摘要:

AbstractIn order to determine the biological functions of moderately abundant, high mobility group (HMG)‐like nuclear proteins, a genetic approach has been taken. The gene for one such protein, NHP2, has been cloned and characterized fromSaccharomyces cerevisiae. NHP2 has been called ‘HMG‐like’ because of the physical/chemical properties it shares with the HMG proteins from higher eukaryotic cells. However, nucleotide sequence analysis revealed thatNHP2could encode a 17·1 kilodalton basic protein which was not significantly homologous to any previously sequenced HMG proteins. Thus NHP2 defines a new member of the HMG class of proteins. A search of protein databases showed that the amino acid sequence of NHP2 shared significant identities with two ribosomal proteins; the acidic ribosomal protein S6 fromHalobacterium marismoriumand protein L7a from mammals. The biological relevance of these homologies is nuclear since previous biochemical results indicated that NHP2 was not a ribosomal protein. S1 nuclease analysis indicated that the gene contained no introns but had multiple transcription initiation sites 20 to 40 bases before the ATG codon. Finally, NHP2 has been shown to have a critical role in the cell; when a diploid yeast strain deleted of one copy of theNHP2gene was sporulated and dissected, only half of the spores grew into normal colonies. The rest of the spors germinated, but only formed microcolonies containing 12 to 40 cells. None of the spores which grew into normal‐sized colonies contained the mutantNHP2gene, thus demonstrating that the NHP2 protein has an essential physiologica

ISSN:0749-503X    

DOI:10.1002/yea.320070202

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe physiological effects on brewing yeast, growing in semi‐defined wort medium, of a sudden transition from aerobiosis to anaerobiosis were studied. Two yeast strains were examined, used for ale and lager fermentations respectively. The reverse transition (from anaerobiosis to aerobiosis) was also examined. Transitions were applied by changing the sparging gas during growth or in stationary phase, and the effects on the specific activities of certain key enzymes and on the viability of the cultures were examined.Neither type of transition led to significant changes in growth rate, the rate of ethanol production or the specific activities of alcohol dehydrogenase and pyruvate decarboxylase. The most significant change was in the specific activity of CuZn‐superoxide dismutase, which showed a rapid increase in activity on transition from anaerobiosis to aerobiosis, and a decrease in activity on the reverse transition. Catalase activity in the ale yeast generally followed that of CuZn‐superoxide dismutase, whereas in the lager yeast it remained unchanged by the transitions. The transition from anaerobiosis to aerobiosis caused increases in citrate synthase and Mn‐superoxide dismutase, though only after a significant lag period. Aerobic to anaerobic transitions caused a decrease in Mn‐superoxide dismutase activity, while citrate synthase remained unchanged.Anaerobically grown cells showed a rapid loss in viability on exposure to oxygen (5–7% in the first hour), while aerobically grown cells were unaffected. When anaerobically grown cells were exposed to 0·25 mM‐potassium superoxide, there was an 8% loss of viability within 10 min, whereas aerobic cells were not affected.It is concluded that the toxic effect of oxygen is due to superoxide (or a species derived from it) and that the CuZn‐superoxide dismutase (but not the Mn‐isoenzyme) plays a role in protecting the cells. Thede novosynthesis of the CuZn‐enzyme is not always rapid enough to co

ISSN:0749-503X    

DOI:10.1002/yea.320070203

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractSpontaneous mutation of some genes was studied in haploid adenine and leucine auxotrophic yeastSaccharomyces.It was shown that a decrease in the amount of adenine (from 500 to 0 mgl−1) or leucine (from 300 to 0·3 mgl−1) in the medium, simultaneously with the transition from repression to derepression of the biosynthesis of these metabolites, resulted in a 15‐ to 150‐fold increase in the reversion rate of genesade2andleu2, respectively, for different strains. At the same time the mutation rate of suppressor genes varied relatively little (up to five‐fold), and that of genelys1did not change at all. It was also demonstrated (on geneleu2) that the mutation rate is determined by the composition of the nutrient medium at the time of the S‐phase of the cell cycle and it does not depend on the cultivation conditions during the presynthetic period.We discuss the hypothesis that derepressed genes mutate with a significantly higher rate than genes in the rep

