Williams J A, Schlichter D, Wicks W D
Department of Biochemistry, LSU Medical Center, New Orleans.
Second Messengers Phosphoproteins. 1988;12(5-6):261-70.
Stage VI Xenopus oocytes were injected with a plasmid (pBB0.6-CAT) which contains the cAMP regulatory element (CRE) from the rat liver phosphoenolpyruvate carboxykinase (PEPCK) gene fused upstream from a reporter gene [chloramphenicol acetyltransferase (CAT)]. Inhibition of the expression of the reporter gene (average = 51%) was observed in the presence of 10 microM progesterone, which is known to lead to inactivation of the oocyte cAMP dependent protein kinase (A kinase). In contrast, oocytes injected with a control plasmid (pSV2CAT), which contains no CRE, exhibited a variable increase (average = 31%) in CAT activity after progesterone treatment. Injection of the purified bovine cardiac A kinase catalytic subunit prior to exposure of oocytes injected with pBB0.6 CAT to progesterone prevents the loss of CAT activity generated by incubation with the steroid. Gel retardation analyses with oocyte lysates and a labeled synthetic oligonucleotide fragment containing the CRE from the PEPCK gene showed the existence of a complex with the same Rf and specificity as that formed with rat liver extracts. Subsequent exposure to progesterone, however, led to a rapid and extensive decrease in this binding activity. Taken together, these results are consistent with but do not prove the hypothesis that progesterone treatment and A kinase inactivation lead to a decrease in pBB0.6 CAT expression by virtue of a decline in the binding activity of an oocyte factor(s) to the CRE of the PEPCK fragment in pBB0.6-CAT, thereby decreasing transcription of the CAT gene.
将含有大鼠肝脏磷酸烯醇式丙酮酸羧激酶（PEPCK）基因的环磷酸腺苷（cAMP）调节元件（CRE）的质粒（pBB0.6-CAT）注射到Ⅵ期非洲爪蟾卵母细胞中，该调节元件融合在报告基因[氯霉素乙酰转移酶（CAT）]的上游。在10微摩尔孕酮存在的情况下，观察到报告基因的表达受到抑制（平均为51%），已知孕酮会导致卵母细胞cAMP依赖性蛋白激酶（A激酶）失活。相比之下，注射不含CRE的对照质粒（pSV2CAT）的卵母细胞在孕酮处理后，CAT活性出现了不同程度的增加（平均为31%）。在用孕酮处理注射了pBB0.6 CAT的卵母细胞之前，先注射纯化的牛心脏A激酶催化亚基，可以防止与类固醇孵育产生的CAT活性丧失。用卵母细胞裂解物和含有PEPCK基因CRE的标记合成寡核苷酸片段进行凝胶阻滞分析，结果显示存在一种复合物，其迁移率（Rf）和特异性与用大鼠肝脏提取物形成的复合物相同。然而，随后暴露于孕酮会导致这种结合活性迅速且大幅下降。综上所述，这些结果与以下假设一致，但并未证明该假设：孕酮处理和A激酶失活导致pBB0.6 CAT表达下降，原因是卵母细胞因子与pBB0.6-CAT中PEPCK片段的CRE结合活性下降，从而降低了CAT基因的转录。
