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J F Spencer

Publications and source records attributed to J F Spencer.

At least 19 recordsLinked to original sources

Modulation of sporulation and metabolic fluxes in Saccharomyces cerevisiae by 2 deoxy glucose.

Quantitative studies of metabolic fluxes during Saccharomyces cerevisiae sporulation on acetate in the presence of the glucose analog, 2-deoxy glucose (2dG) are reported. We have studied the inhibition of sporulation and associated catabolic or anabolic fluxes by 2dG. Sporulation frequencies decreased from 50% to 2% asci per cell at 2dG concentrations in the range of 0.03 to 0.30 g l-1, respectively. Under the same conditions, the acetate consumption flux was inhibited up to 60% and the glyoxylate cycle and gluconeogenic fluxes decreased from 0.7 and 0.3 mmol h-1 g-1 dw, respectively, to negligible values. We observed a linear correlation of the acetate consumption rate with the sporulation frequency by varying the 2dG concentration. The linear correlation was also verified between the frequency of sporulation and the fluxes through glyoxylate cycle and gluconeogenic pathways. In addition, the same association of inhibition of sporulation and metabolic fluxes was found in other S. cerevisiae strains displaying different potentials of sporulation. The results presented suggest that inhibition of sporulation in the presence of the glucose analog may be attributed, at least in part, to the inhibition of anabolic fluxes and might be associated with catabolite repression.

Acetates

Meiotic analysis.

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Ascomycota

Hybrids obtained by protoplast fusion with a salt-tolerant yeast.

An industrial strain of Saccharomyces cerevisiae was fused with an osmotolerant yeast, Debaryomyces hansenii, to obtain hybrids having increased tolerance to elevated salt concentrations. The hybrids were intermediate to parent species in production of ethanol and polyols.

Alcohols

Transfer of genes for utilization of starch (sta2) and melibiose (mel) to industrial strains of Saccharomyces cerevisiae by single-chromosome transfer, using a kar1 mutant as vector.

A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector. The sta2 gene, conferring the ability to metabolize starch was transferred from an auxotrophic haploid strain of S. cerevisiae (S. diastaticus) and the melibiose-metabolism (mel) gene(s), from S. kluyveri, to the kar1-1 mutant [K5-5A; (alpha ade2 his4 can1 gal) by normal mating and protoplast fusion. From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating. Strains that utilized starch or melibiose were obtained by all three methods. Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids. The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S. cerevisiae. The transfer of the genes is probably accomplished through formation of disomic strains and then, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.

Chromosomes, Fungal

Effects of changes in the mitochondrial genome on the performance of baking yeasts.

Modifications to the mitochondria in baking yeasts affect their performance in dough-raising tests significantly. Conversion of a respiratory-competent baking strain, sensitive to glucose repression, to the petite mutant, yielded a strain which was released from this effect, as shown by the increased development of the cytochrome c peak in the cytochrome spectrum, and which showed a comparably improved dough-raising performance, approaching the levels found for a respiratory-competent strain which was fully derepressed. Replacement of the mitochondria of one strain by those from another, in some cases, improved the performance of the recipient strain, especially if the latter was sensitive to glucose repression. In addition, reduction of glucose repression in single-spore clones derived from the composite strains, as determined by the increased size of the cytochrome c peak, usually segregated 2:2 both for this character and for improved dough-raising capability as well.

Genes, Fungal

Genetic manipulation of non-conventional yeasts by conventional and non-conventional methods.

In recent years, yeasts other than those belonging to the species Saccharomyces cerevisiae and Schizosaccharomyces pombe have become increasingly important in industrial processes. Species such as Pichia stipitis, Hansenula polymorpha, Zygosaccharomyces rouxii, Saccharomyces exiguus, Torulaspora delbrueckii, Yarrowia lipolytica and others whose perfect stage is known, can be manipulated genetically by classical methods, but those belonging to the genera Candida (C. utilis, C. tropicalis, C. bombicola, C. zeylanoides, C. boidinii, etc.), Brettanomyces, Cryptococcus, Rhodotorula, and others of the different form genera, cannot be treated in this way. Some, such as Schwanniomyces and Debaryomyces spp., which have a perfect stage, are still difficult to manipulate by conventional means. Genetic manipulation of these yeasts can be approached from two points of view; the first involving improvement of strains by cross-breeding within one species, and the second, the introduction of desirable genes from unrelated species and even from plants or animals. Two techniques are available for construction of industrially-useful strains from these yeasts: protoplast fusion and transformation with chimaeric plasmids containing the gene(s) it is desired to introduce into the recipient strain. The methods for the latter procedure are well known but can be laborious and time-consuming, especially if it is desired to introduce genes from plant or animal sources for production of enzymes, hormones, vaccines and similar products. Protoplast fusion is a simple technique which can be utilized in most laboratories and used for construction of improved yeast strains for brewing, baking, ethanol production and wine-making, either by the fusion of desirable strains of the same species which do not sporulate, or by introduction of genes from non-Saccharomyces species. Methods for fusion of species from different genera and isolation of the desired hybrids have been improved considerably in recent years. We have developed a method for isolation of strains carrying the desired genes by fusing a non-Saccharomyces species with an auxotrophic strain of Saccharomyces cerevisiae and selecting hybrids having the desired characteristics on appropriate media, after which the genes are transferred to the industrial strain by rare-mating, repeated protoplast fusion, or classical mating as required. The advantages and limitations of the method are under investigation.

Genetic Engineering

The use of mitochondrial mutants in the isolation of hybrids involving industrial yeast strains.

Methods for the isolation of hybrids in which one or both of the parental strains are industrial yeasts, using mitochondrial mutations as markers for the selection and isolation of the hybrids, are described. The systems used included crosses of industrial strains with auxotrophic laboratory strains which also carried a mitochondrial antibiotic resistance mutation, crosses using an auxotrophic laboratory strain and a petite mutant of an industrial strain carrying a rescuable antibiotic resistance mutation, and crosses using a petite mutant of an industrial strain, carrying a rescuable mitochondrial mutation for antibiotic resistance and a respiratory-competent industrial strain which carried some other marker.

DNA, Mitochondrial

Apparent bisexual behavior of yeast strains obtained from hybridization of industrial yeasts of the genus Saccharomyces with auxotrophic diploids.

During a genetic study of some hybrids of brewer's and distiller's yeast strains with impaired sporulation characteristics and genetically marked auxotrophic aa and alpha alpha diploids, strains which showed positive mating reactions with both a and alpha haploid tester strains were observed. These strains proved to be homothallic and sporulated freely. The original hybrids, which appeared to be tetraploid, usually yielded sporulating single-spore clones on dissection of asci formed from them, with few or no mating strains among them. Dissection of asci from these clones yielded some single-spore clones which showed mating reactions with one or the other or both haploid tester strains, and further selection produced strains which on sporulation and dissection yielded single-spore clones which were apparently bisexual and sporulated freely. These strains proved to be homothallic, yielding single-spore clones which were all of the a mating type, and in which the mating reaction and, possibly, the action of the genes for homothallism were impaired, so that sporulating, non-mating diploids and haploids of both mating types were present in cultures originally obtained as single-spore clones.

Hybridization, Genetic