Regulation of malic enzyme expression and the molecular basis for a cytosolic malic enzyme null mutation

Abstract

Many tissues from wild type mice express cytosolic malic enzyme activity and contain two mRNAs (2.0 and 3.1 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. The two malic enzyme mRNAs are induced in the livers of wild type and MOD-1 null mice by a starvation/carbohydrate feeding regimen. In order to investigate the basis for the MOD-1 null mutation, a {dollar}\lambda{dollar}gt 11 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. 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. 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 {dollar}\sp{lcub}32{rcub}{dollar}P-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. A 32P-labeled anti-sense RNA that includes 302 nucleotides of normal sequence and 221 nucleotides of the aberrant duplication was fully resistant to RNAse digestion only after hybridization with MOD-1 null mRNA. These observations demonstrate directly that both MOD-1 null malic enzyme mRNAs contain the duplication deduced from cDNA sequence analyses. A hypothetical explanation for the origin of the MOD-1 null mutation is that an unequal cross-over occurred between homologous regions of two different introns in the malic enzyme gene, thereby causing the duplication of one or more exons.