Genetic and mutational tools for investigating the genetics and molecular biology of trichothecene production in Gibberella pulicaris (Fusarium sambucinum).

Gibberella pulicaris (Fusarium sambucinum) is a promising organism for studying the genetics and regulation of trichothecene biosynthesis; conditions for obtaining fertile crosses have been defined (Desjardins & Beremand, 1987) and crosses between natural variants have provided some information about the number, location, arrangement, and role of genes which determine trichothecene production (Desjardins & Beremand, 1987; Beremand & Desjardins, 1988). The development of some additional experimental tools and methodologies required for the further genetic analysis of trichothecene production in G. pulicaris are described in the present study.

Effect of alpha-tocopherol on the volatile thermal decomposition products of methyl linoleate hydroperoxides.

alpha-Tocopherol and 1,4-cyclohexadiene were tested for their effect on the thermal decomposition of methyl linoleate hydroperoxide isomers. The volatiles generated by thermolysis in the injector port of a gas chromatograph at 180 degrees C were analyzed by capillary gas chromatography. In the presence of either alpha-tocopherol or 1,4-cyclohexadiene, which are effective donors of hydrogen by radical abstraction, volatile formation decreased in all tests, and significant shifts were observed in the relative distribution of products in certain hydroperoxide samples.

Purification and characterization of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti.

The sesquiterpene cyclase, aristolochene synthase, has been purified from Penicillium roqueforti by gel filtration and anion-exchange chromatography. Isolation was facilitated by a change in the elution behavior of the enzyme during gel filtration at different steps in the purification. The purified enzyme had a specific activity of 70 nmol/min/mg protein.

Isolation and nucleotide sequence of a sesquiterpene cyclase gene from the trichothecene-producing fungus Fusarium sporotrichioides.

The trichodiene synthase gene (Tox5) has been isolated from the fungus Fusarium sporotrichioides, and its nucleotide (nt) sequence determined. A lambda gt11 library of F. sporotrichioides DNA was screened with antiserum against trichodiene synthase (TS).

Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature.

Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35 degrees C.

Regulation of Trichodiene Synthase in Fusarium sporotrichioides and Gibberella pulicaris (Fusarium sambucinum).

The regulation of trichodiene synthase (TS) and its relationship to trichothecene biosynthesis was investigated in Fusarium sporotrichioides NRRL 3299 and Gibberella pulicaris R-6380. Cultures were analyzed for the presence of TS activity, trichothecenes, and immunodetectable TS polypeptide over a time period of 144 h. Enzyme activity increased from barely detectable to maximum levels over a period of 3 h for F.

Detection of lignin peroxidase and xylanase by immunocytochemical labeling in wood decayed by basidiomycetes.

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay.

Estimation of phylogenetic distances among ascomycetous yeasts from partial sequencing of ribosomal RNA.

Extent of taxonomic resolution obtained from nuclear DNA complementarity and ribosomal RNA sequencing is discussed. The phylogenetic relationships of Schizosaccharomyces pombe, Pichia stipitis, Pachysolen tannophilus, and Saccharomyces cerevisiae are compared from partial sequences of 18S and 26S ribosomal RNA.

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23.

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E.

Effects of zinc, iron, cobalt, and manganese on Fusarium moniliforme NRRL 13616 growth and fusarin C biosynthesis in submerged cultures.

The influence of zinc, iron, cobalt, and manganese on submerged cultures of Fusarium moniliforme NRRL 13616 was assessed by measuring dry weight accumulation, fusarin C biosynthesis, and ammonia assimilation. Shake flask cultures were grown in a nitrogen-limited defined medium supplemented with various combinations of metal ions according to partial-factorial experimental designs. Zinc (26 to 3,200 ppb [26 to 3,200 ng/ml]) inhibited fusarin C biosynthesis, increased dry weight accumulation, and increased ammonia assimilation.

Incorporation of deuterium-labeled fatty acids into human milk, plasma, and lipoprotein phospholipids and cholesteryl esters.

Fatty acid metabolism and the contribution of dietary fatty acids to milk cholesteryl ester (CE) and phospholipid (PL) were investigated in normal lactating mothers. The approach used was to feed mixtures of triglycerides containing deuterium-labeled palmitic acid (16:0-2H2), oleic acid (18:1-2H6), and linoleic acid (18:2-2H4). Milk and plasma samples were collected for 72 hr.

Purification of cyclopiazonic acid by liquid chromatography.

A purification procedure for cyclopiazonic acid has been developed, using sequential preparative and semi-preparative liquid chromatography. Crude cyclopiazonic acid (324 mg) was extracted from a 1 L fermentation medium with chloroform-methanol (80 + 20), dried, dissolved in chloroform, and chromatographed on an oxalic acid/silica preparative column with chloroform-methanol (99 + 1) as the eluant. A semi-preparative oxalic acid/silica column and chloroform-methanol (99.

Soybean lipoxygenase-1 enzymically forms both (9S)- and (13S)-hydroperoxides from linoleic acid by a pH-dependent mechanism.

Soybean lipoxygenase-1 produces a preponderance of two chiral products from linoleic acid, (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid and (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid. The former of these hydroperoxides was generated at all pH values, but in the presence of Tween 20, the latter product did not form at pH values above 8.5.

