Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa.
A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suco and N. crassa inv strains transformed with pNC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suco (pNC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa, although S. cerevisiae suc+ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI-restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv- to Inv+ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.
Carú
M
M
Departmento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago.
Cifuentes
V
V
Pincheira
G
G
Jiménez
A
A
D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000235:genetics*; D005779:Immunodiffusion; D009491:Neurospora / Q000235:genetics*; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014170:Transformation, Genetic; D043324:beta-Fructofuranosidase
D004271:DNA, Fungal; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase