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R Lightowlers

Publications and source records attributed to R Lightowlers.

9 recordsLinked to original sources
PubMed1996

A low-temperature-responsive gene from barley encodes a protein with single-stranded nucleic acid-binding activity which is phosphorylated in vitro.

A low-temperature-responsive gene, blt 801, isolated from a winter barley (Hordeum vulgare L.) cDNA library prepared from leaf meristematic tissue, was sequenced. The deduced amino acid sequence predicts a glycine-rich RNA-binding protein (GR-RNP) which was homology to stress-responsive GR-RNPs from several other plant species. BLT 801 is a two-domain protein, the amino-terminal domain comprises a consensus RNA-binding domain similar to that found in many eukaryotic genes and the carboxy-terminal domain is extremely glycine-rich (68.5% glycine). Blt 801 mRNA also accumulates in response to the phytohormone abscisic acid. The protein encoded by blt 801 has been produced as a recombinant fusion protein using a bacterial expression vector. The fusion protein, a chimaera of glutathione S-transferase and BLT 801, has been used in studies to determine nucleic acid binding and other characteristics. Binding studies with single-stranded nucleic acids show that BLT 801 has affinity for homoribopolymers G, A and U but not C, it also binds to single-stranded DNA and selects RNA molecules containing open loop structures enriched in adenine but low in cytosine. Blt 801 has a consensus motif for phosphorylation by cAMP protein kinase (PKA) at the junction between the two domains which can be phosphorylated by PKA in vitro and which, by analogy to animal studies, may have significance for controlling enzyme function.

Amino Acid Sequence↗
PubMed1991

Human cytochrome c oxidase subunit VIb: characterization and mapping of a multigene family.

We report the isolation and sequence of a human heart cDNA coding for cytochrome c oxidase (COX) subunit VIb (COX VIb). This cDNA extends 50 bp upstream from the region coding for the mature peptide. By Northern analysis, a single transcript of approx. 550 nucleotides (nt) has been identified in six human tissues. Southern analysis of human genomic DNA demonstrates the presence of multiple loci that show high homology to the cDNA. These loci cosegregate with either five or six different human chromosomes in human-rodent somatic cell hybrids. Using the COX6b cDNA, genomic sequences representing two of these loci have been isolated and characterized. The nt sequence analysis suggests that both loci represent COX6b pseudogenes.

Amino Acid Sequence↗
PubMed1991

Subunit function in eukaryote cytochrome c oxidase. A mutation in the nuclear-coded subunit IV allows assembly but alters the function and stability of yeast cytochrome c oxidase.

Strains of the yeast Saccharomyces cerevisiae disrupted in YCOX4, the nuclear gene encoding cytochrome c oxidase subunit IV, do not assemble a functional or spectrally visible oxidase. We report the characterization of a yeast strain, RM1, expressing a mutated YCOX4 gene which is temperature sensitive for respiration at 37 degrees C, but incorporates cytochrome aa3 over all growth temperatures. The mutant enzyme is less stable than the wild type, with subunit IV readily proteolyzed without gross denaturation of the complex but with a concomitant loss of oxidase activity. When grown fermentatively at 37 degrees C, cytochrome c oxidase from the mutant strain had a turnover number of less than 3% of the normal complex, while Km values and subunit levels were comparable to normal. Thus alterations in subunit IV can perturb the enzyme structure and alter its catalytic rate, implying a role for this subunit in cytochrome c oxidase function as distinct from assembly.

Base Sequence↗
PubMed1990

Isolation and characterization of the cDNAs encoding two isoforms of subunit CIX of bovine cytochrome c oxidase.

The smallest subunit of bovine cytochrome c oxidase (CIX or VIII in different nomenclatures) occurs in two isoforms, a heart (H) form and a liver (L) form. The cDNAs for both of these forms have been isolated and sequenced. The cDNA for the H form encodes a protein 70 amino acids long with a 24-residue presequence and a mature polypeptide of 46 amino acids; that of liver encodes a protein of 69 amino acids, a 25-residue presequence and a mature polypeptide of 44 amino acids. The leader sequences of the H and L forms are 40% homologous with an abundance of positively charged residues but no negatively charged amino acids. These features are typical of polypeptides targeted to the mitochondrion for processing in the matrix space. The homology of the two isoforms is 52% in the mature subunit with most of the differences occurring in the N-terminal hydrophilic domain of the protein. Evidence has been obtained of polymorphisms of both the H and L forms of the subunit. Protein chemical analyses show that the H isoform is the predominant if not the exclusive form of subunit CIX in heart and skeletal muscle tissue. The L form is the predominant form in liver, kidney, and brain. Northern analyses, using cDNAs to the two forms to screen whole cell RNA preparations, show that the transcript of the H isoform is present in heart and skeletal muscle but not in other tissues examined. The mRNA of the L form was found in brain, kidney, and liver and also in heart and skeletal muscle. These results indicate that the synthesis of the H isoform of CIX is controlled transcriptionally while the L form is under post-transcriptional regulation at least in heart and muscle tissue.

