A novel plant cysteine-rich peptide family conferring cadmium tolerance to yeast and plants

We have identified a novel cDNA clone, termed DcCDT1, from Digitaria ciliaris, that confers cadmium (Cd)-tolerance to yeast (Saccharomyces cerevisiae). The gene encodes a predicted peptide of 55 amino acid residues of which 15 (27.3%) are cysteine residues. We found that monocotyledonous plants possess multiple DcCDT1 homologues, for example rice contains five DcCDT1 homologues (designated OsCDT1∼5), whereas dicotyledonous plants, including Arabidopsis thaliana, Brassica rapa, poplar (Populus tremula × Populus alba) and Picea sitchensis, appear to possess only a single homologue. GFP fusion experiments demonstrate that DcCDT1 and OsCDT1 are targeted to both the plant cytoplasmic membranes and cell walls. Constitutive expression of DcCDT1 or OsCDT1 confers Cd-tolerance to transgenic A. thaliana plants by lowering the accumulation of Cd in the cells. The functions of the DcCDT1 family members are discussed in the light of these findings.Key words: cadmium, cysteine-rich peptide, hypoaccumulation, phytoremediation, toleranceCadmium (Cd) is a highly toxic transition metal, and is nonessential for almost all living organisms.¹ Therefore, Cd pollution of the earth''s environment could potentially cause serious problems for both the global ecosystem and human health.² Indeed, we have a tragic history brought about by Cd poisoning, with patients suffering kidney failure and unbearable pain in the joints and spine due to bone softening.^3,4 Despite the numerous laws that have been enacted to prevent further Cd contamination, yet pollution with heavy metals including Cd is still increasing on a global scale. As the control of Cd contamination of foodstuffs is expected to become extremely severe, remediation of Cd-contaminated soils is an issue that needs to be urgently addressed. One countermeasure strategy is the use of plants in phytoremediation⁵ which, despite its tremendous potential, still requires vast improvements before it can be promoted as an effective and established technology. Such improvements will necessitate the identification and development of novel and useful ‘molecular resources’. As a step towards this aim, we have screened cDNA libraries derived from natural habitat plants growing in a former mining site and isolated numerous candidate genes that could confer Cd tolerance to Cd-hypersensitive yeast mutant cells.⁶ Of these genes, we chose DcCDT1 (Digitaria ciliaris cadmium tolerance 1) for further analysis as it encodes a novel 55-amino acid-peptide product containing 15 cysteine (Cys) residues, and because several other Cys- rich peptides are known to function as heavy metal chelators.^7,8 Rice plants possess five DcCDT1 homologues, designated OsCDT1∼5, while other monocotyledonous plant species, such as maize and barley, also contain multiple DcCDT1 homologues. In contrast, Arabidopsis thaliana appears to contain only a single DcCDT1 homologue (accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_202281","term_id":"1063678830"}}NM_202281, At1g52827) with an open-reading frame (nucleotide positions 29–178) encoding a 49-amino acid peptide of sequence,MKAPPQQEMTYYDNVKKRQDEQGCLFATFYALFCCCCCYEKCKCCCCCV,whereas the database predicts a peptide (encoded by an alternative open-reading frame between nucleotides 63–251) consisting of 62-amino acids, MTM SRN GKT NKA AYS QRF TRC SVA VAA TRS ASV VAA AFD FYI CII IST LLS LIV SLA SQL LF, which is of unknown function and shows no similarity to DcCDT1. The 49-amino acid-peptide sequence (here termed AtCDT1) shares a high level of identity with DcCDT1 and OsCDT1, and these all contain 11 conserved Cys residues clustered in their carboxy-distal regions (Fig. 1A). Furthermore, as with DcCDT1 and OsCDT1, constitutive expression of AtCDT1 confers Cd-tolerance to S. cerevisiae (Fig. 1B), confirming that A. thaliana possesses a functional DcCDT1 counterpart. The question thus arises if other dicotyledonous plants also possess homologous gene(s). It appears that they do