GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

环磷酸腺苷（cAMP）在细胞内起重要作用， 分化和恶性转化。我们之前已经展示了 产生中国仓鼠卵巢（CHO）细胞的一系列突变体， 事实上，cAMP对CHO细胞的所有作用都是通过 cAMP依赖性蛋白激酶。显性负突变体影响 这种激酶的调节（RI）亚基很容易在这种 系统，因为全酶cAMP依赖性蛋白激酶通常由 两个RI和两个催化（C）亚基，其中C的活性 RI抑制亚基的表达。任何阻断RI能力的突变 与cAMP相互作用，从而抑制cAMP 解离复合物，释放活性C，或增加 RI和C的联合将作为显性负突变体并阻止 cAMP效应。可以实现cAMP效应的类似消融 使用允许酵母磷酸二酯酶过表达的载体， 在cAMP形成时降解cAMP。为了研究RI与C的相互作用， 在E. 大肠杆菌，并证明了活性，相互作用的亚基可以合成 在这个系统中。多种影响cAMP与RI结合的突变， 在该体系中研究了RI与C相互作用。在 此外，我们还发现RI和C在E.杆菌 导致细菌的生长抑制。这种生长抑制 需要功能性RI和活性C以及cAMP，因此模拟cAMP 对哺乳动物细胞的影响。我们已经用这个系统开始分离 RI的突变体，目的是生成详细的功能和 这个重要的调节蛋白的非功能区域。此外，委员会认为， 我们已经开发了一种突变型RI的表达系统， 在哺乳动物细胞中选择显性负RI突变体。最后， 在转基因小鼠中过度表达酵母磷酸二酯酶的努力已经导致 一个意想不到的发现：我们的转基因小鼠之一， 当作为纯合子繁殖时免疫缺陷，表明插入 免疫系统正常发育所必需的基因失活 系统

Cyclic adenosine-5'-monophosphate (cAMP) plays an important role in cell differentiation and malignant transformation. We have previously shown by generating a series of mutants of Chinese hamster ovary (CHO) cells that virtually all of the effects of cAMP on CHO cells are mediated through cAMP-dependent protein kinase. Dominant negative mutants affecting the regulatory (RI) subunits of this kinase are easily obtained in this system, since holoenzyme cAMP-dependent protein kinase normally consists of two RI and two catalytic (C) subunits in which the activity of the C subunits is inhibited by RI. Any mutation which blocks the ability of RI to interact with cAMP, thereby inhibiting the ability of cAMP to dissociate the complex, which releases active C, or which increases the association of RI and C will act as a dominant negative mutant and block cAMP effects. A similar ablation of the effect of cAMP can be achieved with vectors which allow overexpression of yeast phosphodiesterase, which degrades cAMP as it is formed. To study the interaction of RI with C we have expressed the Chinese hamster recombinant RI and C proteins in E. coli and demonstrated that active, interactive subunits can be synthesized in this system. A variety of mutations which affect cAMP binding to RI and interaction of RI with C have been characterized in this system. In addition, we have discovered that co-expression of RI and C in E. coli results in growth inhibition of the bacteria. This growth inhibition requires functional RI and active C as well as cAMP, and hence mimics cAMP effects in mammalian cells. We have used this system to begin to isolate mutants of RI with the goal of generating a detailed map of functional and non-functional regions of this important regulatory protein. Furthermore, we have developed an expression system for mutant RIs which allows direct selection of dominant negative RI mutants in mammalian cells. Finally, efforts to overexpress yeast phosphodiesterase in transgenic mice have led to an unexpected finding: one of our transgenic mice is severely immunodeficient when bred as a homozygote, suggesting insertional inactivation of a gene essential for normal development of the immune system.