| Project/Area Number |
63570880
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional basic dentistry
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| Research Institution | Nippon Dental University |
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Principal Investigator |
AOKI Shigeji Nippon Dental University, School of Dentistry at Niigata, General Research Institute. Associate professor, 新潟歯学部, 助教授 (20095045)
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| Co-Investigator(Kenkyū-buntansha) |
ITO-KUWA Shoko Nippon Dental University, School of Dentistry at Niigata, General Research Insti, 新潟歯学部, 助手 (40095063)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1989: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1988: ¥600,000 (Direct Cost: ¥600,000)
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| Keywords | Candida albicans / Petite mutant / Mitochondrial DNA / Restriction map |
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| Research Abstract |
We have been able to isolate respiration-deficient (petite) mutants of the opportunistic fungus Candida albicans. The petite mutants are deficient in cytochrome a and their mitochondria lack cristae. We attempted to compare molecular structures of mitochondrial DNA of a petite mutant (KRD- 19) with those of its parent, wild-type strain (K) In this study. It is important for analysis of mitochondrial DNA to prepare mitochondria free from nuclei. Two methods for isolating mitochondria were examined using the parent strain. Protoplasts were prepared by treatment with Zymolyase, and then (1) mitochondria were purified by repeated differential centrifugation, or (2) mitochondria purified by differential centrifugation were treated with DNase to digest nuclear DNA. DNA extracted by a SDS-phenol method from the above two mitochondrial preparations gave an electrophoretically high molecular, single band, showing both the methods were useful for preparing mitochondrial DNA.
DNA fragments produce
… More
d by digestion of mitochondrial DNA of the strain K with EcoRI, PvuII or EcoRI plus PvuII were almost completely identical to those described previously for mitochondrial DNA of an isolate in USA by Wills (1985), and the restriction map of circular mitochondrial DNA (about 40 kbp) of the wild-type parent strain K was reconstructed.
Mitochondria of the petite mutant KRD-19 were purified according to the above two different methods, and their DNA preparations were analyzed by agarose gel electrophoresis. Mitochondrial DNA before digestion with restriction enzymes showed a broad single band with a low mobility in agarose get. When the mitochondrial DNA was digested with EcoRI or PvuII, most of the high molecular weight DNA disappeared, but DNA fragments were not detected as separated bands. Repeated trials also failed to purify mitochondrial DNA of the petite mutant KRD-19 unfortunately.
These results obtained with the petite mutant suggest that the method developed for isolating mitochondria of the parent strain is not applicable to the petite mutant and its mitochondrial preparations contain nuclei to some extent. Accordingly, we are purifying mitochondrial DNA of the petite mutant using an alternate method: DNA extracted from mitochondria is ultracentrifuged in cesium chloride-density gradient and mitochondrial DNA is separated from nuclear DNA due to difference in the density. It is expected that mitochondrial DNA would be purified and its restriction map would be reconstructed using this method. Less
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