1,3-β-Glucan synthase from Citrus phloem

1,3-β-Glucan synthase activity has been demonstrated in particulate fractions of bark extracts from Mexican lime. With respect to substrate, the enzyme kinetics did not conform to the Michaelis-Menten equation. The value of the Hill coefficient was 1.2 and S_0.5 is 1.1 mM. The enzyme had an optimum pH of 7.5. Maltose, sucrose, and especially cellobiose and glucose, were enzyme activators when tested at physiological concentrations. In the presence of 15 mM MgCl₂ the enzymic activity was stimulated at 10 μM UDP-glucose but decreased at 1 mM UDP-glucose, suggesting a minor 1,4-β-glucan synthase activity.     

Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids

A nonselectable gene carried on a poorly selectable recombinant plasmid has been physically mapped by deletion analysis. Our method involved cloning the plasmid into a coliphage lambda vector and treating the recombinant phage with a chelator. Virtually all particles surviving this treatment carried large deletions within the plasmid insert. Further deletion analysis was done by inserting a selectable lambda sequence into one such deletion derivative and repeating the chelator selection. Chelator selection was also used to isolate deletions constructed in vitro. The deleted phage are readily characterized by restriction mapping, and the gene in question scored after infection of a mutant host strain. These techniques have enabled us to physically assign the cyclopropane fatty acid synthase gene of Escherichia coli to 0.8 kb of a 16-kb segment after characterizing only a small number of isolates. This approach should be generally useful in the mapping of plasmids for which no convenient method exists for selecting or scoring the gene in question.     

Effect of papulacandin B on β-glucan synthesis in Schizosaccharomyces pombe

Abstract The antifungal antibiotic papulacandin β inhibited B(1,3)glucan-synthase activity, in vitro, from Schizosaccharomyces pombe . Levels of β(1,3)glucan-synthase from antibiotic-treated cultures were lower than the control cultures whereas mannan-synthase and β(1,3)glucanase activities were almost unaffected. The presence of an osmotic stabilizer reduced the inhibition of growth caused by the antibiotic. Addition of papulacandin β to a culture of S. pombe specifically inhibited incorporation of glucose into the β-glucan cell wall fraction. The fatty acids as well as the hydroxyl groups on the phenol residue of the papulacandin β molecule were essential for the inhibitory activity.     

Studies on some enzymes relevant to citric acid accumulation by Aspergillus niger

Enzymatic studies have been performed on a local strain of Aspergillus niger to find a correlation with citric acid accumulation. The activity of aconitase [aconitate hydratase, citrate(isocitrate) hydrolyase, EC 4.2.1.3] and isocitrate dehydrogenase (NADP⁺) [threo-d_s-isocitrate:NADP⁺ oxidoreductase (decarboxylating) EC 1.1.1.42] decreased after 4 days whereas that of citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH₂COO^?→acetylCoA), EC 4.1.3.7] did so after 8 days, when citric acid accumulation in the medium reached a maximum (45.9 mg ml^?1). In vitro studies with mycelial cell-free extracts demonstrated inhibition of citrate synthase activity by sodium azide and potassium ferricyanide on both the 4th and 8th days. Aconitase was inhibited by sodium arsenate, sodium fluoride, iodoacetic acid and potassium ferricyanide only on the 4th day. Isocitrate dehydrogenase (NADP⁺) activity on the 4th and 8th days was inhibited by iodoacetic acid but was stimulated by potassium ferricyanide. The possible existence of isozyme species of these enzymes is discussed.     

Structural similarities between a mitochondrially encoded polypeptide and a family of prokaryotic respiratory toxins involved in plasmid maintenance suggest a novel mechanism for the evolutionary maintenance of mitochondrial DNA

Summary Subunit 8 of mitochondrial ATP synthase (A8), a mitochondrially encoded polypeptide, has no known homologue in any prokaryotic or plastid ATP synthase, suggesting that it has been recruited to its present role in the enzyme from an extraneous source. The polypeptide is poorly conserved at the primary sequence level, but shows a well-conserved hydropathy profile. The hydropathy profiles of A8 from diverse taxa were compared with those of thehok family of prokaryotic respiratory toxins, some of whose members are involved in plasmid maintenance, through postsegregational killing of cells that lose the plasmid at cell division. Such comparisons revealed a highly significant degree of similarity, suggesting a functional relationship. Based on these findings, it is proposed that A8 evolved from ahok-like protein, whose original role was the maintenance of an extrachromosomal replicon in the endosymbiont ancestor of mitochondria. An aggressive mechanism for the evolutionary maintenance of mitochondrial DNA overcomes many of the failings of traditional explanations for its retention as a separate genome.     

Molecular Analysis of Plant Defense Responses to Plant Pathogens

A number of inducible plant responses are believed to contribute to disease resistance. These responses include the hypersensitive reaction, phytoalexin synthesis, and the production of chitinase, glucanase, and hydroxyproline-rich glycoproteins. Because of the coordinate induction of these responses, it has been difficult to determine whether they are functional defense responses, and if they are, how they specifically contribute to disease resistance. Recent developments in molecular biology have provided experimental techniques that will reveal the specific contribution of each response to disease resistance. In this paper, we describe a strategy to determine if the hypersensitive reaction is a functional plant defense mechanism.     

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis

Summary Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 by fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1–5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS : a heterodimeric -ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a -ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORFl-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM 160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.     

Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.     

The purification and characterisation of the two forms of soluble starch synthase from developing pea embryos

Soluble starch synthase was purified 10000-fold from developing embryos of pea (Pisum sativum L.). The activity was resolved into two forms which together account for most if not all of the soluble starchsynthase activity in the embryo. The two isoforms differ in their molecular weights but are similar in many other respects. Their kinetic properties are similar, neither isoform is active in the absence of primer, and both are unstable at high temperatures, the activity being abolished by a 20-min incubation at 45° C. Both isoforms are recognised by antibodies raised to the granule-bound starch synthase of pea. Isoform II, which has the same molecular weight (77 kDa) as the granulebound enzyme, is recognised more strongly than Isoform I.