Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.)

Sesquiterpenoid lactones (SLs) from lettuce (Lactuca sativa L.) include constitutive components of latex such as lactucin and the induced phytoalexin, lettucenin A. A redundant primer strategy was used to recover two full length cDNA clones (LTC1 and LTC2) encoding sesquiterpene synthases from a cDNA library derived from seedlings with the red spot disorder, which accumulate phytoalexins. Recombinant enzymes produced from LTC1 and LTC2 in Escherichia coli catalysed the cyclisation of farnesyl diphosphate to germacrene A, potentially an early step in the biosynthesis of SLs. RT-PCR analysis showed LTC1 and LTC2 were expressed constitutively in roots, hypocotyls and true leaves but not in cotyledons. Expression in cotyledons was induced by challenge with the downy mildew pathogen Bremia lactucae in the disease resistant cultivar Diana. Southern hybridisation experiments showed that LTC1 and LTC2 were not part of a multigene family. The germacrene A synthases provide targets for modified expression to generate beneficial modifications to the SL profile in lettuce.     

Gibberellin A58 and ent-6α,7α,12α-trihydroxykaur-16-en-19-oic acid from seeds of cucurbita maxima

The isolation of gibberellin A₅₈ and ent-6α,7α,12α-trihydroxykaurenoic acid from a cellulase-hydrolysed extract of endosperm ofCucurbita maxima is described. The two compounds are characterized by their MS,¹H NMR and ¹³C NMR.     

Chemical synthesis of 4,5-dioxovaleric acid and its nonenzymatic transamination to 5-aminolevulinic acid

4,5-Dioxovaleric acid (DOVA) was synthesized from 5-bromolevulinic acid via formation of the pyridinium bromide of 5-bromolevulinic acid, followed by nitrone formation with p-nitrosodimethylaniline, and hydrolysis of the nitrone to yield DOVA. Partial purification of DOVA was obtained by passage of the reaction mixture through a cation exchange column. DOVA was identified by paper electrophoresis and by a specific fluorometric assay. DOVA was nonenzymatically transaminated to 5-aminolevulinic acid (ALA) with glycine serving as the amino donor. Other compounds tested were less effective amino donors. Glyoxylic acid was identified as a reaction product by paper electrophoresis and a specific calorimetric test. ALA was identified by paper electrophoresis, paper chromatography of a pyrrole derivative, reaction with Ehrlich reagent, and by its enzymatic conversion by a barley extract to porphobilinogen and uroporphyrin. The nonenzymatic transamination was inhibited by Tris and was stimulated by high pH. The existence of this nonenzymatic activity is discussed in relation to previous reports of dova transaminase activity in cell extracts.     

Gibberellin-photoaffinity labelling of two polypeptides in plant plasma membranes

Two polypeptides of M _r 68 kDa and 18 kDa were gibberellin (GA)-photoaffinity labelled in vitro in plasma membrane preparations from oat ( Avena sativa L.) aleurone and from leaves and stems of wild-type and GA-sensitivity mutants of different species. Labelling of these polypeptides could be competed by biologically active, but not by inactive, GAs, indicating the likely biological significance of these interactions. On 2-dimensional gels the radiolabelled polypeptides were each resolved as one intensely labelled low abundance spot with a slightly lower pI form adjacent to it. There was a strong pH dependency for both labelling events, which correlated well with pH values at which GA are known to be most biologically active. A semi-dwarf GA-sensitivity mutant of sweet pea ( Lathyrus odoratus L.), lb , showed reduced photoaffinity labelling of both polypeptides compared with the wild type, Lb . In the GA-insensitive Arabidopsis thaliana mutant, gai , the level of labelling was the same as in wild type, GAI . This is the first report of GA-binding proteins in plant plasma membranes. Some preliminary sequence data are given for one of the labelled polypeptides. We discuss these mutants and consider their possible roles in GA perception or action.     

Evidence that the Yb subunits of hepatic glutathione transferases represent two different but related families of polypeptides

Three soluble rat liver glutathione (GSH) transferases A, C and one referred to as 'D', all of which are dimers of Yb subunits [Bass et al. (1977) Biochim. Biophys. Acta, 492, 163-175], have been compared with respect to C-terminal amino acids and tryptic peptide maps. GSH transferases A and 'D' gave different tryptic peptide maps and different C-terminal amino acids, lysine and proline respectively. In each case the number of tryptic peptides is about half of that expected from their lysine and arginine content, and there are 2 mol C-terminal amino acid/mol enzyme. This indicates that GSH transferases A and 'D' represent two different Yb homodimers, which we refer to here as Y1bY1b and Y2bY2b respectively. GSH transferase C is the corresponding heterodimer Y1bY2b since it gives all the tryptic peptides which arise from GSH transferase A and GSH transferase 'D' and also contains both C-terminal lysine and proline. These results provide a structural basis to similar conclusions drawn by Mannervik and Jensson [(1980) J. Biol. Chem. 257, 9909-9912] based on enzymic and immunological comparisons. Tryptic peptide maps show that GSH transferases A and 'D' have considerable homology since there are 23 peptides common to both, 12 peptides unique to A and 8 peptides unique to 'D'. Even so GSH transferase A is selectively induced by a phenobarbitone regime. It is, therefore, concluded that Y1b and Y2b are derived from separate but related genes. A similar conclusion has been drawn concerning the Ya and Yc subunits [Beale et al. (1982) Eur. J. Biochem. 126, 459-463], and a comparison of amino acid compositions, presented here, further suggests a genetic relationship between both pairs of subunits.     

Regulation of phosphoenolpyruvate carboxykinase mRNA in mouse liver, kidney, and fat tissues by fasting, diabetes, and insulin.

Previous investigations of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been conducted using rats. In a recent comparative study, we investigated, for the first time, the effects of fasting, refeeding, alloxan-induced diabetes, and insulin treatment on the levels of PEPCK mRNA in mouse liver, kidney, and adipose tissues. As in rats, fasting and diabetes induced, while insulin repressed, hepatic PEPCK mRNA. In contrast, the response of renal PEPCK mRNA to fasting, refeeding, and diabetes in mice differed quantitatively with that in rats: fasting caused a twofold increase in mice and a fourfold increase in rats. Moreover, diabetes, which induces renal PEPCK mRNA indirectly by causing acidosis in rats, was without effect in mice. In adipose tissue, the results of previous studies in both rats and mice have shown that the amount of PEPCK protein and its rate of synthesis are increased by fasting and diabetes and decreased by refeeding and insulin treatment. Thus, it was surprising to find that fasting, refeeding, alloxan-induced diabetes, and insulin treatment had no effect on adipose tissue PEPCK mRNA in either rats or mice.     

Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP

The effect of N6,O2'-dibutyryl cyclic adenosine monophosphate (Bt2cAMP) on the induction of the mRNA coding for the enzyme phosphoenolpyruvate carboxykinase was examined in H4-II-E cells. this mRNA comprised about 0.1% of total cellular poly(A)+RNA activity in uninduced cells and was increased 5- to 7-fold by the cyclic nucleotide. The maximal level was reached 3 h after addition of the nucleotide to the cell culture. This induction is attributed to cAMP since the nonmetabolizable analogs 8-bromocAMP and 8-(4-chlorophenylthio)cAMP produce inductions comparable to Bt2cAMP while sodium butyrate and dibutyryl cyclic GMP had little effect. The increased translational activity correlated well with a proportionate increase in the amount of phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) mRNA sequences which were hybridizable to a specific cDNA probe. Blot hybridization of total nuclear RNA isolated from uninduced H4-II-E cells revealed eight P-enolpyruvate carboxykinase RNA sequence species ranging in size from 1.8 to 6.9 kilobases. Treatment with Bt2cAMP increased the amount of all eight of these forms. This increase became maximal by 45-60 min and was maintained for at least 1 h. In contrast, analysis of cytoplasmic RNA showed a single 3.2-kilobase (23 S) band, which was still increasing in amount 2 h after Bt2cAMP treatment. Thus, Bt2cAMP resulted in a sequential induction of nuclear P-enolpyruvate carboxykinase RNA sequences followed by an increase in cytoplasmic phosphoenolpyruvate carboxykinase mRNA. We conclude that cyclic AMP exerts its main effect on P-enolpyruvate carboxykinase induction at the nuclear level.     

