Comprehensive characterization of the Drosophila transcriptome

描述：我们的主要目标是全面鉴定模式生物果蝇的功能转录序列，包括蛋白质编码和非蛋白质编码。我们将提供完整的基因结构的描述，包括转录起始点、多聚腺苷酸化位点和所有可检测到的可选转录本。我们计划调查整个发育过程中具有代表性的时间点，各种组织类型和特征良好的新细胞系。从这些样本中提取的RNA将用于转录组的高分辨率表达谱分析，方法包括全基因组拼接阵列、RNA连接酶介导的cDNA端快速扩增、RT-PCR和cDNA文库构建。RT-PCR将被用来鉴定和分离中小型很少表达的基因的转录本。将使用有针对性的方法来筛选cDNA文库，以识别和分离中到大的转录本。对小RNA(100个碱基)的表征需要创新的策略，我们将使用高分辨率(5个碱基)全基因组拼接阵列和454个测序。与这些研究同时进行的还有一项生物信息学分析，以确定新的未注释基因，这将首次利用合成表达、比较序列和基因预测的算法。此外，我们计划利用体内剪接报告分析、RNAi和计算分析来表征和注释用于产生蛋白质异构体的剪接变异的程度，并识别受调控的替代剪接所需的序列。最后，将使用体内组织培养试验来验证ncRNAs的表达和功能。这些研究的范围是史无前例的，将为任何生物体提供一套最全面的转录实验证据。作为一种公共资源，这些研究是了解正常生长和分化的先决条件，这将有助于了解包括人类在内的其他生物的这些过程。果蝇模型已经被开发出来用于老年痴呆症、神经退行性疾病和癌症。此外，首先被发现在果蝇发育中发挥作用的基因往往是保守的调控网络的组成部分，这些网络在动物发育过程中发挥着重要作用，并已在人类身上被发现有助于各种人类癌症的发展。

DESCRIPTION: Our primary goal is to comprehensively identify the functional transcribed sequences, both protein coding and non-protein coding in the model organism Drosophila melanogaster. We will provide a description of the complete gene structures with transcription start sites, polyadenylation sites, and all detectable alternative transcripts. We plan to survey representative time points throughout development, a wide-variety of tissue types and well-characterized and novel cell lines. RNAs from these samples will be used for high-resolution expression profiling of the transcriptome using whole-genome tiling arrays, RNA ligase mediated Rapid Amplification of cDNA ends, RT-PCR and cDNA library construction. RT-PCR will be used to identify and isolate transcripts for rarely expressed genes of small to medium size. The cDNA libraries will be screened using a targeted approach to identify and isolate medium to large transcripts. Characterization of small RNAs (<100 bp) requires innovative strategies and we will use high-resolution (5bp) whole-genome tiling arrays and 454 sequencing. Concurrent with these studies will be a bioinformatic analysis to identify novel unannotated genes that for the first time utilizes algorithms that synthesize expression, comparative sequence and gene prediction. Further, we plan to characterize and annotate the extent of splice variation used to generate protein isoforms and identify the sequences necessary for regulated alternative splicing utilizing in vivo splicing reporter assays, RNAi and computational analysis. Finally, ncRNAs will be validated using in vivo tissue culture assays for expression and function. The scope of these studies is unprecedented and will provide the most comprehensive set of experimental evidence of transcription for any organism. As a public resource, these studies are a prerequisite for understanding normal growth and differentiation and that will aid in understanding these processes in other organisms, including humans. Drosophila models have been developed for Alzheimers, neurodegenerative diseases and cancer. In addition, genes first identified to play a role in Drosophila development are often components of conserved regulatory networks that play important roles during animal development and have been found, in humans, to contribute to the development of a variety of human cancers.