Huang M E, Chuat J C, Thierry A, Dujon B, Galibert F
Laboratoire de Biochimie et Biologie Moléculaire, Unité de Recombinaisons Génétiques (UPR 41-CNRS), Faculté de Médecine, Rennes, France.
DNA Seq. 1994;4(5):293-300. doi: 10.3109/10425179409020855.
We report here the construction of a complete physical map of the chromosome X of yeast Saccharomyces cerevisiae. Fragments resulting from partial Sau3AI digestion of DNA from a diploid strain derived from S288C were ligated to linearized pWE15, a cosmid vector with T3 and T7 promoters. Another library, made in the cosmid vector pOU61 cos, that lacks T3 and T7 promoters, was also used as a source of target clones. Chromosome-X-specific clones were sorted out by hybridization with radiolabelled pulse-field-gel-purified chromosome X as a probe. Then, 254 cosmids were ordered by walking from one to another by hybridization with end-specific T3 or T7 RNA transcripts as probes. The construction was put to the test by hybridization with a battery of chromosome X gene markers, that showed that the physical map and the genetic map were colinear. The validity of the contig was further strengthened by the results of chromosome nested fractionation with meganuclease I-SceI. An EcoRI restriction map of the contig enabled further verification and measurement of the total length of the contig, that was found to be approximately 700 kb in size. In addition to providing a base for the ongoing yeast genome sequencing project, the physical map can be used to map any sequence belonging to chromosome X.
我们在此报告酿酒酵母X染色体完整物理图谱的构建。对源自S288C的二倍体菌株的DNA进行部分Sau3AI消化产生的片段，与线性化的pWE15连接，pWE15是一种带有T3和T7启动子的黏粒载体。另一个用缺乏T3和T7启动子的黏粒载体pOU61 cos构建的文库，也用作目标克隆的来源。通过与放射性标记的脉冲场凝胶纯化的X染色体作为探针杂交，筛选出X染色体特异性克隆。然后，以末端特异性T3或T7 RNA转录本作为探针，通过杂交从一个克隆依次步移到另一个克隆，对254个黏粒进行了排序。通过与一系列X染色体基因标记杂交对构建结果进行检验，结果表明物理图谱和遗传图谱是共线的。巨核酸酶I-SceI的染色体嵌套分级分离结果进一步强化了重叠群的有效性。重叠群的EcoRI限制酶图谱能够进一步验证和测量重叠群的总长度，发现其大小约为700 kb。除了为正在进行的酵母基因组测序项目提供基础外，该物理图谱还可用于定位属于X染色体的任何序列。
