Gene acquisition by large DNA viruses

背景--大型DNA病毒编码的基因似乎来自细胞有机体。这些病毒很可能是从宿主那里获得的，因为不同的病毒和它们感染的有机体经过数十亿年的共同进化。例如，粘液瘤病毒编码一种酶，保护这些昆虫传播的痘病毒免受紫外线的伤害。最相似的蛋白质是在植物和昆虫中编码的。这些病毒基因从哪里来，它们是如何到达那里的？我们希望找到这个问题的答案。基因重组是一个用来描述不同DNA交换和连接的术语，它创造了具有新生物特性的生物体。我们研究了痘病毒是如何重组DNA的，这就产生了克隆DNA和基因工程病毒的商业工具。我们已经测试了痘苗病毒(VAC)和输入DNA之间的重组需要多大的相似性，虽然随着相似性的降低DNA交换减少，但与无关DNA的DNA交换并不会降至零。我们已经对17个VAC克隆进行了测序，这些克隆捕获的DNA与病毒DNA没有相似之处。这些病毒在随机整合位点附近表现出复杂的基因重排。因此，我认为这个过程提供了一条途径，让大型DNA病毒能够捕获宿主基因的拷贝。这一过程可能只涉及DNA片段的端到端连接，也可能使用与逆转录病毒和逆转录转座子运动有关的机制。后一种方案的优势在于，它可以以一种能够使病毒表达的方式修剪基因拷贝(移除内含子)。AIMS-有三种AIMS转导/定位。我们将把编码药物可选择的报告蛋白的DNA导入VAC感染的细胞，然后分离和测序得到的重组体。这些研究将确定DNA的去向以及这些事件如何改变病毒基因组。双链断裂。我们将在复制VAC DNA中引入双链断裂，然后测试这是否会增加捕获报告基因的病毒的产量。这样的研究将更好地定义支持DNA捕获的步骤。逆转录病毒/反转录转座子。逆转座反应将RNA复制到DNA中，然后将复制的DNA插入基因组中的新位置。我们将测试报告基因是否可以用催化人类LINE 1反转录转座子移动的蛋白质或包装成逆转录病毒颗粒的基因“逆转录”到VAC中。培训-学员将在先进基础设施的支持下，在刺激的学术环境中获得高度可取的技能，学习基因组学、病毒学和分子生物学。结论--目前尚不清楚有多大的DNA病毒能从细胞宿主中捕获基因，但这一过程可能在创造具有新特性的新病毒方面发挥关键作用。这项工作将提供对负责病毒生物学这一迷人特征的过程的原创性见解，并提供HQP在生物技术方面的杰出培训。

Background - Large DNA viruses encode genes that seem to have come from cellular organisms. These have probably been acquired from their hosts as the different viruses and the organisms they infect have co-evolved over billions of years. For example, myxoma virus encodes an enzyme that protects these insect-vectored poxviruses from UV-light. The closest similar proteins are encoded in plants and insects. Where do these virus genes come from and how did they get there? We hope to find answers to this question.      "Genetic recombination" is a term used to describe the swapping and joining of different DNAs and it creates organisms with new biological properties. We've studied how poxviruses recombine DNA and this has produced commercial tools for cloning DNA and genetically-engineering viruses. We've tested how much similarity is needed for recombination between vaccinia virus (VAC) and input DNA and while there is less DNA swapping as the resemblance decreases, it doesn't drop to zero with unrelated DNAs. We've sequenced 17 VAC clones that have captured DNA bearing no resemblance to the virus DNA. These viruses exhibit complex gene rearrangements near random integration sites.      I propose that this process offers a route by which large DNA viruses can capture copies of host genes. The process could simply involve end-to-end joining of DNA fragments or might use a mechanism related to the movement of retroviruses and retrotransposons. The latter scheme has an advantage in that it can prune the gene copy (remove introns) in a way that would enable virus expression. Aims - there are three aims Transfection/mapping. We'll transfect DNA encoding a drug-selectable reporter protein into VAC-infected cells, and then isolate and sequence the resulting recombinants. These studies will determine where the DNA goes and how the virus genome is altered by these events. Double-stranded breaks. We'll introduce double-stranded breaks into replicating VAC DNA and then test if this increases the yield of viruses that have captured a reporter gene. Such studies will better define the steps underpinning DNA capture. Retroviruses/retrotransposons. Retrotransposition reactions copy RNA into DNA and then insert the copied DNA into a new site in the genome. We'll test whether reporter genes can be "retroposed" into VAC using either the proteins that catalyze movement of human LINE 1 retrotransposons or as genes packaged into retrovirus particles. Training - Trainees will acquire highly-desirable skills studying genomics, virology and molecular biology in a stimulating academic environment supported by advanced infrastructure. Conclusions - It is unknown how large DNA viruses capture genes from cellular hosts, but the process could play a key role in creating new viruses with novel properties. This work will provide original insights into the process(es) responsible for this fascinating feature of virus biology and offer HQP outstanding training in biotechnology.