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Previously, we isolated amylase negative mutants from Bacillus subtilis and described the results of genetic analysis of the allelic genes specifying different types of amylase, studied by the employment of transformation (Yuki 1967). Since no marker linked to amy gene was available at that time, the genetic analysis were imperfect.Later, we attempted to find markers linked to amy gene by the transformation technique, and finally discovered two markers of such.By using these markers, fine mapping experiments of amy gene were conducted.The results, with which this paper deals, will be of use in the detailed genetic analysis of amylase production in B. subtilis. MATERIALS AND METHODSStrains of Bacillus subtilis used in this study are listed in Table 1. The composition of the media used has been described in the previous paper (Yuki 1967). To tell amy+ colonies from amy-colonies, agar plates containing 0.2 % starch were used. Extraction of transforming DNA and preparation of competent cells were performed as described previouly (Yuki 1967). For the isolation of mutants, the cells that had been suspended in minimal medium without glucose were treated with 200 tCg/ml of N-methyl-N'-nitro-N-nitrosoguanidine (NG) for 60 minutes at 37°C.The treated cells were plated on nutrient agar plates and, when colonies developed, auxotrophic mutants were isolated by the replica method. When amylase negative mutants were to be isolated, the mutagen-treated cells were plated on nutrient agar plates containing 0.2 % starch.An iodine solution was flooded on the plate when colonies developed, and colonies were searched for amylase negative colonies.

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