Showing papers on "Yeast published in 1983"

Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast.

Abstract: Publisher Summary This chapter describes plasmids and methods for constructing fusions of any cloned gene to lacZ for study in yeast. If the gene to be fused to lacZ contains no appropriate Sau3A sites, alternative strategies to the above must be employed. If the DNA sequence of the gene to be fused to lacZ is known, inframe fusions can be made simply by choosing the appropriate gene fragment. The vector sequences that precede the yeast DNA is a yeast gene–lacZ fusion may affect gene regulation. Plasmids may be used to probe the signals that govern the initiation of transcription and translation in S. cerevisiae. These methods provide powerful tools in the analysis of the expression of yeast genes. Further, the plasmids should facilitate the expression in yeast of any cloned gene to produce the native, unfused product.

By-product inhibition effects on ethanolic fermentation by Saccharomyces cerevisiae.

Abstract: Inhibition by secondary fermentation products may limit the ultimate productivity of new glucose to ethanol fermentation processes. New processes are under development whereby ethanol is selectively removed from the fermenting broth to eliminate ethanol inhibition effects. These processes can concentrate minor secondary products to the point where they become toxic to the yeast. Vacuum fermentation selectively concentrates nonvolatile products in the fermentation broth. Membrane fermentation systems may concentrate large molecules which are sterically blocked from membrane transport. Extractive fermentation systems, employing nonpolar solvents, may concentrate small organic acids. By-product production rates and inhibition levels in continuous fermentation with Saccharomyces cerevisiae have been determined for acetaldehyde, glycerol, formic, lactic, and acetic acids, 1-propanol, 2-methyl-1-butanol, and 2,3-butanediol to assess the potential effects of these by-products on new fermentation processes. Mechanisms are proposed for the various inhibition effects observed.

Expression of hepatitis B surface antigen gene in yeast

Abstract: The DNA sequence coding for hepatitis B virus surface antigen (HBsAg) was placed under control of the repressible acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both yeast and Escherichia coli. Yeast transformed by this plasmid synthesized up to 5 X 10(5) molecules per cell of immunologically active HBsAg polypeptide in phosphate-free medium. The HBsAg polypeptides produced in the yeast cells were assembled into 20- to 22-nm spherical or oval particles and were immunogenic.

The role of redox balances in the anaerobic fermentation of xylose by yeasts

Abstract: The kinetics of glucose and xylose utilization by batch cultures of Candida utilis were studied under aerobic and anaerobic conditions during growth in complex media. Rapid ethanol formation occurred during growth on glucose when aerobic cultures were shifted to anaerobic conditions. However, with xylose as a substrate, transfer to anaerobiosis resulted in an immediate cessation of metabolic activity, as evidenced by the absence of both ethanol formation and xylose utilization. The inability of the yeast to ferment xylose anaerobically was not due to the absence of key enzymes of the fermentation pathway, since the addition of glucose to such cultures resulted in the immediate conversion of glucose to ethanol. Furthermore, when the enzyme xylose isomerase was added to an anaerobic xylose culture, immediate conversion of xylose to ethanol was observed. This indicates that the inability of the yeast to form ethanol from xylose under anaerobic conditions is caused by metabolic events associated with the conversion of xylose to xylulose. A hypothesis is put forward which explains that ethanol production from xylose by yeast under anaerobic conditions is negligible. It is suggested that the failure to ferment xylose anaerobically is due to a discrepancy between production and consumption of NADH in the overall conversion of xylose to ethanol. When a hydrogen acceptor (i.e. acetoin) was added to anaerobic cultures of C. utilis, xylose utilization resumed, and ethanol and acetate were produced with the concomitant stoicheiometric reduction of acetoin to 2,3-butanediol.

Eviction and transplacement of mutant genes in yeast.

Abstract: Techniques for high frequency yeast transformation have been described. A double-strand break introduced by restriction enzyme cleavage can be used to direct a plasmid to integrate into a particular chromosomal locus. Plasmids containing a double-strand gap can be used in a straightforward method for the isolation and mapping of chromosomal alleles. These techniques extend the genetic applications of yeast transformation.

