Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization.
Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization.
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通过高灵敏原位杂交方法定位叙利亚仓鼠染色体中的单拷贝和扩增 CAD 基因。
DOI：
10.1128/mcb.2.3.308-319.1982
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发表时间：
1982
影响因子：
5.3
通讯作者：
Stark,GR
中科院分区：
文献类型：
--
作者：
Wahl,GM;Vitto,L;Padgett,RA;Stark,GR
叙利亚仓鼠细胞对多功能蛋白CAD的天冬氨酸转氨酶活性的特异性抑制剂TON-(膦乙酰基)-L-天冬氨酸(PALA)具有抗性，由于CAD基因的扩增，导致该蛋白的过度生产。我们使用了一种灵敏的原位杂交技术，从几个独立的PALA抗性品系和野生型PALA敏感细胞的中期染色体的扩散中定位CAD基因组。扩增的基因总是在染色体中发现的，通常在B9染色体短臂的扩展区域。在野生型细胞中，CAD基因也位于染色体B9的短臂上。在一个突变系中，在一个扩展的B9染色体中发现了90到100个CAD基因，还有10到15个基因位于几个不同染色体的一条手臂的远端附近。另一株系含有端粒染色体或大染色体片段中的大部分基因。扩增的基因位于染色体区域，经胰酶-Giemsa染色呈带状。在所研究的总染色体中，只有很小一部分观察到了一些双微小染色体。原位杂交是在10%的右旋糖醛硫酸盐500存在的情况下进行的，这使信号增加了100倍。用[125I]dCTP缺口翻译成高比活度的重组DNA质粒，在放射自显影照射1周后，在单个染色体区域发现了10个CAD基因，1个月后可以看到唯一基因的位置。
Syrian hamster cells resistant toN-(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.