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: 5210519
• 负责人: JERRY BROWN
• 金额: --
• 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/5210519
• 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体的计划 我们最近分离的大鼠肝癌细胞系 Fao 的 (RSP-)。 然而，RSP- 无法分泌一小组（至少四种）血清 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的蛋白质。 对分离的 DNA 和 RNA 进行 Southern 杂交和狭缝印迹杂交分析 这些细胞显示它们含有完整的白蛋白基因并且 该基因不表达。 其他测量相对值的实验 大鼠血清白蛋白启动子驱动的效率 Fao 和 RSP 中荧光素酶基因的表达表明反式 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 肝细胞中产生的因子似乎是维持肝脏功能所需的 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，非反式作用因子产生 已经描述了肝细胞抑制特定组的表达 肝脏特异性基因。 因此，RSP-表型似乎有可能 由编码蛋白质的通常沉默基因激活引起 参与抑制一组基因的表达，这些基因指定了四种 无法分泌的蛋白质或特定基因的突变 激活一小部分肝脏特异性基因的反式作用因子 发生。 确定这些解释中哪一个（如果有的话）是正确的 我将：1）利用 DNase I 足迹分析来识别 白蛋白和 Gc 球蛋白基因的启动子区域 受到从 Fao 和 RSP 中提取的核蛋白的差异保护 细胞和 2) 确定是否通过融合形成体细胞杂交体 RSP-和Hep G2（人类肝癌细胞系）保留了RSP-表型。 这 这些实验的结果将有助于评估以下可能性： 反式作用因子在 Fao 和 RSP-细胞中差异表达 如果是这样，请证明该因素是否起到积极或积极的作用 负调节器。