Positive regulation in the general amino acid control of Saccharomyces cerevisiae.

Abstract: Starvation of yeast for a single amino acid leads to derepression of enzymes in many different amino acid biosynthetic pathways. This general control is regulated by several transacting genes. Mutations in the TRA3 gene result in constitutive derepression, whereas mutations in AAS genes lead to the inability to derepress. We have isolated aas mutations as suppressors of the tra3-1 mutation. Some of these suppressors are alleles of AAS2 and others define a heretofore unidentified gene, AAS3. We have studied the regulatory behavior of strains containing both aas and tra3 mutations and strains containing the cloned AAS genes in high copy number. Either aas1- or aas2- in combination with tra3- has the Tra- phenotype, whereas aas3- in combination with tra3- has the Aas- phenotype. These interactions suggest that the AAS1 and AAS2 products act indirectly to bring about derepression by disabling the repressive effect of TRA3, whereas the AAS3 product functions more directly and is required even in the absence of the TRA3 function. When present in high copy number, the AAS3 gene complements mutations in AAS1 and AAS2, whereas AAS1 and AAS2 only complement their cognate mutations. Taken together these data suggest that AAS1 and AAS2 are negative regulators of TRA3, which in turn is a negative regulator of AAS3. AAS3 is a positive regulator, which is required for the general control response. This model of negative and positive interactions is formally identical to those proposed for the regulation of the galactose and phosphatase systems in yeast.

Fermentation of D-xylose to ethanol by a strain ofCandida shehatae

Abstract: A strain ofCandida shehatae fermented 9% D-xylose directly to ethanol within 40 hours with a yield coefficient of 0.29. The specific rate of ethanol production attained a maximum value of 0.28 g ethanol (g cells h)−1. The aeration rate greatly influenced the fermentation parameters. The performance of this yeast compared favourably with the published data for other xylose-fermenting microorganisms.

Secretion of human interferons by yeast.

Abstract: Plasmids were constructed to direct synthesis of the human interferons IFN-alpha 1, IFN-alpha 2, and IFN-gamma in the yeast Saccharomyces cerevisiae. Expression of IFN genes containing coding sequences for secretion signals resulted in the secretion of IFN activity. A large proportion of the IFN-alpha 1 and IFN-alpha 2 isolated from the yeast cell growth media had the same amino termini as the natural mature interferons, suggesting a removal of the signal sequences identical to that of human cells. These results show that a lower eukaryote, such as yeast, can utilize and process a human signal sequence.

The new yeast genetics

Abstract: Gene cloning and yeast DNA transformation techniques have greatly enhanced the power of classical yeast genetics. It is now possible to isolate any classically defined gene, to alter the yeast genome at will by replacing normal chromosomal sequences with mutated derivatives produced in vitro, and to create DNA molecules that behave as autonomous replicons or minichromosomes. These unique features of the new yeast genetics have been used to study many problems in eukaryotic molecular biology.

Cell Wall Receptor for Yeast Killer Toxin: Involvement of (1 → 6)-β-d-Glucan

Abstract: The linear (1 → 6)-β-d-glucans pustulan and luteose were effective competitive inhibitors of killer toxin action. Affinity chromatography of killer toxin on a pustulan-Sepharose column showed that toxin bound directly to a (1 → 6)-β-linked polysaccharide. Other polysaccharides found in yeast cell walls, including (1 → 3)-β-d-glucan, mannan, chitin, and glycogen, were not effective as inhibitors of toxin. Fractionation of yeast cell walls was attempted to identify the toxin receptor in sensitive Saccharomyces cerevisiae . The receptor activity was retained among the insoluble glucans in alkali-washed cells; yeast mannan and alkali-soluble glucan had little receptor activity. A minor fraction of receptor activity was removed from alkali-washed cells by hot acetic acid extraction, a procedure which solubilized some (1 → 6)-β-d-glucan and glycogen. The major fraction (>70%) of receptor activity remained with the acid-insoluble (1 → 6)-β-and (1 → 3)-β-glucans. Zymolyase, an endo-(1 → 3)-β-d-glucanase, solubilized a substantial fraction of the receptor activity in the acid-insoluble glucans. The receptor activity in yeast cell walls was periodate and (1 → 6)-β-d-glucanase sensitive, but was resistant to (1 → 3)-β-d-glucanase and α-amylase. The acid-soluble glucan fractions of a sensitive strain and a krel-l receptor-defective toxin-resistant mutant were examined. The krel-l strain had a reduced amount (ca. 50%) of (1 → 6)-β-d-glucan compared with the sensitive parent strain. A sensitive revertant of the krel-l strain regained the parental level of glucan. These results implicate (1 → 6)-β-d-glucan as a component of the yeast cell wall receptor for killer toxin. Images

Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast.

Abstract: We have chemically synthesized and expressed in yeast a gene coding for human epidermal growth factor (urogastrone), a 53-amino-acid polypeptide that has been shown to promote epithelial cell proliferation and to inhibit gastric acid secretion. The synthetic gene, consisting of 170 base pairs, was designed with yeast-preferred codons and assembled by enzymatic ligation of synthetic fragments produced by phosphoramidite chemistry. The DNA synthesis protocol used allows for facile synthesis of oligonucleotides larger than 50 bases. Yeast cells were transformed with plasmids containing the synthetic gene under control of a yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter and were shown to synthesize a biologically active human epidermal growth factor.

Cell Interactions and Regulation of Cell Type in the Yeast Saccharomyces Cerevisiae

Abstract: Examination of the control of cell type in yeast at the molecular level and understanding of the biochemical basis of the cell-cell interactions involved in the mating process are clearly entering an extremely productive and exciting period. The tools and opportunities are now available to answer fundamental questions with regard to the mechanism of differential gene expression in eukaryotic cells by using cloned a-specific, alpha-specific, and haploid-specific genes as the probes. Basic questions concerning eukaryotic chromosome structure and organization can be addressed by elucidating the properties of the SIR/MAR regulators and their mode of action. Furthermore, the availability both of cloned MAT, HML, and HMR regions and of the HO gene will provide the material for unravelling the enzymology of the DNA transposition that occurs during mating type interconversion. The isolation of the structural genes for the pheromones and mutations that block pheromone production will provide useful information on how extracellular hormones are synthesized, processed, and secreted by eukaryotic cells. Moreover, the apparent mode of action of the phermonones through cyclic AMP as an intracellular "second messenger," and the genetic and biochemical tractability of yeast cells, may allow tracing of the entire pathway of hormonal regulation of a eukaryotic cell division cycle. These and other studies of the developmental biology of yeast cells will provide more important insights into fundamental aspects of the genetic control of developmental processes in eukaryotic cells.

The protein products of the myc and myb oncogenes and adenovirus E1a are structurally related.

Abstract: Structural and functional homologies have been found among proteins encoded by several retroviral oncogenes, demonstrating the existence of families of these genes1. Because the retroviral oncogenes have cellular homologues1,2, the existence of similar families among these ‘cellular oncogenes’ is also implied (for a review, see ref. 2). Cellular genes belonging to these families have been found in such evolutionarily distant species as humans3,4, fruit flies5,6, nematodes5 and brewer's yeast (E. Scolnick and S. Reed, personal communications), consistent with the hypothesis that these genes have evolved from a small number of ancestral sequences2. We extend these observations by showing here that the proteins encoded by the oncogenes myc, myb and adenovirus E1a are structurally related. Our findings suggest that oncogenes of RNA and DNA tumour viruses may in at least some instances share evolutionary origins and function according to common principles.

