Grill E, Somerville C
MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing 48824.
Mol Gen Genet. 1991 May;226(3):484-90. doi: 10.1007/BF00260662.
A yeast artificial chromosome (YAC) genomic library of Arabidopsis thaliana was constructed in a derivative of the vector pYAC4 which was modified to facilitate the production of end-specific probes for chromosome walking. Experiments in which a subset of 2300 clones from the library were probed with 30 restriction fragment length polymorphism (RFLP) markers indicated that, on the average, the entire genome is represented once in each 800 YAC clones. Thus, the complete library of more than 20,000 YACs is expected to contain most or all of the Arabidopsis genome with a high probability. The YAC clones examined in the sample had an average insert size of approximately 150 kb +/- 10 and represented more than 5% of the Arabidopsis genome. Based on the properties of the library and the currently available RFLP maps for Arabidopsis, only one or two steps from flanking RFLPs should be sufficient to isolate an average gene in Arabidopsis by chromosome walking with the YACs. In order to facilitate chromosome walking, a method for the production of hybridization probes from the ends of the inserts was employed that is based on a combination of pre-amplification of the vector/insert junction sequences via the polymerase chain reaction and specific transcription from T3 or T7 RNA polymerase promoters flanking the cloning site in the YAC vector.
构建了拟南芥的酵母人工染色体（YAC）基因组文库，该文库基于载体pYAC4的衍生物构建，pYAC4经过修饰以利于产生用于染色体步移的末端特异性探针。用30个限制性片段长度多态性（RFLP）标记对文库中2300个克隆的子集进行探测的实验表明，平均而言，每800个YAC克隆中就有一个代表整个基因组。因此，超过20000个YAC的完整文库很有可能包含大部分或全部拟南芥基因组。样本中检测的YAC克隆平均插入片段大小约为150 kb±10，代表了拟南芥基因组的5%以上。基于该文库的特性以及目前可用的拟南芥RFLP图谱，通过用YAC进行染色体步移，从侧翼RFLP只需一步或两步就足以分离拟南芥中的一个平均基因。为了便于染色体步移，采用了一种从插入片段末端产生杂交探针的方法，该方法基于通过聚合酶链反应对载体/插入片段连接序列进行预扩增以及从YAC载体中克隆位点侧翼的T3或T7 RNA聚合酶启动子进行特异性转录的组合。