since we found two other examples, a Populus EST clone (accession number {"type":"entrez-nucleotide","attrs":{"text":"CU225257","term_id":"160955042"}}CU225257) and also a Brassica rapa subsp. pekinensis clone (accession number {"type":"entrez-nucleotide","attrs":{"text":"AC189268","term_id":"199579954"}}AC189268), that encode DcCDT1 homologues. However, further analyses will be required to determine whether these plants carry a single gene like Arabidopsis or a small gene family as in rice plants.Open in a separate windowFigure 1DcCDT1 and its homologues are a highly-conserved, Cys-rich family of proteins and the Arabidopsis homologue, AtCDT1, confers Cd-tolerant to yeast cells. (A) Amino acid alignment of DcCDT1, OsCDT1 and AtCDT1. OsCDT1 is one of the rice DcCDT1 homologues, and AtCDT1 is the Arabidopsis thaliana-derived 49-amino acid-peptide. Identical and conserved amino acid residues are highlighted in black and gray backgrounds, respectively. Conserved Cys residues are marked with an asterisk. (B) Constitutive expression of the nucleotide sequence encoding AtCDT1 confers Cd tolerance to yeast cells. S. cerevisiae strain, DTY165 (genotypic markers MATα, ura3-52, leu2-3,-112, his3-delta200, trp1-delta901, lys2-801, suc2-delta9), and its ycf1^6,17-mutant strain, DTY167 (genotypic markers MATα, ura3-52, leu2-3,-112, his3-delta200, trp1-delta901, lys2-801, suc2-delta9, ycf1::hisG), cells were transformed with the control vector p112A1NE or p112A1NE::AtCDT1.¹⁸ The transformants obtained were grown in yeast liquid culture media, and the cell density of the cultures then adjusted to an optical density of 1.0 at 600 nm (OD₆₀₀). Aliquots of 5 µl of the serial dilutions (1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵) were then plated onto Synthetic Dropout (SD)-Trp media without CdCl₂ (left) or containing 60 µM CdCl₂ (right).Several different plant components contribute to heavy metal homeostasis and detoxification. Among these are the low molecular mass (4–14 kDa) metallothioneins that contain a high ratio of Cys residues, and the small peptides known as phytochelatins that have the general structure, (γ-Glu-Cys)_n-Gly.^7–10 Other Cys-rich plant proteins involved in Cd tolerance and detoxification have also been reported.^11,12 However, compared to all these, DcCDT1 and its homologues are unique and distinctive in both their peptide lengths (49–60 amino acids) and arrangement of Cys residues in the CL-(Y/F)-A-(C/T)-X5-CC-(F/C)-CCYE-(T/K)-C-(E/K)-C( CLDCL or delete)-CCCC consensus sequence.As with other non-essential heavy metals, Cd can be detoxified by a variety of mechanisms, including secretion, compartmentalization, or chelation by metal ligands.^13–16 DcCDT1 and OsCDT1 confer Cd-tolerance to both yeast and Arabidopsis via a reduction in their cellular Cd contents. Both proteins also appear to be localized to the plant cell surface, including the cell walls, as judged by our GFP-fusion experiments. Based on these findings, we propose several possible functions for this novel peptide family. In one mechanism, the DcCDT1 family proteins chelate Cd at the cellular surface and prevent further Cd entry into the cells. In an alternative mechanism, the intracellular-formed DcCDT1-Cd complex is secreted out from the cells via an unknown mechanism. Induced expression of DcCDT1 in ¹⁰⁹Cd-preloaded cells may well allow us to distinguish between these two possibilities.A final question that needs to be addressed is whether the genes encoding this family of proteins can be potentially useful genetic resources for phytoremediation. OsCDT1, one of the five rice DcCDT1 homologues, reduces the Cd contents of yeast and Arabidopsis cells. Therefore, from the viewpoint of food safety, this protein family may be useful since constitutive root-specific expression of the genes may contribute to reduced Cd accumulation in the edible plant parts. Alternatively, silencing of all DcCDT1 homologues in rice could plausibly result in the hyperaccumulation of Cd and the use of these plants in phytoremediation strategies.     

