PubMed Health⌕ Search

Biomedical subjects

C Somerville

Publications and source records attributed to C Somerville.

At least 37 records · Page 2Linked to original sources
PubMed1996

Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases.

The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.

Alleles↗
PubMed1995

An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog.

Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with approximately 67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution.

Base Sequence↗
PubMed1995

Synthesis of high-molecular-weight poly([R]-(-)-3-hydroxybutyrate) in transgenic Arabidopsis thaliana plant cells.

High-molecular-weight poly([R]-(-)-3-hydroxybutyrate) (PHB), a biodegradable thermoplastic, was produced from a suspension culture of transgenic Arabidopsis thaliana plant cells expressing two genes from the bacterium Alcaligenes eutrophus involved in the synthesis of PHB. The molecular structure of the plant-produced polymer was analysed by gas chromatography, mass spectrometry, proton nuclear magnetic resonance spectroscopy, infra-red spectroscopy, spectropolarimetry, differential scanning calorimetry, X-ray diffraction and size exclusion chromatography. The results indicate that the polymer from transgenic plants appears to have a chemical structure identical to that of PHB produced by bacteria. However, the molecular weight distribution of the plant-produced PHB was much broader than that of typical bacterial PHB.

Alcaligenes↗
PubMed1995

Production of polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants.

In response to problems associated with plastic waste and its effect on the environment, there has been considerable interest in the development and production of biodegradable plastics. Polyhydroxyalkanoates (PHAs) are polyesters that accumulate as inclusions in a wide variety of bacteria. These bacterial polymers have properties ranging from stiff and brittle plastics to rubber-like materials. Because of their inherent biodegradability, PHAs are regarded as an attractive source of nonpolluting plastics and elastomers that can be used for specialty and commodity products. The possibility of producing PHAs in large scale and at a cost comparable to synthetic plastics has arisen from the demonstration of PHA accumulation in transgenic Arabidopsis plants expressing the bacterial PHA biosynthetic genes. Synergism between knowledge of the enzymes and genes contributing to PHA synthesis in bacteria and engineering of plant metabolic pathways will be necessary for the development of crop plants that produce biodegradable plastics.

Acids, Acyclic↗
PubMed1995

Functional assessment of CD2, CD3 and CD28 on the surface of peripheral blood T-cells from infants at low versus high genetic risk for atopy.

Recent studies from several laboratories suggest that the rate of postnatal maturation of T-cell function(s) associated with in vitro activation may be slower in children at high genetic risk for atopy (HR), compared to their normal (low risk; LR) counterparts. The present study compared the in vitro activity of the function-associated surface molecules CD2, CD3 and CD28 in panels of 27 HR and 13 LR infants, with a reference panel of 10 adults, employing assay systems involving T-cell stimulation with MoAbs against these molecules. The response maxima induced by saturating levels of the MoAbs were equivalent in all 3 groups, but T-cells from the HR infants required 10-50 fold higher levels of anti-CD3 stimulation to attain their maximum response, relative to adults (p = 0.02); T-cells from LR infants were also less responsive to anti-CD3 than adults, but these differences were smaller and did not attain statistical significance. It is suggested that these differences are attributable to varying proportions of competent T-memory cells (which respond to low levels of anti-CD3) in PBL from these populations, the postnatal accumulation of which proceeds slowest in the HR group.

Adult↗
PubMed1995

TOM, a new aromatic degradative plasmid from Burkholderia (Pseudomonas) cepacia G4.

Burkholderia (Pseudomonas) cepacia PR1(23) has been shown to constitutively express to toluene catabolic pathway distinguished by a unique toluene ortho-monooxygenase (Tom). This strain has also been shown to contain two extrachromosomal elements of < 70 and > 100 kb. A derivative strain cured of the largest plasmid, PR1(23) Cure, was unable to grow on phenol or toluene as the sole source of carbon and energy, which requires expression of the Tom pathway. Transfer of the larger plasmid from strain G4 (the parent strain inducible for Tom) enabled PR1(23) Cure to grow on toluene or phenol via inducible Tom pathway expression. Conjugal transfer of TOM23c from PR1(23) to an antibiotic-resistant derivative of PR1(23) Cure enabled the transconjugant to grow with either phenol or toluene as the sole source of carbon and energy through constitutive expression of the Tom pathway. A cloned 11.2-kb EcoRI restriction fragment of TOM23c resulted in the expression of both Tom and catechol 2,3-dioxygenase in Escherichia coli, as evidenced by its ability to oxidize trichloroethylene, toluene, m-cresol, o-cresol, phenol, and catechol. The largest resident plasmid of PR1 was identified as the source of these genes by DNA hybridization. These results indicate that the genes which encode Tom and catechol 2,3-dioxygenase are located on TOM, an approximately 108-kb degradative plasmid of B. cepacia G4.

Biodegradation, Environmental↗
PubMed1994

Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation.

In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plants accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-micron granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB that accumulated. We conclude that there does not appear to be any biological barrier to high-level production of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand.

Acyltransferases↗
PubMed1994

Tissue-specific expression of a gene encoding a cell wall-localized lipid transfer protein from Arabidopsis.

