Ray P N, Siminovitch L, Andrulis I L
Gene. 1984 Oct;30(1-3):1-9. doi: 10.1016/0378-1119(84)90098-2.
In previous reports we have described the isolation and characterization of a number of Chinese hamster ovary (CHO) cell mutants resistant to the amino acid analogue albizziin (Alb). Multistep mutants were derived which showed a high degree of drug resistance and expressed increased levels of asparagine synthetase (AS) levels up to 300-fold over that of the parental cell line. Karyotypic analysis of these mutants revealed homogeneously staining regions (HSRs) usually indicative of gene amplification. In the present work, we provide further proof for gene amplification by showing that the mutants greatly overproduce functional AS mRNA, as evidenced by in vitro translation of purified mRNA and immunoprecipitation of AS. By using these overproducing mutants as sources of mRNA coupled with velocity centrifugation, we have been able to greatly enrich for AS sequences in our mRNA preparations to the point where they represent 1-5% of the total message. This facilitated cloning and selection of the cDNA sequences complementary to the gene. Utilizing these cloned cDNAs, we have demonstrated a correlation between gene copy number and enzyme expression in the parent and Alb-resistant mutants, thus providing direct evidence that drug resistance is due to gene amplification.
在之前的报告中，我们描述了一些对氨基酸类似物合欢氨酸（Alb）具有抗性的中国仓鼠卵巢（CHO）细胞突变体的分离和特性。衍生出了多步突变体，这些突变体显示出高度的耐药性，并且天冬酰胺合成酶（AS）的表达水平比亲代细胞系高出300倍。对这些突变体的核型分析揭示了通常指示基因扩增的均匀染色区（HSR）。在本研究中，我们通过证明突变体大量过量产生功能性AS mRNA，进一步证明了基因扩增，这通过纯化mRNA的体外翻译和AS的免疫沉淀得以证实。通过使用这些过量产生的突变体作为mRNA来源并结合速度离心，我们能够在mRNA制剂中极大地富集AS序列，使其占总信息的1 - ５％。这有助于克隆和选择与该基因互补的cDNA序列。利用这些克隆的cDNA，我们已经证明了亲代和Alb抗性突变体中基因拷贝数与酶表达之间的相关性，从而提供了耐药性是由于基因扩增的直接证据。
