确定其是否能克服Flam+ 对本土吉普赛元素的抑制。如果不是，则表示其他 整合酶、逆转录酶和RNAseH等吉普赛活性 将添加热冲击控制，以尝试识别限制组件 在flam+基因型别中。第二个是表征插入的时间 通过聚合酶链式反应并试图将这些信息与 利用抗体表达各种蛋白质。三是评估 应用EM抗体染色技术研究病毒颗粒的精确作用 在卵母细胞中寻找与卵黄有关的颗粒的切片 颗粒(测试吉普赛人是从卵泡转移到 与卵黄有关)和在卵黄周隙中 受精，以及是否需要从卵泡移植卵黄 到卵母细胞。四是评估吉普赛人动员的机制 通过检测吉普赛包膜蛋白与其他反转录转座子的相关性 其他元素的RNAs将指示 杂交颗粒和环境蛋白是一个普遍的限制因素，以及 检测吉普赛整合酶过表达对插入片段的刺激作用 这与整合酶是一种常见的限制功能是一致的。 最后，将提纯的吉普赛颗粒喂给其他果蝇的果蝇 物种是将吉普赛人发展为普通昆虫的第一步 变换向量。