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J A Napier

Publications and source records attributed to J A Napier.

At least 37 records · Page 2Linked to original sources
PubMed1999

Plant desaturases: harvesting the fat of the land.

The past few years have witnessed a major upsurge in research towards the goal of modifying the lipid composition of plants. Genes encoding a range of different fatty acid desaturase activities have been cloned, and the evolutionary relationships between and within different classes of enzymes have tentatively been established. The effects of expressing some of these desaturases in heterologous hosts have also been studied, often producing unexpected results which contribute further to our understanding of plant lipid modification. It is to be hoped that, in the near future, the goal of producing unusual and valuable fatty acids in transgenic oilseeds will be achieved on a commercial scale.

Arabidopsis↗
PubMed1999

Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.

Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.

Amino Acid Sequence↗
PubMed1998

Functional identification of a fatty acid delta5 desaturase gene from Caenorhabditis elegans.

We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes a fatty acid delta5 desaturase. Saccharomyces cerevisiae expressing the full-length cDNA was able to convert di-homo-gamma-linolenic acid to arachidonic acid, thus confirming delta5 desaturation. The 1341 bp delta5 desaturase sequence contained an N-terminal cytochrome b5 domain and was located within a kilobase of the C. elegans delta6 desaturase on chromosome IV. With an amino acid identity of 45% it is possible that one of these genes arose from the other by gene duplication. This is the first example of a delta5 desaturase gene isolated from an animal.

8,11,14-Eicosatrienoic Acid↗
PubMed1998

Secondary structure of oleosins in oil bodies isolated from seeds of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.).

Oil bodies were isolated from mature seeds of sunflower (Helianthus annuus L.) and safflower (Carthamus tinctorius L.). Oil body preparations containing only oleosin proteins could be obtained from safflower seeds by salt-washing followed by centrifugation on discontinuous sucrose density gradients. However, it was necessary to treat sunflower oil bodies with urea to obtain preparations of similar purity. Incubation of the oil bodies with proteinases gave two fragments with molecular masses of 6 and 8 kDa which were protected from digestion. These fragments represented the hydrophobic domain of the oleosins, as determined by N-terminal sequencing. Intact and proteinase-treated oil bodies of both species were analysed by Fourier-transform infrared spectroscopy, as dry films and in aqueous medium, the spectra being compared with those obtained for pure oil samples in order to identify the bands resulting from the oleosin proteins and protected peptides. This investigation showed that the hydrophobic domain of the oleosins in intact oil bodies is predominantly alpha-helical in structure and that the conformation was not greatly affected by washing the oil bodies with urea during preparation.

Amino Acid Sequence↗
PubMed1998

Isolation of a Delta5-fatty acid desaturase gene from Mortierella alpina.

Arachidonic acid (C20:4 Delta5,8,11,14) is a polyunsaturated fatty acid synthesized by the Delta5-fatty acid desaturation of di-homo-gamma-linolenic acid (C20:3 Delta8,11,14). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungus Mortierella alpina. We have isolated a cDNA encoding the Delta5-fatty acid desaturase from M. alpina via a polymerase chain reaction-based strategy using primers designed to the conserved histidine box regions of microsomal desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic acid. The M. alpina Delta5-desaturase is the first example of a cloned Delta5-desaturase, and differs from other fungal desaturases previously characterized by the presence of an N-terminal domain related to cytochrome b5.

Amino Acid Sequence↗
PubMed1998

Identification of a Caenorhabditis elegans Delta6-fatty-acid-desaturase by heterologous expression in Saccharomyces cerevisiae.

We identified a cDNA expressed sequence tag from an animal (the nematode worm Caenorhabditis elegans) that showed weak similarity to a higher-plant microsomal Delta6-desaturase. A full-length cDNA clone was isolated and expressed in the yeast Saccharomyces cerevisiae. This demonstrated that the protein encoded by the C. elegans cDNA was that of a fatty acid Delta6-desaturase, as determined by the accumulation of gamma-linolenic acid. The C. elegans Delta6-desaturase contained an N-terminalcytochrome b5 domain, indicating that it had a similar structure to that of the higher-plant Delta6-desaturase. The C. elegans Delta6-desaturase mapped to cosmid W08D2, a region of chromosome III. This is the first example of a Delta6-desaturase isolated from an animal and also the first example of an animal desaturase containing a cytochrome b5 domain.

Amino Acid Sequence↗
PubMed1998

Clinical use of FFP: results of a retrospective process and outcome audit.

An audit has been carried out on the usage of 216 units of fresh frozen plasma (FFP) issued to 41 patients. This involved the systems of FFP issue, the appropriateness of prescription as well as the recorded benefits. Sixty-six per cent of the initial requests for FFP appeared to satisfy criteria for appropriate use. Review of the case notes resulted in some changes to earlier decisions and a slight increase to 73% of those accepted as valid. Only 94% of the FFP issued could be proved to have been given to identified patients and only 88% of case notes showed the reason for treatment. Pre- and post-treatment coagulation results were available for all patients. In 15% of cases, pretreatment results were not significantly abnormal, and consequently no post-treatment improvement found. Coagulation improvement was documented in 78% of cases, and clinical statements on patient progress noted in 40%. In 80% of cases, the patient received other blood products carrying a potential virus risk. Six of the remaining eight patients were treated to stabilize oral anticoagulation. For these patients, virus-inactivated prothrombin concentrates could have been used, resulting in almost the same reduction in virus transmission risk for the group of 41 patients as could be obtained by using virus-inactivated FFP.

Blood Coagulation Disorders↗
PubMed1997

Chloroplast SecA functions as a membrane-associated component of the Sec-like protein translocase of pea chloroplasts.

