Diaz-Perez S V, Alatriste-Mondragon F, Hernandez R, Birren B, Gunsalus R P
Department of Microbiology and Molecular Genetics, University of California Los Angeles, USA.
Microb Comp Genomics. 1997;2(4):275-86. doi: 10.1089/omi.1.1997.2.275.
We have used a variety of methods to characterize the genome of the archaeon Methanosarcina thermophila TM-1. Pulsed-field gel analysis indicates a genome size of 2.8 Mb. We have constructed a bacterial artificial chromosome (BAC) library of M. thermophila and have used it to generate physical maps for this organism. The library is made up of 384 clones with an average insert size of 58 kb representing 8.0 genome equivalents. The utility of the library for low-resolution physical mapping was shown by identifying NotI linking clones and using these to order the NotI macrorestriction fragments of M. thermophila into a 2.8 Mb map. Hybridization of nine single copy genes and a 16S rRNA sequence to these macrorestriction fragments forms the basis for the first genetic map in this organism. High-resolution physical maps, consisting of overlapping clones, have been created using HindIII fingerprints of BAC clones. In this way, we identified a minimal path of five clones that span a 270 kb NotI fragment. The ease of manipulating BAC clones makes the BAC system an excellent choice for the construction of low-resolution and high-resolution physical and genetic maps of archaeal genomes. It also provides a substrate for future genome-sequencing efforts.
我们运用了多种方法来表征嗜热甲烷八叠球菌TM-1的基因组。脉冲场凝胶分析表明其基因组大小为2.8 Mb。我们构建了嗜热甲烷八叠球菌的细菌人工染色体（BAC）文库，并利用该文库为这种生物构建物理图谱。该文库由384个克隆组成，平均插入片段大小为58 kb，代表8.0个基因组当量。通过鉴定NotI连接克隆并利用这些克隆将嗜热甲烷八叠球菌的NotI宏观限制性片段排列成一个2.8 Mb的图谱，展示了该文库在低分辨率物理图谱构建中的实用性。九个单拷贝基因和一个16S rRNA序列与这些宏观限制性片段的杂交构成了该生物首张遗传图谱的基础。利用BAC克隆的HindIII指纹图谱创建了由重叠克隆组成的高分辨率物理图谱。通过这种方式，我们确定了跨越一个270 kb NotI片段的五个克隆的最小路径。BAC克隆易于操作，这使得BAC系统成为构建古菌基因组低分辨率和高分辨率物理图谱及遗传图谱的绝佳选择。它还为未来的基因组测序工作提供了一个基础。
