The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.

In yeasts, pyruvate is located at a major junction of assimilatory and dissimilatory reactions as well as at the branch-point between respiratory dissimilation of sugars and alcoholic fermentation. This review deals with the enzymology, physiological function and regulation of three key reactions occurring at the pyruvate branch-point in the yeast Saccharomyces cerevisiae: (i) the direct oxidative decarboxylation of pyruvate to acetyl-CoA, catalysed by the pyruvate dehydrogenase complex, (ii) decarboxylation of pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase, and (iii) the anaplerotic carboxylation of pyruvate to oxaloacetate, catalysed by pyruvate carboxylase. Special attention is devoted to physiological studies on S. cerevisiue strains in which structural genes encoding these key enzymes have been inactivated by gene disruption.

The 14-3-3 proteins constitute a family of conserved proteins present in all eukaryotic organisms so far investigated. These proteins have attracted interest because they are involved in important cellular processes such as signal transduction, cell-cycle control, apoptosis, stress response and malignant transformation and because at least 100 different binding partners for the 14-3-3 proteins have been reported. Although the exact function of 14-3-3 proteins is still unknown, they are known to (1) act as adaptor molecules stimulating protein-protein interactions, (2) regulate the subcellular localisation of proteins and (3) activate or inhibit enzymes. In this review, we discuss the role of the 14-3-3 proteins in three cellular processes: cell cycle control, signal transduction and apoptosis. These processes are regulated by the 14-3-3 proteins at multiple steps. The 14-3-3 proteins have an overall inhibitory effect on cell cycle progression and apoptosis, whereas in signal transduction they may act as stimulatory or inhibitory factors. This article contains supplementary material which may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/Suppmat/23/v23_10.936.

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

The quality of brewing strains is, in large part, determined by their flocculation properties. By classical genetics, several dominant, semidominant and recessive flocculation genes have been recognized. Recent results of experiments to localize the flocculation genes FLO5 and FLO8, combined with the in silicio analysis of the available sequence data of the yeast genome, have revealed that the flocculation genes belong to a family which comprises at least four genes and three pseudogenes. All members of this gene family are located near the end of chromosomes, just like the SUC, MEL and MAL genes, which are also important for good quality baking or brewing strains. Transcription of the flocculation genes is repressed by several regulatory genes. In addition, a number of genes have been found which cause cell aggregation upon disruption or overexpression in an as yet unknown manner. In total, 33 genes have been reported that are involved in flocculation or cell aggregation.

Saccharomyces cerevisiae contains two structural genes, ACS1 and ACS2, each encoding an active acetylcoenzyme A synthetase. Characterization of enzyme activities in cell-free extracts from strains expressing either of the two genes revealed differences in the catalytic properties of the two enzymes. The K m for acetate of Acs1p was about 30-fold lower than that of Acs2p and Acs1p, but not Acs2p, could use propionate as a substrate. Enzyme activity measurements and mRNA analyses showed that ACS1 and ACS2 were both expressed during carbon-limited growth on glucose, ethanol, and acetate in aerobic chemostat cultures. In anaerobic glucose-limited cultures, only the ACS2 gene was expressed. Based on these facts, the products of the ACS1 and ACS2 genes were identified as the previously described "aerobic" and "non-aerobic" forms of acetylcoenzyme A synthetase, respectively. Batch and glucosepulse experiments revealed that transcription of ACS1 is subject to glucose repression. A mutant strain lacking Acs2p was unable to grow on glucose in batch cultures, but grew readily in aerobic glucose-limited chemostat cultures, in which the low residual glucose concentration alleviated glucose repression. Experiments in which ethanol was pulsed to aerobic ethanol-limited chemostat cultures indicated that, in addition to glucose, ethanol also repressed ACS1 transcription, although to a lesser extent. In contrast, transcription of ACS2 was slightly induced by ethanol and glucose. Absence of ACS2 prevented complete glucose repression of ACS1, indicating that ACS2 (in)directly is involved in the transcriptional regulation of ACS1.When Saccharomyces cerevisiae grows on acetate or ethanol, ATP-dependent activation of acetate to acetyl-coenzyme A is catalyzed by acetyl-coenzyme A synthetase (EC 6.2.1.1). In addition to serving as the fuel for the citric acid cycle, acetylcoenzyme A is an essential building block for the synthesis of lipids and some amino acids. During growth on glucose, direct formation of acetyl-coenzyme A from pyruvate is catalyzed by the mitochondrial pyruvate dehydrogenase complex. Alternatively, conversion of pyruvate into acetyl-coenzyme A can be accomplished by the concerted action of the enzymes of the pyruvate dehydrogenase bypass: pyruvate decarboxylase, acetaldehyde dehydrogenase, and acetyl-coenzyme A synthetase (1, 2). Recent work has indicated that the latter pathway is essential for growth, probably for the provision of cytosolic acetylcoenzyme A required for lipid synthesis (3, 4).In S. cerevisiae, a completely respiratory sugar metabolism is only observed at relatively low growth rates in aerobic, sugarlimited cultures (e.g. chemostat cultures). Upon exposure of such cultures to high sugar concentrations, metabolism becomes respirofermentative and pyruvate metabolism occurs predominantly via pyruvate decarboxylase (5, 6). Under such conditions acetate is formed as a by-product, indicating that the in vivo activity of acetaldehyde dehydrogenase exceeds that of acetyl-coenzyme A synthetase. Acetate produ...

In Sacchuromyces cerevisiue, the structural genes PDCI, PDCS and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild-type strain, using the dominant marker genes APT1 and TnSble. A pyruvate-decarboxylase-negative (Pdc-) mutant lacking all three PDC genes exhibited a threefold lower growth rate in complex medium with glucose than the isogenic wild-type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc-strains. Furthermore, in ethanol-limited chemostat cultures, the biomass yield of Pdc-and wild-type S. cerevisiae were identical. However, Pdc-S. cerevisiue was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol-limited chemostat cultures (D = 0.10 h-') were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed-substrate cultures were switched to a feed containing glucose as the sole carbon source, wash-out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl-CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose-limited chemostat cultures.

Maltotriose is the second most abundant fermentable sugar in wort and, due to incomplete fermentation, residual maltotriose in beer causes both quality and economic problems in the brewing industry. To identify genes that might improve utilization of maltotriose, we developed a library containing genomic DNA from four lager strains and a laboratory Saccharomyces cerevisiae strain and isolated transformants that could grow on YP/2% maltotriose in the presence of 3 mg/l of the respiratory inhibitor antimycin A. In this way we found a gene which shared 74% similarity with MPH2 and MPH3, 62% similarity with AGT1 and 91% similarity with MAL61 and MAL31, all encoding known maltose transporters. Moreover, the gene shared an even higher similarity (98%) with the uncharacterized Saccharomyces pastorianus mty1 gene (M. Salema-Oom, unpublished; NCBI Accession No. AJ491328). Therefore, we named the gene MTT1 (mty1-like transporter). We showed that the gene was present in four different lager strains but was absent from the laboratory strain CEN.PK113-7D. The ORF in the plasmid isolated from the library lacks 66 base pairs from the 3 -end of MTT1 but instead contains 54 bp of the vector. We named this ORF MTT1alt (NCBI Accession No. DQ010174).14 C-Maltose and repurified 14 C-maltotriose were used to show that MTT1 and, especially, MTT1alt, encode maltose transporters for which the ratio between activities to maltotriose and maltose is higher than for most known maltose transporters. Introduction of MTT1 or MTT1alt into lager strain A15 raised maltotriose uptake by about 17% or 105%, respectively.