Sheppard P O, Grant F J, Oort P J, Sprecher C A, Foster D C, Hagen F S, Upshall A, McKnight G L, O'Hara P J
ZymoGenetics Inc., Seattle, WA 98102.
Gene. 1994 Dec 2;150(1):163-7. doi: 10.1016/0378-1119(94)90878-8.
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.
已克隆并测序了来自纤维素分解真菌尖孢镰刀菌的五个cDNA，它们编码五种不同的纤维素酶同源物。克隆策略利用了基于疏水簇分析的纤维素酶家族分类方法（由Henrissat和Bairoch提出[《生物化学杂志》293卷（1993年）781 - 788页]）来设计简并寡脱氧核糖核苷酸（寡核苷酸），这些寡核苷酸编码的氨基酸序列在不同物种的纤维素酶中以家族内而非家族间保守的方式存在。使用这些“家族特异性”寡核苷酸作为引物，以尖孢镰刀菌基因组DNA进行聚合酶链反应（PCR）实验，快速生成PCR片段，这些片段进而用于探测cDNA文库。通过这种方式，分离出了两个编码纤维素酶C家族同源物的不同cDNA，以及各一个分别编码B、F和K家族同源物的cDNA。这种方法是多重序列分析强大功能的一个实例，它能够生成跨物种的、基于同源性的探针，从而快速克隆感兴趣物种中的同源物。