Expression of Arabidopsis SR-like splicing proteins confers salt tolerance to yeast and transgenic plants

Searching for novel targets of salt toxicity in eukaryotic cells, we have screened an Arabidopsis thaliana cDNA library to isolate genes conferring increased tolerance to salt stress when expressed in the yeast Saccharomyces cerevisiae. Here we show that expression of the 'alternating arginine-rich' (or RS) domains of two different SR-like, putative splicing proteins from Arabidopsis allows yeast cells to tolerate higher lithium and sodium concentrations. Protection against salt stress appears to require the in vivo phosphorylation of these plant polypeptides, since the yeast SR protein kinase Sky1p, which was able to phosphorylate in vitro at least one of them, also proved to be essential for the observed salt tolerance phenotype. In addition, a clone encoding the U1A protein, a previously characterised Arabidopsis splicing factor, was also isolated in the screening. No significant decrease in the intracellular concentration of lithium was observed in yeast cells incubated in the presence of LiCl upon expression of any of the Arabidopsis proteins, suggesting that their effects are not mediated by the stimulation of ion transport. In support of the general significance of these data, we also show that the expression of the RS domain of one of the SR-like proteins in transgenic Arabidopsis plants increases their tolerance to LiCl and NaCl. These results point to an important role of pre-mRNA splicing and SR-like proteins in the salt tolerance of eukaryotic cells, offering a novel route to improve this important trait in crop plants.     

Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants

The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants. Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments. Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants. The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.     

Isolation and characterization of the genes encoding delta(8)-sphingolipid desaturase from Saccharomyces kluyveri and Kluyveromyces lactis

Saccharomyces kluyveri IFO 1685 and Kluyveromyces lactis IFO 1090 synthesize cerebroside containing 9-methyl- trans-4, trans-8-sphingadienine as a sphingoid base. From the genome of the two strains, the regions encompassing Delta(8)-sphingolipid desaturase were amplified and sequenced. The nucleotide sequences of these regions revealed single open reading frames of 1707 bp for S. kluyveri and 1722 bp for K. lactis, encoding polypeptides of 568 and 573 amino acids with molecular weights of 66.5 and 67.1 kDa, respectively. Conversion of 4-hydroxysphinganine to 4-hydroxy- trans-8-sphingenine in the cells of Saccharomyces cerevisiae was observed by the expressed gene from K. lactis and not by that from S. kluyveri. These findings may be explained by the difference in substrate specificity for the sphingoid base moiety between Delta(8)-sphingolipid desaturases of S. kluyveri and K. lactis.     

Expression and regulation of delta5-desaturase,delta6-desaturase,stearoyl-coenzyme A (CoA) desaturase 1, and stearoyl-CoA desaturase 2 in rat testis

In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and Delta5- and Delta6-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of Delta5- and Delta6-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, Delta5- and Delta6-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. Delta5- and Delta6-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)2cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.     

A combination of five mechanisms confers a high tolerance for aluminum to a wild species of Poaceae,Andropogon virginicus L.

How can high tolerance against aluminum (Al) toxicity be obtained in plants? To address this question, tolerant mechanisms were characterized in a highly Al tolerant wild species of Poaceae, Andropogon virginicus L. A. virginicus showed an Al-stress-induced synthesis and secretion of citrate and malate in roots. This mechanism may help to suppress an increase of toxic Al ions in the root region. Microscopic observation of the morin-stained leaves indicated that the Al transferred to shoots was specifically accumulated in the trichomes and spikes of the leaves and that some portion of the accumulated Al was furthermore secreted as sap from the tips of trichomes. Al-induced synthesis of poly-phenolic compounds including anthocyanin also occurred in roots as a long term response to Al toxicity and anthocyanin production did not co-localize with either Al accumulation, nitric oxide (NO) production or lipid peroxides production in the roots. It was suggested that oxidative damage caused by Al stress was suppressed in these areas where anthocyanin was localized. Moreover, induction of NO production occurred in roots within 24 h of Al treatment. Our results suggested that NO could not efficiently ameliorate the Al-dependent nuclei deformation and DNA fragmentation, but could function as a trigger to stimulate anti-peroxidation enzymes under Al stress. Collectively the results suggested that A. virginicus manifests its high Al tolerance by a unique combination of effective mechanisms.     

