Brosch R, Gordon S V, Billault A, Garnier T, Eiglmeier K, Soravito C, Barrell B G, Cole S T
Unité de Génétique Moléculaire Bacteriénne, Institut Pasteur, Paris, France.
Infect Immun. 1998 May;66(5):2221-9. doi: 10.1128/IAI.66.5.2221-2229.1998.
The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosis genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of approximately 150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.
细菌人工染色体（BAC）克隆系统能够在大肠杆菌中稳定地扩增大型复杂的DNA插入片段。作为结核分枝杆菌H37Rv基因组测序项目的一部分，构建了一个以pBeloBAC11载体为基础的BAC文库，并将其用于基因组图谱绘制、序列组装确认和测序。该文库包含约5000个BAC克隆，插入片段大小在25至104 kb之间，理论上代表了结核分枝杆菌基因组（4.4 Mb）70倍的覆盖率。共测定了420个BAC的T7和SP6末端的840个序列，并与部分基因组数据库的序列进行了比较。这些序列显示，BAC克隆的估计大小和位置与先前测序的黏粒及由此产生的重叠群的大小和位置之间具有极好的相关性。许多BAC克隆代表了已测序黏粒之间的连接克隆，从而实现了对H37Rv染色体的全面覆盖，目前它们正在H37Rv测序项目的框架内进行鸟枪法测序。此外，未检测到嵌合、缺失或重排的BAC克隆，这对于H37Rv序列的正确图谱绘制和组装至关重要。最小重叠集包含68个独特的BAC克隆，除了一个约150 kb的单一缺口外，覆盖了整个H37Rv染色体。作为一种后基因组应用，标准BAC集被用于一项比较研究，以揭示结核分枝杆菌、牛分枝杆菌和卡介苗巴斯德株之间的染色体多态性，并对结核分枝杆菌中存在但牛分枝杆菌和卡介苗巴斯德株中不存在的一个新的12.7 kb片段进行了表征。该区域包含一组基因，其产物与参与多糖生物合成的蛋白质具有较低的相似性。因此，H37Rv BAC文库为我们提供了一个强大的工具，可用于生成和确认序列数据以及进行比较基因组学和其他后基因组应用。它代表了当前和未来结核分枝杆菌研究项目的一项重要资源。
