Some bacteria belonging to the genera Arthrobacter, Brevibacterium and Corynebac-.terium, so-called glutamic acid-producing bacteria, are highly important as they are used for the industrial fermentation production of various amino acids. The genetic breeding of such amino acid-producing strains has so far been carried out by conventional mutation and selection methods. After a phenol treatment of the supernatant, DNAwas recovered as ethanol precipitation. These DNA samples were analysed by agarose gel electrophoresis with a Tris-acetate buffer system as described by Sharp et al.7) As a result of the screening, two low-molecular-weight plasmids were found. One was found in Brevibacterium lactofermentum ATCC13869
Recentlyand named pAM330.The other was found in Corynebacterium glutamicum ATCC1 3058 and named pHM1519. They were purified.by the CsCl-EtBr equilibrium density gradient centrifugation method, and were confirmed to exist as closed circular molecules. The molecular weights of pAM330 and pHM1519 were determined as 3.0 Md and 1.8 Md, respectively, from electrophoretic mobility in a 0.8% agarose gel. Restriction enzyme analysis was carried out on those plasmids, and the results are shown in Fig. 1. The contents of pAM330 and pHM1519 were roughly analysed by the agarose gel electrophoresis method8'9* as follows: Cells were cultured in 4ml of the CM2Gmediumto the exponential growth phase, and 600 jig/ml of ampicillin was added to the culture medium to ensure the lysozyme digestion. The cells were harvested at 2.5 hr after the addition of ampicillin, and were lysed with lysozyme (lOmg/ml, 37°C 2hr) and SDS (4%). The lysis was completed by heating at 65°C for lOmin, followed by phenol extraction and ethanol precipitation. The DNAsamples were dissolved in TEN buffer and treated with RNase (20/ig/ml, 37°C fir 20min) and pronase (100/^g/ml, 37°C for 20min) sequentially.After phenol extraction and ethanol precipitation, the recovered DNAwas dissolved in TENbuffer, and electrophoresed in a 0.8% agarose gel. The photographs of the gel were taken under irradiation by ultra-violet light, and the negative films of them were analysed by a densitometer (Shimadzu CS-910). As a result, the amounts ofpAM330 and pHM1519 were approximately 1.4% and 9.1% of the total DNA, respectively.To estimate the
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