Response to light of a specific aminotransferase for delta-aminolevulinate formation in higher plants

It is thought that the C-5 pathway is the major, possibly the sole, route for the formation of delta-aminolevulinic acid for the biosynthesis of tetrapyrroles, including chlorophylls, in higher plants; a route involving 4,5-dioxovalerate as an intermediate followed by transamination to delta-aminolevulinic acid has been supported as one of the C-5 pathways (Granick, S., and Beale, S. I. (1978) Adv. Enzymol. Relat. Areas Mol. Biol. 46, 33-203). A specific aminotransferase for L-alanine and 4,5-dioxovalerate was found in the cucumber seeds. In dark-grown cucumber seedlings, alanine:4,5-dioxovalerate aminotransferase activity in the transitional region between shoot and root was remarkably high compared with that in the cotyledons. The exposure of the dark-grown seedlings to illumination resulted in a rapid and dramatic increase in the activity only in this transitional region. In contrast, the enzyme in the cotyledons, stem, and roots did not respond to illumination. After a 27-h illumination, the enzyme activity in the transitional region was 100-fold higher than that in the cotyledons. Other aminotransferases assayed in the transitional region did not respond to illumination. Alanine:4,5-dioxovalerate aminotransferase in the transitional region was also specific for L-alanine and 4,5-dioxovalerate.     

Altered fractionation profiles of higher plant poly(A)-containing RNA by selective formation of a complex with a hydrophobic impurity

Synthetic polyadenylate (poly(A)) which originally sedimented at 6–7 S in a sucrose density gradient did so at 15–19 S in the presence of phenol-extracted nucleic acids from higher plant tissues. After centrifugation, the input poly(A) was found to be insensitive to RNase T2 (EC 3.129.1), suggesting a newly-formed complex with a hydrophobic substance contaminating the nucleic acid preparation. The artifically-formed poly(A)-impurity complex migrated more slowly than unbound poly(A) during gel electrophoresis. The complex was retained rather effectively by an oligodeoxythymidylate (oligo-(dT))-cellulose column and a polyuridylate (poly(U))-glassfiber filter. However, a nitrocellulose membrane filter retained the poly(A)-complex to various extents depending on plant materials, suggesting different varieties of the poly(A)-binding impurity in the higher plant kingdom. Fractionation of mRNA by these ordinarily used procedures is suggested to be variously altered by complex formation with this hydrophobic impurity.     

Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance

The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)). Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.     

A systematic search for positive selection in higher plants (Embryophytes)

Background  

Previously, a database characterizing examples of Embryophyte gene family lineages showing evidence of positive selection was reported. Of the gene family trees, 138 Embryophyte branches showed Ka/Ks>>1 and are candidates for functional adaptation. The database and these examples have now been studied in further detail to better understand the molecular basis for plant genome evolution.     

An osmotin from the resurrection plant Tripogon loliiformis (TlOsm) confers tolerance to multiple abiotic stresses in transgenic rice

Osmotin is a key protein associated with abiotic and biotic stress response in plants. In this study, an osmotin from the resurrection plant Tripogon loliiformis (TlOsm) was characterized and functionally analyzed under abiotic stress conditions in T. loliiformis as well as in transgenic Nicotiana tabacum (tobacco) and Oryza sativa (rice) plants. Real‐time PCR analysis on mixed elicitor cDNA libraries from T. loliiformis showed that TlOsm was upregulated a 1000‐fold during the early stages of osmotic stresses (cold, drought, and salinity) in both shoots and roots but downregulated in shoots during heat stress. There was no change in TlOsm gene expression in roots of heat‐stressed plants and during plant development. The plasma membrane localization of TlOsm was showed in fluorescent‐tagged TlOsm tobacco plants using confocal laser scanning microscopic analysis. Transgenic rice plants expressing TlOsm were assessed for enhanced tolerance to salinity, drought and cold stresses. Constitutively expressed TlOsm in transgenic rice plants showed increased tolerance to cold, drought and salinity stress when compared with the wild‐type and vector control counterparts. This was evidenced by maintained growth, retained higher water content and membrane integrity, and improved survival rate of TlOsm‐expressing plants. The results thus indicate the involvement of TlOsm in plant response to multiple abiotic stresses, possibly through the signaling pathway, and highlight its potential applications for engineering crops with improved tolerance to cold, drought and salinity stress.     

