MURINE ERYTHROPOIETIN--RESPONSE AND RECEPTORS

鼠促红细胞生成素——反应和受体

• 批准号: 3239910
• 负责人: SAMUEL H BOYER
• 金额: $ 30.4万
• 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/3239910
• 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Epo（rEpo）已经可用， 已经开发了红系祖细胞 几种来源的细胞可以以接近纯度收获。 我们 对《纪元》接受的初步研究。 80%纯鼠 红系集落形成单位（CFUe）和S8小鼠 红白血病（MEL）细胞系，以及附带研究 其他人的纯化脾细胞来源于感染 Friend病毒贫血株（FVA）表明， 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，另一个具有 Kd约为400 pM。 因为我们不断地 观察到红系成熟在病毒中可能是非典型的， 感染的细胞，我们假设（i）高亲和力 （约40 pM）EpoR是病毒输入或病毒输入。 受病毒转化调节和（ii）低亲和力 （约400 pM）EpoR是在大肠杆菌中天然表达的EpoR。 小鼠CFUe。 我们的目的是验证/证伪这些假设， 同时，回答初步研究中提出的问题， CFUe的Epo-response和CFUe中EpoR生物学研究 和S8-MEL细胞。 我们希望借此澄清时间问题 Epo-添加与珠蛋白基因应答的关系; 解决了为什么CFUe-EpoR位点似乎丰富的奥秘， 超越功能需求;建立一些EpoR可以 规范;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;在大规模的工作中，将亲和方法应用于 EpoR的分离和免疫学方法的分离 重组cDNA克隆编码它;和，在一个轻快，但短- 活的事业，分离“早期反应”基因的cDNA， 表达依赖于Epo，此后，使用产生的cDNA 克隆人来检查迄今为止未知的Epo反应方面 几个EpoR。