Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.     

Heterokaryosis inPolyporus ostreiformis for improved yield of exo-1,4-α-d-glucosidase

An amylolytic strain ofPolyporus ostreiformis was subjected to multistep mutagenic treatment and a heterokaryotic mutant was finally selected which produced exo-1,4-α-d-glucosidase in a yield of 2.54 U/mL against the parental yield of 0.42 U/mL. The crude enzyme extract of the mutant was used to hydrolyze soluble starch, maltose and amylopectin and chromatography of the hydrolyzates revealed exo-1,4-α-D-glucosidase activity predominant in the enzyme system.     

Adaptation of Aquatic Microbial Communities to Quaternary Ammonium Compounds

The effects of long-chain (C₁₂ to C₁₈) quaternary ammonium compounds (QACs) on the density, heterotrophic activity, and biodegradation capabilities of heterotrophic bacteria were examined in situ in a lake ecosystem. Monoalkyl and dialkyl substituted QACs were tested over a range of concentrations (0.001 to 10 mg/liter) in both acute (3 h) and chronic (21 day) exposures. In general, none of the QACs tested had significant adverse effects on bacterial densities in either acute or chronic studies. However, significant decreases in bacterial heterotrophic activity were noted in acute studies at QAC concentrations from 0.1 to 10 mg/liter. Chronic exposure of lake microbial communities to a specific monoalkyl QAC resulted in an adaptive response and recovery of heterotrophic activity. No-observable-effect level in the adapted populations was >10 mg/liter. Chronic exposure also resulted in significant increases in the number and activity of bacteria capable of biodegrading the material. The increase in biodegradation capability was observed at low (microgram per liter) concentrations which are approximately the same as realistic environmental levels. In general, our studies indicated that exposure of lake microbial communities to QACs results in the development of adapted communities which are less sensitive to potential toxic effects and more active in the biodegradation of these materials.     

Genetic and developmental characterization of the aldox-2 locus of Drosophila melanogaster

The aldox-2 locus in Drosophila melanogaster has been shown to affect differentially three molybdoenzymes, aldehyde oxidase, pyridoxal oxidase, and xanthine dehydrogenase. These effects are most obvious at times surrounding the pupal-adult boundary, when the normal organism accumulates large amounts of these enzymes in their active form. This locus has been more precisely mapped genetically to 2-82.9 +/- 2.1, with complete concordance between the effects of all recombinant chromosomes on all three enzymes. The cytogenetic location has also been determined to be between 52E and 54E8, with the likelihood that it lies within the region 54B1-54E8. The aldox-2 mutant allele has no visible phenotype and is completely recessive for enzyme effects at all stages tested. Segmental duplication of this region, including the aldox-2+ allele, has no apparent effect on the visible phenotype or the enzymatic activity. The mutant aldox-2 allele has no effect on the developmental expression of two unrelated enzymes, 6-phosphogluconate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. The effects of this locus on aldehyde oxidase, xanthine dehydrogenase, and pyridoxal oxidase suggest that this locus may code for a product involved in the synthesis of the molybdenum cofactor common to these enzymes.     

Direct transesterification of all classes of lipids in a one-step reaction

Conventional techniques for the determination of fatty acid composition of lipids require solvent extraction, purification, hydrolysis, and derivatization procedures that are both lengthy and cumbersome. A 1-hr direct transesterification procedure carried out in methanol-benzene 4:1 with acetyl chloride circumvented all these steps and was applicable for analysis of both simple (triglycerides) and complex lipids (cholesteryl esters, phospholipids, and sphingomyelin). Recoveries (greater than 95%) of standards unaffected by the presence of 5% water and 200 mg of silica suggested that the technique could be used for the quantitative analysis of total fatty acids as well as of fatty acids in classes of lipids separated on silica from biological samples. When compared to the Folch procedure, the technique led to a 20.1% increase in total fatty acids for plasma, 3.9% for feces, 7.4% for bile, and 9.7% for rat liver. We therefore conclude that this one-step direct transesterification procedure is superior to currently used methods, not only because of its simplicity and speed, but also because of its added precision.     

