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48. Construction of EST Database for Genome Analysis of Bombyx mori
Kazuei Mita, Mitsuoki Morimyo, Kazuhiro Okano1, Susumu Maeda1 and Toru Shimada2
(1RIKEN; 2Univ. of Tokyo)
Keywords: cDNA library, Bombyx mori, EST database, multi-cellular organism
The silkworm, Bombyx mori, is an established model system for the study of insect physiology, biochemistry, development, metamorphosis and so on. Although numerous strains have been isolated, molecular biological information on the silkworm genome is limited. In order to study the gene expression patterns in the major organs of the silkworm, we initiated the construction of a cDNA database. This cDNA project is the first step in the genome analysis of Bombyx mori.
The gene expression patterns significantly depend on tissues as well as developmental stages in multi-cellular organisms, 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 cloning method. 1000 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 public protein databases. 1000 cDNA clones are enough to configure the abundantly expressed genes in the tissue from which the cDNA library is constructed, and the 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 cDNA catalog is to configure all members of related genes and display the whole pathway that the cells (or tissues) employ.
So far, we have performed the analyses of 22 cDNA libraries: a culture cell (as a control), baculovirus-infected culture cell (2h post-infection, 6h p.i. and 12h p.i.), male and female fat bodies at the 5th instar, the 5th instar midgut, the brain (adult), the prothoracic gland at the 5th instar, the wing disc at the 5th instar day 4, the beginning of spinning and 2 days after spinning, pheromone gland (adult), the testis of the 5th instar larva and pupa, the embryo at 40 h and 96 h after fertilization and so on. We determined more than 16,000 cDNAs, and identified 6,000 genes that will cover a third of the total genes of Bombyx mori.
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 11 times redundancy. The genome size of B. mori was estimated to be 530 Mb. If 6,200 independent ESTs are available, one BAC clone 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 initiated the construction of BAC contigs by hybridization using independent ESTs as probes.
Publications:
Mita, K., Morimyo, M., Okano, K., Shimada, T. and Maeda, S.: RIKEN Review, 22, 63-67, 1999.
