The P. stipitis XDH gene, XD, was also amplified by PCR. The forward primer
59-TCTAGACCACCCTAAGTCG-39 and the reverse primer 59-GGATCCACT
ATAGTCGAAG-39 were used to amplify the intact XD gene (with its original 59
and 39 control sequences), and the forward primer 59-CACACAATTAAAATG
A-39 and the reverse primer 59-GGATCCACTATAGTCGAAG-39 were used to
amplify the promoterless XD gene (without the 59 noncoding sequence) from the
Pichia chromosomal DNA by using the previously published sequence of the
Pichia XD gene (20). The amplified XD gene was first cloned and stored in
pUC19. The original 59 control region of XD was subsequently replaced by the
intact 59 control region (positions 21 to 2911 of the noncoding sequence)