Kamoda S, Saburi Y
Department of Forest Products, Faculty of Agriculture, University of Tokyo, Japan.
Biosci Biotechnol Biochem. 1995 Oct;59(10):1866-8. doi: 10.1271/bbb.59.1866.
A genomic DNA library of Pseudomonas paucimobilis TMY1009 was constructed using a cosmid vector pWE15. Screening of the library for lignostilbene-alpha,beta-dioxygenase (LSD) isozyme genes was done with a common probe for the alpha, beta, and gamma subunits, which composed LSD isozymes. The positive clones obtained by colony hybridization were further confirmed by Southern hybridization. A 4.2-kb BamHI-HindIII fragment hybridized with the probe was subcloned into pUC118 to yield the plasmid pKHE1700. Escherichia coli MV1184 carrying pKHE1700 produced one of the LSD isozymes. The cloned LSD was purified and compared with LSD isozymes from P. paucimobilis TMY1009. The behavior on column chromatographies through all purification steps accorded with that of LSD-III. Furthermore, the mobility on polyacrylamide gel electrophoresis, the elution profile on reversed-phase HPLC, and the partial amino acid sequence were common to the cloned LSD and the native LSD-III. Thus the cloned LSD could be identified as LSD-III. It was found that the gene of LSD-III (lsdB) was composed of 489 codons.
使用黏粒载体pWE15构建了少动假单胞菌TMY1009的基因组DNA文库。用组成木质素芪α,β -双加氧酶（LSD）同工酶的α、β和γ亚基的通用探针筛选该文库中的LSD同工酶基因。通过菌落杂交获得的阳性克隆进一步通过Southern杂交进行确认。将与探针杂交的4.2 kb BamHI - HindIII片段亚克隆到pUC118中，得到质粒pKHE1700。携带pKHE1700的大肠杆菌MV1184产生了一种LSD同工酶。对克隆的LSD进行纯化，并与来自少动假单胞菌TMY1009的LSD同工酶进行比较。在所有纯化步骤中，柱色谱行为与LSD - III一致。此外，克隆的LSD和天然LSD - III在聚丙烯酰胺凝胶电泳上的迁移率、反相高效液相色谱上的洗脱图谱以及部分氨基酸序列均相同。因此，克隆的LSD可被鉴定为LSD - III。发现LSD - III（lsdB）基因由489个密码子组成。
