MOLECULAR BASIS OF RETROTRANSPOSON MOBILIZATION

描述：（改编自研究者摘要）吉普赛是一个7 kb的 具有类似于逆转录病毒前病毒的结构的元件， gag、pol和env基因，这些特征可能指示转座 该机制涉及逆转录和cDNA的插入。 逆转录转座一直难以研究后生动物，因为低 插入率。 然而，X-连锁遗传纯合子雌性的后代， 突变弗拉门戈（flam）经历吉普赛插入率高，可以 通过显性雌性不育ovoD突变的回复（卵 基因已被证明是反转录转座子插入的热点），这已经 允许系统研究与吉普赛人有关的分子事件， 插入。 Corces博士报告了一些有趣的发现， 火焰应变。 主要有：1）gypsy RNA在卵泡中产生 细胞和一些其它体细胞组织但不存在于卵母细胞中， 表明插入需要从索马到胚芽的运输或感染 线; 2）在ovoD测定中检测到的插入事件相对于 在后代的生殖系晚期（因为它们不成簇）， 而不是在体细胞中，这表明对加工的第二层次控制 RNA插入3）吉普赛人的流动性与存在 母亲卵泡细胞中的env多肽（和相应的2kb RNA） 提示病毒样阶段; 4）在EM中可以看到病毒样颗粒 在火焰卵巢的分级提取物和 第10阶段卵泡的膜;此外， 含有颗粒也具有可测量的逆转录酶活性; 5） 将活性菌株的提取物饲喂缺乏Flam（而不是+）的果蝇 内源性活性元件导致高比率的插入突变， 它们的后代，表明病毒颗粒具有感染性， 可能通过肠道摄入; 6）新的插入涉及许多其他 反转录转座子，表明吉普赛感染触发了一个 逆转录转座子的普遍动员，并表明限制 该组分可以是所有反转录转座子共有的。 受青睐的模式 博士Corces是感染性颗粒产生于卵泡细胞（在 缺乏flam+蛋白，其可能通过降解RNA起作用）;它们 感染卵母细胞，在这个过程中失去env蛋白，然后RNA变成 在那里它被逆转录并插入到极细胞中， 后期阶段。 还考虑了几种可行的替代方案。 实验主要围绕5个目标展开。 第一是化验 通过测定吉普赛人env蛋白是否能 替代逆转录病毒的相应蛋白质，并通过工程改造 一个单独的吉普赛元件的env编码区中的突变， 失去活动。 此外，env蛋白将从一个 热休克促进剂在不同阶段，以确定它是否可以克服火焰+ 抑制原生吉普赛元素。 如果不是，则表达其他 吉普赛人的活动，如整合酶，逆转录酶和RNaseH下， 将增加热冲击控制，以尝试识别限制组件 flam+基因型。 二是表征插入时机 并试图将这些信息与 使用抗体表达各种蛋白质。 三是考核 用抗体电镜染色法对病毒颗粒的精确作用 切片以寻找卵母细胞中与蛋黄相关的颗粒 颗粒（以测试吉普赛是从卵泡转移到 与卵黄结合），并在卵黄周间隙， 受精，以及插入是否需要从卵泡转移卵黄 到卵母细胞 第四，评估吉普赛人的动员机制， 通过测试吉普赛env蛋白与 其他元件的RNA，这将是形成的指示， 杂合颗粒和env蛋白是通常的限制因素，和 通过吉普赛整合酶的过表达测试插入的刺激 这与整合酶是一种常见的限制功能相一致。 最后将纯化的吉普赛颗粒喂给其他果蝇 物种作为第一步发展吉普赛作为一般昆虫 变换向量

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.