REARRANGEMENT MECHANISMS OF CRITHIDIA RETROTRANSPOSONS

我们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 在昆虫锥虫Crithidia中分析这种现象 fasciculata，一种原生动物寄生虫，作为该网站的宿主， 特异性反转录转座子CRE 1和CRE 2。CRE-elements是 一个鲜为人知的移动的基因家族，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 这些不同的过程，如基因组进化，假基因形成， 人类遗传疾病的起源，但几乎一无所知， 这些元素转换的机制。CRE 1和CRE 2被插入 在串联排列的小外显子中的一个特定的高度保守位点上， 基因. CRE 1在束草小外显子基因座内快速重排， 编码新的RT活性。这项建议的目的是扩大和 探讨这些意见，侧重于有关机制的问题， 潜在的CRE 1基因组重排和逆转录转座子的程度 短尾虫基因组的入侵 具体目标的实现将促进我们对 这种复制机制知之甚少， 生物学上重要的一类反转录转座子。表征 小外显子基因座内发生的遗传事件可能 对非洲昏睡病和南美锥虫病的治疗意义 疾病在更广泛的范围内，研究结果将与以下问题相关： 关于真核生物基因组的组织和进化，以及 人类遗传性疾病的发病机制。 我们建议：（1）表征由CRE 1编码的RT活性， 就其功能结构域及其引物和模板特异性而言， 使用靶向融合蛋白的蛋白表达系统， 酵母Ty 1病毒样颗粒（VLP）和导致 酵母中CRE 1 RT介导的假基因形成;（2）鉴定和 表征插入到细胞中的另外的位点特异性反转录转座子， （3）确定簇毛麦的基因组结构 Crithidia miniexon中的CRE 1和相关位点特异性反转录转座子 阵列，并剖析遗传机制，包括特定地点 通过转座和同源重组，CRE 1重新排列 在C. fasciculata的基因组中。

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.