Digital cloning: Identification of human cDNAs homologous to novel kinases through expressed sequence tag database searching |
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Authors: | Dr. Hua-Chien Chen Hsing-Jien Kung Dan Robinson |
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Affiliation: | (1) Molecular and Genomic Medicine Division, National Health Research Institutes, 128 Yen-Chiu-Yuan Road, Sec. 2, 115 Taipei, Taiwan (ROC);(2) Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA |
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Abstract: | Identification of novel kinases based on their sequence conservation within kinase catalytic domain has relied so far on two major approaches, low-stringency hybridization of cDNA libraries, and PCR method using degenerate primers. Both of these approaches at times are technically difficult and time-consuming. We have developed a procedure that can significantly reduce the time and effort involved in searching for novel kinases and increase the sensitivity of the analysis. This procedure exploits the computer analysis of a vast resource of human cDNA sequences represented in the expressed sequence tag (EST) database. Seventeen novel human cDNA clones showing significant homology to serine/threonine kinases, including STE-20, CDK- and YAK-related family kinases, were identified by searching EST database. Further sequence analysis of these novel kinases obtained either directly from EST clones or from PCR-RACE products confirmed their identity as protein kinases. Given the rapid accumulation of the EST database and the advent of powerful computer analysis software, this approach provides a fast, sensitive, and economical way to identify novel kinases as well as other genes from EST database. |
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Keywords: | EST database Protein kinase YAK STE-20 CDK ERK PKC PKA |
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