Flipphi M J, van Heuvel M, van der Veen P, Visser J, de Graaff L H
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, The Netherlands.
Curr Genet. 1993 Dec;24(6):525-32. doi: 10.1007/BF00351717.
Based on amino-acid sequence data from Aspergillus niger alpha-L-arabinofuranosidase B (ABF B), and cyanogen bromide fragments derived thereof, deoxyoligonucleotide mixtures were designed to be employed as primers in a polymerase chain reaction (PCR) on A. niger genomic DNA. This resulted in amplification of three related PCR products. The abfB gene encoding ABF B was isolated from a genomic library using such an amplification product as a probe. A 5.1-kb BamHI fragment was subcloned to result in plasmid pIM991. Upon introduction by co-transformation into both A. niger and A. nidulans uridine auxotrophic strains, pIM991 was shown to contain the functional gene since prototrophic transformants overproduced ABF B upon growth on the inducing carbon source sugar beet pulp. A plate assay was developed enabling quick selection of ABF B-overproducing transformants. The sequence of a 4122-bp long BamHI/SstI fragment was determined. The abfB gene does not contain introns and codes for a protein of 499 amino acids. The mature ABF B, 481 amino acids in length, has a deduced molecular weight of 50.7 kDa. A. niger abfB is the first eukaryotic gene encoding an ABF to be characterized.
基于黑曲霉α-L-阿拉伯呋喃糖苷酶B（ABF B）的氨基酸序列数据及其衍生的溴化氰片段，设计了脱氧寡核苷酸混合物，用作黑曲霉基因组DNA聚合酶链反应（PCR）中的引物。这导致扩增出三种相关的PCR产物。使用这样的扩增产物作为探针，从基因组文库中分离出编码ABF B的abfB基因。将一个5.1 kb的BamHI片段亚克隆，得到质粒pIM991。通过共转化导入黑曲霉和构巢曲霉尿嘧啶营养缺陷型菌株后，pIM991被证明含有功能基因，因为原养型转化体在诱导碳源甜菜浆上生长时过量产生ABF B。开发了一种平板测定法，能够快速筛选出过量产生ABF B的转化体。测定了一个4122 bp长的BamHI/SstI片段的序列。abfB基因不含内含子，编码一个由499个氨基酸组成的蛋白质。成熟的ABF B长度为481个氨基酸，推导分子量为50.7 kDa。黑曲霉abfB是第一个被表征的编码ABF的真核基因。
