• The Korean Journal of Mycology
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• v.25 no.1 s.80
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• pp.26-29
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• 1997

Pyrimidine auxotrophic basidiospores of Pleurotus sajor-caju were selected using positive selection method. Wild type basidiospores could not grow on minimal medium containing the pyrimidine analog 5'-fluoro-orotic acid (5'-FOA) whereas pyrimidine auxotrophs grew normally. After treatment of basidiospores with ultraviolet light, a total of 13 pyrimidine auxotrophic basidiospores were isolated among 24 5'-FOA resistant mutants. These mutants require the pyrimidine such as uracil, cytosine, thymine. Mating type group and growth rate of their mutants were determined.

• Journal of Life Science
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• v.21 no.10
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• pp.1370-1376
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• 2011

To study the functional genomic analysis of a crenachaeon Sulfolobus acidocaldarius, we have constructed an auxotrophic mutant based on pyrEF, which encodes the pyrimidine biosynthetic enzymes orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. S. acidocaldarius was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can be selected for mutations in pyrEF genes within a pyrimidine biosynthesis cluster. Spontaneous 5-FOA-resistant mutants by ultraviolet, KH1U and KH2U, were found to contain two point mutations and a frame shift mutation in pyrE, respectively. Mutations at these sites from KH1U and KH2U decreased the activity of orotate phosphoribosyltransferase encoded by the pyrE gene and blocked the degradation of 5-FOA into toxic 5-FOMP and 5-FUMP that kill the cells. Therefore, KH1U and KH2U were uracil auxotrophs. Transformation of Sulfolobus-Escherichia coli shuttle vector pC bearing pyrEF genes from S. solfataricus P2 into S. acidocaldarius mutant KH2U restored 5-FOA sensitivity and overcame the uracil auxotrophy. This study establishes an efficient genetic strategy towards the systematic knockout of genes in S. acidocaldarius.