PILOT STUDY--REGULATION OF EXPRESSION OF SETS OF LIVER SPECIFIC GENES

试点研究--肝脏特异性基因组表达的调节

• 批准号: 3732960
• 负责人: JERRY BROWN
• 金额: --
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 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/3732960
• 关键词: DNA footprinting; biomedical facility; blood proteins; genes; genetic regulation; hybrid cells; liver; neoplastic cell culture for noncancer research; nucleic acid hybridization; tissue /cell culture

This proposal describes plans to characterize a well differentiated variant (RSP-) of the rat hepatoma cell line Fao that we have recently isolated. RSP-, however, fails to secrete a small set, at least four, of serum proteins that are secreted by the parental cell line. These proteins include albumin, Gc globulin and two other, as yet, unidentified proteins. Southern and slot blot hybridization analyses of the DNA and RNA isolated from these cells show that they contain an intact albumin gene and that this gene is not expressed. Other experiments measuring the relative efficiencies with which the rat serum albumin promoter drives the expression of the luciferase gene in Fao and RSP- indicate that a trans acting factor that mediates expression of the albumin gene is differentially expressed in these cell lines. The known trans acting factors produced in liver cells that appear to be required to maintain the differentiated state seem to affect expression of most if not all liver specific genes. On the other hand, trans acting factors produced in non- liver cells have been described which repress expression of defined sets of liver specific genes. Thus it seems possible that the RSP- phenotype may result from activation of a normally silent gene that encodes a protein involved in repressing expression of a set of genes specifying the four proteins that fail to be secreted or that a mutation of a gene specifying a trans acting factor that activates a small set of liver specific genes has occurred. To establish which, if either, of these explanations is correct I will: 1) utilize DNase I footprint analyses to identify sequences in the promoter regions of the albumin and Gc globulin genes that are differentially protected by nuclear proteins extracted from Fao and RSP- cells and 2) establish whether somatic cell hybrids formed by fusion of RSP- and Hep G2 (a human hepatoma line) retain the RSP- phenotype. The results of these experiments will allow evaluation of the possibility that a trans acting factor is differentially expressed in Fao and RSP- cells and, if so, demonstrate whether or not this factor acts as a positive or a negative regulator.

该提案描述了表征差异化变异的计划 （RSP-）的大鼠肝癌细胞系Fao，我们最近分离。 然而，RSP-不能分泌一小部分，至少四个， 由亲本细胞系分泌的蛋白质。 这些蛋白质 包括白蛋白、Gc球蛋白和另外两种尚未鉴定蛋白质。 DNA和RNA的Southern杂交和狭缝杂交分析 显示它们含有完整的白蛋白基因， 该基因不表达。 其他实验测量相对 大鼠血清白蛋白启动子驱动 荧光素酶基因在Fao和RSP中的表达表明， 介导白蛋白基因表达的作用因子是 在这些细胞系中差异表达。 已知的反式作用 肝细胞中产生的似乎是维持 分化状态似乎影响大多数（如果不是全部）肝脏的表达 特定基因 另一方面，非物质文化遗产中产生的反作用因素， 已经描述了肝细胞，其抑制特定组的 肝脏特异性基因 因此，RSP-表型可能 是由一个通常沉默的编码蛋白质的基因的激活引起的 参与抑制一组基因的表达，这些基因指定了四种 不能分泌的蛋白质，或者指定蛋白质的基因突变， 激活一小部分肝脏特异性基因的反式作用因子， 发生了。 为了确定这些解释中哪一个是正确的， 我将：1）利用DNA酶I足迹分析来鉴定DNA中的序列。 白蛋白和Gc球蛋白基因的启动子区， 受到从Fao和RSP中提取的核蛋白的差异保护- 细胞和2）确定体细胞杂种是否通过融合 RSP-和Hep G2（人肝癌细胞系）保留RSP-表型。 的 这些实验的结果将允许评估的可能性， 反式作用因子在Fao和RSP-细胞中的差异表达 如果是的话，说明这一因素是否起到积极作用， 负调节器