Tauch Andreas, Homann Iris, Mormann Sascha, Rüberg Silvia, Billault Alain, Bathe Brigitte, Brand Sven, Brockmann-Gretza Olaf, Rückert Christian, Schischka Natalie, Wrenger Carsten, Hoheisel Jörg, Möckel Bettina, Huthmacher Klaus, Pfefferle Walter, Pühler Alfred, Kalinowski Jörn
Zentrum für Genomforschung, Universität Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany.
J Biotechnol. 2002 Apr 25;95(1):25-38. doi: 10.1016/s0168-1656(01)00443-6.
The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.
谷氨酸棒杆菌基因组计划的初始策略是对有序黏粒文库的重叠插入片段进行测序。利用约28个基因组当量的高密度菌落网格，通过Southern杂交鉴定重叠克隆。总共组装了18个连续的基因组片段，包含95个重叠黏粒。对组装好的黏粒集进行系统鸟枪法测序发现，黏粒文库仅覆盖了谷氨酸棒杆菌基因组的2.84 Mb（86.6%）。为了获得完整的基因组覆盖，在pBeloBAC11中构建了谷氨酸棒杆菌染色体的细菌人工染色体（BAC）文库，并用于基因组图谱绘制。BAC文库由3168个BAC组成，理论上覆盖谷氨酸棒杆菌基因组（3.28 Mb）63倍。用PCR扩增的染色体标记对2304个BAC克隆进行Southern筛选，随后对插入片段末端进行测序，从而鉴定出119个覆盖谷氨酸棒杆菌整个染色体的BAC。代表100%基因组覆盖的最小集合包含44个独特的BAC克隆，平均重叠22 kb。共有21个BAC代表了先前测序的黏粒重叠群之间的连接克隆，为完成谷氨酸棒杆菌的基因组序列提供了有价值的工具。
