The molecular basis for a cytosolic malic enzyme null mutation. Malic enzyme mRNA from MOD-1 null mice contains an internal in-frame duplication that extends the coding sequence by 522 nucleotides

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.     

Genomic sequence analysis of the 238-kb swine segment with a cluster of TRIM and olfactory receptor genes located, but with no class I genes, at the distal end of the SLA class I region

Continuous genomic sequence has been previously determined for the swine leukocyte antigen (SLA) class I region from the TNF gene cluster at the border between the major histocompatibility complex (MHC) class III and class I regions to the UBD gene at the telomeric end of the classical class I gene cluster (SLA-1 to SLA-5, SLA-9, SLA-11). To complete the genomic sequence of the entire SLA class I genomic region, we have analyzed the genomic sequences of two BAC clones carrying a continuous 237,633-bp-long segment spanning from the TRIM15 gene to the UBD gene located on the telomeric side of the classical SLA class I gene cluster. Fifteen non-class I genes, including the zinc finger and the tripartite motif (TRIM) ring-finger-related family genes and olfactory receptor genes, were identified in the 238-kilobase (kb) segment, and their location in the segment was similar to their apparent human homologs. In contrast, a human segment (alpha block) spanning about 375 kb from the gene ETF1P1 and from the HLA-J to HLA-F genes was absent from the 238-kb swine segment. We conclude that the gene organization of the MHC non-class I genes located in the telomeric side of the classical SLA class I gene cluster is remarkably similar between the swine and the human segments, although the swine lacks a 375-kb segment corresponding to the human alpha block. The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL, and GenBank databases under accession numbers AB158486 and AB158487     

Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli

Mitochondrial NAD(+)-dependent malic enzyme (EC 1.1.1.40) is expressed in rapidly proliferating cells and tumor cells, where it is probably linked to the conversion of amino acid carbon to pyruvate. In this paper, we report the cDNA cloning, amino acid sequence, and expression in Escherichia coli of functional human NAD(+)-dependent mitochondrial malic enzyme. The cDNA is 1,923 base pairs long and contains an open reading frame coding for a 584-amino acid protein. The molecular mass is 65.4 kDa for the unprocessed precursor protein. Comparison of the amino acid sequence of the human protein with the published NADP(+)-dependent mammalian cytosolic or plant chloroplast malic enzymes reveals highly conserved regions interrupted with long stretches of amino acids without significant homology. Expression of the processed protein in E. coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells.     

Structure and expression of murine malic enzyme mRNA. Differentiation-dependent accumulation of two forms of malic enzyme mRNA in 3T3-L1 cells

Many murine cells express two mRNAs with markedly different sizes (2.0 and 3.1 kilobases (kb)) that hybridize with cDNA probes for cytosolic malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40). A series of overlapping cDNA clones corresponding to 3129 nucleotides of malic enzyme mRNA was isolated and sequenced to determine the relationship between the two mRNAs and establish the primary structure of mouse malic enzyme. The larger mRNA has an open reading frame of 1716 nucleotides followed by a 3' untranslated region of 1348 nucleotides. The sequence of an exceptionally G/C-rich (88%) portion (65 nucleotides) of the 5' noncoding region was also established. An uncommon poly A addition signal (AUUAAA) is used during the processing of the 3.1-kb mRNA. The 2.0-kb mRNA results from the utilization of another poly A addition signal that truncates the 3' noncoding sequence by approximately 1 kb. The mRNA coding sequence indicates that the malic enzyme subunit contains 572 amino acid residues and has a Mr of 64,000. Two putative components of an NADP-binding domain are located between residues 100 and 165. During the differentiation of 3T3-L1 preadipocytes into adipocytes both the rate of synthesis and relative mRNA concentration for malic enzyme and another lipogenic enzyme, ATP-citrate lyase, are coordinately increased 5-7-fold. However, as preadipocytes approach confluence, the mRNA levels for both lipogenic enzymes transiently increase 3-4-fold, whereas the rates of synthesis of the two proteins are only slightly elevated. Thus, lipogenic enzyme expression is controlled at a pretranslational level during adipogenesis, but the accumulation of the same enzymes may also be subject to translational control in the fibroblast-like preadipocytes. In contrast, mRNA coding for a third enzyme required for lipogenesis, glycerol-3-phosphate dehydrogenase, is not detected in 3T3-L1 preadipocytes, but rapidly accumulates during adipocyte development.     

