Lloyd C E, Kalinyak J E, Hutson S M, Jefferson L S
Am J Physiol. 1987 Feb;252(2 Pt 1):C205-14. doi: 10.1152/ajpcell.1987.252.2.C205.
The first goal of the work reported here was to prepare single-stranded DNA sequences for use in studies on the regulation of albumin gene expression. A double-stranded rat albumin cDNA clone was subcloned into the bacteriophage vector M13mp7. Single-stranded recombinant clones were screened for albumin sequences containing either the mRNA strand or the complementary strand. Two clones were selected that contained the 1,200 nucleotide long 3' end of the albumin sequence. DNA from the clone containing the mRNA strand was used as a template for DNA polymerase I to prepare a radiolabeled, single-stranded cDNA to albumin mRNA. This radiolabeled cDNA probe was used to quantitate the relative abundance of albumin mRNA in samples of total cellular RNA. DNA from the clone containing the complementary strand was used to measure relative rates of albumin gene transcription in isolated nuclei. The second goal was to use the single-stranded DNA probes to investigate the mechanism of the insulin-mediated stimulation of albumin synthesis in primary cultures of rat hepatocytes. Addition of insulin to hepatocytes maintained in hepatocytes. Addition of insulin to hepatocytes maintained in a chemically defined, serum-free medium for 40 h in the absence of any hormones resulted in a specific 1.5- to 2.5-fold stimulation of albumin gene transcription that was maximal at 3 h and was maintained above control values for at least 24 h. The relative abundance of albumin mRNA and albumin secretion increased correspondingly within 24 to 30 h. These parameters remained above control levels for at least 60 h after addition of insulin. Maximal responses were attained at an insulin concentration of 100 nM and there was a close correspondence between albumin gene transcription and albumin secretion at each concentration tested. The rate of albumin gene transcription in nuclei isolated from livers of diabetic rats was reduced to 50% of the value recorded in control nuclei. Taken together, these findings demonstrate that insulin regulates synthesis of albumin at the level of gene transcription.
本文报道的研究工作的首要目标是制备单链DNA序列，用于白蛋白基因表达调控的研究。将双链大鼠白蛋白cDNA克隆亚克隆到噬菌体载体M13mp7中。筛选单链重组克隆，寻找含有mRNA链或互补链的白蛋白序列。挑选出两个克隆，它们含有白蛋白序列1200个核苷酸长的3'端。以含有mRNA链的克隆的DNA为模板，用DNA聚合酶I制备白蛋白mRNA的放射性标记单链cDNA。该放射性标记的cDNA探针用于定量总细胞RNA样品中白蛋白mRNA的相对丰度。以含有互补链的克隆的DNA来测量分离细胞核中白蛋白基因的相对转录速率。第二个目标是使用单链DNA探针研究胰岛素介导的大鼠原代肝细胞白蛋白合成刺激机制。在无任何激素的情况下，将胰岛素添加到在化学限定的无血清培养基中培养40小时的肝细胞中，导致白蛋白基因转录特异性地增加1.5至2.5倍，在3小时时达到最大值，并在至少24小时内维持在对照值以上。白蛋白mRNA的相对丰度和白蛋白分泌在24至30小时内相应增加。添加胰岛素后，这些参数至少在60小时内保持在对照水平以上。在胰岛素浓度为100 nM时达到最大反应，并且在每个测试浓度下白蛋白基因转录和白蛋白分泌之间存在密切对应关系。从糖尿病大鼠肝脏分离的细胞核中白蛋白基因转录速率降至对照细胞核中记录值的50%。综上所述，这些发现表明胰岛素在基因转录水平上调节白蛋白的合成。
