REARRANGEMENT MECHANISMS OF CRITHIDIA RETROTRANSPOSONS

我们这项研究的总体目标是研究 逆转录酶(RT)介导的真核生物转座。我们是 昆虫锥虫甲虫中的这种现象分析 束毛虫是一种原生动物寄生虫，它是该站点的宿主- 特异性逆转录转座子CRE1和CRE2。CRE元素是 鲜为人知的可动基因家族，称为非LTR反转录转座子 广泛分布于哺乳动物、昆虫、植物、锥虫、 和真菌。有证据表明，这类转座子参与了 不同的过程，如基因组进化、假基因形成和 人类遗传病的起源，但几乎对 这些元素转置的机制。插入CRE1和CRE2 在排列紧密的小外显子中的一个特定的高度保守的位置 基因。CRE1在簇毛锦鸡儿微小外显子基因座内快速重排 编码一种新的RT活性。这项提议的目的是延长和 探索这些观察结果，重点关注有关机制的问题 CRE1基因重排与反转录转座子的程度 克里西迪亚虫基因组的入侵。 具体目标的实现将增进我们对 复制这种病毒的机制鲜为人知，但 生物学上重要的一类反转录转座子。特征描述 发生在小外显子基因座内的遗传事件具有潜在的 非洲昏睡病和查加斯病的治疗意义 疾病。在更广泛的范围内，结果将与问题相关 关于真核基因组的组织和进化，以及 人类遗传病的发病机制。 我们建议：(1)刻画CRE1编码的RT活性 根据其功能区以及其引物和模板专一性， 使用针对融合蛋白的蛋白质表达系统 酵母Ty1病毒样颗粒(VLP)和一种导致 CRE1RT介导的酵母假基因形成；(2)鉴定和鉴定 鉴定插入到基因中的额外的位点特异性逆转座子 簇毛锦鸡儿微外显子阵列；(3)确定 克里斯蒂亚目微小外显子中的CRE1及其相关的位置特异性反转录转座子 阵列，并剖析了遗传机制，包括特定于部位的 转座和同源重组，通过它进行CRE1的重排 在丛枝锦鸡儿基因组中。

The overall goal of our research is to study the molecular basis of reverse transcriptase (RT)-mediated transposition in eukaryotes. We are analyzing this phenomenon in the insect trypanosomatid Crithidia fasciculata, a protozoan parasite which serves as host to the site- specific retrotransposons CRE1 and CRE2. CRE-elements are members of the little understood family of mobile genes known as non-LTR retrotransposons that are widely distributed in mammals, insects, plants, trypanosomatids, and fungi. Evidence suggests that this class of transposon is involved in such diverse processes as genome evolution, pseudogene formation, and human genetic disease initiation, yet almost nothing is known about the mechanisms by which these elements transpose. CRE1 and CRE2 are inserted at a specific highly conserved site in the tandemly arrayed miniexon genes. CRE1 rapidly rearranges within the C.fasciculata miniexon locus and encodes a novel RT activity. The purpose of this proposal is to extend and explore these observations, focusing on questions regarding the mechanisms underlying CRE1 genomic rearrangement and the extent of retrotransposon invasion of the Crithidia genome. Accomplishment of the specific aims will advance our knowledge of the mechanisms involved in the replication of this poorly understood but biologically important class of retrotransposons. Characterization of genetic events occurring within the miniexon gene locus has potential therapeutic implications for both African sleeping sickness and Chagas' disease. On a broader scale, the results will be relevant to questions concerning eukaryotic genome organization and evolution, as well as the pathogenesis of human genetic disease. We propose to: (1) Characterize the RT activity encoded by CRE1 both in terms of its functional domains and its primer and template specificity, using both a protein expression system that targets fusion proteins to yeast Ty1 virus-like particles (VLPs) and a genetic assay that results in CRE1 RT-mediated pseudogene formation in yeast; (2) Identify and characterize additional site-specific retrotransposons inserted into the C.fasciculata miniexon array; (3) Determine the genomic organization of CRE1 and related site-specific retrotransposons in the Crithidia miniexon array, and dissect the genetic mechanisms, including site-specific transposition and homologous recombination, by which CRE1 rearranges within the genome of C.fasciculata.