Gnirke A, Barnes T S, Patterson D, Schild D, Featherstone T, Olson M V
Department of Genetics, Washington University School of Medicine, St Louis, MO 63110.
EMBO J. 1991 Jul;10(7):1629-34. doi: 10.1002/j.1460-2075.1991.tb07685.x.
Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC.
分离出了两个酵母人工染色体（YAC），每个都含有编码包含磷酸核糖甘氨酰胺合成酶（GARS）、磷酸核糖甘氨酰胺甲酰基转移酶（GART）和磷酸核糖氨基咪唑合成酶（AIRS）的三功能蛋白的人类基因的全长拷贝。通过限制性图谱分析以及将包含YAC末端的黏粒亚克隆与人类中期染色体进行原位杂交，对这些YAC进行了表征。其中一个YAC包含共克隆的非连续DNA，而另一个似乎有一个来自21q22.1（GART基因的位置）的600kbp单一插入片段。从两个共同跨越40kb基因的黏粒亚克隆中获得了该基因的限制性图谱。当将YAC DNA转入GARS缺陷型或GARS和AIRS双缺陷型中国仓鼠卵巢细胞时，该基因具有功能。基因转移通过使用纯化酵母DNA的脂质体转染以及酵母原生质体与仓鼠细胞之间的融合来进行。对嘌呤营养缺陷型由YAC互补的细胞系的DNA进行限制性分析表明，无论采用哪种方法，都可以转移该基因的完整且未重排的拷贝。大多数融合细胞系似乎至少含有80%的YAC。