Linkages among zein genes determined by isoelectric focusing.

Genetic control of the major zein polypeptides in maize (Zea mays L.) was studied by isoelectric focusing (IEF) in agarose. Linkage relationships were determined by making a number of crosses, then determining the expression of zein polypeptides in backcross seeds.

Measurement of oxygen solubility in fermentation media: a colorimetric method.

Methods of measuring oxygen solubility in culture media are scarce, and those available are tedious to apply. A simple colorimetric assay was developed and applied to the analysis of oxygen solubility during alcoholic fermentation. The method was based on the consumption of oxygen by glucose oxidase activity and the production of the pink quinone of syringaldazine by coupled peroxidase activity.

Expression of an active spinach acyl carrier protein-I/protein-A gene fusion.

A synthetic gene encoding spinach acyl carrier protein I (ACP-I) was fused to a gene encoding the Fc-binding portion of staphylococcal protein A. This gene fusion, under the control of the λPR promoter, was expressed at high levels in Escherichia coli producing a 42 kDa fusion protein. This fusion protein was phosphopantethenylated in E.

Fusaric acid : A secondary fungal metabolite that synergizes toxicity of cooccurring host allelochemicals to the corn earworm,Heliothis zea (Lepidoptera).

The ability of naturally occurring levels of theFusarium spp. fungal metabolite fusaric acid to synergize the toxicity of the allelochemicals gossypol, a saponin, and 6-methoxy-2-benzoxazolinone to larvae ofHeliothis zea (Boddie) was tested. Levels of fusaric acid comparable to those found near the fungus increased mortality ofH.

Effect of meliaceous seed extracts on growth and survival ofSpodoptera frugiperda (J.E. Smith).

Hexane and ethanol extracts of seeds from 10 plant species (including neem-Azadirachta indica A. Juss.) of the family Meliaceae were incorporated into artificial diet at various doses and fed to fall armyworm [Spodoptera frugiperda (J.

Incorporation of trans-8- and cis-8-octadecenoic acid isomers in human plasma and lipoprotein lipids.

Mixtures of deuterium-labeled trans-8, cis-8 and cis-9-octadecenoic acids (8t-18:1, 8c-18:1, 9c-18:1) were fed as triglycerides (TG) to two adult male subjects. Blood samples were collected sequentially over a 48-hour period. Plasma and lipoprotein lipids were separated by thin layer chromatography and analyzed by gas chromatography-mass spectroscopy.

Soybean metal-binding proteins: calmodulin purification by hydrophobic interaction with polymer 3520.

Polymer 3520, a non-polar styrene divinylbenzene polymer, provides a simple way to purify calmodulin (CAM) from soybeans. This polymer, which selectively adsorbs CAM by hydrophobic interaction within the polymer matrix, contains no exchangeable groups; thus, interaction with CAM requires no Ca++ ions, and elution is achieved with 50% ethanol. Purification by this form of reversed-phase liquid chromatography is a substantial improvement over the conventional method, which requires high salt in elution buffers.

Oxygen radical chemistry of polyunsaturated fatty acids.

Polyunsaturated fatty acids (PUFA) are readily susceptible to autoxidation. A chain oxidation of PUFA is initiated by hydrogen abstraction from allylic or bis-allylic positions leading to oxygenation and subsequent formation of peroxyl radicals. In media of low hydrogen-donating capacity the peroxyl radical is free to react further by competitive pathways resulting in cyclic peroxides, double bond isomerization and formation of dimers and oligomers.

The antioxidant and nutritional effects of tocopherols, ascorbic acid and beta-carotene in relation to processing of edible oils.

Tocopherols belong to a class of phenolic antioxidants which can inhibit lipid autoxidation by scavenging free radicals and by reacting with singlet oxygen. In vegetable oils alpha-tocopherol inhibits the effects of singlet oxygen during sensitized photoxidation. Ascorbic acid has a complex multi-function, acting as a hydrogen donor, as a metal inactivator, and as a peroxide destroyer.

Energy-dispersive X-ray analysis of the mineral content of corn bran treated in vitro and by passage through the pig gastrointestinal tract.

Energy-dispersive X-ray (EDX) analysis was tested as a method for examining the mineral contents of corn bran loaded in vitro or passed through the GI tract of pigs. Particles of dry-milled corn pericarp treated in vitro or retrieved from the stomach, ileum, and colon of killed pigs were prepared as microtomed bulk specimens directly embedded in resin. Because of heterogeneity caused by differences in substrate cell density and mineral content, X-ray count averages for a number of different specimens had a coefficient of variation greater than or equal to 0.

4-O-(1-carboxyethyl)-D-galactose. A new acidic sugar from the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain 49.

The structure of a new acidic sugar from the extracellular polysaccharide of Butyrivibrio fibrisolvens strain 49 was determined as 4-O-(1-carboxyethyl)-D-galactose on the basis of 13C-n.m.r.

Sugar uptake by maize endosperm suspension cultures.

Maize (Zea mays L.) endosperm suspension cultures are a useful model system for studying biochemical and physiological events in developing maize endosperm. In this report, sugar uptake by the cultures is characterized.