Expression of hepatitis B virus surface antigen in yeast

Abstract: The structural gene of Hepatitis B virus surface protein (HBsAg) was introduced into a plasmid capable of autonomous replication and selection in both the yeast Saccharomyces cerevisiae and E. coli. In this plasmid transcription of the HBsAg is initiated by the 5'-flanking sequence of the yeast 3-phosphoglycerate kinase (PGK) gene and terminated by the 3'-flanking region of the yeast TRP1 gene. Yeast cells containing this plasmid produce a new major species of mRNA of 1200 nucleotides in length coding for HBsAg. Viral surface antigen is made in nonglycosylated form at a level of about 1-2 percent of total yeast protein. A small fraction of this polypeptide (2-5 percent) is found in aggregated form upon yeast cell disruption by glass beads. This material is similar in size, density, and shape to the 22nm particle, isolated from the plasma of human hepatitis carriers, and induced comparable levels of HBsAg antibodies in mice when compared with the natural particle.

Isolation of the yeast structural gene for the membrane-associated enzyme phosphatidylserine synthase

Abstract: The structural gene (CHO1) for phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was isolated by genetic complementation in Saccharomyces cerevisiae from a bank of yeast genomic DNA on a chimeric plasmid. The cloned DNA (4.0 kilobases long) was shown to represent a unique sequence in the yeast genome. The DNA sequence on an integrative plasmid was shown to recombine into the CHO1 locus, confirming its genetic identity. The cho1 yeast strain transformed with this gene on an autonomously replicating plasmid had significantly increased activity of the regulated membrane-associated enzyme phosphatidylserine synthase. Partial purification of phosphatidylserine synthase from microsomes of this transformed strain confirmed that the membrane-bound enzyme was overproduced 6- to 7-fold as compared with the wild-type strain. The strain also synthesized the product phospholipid, phosphatidylserine, at an increased rate. The transformed strain had altered proportions of a variety of other phospholipids, suggesting that their synthesis is affected by the rate of synthesis of phosphatidylserine in yeast.

Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.

Abstract: Bud scar analysis integrated with mathematical analysis of DNA and protein distributions obtained by flow microfluorometry have been used to analyze the cell cycle of the budding yeast Saccharomyces cerevisiae. In populations of this yeast growing exponentially in batch at 30 degrees C on different carbon and nitrogen sources with duplication times between 75 and 314 min, the budded period is always shorter (approximately 5 to 10 min) than the sum of the S + G2 + M + G1* phases (determined by the Fried analysis of DNA distributions), and parent cells always show a prereplicative unbudded period. The analysis of protein distributions obtained by flow microfluorometry indicates that the protein level per cell required for bud emergence increases at each new generation of parent cells, as observed previously for cell volume. A wide heterogeneity of cell populations derives from this pattern of budding, since older (and less frequent) parent cells have shorter generation times and produce larger (and with shorter cycle times) daughter cells. A possible molecular mechanism for the observed increase with genealogical age of the critical protein level required for bud emergence is discussed.

An analysis of the metabolism and cell wall composition of Candida albicans during germ-tube formation.

Abstract: The uptake of nutrients (glucose, glutamine, and N-acetylglucosamine), the intracellular concentrations of metabolites (glucose-6-phosphate, cyclic AMP, amino acids, trehalose, and glycogen) and cell wall composition were studied in Candida albicans. These analyses were carried out with exponential-phase, stationary-phase, and starved yeast cells, and during germ-tube formation. Germ tubes formed during a 3-h incubation of starved yeast cells (0.8 × 108 cells/mL) at 37 °C during which time the nutrients glucose plus glutamine or N-acetylglucosamine (2.5 mM of each) were completely utilized. Control incubations with these nutrients at 28 °C did not form germ tubes. Uptake of N-acetylglucosamine and glutamine was inhibited by cycloheximide which suggests that de novo protein synthesis was required for the induction of these uptake systems. The glucose-6-phosphate content varied from 0.4 nmol/mg dry weight for starved cells to 2–3 nmol/mg dry weight for growing yeast cells and germ tube forming cells. Trehal...

Conversion of pentoses by yeasts

Abstract: The utilization and conversion of D-xylose, D-xylulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: (1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. (2)The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol, D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. (3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. (4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. (5) Of the four substrates examined, D-xylulose was the perferred substrate, followed by D-xylose, L-arabinose, and xylitol. (6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.