Nonspecific lipid transfer proteins (LTPs) from plants are characterized by their ability to stimulate phospholipid transfer between membranes in vitro. However, because these proteins are generally located outside of the plasma membrane, it is unlikely that they have a similar role in vivo. As a step toward identifying the function of these proteins, one of several LTP genes from Arabidoposis has been cloned and the expression pattern of the gene has been examined by analysis of the tissue specificity of beta-glucuronidase (GUS) activity in transgenic plants containing LTP promoter-GUS fusions and by in situ mRNA localization. The LTP1 promoter was active early in development in protoderm cells of embryos, vascular tissues, lignified tips of cotyledons, shoot meristem, and stipules. In adult plants, the gene was expressed in epidermal cells of young leaves and the stem. In flowers, expression was observed in the epidermis of all developing influorescence and flower organ primordia, the epidermis of the siliques and the outer ovule wall, the stigma, petal tips, and floral nectaries of mature flowers, and the petal/sepal abscission zone of mature siliques. The presence of GUS activity in guard cells, lateral roots, pollen grains, leaf vascular tissue, and internal cells of stipules and nectaries was not confirmed by in situ hybridizations, supporting previous observations that suggest that the reporter gene is subject to artifactual expression. These results are consistent with a role for the LTP1 gene product in some aspect of secretion or deposition of lipophilic substances in the cell walls of expanding epidermal cells and certain secretory tissues. The LTP1 promoter region contained sequences homologous to putative regulatory elements of genes in the phenylpropanoid biosynthetic pathway, suggesting that the expression of the LTP1 gene may be regulated by the same or similar mechanisms as genes in the phenylpropanoid pathway.

Amino Acid Sequence↗
PubMed1994

Identification of a gene that complements an Arabidopsis mutant deficient in chloroplast omega 6 desaturase activity.

Membrane lipids of the fad6 (formerly fadC) mutant of Arabidopsis, which is deficient in chloroplast omega 6 desaturase activity, have increased levels of monounsaturated fatty acids and are deficient in trienoic fatty acids. A putative fad6 cDNA clone was isolated by probing a cDNA library with a degenerate oligonucleotide based on a conserved region within known omega 3 desaturase genes. Expression of the cDNA in transgenic plants of a fad6 mutant restored normal levels of all fatty acids. When used as a hybridization probe, the cDNA identified a restriction fragment-length polymorphism that co-segregated with the fad6 mutation. Thus, on the basis of a genetic complementation test and genetic map position, the fad6 gene is encoded by the cDNA. The cDNA encoded a 418-amino acid polypeptide of 47,727 D that displayed a high degree of sequence similarity to a delta 12 desaturase from the cyanobacterium Synechocystis. The fad6 gene exhibited less sequence homology to any known higher plant desaturase, including an endoplasmic reticulum-localized omega 6 desaturase corresponding to the Arabidopsis fad2 gene.

Amino Acid Sequence↗
PubMed1994

Cloning of a temperature-regulated gene encoding a chloroplast omega-3 desaturase from Arabidopsis thaliana.

Previous genetic evidence suggested that the fad8 and fad7 genes of Arabidopsis thaliana encode chloroplast membrane-associated omega-3 desaturases. A putative fad8 cDNA was isolated by heterologous hybridization using a gene encoding an endoplasmic reticulum-localized omega-3 desaturase (fad3) as a probe. The cDNA encodes a protein of 435 amino acid residues with a molecular mass of 50,134 D. Constitutive expression of the cDNA in transgenic plants of a fad7 mutant resulted in genetic complementation of the mutation, indicating that the fad7 and fad8 gene products are functionally equivalent. Expression of the fad8 cDNA in transgenic plants often resulted in the co-suppression of both the endogenous fad7 and fad8 genes in spite of the fact that these two genes share only about 75% nucleotide identity. In contrast to all other known plant desaturases, including fad7, the steady-state level of fad8 mRNA is strongly increased in plants grown at low temperature. This suggests that the role of fad8 is to provide increased omega-3 desaturase activity in plants that are exposed to low growth temperature. The fad8-1 mutation created a premature stop codon 149 amino acids from the amino-terminal end of the fad8 open reading frame, suggesting that this mutation results in a complete loss of fad8 activity.

Amino Acid Sequence↗
PubMed1993

A gene encoding a chloroplast omega-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana.

Mutations at the fad7 locus of Arabidopsis thaliana (previously called fadD) cause decreased desaturation of dienoic fatty acids in chloroplast lipids in plants grown at elevated temperatures. This suggested that the fad7 locus encodes a chloroplast omega-3 desaturase that catalyzes the desaturation of lipid-linked 18:2 and 16:2 fatty acids. In order to clone the fad7 gene, it was first genetically mapped relative to the flanking restriction fragment length polymorphism markers 4547 and 2488A on chromosome 3, and yeast artificial chromosomes covering the locus were identified. A putative desaturase cDNA clone that was isolated by low stringency heterologous probing with a cDNA for an endoplasmic reticulum-localized omega-3 desaturase (fad3) hybridized to the yeast artificial chromosomes and could not be resolved from the locus by restriction fragment length polymorphism mapping. Expression of the cDNA in transgenic fad7 mutant plants resulted in restoration of wild type fatty acid composition and suppression of a previously observed effect of the fad7 mutation on chloroplast number, indicating genetic complementation. The structural gene contained seven introns within a coding sequence of 1338 base pairs, which encodes a 446-amino acid polypeptide of 51,172 daltons. The amino-terminal region of the fad7 gene product contained a consensus chloroplast transit peptide. Except for the amino-terminal domain, the deduced amino acid sequence of the fad7 gene product had high homology to the fad3 gene product, indicating that fad7 encodes an omega-3 desaturase and that the two genes arose from a common ancestral gene. There was no apparent effect of growth temperature on the steady-state levels of fad7 mRNA in wild type plants.

Amino Acid Sequence↗
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↗