ESTROGEN ACTION IN XENOPUS LIVER

雌激素给雄性非洲爪蟾会导致后 血清白蛋白mRNA的转录抑制 雌激素受体依赖机制，发生在数小时内 在卵黄基因的转录诱导后 蛋白质前体卵黄原蛋白。 本提案的具体目标 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 选择性地在细胞核或细胞质中降解，如果这是 核以检查多聚腺苷酸化和/或RNA （2）在这一过程中的变化;（3）在这两个稳定的变化。 白蛋白mRNA的状态水平和周转 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 需要合成新的基因产物; 3）研究 血清蛋白协同转录后调节 为了鉴定共有序列或二级序列， 定义目标特异性的结构。 长期目标是 来识别雌激素的分子和机制 依赖于基因表达的转录后调节。 雌激素诱导的白蛋白核前体RNA的变化将 最初通过北方印迹分析进行检测， 从白蛋白基因克隆cDNA和克隆内含子。 核前体RNA的定量变化将 通过S1保护实验确定。 的作用 多聚腺苷酸化将通过细胞核的层析来检查。 前体RNA的聚（U）-纤维素和通过直接分析 雌激素治疗后白蛋白mRNA上的poly（A）长度 局 任一纯外显子的亚克隆片段 序列或跨越内含子/外显子边界将用于S1 保护实验来确定雌激素是否会导致 白蛋白mRNA剪接的改变。 详细研究将在 在培养的肝细胞中进行白蛋白mRNA周转 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物 白蛋白mRNA。 最后，如果数据表明细胞质 机制的重点将转移到协调调节血清 蛋白质mRNAs寻找共同的特征，可能发挥作用， 在mRNA靶向稳定性变化中的作用。 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测定 被鉴定为白蛋白基因调节的组分 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.