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C Somerville

Publications and source records attributed to C Somerville.

At least 55 records · Page 3Linked to original sources
PubMed1993

Stearoyl-acyl carrier protein delta 9 desaturase from Ricinus communis is a diiron-oxo protein.

A gene encoding stearoyl-acyl carrier protein delta 9 desaturase (EC 1.14.99.6) from castor was expressed in Escherichia coli. The purified catalytically active enzyme contained four atoms of iron per homodimer. The desaturase was studied in two oxidation states with Mössbauer spectroscopy in applied fields up to 6.0 T. These studies show conclusively that the oxidized enzyme contains two (identical) clusters consisting of a pair of antiferromagnetically coupled (J > 60 cm-1, H = JS1.S2) Fe3+ sites. The diferric cluster exhibited absorption bands from 300 to 355 nm; addition of azide elicited a charge transfer band at 450 nm. In the presence of dithionite, the clusters were reduced to the diferrous state. Addition of stearoyl-CoA and O2 returned the clusters to the diferric state. These properties are consistent with assigning the desaturase to the class of O2-activating proteins containing diiron-oxo clusters, most notably ribonucleotide reductase and methane monooxygenase hydroxylase. Comparison of the primary structures for these three catalytically diverse proteins revealed a conserved pair of the amino acid sequence -(Asp/Glu)-Glu-Xaa-Arg-His- separated by approximately 100 amino acids. Since each of these proteins can catalyze O2-dependent cleavage of unactivated C--H bonds, we propose that these amino acid sequences represent a biological motif used for the creation of reactive catalytic intermediates. Thus, eukaryotic fatty acid desaturation may proceed via enzymatic generation of a high-valent iron-oxo species derived from the diiron cluster.

Amino Acid Sequence↗
PubMed1993

Isolating plant genes.

The genetic transformation of most agriculturally important plant species is now possible. However, the application of this technology to rational plant-improvement is currently limited by a shortage of cloned genes for important traits. Recent technological advances in plant-gene isolation and identification, such as map-based cloning, insertional mutagenesis and large-scale cDNA sequencing, have accelerated the rate of gene isolation and significantly expanded the opportunities for genetic engineering of crop plants.

Biotechnology↗
PubMed1993

A non-specific lipid transfer protein from Arabidopsis is a cell wall protein.

Lipid transfer proteins (LTPs), mediate the transfer of phospholipids between membranes in vitro. However, the in vivo function of LTPs is not known. To determine the precise location of a non-specific LTP from Arabidopsis, a cDNA clone was used to produce an Arabidopsis LTP:protein A fusion. Antibodies raised against the fusion were used to localize the Arabidopsis LTP by immunoelectron microscopy. LTP was found to be located in the cell wall, mainly in epidermal cells. This location appears to be inconsistent with the proposed role of the protein in intracellular lipid transfer.

Antigens, Plant↗
PubMed1993

Wiskott Aldrich syndrome: an immunodeficiency syndrome not rare in Western Australia.

Wiskott Aldrich syndrome, a combined cellular and humoral X-linked immunodeficiency, is generally considered to be rare. The aim of this study was to ascertain the true prevalence in the paediatric population in Western Australia, describe the clinical features, and summarise the current literature on this unusual condition. All cases of Wiskott Aldrich syndrome presenting to Princess Margaret Hospital in Perth during the period from January 1960 to January 1990 were identified by a retrospective review of case records and by interviewing hospital immunology, haematology and general clinical staff. Nine cases of Wiskott Aldrich syndrome are described, demonstrating that the prevalence of Wiskott Aldrich syndrome in Western Australia is nine times that expected from previous reports. Death occurred in a number of patients before the correct diagnosis was recognised. The clinical features in this group are quite variable. Low isohaemagglutinins, elevated IgE, blunted DTH skin multitest, and very low CD8 numbers are however consistent features. Wiskott Aldrich syndrome may be more prevalent than previously recognised, and should be considered in males with thrombocytopenia and infection.

Adolescent↗
PubMed1992

Preliminary crystallographic data for stearoyl-acyl carrier protein desaturase from castor seed.

Recombinant stearoyl-acyl carrier protein desaturase (EC 1.14.99.6) from castor seed has been crystallized with polyethylene glycol 8000 as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 81.3, b = 146.4 and c = 197.7 A. The observed diffraction pattern extends to at least 2.5 A resolution. Rotation function calculations indicate a non-crystallographic 3-fold rotation axis parallel to the crystallographic a-axis. Perpendicular to this axis, 2-fold rotation axes were found at 30 degrees intervals, i.e. maxima at kappa = 180 degrees, phi = 90 degrees and omega = 30 degrees and 60 degrees, respectively. Together with the packing density of the crystals (Vm = 2.4 A3/Da for n = 6), these results suggest, that the crystal asymmetric unit most likely contains a hexamer of desaturase subunits.

Crystallization↗
PubMed1992

A role for membrane lipid polyunsaturation in chloroplast biogenesis at low temperature.

Two different mutants of Arabidopsis thaliana deficient in chloroplast membrane lipid polyunsaturation were indistinguishable in appearance from the wild-type when grown at 22 degrees C. By contrast, leaf tissues of the mutants that developed during growth at 5 degrees C were chlorotic, whereas the wild type was not. This is the first direct evidence that chloroplast lipid polyunsaturation contributes to low-temperature fitness. Chloroplasts from mutant lines grown at 5 degrees C were much smaller than those of the wild-type, and the thylakoid membrane content was reduced by up to 70%. However, there was no discernible effect of low temperature on chloroplasts that developed prior to exposure to low temperatures. These and related observations suggest that the high degree of chloroplast membrane lipid polyunsaturation is required for some aspect of chloroplast biogenesis.

Journal Article↗
PubMed1991

Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs.

Stearoyl-acyl-carrier-protein (ACP) desaturase (EC 1.14.99.6) was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was no detectable identity between the deduced amino acid sequences of the castor delta 9-stearoyl-ACP desaturase and either the delta 9-stearoyl-CoA desaturase from rat or yeast or the delta 12 desaturase from Synechocystis, suggesting that these enzymes may have evolved independently. However, there was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the delta 9 desaturase is developmentally regulated.

Amino Acid Sequence↗
PubMed1991

Construction and characterization of a yeast artificial chromosome library of Arabidopsis which is suitable for chromosome walking.

A yeast artificial chromosome (YAC) genomic library of Arabidopsis thaliana was constructed in a derivative of the vector pYAC4 which was modified to facilitate the production of end-specific probes for chromosome walking. Experiments in which a subset of 2300 clones from the library were probed with 30 restriction fragment length polymorphism (RFLP) markers indicated that, on the average, the entire genome is represented once in each 800 YAC clones. Thus, the complete library of more than 20,000 YACs is expected to contain most or all of the Arabidopsis genome with a high probability. The YAC clones examined in the sample had an average insert size of approximately 150 kb +/- 10 and represented more than 5% of the Arabidopsis genome. Based on the properties of the library and the currently available RFLP maps for Arabidopsis, only one or two steps from flanking RFLPs should be sufficient to isolate an average gene in Arabidopsis by chromosome walking with the YACs. In order to facilitate chromosome walking, a method for the production of hybridization probes from the ends of the inserts was employed that is based on a combination of pre-amplification of the vector/insert junction sequences via the polymerase chain reaction and specific transcription from T3 or T7 RNA polymerase promoters flanking the cloning site in the YAC vector.

Base Sequence↗
PubMed1991

Mutants of Arabidopsis with altered regulation of starch degradation.

Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes alpha-amylase, beta-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutation at a locus designated sex1 at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation.

Journal Article↗
PubMed1991

Mutant of Arabidopsis deficient in xylem loading of phosphate.

A mutant of Arabidopsis thaliana deficient in the accumulation of inorganic phosphate has been isolated by screening directly for plants with altered quantities of total leaf phosphate. The mutant plants accumulate approximately 5% as much inorganic phosphate, and 24 to 44% as much total phosphate, as wild-type plants in aerial portions of the plant. Growth of the mutant is reduced, relative to wild type, and it exhibits other symptoms normally associated with phosphate deficiency. The phosphate deficiency is caused by a single nuclear recessive mutation at a locus designated pho1. The rate of phosphate uptake into the roots was similar between mutant and wild-type plants over a wide range of external phosphate concentrations. In contrast, when plants were grown in media containing 200 micromolar phosphate or less, phosphate transfer to the shoots of the mutant was reduced to 3 to 10% of the wild-type levels. The defect in phosphate transfer to the shoots could be overcome by providing higher levels of phosphate. Transfer of sulfate to the shoots was essentially normal in the mutant, indicating that the pho1 lesion was not a general defect in anion transport. Movement of phosphate through the xylem of the shoots was not impaired. The results suggest that the mutant is deficient in activity of a protein required to load phosphate into the xylem.

Journal Article↗
PubMed1991

Development of a system for efficient chromosome walking in Arabidopsis.

The small genome size of Arabidopsis and the low level of repetitive DNA sequences make this crucifer an attractive system for chromosome walking to isolate genes. Mapping of a mutant locus relative to restriction fragment length polymorphism (RFLP) markers provides the first step towards isolating the corresponding gene. The RFLP marker closest to the target gene serves as a starting point. The distance between gene and marker is generally in the range of 50-200 kb. In order to facilitate chromosome walking of this magnitude, we constructed a yeast artificial chromosome (YAC) library of Arabidopsis. Large fragments of Arabidopsis DNA were cloned into a YAC vector and transformed into yeast. The library contains more than 10 equivalents of the Arabidopsis genome. YACs containing sequences of RFLP markers of Arabidopsis revealed an average insert size of 150 kb. Thus, 1-3 (contiguous) YACs should be sufficient to clone genes from Arabidopsis by chromosome walking. In order to use the system to isolate genes involved in the signal transduction of abscisic acid, we fine-mapped the mutant loci abi-1 and abi-2, which confer abscisic acid insensitivity, relative to RFLPs and isolated the corresponding YACs.

Abscisic Acid↗
PubMed1990

Soluble and membrane-bound forms of cytochrome b5 are the products of a single gene in chicken.

In order to determine the relationship of the soluble cytochrome b5 found in erythrocytes to the membrane-bound form found in other tissues, a cDNA clone encoding cytochrome b5 in chicken erythrocytes was isolated by using mixed oligonucleotides based on a partial amino acid sequence of the protein. Complete nucleotide sequence identity between the erythrocyte cDNA and the sequence of a cDNA clone of the liver protein suggests that they are transcribed from the same gene. The isolation and structural analysis of genomic clones was also consistent with the presence of only one cytochrome b5 gene in chicken. These results suggest that the formation of soluble erythrocyte cytochrome b5 occurs by proteolytic processing of the membrane-bound form. Thus, previous reports indicating that the carboxyl terminal amino acid residue of the erythrocyte form differs from the corresponding residue of the membrane-bound form may suggest the existence of a novel post-translational modification.

Amino Acid Sequence↗
PubMed1990

A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism.

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18 degrees C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury-leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of (14)CO(2) into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury. These results suggest one of two possible explanations: either the mutation directly affects sterol metabolism, which in turn leads to chilling sensitivity, or the mutation affects another unidentified function and the accumulation of radioactivity in steryl-esters is a secondary consequence of chilling injury.

Journal Article↗
PubMed1989

Altered regulation of beta-amylase activity in mutants of Arabidopsis with lesions in starch metabolism.

Three classes of mutants of Arabidopsis thaliana (L.) Heynhold with alterations in starch metabolism were found to have higher levels of leaf amylase activity than the wild type when grown in a 12-hr photoperiod. This effect was dependent upon the developmental stage of the plants and was largely suppressed during growth in continuous light. The various amylolytic activities in crude extracts were separated by electrophoresis in nondenaturing polyacrylamide gels and visualized by activity staining. The increased amylase activity in the mutants was due to an up to 40-fold increase in the activity of an extrachloroplast beta-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of beta-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that beta-amylase appears to be a highly regulated enzyme, but as yet no physiologically relevant function can be assigned to this enzyme due to the absence of starch in the cytoplasmic compartment of leaf cells.

Journal Article↗
PubMed1989

A mutant of Arabidopsis deficient in the chloroplast 16:1/18:1 desaturase.

Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 18-carbon and 16-carbon polyunsaturated fatty acids and increased levels of the 18:1 and cis-16:1 precursors due to a single nuclear mutation at a locus designated fadC. Analysis of the fatty acid compositions of individual lipids and the kinetics of lipid labeling with [(14)C]acetate in vivo indicate that the mutant lacks activity of the chloroplast glycerolipid omega-6 desaturase. As a result, lipids synthesized by the prokaryotic pathway are not desaturated further than 18:1 and 16:1. Lipids derived from the eukaryotic pathway are desaturated-presumably by the endoplasmic reticulum 18:1 phosphatidylcholine desaturase. However, an increase in the level of 18:1 on all the phospholipids derived from the eukaryotic pathway in leaves of the mutant suggests that the mutation does exert an effect on the composition of extrachloroplast membranes. Synthesis of monogalactosyldiacylglycerol (MGD) by the prokaryotic pathway is reduced 30 to 35% in the mutant and there is a corresponding increase in MGD synthesis by the eukaryotic pathway. This shift in metabolism which results in a more unsaturated MGD pool, may reflect the existence of a regulatory mechanism which apportions lipid synthesis between the two pathways in response to alterations in the physical properties of the chloroplast membranes.

Journal Article↗
PubMed1989

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation.

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C(16:1) and C(18:1) lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.

Journal Article↗