Glucose-transport-deficient mutants of Schizosaccharomyces pombe: phenotype, genetics and use for genetic complementation
1994
Milbradt, B. | Hofer, M.
Glucose-transport-deficient mutants of Schizosaccharomyces pombe were obtained by treatment of wild-type cells (972h-) with N-methyl-N'-nitro-N-nitrosoguanidine, and by selection of resulting mutants on gluconate medium containing 0.05% 2-deoxy-D-glucose (2DG). One mutant, designated YGS-B22, was unable to grow on D-glucose and/or D-fructose as a carbon source (Glc/Fru-), and was resistant to 2DG; hence, none of the three sugars was taken up by the mutant cells. The hexokinase activity in the wild-type and the mutant cells was equal. Genetic purification of YGS-B22 by back-crossing with a leucine-auxotrophic mutant and the wild-type resulted in two strains: YGS-4, with reduced 2DG resistance, and YGS-5, which had lost 2DG-resistance. YGS-5 grew in D-glucose-containing media, albeit very slowly. No measurable sugar uptake was detectable in either of the two mutants within the 1 h test interval. Tetrad analyses proved a Mendelian segregation of growth on D-glucose and leucine auxotrophy. However, 2DG resistance did not co-segregate with the Glc/Fru- phenotype, indicating that the transport deficiency and 2DG resistance characters are not encoded on the same genomic locus. Using a genomic bank of Sch. pombe, two transformants, YGS-5-G7 and YGS-5-G12, were found which had regained the wild-type growth and transport phenotype by complementation. Correspondingly, both D-glucose uptake and 2DG accumulation were restored in the transformed strains. Restriction analysis and Northern blots suggested that the G7 genomic fragment and the 4.1 kb SalI restriction fragment of the G12 genomic fragment both contain a complete structural symporter gene.
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