Brown M L, Wise L S, Rubin C S
Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1988 Mar 25;263(9):4494-9.
Many tissues from wild type mice express cytosolic malic enzyme activity and contain two mRNAs (2.0 and 3.1 kilobases (kb)) that encode a single 64-kDa malic enzyme subunit polypeptide. MOD-1 null mutant mice lack cytosolic malic enzyme activity but express 2.5- and 3.6-kb mRNAs that hybridize with wild type malic enzyme cDNAs and are induced in liver by a starvation/carbohydrate refeeding regimen. To investigate the basis of the MOD-1 null mutation, a lambda gt11 cDNA library was constructed using mRNA from the livers of induced MOD-1 null mice as a template. A recombinant phage with a 2-kb insert was isolated by screening with wild type malic enzyme cDNA probes. The subcloned insert exhibited an atypical (non-wild type) restriction pattern and was subjected to sequence analysis. MOD-1 null malic enzyme cDNA contains an internal tandemly duplicated sequence that corresponds to nucleotides 1027-1548 in the coding region of wild type murine malic enzyme cDNA (Bagchi, S., Wise, L. S., Brown, M. L., Bregman, D., Sul, H. S., and Rubin, C. S. (1987) J. Biol. Chem. 262, 1558-1565). An open reading frame is retained throughout the duplicated sequence. The discovery of a 522-nucleotide in-frame duplication accounts for the increased size of MOD-1 null malic enzyme mRNAs and suggests that a variant malic enzyme polypeptide that is 19 kDa larger than the wild type subunit might be found in mutant mice. Western immunoblot analysis disclosed that MOD-1 null liver cytosol contains an 82-kDa protein that is recognized by anti-malic enzyme antibodies. Under stringent conditions, an anti-sense 32P-oligonucleotide that spans the abnormal junction between the reiterated sequences hybridized with the 2.5 and 3.6-kb MOD-1 null malic enzyme mRNAs but failed to form stable complexes with wild type malic enzyme mRNAs. Thus, both MOD-1 null malic enzyme mRNAs contain the duplication deduced from cDNA sequence analyses. The MOD-1 null mutation might originate from an unequal crossover between homologous regions of two different introns in the malic enzyme gene, thereby causing the duplication of one or more exons.
许多野生型小鼠组织表达胞质苹果酸酶活性，并含有两种mRNA（2.0和3.1千碱基（kb）），它们编码一个单一的64-kDa苹果酸酶亚基多肽。MOD-1基因敲除突变小鼠缺乏胞质苹果酸酶活性，但表达与野生型苹果酸酶cDNA杂交的2.5-kb和3.6-kb mRNA，并在饥饿/碳水化合物再喂养方案诱导下在肝脏中表达。为了研究MOD-1基因敲除突变的基础，以诱导的MOD-1基因敲除小鼠肝脏的mRNA为模板构建了一个λgt11 cDNA文库。用野生型苹果酸酶cDNA探针筛选分离出一个带有2-kb插入片段的重组噬菌体。亚克隆的插入片段表现出非典型（非野生型）的限制性图谱，并进行了序列分析。MOD-1基因敲除的苹果酸酶cDNA包含一个内部串联重复序列，该序列对应于野生型小鼠苹果酸酶cDNA编码区的核苷酸1027-1548（Bagchi, S., Wise, L. S., Brown, M. L., Bregman, D., Sul, H. S., and Rubin, C. S. (1987) J. Biol. Chem. 262, 1558-1565）。在整个重复序列中保留了一个开放阅读框。一个522个核苷酸的框内重复序列的发现解释了MOD-1基因敲除的苹果酸酶mRNA大小的增加，并表明在突变小鼠中可能发现一种比野生型亚基大19 kDa的变异苹果酸酶多肽。蛋白质免疫印迹分析显示，MOD-1基因敲除的肝脏胞质溶胶中含有一种82-kDa的蛋白质，该蛋白质可被抗苹果酸酶抗体识别。在严格条件下，一个跨越重复序列之间异常连接的反义32P-寡核苷酸与2.5-kb和3.6-kb的MOD-1基因敲除的苹果酸酶mRNA杂交，但未能与野生型苹果酸酶mRNA形成稳定复合物。因此，两种MOD-1基因敲除的苹果酸酶mRNA都包含从cDNA序列分析推断出的重复序列。MOD-1基因敲除突变可能源于苹果酸酶基因中两个不同内含子同源区域之间的不等交换，从而导致一个或多个外显子的重复。
