摘要:

AbstractAn expression system has been developed for the methylotrophic yeastHansenula polymorphaand used to co‐express both the L (preS1‐S2‐S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol‐inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of thisH. polymorphaexpression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5–8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle‐specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. TheseH. polymorpha‐derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible

ISSN:0749-503X    

DOI:10.1002/yea.320070502

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe α‐glucosidase gene ofCandida tsukubaensisis contained within a 3·47 kbBamH1‐Mlu1 fragment which, when introduced intoSaccharomyces cerevisiaeAH22 on a yeast–Escherichia colishuttle vector, allows the transformants to utilize maltose as sole carbon source. Thus, the cloned gene confers a dominant selectable phenotype on transformed strains ofS. cerevisiaewhich are otherwise unable to grow in nutrient media containing maltose, dextrin or other α‐1,4‐linked α‐D‐glucopyranosides, specifically hydrolysed by the α‐glucosidase. The cloned enzyme expressed in yeast is secreted into the extracellular medium in a glycosylated form which accounts for up to 60% of the secreted protein and has a molecular size of 70–80 kilodalton (kDa). Deglycosylation of the α‐glucosidase showed that the enzyme is composed of two distinct polypeptides with subunit molecular weights of 63–65 kDa (peptide 1) and 50–52 kDa (peptide 2). An increase in the level of expression of the α‐glucosidase by yeast transformants in selective minimal medium was obtained by using a vector with increased copy number containing theleu2‐dgene as selectable marker. The α‐glucosidase gene promoter functions more effectively than theGal1–10promoter in directing α‐glucosidase expression inS. cerevisiae. It also directs the expression of high levels of β‐galactosidase activity in yeast when fused to a promoterlessE. coli lacZgene. Expression of the α‐glucosidase gene under the control of its own promoter is constitutive, orientation de

ISSN:0749-503X    

DOI:10.1002/yea.320070503

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe phenotype of VY1160 fragileSaccharomyces cerevisiaemutant is characterized by cell lysis upon transfer to hypotonic solutions and increased permeability of cells growing in osmotically stabilized media. Two mutations,srb1andts1, have been identified in VY1160 cells and previous studies have shown that the increased permeability is due to thets1mutation which causes a shortening of mannan side‐chains. Here we report that thesrb1mutation, which is the genetic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic single mutation strains, genetic studies coupled with quantitative measurements of glucan content per cell, and methylation analysis of glucans provide evidence thatsrb1mutation leads to (i) formation of mechanically unstable cell wall network made of insoluble glucan fibrils which are shorter and contain β(1–6) inter‐residue linkages and (ii) insufficient filling of the space between the fibrils due to a shortage of the alkali‐soluble glucan. Although growing exponentially in osmotically stabilized media, thesrb1cells cannot resist an osmotic shock and, hence, burst imm

ISSN:0749-503X    

DOI:10.1002/yea.320070504

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe methylotrophic yeastHansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the α‐galactosidase from the plantCyamopsis tetragonoloba(guar). The yeast/Escherichia colishuttle expression vector used is based on the origin of replication of the endogenous 2 μm plasmid ofSaccharomyces cerevisiaeand theLEU2gene ofS. cerevisiaefor selection inH. polymorpha. In the expression vector, the α‐galactosidase is controlled by the methanol‐regulated promoter from the methanol oxidase gene,MOX, ofH. polymorpha. The signal sequence ofSUC2(invertase) from the yeastS. cerevisiae, was used to ensure secretion of the α‐galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active α‐galactosidase enzyme was efficiently secreted (>85%) and after methanol induction, the expression level was 42 mg/l. Amino‐terminal sequencing of the purified α‐galactosidase enzyme synthesized byH. polymorphashowed that theS. cerevisiaeinvertase signal sequence was correctly processed byH. polymorpha. The secreted α‐galactosidase was glycosylated and had a sugar content of 9·5%. The specific activity of the α‐galactosidase produced byH. polymorphawas 38 U mg−1compared to 100 U mg−1for guar α‐galactosidase. Deglycosylation of theH. polymorphaα‐galactosidase resto

ISSN:0749-503X    

DOI:10.1002/yea.320070505

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe YDp plasmids (YeastDisruptionplasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3,LEU2,LYS2,TRP1orURA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.

