Cytoplasmic inheritance of oligomycin resistance in Chinese hamster ovary cells

Oligomycin-resistant clones were isolated from Chinese hamster ovary cells by treatment of cells with ethidium bromide, followed by mutagenesis with ethylmethane sulfonate and selection in oligomycin. One clone (Olir 8.1) was chosen for further study. Olir 8.1 cells grow with doubling time similar to that of wild-type cells, whether grown in the presence or absence of drug (doubling time of 13-14 h). In plating efficiency experiments, Olir 8.1 cells are approximately 100-fold more resistant to oligomycin than are wild-type cells. There is approximately a 32-fold increase in the resistance to inhibition by oligomycin of the mitochondrial ATPase from Olir 8.1 cells. The electron transport chain is functional in Olir 8.1 cells. Oligomycin resistance is stable in the absence of selective pressure. There is little or no cross-resistance of Olir 8.1 cells to venturicidin and dicyclohexylcarbodiimide, other inhibitors of the mitochondrial ATPase, or to chloramphenicol, an inhibitor of mitochondrial protein synthesis. Oligomycin resistance is dominant in hybrids between Olir 8.1 cells and wild-type cells. Fusions of enucleated Olir 8.1 cells with sensitive cells and characterization of the resulting "cybrid" clones indicates that oligomycin resistance in Olir 8.1 cells is cytoplasmically inherited.     

Substructural studies on sporulation of Saccharomycopsis lipolytica

During sporulation of diploids from crosses between different strains of the yeast Saccharomycopsis (Candida) lipolytica irregular numbers of ascospores per ascus have been observed. Using the serial section method it could be shown now by means of electron microscopy that in one-, two-, and three-spored asci unenclosed "naked" nuclei occur additionally to nuclei incorporated in mature spores. It was demonstrated that the production of less than four spores per ascus in this yeast is not the result of a lack of meiotic products but of the nonutilization of nuclei from meiosis. In 2--4 spored asci usually four products of meiosis in form of enclosed and free nuclei could be demonstrated which indicate a normal meiotic division. All ascospores derived from asci with different spore numbers are uninuclear. It is assumed that a defect in spore formation caused by structural changes of chromosomes or aneuploidy should give rise to the occurrence of non incorporated nuclei and spore irregularity. It was concluded that meiosis and spore formation in Saccharomycopsis lipolytica seem to represent parallel and coordinated processes which generally resemble those recorded for Saccharomyces cerevisiae and Hansenula species.     

Glycogen-deficient Mutants of the Yeast Saccharomycopsis lipolytica

Following ultra-violet irradiation of the hydrocarbon-utilizing yeast, Saccharomycopsis lipolytica , a number of mutant strains were isolated which failed to show the normal staining reaction with iodine. Exponential phase cells of the mutant strains were found to contain less carbohydrate and more crude protein than wild type cells in the case of both glucose-grown and n -alkane-grown cultures. The difference between wild type and mutant carbohydrate levels was greater for glucose-grown than for n -alkane-grown cells. Carbohydrate fractionation revealed that the mutant cells were deficient in glycogen, particularly the acid-soluble fraction.     

Interspecific T-DNA transfer through plant protoplast fusion

Summary An attempt was made to transfer the T-DNA of Agrobacterium tumefaciens, previously introduced into plant cells, via protoplast fusion from one species into another. For the experiments two cell lines were used: firstly, a Nicotiana paniculata cell line transformed with the Agrobacterium strain B6S3. This cell line exhibits both hormone independent growth and synthesis of octopine as a result of the incorporated T-DNA from Agrobacterium. These two markers are dominant. The second cell line was the nitrate reductase deficient cnx-68 cell line of N. tabacum which contains an intracellular calcium oxalate druse. These two markers are recessive. Isolated protoplasts of the donor cell line N. paniculata B6S3 were mitotically inactivated by X rays and fused with protoplasts of the cell line cnx-68. Asymmetric somatic hybrids were selected on hormone free agar medium supplemented with 50 mM KClO₃. This compound is toxic for cells possessing nitrate reductase activity. From about 1.1×10⁷ cultivated protoplasts 18 cell lines survived the selection treatment. Of these seven exhibited the two dominant and the two recessive markers, whereas the others showed either only one or none of the recessive or only one of the dominant markers. In dot-blot experiments using species specific DNA clones of the donor and the recipient plant species it was confirmed that besides the T-DNA other nuclear genomic DNA of the donor species had also been transferred in various amounts. The possible consequences of these results for plant breeding programmes are discussed.     

Batch growth of Saccharomycopsis lipolytica on animal fats

Summary Solid animal fats aggregated when first added to aqueous media and strong agitation was necessary to accomplish and maintain their dispersion. The growth rate of Saccharomycopsis lipolytica accelerated as fat dispersion proceeded until similar rates of exponential growth were attained with either lard, mutton tallow or beef tallow as sole carbon source. The major fatty acids in all substances were oleic, palmitic, and stearic. A major proportion of both saturated acids were consumed during the yeast's growth on animal fats, but the growth rates were greatly reduced after exhaustion of the preferentially consumed unsaturated acid. At this time, substantial amounts of saturated acids, present both as free fatty acid and in glycerides, remained. The amounts of these residual acids were markedly affected by the distribution of acyl groups within the original triglycerides. With individual fatty acids as the sole carbon source, the yeast grew at comparable rates on palmitic and oleic acids but did not grow on stearic acid.     

