Watanabe Y, Matsumoto N, Ohta T, Tsujita T, Niikawa N
Department of Human Genetics, Nagasaki University School of Medicine, Japan.
Jpn J Hum Genet. 1996 Mar;41(1):149-58. doi: 10.1007/BF01892622.
We previously mapped the putative human HYRC (the hyper-radiosensitivity of the scid mutation, complementing gene) to human chromosome 8q11.1 by fluorescence in situ hybridization (FISH) using Alu-based PCR products from a mouse-human scid radiation cell hybrid (RD15/5) as probes. From a cosmid library constructed from RD15/5, 57 cosmid clones containing human DNA inserts were isolated. 18 of which were mapped to 8q11. Based on the sequences of plasmid subclones of the 18 cosmids, five novel sequence-tagged-sites (STSs) were made. By a screening of the CEPH-YAC library with these STSs, five yeast artificial chromosome (YAC) clones were isolated. All these YAC clones were confirmed not to be chimeric by FISH, but two of them showed deleted human insert DNAs. Using the other 3 non-deleted YACs, we constructed a physical map covering the HYRC region. We confirmed that the recently isolated gene (the DNA-PKcs gene) which is a strong candidate for HYRC is located within the present contig and spans less than 200 kb. This map will be useful for the analysis of the genomic structure of the DNA-PKcs gene and for isolation of other complementing genes in the HYRC region.
我们先前通过荧光原位杂交（FISH），以源自小鼠-人scid辐射细胞杂种（RD15/5）的基于Alu的PCR产物作为探针，将假定的人类HYRC（scid突变的超放射敏感性互补基因）定位到人类染色体8q11.1。从由RD15/5构建的黏粒文库中，分离出57个含有人类DNA插入片段的黏粒克隆。其中18个被定位到8q11。基于这18个黏粒的质粒亚克隆序列，制作了5个新的序列标签位点（STS）。通过用这些STS筛选CEPH-YAC文库，分离出5个酵母人工染色体（YAC）克隆。通过FISH证实所有这些YAC克隆均非嵌合体，但其中两个显示人类插入DNA缺失。利用另外3个未缺失的YAC，我们构建了覆盖HYRC区域的物理图谱。我们证实，最近分离出的作为HYRC有力候选基因的基因（DNA-PKcs基因）位于当前重叠群内，跨度小于200 kb。该图谱将有助于分析DNA-PKcs基因的基因组结构以及分离HYRC区域中的其他互补基因。