A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance

The metal tolerance of metal hyper-accumulating plants is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the Zn hyper-accumulating plant Arabidopsis halleri ssp. halleri, we carried out a functional screening of an A. halleri cDNA library in the yeast Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. The screening revealed four A. halleri defensin genes (AhPDFs), which induced Zn but not cadmium (Cd) tolerance in yeast. The expression of AhPDF1.1 under the control of the 35S promoter in A. thaliana made the transgenic plants more tolerant to Zn than wild-type plants, but did not change the tolerance to Cd, copper (Cu), cobalt (Co), iron (Fe) or sodium (Na). Thus, AhPDF1.1 is able to confer Zn tolerance both to yeast and plants. In A. halleri, defensins are constitutively accumulated at a higher level in shoots than in A. thaliana. A. halleri defensin pools are Zn-responsive, both at the mRNA and protein levels. In A. thaliana, some but not all defensin genes are induced by ZnCl2 treatment, and these genes are not induced by NaCl treatment. Defensins, found in a very large number of organisms, are known to be involved in the innate immune system but have never been found to play any role in metal physiology. Our results support the proposition that defensins could be involved in Zn tolerance in A. halleri, and that a role for plant defensins in metal physiology should be considered.     

Genetic basis of sodium exclusion and sodium tolerance in yeast. A model for plants

A yeast strain carrying disruptions in TRK1 and ENA genes was very sensitive to Na⁺ because uptake discriminated poorly between K⁺ and Na⁺, and Na⁺ efflux was insignificant. Transformation with TRK1 and ENA1 restored discrimination, Na⁺ efflux and Na⁺ tolerance. Increasing external Ca²⁺ increased Na⁺ tolerance almost in the same proportion in TRK1 enal cells and in trkl ENAI cells, suggesting an unspecific effect of this cation. By using a vacuolar ATPase mutant, the role of the vacuole in Na⁺ tolerance was also demonstrated. The yeast model of Na⁺ exclusion and Na⁺ tolerance may be extended to plants.     

Increased secondary product formation in plant cell suspension cultures after treatment with a yeast carbohydrate preparation (elicitor)

A carbohydrate fraction isolated from yeast extract by ethanolic precipitation was used as an elicitor to induce secondary product formation in plant cell suspension cultures. The elicitor preparation is effective in inducing glyceollin isomer synthesis (up to 200 μg glyceollin per g dry wt) in cells of Glycine max and enhancing berberine biosynthesis (up to four-fold) in cells of Thalictrum rugosum. The response of the cell cultures to the elicitor treatment is dependent on the amount of carbohydrate per unit of biomass and on the physiological state of the cells. Cells are optimally induced in late exponential or early stationary growth phases.     

Level of M(IP)2C sphingolipid affects plant defensin sensitivity, oxidative stress resistance and chronological life-span in yeast

The antifungal plant defensin DmAMP1 interacts with fungal sphingolipids of mannosyldiinositolphosphorylceramide (M(IP)2C) class. We screened a Saccharomyces cerevisiae transposon (Tn) mutant library against DmAMP1 and identified one DmAMP1-resistant mutant with the Tn inserted in the M(IP)2C biosynthesis gene IPT1 (DmTn11) and one DmAMP1-hypersensitive mutant with the Tn inserted in rDNA (HsTnII). However, tetrad analysis pointed to HsTnII as a spontaneous mutant. Apparently, membranes of DmTn11 lack M(IP)2C, whereas membranes of HsTnII have increased M(IP)2C levels. In addition, DmTn11 and HsTnII are characterized by increased and reduced oxidative stress resistance/chronological life-span (CL), respectively. A putative involvement of M(IP)2C in oxidative stress and CL in yeast is discussed.     

Comparative expression in Escherichia coli of the native and hybrid genes for acyl-lipid delta(9) desaturase

Expression of the desC gene coding for acyl-lipid delta(9) desaturase of thermophilic cyanobacterium Synechocystis sp. PCC6803 was studied in Escherichia coli cells. In a hybrid gene constructed (desC-licBM3), a sequence of the native acyl-lipid delta(9) desaturase was fused in frame with the reporter gene coding for thermostable lichenase. Lichenase contained in the hybrid protein simplified selection and analysis of the expression of membrane desaturase in the heterologous host. Comparisons of the expression for the native and hybrid genes in bacterial cells showed that lichenase remained active and thermostable in the hybrid protein, while desaturase retains the capability of introducing a double bound in the corresponding position of fatty acids.