Cloning and molecular analysis of cDNA encoding a carboxymethylcellulase of the yeast Cryptococcus flavus.
A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeast Cryptococcus flavus was cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35,698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with the N-terminal amino acid sequence of secreted CMCase 1. No potential N-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that the C. flavus CMCase 1 is partially homologous to the Trichoderma reesei endoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced into E. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Japan.
Z
Z
Cui
D
D
Mochizuki
Y
Y
Matsuno
T
T
Nakamura
Y
Y
Liu
T
T
Hatano
S
S
Fukui
T
T
Miyakawa
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002480:Cellulase; D003001:Cloning, Molecular; D003062:Codon; D003454:Cryptococcus / Q000201:enzymology*; D004271:DNA, Fungal / Q000302:isolation & purification; D004926:Escherichia coli; D006026:Glycoside Hydrolases / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D003062:Codon; D004271:DNA, Fungal; D006026:Glycoside Hydrolases; D002480:Cellulase; C019826:carboxymethylcellulase