MESSENGER RNA METABOLISM IN DICTYOSTELIUM AND YEAST

这一建议涉及转录后的两个方面， 真核生物中基因表达的调节：a） mRNA稳定性的决定因素和B）细胞质功能 mRNA上的3 '-poly（A）束。 决定因素 mRNA稳定性：最近的实验使我们得出结论， 随机的核分解过程可能会对 在确定特定mRNA降解速率中的作用。 因此，我们建议解决具体的 不稳定的网基网柄藻的元件（序列或结构）， 酵母mRNA是造成这些mRNA不稳定的原因。 为了识别这种不稳定因素，我们将构建： 来自编码稳定和不稳定mRNA的基因的嵌合体，和B） 缺失编码不稳定mRNA的基因的特定区域。 这些经过修饰的基因将通过DNA导入细胞 介导的转化和编码的mRNA的稳定性 在体内测定。 体外诱变程序将 随后被用来描绘不稳定因素 更准确地说。 将使用体外系统来确定 不稳定元件是否是特定核酸酶的靶。 预计，了解的结构基础， 特定酵母mRNA的不稳定性将有助于我们设计 mRNA稳定性缺陷突变体的遗传选择 调控 Poly（A）的细胞质功能：大量间接实验 从这个实验室和其他实验室的研究表明，聚（A）在 翻译起始可能是由细胞质内的 多聚腺苷酸结合蛋白。 我们建议利用无细胞 翻译系统来直接解决这个假设。 我们将 酵母poly（A）结合蛋白，或者用特异性 多克隆抗体或通过使用温度敏感突变体， 并评估其对翻译的影响。 此外，我们将 转录具有不同离散poly（A）尾长度的mRNA 从我们构建的修饰的SP 6载体， 确定他们的招聘相对效率， 多核糖体 长度的特定减少的可能性 一个给定的mRNA上的poly（A）尾与转移相关， 较小的多聚核糖体，或多聚核糖体的移位，也将是 通过在酵母中的体内脉冲追踪实验来解决。

This proposal addresses two aspects of the post-transcriptional regulation of gene expression in eukaryotic organisms: a) the determinants of mRNA stability and b) the cytoplasmic function of the 3'-poly(A) tracts present on mRNA. Determinants of mRNA Stability: Recent experiments have led us to conclude that random nucleolytic processes are likely to play a significant role in determining the rates of degradation of specific mRNAs. Therefore, we propose to address the possibility that specific elements (sequences or structures) of unstable Dictyostelium and yeast mRNAs are responsible for the instability of those mRNAs. To identify such instability elements we will construct: a) chimeras from genes encoding stable and unstable mRNAs and b) deletions of specific regions of genes encoding unstable mRNAs. These modified genes will be introduced into cells by DNA mediated transformation and the stability of the encoded mRNAs determined in vivo. In vitro mutagenesis procedures will subsequently be utilized to delineate the instability elements more precisely. An in vitro system will be utilized to determine whether instability elements are the targets of specific nucleases. It is anticipated that an understanding of the structural basis of the instability of specific yeast mRNAs will aid us in devising genetic selections for mutants defective in mRNA stability regulation. Cytoplasmic Function of Poly(A): Numerous indirect experiments from this lab and others suggest that poly(A) has a role in translational initiation which may be mediated by the cytoplasmic poly(A)-binding protein(s). We propose to utilize cell-free translation systems to address this hypothesis directly. We will inactivate the yeast poly(A)-binding protein, either with specific polyclonal antibodies or by using a temperature-sensitive mutant, and evaluate the effects on translation. Further, we will transcribe mRNAs with different discrete poly(A) tail lengths from modified SP6 vectors which we have constructed and determine their relative efficiencies of recruitment into polysomes. The possibility that a specific reduction in the length of the poly(A) tail on a given mRNA correlates with a shift to smaller polysomes, or with a shift off of polysomes, will also be addressed by an in vivo pulse-chase experiment in yeast.