The complete nucleotide sequence of the rat 18S ribosomal RNA gene and comparison with the respective yeast and frog genes

Abstract: The complete nucleotide sequence of the rat 18S ribosomal RNA gene has been determined. A comparison of the rat 18S ribosomal RNA gene sequence with the known sequences of yeast and frog revealed three conserved (stable) regions, two unstable regions, and three large inserts. (A,T) leads to (G,C) changes were more frequent than (G,C) leads to (A,T) changes for three comparisons (yeast leads to frog, frog leads to rat, and yeast leads to rat). GC pairs were inserted preferentially over AT pairs for the same three comparisons. These two factors contribute to the progressively higher GC content of 18S ribosomal RNA of yeast, frog, and rat.

Polycaprolactone degradation by mixed and pure cultures of bacteria and a yeast

Abstract: The degradability of three high molecular weight polycaprolactones (Mw = 35,000, 18,600, and 7,130) and one low molecular weight polycaprolactone diol(Mw = 2060) by mixed and pure cultures of microorganisms was assayed. A yeast, Cryptococcus laurentii, a gram-negative rod, Acinetobacter calcoaceticus var. lwoffi, and a gram-positive coryneform rod were used in the pure culture assays. The analysis of degradation by gel permation chromatography (GPC) allowed for quantitation independent of the growth of the organisms or the addition of supplementary growth factors. GPC analysis showed that the degradation effected by pure cultures was often enhanced when alternate carbon sources were present. This was not the case for mixed cultures. Mixed cultures. Mixed cultures completely metabolized polymer breakdown products while in some cases pure cultures did not.

Expression, processing and secretion of heterologous protein by yeast

Abstract: A yeast host organism is genetically altered via recombinant DNA technology so as to be directed to express, process and secrete a polypeptide ordinarily heterologous to the host which is recoverable in discrete form, free from unwanted polypeptide presequences or other artifact of expression.

Vectors for high-level, inducible expression of cloned genes in yeast.

Abstract: Publisher Summary This chapter describes the design and application of vectors that permit high-level, inducible synthesis of the product of a cloned gene in yeast. These vectors have been developed in recognition of the increasing need for obtaining reasonable amounts of the product of a cloned gene in research projects. For instance, given the variety of cloning strategies currently available, it is often easier to clone DNA encoding a particular gene than to purify the product of that gene. However, appreciation of the biological function of such a gene usually requires the subsequent identification and characterization of its product. There are a number of host-vector systems currently available for the high-level expression of cloned genes. However, there are a number of reasons why yeast systems should be included in the arsenal of genetic engineers. First, the codon bias in yeast is significantly different from that in Escherichia coli, as is, undoubtedly, the spectrum of endogenous proteolytic enzymes. In addition, yeast is capable of promoting glycosylation of newly synthesized proteins.

Affinity partitioning of phosphofructokinase from baker's yeast using polymer-bound Cibacron blue F3G-A.

Abstract: 1 Phosphofructokinase from baker's yeast is partitioned between the phases of an aqueous two-phase system, containing dextran (Mr= 500000) and poly(ethyleneglycol) (Mr= 6000), in favour of the dextran-rich phase. By covalent binding of the dye Cibacron blue F3G-A to poly(ethyleneglycol) the enzyme can be extracted to the phase rich in this polymer, i.e. affinity partitioning. 2 The affinity partitioning effect, measured as the logarithmic increase of the partition coefficient by introducing polymer-bound Cibacron blue, depends on several factors. The influence of dye-polymer concentration, polymer concentration, polymer molecular weight, kind of salt and salt concentration, pH and temperature has been studied. 3 The effect of ATP, ADP, AMP, ITP, fructose 1,6-bis-phosphate and fructose 6-phosphate show large differences in the binding strength of these substances to the Cibacron blue binding sites. AMP cannot compete with Cibacron blue while ATP is strongly competing. 4 The use of affinity partitioning for enzyme isolation and determination of ligand binding is discussed, as well as possible mechanisms concerning this type of liquid/liquid extraction.