TKL2, a second transketolase gene of Saccharomyces cerevisiae. Cloning, sequence and deletion analysis of the gene.
Transketolase activity is indispensable for the generation of erythrose 4-phosphate and therefore necessary for the biosynthesis of the aromatic amino acids. Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells. Here we ... describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL2 demonstrates 71% identity with TKL1 [Sundstroem, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity. Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti-transketolase serum in Western blot analysis. In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids.
Mesh Terms:
Amino Acid Sequence, Blotting, Western, Cloning, Molecular, Gene Deletion, Gene Expression, Genes, Fungal, Molecular Sequence Data, Mutagenesis, Restriction Mapping, Saccharomyces cerevisiae, Sequence Analysis, Transformation, Genetic, Transketolase
Amino Acid Sequence, Blotting, Western, Cloning, Molecular, Gene Deletion, Gene Expression, Genes, Fungal, Molecular Sequence Data, Mutagenesis, Restriction Mapping, Saccharomyces cerevisiae, Sequence Analysis, Transformation, Genetic, Transketolase
Eur. J. Biochem.
Date: Oct. 01, 1993
PubMed ID: 7916691
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