Primary structure, genomic organization and expression of the major secretory protein of murine epididymis, ME1

The mouse cDNA and its genomic clones encoding the epididymal secretory glycoprotein ME1 were identified. The Me1 gene spans 15kb with four exons and three introns. The deduced amino-acid sequence of the ME1 cDNA revealed that it consists of 149 amino acid residues, which contain a signal peptide characteristic of secretory proteins, six cysteine residues and a proline-rich region conserved in the orthologous proteins. Northern blot analysis revealed that 1.3kb ME1 mRNA is highly expressed in the mouse epididymis. The polyclonal antibodies generated against human HE1 (ME1 orthologous protein) expressed in bacteria reacted with approximately 17 to 25kDa components in mouse epididymis crude extract. The reduction of the molecular mass of the recombinant ME1 protein with the digestion of glycopeptidase A indicated that it is modified by Asn-linked glycosylation.     

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.     

Primary structure of the maize NADP-dependent malic enzyme

Chloroplast-localized NADP-dependent malic enzyme (EC 1.1.1.40) (NADP-ME) provides a key activity for the carbon 4 fixation pathway. In maize, nuclear encoded NADP-ME is synthesized in the cytoplasm as a precursor with a transit peptide that is removed upon transport into the chloroplast stroma. We present here the complete nucleotide sequence for a 2184-base pair full-length maize NADP-ME cDNA. The predicted precursor protein is 636 amino acids long with a Mr of 69,800. There is a strong codon bias found in the amino-terminal portion of NADP-ME that is present in genes for the other enzymes of the C-4 photosynthetic pathway. The NADP-ME transit peptide has general features common to other known chloroplast stroma transit peptides. Comparison of mature maize NADP-ME to the amino acid sequences of known malic enzymes shows two conserved dinucleotide-binding sites. There is a third highly conserved region of unknown function. On the basis of amino acid sequence similarity, the maize chloroplastic enzyme is more closely related to eukaryotic cytosolic isoforms of malic enzyme than to prokaryotic isoforms. We discuss the functional and evolutionary relationship between the chloroplastic and cytosolic forms of NADP-ME.     

BSMAP, a novel protein expressed specifically in the brain whose gene is localized on chromosome 19p12.

Using the sequence of cosmids derived from chromosome 19p12, we have identified a gene encoding a novel protein, BSMAP (brain-specific membrane-anchored protein) and cloned cDNA encoding the full-length open reading frame. Northern blot analysis revealed that BSMAP mRNA is preferentially expressed at a high level in the brain. BSMAP has a putative transmembrane domain and is predicted to be a type-I membrane glycoprotein. Genomic sequence analysis revealed that the gene encoding BSMAP consists of eight exons spanning approximately 8 kb and lies 6 kb away from the gene encoding CLF-1 in a reverse orientation. Although no candidate genetic disorders were found to map either to this precise region of chromosome 19 or to the syntenic region of the mouse genome, the highly specific expression of BSMAP mRNA suggests a role for the protein in CNS function.     

The swine steroid 21-hydroxylase gene (CYP21): cloning and mapping within the swine leucocyte antigen complex

A swine genomic cosmid library constructed from a genotypically SLA homozygous Large White individual was screened with a murine genomic 21-hydroxylase probe. A clone which contained a pig 21-hydroxylase gene was isolated and after subcloning, the 5' region of the gene was sequenced. The deduced amino acid sequence corresponded almost exactly to the NH2 terminal portion of the steroid 21-hydroxylase from porcine adrenal microsomes. Comparison of the first 99 amino acid residues of both sequences revealed three substitutions comprising two leucine residues in positions 10 and 13, and one arginine residue in position 55 for our sequence, instead of threonine in position 10 and lysine in position 13 and 55 for the isolated enzyme. A swine homologous probe was derived from the isolated 21-hydroxylase gene and used for gene assignment by RFLP studies in two swine leucocyte antigen (SLA) informative families. The results demonstrate that the swine 21-hydroxylase gene is located within or close to the swine MHC. Taken together, the present results suggest the existence of a single 21-hydroxylase gene per haploid genome.     

Chronic Reduction of the Cytosolic or Mitochondrial NAD(P)-malic Enzyme Does Not Affect Insulin Secretion in a Rat Insulinoma Cell Line

The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80–86% and ME1 enzyme activity by 78–86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/or cytosolic NADPH production in insulinoma cells.