Itaya M
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Mol Gen Genet. 1995 Jul 22;248(1):9-16. doi: 10.1007/BF02456608.
A novel approach to the cloning large DNAs in the Bacillus subtilis chromosome was examined. An Escherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome of B. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In the B. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kb B. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in the B. subtilis chromosome and possible applications of this technique are discussed.
研究了一种在枯草芽孢杆菌染色体中克隆大DNA的新方法。将大肠杆菌原噬菌体λDNA（48.5 kb）组装到枯草芽孢杆菌的染色体中。首先将λDNA亚克隆为四个片段，这些片段具有部分重叠区域。通过使用三种不同的DNA整合模式（重叠延伸、坎贝尔型整合和缺口填充）进行连续转化，实现了完整原噬菌体的组装。在枯草芽孢杆菌染色体中，通过重叠延伸利用相邻的DNA片段使DNA得以延伸。通过坎贝尔型整合实现了从一段相邻DNA延伸的一端跳跃到另一段。剩余的缺口通过缺口填充进行封闭。如此组装而成的整合λDNA在非选择性条件下作为4188 kb枯草芽孢杆菌染色体的一部分稳定复制。本方法可用于在枯草芽孢杆菌染色体中容纳更大的DNA，并讨论了该技术的可能应用。
