GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

我们正在利用中国仓鼠卵巢（CHO）成纤维细胞研究 遗传学和生物化学的某些方面的行为培养 细胞 我们的工作重点是形态学及其与生长的关系 对照和对环AMP的反应。 我们已经分离出了 具有改变的微管的不同突变体，其表达突变的α或 β-微管蛋白亚基，并使用这些突变体来研究纺锤体的形成， 抗微管药物的作用机制。 这些变种人有缺陷 因为突变的微管蛋白存在于纺锤体中， 并干扰其正常功能。 一种β-微管蛋白突变 通过DNA介导的基因转移转移到野生型细胞中， 被扩增，过表达，并使细胞依赖于秋水仙胺， 成长37度 我们还建立了两个细胞系统， 研究AMP可以正向和负向调节细胞的方式 增长 CHO细胞生长受cAMP抑制;选择突变体 对生长抑制的抗性具有缺陷的cAMP依赖性蛋白 激酶。 我们使用了来自携带显性cAMP抗性基因的细胞的DNA， 将cAMP抗性表型转移到敏感细胞的缺陷， 从我们的突变细胞制备的粘粒文库中克隆这些基因 线 由RSV恶性转化的CHO细胞也具有cAMP抗性。 CHO-RSV细胞和人黑色素瘤细胞的肿瘤形成依赖于 在先前的治疗与霍乱毒素，提高cAMP水平内 细胞 这种增加的致瘤性是正调控的一个例子 细胞的生长。

We are utilizing the Chinese hamster ovary (CHO) fibroblast to study the genetics and biochemistry of some aspects of the behavior of cultured cells. Our work has emphasized morphology and its relationship to growth control, and response to cyclic AMP. We have isolated a variety of different mutants with altered microtubules which express mutated Alpha or Beta-tubulin subunits and used these mutants to study spindle formation and the mechanism of anti-microtubule drug action. These mutants are defective in spindle formation because the mutant tubulins are present in spindles and interfere with their normal function. One Beta-tubulin mutation has been transferred by DNA mediated gene transfer to wild-type cells where it is amplified, overexpressed and renders the cells dependent on Colcemid for growth at 37 degrees. We have also established two cell systems for examining the ways in which AMP can positively and negatively regulate cell growth. CHO cell growth is inhibited by cAMP; mutants selected for resistance to growth inhibition have defective cAMP dependent protein kinases. We have used DNA from cells carrying dominant cAMP-resistant defects to transfer the cAMP-resistance phenotype to sensitive cells and are cloning these genes from cosmid libraries prepared from our mutant cell lines. CHO cells malignantly transformed by RSV are also cAMP-resistant. Formation of tumors by CHO-RSV cells and human melanoma calls is dependent on prior treatment with cholera toxin which raises cAMP levels within the cells. This increased tumorigenicity is an example of positive regulation of cell growth by cAMP.