9. 3  Exploring the Mechanisms of DNA Damage Repair with an Extremely Radioresistant Bacterium
 

Fig. 9-5 Electron microscopic image of the extremely radioresistant bacterium, D. radiodurans D. radiodurans is a bacterium which shows about 1000 times greater radiation tolerance than that of a human cell.

Fig. 9-6 Disruption of a specific gene with the KatCAT cassette In order to disrupt the specific gene of D. radiodurans , a gene cassette KatCAT was constructed. The gene cassette is made from a combination of the drug tolerance enzyme gene (cat) from E. coli and the gene expression signal sequence (katA promoter) from D. radiodurans . To disrupt the target gene, we constructed an artificial unfunctional gene by inserting the KatCAT cassette into the structural gene of the target sequence. The constructed gene was transfected into the D. radiodurans to replace the normal target gene with an unfunctional one.

Fig. 9-7 Prediction of another radiosensitive mutation in the radiosensitive mutant strain KR4128 The recN disrupted strain exhibited higher radiotolerance than the radiosensitive mutant strain KR4128. This result indicates that the mutant KR4128 has a mutation other than that of the recN gene.

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Various ventures of post-genomic research are now in progress, which solve the function of genes on the basis of the genomic information acquired from a series of genome projects that started in the second half of the 1990s. The complete genome sequence of Deinococcus radiodurans (D. radiodurans), which we are studying, was also determined (Fig. 9-5). Before completion of the genome project, we examined prospective strategies for post-genomic research, and then established a method for disrupting the specific gene of D. radiodurans with the objective of analyzing the function of genes discovered during the genome project. The first step in disrupting the target gene is to construct an artificial unfunctional gene using a drug-resistant gene cassette (Fig. 9-6). The unfunctional gene so constructed was then transfected into the bacterial cell, and recombined with the normal (functional) target gene in the bacterial genome. Our results indicate that this procedure provides a high efficiency method for obtaining a specific gene-disrupted strain of D. radiodurans . With this method, we have discovered that the radiosensitive mutant strain KR4128 has another mutation in the DNA repair gene apart from recN. This technique will enable us to analyze the function of genes one by one through disruption of the D. radiodurans genes whose functions are still unknown. We believe that through this analysis we will soon be able to discover the entire mechanism of DNA tolerance of D. radiodurans . Results from this work can be utilized for applications to radioprotection and biotechnology.

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Reference T. Funayama et al., Identification and Disruption Analysis of recN Gene in the Extremely Radioresistant Bacterium Deinococcus radiodurans, Mutat. Res. DNA Repair, 435, 151 (1999).

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Persistent Quest - Research Activities 2001 Copyright(c) Japan Atomic Energy Research Institute