Molecular cloning and nucleotide sequence of the cDNA for sperm-specific lactate dehydrogenase-C from mouse.

Mouse sperm-specific lactate dehydrogenase-C (LDH-C) cDNA was cloned and sequenced from lambda gt11 expression library. The LDH-C cDNA insert of 1236 bp consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (113 bp) non-coding regions, and the poly(A) tail (70 bp). The Northern blot analysis of poly(A)-containing RNAs from mouse testes and liver indicates that the LDH-C gene is expressed in testes but not in liver, and that its mRNA is approx. 1400 nucleotides in length. The nucleotide and amino acid sequences of the mouse LDH-C cDNA show 73% and 72% homologies, respectively, with those of the mouse LDH-A. The Southern blot analysis of genomic DNAs from mouse liver and human placenta indicates the presence of multiple LDH-C gene-related sequences.     

cDNA cloning of human calpastatin: sequence homology among human, pig, and rabbit calpastatins

cDNA of human calpastatin, an inhibitor protein specific for calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase) was isolated by screening of a library prepared from human liver mRNA with pig calpastatin cDNA fragment as a probe. The primary structure of human calpastatin was deduced from the nucleotide sequence of the cDNA and compared with that of pig and rabbit calpastatins already reported. Human calpastatin consisted of 673 amino acid residues and had 78% and 77% identity to pig or rabbit calpastatins, respectively. Human calpastatin had a domain structure with four internally repetitive sequences and one N-terminal non-homologous sequence like the other calpastatins. Human calpastatin had two deletions, 22 and 13 residues long in domain L and domain 1, respectively, compared to pig or rabbit calpastatins.     

cDNA cloning of rice lipoxygenase L-2 and characterization using an active enzyme expressed from the cDNA in Escherichia coli.

A full-length cDNA of rice lipoxygenase L-2 was cloned from 3-day-old seedlings. The identity of the clone was determined by amino acid sequencing of selected peptides of the purified enzyme and immunological characterization of an active enzyme that was produced from the cDNA in Escherichia coli by cultivation at 15 degrees C. The nucleotide sequence showed a strong bias toward G and C in the selection of nucleotides, especially at the third position of the codons (93% G/C). The complete amino acid sequence of the enzyme was deduced from the nucleotide sequence. The molecular mass of the enzyme was calculated to be 96,657 Da based on 865 amino acids. The amino acid sequence shares similarity with those of dicot lipoxygenases throughout the enzyme at a level of 50%. A hydropathy profile calculated from the amino acid sequence resembled those of dicot lipoxygenases, suggesting conservation of the secondary structure of these enzymes. The active enzyme, expressed in Escherichia coli, was characterized for pH dependence of the enzyme activity, intramolecular specificity, heat stability and Km. The enzyme had the same properties as the L-2 enzyme that was isolated from seedlings, but differed from the lipoxygenase L-3 isolated from mature plants.     

Yak lactate dehydgogenase A4: purification, properties, and cDNA cloning

Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.     

cDNA cloning, genomic structure and polymorphism of the porcine FHL3 gene

LIM domain proteins are important regulators of the growth, determination and differentiation of cells. Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). In this study, we have determined the complete coding sequence of pig FHL3 which encodes a 280 amino acid protein. The coding region of the pig FHL3 gene is organized in five exons and spans an approximately 2.1-kb genomic region. Comparative sequencing of six pig breeds revealed three single nucleotide polymorphisms (SNPs) within exon 2 of which an A-->G substitution at position 313 changes a codon for arginine into a codon for glycine. The substitution was situated within a PstI recognition site and developed as a PCR-RFLP marker for further use in population variation investigations and association analysis. The A/G polymorphism was segregating only in Landrace pigs. Association studies of the FHL3 polymorphism with carcass traits provided preliminary evidence that the PstI PCR-restriction fragment length polymorphism (RFLP) genotype may be associated with variation in several carcass traits of interest for pig breeding. Further investigations in more Landrace pigs are needed to confirm this.     

cDNA cloning and expression of pig cytosolic aspartate aminotransferase in Escherichia coli: amino-terminal heterogeneity of expressed products and lack of its correlation with enzyme function