Amino Acid Sequence↗
PubMed1990

Isoforms of mammalian cytochrome c oxidase: correlation with human cytochrome c oxidase deficiency.

We have reviewed the structure, function, and biogenesis of mammalian cytochrome c oxidase, examined the tissue-specific expression of isoforms of cytochrome c oxidase subunits in different mammals, and attempted to correlate the data with our knowledge of cytochrome c oxidase deficiency, illustrated by one particular patient. Cytochrome c oxidase was isolated from bovine tissues, and individual subunits examined by SDS-PAGE, N-terminal peptide sequencing, and antibody binding. Isoforms of subunits VIa, VIIa, and VIII were identified, manifesting one pattern of expression in heart and skeletal muscle, and another in liver, kidney, and brain. In rat heart and liver, only one form of subunit VIIa was identified. Northern analysis of bovine and rat tissues suggested that the tissue-specific expression of subunits VIa and VIII is regulated transcriptionally in liver, kidney, and brain, and posttranscriptionally in heart and skeletal muscle. In humans, antibody binding documented isoforms of subunits VIa and VIIa, with the pattern of expression in heart and skeletal muscle differing from that in liver, kidney, and brain; our data suggested that both isoforms of subunit VIa may be expressed in human heart. In a patient with cytochrome c oxidase deficiency, the clinical, morphologic, and biochemical manifestations were much more severe in heart than in skeletal muscle. Antibody binding suggested partial assembly of the enzyme in heart. These and other data suggest considerably more variability in the tissue-specific expression of isoforms of cytochrome c oxidase subunits than previously recognized.

Amino Acid Sequence↗
PubMed1990

The use of skin fibroblast cultures in the detection of respiratory chain defects in patients with lacticacidemia.

Cultured skin fibroblasts from patients with lacticacidemia were incubated with glucose for 1 h and the lactate and pyruvate production measured. Those patients with increased lactate to pyruvate ratios were further analyzed for the cause of the abnormal redox state. Two categories of patients are described. The first contains patients with either severe or partial cytochrome oxidase deficiency; this group can be broken down further into patients with Leigh's disease, Kearns-Sayre syndrome, and liver-specific cytochrome oxidase deficiency. In this group, the rise in lactate to pyruvate ratio roughly correlated with the severity of the defect. The second patient category had defects located in complex I of the mitochondrial respiratory chain. This is easily demonstrated in the most severely affected patients with the fatal infantile form of the disease. Patients with severe defects in either complex I or cytochrome oxidase had complexes that were only partially assembled. Patients with mitochondrial encephalopathy with lactic acidosis and stroke-like episodes demonstrated only minor changes in redox state and in the behavior of the mitochondrial respiratory chain.

Acidosis, Lactic↗
PubMed1989

Cloning and sequencing of the cDNA for a 13th different subunit (IHQ) of beef heart cytochrome c oxidase.

The mammalian cytochrome c oxidase is a complex of 13 different subunits. We present the full amino acid sequence of the one remaining uncharacterized subunit, subunit IHQ in the nomenclature used here, VIIb in the numbering system of Kadenbach and colleagues (e.g. Kadenbach, B., and Merle, P. (1981) FEBS Lett. 135, 1-11). A partial protein sequence was obtained from the purified subunit isolated by gel filtration procedures. This information was used to synthesize an oligonucleotide probe which was then used to isolate a cDNA clone encoding the subunit. This cDNA for subunit IHQ is 480 base pairs long and encodes a polypeptide which is either 83 or 88 amino acids long, including an N-terminal leader sequence of either 27 or 32 residues. The molecular weight of the mature subunit IHQ is 6350 based on the amino acid sequence deduced from the gene. The leader sequence is typical of other mitochondrial target sequences in having several positively charged residues but no negatively charged side chains.

Amino Acid Sequence↗
PubMed1987

Altered translation of the uncC gene coding for the epsilon subunit of the F1F0-ATPase of Escherichia coli.

The nucleotide sequence of the previously described uncC424 allele was determined and found to be the same as that of a wild-type uncC gene. However, a G----A change occurred 7 nucleotides upstream from the translation start codon, changing the putative Shine-Dalgarno sequence from GAGG to GAAG. Four revertant strains were examined. In one revertant, which had normal growth and membrane properties, a single base deletion had occurred to re-form the Shine-Dalgarno sequence GAGG 1 nucleotide closer to the translation start codon. A second revertant had a single base deletion in the preceding uncD gene, causing an extension of the beta subunit by 6 amino acids and an increase, presumably by translational coupling, in the amount of epsilon subunit. The third and fourth revertant strains were phenotypically similar and had either C----T or G----T changes 18 or 19 nucleotides, respectively, upstream from the translation start codon.

Base Sequence↗