STRUCTURE AND EXPRESSION OF HORMONALLY REGULATED GENES

植物激素生长素的主要作用机制很差。 然而，最近的实验证据表明， 荷尔蒙能够在转录或转录时非常迅速地起作用 转录后水平。细胞的结构和调控研究 本文提出了豌豆茎组织中生长素诱导的基因，以获得 深入了解调节它们的生物化学机制的细节 表达及其在植物细胞生长中的作用。这样做的具体目的是 建议包括： 1.早期基因组序列的分离和结构特征 豌豆组织中生长素诱导的mRNAs。 2.体内激素调控基因的表达调控 在分离的豌豆核中。 3.编码蛋白的纯化和亚细胞定位 生长素调节基因。 为了实现上述目标，提出了以下实验。基因组学 文库将被构建到克隆载体EMBL3中 黄化的豌豆幼苗。早期的基因组序列 生长素调节的mRNAs将通过斑块过滤器杂交来分离 已分离的cDNA克隆pIAA4/5和pIAA6作为探针。这个 基因的组织和结构分析将在#年进行调查。 细节。为了确定生长素是在转录还是在转录中起作用 转录后水平，即可诱导的mRNAs的稳定性，将 活体检查。这些实验将在体外补充 在分离的细胞核中转录。最后，生长素的cdna克隆将 导入Lambda表达载体生产融合蛋白 β-半乳糖苷酶。针对杂交蛋白的抗体将 然后被用来提纯蛋白质，在亚细胞水平上定位它们 并测定它们在细胞生长过程中积累的动力学。

The primary mechanism of action of the plant hormone auxin is poorly understood; however, recent experimental evidence indicates that the hormone has the capacity to act very rapidly at the transcriptional or post-transcriptional level. Studies on the structure and regulation of the auxin-inducible genes in pea stem tissue are proposed herein to gain insight into the details of the biochemical machinery that regulates their expression and their role in plant cell growth. The specific aims of this proposal are: 1. Isolation and structural characterization of genomic sequences to early auxin-inducible mRNAs from pea tissue. 2. Regulation of expression of the hormonally regulated genes in vivo and in isolated pea nuclei. 3. Purification and subcellular localization of the proteins coded by the auxin-regulated genes. To achieve the above goals the following experiments are proposed. Genomic libraries will be constructed into the cloning vector EMBL 3 using DNA from etiolated pea seedlings. The genomic sequences of the early auxin-regulated mRNAs will be isolated by plaque filter hybridization using the already isolated cDNA clones pIAA4/5 and pIAA6 as probes. The organization and structural analysis of the genes will be investigated in detail. To determine whether auxin acts at the transcriptional or post-transcriptional level, the stability of the inducible mRNAs, will be examined in vivo. These experiments will be supplemented with in vitro transcription in isolated nuclei. Finally, the auxin cDNA clones will be introduced into Lambda expression vectors to produce fused proteins with Beta-galactosidase. Antibodies directed toward the hybrid proteins will then be used to purify the proteins, localize them at the subcellular level and determine the kinetics of their accumulation during cell growth.