1. The use of conserved cellulase family-specific sequences to clone cellulase homologue cDNAs from Fusarium oxysporum.
- Author
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Sheppard PO, Grant FJ, Oort PJ, Sprecher CA, Foster DC, Hagen FS, Upshall A, McKnight GL, and O'Hara PJ
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular methods, DNA, Fungal, Fusarium enzymology, Molecular Sequence Data, Sequence Homology, Amino Acid, Cellulase genetics, Conserved Sequence, Fusarium genetics
- Abstract
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.
- Published
- 1994
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