ESTROGEN ACTION IN XENOPUS LIVER

给雄性非洲爪哇注射雌激素会导致后 人血清白蛋白基因转录抑制的研究 在数小时内发生的雌激素受体依赖机制 在蛋黄基因转录诱导后 蛋白前体卵黄蛋白原。这项建议的具体目的 每一个都解决了一个关于转录后的基本问题 雌激素的调节：1)确定白蛋白mRNA是否 在细胞核或细胞质中选择性降解，如果这是 核检测多聚腺苷酸化和/或RNA的作用 这一过程中的剪接；2)检查在稳定的- 白蛋白mRNA在培养细胞中的状态和周转 肝细胞进一步确定雌激素受体的作用 并确定白蛋白mRNA的抑制是否 需要合成一种新的基因产物；3)研究 协调血清蛋白的转录后调控 为了识别一致的序列或次要的mRNAs 定义目标专一性的结构。远景目标是 为了确定涉及雌激素的分子和机制- 依赖于基因表达的转录后调控。 雌激素诱导白蛋白核前体RNA的变化 初步用Northern印迹分析作为探针 克隆了白蛋白基因的cDNA1个内含子。 核前体RNA的数量变化将是 由S1保护实验确定。的作用 多聚腺苷酸化将通过核层析进行检测 聚(U)-纤维素上的前体RNA及其直接分析 雌激素作用后不同时间段白蛋白基因的Poly(A)长度 行政管理。亚克隆的任一纯外显子片段 S1将使用序列或跨越内含子/外显子边界 保护性实验以确定雌激素是否可能导致 白蛋白基因剪接的改变。详细的研究将在 对培养肝细胞白蛋白信使核糖核酸周转的影响 蛋白质和RNA合成的抑制剂将用于 确定是否需要新的基因产品来破坏稳定 白蛋白基因。最后，如果数据表明细胞质 机制重点将转移到协调血清调节上 蛋白质mRNAs来寻找可能发挥作用的共同特征 在靶向稳定变化的信使核糖核酸中的作用。未来 研究将利用DNA介导的基因转移到培养的 肝细胞作为任何特定序列的功能分析 白蛋白基因调控成分的鉴定 表情。

Estrogen administration to male Xenopus laevis causes the post- transcriptional suppression of serum albumin mRNA via an estrogen receptor-dependent mechanism that occurs within hours after the transcriptional induction of the genes for the yolk protein precursor vitellogenin. The specific aims of this proposal each address a fundamental question about post-transcriptional regulation by estrogen: 1) To determine if albumin mRNA is selectively degraded in the nucleus or the cytoplasm, and if this is nuclear to examine the role of polyadenylation and/or RNA splicing in this process; 2) To examine changes in both steady- state levels and turnover of albumin mRNA in cultured hepatocytes to further define the role of the estrogen receptor and to determine whether the suppression of albumin mRNA requires the synthesis of a new gene product; 3) To investigate coordinate post-transcriptional regulation of serum protein mRNAs in order to identify a consensus sequence or secondary structure that defines target specificity. The long range goal is to identify the molecules and mechanisms involved in estrogen- dependent post-transcriptional regulation of gene expression. Estrogen-induced changes in albumin nuclear precursor RNA will examined initially by Northern blot analysis using as probes the cloned cDNA and a cloned intron from the albumin gene. Quantitative changes in nuclear precusor RNA will then be determined by S1 protection experiments. The role of polyadenylation will be examined by chromatography of nuclear precursor RNA on poly(U)-cellulose and by direct analysis of poly(A) length on albumin mRNA at intervals after estrogen administration. Subcloned fragments of either pure exon sequence or spanning intron/exon boundaries will be used in S1 protection experiments to determine if estrogen might cause alterations in albumin mRNA splicing. Detailed studies will be performed on albumin mRNA turnover in cultured hepatocytes and inhibitors of protein and RNA synthesis will be used to determine if new gene products are required to destabilize albumin mRNA. Finally, if the data indicate a cytoplasmic mechanism the focus will shift to coordinate regulation of serum protein mRNAs to search for common features that may play a role in the targeting of mRNA for changes in stability. Future studies will utilize DNA-mediated gene transfer into cultured hepatocytes as a functional assay for any specific sequences identified as components in the regulation of albumin gene expression.