ISSN:0749-503X    

DOI:10.1002/yea.320070204

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThree modes of production of the extracellular glucoamylase (GA) inSaccharomyces cerevisiaehave been identified: repressed, basal and induced. The repressed mode is found with cells grown in rich media containing non‐limiting concentrations of monosaccharides or disaccharides, including GA‐hydrolysable maltose, as a sole carbon source. Both the basal and the induced modes (spanned by some seven‐fold difference in the rate of GA production) can be displayed by either glucose‐limited or glycerol‐ plus ethanol‐consuming cultures: the induced mode is switched over to the basal one due to a feed‐back inhibition by extracellularly accumulated GA. It is proposed that the feed‐back control involved in GA production can be attenuated by starch which can thus ‘induce’ higher rates of GA production compare

ISSN:0749-503X    

DOI:10.1002/yea.320070205

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractShuttle plasmids, pE1.CUP1B and pE1.CUP1E of 10·6 kb, have been constructed between the metallothioneinencodingCUP1gene ofSaccharomyces cerevisiaeand a vector capable of replication inKluyveromyces lactis. Introduction of these plasmids intoK. lactisconfers resistance to copper as well as to cadmium and silver. Resistance to these latter metal ions, in the absence of induction by copper, suggested that theCUP1gene is constitutively expressed in the foreign background. Introduction of thelacZreporter gene fromEscherichia coliinto a cloning site downstream from theCUP1promoter showed that expression of this gene is constitutive inK. lactisbut inS. cerevisiaeinduction by copper is necessary. Sequences upstream from theCUP1promoter are involved in the constitutive expression since deletion of 91 nucleotides from this region abolishes metal resistance. It is suggested that aK. lactisprotein, normally involved in activating transcription of the residentCUP1gene in the presence of copper, can promote transcription in the absence of metal ion by binding to the upstream activation sequence of the introducedCUP1gene

ISSN:0749-503X    

DOI:10.1002/yea.320070206

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractChemostat cultures of a catalase‐negative mutant ofHansenula polymorphaCBS 4732 were able to decompose hydrogen peroxide at a high rate. This was apparent from experiments in which yeast was grown under carbon limitation in chemostat culture on mixtures of glucose and H2O2. The enzyme responsible for H2O2degradation is probably the mitochondrial enzyme cytochromecperoxidase (CCP), which was present at very high activities. This enzyme was partially purified and shown to be specific for reduced cytochromecas an electron donor; no reaction was observed with NAD(P)H. Thus, reducing equivalents for H2O2degradation by CCP must be provided by the respiratory chain.That H2O2can act as an electron acceptor for reducing equivalents could be confirmed with experiments in which cells were incubated with ethanol and H2O2in the absence of oxygen. This resulted in oxidation of ethanol to equimolar amounts of acetate.Energetic aspects of mitochondrial H2O2decomposition via CCP and the physiological function of CCP in yeasts are discusse

