• The Korean Journal of Mycology
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• v.25 no.2 s.81
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• pp.85-90
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• 1997

The Isozyme activities of Lentinula edodes were studied as a preliminary study for genetic analysis after protoplast fusion. The presence of peroxidase, esterase, superoxide dismutase, acid phosphatase, alkaline phosphatase, alcohol dehydrogenase and ${\alpha}-amylase$ was examined. An intracellular buffer-soluble protein from the mycelia was used for enzyme analysis on nondenaturing polyacrylamide gels. The auxotrophs of Lentinula edodes were positive for peroxidase, esterase, superoxide dismutase and acid phosphatase. However, alkaline phosphatase, alcohol dehydrogenase and ${\alpha}-amylase$ were not detected. The esterase and peroxidase were not affected by the various culture age. Isozyme identification may be a useful tool after protoplast fusion.

• The Korean Journal of Mycology
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• v.14 no.2
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• pp.185-188
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• 1986

After treatment of basidiospores of P. cornucopiae with ultraviolet light, 84 putative mutants from 4671 isolates were obtained. The highest proportion of auxotrophic mutants was obtained from the isolates irradiated to give $0.4{\sim}1.0%$ survival. Fourteen auxotrophs were selected for protoplast fusion and each genetic marker was identified.

• Applied Biological Chemistry
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• v.40 no.3
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• pp.173-177
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• 1997

Bacillus licheniformis 9945a releases a natural ${\gamma}-poly(glutamic\;acid)({\gamma}-PGA)$ into fermentation broth and shows a mucoid phenotype on the solid agar medium. Transformation of mucoid cells of Bacillus species has not been simple and straightforward. The transpositional activity of Tn10 in B. licheniformis also has not been own either. Thus, a spontaneous non-mucoid derivative of the B. licheniformis was obtained first. Shuttle vector pHV1248 containing mini-Tn10 was introduced into the non-mucoid derivative by the method of protoplast transformation. The resulting transformant was reverted to the wild mucoid phenotype, and then mutated randomly with the mini-transposon by heat induction. Auxotrophs requiring arginine, lysine, or tryptophan were isolated by replica plating method. Southern blotting and DNA-DNA hybridzation analysis showed that these auxotrophs were generated by mini-Tn10 insertion into the chromosomal DNA. This method of transformation and mutation using pHV1248 would be useful for the generation of diverse mutants of B. licheniformis 9945a.(Received January 24,1997; accepted March 10, 1997)

• Korean Journal of Microbiology
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• v.25 no.4
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• pp.274-281
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• 1987

The effects of bovine serum albumin, myoinositol and ergosterol on protoplast formation, regeneration and fusion from auxotrophic mutants of Candida pseudotropicalis were examined. Frequency of protoplast formation ranged from 48 to 98% depending on auxotrophic types. When myoinositol (0.5mg/ml) and ergosterol (0.1mg/ml) were supplemented in the medium of cell growth, and bovine serum albumin (4mg/ml)was added to protoplasting buffer, 50-100% of cells were converted to protoplasts. Such a treatment of three additives improved 2.2-3.0 fold of regeneration rate of protoplasts. The fusion frequencies between complementary auxotrophs ranged from $7.0\times 10^{-4}$ to $1.5\times 10^{-3}$ in the optimal conditions. These values showed 1.9-2.3 fold increase when compared with fusion frequencies obtained without the treatment of additives. These results suggested that these comsion frequencies obtained without the treatment of additives. These results suggested that these xompounds may improve protoplast regeneration and fusion between complementary auxotrophs used in this study.

• Archives of Pharmacal Research
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• v.20 no.5
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• pp.448-453
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• 1997

Protoplast fusion between isoleucine-, argihine- and thymidine-requiring auxotroph $(Ile^{-}, Arg^{-}, Thy^{-})$ of Lentinula edodes and arginine-requiring auxotroph $(Arg^-)$ of Coriolus versicolor has been achieved using 30% polyethylene glycol (M.W.4000) in 10 mM $CaCl_2$-glycine solution (pH 8.0). Fusion hybrids were selected in the 0.6 M sucrose supplemented minimal media on the basis of nutritional complementation with fusion frequency of $7.4{\times}10{-6}$ The hybrids included both parental and non-parental types in colony morphology, growth rate and isozyme patterns. We succeeded inter-order protoplast fusion between the auxotrophs of Lentinula edodes and Coriolus versicolor overcoming the natural barriers of incompatibility. We examined the characteristics of the hybrids and clarified the fusion rocess using electron microscopy.

