Identification of novel genes in human genome

描述（由申请人提供）：要全面了解发育和分化过程中基因表达的复杂性，需要鉴定所有mRNA转录本。SAGE（基因表达序列分析）技术是目前实现这一目标的最全面的方法之一。由于SAGE不需要事先了解样本中表达的转录本，因此SAGE可以识别新的转录本。我们已经从造血细胞中鉴定出大量新的SAGE标签。我们对新SAGE标签性质的分析表明，大多数新SAGE标签来自新转录本，其中许多新转录本可能代表人类基因组中未发现的新基因。在他的建议中，我们设定了三个特定目标，从新的SAGE标签开始分离全长新型cdna: specific Aim 1。将人类CD34+干细胞/祖细胞中鉴定的10,000个新的SAGE标签转化为3' cdna；具体目标2。将从Specific Aim 1产生的大约6000个新的3' cdna转化为8000到10000个全长cdna，包括选择性剪接的变体；3.具体目标将Specific Aim 2生成的全长新cdna注释到人类基因组序列上。我们开发了一个高通量系统来实现这些目标。在这个系统中，一个包含14个碱基的SAGE标签被延伸到cDNA的3'端，平均140个碱基可以映射到人类基因组序列。对于显示新颖性的映射序列，我们将使用计算程序first EF预测的假定的第一个外显子来设计一个引物。结合基于3′cDNA设计的反义引物，我们将扩增出以SAGE为代表的全长cDNA，并将其整合到人类基因组中。我们的分析应该揭示大量的新基因、来自新基因或已知基因的新选择性剪接转录物、非编码rna和来自假基因的转录物。本研究的成功将为人类基因组的注释做出重要贡献。为研究正常和异常造血功能提供参考。这些信息对改进基因组注释算法也有一定的参考价值。我们的方法也应该适用于对人类和非人类起源的其他组织的研究。

DESCRIPTION (provided by applicant): A comprehensive understanding of the complexity of gene expression during development and differentiation requires the identification of all of the mRNA transcripts. The SAGE (serial analysis of gene expression) technique is one of the most comprehensive methods presently available to achieve this goal. Because it does not require the prior knowledge of the expressed transcripts present in a sample, SAGE can identify novel transcripts. We have identified a large number of novel SAGE tags from hematopoietic cells. Our analysis of the nature of the novel SAGE tags indicates that most of the novel SAGE tags are derived from novel transcripts, and many of these novel transcripts may represent novel genes not identified in the human genome. In his proposal, we have set three specific aims to isolate full-length novel cDNAs starting from novel SAGE tags: Specific Aim 1. Convert 10,000 novel SAGE tags identified in human CD34+ stem/progenitor cells into 3' cDNAs; Specific Aim 2. Convert about 6,000 novel 3' cDNAs generated from Specific Aim 1 into 8,000 to 10,000 full-length cDNAs including alternatively spliced variants; and Specific Aim 3. Annotate full-length novel cDNAs generated by Specific Aim 2 to the human genome sequence. We have developed a high-throughput system to achieve these aims. In this system, a SAGE tag of 14 bases is extended to the 3' end of the cDNA averaging 140 bases that can be mapped to the human genomic sequences. For the mapped sequences that show novelty, we will use the putative first exon predicted by the computational program First EF to design a sense primer. Together with the antisense primer designed based on the 3' cDNA, we will amplify the full-length cDNA represented by the novel SAGE tag and integrate the information into the human genome. Our analysis should reveal a significant number of novel genes, novel alternatively spliced transcripts originating from novel genes or known genes, non-coding RNAs, and transcripts from pseudogenes. The success of our proposal should make a significant contribution for the annotation of human genome. It should also provide information for studying normal and abnormal hematopoiesis. The information should also be useful for the improvement of genome annotation algorithms. Our approach should also be applicable for studies on other tissues of both human and non-human origins.