The aconitase of yeast. II. Crystallization and general properties of yeast aconitase.

Yeast aconitase [citrate (isocitrate) hydro-lyase, ED 4.2.1.3], inductively formed by Candida iipolytica in the presence of fluoroacetate, was purified approximately 100-fold by Sephadex G-100 gel filtration and DEAE-Sephadex column chromatography, yielding dark-brown needle crystals. The crystalline aconitase was homogenious as judged by polyacrylamide gel electrophoresis and sedimentation by ultracentrifugation. The enzyme showed maximal activity at pH 8.0 and at 55 degrees. It has an S20, W of 5.03 S, a molecular weight of 68,500 and an isolectric point of pH 4.2. The presence of 2.10 moles of iron per mole of the enzyme was demonstrated by atomic absorption spectroscopy.     

Fusion of yeast spheroplasts.

Spheroplasts of two different auxotrophic strains of Saccharomyces cerevisiae, both of mating type a, were fused with the aid of polyethylene glycol and calcium ions. After reversion to vegetative cells in solid media, the resulting zygotes were shown to be diploid cells of mating type aa.     

Antimutagenicity in yeast.

In recent years there has been increasing interest in antimutagenesis, and studies have been done using both prokaryotic and eukaryotic systems. In eukaryotic systems the first studies were performed with different strains of Schizosaccharomyces pombe. In particular, caffeine and L-methionine were investigated. Different strains of Saccharomyces cerevisiae were employed in studies of a wide variety of compounds, including acridine, saccharin, salts, tumor promoters and co-carcinogens. Strain D7 was widely employed and antimutagenic activity of spermine, chlorophyllin, cobaltous chloride and fermented milk is reported.     

Preparation of an inner membrane-matrix fraction from yeast yeast mitochondria.

Treatment of yeast mitochondria with digitonin was used in order to prepare an inner membrane-matrix fraction preserving its permeability properties. The incubation time of mitochondria with digitonin was an essential parameter for the selective solubilization of the outer membrane. The incubation of mitochondria for l min at different concentrations of digitonin led to a three-step release of mitochondrial enzymes: (a) at low concentrations of digitonin, adenylate kinase was released; (b) higher concentrations were required to solubilize kynurenine hydroxylase, an outer membrane marker; (c) inner membrane markers (succinate dehydrogenase and oligomycin-sensitive adenosine triphosphatase) and matrix markers (fumarase and isocitrate dehydrogenase) were significantly released at concentrations of digitonin higher than 0.4 mg/mg of protein. The electron microscopic aspects of yeast mitoplasts (inner membrane-matrix fraction obtained by treatment with 0.4 mg of digitonin) showed an orthodox and a twisted configuration. These new organelles retained respiratory control when assayed with ethanol as the substrate. Their selective permeability properties were preserved as shown by isoosmotic swelling in potassium or ammonium salt solutions.     

Transfer of yeast artificial chromosomes from yeast to mammalian cells.

Human DNA can be cloned as yeast artificial chromosomes (YACs), each of which contains several hundred kilobases of human DNA. This DNA can be manipulated in the yeast host using homologous recombination and yeast selectable markers. In relatively few steps it is possible to make virtually any change in the cloned human DNA from single base pair changes to deletions and insertions. In order to study the function of the cloned DNA and the effects of the changes made in the yeast, the human DNA must be transferred back into mammalian cells. Recent experiments indicate that large genes can be transferred from the yeast host to mammalian cells in tissue culture and that the genes are transferred intact and are expressed. Using the same methods it may soon be possible to transfer YAC DNA into the mouse germ line so that the expression and function of genes cloned in YACs can be studied in developing and adult mammalian animals.     

