37. Bombyx EST Database, 'SilkBase', for Genome Analysis of Bombyx mori
Kazuei Mita, Mitsuoki Morimyo Yoshiko Koike1, Junko Nohata, Masataka Suzuki1, Kazuhiro Okano2 Susumu Maeda2 and Toru Shimada1 (1Univ. of Tokyo; 2RIKEN)
Keywords: cDNA library, Bombyx mori, EST database, multi-cellular organism, BAC library
Aiming at the genome analysis of Bombyx mori, we are constructing the EST database through the analysis of cDNA libraries. The gene expression patterns significantly depend on tissues as well as developmental stages in multi-cellular organnsms, unlike the case of uni-cellular organisms such as yeast. The cDNAs from which the ESTs are derived are present in libraries in proportion to the level of mRNA in the tissues from which the libraries were prepared. Thus, ESTs are subject to 'expression bias' for multi-cellular organisms. Therefore, we took the following strategy: cDNA libraries of various tissues (and different'stages) were constructed by the directional atoning method. 1,000 cDNA clones were chosen at random from each library and around 700-base nucleotide sequences from the 5 end of the cDNA were determined, followed by gene identification with a protein homology search in publie protein databases. Random sequencing of approximately 1,000 cDNA clones is effective to configure the abundantly expressed genes in the ttssue from which the cDNA library is constructed, and analyses of various tissues (and different stages) will provide a sufficient amount of ESTs for genome analysis. In addition, this approach explicitly represents the gene expression patterns of all genes identified. Another advantage of the eDNA catalog is to figure out all members of related genes and display the whole pathway that the cells (or tissues) employ.
So far we have determined over 20,000 cDNAs derived from 29 cDNA libraries and identified more than 7,600 independent ESTs which cover about 40% of the total genes of B. mori. All ESTs are compiled into 'SilkBase' at the WEB site http://www.ab.a.u-tokyo.ac.jp/silkbase/
Recently, we have succeeded in the construction of a high quality B. mori BAC library in collaboration with Dr. Pieter de Jong's group at Roswell Park Cancer institute. Its average insertion size was estimated to be 168 kb with 1 1 times redundancy. The genome size of B. mori was estimated to be 530 Mb. If 6,200 independent ESTs are available, one BAC alone will have 2-3 EST markers on an average. Therefore, more than 6,200 independent ESTs are needed for the construction of BAC contigs. We proceeded the construction of BAC contigs by hybridization using independent ESTs as probes.
Publications:
1)Mita, K., Morimyo, M., Okano, K., Shimada, T and Maeda, S.: RIKEN Review, 22, 63-67, 1999
2)Lee, A. J., Hahn, Y., Yun, J. H., Mita, K. and Chung, J. H.: Biochim. Biophys. Acta 1491, 355363, 2000 3)Yoshiga, T., Okano, K., Mita, K., Shimada, T and Matsumoto, S.: Gene in press.