Cloning and characterisation of a glucoamylase gene (GlaM) from the dimorphic zygomycete Mucor circinelloides.
This article reports a novel strategy for the cloning of glucoamylase genes using conserved sequences and semi-nested PCR and its application in cloning the GlaM glucoamylase gene and cDNA from the dimorphic zygomycete Mucor circinelloides. The deduced 609-amino-acid enzyme (including signal peptide) is 63% identical to the Rhizopus oryzae raw starch-degrading glucoamylase and is the third glucoamylase reported to have the putative starch-binding domain placed N-terminally. The C-terminal catalytic domain is separated from the starch-binding domain by a serine/threonine-rich linker. An alignment of the cloned gene and cDNA sequences showed that the gene contains three introns. The transcriptional start site and the site of polyadenylation were defined by primer extension and 3'RACE, respectively. The atypical Kozak sequence is identical to the one used in R. oryzae in positions -1 to -4. Northern slot blots revealed that glucoamylase transcription is induced during growth on starch and repressed by glucose. In silico analysis of the 1.9-kb promoter sequence cloned by inverse PCR revealed the presence of several putative regulatory elements, most notably a 19-bp sequence containing six overlapping copies of the Saccharomyces cerevisiae Nrg1p binding sequence.
Houghton-Larsen
J
J
Biomembrane Research Centre, August Krogh Institute, University of Copenhagen, Universitetsparken 13, 2100 OE, Copenhagen, Denmark.
Pedersen
P A
PA
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D005809:Genes, Regulator; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D013728:Terminator Regions, Genetic
D018076:DNA, Complementary; D004271:DNA, Fungal; D005087:Glucan 1,4-alpha-Glucosidase