Gene cloning and nucleotide sequence analysis of eclosion hormone of the silkworm, Bombyx mori

Research Project

Project/Area Number	62560117
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	製造化学・食品
Research Institution	Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo
Principal Investigator	NAGASAWA Hiromichi Faculty of Agriculture, The University of Tokyo, 農学部, 助手 (60134508)
Co-Investigator(Kenkyū-buntansha)	SUZUKI Akinori Faculty of Agriculture, The University of Tokyo, 農学部, 教授 (90011907)
Project Period (FY)	1987 – 1988
Project Status	Completed (Fiscal Year 1988)
Budget Amount *help	¥1,900,000 (Direct Cost: ¥1,900,000) Fiscal Year 1988: ¥400,000 (Direct Cost: ¥400,000) Fiscal Year 1987: ¥1,500,000 (Direct Cost: ¥1,500,000)
Keywords	eclosion hormone / silkworm / DNA cloning / nucleotide sequence analysis / ペプチドホルモン / 神経ペプチド / アミノ酸配列 / DNAの塩基配列分析
Research Abstract	Eclosion hormone (EH), an insect neuropeptide, triggers behavior of larval, pupal and adult ecdyses. We have purified EH to a homogeneous state from pupal and adult heads of the silkworm, Bombyx mori, and proposed its amino-terminal 61 amino acid residues. However, we could not establish the complete amino acid sequence of EH because of the limitted amount of the purified peptide. In order to estimate the whole amino acid sequence of EH we tried to isolate a genomic DNA encoding for EH. A DNA fraction was prepared from whole bodies of 3rd instar larvae of B. mori. It was digested with Sau3AI and the resulting DNA fragments (9-23 kb) were inserted into EMBL3 phage to construct a genomic library. Three unique oligonucleotide probes (54mer, 33mer and 36mer) and mix probe (17mer) were synthesized according to the amino acid sequence. Screening of 1.2 x 10^5 clones with ^<32>P-labled probes permitted us to find out only one clone, which hybridized with 54mer, 33mer and 17mer probes. A XbaI-EcoRI fragment (380 bp) of this clone, which hybridized with all the three probes, was sequenced by the dideoxy method. A portion of the translated sequence matches exactly with the known peptide sequence, ALa(5)-LYs(61). The DNA sequence of this portion is followed by the sequence of CTT(Leu)-TAA(stop codon). On the other hand, the sequence encoding for Ser(1)-Ile(4) could not be found in this fragment and in addition, there was a sequence similar to the consensus sequence of the intron acceptor just upstream of Ala(5), suggesting that there is an intron between Ile(4) and Ala(5). Thus, EH was estimated to be a 62-residue peptide with a Leu residue at the carboxyl-terminus. We are now searching for the sequence encoding for the signal peptide and EH(1-4).