Makeh I, Thomas M, Hardelin J P, Briand P, Kahn A, Skala H
Laboratoire de Recherche en Génétique et Pathologie Expérimentales, Institut National de la Santé de de la Recherche Médicale (INSERM) Unité 380, Paris, France.
J Biol Chem. 1994 Feb 11;269(6):4194-200.
Aldolase C mRNA is detected by Northern blot in all fetal tissues in rat; it is very abundant in the adult brain and undetectable in the other adult tissues. However, reverse transcriptase polymerase chain reaction amplification indicates that this gene is not totally repressed in these tissues. A DNase-I hypersensitivity site located in a 115-base pair proximal promoter fragment is detectable in the brain as well as in other adult tissues. Two MspI/HpaII restriction sites located at -3800 and -450 base pairs are demethylated in the brain and totally or partially methylated in other tissues. In transgenic mice, a 12.5-kilobase genomic fragment is strongly and tissue specifically expressed in different lines, with conservation of a methylation pattern similar to that of the endogenous gene. A chloramphenicol acetyltransferase gene directed by either 800 or 115 base pairs of aldolase C 5'-flanking sequences is tissue specifically expressed in transgenic mice, but the level of expression is very low. This level is greatly increased when the transgene consists of a chloramphenicol acetyltransferase hybrid gene directed by 5.5 kilobases of aldolase C 5'-flanking sequences. We propose therefore that the chromatin structure around the aldolase C promoter is accessible in fetal tissues, then remains open in the adult brain, where the gene is very active, as well as in tissues in which it is practically inactive. The specificity of expression in the brain is conferred by a short 115-base pair proximal promoter fragment that needs more upstream sequences to be fully active.
通过Northern印迹法在大鼠所有胎儿组织中检测到醛缩酶C信使核糖核酸；它在成年大脑中非常丰富，而在其他成年组织中检测不到。然而，逆转录酶聚合酶链反应扩增表明该基因在这些组织中并未完全被抑制。位于115个碱基对近端启动子片段中的一个脱氧核糖核酸酶I超敏位点在大脑以及其他成年组织中均可检测到。位于-3800和-450碱基对处的两个MspI/HpaII限制性位点在大脑中去甲基化，而在其他组织中完全或部分甲基化。在转基因小鼠中，一个12.5千碱基的基因组片段在不同品系中强烈且组织特异性表达，其甲基化模式与内源基因相似且得以保留。由醛缩酶C 5'侧翼序列的800或115个碱基对指导的氯霉素乙酰转移酶基因在转基因小鼠中组织特异性表达，但表达水平非常低。当转基因由醛缩酶C 5'侧翼序列的5.5千碱基指导的氯霉素乙酰转移酶杂交基因组成时，该水平会大幅提高。因此我们提出，醛缩酶C启动子周围的染色质结构在胎儿组织中是可及的，然后在成年大脑中保持开放，在成年大脑中该基因非常活跃，在实际上不活跃的组织中也是如此。大脑中的表达特异性由一个短的115个碱基对近端启动子片段赋予，该片段需要更多上游序列才能完全激活。