Vicilin genes ofPisum

Summary We describe four genomic clones of pea 7S storage protein gene, one of which corresponds to convicilin, and the others to vicilin. Hybridization studies exploiting these clones, and previously identified cDNA clones, have enabled us to define six different loci. Three of these loci have been mapped to positions on chromosome 7.     

Purification and characterization of a mitochondrial isozyme of C1-tetrahydrofolate synthase from Saccharomyces cerevisiae

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is encoded by the ADE3 locus, yet ade3 mutants have low but detectable levels of these enzyme activities. Synthetase, cyclohydrolase, and dehydrogenase activities in an ade3 deletion strain co-purify 4,000-fold to yield a single protein species as seen on sodium dodecyl sulfate-polyacrylamide gels. The native molecular weight of the isozyme (Mr = 200,000 by gel exclusion chromatography) and the size of its subunits (Mr = 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are similar to those of C1-tetrahydrofolate synthase. Cell fractionation experiments show that the isozyme, but not C1-tetrahydrofolate synthase, is localized in the mitochondria. Genetic studies indicate that the isozyme is encoded in the nuclear genome. Peptide mapping experiments show that C1-tetrahydrofolate synthase and the isozyme are not structurally identical. However, immunotitration experiments and amino acid sequence analysis suggest that C1-tetrahydrofolate synthase and the isozyme are structurally related. We propose to call the isozyme "mitochondrial C1-tetrahydrofolate synthase."     

Exogenous Tryptophan Increases Synthesis, Storage, and Intraneuronal Metabolism of 5-Hydroxytryptamine in the Rat Hypothalamus

The effects of tryptophan administration on neurochemical estimates of synthesis [5-hydroxytryptophan (5-HTP) accumulation following administration of a decarboxylase inhibitor], storage [5-hydroxytryptamine (5-HT) concentrations], and metabolism [5-hydroxyindoleacetic acid (5-HIAA) concentrations] of 5-HT in selected regions of the hypothalamus were determined using HPLC coupled to an electrochemical detector. Tryptophan methyl ester HCl (30-300 mg/kg i.p.) produced a dose-dependent increase in the rate of 5-HTP accumulation throughout the hypothalamus but had no effect on the rate of accumulation of 3,4-dihydroxyphenylalanine. Peak 5-HTP levels were attained by 30 min following administration of tryptophan (100 mg/kg i.p.) and were maintained for an additional 60 min. Tryptophan also produced concomitant dose-dependent increases in 5-HT and 5-HIAA concentrations in these same regions without changes in the 5-HIAA/5-HT ratio. These results indicate that exogenous tryptophan administration selectively increases the synthesis, storage, and metabolism of 5-HT in the hypothalamus without altering the synthesis of catecholamines. Inhibition of 5-HT uptake with chlorimipramine or fluoxetine produced modest (10-40%) reductions in 5-HIAA concentrations throughout the hypothalamus, revealing that only a minor portion of 5-HIAA is derived from released and recaptured 5-HT, whereas the major portion of this metabolite reflects intraneuronal metabolism of unreleased 5-HT. In both chlorimipramine- and fluoxetine-treated rats, 5-HIAA concentrations were significantly increased by tryptophan administration, indicating that the increase in synthesis of 5-HT following precursor loading is accompanied by an increase in the intraneuronal metabolism of 5-HT.     

Differences in adhesiveness among type 1 fimbriate strains of Enterobacteriaceae revealed by an in vitro HEp2 cell adhesion model

Ten type 1 fimbriate strains of Enterobacteriaceae were examined in an in vitro adhesion assay with HEp2 epithelial cells. The range of HEp2 cell adhesiveness, which was characteristic for each strain, was affected by motility, type 1 fimbriation and production of mannose sensitive haemagglutinin. Nevertheless, not all type 1 fimbriate strains adhered well in this model. The findings are discussed with regard to the possibility that different type 1 fimbriate enterobacteria, though all are mannose sensitive, recognize different mannose-containing receptors present or available on the surfaces of the HEp2 cells.