1988 Fiscal Year Final Research Report Summary
Induction of Gene Amplification on the Chromosome of Bacillus subtilis and Its Application
| Project/Area Number |
62470121
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵・醸造
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| Research Institution | The University of Tokyo |
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Principal Investigator |
YAMASAKI Makari Faculty of Agriculture, the University of Tokyo, 農学部, 教授 (60011889)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Bacillus subtilis / Gene Amplification / Tunicamycin Resistance Gene / ツニカマイシン |
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| Research Abstract |
We discovered a gene amplification occurred on the chromosome of Bacillus subtilis in the course of the study of a tunicamycin-resistant and amylase hyper-producing mutant. We found that the same gene amplification can be induced on the chromosome of the recipient bacteria by competence transformation and revealed the essential structure of the transforming DNA fragment. The objectives of this study were to induce amplification of foreign genes on the chromosome of B. subtilis and to analyse the function of the tunicamycin-resistant gene, tmrB,used as the selective marker of gene amplification.
In the first year, we aimed to induce amplification of foreign genes. We have cloned a 6.4kb EcoRI fragment which contains the tmrB gene and a part of the amyE gene for alpha-amylase. This fragment can induce gene amplification by competence transformation. We inserted the chloramphenicol acetyltransferase gene, cat, of the plasmid pC194 in the middle of the 6.4kb EcoRI fragment. We transformed a amyE07 mutant with this fragment and selected amylase-positive and tunicamycin-resistant transformants. These transformants had about 20 copies of the cat gene on the chromosome and were highly resistant to chloramphenicol (40 mcg/ml).
In the last year, we aimed to analyze the possible function of the tmrB gene. We determined the initiation base of transcription of tmrB gene by Sl mapping and confirmed that the gene is actually transcribed. We examined if any degradation or modification of tunicamycin occurred in the resistant mutants but failed to find any significance. The target enzyme of tunicamycin also had any difference between the resistant and sensitive cells.
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