Zhang Z F, Kelly D P, Kim J J, Zhou Y Q, Ogden M L, Whelan A J, Strauss A W
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Biochemistry. 1992 Jan 14;31(1):81-9. doi: 10.1021/bi00116a013.
Medium-chain acyl-CoA dehydrogenase (MCAD) is a highly regulated mitochondrial flavo-enzyme that catalyzes the initial reaction in fatty acid beta-oxidation. Deficiency of MCAD is a common inherited defect in energy metabolism. We have previously shown that the mRNA encoding MCAD in an MCAD-deficient child is homozygous for the point mutation A985 to G [Kelly et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9236-9420]. To define the molecular basis of MCAD deficiency and as an initial step in the study of the regulation of MCAD gene expression, we determined the structure and organization of the human MCAD gene. The gene is comprised of 12 exons which span 44 kb of DNA. Comparison of the MCAD gene to MCAD mRNAs from the MCAD-deficient child revealed that missplicing was common, resulting in a variety of exon deletions and intron insertions. The MCAD gene promoter region is extremely GC-rich and lacks prototypical TATA and CAAT boxes. Several regions upstream of the promoter are homologous with mitochondrial enhancers purportedly involved in coordinate expression of nuclear genes encoding mitochondrial proteins. Transfection of chimeric plasmid constructs with 299 bp of upstream sequence into HepG2 cells revealed high-level transcriptional activity. We conclude that the precursor MCAD mRNA is misspliced to a high degree and complexity in association with the G985 mutation and the MCAD gene contains a strong promoter which shares some structural features with other "housekeeping" genes encoding mitochondrial proteins.
中链酰基辅酶A脱氢酶（MCAD）是一种受到高度调控的线粒体黄素酶，它催化脂肪酸β-氧化的起始反应。MCAD缺乏是能量代谢中常见的遗传性缺陷。我们之前已经表明，一名MCAD缺乏儿童中编码MCAD的mRNA在A985到G的点突变上是纯合的[凯利等人（1990年），《美国国家科学院院刊》87卷，9236 - 9420页]。为了确定MCAD缺乏的分子基础，并作为研究MCAD基因表达调控的第一步，我们确定了人类MCAD基因的结构和组织。该基因由12个外显子组成，跨越44 kb的DNA。将MCAD基因与来自该MCAD缺乏儿童的MCAD mRNA进行比较，发现错误剪接很常见，导致多种外显子缺失和内含子插入。MCAD基因启动子区域富含GC，并且缺乏典型的TATA盒和CAAT盒。启动子上游的几个区域与据称参与编码线粒体蛋白的核基因协同表达的线粒体增强子同源。将含有299 bp上游序列的嵌合质粒构建体转染到HepG2细胞中显示出高水平的转录活性。我们得出结论，前体MCAD mRNA与G985突变相关联，发生了高度且复杂的错误剪接，并且MCAD基因包含一个强启动子，该启动子与其他编码线粒体蛋白的“管家”基因具有一些结构特征。