ISSN:0749-503X    

DOI:10.1002/yea.320070506

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe glucose‐fermenting yeast,Candida utiliscannot use the β‐D‐glucoside, cellobiose, anaerobically, although it is able to do so aerobically. β‐Glucoside transport and hydrolysis and pyruvate decarboxylase activities of this yeast were measured aerobically and anaerobically. β‐Glucoside transport was five‐fold faster aerobically than anaerobically, but there was no corresponding difference in β‐glucosidase activity. Pyruvate decarboxylase activity varied greatly, being synthesizedde novoin response to the presence ofD‐glucose and anaerobic conditions and about 50% deactivated on the removal ofD‐glucose or the addition of air. Activation and deactivation were rapidly reversible. Failure to utilize cellobiose anaerobically, in particular, and the Kluyver effect, in general, probably depends on much reduced glycolytic flux, associated under anaerobic conditions, with (i) lower transport rate, (ii) low substrate affinity of the relevant glycosidase and (iii) deactivation of pyruvate decarboxylase. So, in addition to the complex effects of oxygen, anaerobiosis and specific sugars on induction, repression and derepression, there are fine controls on pyruvate decarboxylase activity, leading to fast activation or deact

ISSN:0749-503X    

DOI:10.1002/yea.320070507

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractSaccharomyces cerevisiaewas grown anaerobically in media supplemented with myritoleic 14:1(9c), palmitoleic 16:1(9c), oleic 18:1(9c), linoleic 18:2(9,12c), γ‐linolenic 18:3(9,12,15c) or eicosenoic 20:1(11c) acid. Cells from exponential‐phase cultures contained approximately the same proportions of the major phospholipid classes, namely phosphatidycholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, the greatest differences being detected in cells grown in the presence of 14:1(9c) or 20:1(11c) acids. The extent to which phospholipids from cells were enriched with residues of the exogenously supplied acid varied from 52% in cells grown in the presence of 14:1(9c) acid to 13% in cells grown in media supplemented with 20:1(11c) acid. Analysis of the fatty‐acyl composition of the four major phospholipid classes revealed that the degree of unsaturation varied considerably in three of the classes, while phosphatidylinositol conserved a high degree of saturation. The possible significance of the latter finding in relation to the physiological role of phosphatidylinositol in the plasma membrane is dis

ISSN:0749-503X    

DOI:10.1002/yea.320070508

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractIsolated vacuoles of the yeastSaccharomyces pastorianusaccumulate citrate, α‐ketoglutarate, malate and guanosine. This accumulation is Mg ATP‐dependent and inhibited by protonophores. The ionophores monensin and A23187 (electroneutral Men+/nH+‐exchange) inhibit guanosine accumulation but fail to block citrate uptake. Mg2+ions (2 mM) increase the values of both Δ\documentclass{article}\pagestyle{empty}\begin{document}$ \tilde \mu $\end{document}H+components and stimulate the uptake of all the above compounds. Ca2+ions (1 mM), hyperpolarizing the yeast vacuolar membrane and dissipating the pH gradient, inhibit guanosine uptake and stimulate that of citrate. It is concluded that guanosine is transported into yeast vacuoles by an H+/guanosine antiporter while citrate, malate and α‐ketoglutarate are translocated by a uniporter(s) at the expense of the membrane potential (positi

ISSN:0749-503X    

DOI:10.1002/yea.320070509

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe peroxisomes of the asporogenic yeastCandida tropicaliscontain about 20 major polypeptides (PXPs). We have isolated a number of genes encoding them; 11POXgenes encoded independent PXPs and threePOYgenes were likely to encode three other PXPs. To locate these genes on the chromosomes, chromosomes ofC. tropicaliswere separated by pulsed‐field gel electrophoresis. Eight chromosomal bands were observed over the range of 1·0 Mbp (band I) to 2·8 Mbp (band VIII); the genome size was estimated to be about 20 Mbp. Southern blot analysis showed that ten genes were on band V, three genes were on band IV, and the other gene was on band VI. Three genes gave hybridization signals of nearly equal intensity on two different chromosomal bands:POX6AandPOX8B, on bands V and VII; andPOX8A, on bands IV and VI. Ribosomal RNA genes also hybridized to two bands, VI and VII. Most genes assigned to only one band hybridized to two restriction fragments produced by eitherNotIorSfiIendonuclease. The results suggested thatC. tropicaliswas diploid and that restriction sites were conserved little between homologues. The threePOXgenes that were found on two chromosomal bands hybridized to not more than two restriction fragments, implying that the allelic genes were present on different chromosomal ba

ISSN:0749-503X    

DOI:10.1002/yea.320070510

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractA transformation system for the filamentous yeastTrichosporon cutaneumbased on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully in spheroplast transformation experiments using the clonedAspergillus nidulansornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5–100 transformants per μg of DNA. In these transformants the heterologousargB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non‐selective media. The same mutant could be complemented by aT. cutaneumcosmid gene library and a complementing cosmid was subsequently isolated from this library by a sib‐selection strategy. This cosmid transformed.T. cutaneumspheroplasts with an efficiency of 50–200 colonies per μg of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homolo

ISSN:0749-503X    

DOI:10.1002/yea.320070511

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY