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The gaoA gene, encoding the secreted copper-containing enzyme galactose oxidase,
has been isolated from the Deuteromycete fungus Dactylium dendroides. Degenerate
oligonucleotide primers were designed from amino acid sequence data for use in
the polymerase chain reaction. A 1.4-kilobase DNA fragment amplified from
genomic DNA was used to screen a genomic library constructed in ZAP. A strongly
hybridizing clone was rescued as a pBluescript derivative, pGAO9, by in vivo
excision. The sequence of 3466 nucleotides of pGAO9 insert DNA was determined by
progressively designing sequencing primers. The translation product of the
single long open reading frame matches the available galactose oxidase peptide
sequence data, which represents 42% of the residues in the protein. The mature
enzyme has 639 residues, which have been assigned to a 1.7-A electron density
map (Ito, N., Phillips, S. E. V., Stevens, C., Ogel, Z. B., McPherson, M. J.,
Keen, J. N., Yadav, K. D. S., and Knowles, P. F. (1991) Nature 350, 87-90). The
gene lacks introns and encodes an mRNA of approximately 2.5 kilobases with three
transcription initiation start points at least 324 nucleotides upstream of the
translation start site. Multiple ATG codons are present between the
transcription initiation region and the start of the mature protein; two
in-frame ATGs could encode the initiating Met residue to give proteins with 89
or 41 residue N-terminal leader peptides. The shorter potential leader has
N-terminal features characteristic of a secretion signal sequence and may also
contain a pro-sequence processed by an enzyme specific for a monobasic
(arginine) cleavage site, as proposed for other fungal genes. The codon bias of
gaoA is characteristic of other filamentous fungal genes. No significant
homologies exist between galactose oxidase and other protein sequences available
in data bases.
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