CLONING OF THE HUMAN RECEPTOR FOR ALPHA-2-INTERFERON

干扰素具有许多生物功能，包括 诱导分化，减少细胞生长，逆转 几种癌基因的作用；它们的作用是负生长 监管者。尽管肿瘤细胞上存在干扰素受体 而许多肿瘤外植体，大多数癌细胞都不生长-- 受到干扰素的抑制，表明有能力绕过 α2-干扰素等负性生长因子的作用 可能是恶变的重要组成部分。至 了解干扰素的作用机制，我们建议 克隆编码α2干扰素受体的人类基因。 我们将首先从培养物中提纯膜受体 Daudi(或HL-60，见第26页)细胞的亲和力 层析；我们将制备干扰素亲和柱 使用重组人α2-干扰素和第二亲和力 使用针对该病毒的单抗的柱子 干扰素受体。N-氨基酸序列分析 将使用末端和胰蛋白酶多肽来制备 以该序列为基础的寡核苷酸探针。我们将使用 以寡核苷酸为探针筛选沙门氏菌基因组文库 人DNA(或从Daudi细胞制备的cDNA文库) 分离该受体的编码基因。或者， 针对该受体的单抗将被用于筛选 在大肠杆菌中构建的表达文库。该基因将被克隆并 用于分析DNA和RNA的测序和基因片段 在正常和癌变的人体组织中确定 干扰素作用的敏感性和耐受性机制。 这些研究可能会允许干扰素在更合理的 治疗人类肿瘤的方式。 这些研究将由荣誉学院的林维尔·M·梅多斯博士领导 毕业于北卡罗来纳大学医学院。 在医学院之前，梅多斯医生在医院工作了5年。 杜克大学研究肿瘤的技术人员 免疫学；在他#年在杜克大学接受团契训练期间 他开始研究血液学/肿瘤学的分子生物学 人类肿瘤，特别是α-干扰素对肿瘤细胞的影响 原癌基因c-myc的表达。梅多斯医生已经 最近加入内科肿瘤科的工作人员 北卡罗来纳大学在博士的指导下。 霍华德·奥泽，这些研究将在那里进行。奥泽医生 他们的兴趣集中在干扰素对免疫系统的影响上 系统，其实验室一直在积极开发一种 抗体的α-干扰素结合部位，将作为发起人。 大卫·李博士，莱恩伯格癌症的分子生物学家 中心，其利益包括人类成长因素，将采取行动 作为联合发起人，为分子克隆提供监督 学习。医学院内的研究设施和 莱恩伯格癌症中心将提供极好的 梅多斯博士完成基础研究的环境 分子生物学方面的培训。

Interferons possess many biologic functions, including the ability to induce differentiation, decrease cell growth, and reverse the action of several oncogenes; they function as negative growth regulators. In spite of receptors for interferon on tumor cell lines and many tumor explants, most cancer cells are not growth- inhibited by interferons, suggesting that the ability to circumvent the effects of negative growth factors such as alpha2-interferon may be an essential component of malignant transformation. To understand the mechanism of interferon's action, we propose to clone the human gene coding for the alpha2-interferon receptor. We will begin by purifying the membrane receptor from cultures of Daudi (or HL-60, see p.26) cells utilizing affinity chromatography; we will prepare an interferon affinity column using recombinant human alpha2-interferon, and a second affinity column using a monoclonal antibody directed against the interferon receptor. Amino acid sequence analysis of the N- terminus and of tryptic peptides will be used to prepare oligonucleotide probes predicated on that sequence. We will use the oligonucleotides as probes to screen a genomic library of human DNA (or a cDNA library prepared from Daudi cells) to isolate the gene coding for the receptor. Alternatively, monoclonal antibodies to the receptor will be used to screen a cDNA expression library in E. coli. The gene will be cloned and sequenced and gene fragments used to analyze the DNA and RNA in normal and cancerous human tissues to determine the mechanism of susceptibility and resistance to interferon's action. These studies may allow interferon to be used in a more rational manner in the treatment of human tumors. These studies will be led by Dr. Linville M. Meadows, an Honors graduate of the University of North Carolina School of Medicine. Prior to medical school, Dr. Meadows worked for 5 years as a research technician at Duke University studying tumor immunology; during his fellowship training at Duke in Hematology/Oncology he began studying the molecular biology of human tumors, specifically the effects of alpha-interferon on the expression of the proto-oncogene c-myc. Dr. Meadows has recently joined the staff of the Division of Medical Oncology at the University of North Carolina under the direction of Dr. Howard Ozer, where these studies will be performed. Dr. Ozer, whose interests center on the effects of interferon on the immune system, and whose laboratory has been actively developing an antibody to the alpha-interferon binding site, will act as Sponsor. Dr. David Lee, a molecular biologist in the Lineberger Cancer Center and whose interests include human growth factors, will act as Co-sponsor, providing supervision for the molecular cloning studies. The research facilities within the School of Medicine and the Lineberger Cancer Center will provide an excellent environment for Dr. Meadows to complete his basic research training in molecular biology.