Insulin decreases phosphoenolpyruvate carboxykinase (GTP) mRNA activity by a receptor-mediated process

The mRNA that codes for phosphoenolpyruvate carboxykinase accounts for approximately 0.2% of the protein synthesized in H4IIEC3 hepatoma cells maintained for 24 h in serum-free medium containing N6,O2'-dibutyryl cAMP and theophylline. This value decreases to 0.04% within 3 h after the addition of insulin. Maximal effects are produced by 10(-10) M insulin, and half-maximal deinduction of both the relative rate of synthesis of P-enolpyruvate carboxykinase and mRNA coding for P-enolpyruvate carboxykinase activity occurs at approximately 2 X 10(-12) M insulin. Porcine proinsulin is 4% as potent as porcine insulin since half-maximal deinduction of mRNA coding for P-enolpyruvate carboxykinase occurs at 5 X 10(-11) M. The concentration of proinsulin required to inhibit 125I-insulin binding by 50% is 2 X 10(-7) M, as compared to 6 X 10(-9) M for insulin; thus, the decreased sensitivity of this deinduction to proinsulin parallels the decreased binding affinity H4IIEC3 cells have for proinsulin as compared to insulin. These data indicate that insulin regulates P-enolpyruvate carboxykinase synthesis through a receptor-mediated process, that the effect occurs when less than 2% of the insulin receptors are occupied, and that this effect is exerted prior to the level of mRNA translation.     

Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo

To investigate the extent to which in vivo mutation spectra might reflect the intrinsic specificities of active mutators, genetic and biochemical assays were used to analyse the DNA target specificities of cytidine deaminases of the APOBEC family. The results reveal the critical importance of nucleotides immediately 5' of the targeted C for the specificity of all three enzymes studied (AID, APOBEC1 and APOBEC3G). At position -1, APOBEC1 showed a marked preference for dT, AID for dA/dG and APOBEC3G a strong preference for dC. Furthermore, AID and APOBEC3G showed distinct dependence on the nucleotide at position -2 with dA/dT being favoured by AID and dC by APOBEC3G. Most if not all activity of the recombinant deaminases on free dC could be attributed to low-level contamination by host enzymes. The target preference of APOBEC3G supports it being a major but possibly not sole contributor to HIV hypermutation without making it a dominant contribution to general HIV sequence variation. The specificity of AID as deduced from the genetic assay (which relies on inactivation of sacB of Bacillus subtilis) agrees well with that deduced by Pham et al. using an in vitro assay although we postulate that major intrinsic mutational hotspots in immunoglobulin V genes in vivo might reflect favoured sites of AID action being generated by proximal DNA targets located on opposite DNA strands. The target specificity of AID also accords with the spectrum of mutations observed in B lymphoma-associated oncogenes. The possibility of deaminase involvement in non-lymphoid human tumours is hinted at by tissue-specific differences in the spectra of dC transitions in tumour-suppressor genes. Thus, the patterns of hypermutation in antibodies and retroviruses owe much to the intrinsic sequence preferences of the AID/APOBEC family of DNA deaminases: analogous biases might also contribute to the spectra of cancer-associated mutation.     

Chickpea wilt: identification and toxicity of 8-O-methyl-fusarubin from Fusarium acutatum

Fusarium acutatum was isolated from wilting chickpea plants in Pakistan. Filtrates from cultures grown on a defined liquid medium caused permanent wilting of chickpea cuttings and killed cells, isolated enzymically from healthy plants, in a bioassay. Toxic activity was retained by a cyano solid phase extraction cartridge and the toxin was isolated by elution from the cartridge in acetonitrile and Si-gel thin layer chromatography of the eluate. Analytical HPLC of the compound on a cyano column with diode array detection gave a single peak with a homogeneous spectrum and lambda(max) at 224 and 281 nm. NMR and mass spectral studies showed that the toxin was 8-O-methyl-fusarubin. The pure compound caused permanent wilting of chickpea cuttings and the LD50 value in the cell bioassay was 327 ng/ml.