|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1991 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1990 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Five phenotypes of mutants which have a mutation in gene 18 have been reported, namely amber (am), temperature sensitive (ts : grow at 30ﾟC, but not at 42ﾟC), cold sensitive (cs : grow at 37ﾟC, but not at 25ﾟC), heat labile or sensitive (hs) and polyethylene glycol insensitive (CBW). We collected these mutants from original authors and have identified the mutation sites on gene 18. In order to determine the mutation sites, the mutant gene 18 were amplified by the asymmetric PCR method and directly sequenced by the dideoxy method. Mutation sites of 17 am, 3 cs, 3 ts, 3 CBW and 4 hs mutants have thus been identified. Amber mutations were mapped both in N-terminal and in C-terminal half of the molecule. On the other hand, hs and cs mutations which are considered to be related to inter-subunit interactions were mapped in a rather restricted region in the C-terminal half of the molecule except for one hs mutant which was mapped close to the N-terminus of gp18 molecule. Ts mutations were mapped in the N-terminal half of the molecule. For amber mutants, E. coli natural and artificial suppressor strains were used to insert an amino acid (Ser, Gln, Tyr, Ala, Cys, Glu, His, Phe, Pro or Arg) into each amber site and recovery of the function and its temperature dependance were examined. For missense mutants (cs, ts, hs and CBW), the relationship between each phenotype and the amino acid replacement were reinvestigated. The results were all consistent with our model of the tertiary and quartenary structure of gp18, in which the protease-resistant domain (residue numbers between ca. 80 and 320) constitutes the protruding part of the tail sheath and the N-terminal part (ca. 1-80) and the C-terminal half of the molecule (ca. 320 - 658) constitutes the inner part of the tail sheath in contact with other protomers of gp18 and the tube structure.