The use of conserved cellulase family-specific sequences to clone cellulase homologue cDNAs from Fusarium oxysporum.
Five cDNAs from the cellulolytic fungi Fusarium oxysporum that code for five distinct cellulase homologues have been cloned and sequenced. The cloning strategy exploited the hydrophobic cluster analysis-based cellulase family classification of Henrissat and Bairoch [Biochem. J. 293 (1993) 781-788] to design degenerate oligodeoxyribonucleotides (oligos) that encoded amino-acid sequences conserved in an intra-family, but not inter-family, manner among cellulases from different species. Polymerase chain reaction (PCR) experiments using F. oxysporum genomic DNA primed with these 'family-specific' oligos were used to rapidly generate PCR fragments which were in turn used to probe cDNA libraries. Two distinct cDNAs coding for cellulase C-family homologues and one cDNA each coding for homologues to the B, F and K families, were isolated in this manner. This approach is an example of the power of multiple sequence analysis to generate cross-species, homology-based probes to rapidly clone homologues in a species of interest.
ZymoGenetics Inc., Seattle, WA 98102.
P O
PO
Sheppard
F J
FJ
Grant
P J
PJ
Oort
C A
CA
Sprecher
D C
DC
Foster
F S
FS
Hagen
A
A
Upshall
G L
GL
McKnight
P J
PJ
O'Hara
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence; D004271:DNA, Fungal; D005670:Fusarium / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D002480:Cellulase