Thierry A, Gaillon L, Galibert F, Dujon B
Départment de Biologie Moléculaire (URA 1149 du CNRS and UFR927 Université P.M. Curie), Institut Pasteur, Paris, France.
Yeast. 1995 Feb;11(2):121-35. doi: 10.1002/yea.320110204.
A consortium of European laboratories has been organized to systematically sequence the genome of Saccharomyces cerevisiae. As part of the BIOTECH program aimed at sequencing chromosomes XI and II, we have constructed a total genomic library of yeast strain FY1679 (a direct S288C derivative) into cosmid vectors pWE15 and pOU61cos. Primary clones from four independent libraries totalling 190 genome equivalents have been stored at -80 degrees C. A subset of 1939 independent clones (six genome equivalents) was hybridized using purified chromosomes XI and X as probes. A total of 147 chromosome XI-specific cosmid clones was used to construct the physical map of that chromosome. Mapping methods included a combination of classical bottom-up strategies (fingerprinting, hybridizations) and a novel top-down strategy using I-SceI chromosome fragmentation. The 147 cosmid clones form a unique contig covering the entire chromosome XI (666 kb) with the sole exceptions of the (C1-3A)n repeats of the telomeres. Colinearity of cosmid inserts with yeast DNA was directly verified. A complete EcoRI map of chromosome XI was deduced from partial overlaps of cosmids and used for the sequencing program. Comparison of this map with the genetic map shows unexpected divergences that have been solved by subsequent genetic analysis, yet underline the necessity of independent physical mapping in genome projects.
一个欧洲实验室联盟已组织起来，对酿酒酵母的基因组进行系统测序。作为旨在对第十一条和第二条染色体进行测序的生物技术计划的一部分，我们已将酵母菌株FY1679（S288C的直接衍生物）的全基因组文库构建到黏粒载体pWE15和pOU61cos中。来自四个独立文库的总计190个基因组当量的初级克隆已保存在-80℃。使用纯化的第十一条和第十条染色体作为探针，对1939个独立克隆（六个基因组当量）的一个子集进行杂交。总共147个第十一条染色体特异性黏粒克隆用于构建该染色体的物理图谱。作图方法包括经典的自下而上策略（指纹图谱、杂交）和使用I-SceI染色体片段化的新型自上而下策略的组合。这147个黏粒克隆形成了一个独特的重叠群，覆盖了整个第十一条染色体（666 kb），唯一的例外是端粒的（C1-3A）n重复序列。直接验证了黏粒插入片段与酵母DNA的共线性。第十一条染色体的完整EcoRI图谱是从黏粒的部分重叠推导出来的，并用于测序计划。将该图谱与遗传图谱进行比较，发现了一些意外的差异，这些差异已通过后续的遗传分析得到解决，但也强调了在基因组计划中进行独立物理作图的必要性。
