Wahl G M, Vitto L, Padgett R A, Stark G R
Mol Cell Biol. 1982 Mar;2(3):308-19. doi: 10.1128/mcb.2.3.308-319.1982.
Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.
叙利亚仓鼠细胞对N-（膦酰基乙酰基）-L-天冬氨酸（PALA）具有抗性，PALA是多功能蛋白CAD中天冬氨酸转氨甲酰酶活性的特异性抑制剂，由于CAD基因的扩增，这些细胞过量产生该蛋白。我们使用了一种灵敏的原位杂交技术，在来自几个独立的PALA抗性品系和野生型PALA敏感细胞的中期染色体铺片中定位CAD基因组。扩增的基因总是在染色体内发现，通常位于B9染色体短臂的一个扩展区域。在野生型细胞中，CAD基因也位于B9染色体的短臂上。在一个突变品系中，在一条扩展的B9染色体上发现了90至100个CAD基因，在几个不同染色体的一条臂的远端附近还发现了另外10至15个。另一个品系的大多数基因存在于端粒染色体或大的染色体片段中。扩增的基因位于经胰蛋白酶-吉姆萨染色呈带状的染色体区域。在所检查的全部铺片的极小部分中观察到了一些双微小染色体。原位杂交是在10%硫酸葡聚糖500存在的情况下进行的，它可使信号增强多达100倍。使用用[125I]dCTP缺口平移至高比放射性的重组DNA质粒，放射自显影片曝光1周后可显示单个染色体区域中的10个CAD基因，1个月后可看到单一基因的位置。