Genetic control of lysine permeases in Saccharomycopsis lipolytica

In order to obtain strains of Saccharomycopsis lipolytica impaired in the active transport of l-lysine, mutants resistant to a mixture of l-canavanine, l-4-5-transdehydrolysine and l-S-amino ethylcysteine, taken either all three or two by two, were isolated. These compounds were shown previously to be competitive inhibitors of l-lysine uptake.The resistance patterns and excretion capacity of the mutants were established. All mutants behaved as monogenic. Recombination tests indicated that four genes at least were involved. All mutants were impaired in both high and low affinity l-lysine transport systems.Several hypotheses on the functions of these genes are put forward and discussed.     

Physiology of lysine permeases in Saccharomycopsis lipolytica.

Two active lysine transport systems were detected in Saccharomycopsis lipolytica. No excretion of lysine out of the cells could be obtained, even by chasing with L-lysine or by poisoning with sodium azide. The kinetic properties of one of the permeases, the high-affinity lysine permease, were studied in detail. Its Km was 1.91 +/- 0.23 X 10(-5) M. It proved highly specific, the only potent competitive inhibitors being (i) arginine and its analogs L-canavanine and L-ornithine, and (ii) the lysine analogs L-5 aminoethylcysteine and L-4,5-transdehydrolysine. It is suggested that the high-affinity lysine permease is common to L-lysine, L-ornithine, and L-arginine. The other amino acids tested behaved as noncompetitive inhibitors. The variation of uptake during a growth cycle was studied on ammonia-rich, ammonia-poor, and ammonia-free media. In each case, the uptake exhibited a peak in the early exponential growth phase. No new permease activity was detected during the lag phase or the stationary phase. Ammonia ions competitively inhibited the uptake and also decreased the Vmax value.     

Regulation of S-amino acids biosynthesis in Saccharomycopsis lipolytica

Molecular Genetics and Genomics - ATP-sulfurylase, cysteine synthase, homocysteine synthase, arylsulfatase and β-cystathionase in Saccharomycopsis lipolytica are repressed on the addition of...     

Intergeneric gene transfer mediated by plant protoplast fusion

Summary In attempts at somatic transfer of plant genomes of reduced size, X-irradiated leaf protoplasts of parsley (Petroselinum hortense, 2n=22) were fused with cell culture protoplasts of a nuclear albino mutant of carrot (Daucus carota, 2n=18). Introduction of genes from the irradiated parsley nuclei into the carrot genome was shown by the correction of the albino defect and by the appearance of parsley isoenzymes in selected green tissues and plants. The cytological studies provided information on significant deviation from the amphidiploid chromosome number. The high frequency of cells with 2n=19, 2n=38 and regeneration of plants with 2n=19 chromosomes can indicate that the elimination of parsley chromosomes is incomplete. A correlation was found between the lethality of selected tissues and differentiated or undifferentiated stages of the cells.     

Mutants of Saccharomycopsis lipolytica defective in lysine catabolism.

Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests.     

Regulation of s-amino acids biosynthesis in Saccharomycopsis lipolytica.

Summary ATP-sulfurylase, cysteine synthase, homocysteine synthase, arylsulfatase and -cystathionase in Saccharomycopsis lipolytica are repressed on the addition of methionine, homocysteine or cysteine to the growth medium. The use of appropriate mutants enabled us to demonstrate that the synthesis of these enzymes is regulated by the system involving at least two low-molecular weight effectors — most likely cysteine and methionine (or their close derivatives).Abbreviations SAM S-adenosylmethionine - OAS O-acetyl-L-serine - OAH O-acetyl-L-homoserine     

Isolation and Identification of Lipase Activators from Saccharomycopsis lipolytica

Three types of lipase activators (α, β, γ) were isolated from the culture broth of Saccharomycopsis lipolytica using high performance liquid chromatography. Activator γ was the most active for the lipase reaction. One of them (β) was identified with a mixture of 3,5-dihydro xy-7-tetradecenoic acid and related compounds by the method of NMR and GC-MS analyses. The free carboxyl group in the compounds was essential for the activation of the lipase reaction.     

Selective transfer of fungal cytoplasmic genetic elements by protoplast fusion

An anucleate small-protoplast fraction was prepared from a respiratory-competentSaccharomyces cerevisiae strain carrying mitochondrially inherited resistance to erythromycin, and used to transfer mitochondria selectively. Polyethylene glycol and Ca²⁺ were applied to induce fusion between these small protoplasts and nucleus-containing protoplasts of a respiratory-deficient ρ° mutant derived from an adenine-requiring strain of the same species. The majority of fusion products were haploid and erythromycin resistant, containing the nucleus of the recipient adenine-requiring strain and the mitochondrial genome from the respiratory-competent donor cells. Selective transfer of mitochondria and other cytoplasmic genetic elements also seems possible in a wide variety of fungal and other cells.     

Purification and Characterization of a Neutral Protease from Saccharomycopsis lipolytica

Saccharomycopsis lipolytica 37-1 produced two inducible extracellular proteases, one under neutral or alkaline growth conditions and the second under acid conditions. Secretion of the neutral protease was repressed in the presence of glycerol or glucose, both of which supported rapid growth of the organism. Ammonium ions also repressed the secretion of the enzyme. The neutral protease activity copurified with esterase activity during ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose, and gel filtration on Sephadex G-150. The molecular weight of the enzyme was estimated to be 42,000 by sucrose density gradient centrifugation and 38,500 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme had a pH optimum of 6.8. Phenylmethylsulfonylfluoride inhibited both protease and esterase activities, indicating the presence of a serine residue in the active center. Protease, but not esterase, activity was sensitive to ethylenediaminetetraacetate and was significantly activated by divalent ions. Dithiothreitol inhibited both protease and esterase activities, indicating the presence of a critical disulfide bridge. The enzyme hydrolyzed casein (K(m) = 25.6 muM) and hemoglobin as well as the nitrophenyl esters of tyrosine (K(m) = 2.4 mM), glycine, tryptophan, and phenylalanine.