A full-length cDNA encoding the pig cytosolic aspartate aminotransferase (EC 2.6.1.1) (cAspAT) was constructed from two overlapping cDNA clones. One clone (Lm pcAAT-8) isolated from a lambda gt10 pig heart cDNA library contained a 3' untranslated sequence, a poly(A) segment, and a part of the coding region for amino acid positions 127-412. Another clone (Lm pcAAT-107) isolated from a lambda gt10 primer extension library contained the coding region for amino acid positions 1-148 and a 5' untranslated sequence. Rejoining of the cDNA inserts of the two clones and recloning into pUC18 gave rise to a cDNA covering an entire coding sequence for pig cAspAT mRNA. Insertion into pKK223-3 yielded an expression plasmid, ppcAAT200. Escherichia coli JM105 cells transfected with ppcAAT200 overproduced pig cAspAT to an extent of about 3% of the total cellular soluble proteins. The expressed product was indistinguishable from the alpha subform of cAspAT isolated from pig heart in terms of specific activity, absorption spectra, molecular size, crystalline form, and immunological reactivity with anti pig cAspAT antibody. Compared with the amino-terminal sequence (Ala-Pro-Pro-) reported for pig heart cAspAT, the recombinant pig cAspAT showed heterogeneity in the amino-terminal sequence: Ala 1 (26%), Pro2 (54%), and Pro3 (19%). Construction of a mutant cAspAT with deletion of residues 1-3 and its comparison with the wild-type enzyme revealed that loss of the three amino-terminal residues does not affect the catalytic activity and structural integrity of the enzyme.     

Purification, characterization, and cDNA cloning of lipoate-activating enzyme from bovine liver

In mammals, lipoate-activating enzyme (LAE) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. The lipoyl moiety is then transferred to the specific lysine residue of lipoate-dependent enzymes by the action of lipoyltransferase. We purified LAE from bovine liver mitochondria to apparent homogeneity. LAE activated lipoate with GTP at a 1000-fold higher rate than with ATP. The reaction absolutely required lipoate, GTP, and Mg(2+) ion, and the reaction product was lipoyl-GMP. LAE activated both (R)- and (S)-lipoate to the respective lipoyl-GMP, although a preference for (R)-lipoate was observed. Similarly, lipoyltransferase equally transferred both the (R)- and (S)-lipoyl moieties from the respectively activated lipoates to apoH-protein. Interestingly, however, only H-protein carrying (R)-lipoate was active in the glycine cleavage reaction. cDNA clones encoding a precursor LAE with a mitochondrial presequence were isolated. The predicted amino acid sequence of LAE is identical with that of xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III, but an amino acid substitution due to a single nucleotide polymorphism was found. These results indicate that the medium-chain acyl-CoA synthetase in mitochondria has a novel function, the activation of lipoate with GTP.     

The cDNA cloning and molecular evolution of reptile and pigeon lactate dehydrogenase isozymes

The cDNAs encoding lactate dehydrogenase isozymes LDH-A (muscle) and LDH-B (heart) from alligator and turtle and LDH-A, LDH-B, and LDH-C (testis) from pigeon were cloned and sequenced. The evolutionary relationships among vertebrate LDH isozymes were analyzed. Contrary to the traditional belief that the turtle lineage branched off before the divergence between the lizard/alligator and bird lineages, the turtle lineage was found to be clustered with either the alligator lineage or the alligator-bird clade, while the lizard lineage was found to have branched off before the divergence between the alligator/turtle and bird lineages. The pigeon testicular LDH-C isozyme was evidently duplicated from LDH-B (heart), so it is not orthologous to the mammalian testicular LDH-C isozymes.      

The oligosaccharyltransferase complex from pig liver: cDNA cloning, expression and functional characterisation

Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc₃-Man₉-GlcNAc₂ from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of 48.9[emsp4 ]kDa (OST48), 68.7[emsp4 ]kDa (ribophorin I) and 69.3[emsp4 ]kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein.Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible 25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the –3/–5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the –3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif.     

Molecular cloning,polymorphism and association analyses of a novel differentially expressed porcine mRNA

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus muscle tissues from Meishan and Large White pigs. One novel mRNA that was differentially expressed was identified through semi-quantitative RT-PCR and the full-length cDNA sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the mRNA is not homologous to any of the known porcine genes. Sequence prediction analysis revealed that this mRNA is no-coding mRNA. Polymorphism analyses revealed that there was a C-T mutation on the position of 505 bp and PCR-HhaI-RFLP analyses revealed that Chinese indigenous pig breeds and exotic pig breeds displayed obvious genotype and allele frequency differences at this locus. Association analyses revealed that this polymorphic locus was significantly associated with the drip loss rate, water holding capacity, dressing percentage, rib numbers, lean meat percentage, estimated lean meat percentage, loin eye width and loin eye area (P < 0.05).     

Enhanced heat, alkaline and tryptic stability of acetamidinated pig heart lactate dehydrogenase.

Modification of 17 from 24 lysine residues in pig heart lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) with methyl aceimidate yields an enzyme derivative with enhanced stability toward meat and alkaline denaturation as well as tryptic digestion. The specific activity of the modified enzyme is only slightly reduced