| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
1 The mechanism of the killing and cytocidal effects produced by HYI killer toxin from Williopsis saturnus var. saturnus, and by the amphiphilic antibiotics aculeacin A and papulacandin B on yeast Saccharomyces bayanus cells was studied. When the yeast cells were treated with these molecules, a discharze of cell materials at the budding position was observed by phase-contrast and scanning electron microscopy. The cytocidal effect of these molecules was most pronounced when the cells were in the logarithmic growth phase. Washing the HYI toxin incubation mixture completely eliminated the killer activity, but in the case of antibiotics, it only partially reduced the cytocidal activity. Full recovery of the killing activity in the supernatant of the washing solution was observed after HYI toxin incubation, but in the case of the antibiotics, the recovery of cytocidal activity was time-dependent. The activity of membrane β-1, 3-glucan synthase was potently inhibited by HYI toxin, and the concentration of this enzyme in the budding tip was observed. These results suggest that HYI toxin and these antibiotics exert a cytocidal effects on the budding of sensitive yeast cells by inhibiting cell wall synthesis.
2 Each of the four arginine residues in the HM-1 killer toxin was replaced by alanine using site-directed mutagenesis. The polymerase chain reaction (PCR)-constructed mutant sene was successfully expressed in HM-1 toxin resistant S.cerevisiae. Among four HM-1 toxin analogues, R82A HM-1 toxin and R86A HM-1 toxin lost killer activity, while R61A HM-1 toxin and R85A HM-1 toxin retained activity. These results strongly indicate the importance of the arginine residues at positions 82 and 86 which are located in the C-terminal region of the HM-1 toxin for the action of killer activity.
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