ISSN:0749-503X    

DOI:10.1002/yea.320070207

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractUsing cells grown in a chemostat at steady state, the levels of various components of the microsomal electron transport chain ofSaccharomyces cerevisiaewere examined. Cytochrome P450 haemoprotein levels measured in cells grown in medium with a dissolved oxygen concentration of 15% were induced between 10‐ and 20‐fold over levels in cells grown in medium containing 70% dissolved oxygen concentation. An increase in the dilution rate of a culture growing in medium containing 15% dissolved oxygen resulted in an increase in the residual glucose concentration of the medium. This was paralleled by an increase in the microsomal levels of cytochrome P450. The rise could not be attributed either to increases in the concentration of ethanol in the chemostat or to an increase in the proportion of energy generated using fermentative pathways. However, this effect was not observed in cells grown in an oxygen concentration of 70%. Cytochromeb5haemoprotein levels were also induced approximately three‐fold by reducing the dissolved oxygen concentration from 70% to 15%. Changes in the medium glucose concentration from 0·03% to 1·6% (w/v) had no effect on the levels of this enzyme. Conversely, levels of cytochrome P450 NADPH reductase appeared lower in cells grown in 15% as opposed to 70% dissolved oxygen concentration. Northern slot blot analysis of total RNA extracted from chemostat‐grown cells, probed with a C‐14 sterol demethylase cytochrome P450 gene (cytochrome P450 LIA1), revealed a pattern of message induction which matched that of the cytochrome P450 haemoprotein, indicating that control of the levels of this enzyme was at least partially transcriptional. Qualitative examination of combined cytochrome P450 apoprotein and haemoprotein levels using Western blot analysis revealed a similar pattern of induction to that observed with Northe

ISSN:0749-503X    

DOI:10.1002/yea.320070208

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractA set of 32 different codons were introduced in alacZexperssion vector (pPTK400) immediately 3′ from the AUG initiation codon. Expression of thelacZgene was determined inSaccharomyces cerevisiaeby measuring the amount of β‐galactosidase fusion protein using immuno‐gel electrophoresis. A 5·3‐fold difference in expression was found among the various constructs. It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression. No correlation could be found between the local secondary structure and expression. When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference. This indicates that in yeast, in contrast toEscherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation. Important determinatnts of the optimal context for an initiation codon in yeast therfore must be located mainly 5′ from

ISSN:0749-503X    

DOI:10.1002/yea.320070209

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractTo test whether DNA probes derived from ribosomal DNA spacer sequences are suitable for rapid and species‐specific yeast identification, a pilot study was undertaken. A 7·7 kb entire ribosomal DNA unit of the type strain ofMetschnikowia reukaufiiwas isolated, cloned and mapped. A 0·65 kbBamHI‐HpaI fragment containing nontranscribed spacer sequences was amplified and selected for testing as a32P hybridization probe with total DNA from the type strains ofM. reukaufii,M. pulcherrima,M. lunata,M. bicuspidata,M. australis,M. zobellii,M. krissii, five other strains identified asM. reukaufiiand strains ofSchizosaccharomyces pombe,Hansenula canadensis,Saccharomyces cerevisiaeandYarrowia lipolytica. The probe hybridized exclsively with DNA from the type strain and four other strains ofM. reukaufii. DNA from one strain labelledM. reukaufiidid not hybridize with the probe. Subsequent % G+C comparison and DNA–DNA reassociation with the type strain revealed that the non‐hybridizing strain does not belong to the speciesM.

ISSN:0749-503X    

DOI:10.1002/yea.320070210

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractA simple quantitativein vivoassay has been developed for measuring the efficiency of translation of one or other of the three termination codons, UAA, UAG and UGA inSaccharomyces cerevisiae. The assay employs a 3‐phosphoglycerate kinase‐β‐galactosidase gene fusion, carried on a multicopy plasmid, in which the otherwise retained reading frame is distupted by one or other of the three termination codons. Termination readthrough is thus quantitated by measuring β‐galactosidase in transformed strains. Using these plasmids to quantitate the endogenous levels of termination readthrough we show that readthrough of all three codons can be detected in a non‐suppressor (sup+) strain ofS. cerevisiae. The efficiency of this endogenous readthrough is much higher in a [psi+] strain than in a [psi−] strain with the UGA codon being the leakiest in the nucleotide context used. The utility of the assay plasmids for studying genetic modifiers of nonsense suppressors is also shown by their use to demonstrate that the cytoplasmic genetic determinant [pse+] broadens the decoding properties of a serine‐inserting UAA suppressor tRNA (SUQ5) to allow it to translate the other two termination codons in the order of efficie

ISSN:0749-503X    

DOI:10.1002/yea.320070211

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY