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C R Somerville

Publications and source records attributed to C R Somerville.

48 records · Page 3Linked to original sources
PubMed1987

The primary structure of spinach glycolate oxidase deduced from the DNA sequence of a cDNA clone.

A cDNA clone encoding the peroxisomal enzyme glycolate oxidase (EC 1.1.3.15) was identified by probing a cDNA library of spinach with synthetic oligonucleotides based on the partial amino acid sequence of the enzyme. Determination of the DNA sequence of the 1526-nucleotide cDNA indicated a 1107-nucleotide open reading frame which encodes a polypeptide of 40,282 daltons. The polypeptide produced by in vitro transcription and translation of the cDNA insert had the same apparent subunit molecular mass as the enzyme purified from leaves, indicating that the cDNA encodes a full-length polypeptide and that no cleavage of the polypeptide is required for uptake of the polypeptide by peroxisomes. Comparison of the deduced amino acid sequence with those of two other plant peroxisomal proteins revealed a region of homology which may be involved in directing proteins to the peroxisome.

Alcohol Oxidoreductases↗
PubMed1987

The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis.

A mutant of Arabidopsis thaliana with reduced content of C(18:3) and C(16:3) fatty acids in membrane lipids exhibited a 45% reduction in the cross-sectional area of chloroplasts and had a decrease of similar magnitude in the amount of chloroplast lamellar membranes. The reduction in chloroplast size was partially compensated by a 45% increase in the number of chloroplasts per cell in the mutant. When expressed on a chlorophyll basis the rates of CO(2)-fixation and photosynthetic electron transport were not affected by these changes. Fluorescence polarization measurements indicated that the fluidity of the thylakoid membranes was not significantly altered by the mutation. Similarly, on the basis of temperature-induced fluorescence yield enhancement measurements, there was no significant effect on the thermal stability of chlorophyll-protein complexes in the mutant. These observations suggest that the high content of trienoic fatty acids in chloroplast lipids may be an important factor regulating organelle biogenesis but is not required to support normal levels of the photosynthetic activities associated with the thylakoid membranes.

Journal Article↗
PubMed1986

Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the '16:3' plant Arabidopsis thaliana.

The kinetics of [1-14C]acetate incorporation in Arabidopsis thaliana L. (Heyn) showed almost equal labelling of phosphatidylcholine (PC) and diacylgalactosylglycerol (DGG) at early times and the transfer of radioactivity from PC to DGG and diacyldigalactosylglycerol (DDG) at longer times. These kinetics demonstrated the parallel operation of the prokaryotic and eukaryotic pathways of lipid synthesis [Roughan & Slack (1982) Annu. Rev. Plant Physiol. 33, 97-132] in this tissue. At 2 h after the application of [1-14C]acetate, more than 85% of the radioactivity at the sn-2 position of each chloroplast lipid was in 16-carbon fatty acids. However, after 60 h, molecular species containing labelled C18 fatty acids at position sn-2 and presumably derived from microsomal PC made a large contribution (20-70%) to each chloroplast lipid except phosphatidylglycerol. These findings are consistent with the contention that the chain length of the fatty acid at the sn-2 position of glycerol is an accurate predictor of whether a particular lipid molecule has been synthesized by the prokaryotic or eukaryotic pathway. At 30 min after the start of [1-14C]acetate labelling, only 12.3% of the radioactivity in PC was in saturated fatty acids, but the proportion increased steadily to 24.3% after 142 h. It is suggested that steps involved in the conversion of PC to chloroplast lipids on the eukaryotic pathway discriminate against palmitate-containing species. The step involved does not appear to be transfer of PC to the chloroplast because extrachloroplastic and chloroplast membranes purified from Arabidopsis mesophyll protoplasts each contained PC with a fatty acid composition similar to that of the same lipid from leaves. Positional analysis of unlabelled lipids, together with the information summarized above, is used to construct a quantitative scheme of the fluxes through the prokaryotic and eukaryotic pathways during lipid synthesis in Arabidopsis. This scheme shows that 38% of the fatty acids synthesized de novo in the chloroplast enter the prokaryotic pathway in the chloroplast envelope. Of the 62% which are exported as acyl-CoA species to enter the eukaryotic pathway, 56% (34% of the total) are returned to complete synthesis of the chloroplast's complement of glycerolipids.

Acetates↗
PubMed1986

Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue.

A procedure which uses hot methanolic HCl to digest fresh tissue and simultaneously convert the fatty acids of the leaf lipids to the corresponding methyl esters is described. Extraction of the fatty acid methyl esters into a small volume (0.3 ml) of hexane means that a sample for GLC analysis can be taken directly from the tube used for the digestion/methylation reaction. The procedure provides a fatty acid analysis which is comparable to that obtained by a more conventional technique involving separate extraction, saponification, and methylation steps, but the overall yield is reduced by 10-20%. The analysis can be made quantitative by including an internal standard with the tissue sample.

Chromatography, Gas↗
PubMed1985

Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7120 expressed in Escherichia coli.

We have placed the genes encoding ribulose-bisphosphate carboxylase/oxygenase from the Anabaena 7120 operon under transcriptional control of the lac promoter carried on the Escherichia coli plasmid pUC19. The genes encoding both the large and small subunit polypeptides (rbcL and rbcS) are transcribed and translated so that approximately 0.6% of the soluble protein in E. coli extracts is a fully functional holoenzyme with a sedimentation coefficient of approximately 18S, which contains stoichiometric amounts of the two subunits. However, expression of the large subunit polypeptide vastly exceeds that of the small subunit because the majority of transcripts terminate in the intergenic region between the rbcL and rbcS genes. As a result, excess large subunit is synthesized and accumulates in E. coli as an insoluble and catalytically inactive form. Because small subunit is found only in the high molecular weight soluble form of ribulosebisphosphate carboxylase/oxygenase, we propose that the small subunit promotes assembly of the hexadecameric form of the enzyme via heterodimers of large and small subunits.

Journal Article↗
PubMed1985

Analysis of Photosynthetic Antenna Function in a Mutant of Arabidopsis thaliana (L.) Lacking trans-Hexadecenoic Acid.

Several lines of evidence support the proposal that the unusual chloroplast-specific lipid acyl group Delta3,trans-hexadecenoic acid (trans-C(16:1)) stimulates the formation or maintenance of the oligomeric form of the light-harvesting chlorophyll a/b complex (LHCP). To assess the functional significance of this apparent association we have analyzed LHCP structure and function in a mutant of Arabidopsis thaliana (L.) which lacks trans-C(16:1) by electrophoretic analysis of the protein-chlorophyll complexes and by measurements of chlorophyll fluorescence under a variety of conditions. By these criteria the putative oligomeric form of LHCP appears to be slightly more labile to detergent-mediated dissociation in the mutant. The oligomeric PSI chlorophyll-protein complex, associated with PSI, was also more labile to detergent-mediated dissociation in the mutant, suggesting a previously unsuspected association of trans-C(16:1) with the PSI complex. However, no significant effect of the mutation on the efficiency of energy transfer from LHCP to the photochemical reaction centers was observed under any of the various conditions imposed. Also, the stability of the chlorophyll-protein complexes to temperature-induced dissociation was unaffected in the mutant. The role of trans-C(16:1) is very subtle or is only conditionally expressed.

Journal Article↗
PubMed1982

Mutants of the cruciferous plant Arabidopsis thaliana lacking glycine decarboxylase activity.

A mutant of Arabidopsis thaliana (L.) Heyn. (a small plant in the crucifer family) that lacks glycine decarboxylase activity owing to a recessive nuclear mutation has been isolated on the basis of a growth requirement for high concentrations of atmospheric CO2. Mitochondria isolated from leaves of the mutant did not exhibit glycine-dependent O2 consumption, did not release 14CO2 from [14C]glycine, and did not catalyse the glycine-bicarbonate exchange reaction that is considered to be the first partial reaction associated with glycine cleavage. Photosynthesis in the mutant was decreased after illumination under atmospheric conditions that promote partitioning of carbon into intermediates of the photorespiratory pathway, but was not impaired under non-photorespiratory conditions. Thus glycine decarboxylase activity is not required for any essential function unrelated to photorespiration. The photosynthetic response of the mutant in photorespiratory conditions is probably caused by an increased rate of glyoxylate oxidation, which results from the sequestering of all readily transferable amino groups in a metabolically inactive glycine pool, and by a depletion of intermediates from the photosynthesis cycle. The rate of release of 14CO2 from exogenously applied [14C]glycollate was 14-fold lower in the mutant than in the wild type, suggesting that glycine decarboxylation is the only significant source of photorespiratory CO2.

Amino Acid Oxidoreductases↗
PubMed1982

A Mutant of Arabidopsis thaliana Which Lacks Activation of RuBP Carboxylase In Vivo.

A mutant of Arabidopsis thaliana has been isolated in which ribulose-1,5-bisphosphate carboxylase is present in a nonactivatable form in vivo. The mutation appears to affect carboxylase activation specifically, and not any other enzyme of the photosynthesis or photorespiratory cycles. The effect of the mutation on carboxylase activation is indirect, inasmuch as the properties of ribulose-1,5-bisphosphate carboxylase purified from the mutant are not distinguishable from those of the wild type enzyme. The mutant requires high levels of atmospheric CO(2) for growth because photosynthesis is severely impaired in atmospheres containing normal levels of CO(2), irrespective of the atmospheric O(2) concentration. In this respect, the mutant is distinguished from previously described high-CO(2) requiring mutants of Arabidopsis which have defects in photorespiratory carbon or nitrogen metabolism.

Journal Article↗
PubMed1981

Photorespiration-deficient Mutants of Arabidopsis thaliana Lacking Mitochondrial Serine Transhydroxymethylase Activity.

Three allelic mutants of Arabidopsis thaliana which lack mitochondrial serine transhydroxymethylase activity due to a recessive nuclear mutation have been characterized. The mutants were shown to be deficient both in glycine decarboxylation and in the conversion of glycine to serine. Glycine accumulated as an end product of photosynthesis in the mutants, largely at the expense of serine, starch, and sucrose formation. The mutants photorespired CO(2) at low rates in the light, but this evolution of photorespiratory CO(2) was abolished by provision of exogenous NH(3). Exogenous NH(3) was required by the mutants for continued synthesis of glycine under photorespiratory conditions. These and related results with wild-type Arabidopsis suggested that glycine decarboxylation is the sole site of photorespiratory CO(2) release in wild-type plants but that depletion of the amino donors required for glyoxylate amination may lead to CO(2) release from direct decarboxylation of glyoxylate. Photosynthetic CO(2) fixation was inhibited in the mutants under atmospheric conditions which promote photorespiration but could be partially restored by exogenous NH(3). The magnitude of the NH(3) stimulation of photosynthesis indicated that the increase was due to the suppression of glyoxylate decarboxylation. The normal growth of the mutants under nonphotorespiratory atmospheric conditions indicates that mitochondrial serine transhydroxymethylase is not required in C(3) plants for any function unrelated to photorespiration.

Journal Article↗
PubMed1980

Photorespiration mutants of Arabidopsis thaliana deficient in serine-glyoxylate aminotransferase activity.

Three mutants of the crucifer Arabidopsis thaliana (Linnaeus) Heynhold were isolated that are completely lacking in activity catalyzed by serine-glyoxylate aminotransferase (EC 2.6.1.45), a peroxisomal enzyme involved in photorespiratory carbon metabolism. These mutants were viable and exhibited normal photosynthesis under conditions that suppressed photorespiration, but they were inviable and photosynthesized at greatly reduced rates under conditions that promoted photorespiration. Serine and glycine accumulated as end products of photosynthesis in the mutants, mostly at the expense of starch and sucrose. The mutants are allelic, and the segregation patterns of plant viability, photosynthetic activity, and enzyme activity in the F(1) and F(2) generations indicated that all the observed effects were caused by a single recessive nuclear mutation. This conclusion was confirmed by the isolation of seven revertants in which viability, photosynthetic capacity, and enzyme activity were simultaneously restored. Mutants of the type described here, in which photorespiration is changed from a merely wasteful process into one that is lethal, may permit the direct selection of secondary mutations that reduce photorespiration.

Journal Article↗
PubMed1979

Mutants of Escherichia coli defective in the degradation of guanosine 5'-triphosphate, 3'-diphosphate (pppGpp).

A new class of mutants of E. coli exhibiting altered metabolism of ppGpp and pppGpp has been isolated, and mapped at a locus designated gpp, near min 83 on the genetic map. These mutants accumulate elevated levels of pppGpp during amino acid starvation or carbon source downshift, and exhibit a reduced rate of pppGpp degradation in vivo. The in vitro evidence suggests that the gpp mutants are defective in a 5'-nucleotidase, which specifically hydrolyzes pppGpp to ppGpp. Certain combinations of gpp and spoT mutations are inviable. A gpp spoT double mutant, constructed by employing a leaky spoT mutation, was found to have a slower rate of pppGpp degradation than the gpp mutant alone. This result indicates that spoT also participates in pppGpp degradation. The inviability of certain gpp spoT combinations is attributed to the inability of the double mutants to degrade pppGpp. This is supported by the observation that selection for increased growth rate on the double mutant results in the recovery of relA mutations. Various effects of the gpp mutation upon the pppGpp and ppGpp pools provide additional support for a scheme in which pppGpp is the major precursor of ppGpp.

Chromosome Mapping↗