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.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG002600-03
Application #
6801841
Study Section
Special Emphasis Panel (ZRG1-SSS-G (90))
Program Officer
Feingold, Elise A
Project Start
2003-09-19
Project End
2006-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
3
Fiscal Year
2004
Total Cost
$574,527
Indirect Cost
Name
Northshore University Healthsystem Research Institute
Department
Type
DUNS #
154538107
City
Evanston
State
IL
Country
United States
Zip Code
60201
Wang, San Ming; Zhang, Michael Q (2010) Transcriptome study for early hematopoiesis--achievement, challenge and new opportunity. J Cell Physiol 223:549-52
Kim, Yeong C; Wu, Qingfa; Chen, Jun et al. (2009) The transcriptome of human CD34+ hematopoietic stem-progenitor cells. Proc Natl Acad Sci U S A 106:8278-83
Li, Zejuan; Luo, Roger T; Mi, Shuangli et al. (2009) Consistent deregulation of gene expression between human and murine MLL rearrangement leukemias. Cancer Res 69:1109-16
Wu, Qingfa; Kim, Yeong C; Lu, Jian et al. (2008) Poly A- transcripts expressed in HeLa cells. PLoS One 3:e2803
Chen, Jun; Kim, Yeong C; Jung, Yong-Chul et al. (2008) Scanning the human genome at kilobase resolution. Genome Res 18:751-62
Ge, Xijin; Jung, Yong-Chul; Wu, Qingfa et al. (2006) Annotating nonspecific SAGE tags with microarray data. Genomics 87:173-80
Kim, Yeong Cheol; Jung, Yong-Chul; Xuan, Zhenyu et al. (2006) Pan-genome isolation of low abundance transcripts using SAGE tag. FEBS Lett 580:6721-9
Xuan, Zhenyu; Zhao, Fang; Wang, Jinhua et al. (2005) Genome-wide promoter extraction and analysis in human, mouse, and rat. Genome Biol 6:R72
Prasanth, Kannanganattu V; Prasanth, Supriya G; Xuan, Zhenyu et al. (2005) Regulating gene expression through RNA nuclear retention. Cell 123:249-63
Zhao, Fang; Xuan, Zhenyu; Liu, Lihua et al. (2005) TRED: a Transcriptional Regulatory Element Database and a platform for in silico gene regulation studies. Nucleic Acids Res 33:D103-7

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