Nussbaum R L, Airhart S D, Ledbetter D H
Am J Med Genet. 1986 Jan-Feb;23(1-2):457-66. doi: 10.1002/ajmg.1320230137.
A somatic cell hybrid containing a single human X chromosome bearing the Xq27 fragile site was lethally irradiated and re-hybridized to its HPRT- Chinese hamster parent. One of 24 colonies surviving selection for HPRT was found to have retained human G6PD but not PGK. This line, X3000-11, which shows Xq24-qter translocated to a hamster chromosome by trypsin G-banding and a single human chromatin fragment corresponding to this segment of the X by G-11 staining, expresses the fragile site on exposure to 5-fluorodeoxyuridine. Dot blots using total human DNA suggest that X3000-11 retains approximately 0.2% of the human genome. By Southern blotting, X3000-11 retains Factor IX, DXS11 and DXS42 but lacks DXYS1, DXS3 and DXS17. This hybrid is being used to construct a cosmid library in the vector pCOS2 from which a sub-library of 500-1000 clones of human origin will be isolated using in vivo recombination with cloned Alu and Kpn family repeats. Such a sub-library will greatly facilitate chromosome walking to the fragile site as well as the testing of individual clones for their ability to create a folate-sensitive fragile site by DNA transfer into permissive Chinese hamster recipient cells.
一个含有一条携带Xq27脆性位点的人类X染色体的体细胞杂种被进行致死性照射，然后与它的次黄嘌呤磷酸核糖转移酶缺陷（HPRT-）中国仓鼠亲本重新杂交。在对HPRT进行选择后存活的24个菌落中，有一个被发现保留了人类葡萄糖-6-磷酸脱氢酶（G6PD）但没有磷酸甘油酸激酶（PGK）。这个细胞系X3000 - 11，通过胰蛋白酶G显带显示Xq24 - qter易位到了一条仓鼠染色体上，并且通过G - 11染色显示对应于X染色体这一区段的单个人类染色质片段，在暴露于5 - 氟脱氧尿苷时表达脆性位点。使用总人类DNA进行的斑点印迹表明X3000 - 11保留了大约0.2%的人类基因组。通过Southern印迹分析，X3000 - 11保留了凝血因子IX、DXS11和DXS42，但缺乏DXYS1、DXS3和DXS17。这个杂种正被用于构建载体为pCOS2的黏粒文库，从中将使用与克隆的Alu和Kpn家族重复序列进行体内重组来分离出一个包含500 - 1000个源自人类的克隆的亚文库。这样一个亚文库将极大地促进向脆性位点进行染色体步移，以及通过将DNA转入许可的中国仓鼠受体细胞来测试单个克隆产生叶酸敏感脆性位点的能力。
