PILOT STUDY--MOLECULAR BASIS OF GH & CSH TISSUE SPECIFIC EXPRESSION

试点研究--GH的分子基础

• 批准号: 3839774
• 负责人: CINDY VNENCAK-JONES
• 金额: --
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3839774
• 关键词: gene expression; genetic manipulation; genetic regulatory element; genetic transcription; human genetic material tag; human tissue; laboratory rat; nutrition; nutrition related tag; site directed mutagenesis; somatomammotropin; somatotropin; transfection

The goals of this project are to identify cis-acting regulatory sequences flanking the GH1, CSHP1, CSH1, GH2 and CSH2 genes in the human growth hormone (hGH) cluster and to determine their role in regulating tissue specific transcription of these genes. To accomplish these goals a series of recombinant plasmids will be constructed by fusing the bacterial gene, chloramphenicol acetyltransferase (CAT), to varying amounts of DNA lying 5' to each of the genes in the hGH gene cluster. Human term and first trimester placental as well as rat anterior pituitary cells will be transfected with these recombinant plasmids by Ca2PO4 precipitation. Positive or negative regulatory regions will be identified by comparative analysis of assayed levels of CAT activity. Specific bases that are essential for these regulatory functions will be identified using plasmid constructs that are modified by Bal 31 exonuclease or oligonucleotide directed site mutagenesis. Tissue specificity will be analyzed by comparing the CAT activity of analogous plasmid constructions corresponding to each gene in each cell type. The importance of spacial arrangements on the ability of 5' sequences to regulate the promoter sequences for these genes will be evaluated by assaying CAT activity from a variety of plasmids in which the normal distances separating these regions have been altered. The information obtained from these studies will contribute to our understanding of the tissue specific transcriptional, regulation of this complex of growth promoting genes which will provide insight into the regulation of other eukaryotic multigene families.

该项目的目标是识别顺式作用调控序列 位于人类生长中的 GH1、CSHP1、CSH1、GH2 和 CSH2 基因的侧翼 激素 (hGH) 簇并确定其在调节组织中的作用 这些基因的特异性转录。 为了实现这些目标一系列 通过融合细菌基因构建重组质粒， 氯霉素乙酰转移酶 (CAT)，位于 5' 处不同数量的 DNA hGH 基因簇中的每个基因。 人类术语和第一 妊娠期胎盘以及大鼠垂体前叶细胞将 通过 Ca2PO4 沉淀转染这些重组质粒。 积极或消极的监管区域将通过比较来确定 分析 CAT 活性的测定水平。 具体的碱基是 将使用质粒鉴定这些调节功能所必需的 由 Bal 31 核酸外切酶或寡核苷酸修饰的构建体 定向位点诱变。 组织特异性将通过以下方式进行分析 比较相应的类似质粒构建体的 CAT 活性 每种细胞类型中的每个基因。 空间布局的重要性 5'序列调节启动子序列的能力 将通过测定各种质粒的 CAT 活性来评估基因 其中分隔这些区域的正常距离已被改变。 从这些研究中获得的信息将有助于我们 了解组织特异性转录及其调节 生长促进基因复合体将提供深入了解 其他真核多基因家族的调控。