GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

我们正在利用中国仓鼠卵巢(CHO)成纤维细胞来研究 培养物行为的某些方面的遗传学和生物化学 细胞。我们的工作强调了形态及其与生长的关系 控制和对环状AMP的反应。我们已经分离出各种不同的 微管改变的不同突变体表达突变的α或 并用这些突变体研究纺锤体的形成和 抗微管药物的作用机制。这些突变体是有缺陷的 因为突变的微管蛋白存在于纺锤体中 并干扰它们的正常功能。一种β-微管蛋白突变 通过DNA介导的基因转移到野生型细胞，在那里它 被扩增、过度表达并使细胞依赖于Colcemid 以37度的速度增长。我们还建立了两个细胞系统，用于 探讨AMP对细胞的正负调节作用 成长。CAMP抑制CHO细胞生长；选择突变体用于 抗生长抑制蛋白具有缺陷的cAMP依赖蛋白 激活剂。我们使用了携带显性cAMP抗性的细胞的DNA 将cAMP抗性表型转移到敏感细胞的缺陷 正在从我们的突变细胞准备的粘粒文库中克隆这些基因 台词。被RSV恶性转化的CHO细胞也是cAMP耐药的。 CHO-RSV细胞和人类黑色素瘤细胞的肿瘤形成是相互依赖的 之前使用霍乱毒素治疗会提高体内cAMP水平 细胞。这种致瘤性的增加是积极调控的一个例子。 CAMP对细胞生长的影响。

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.