Fructose bisphosphatase of Saccharomyces cerevisiae. Cloning, disruption and regulation of the FBP1 structural gene

Abstract

Fructose bisphosphatase catalyzes a key reaction of gluconeogenesis. We have cloned the fructose bisphosphatase (FBP1) structural gene from Saccharomyces cerevisiae by screening a genomic library for complementation of an Escherichia coli fbp deletion mutation. The cloned DNA expresses in E. coli a fructose bisphosphatase activity which is precipitable with antibodies specific for the yeast enzyme and is sensitive to inhibition by fructose 2,6-bisphosphate. Evidence is presented demonstrating that the entire gene, including all cis-acting regulatory sequences, has been cloned. A substitution mutation that disrupts FBP1 was incorporated into the yeast genome by transplacement to construct a fructose bisphosphatase null mutation. The fbp1 mutant strain is a hexose auxotroph, otherwise growing normally. Southern blot hybridization analysis confirmed the structure of the transplacement and demonstrated that FBP1 is present in single copy in the haploid genome. Northern blot hybridization analysis revealed an mRNA of about 1350 nucleotides, whose presence was repressible by glucose in the medium. Fructose bisphosphatase activity was not greatly overproduced when the FBP1 gene was present on a multicopy vector in yeast.