Prototrophic hybrids of bakers yeast. I. Selection of haploids and diploids of prototrophic yeast

153 haploids, including 8 prototrophs, 12 biotin prototrophs and 4 biotin auxotrophs were isolated from Y, F and FB strains of the baker's yeast Saccharomyces cerevisiae. Remaining haploids were dependent on vitamins of B group other than biotin. Obtained haploids were characterized by large cell sizes, a or α mating type, the ability to fermentation of sugars and the assimilation of non-fermented carbon sources. Haploids obtained from the yeast of Y and FB strains a good growth in the synthetic medium. Prototrophic haploids, prototrophic haploids with biotin auxotrophs and biotin prototrophs with biotin auxotrophs were crossed. 89 prototrophic hybrids capable of growth in synthetic medium without vitamins were selected out of 178 hybrids obtained. Hybrids are characterized by features typical for baker's yeast; however, not all of them are capable of sporulation. As result of selection, prototrophic hybrids and 16 hybrids characterized by a good increase of biomass in molasses medium were chosen. The efficiency of the biomass of hybrids designated as YY 3040/2, YY 3040/3 and FFB 1910/2 is considerably higher than one obtained from the cultivation of the industrial strain French Mautner (Mf). All hybrids possess adequate enzymatic activity in anaerobic metabolism of saccharose and maltose. Selected prototrophic hybrids were sent out to two yeast factories in the country for experimental propagation.     

酵母及与藻类搭配对萼花臂尾轮虫饵料效果的研究

研究了２种酵母单独投喂萼花臂尾轮虫的最适密度、饵料效果及与藻类搭配的投喂效果．结果表明,在５种密度下,面包酵母和啤酒酵母的最适密度分别为１０和５×１０６个·ｍｌ－１．用面包酵母和啤酒酵母培养轮虫所得到的种群密度和瞬时增长率分别是用蛋白核小球藻培养的４０．２％、２６．０％和８５．５％、７６．６％．用面包酵母和蛋白核小球藻适当比例搭配投喂轮虫,其效果接近或超过单一用小球藻在最适密度下的培养效果．     

Synthesis of yeast wall glucan.

Saccharomyces cerevisiae was treated with a mixture of toluene and ethanol to make it permeable to small molecules. This treatment unmasked a glucan synthetase activity which was assayed with UDP-[U-14C]glucose. About 60% of the polymer formed was beta-(I leads to 3)glucan. No labelled lipids were detected. The 14C incorporated was recovered in a particulate membrane preparation isolated by differential centrifugation. When the particles themselves were assayed for glucosyl transfer activity none was found. The toluene-treated preparations also catalysed the transfer of mannosyl residues from GDP-mannose to polymeric materials by a process independent of glucosyl transfer.     

Fluorescent derivatives of yeast tRNAPhe.

The preparation of four fluorescent derivatives of tRNAPhe (yeast) and their characterization by chemical, spectroscopic, and biochemical methods is described. The derivatives are prepared by replacing wybutine (position 37 in the anticodon loop) or NaBH4-reduced dihydrouracil (positions 16/17 in the hU loop) with ethidium or proflavine; they are isolated by reversed-phase chromatography (RPC-5). All tRNAPhe-dye derivatives are aminoacylated by yeast phenylalanyl-tRNA synthetase to at least 80% of the charging capacity of the unmodified tRNAPhe with an unchanged Km (0.2 mucroM) and a V lowered by 30--50%. They exhibit good to excellent activity in the aminoacylation assay from synthetase from Escherichia coli. It is concluded that the insertion of the dyes does not seriously disturb essential elements of the native tRNAPhe structure. The dyes are bound via N-ribosylic linkages. The appearance of isomeric tRNAPhe-ethidium derivatives is attributed to the involvement of the different amino groups of ethidium in the condensation. In addition, there are indications for the existence of alpha and beta anomers of the tRNA-dye compounds. The dyes are rigidly fixed to their position in the tRNA molecule by stacking interactions with the neighboring bases. The ethidium probes show Mg2+-induced changes of the tRNA conformation which are paralleled by changes of the rate of aminoacylation. On the basis of this observation it is hypothesized that conformational flexibility of the tRNA molecule is a functionally important feature of the tRNA structure.