MOLECULAR REGULATION OF LIVER REGENERATION

肝脏再生的能力为研究提供了一个独特的系统 体内对细胞增殖和基因表达的调节。这 非凡的过程对它从一些疾病中恢复至关重要 国家，包括手术和化学伤害，并依赖于 使通常静止的细胞重新进入复制状态。这个 这项研究项目正在进行的主要目标是确定 细胞周期和生长相关基因表达的mRNA稳定性 在肝部分切除后再生的大鼠肝脏中。假设是 这种转录后机制主要负责 过即刻-早期(G0-G1)阶段的mRNA表达调控 细胞周期。具体目标是为了检验假设和 鉴定参与体内调节基因的机制 稳定性。第一个具体目标是评估Poly(A)的作用 Tail在调节出现mRNA改变的基因的mRNA衰变中的作用 在没有转录速率变化的情况下保持稳定。首字母 聚腺苷酸化程度和聚(A)尾的速率 将使用体外多聚体衰变提取物和核糖核酸酶来研究缩短 H映射。此外，翻译在调节语速中的作用 还将研究再生过程中聚(A)尾巴的去除情况。这个 第二个具体目标是定义翻译和多音节的作用 在再生过程中调节特定mRNAs衰变的关联性。 完全、游离、细胞骨架和膜结合的梯度离心法 多聚体群体和RNA酶保护分析将用于 确定转录本的相对翻译活性。 将在Poly(A)尾部长度和关联 不同多聚体群体的mRNA。第三个具体目标 目的是研究172 kDa蛋白因子在细胞周期调控中的作用 原癌基因c-myc信使核糖核酸衰退。凝胶位移分析、紫外光交联、RNA 亲和层析和纯化技术用于鉴定和鉴定 分离出70 kDa的c-myc结合蛋白将被利用。的作用 将在体外研究巯基和磷酸化，以确定它们的 对RNA/蛋白质结合复合体的影响。体外衰变分析将是 用于确定172 kDa蛋白在人体内的功能意义 信使核糖核酸代谢。第四个具体目的是探索小说的概念 基因组DNA的甲基化状态调节其转录本 肝脏再生过程中的半衰期。这将使用Southern 印迹和聚合酶链式反应分析、嵌合基因构建和体外甲基化 和转基因研究。该项目的长期目标是 建立转录后事件在mRNA调控中的模型 肝脏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理解细胞生长和 对肝脏恢复能力中基因表达的控制 受伤。

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.