Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha.
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M(r) of 49400. The encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
Rhein Biotech GmbH, Düsseldorf, Germany.
A
A
Phongdara
A
A
Merckelbach
P
P
Keup
G
G
Gellissen
C P
CP
Hollenberg
D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000908:Antibodies, Fungal / Q000096:biosynthesis; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry; D011817:Rabbits; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000908:Antibodies, Fungal; D018076:DNA, Complementary; D012333:RNA, Messenger; D000135:Acid Phosphatase