MURINE ERYTHROPOIETIN--RESPONSE AND RECEPTORS

鼠促红细胞生成素——反应和受体

• 批准号: 3239909
• 负责人: SAMUEL H BOYER
• 金额: $ 29.42万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-30 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3239909
• 关键词: antibody receptor; autoradiography; complementary DNA; cytokine; erythropoiesis; erythropoietin; flow cytometry; gel electrophoresis; gene expression; genetic library; genetic regulation; genetic transcription; human tissue; hybridomas; immunoglobulin genes; laboratory mouse; laboratory rat; messenger RNA; molecular genetics; molecular site; nucleic acid hybridization; nucleic acid sequence; protein biosynthesis; radiotracer; tissue /cell culture

Recombinant human Epo (rEpo) has become available and procedures have been developed whereby erythroid progenitor cells of several provenances can be harvested in near purity. Our preliminary studies of Epo-reception by ca. 80%-pure murine erythroid colony-forming units (CFUe) and by the S8 murine erythroleukemia (MEL) cell line, together with collateral studies by others of purified spleen cells derived from mice infected with the anemia-strain of Friend virus (FVA), indicate that there are at least two kinds of erythropoietin receptors (EpoR): one with a dissociation constant (Kd) of approximately 40 pM, the other with Kd of approximately 400 pM. Conditioned by our repeated observation that erythroid maturation can be atypical in virus- infected cells, we hypothesize that (i) high-affinity (approximately 40 pM) EpoR is either a viral import or one regulated by virus transformation and (ii) low-affinity (approximately 400 pM) EpoR is the one naturally expressed in mouse CFUe. Our intent is to verify/falsify these hypotheses and, concurrently, answer questions arising from preliminary studies of Epo-response by CFUe and from studies of EpoR biology in CFUe and S8-MEL-cells. We hope thereby to clarify the temporal relationship between Epo-addition and globin gene response; resolve the mystery of why CFUe-EpoR sites seem abundant far beyond functional needs; establish some ways in which EpoR can be regulated; refine our understanding of physical features and fate of EpoR, including confirmation/rejection of the possibility that Epo and/or EpoR is bound to DNA or chromatin in S8-MEL cells; in a large-scale undertaking, apply affinity methods to the isolation of EpoR and immunological methods to the isolation of recombinant cDNA clones encoding it; and, in a brisk but short- lived undertaking, isolate cDNA for "early-response" genes whose expression depends upon Epo and, thereafter, use arising cDNA clones to examine heretofore unknown aspects of Epo-responses by the several EpoR.

重组人红细胞生成素 (rEpo) 已上市 已经开发出红细胞祖细胞的程序 可以以接近纯度的方式收获多个来源的细胞。 我们的 约 EPO 接收的初步研究。 80%-纯小鼠 红细胞集落形成单位 (CFUe) 和 S8 小鼠 红白血病 (MEL) 细胞系以及并行研究 由其他人纯化的脾细胞来源于感染了 弗兰德病毒（FVA）贫血株，表明有 至少两种促红细胞生成素受体 (EpoR)：一种具有 解离常数 (Kd) 约为 40 pM，另一个为 Kd 约为 400 pM。 以我们的重复为条件 观察到红细胞成熟在病毒中可能是非典型的 被感染的细胞，我们假设（i）高亲和力 （大约下午 40 点）EpoR 要么是病毒输入，要么是病毒输入 受病毒转化和（ii）低亲和力调节 （约 400 pM）EpoR 是天然表达的一种 小鼠 CFUe。 我们的目的是验证/证伪这些假设，并且， 同时回答初步研究中提出的问题 CFUe 的 Epo 反应以及 CFUe 中 EpoR 生物学研究 和S8-MEL-细胞。 我们希望借此澄清暂时的情况 Epo添加与珠蛋白基因反应之间的关系； 解开为什么 CFUe-EpoR 位点看起来丰富的谜团 超出功能需求；建立一些方法，使 EpoR 能够 受到监管；完善我们对身体特征的理解 EpoR 的命运，包括确认/拒绝可能性 Epo 和/或 EpoR 与 S8-MEL 中的 DNA 或染色质结合 细胞；在大型企业中，应用亲和方法 EpoR 的分离和免疫学方法的分离 编码它的重组cDNA克隆；并且，以轻快但简短的方式—— 活生生的事业，分离“早期反应”基因的 cDNA，其 表达取决于 Epo，然后使用产生的 cDNA 克隆来检查迄今为止未知的 Epo 反应方面 由几个EpoR。