MOLECULAR REGULATION OF LIVER REGENERATION

肝脏的再生能力提供了一个独特的研究系统 细胞增殖和基因表达的体内调节。 这 一个非凡的过程对于它从一些疾病中恢复至关重要 国家，包括手术和化学伤害，并取决于 通常静止的细胞重新进入复制状态。 的 该研究项目的主要目标是确定 细胞周期和生长依赖性基因表达的mRNA稳定性 在部分肝切除后再生的大鼠肝脏中。 这个假设是 这种转录后机制主要负责 mRNA表达的调节超过了即刻早期（G 0-G1）阶段， 细胞周期 具体目标旨在检验假设， 表征参与体内mRNA调节的机制 稳定 第一个具体目标是评估poly（A）的作用 尾在调节表现出改变的mRNA的基因的mRNA衰变中的作用 在没有转录速率变化的情况下稳定。 初始 多聚腺苷酸化的程度和poly（A）尾被 将使用体外多核糖体衰变提取物和核糖核酸酶研究缩短 H映射 此外，翻译在调节速率中的作用 还将研究再生过程中聚（A）尾部的去除。 的 第二个具体目标是定义翻译和多核糖体的作用 在再生过程中调节特定mRNA的衰变。 总的、游离的、细胞骨架结合的和膜结合的梯度离心 多核糖体群体和RNA酶保护分析将用于 确定转录物的相对翻译活性。 将在poly（A）尾长和以下相关性之间进行相关性分析： mRNA与不同的多核糖体群体。 第三个具体目标 是表征172 kDa蛋白因子在调节 原癌基因c-myc mRNA降解。 凝胶位移测定，UV交联，RNA 亲和层析和纯化技术用于鉴定和 将使用分离的70 kDa c-myc结合蛋白。 的作用 巯基和磷酸化将在体外研究，以确定它们的 对RNA/蛋白质结合复合物的影响。 体外衰变试验将 用于确定172 kDa蛋白在 mRNA代谢 第四个具体目标是探讨小说观念 基因组DNA的甲基化状态调节它们的转录 肝再生过程中的半衰期。 这将使用Southern 印迹和PCR分析、嵌合基因构建体和体外甲基化 和转染研究。 该项目的长期目标是 建立了一个mRNA调控的转录后事件模型， 肝再生过程中的稳定性。 拟议的研究将提供 重要的和新的信息，在我们的理解细胞生长和 控制基因表达的能力，肝脏恢复， 损伤

The ability of the liver to regenerate provides a unique system to study the in vivo regulation of cell proliferation and gene expression. This remarkable process is critical for its recovery from a number of disease states, including surgical and chemical injuries, and is dependent on the reentry of normally quiescent cells into a state of replication. The ongoing main objective of this research project is to define the role of mRNA stability in the expression of cell cycle- and growth-dependent genes in the regenerating rat liver after partial hepatectomy. The hypothesis is that this posttranscriptional mechanism is primarily responsible for the regulation of mRNA expression beyond the immediate-early (G0-G1) phase of the cell cycle. The specific aims are designed to test the hypothesis and characterize mechanisms involved in the in vivo regulation of mRNA stability. The first specific aim is to evaluate the role of the poly(A) tail in regulating the mRNA decay of genes which exhibit altered mRNA stability in the absence of transcriptional rate changes. The initial degree of polyadenylation and the rate at which the poly(A) tail is shortened will be studied using in vitro polysome decay extracts and RNase H mapping. In addition, the effect of translation in modulating the rate of poly(A) tail removal during regeneration will also be investigated. The second specific aim is to define the role of translation and polysome association in regulating the decay of specific mRNAs during regeneration. Gradient centrifugation of total, free, cytoskeletal- and membrane-bound polysome populations and RNAase protection analysis will be used to determine the relative translational activity of the transcripts. Correlations will be made between poly(A) tail length and association of the mRNA with the different polysome populations. The third specific aim is to characterize the role of the 172 kDa protein factor in modulation of proto-oncogene c-myc mRNA decay. Gel shift assays, UV crosslinking, RNA affinity chromatography and purification techniques used to identify and isolate the 70 kDa c-myc binding protein will be utilized. The role of sulfhydryls and phosphorylation will be studied in vitro to determine their effects on the RNA/protein binding complex. In vitro decay assays will be used to establish the functional significance of the 172 kDa protein in mRNA metabolism. The fourth specific aim is to explore the novel concept that the methylation status of the genomic DNAs regulate their transcript half-lives during liver regeneration. This will be done using Southern blot and PCR analysis, chimeric gene constructs, and in vitro methylation and transfection studies. The long term goal of the project is to formulate a model of posttranscriptional events in the regulation of mRNA stability during hepatic regeneration. The proposed studies will provide important and new information in our understanding of cell growth and the control of gene expression in the ability of the liver to recover from injury.