Protein cross-linking studies with a thylakoid membrane translocation intermediate were used to demonstrate that chloroplast SecA functions as a membrane-associated component of the Sec-like ATP-dependent protein translocase of pea chloroplasts. In assays with isolated thylakoids, it was observed that translocation of the 33-kDa protein of the oxygen-evolving complex of photosystem II (OE33) decreased when the ATP concentration was low, and that the protein accumulated as a bound precursor. The bound precursor was able to be translocated into the lumen when the ATP concentration was raised, indicating that the precursor was bound to the translocation apparatus. Inclusion of apyrase in the import reaction prevented translocation but did not affect precursor binding to the membrane. When this translocation intermediate was treated with the cross-linking agent disuccinimidyl suberate, a single predominant cross-linked product of 120 kDa was produced. This conjugate could be immunoprecipitated with antibodies to pea chloroplast SecA, identifying the cross-linking partner as SecA. This provides direct evidence for a functional interaction between a thylakoid precursor protein and a component of the thylakoid protein-translocation apparatus.

Adenosine Triphosphate↗
PubMed1997

Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of delta6-desaturated fatty acids in transgenic tobacco.

gamma-Linolenic acid (GLA; C18:3 delta(6,9,12)) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a delta6-fatty acid desaturase using linoleic acid (C18:2 delta(9,12)) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the delta6-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 delta(6,9,12,15)) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage delta6-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b5.

Amino Acid Sequence↗
PubMed1997

Trafficking of wheat gluten proteins in transgenic tobacco plants: gamma-gliadin does not contain an endoplasmic reticulum-retention signal.

Wild-type and mutated forms of the wheat (Triticum aestivum L.) storage protein gamma-gliadin were expressed in transgenic tobacco (Nicotiana tabacum L. cv. NVS) under the control of the 35S cauliflower mosaic virus (CaMV) promoter in order to determine what, if any, endogenous targeting signals are present in the wild-type gamma-gliadin protein. The mutant forms of the protein were modified by the addition of a KDEL or HDEL C-terminal endoplasmic reticulum-retention signal, or the addition of a C-terminal propeptide from barley lectin which has been shown to be necessary and sufficient for targeting to the vacuole. Only modified forms of the protein accumulated in leaves of transgenic tobacco, although the transcript levels were similar for all the constructs. Pulse-chase analysis indicated that whereas the wild-type gamma-gliadin was rapidly turned over in tobacco leaves, KDEL and HDEL forms were highly stable. The vacuolar-signal mutant protein accumulated in tobacco leaves, but migrated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a lower mobility than wild-type gamma-gliadin, due in part to glycosylation of the C-terminal propeptide. The vacuolar-signal mutant protein was turned over slowly in tobacco, perhaps indicating a poor level of transport competence. When pulse-chase analysis was carried out on protoplasts isolated from tobacco plants expressing wild-type gamma-gliadin, but in the presence of Brefeldin A, gamma-gliadin was seen to accumulate. Taken together, these results indicate that gamma-gliadin is targeted to the vacuole in transgenic tobacco plants and does not contain any structural determinants which confer retention in the endoplasmic reticulum.

Brefeldin A↗
PubMed1997

[Cloning and expression of cDNA for tobacco rab1, and structural-functional analysis of the protein].

rab1 cDNA coding for a small GTP binding protein Rab1 was isolated from cDNA library of tobacco (Nicotiana tabacum) leaves. The primary structure of this protein was deduced from the rab1 structure. Tobacco rab1 cDNA was expressed in Escherichia coli, and the product was purified and shown to exhibit GTPase activity. A set of Rab1 mutants with altered GTP binding and/or GTPase activities was obtained. Polyclonal antipeptide antibodies were raised against a sequence in the C-terminal region of the tobacco Rab1 capable of recognizing this protein.

Amino Acid Sequence↗
PubMed1996

Comparison of the expression patterns of genes coding for wheat gluten proteins and proteins involved in the secretory pathway in developing caryopses of wheat.

The synthesis of gluten proteins in the developing caryopsis of wheat is highly coordinated, with mRNAs for the various groups being detected from 11 days after anthesis, and the proteins from about 14 days. In contrast, the levels of transcripts for BiP, PDI and PPI are highest at earlier stages of development. The levels of transcripts for two small GTP binding proteins involved in the secretory pathway (Rab1 and Rab5) are also highest early in development, which is consistent with the retention of most of the gluten proteins within the ER to form protein bodies.

Amino Acid Sequence↗
PubMed1995

Isolation and characterisation of cDNA clones representing the genes encoding the major tuber storage protein (dioscorin) of yam (Dioscorea cayenensis Lam.).

cDNA clones encoding dioscorins, the major tuber storage proteins (M(r) 32,000) of yam (Dioscorea cayenesis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of M(r) 30,015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.

Amino Acid Sequence↗
PubMed1995

Expression and in vitro targeting of a sunflower oleosin.

Two distinct cDNAs encoding oleosins (oil body proteins) have been identified by degenerate PCR as transcripts present in the developing seeds of sunflower (Helianthus annuus L. cv. Dwarf Sunbred). One (pSOM) of these is closely related to a reported sunflower oleosin, whilst the other (pSO5) has not been previously described. Different expression patterns were observed for the two cDNAs, pSO5 being expressed earlier than pSOM in seed maturation and oil deposition. The results support the contention that oleosin proteins are synthesised either during or closely after the formation of the oil body. Translation in vitro of synthetic oleosin transcripts was enhanced by the addition of microsomes but suppressed by the addition of purified signal recognition particle (SRP) complex. Deletion of 62 amino acid residues at the C-terminus of the oleosin did not alter the in vitro targeting of the protein to the microsomal membrane. Taken together these data support the idea that oleosins are targeted to the ER membrane as part of oil body biogenesis.

Amino Acid Sequence↗