• Journal of Microbiology and Biotechnology
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• v.3 no.2
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• pp.91-94
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• 1993

A 3-isopropylmalate dehydrogenase (3-IPMD) gene was cloned from Kluyveromyces fragilis. pJK104 could complement Escherichia coli leuB and Saccharomyces cerevisiae leu2 auxotrophs. The coding region was subcloned and the nucleotide sequence was determined. A 1.8 Kb EcoRI/SphI fragment of pJK104 subcloned in pUC18 could still complement the leuB mutation. An open reading frame of 1164 bp that corresponds to a polypeptide of 387 amino acids was found in the cloned fragment. The homology between the 3-isopropylmalate dehydrogenase of S. cerevisiae and that of K. fragilis was 68.13％ in nucleotides.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.517.2-517
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• 1986

The regulation of 3 ammonia assimilatory enzymes GDH(glutamate dehydrogenase), GS(glutamine synthetase) and GOGAT (glutamate synthase), have been examined in C. glutamicum for the biosynthesis of glutamate and glutmine. The cell free extracts of 3 kinds of arg, his and trp auxotrophs were investigated the activities of -ketoglutarate dehydrogenase, GDH, GS, and GOGAT on the media cultured with nitrogen excess and limiting conditions. Trp and his howed higher level of glutamate and glutamine than that of parental strain. The inhibition of GS activities by ADP suggested that GS is regulated by energy charge in C. glutamicum. The results with his, trp, glyc, ala, ser, and GMP implied that a system of feedback inhibition were effective. Three enzyme biosynthesis is repressed by nitrogen sources such as trp, pro, glyc, ala, ser and tyrosine.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.583-587
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• 1991

A threonine overproducer, E. coli TF427, which is resistant to threonine analogue, a-amino-(3-hydroxyvaleric acid (AHV), and requires both methionine and isoleucine was developed by the mutations of E, coli W3110 using N-methyl-Nf-nitro-N-nitrosoguanidine (NTG) and UV. The E. coli TF427 produced 46.5 gll of threonine in a 5-L jar fermentor after 44 hr cultivation. The aspartokinase I of TF427 was not inhibited by threonine, and its synthesis was not repressed by threonine plus isoleucine.

• Korean Journal of Microbiology
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• v.22 no.3
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• pp.143-150
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• 1984

The effect of growth inhibitors on colonial growth of cellulolytic fungus, genus Trichoderma, was investigated to develop a method for the effective screening of various auxotrophs and hypercellulolytic strains. As the results, it was revealed that non-ionic detergents such as Sodium deoxycholate and Triton X-100 were the better ones as a growth inhibitor than Oxgall which has been used to restrict the colony size in genus Trichoderma. Each individual colony remained distinct on minimal plate supplemented with 0.05% Triton X-100 for as long as two or more weeks. The screening of 150 to 200 colonies simultaneous on a single plate was possible in the presence of Triton X-100. The effect of Triton X-100 on simultaneous screening was higher than that of Oxgall by a factor of two.

• Current Research on Agriculture and Life Sciences
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• v.5
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• pp.119-126
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• 1987

This study was conducted to elucidate the most appropriate method to obtain auxotrophic mutants from Valsa ceratosperma, the causal fungus of apple canker, which may be used as a gene marker in detecting the transfer of the factors of avirulent strains to virulent strains. Among the 3 kinds of synthetic media tested, each have two formula for minimal and complete, the medium which has been used in study of Endothia parasitica (E. P medium) was turned out to be most appropriate for the growth of V. ceratosperma. A medium for single colony formation from pycnidiospore of this fungus was developed by adding 0.5% L - sorbose to the E. P minimal medium. The period of incubation in dark for preventing the photoreactivation after U. V irradiation was estimated as about 60hrs at which most of the spores become binucleate. Largest number of putative auxotrophs were obtained at about 50second of irradiation to the spores smeared on the medium for single colony formation, at which the survival rate of spores was 5 to 6 percent. With these method developed in this experiment, 161 isolates of putative auxotrophs were detected among which the nutrient requirement for 10 isolates were determined. Five out of 10 mutants were still virulent to apple tree and all but one could not sporulate.