Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK
Correspondence
Peter A. Meacock
mea{at}le.ac.uk
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ABSTRACT |
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INTRODUCTION |
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We are interested in looking at the kind of cellular processes conferred by such subtelomeric gene families and in determining their redundancy. In this study, we report on a highly conserved gene family of four members, which, based upon homology to a gene present in Schizosaccharomyces pombe, have a putative role in biosynthesis of the enzyme cofactor thiamin diphosphate (ThdP). This gene family, here collectively termed the THI5 gene family, comprises the subtelomeric open reading frames (ORFs) designated YFL058w (THI5), YJR156c (THI11), YNL332w (THI12) and YDL244w (THI13).
The THI5 gene was originally isolated as a cDNA clone obtained through a screen for genes expressed during entry into stationary phase, when Saccharomyces cerevisiae was grown on industrial molasses medium (Praekelt & Meacock, 1992). Several genes were identified, one of which was originally named MOL1 (molasses inducible). MOL1 was later found to be expressed as a result of the depletion of exogenous thiamin (vitamin B1) and was renamed THI4. Disruption of THI4 resulted in thiamin auxotrophy that was rescuable by supplementation of the growth medium with one of the thiamin precursors, hydroxyethylthiazole (HET) (Praekelt et al., 1994
). A second cDNA identified from that screen, MOL2 (THI5), represented another gene with a putative role in the biosynthesis of the other thiamin precursor, hydroxymethylpyrimidine diphosphate (HMP-PP). This prediction was based upon its homology to the single copy gene of Schizosaccharomyces pombe, termed nmt1 (Maundrell, 1990
). An nmt1-negative mutant strain was reported to be a thiamin auxotroph (Schweingruber et al., 1991
) that could be rescued by the addition of thiamin or hydroxymethylpyrimidine (HMP), but not of HET, to the growth medium.
The biosynthesis of thiamin, vitamin B1, in S. cerevisiae has been reviewed elsewhere (Hohmann & Meacock, 1998). Thiamin is formed by the condensation of its phosphorylated precursors hydroxyethylthiazole phosphate and HMP-PP. HMP-PP is itself derived from pyridoxine (vitamin B6) (Tazuya et al., 1993
). There has been a report of an alternative but unrelated HMP biosynthetic pathway that functions when S. cerevisiae is grown under anaerobic conditions (Tanaka et al., 2000
). This pathway has been shown not to proceed through the pyridoxine intermediate.
Most of the known thiamin biosynthetic genes are under feedback regulation from the active cofactor ThdP. So far, only positive regulators of this thiamin-dependent gene expression have been identified, encoded by THI2, THI3 and PDC2 (Hohmann & Meacock, 1998; Muller et al., 1999
; Nishimura et al., 1992a
, b
), although several so-called det (derepressed expression on thiamin) mutants have been isolated which could represent negatively acting regulators (Burrows et al., 2000
). In Schizosaccharomyces pombe, nmt1 gene expression is totally repressed in the presence of thiamin at concentrations of 0·5 µM or greater (Schweingruber et al., 1991
). In fact, nmt1 was the first fully repressible gene discovered in Schizosaccharomyces pombe and its promoter is extensively used as a tool for the regulated expression of cloned genes (Maundrell, 1993
).
In this study, we examined the distribution of the THI5 multicopy state among the hemiascomycetes in order to establish how widespread this situation is, and to help formulate a hypothesis about the selective advantage it might confer; so far, only the sequenced genome of Saccharomyces cerevisiae S288C has been shown to possess THI5 in more than one copy. We describe the construction of all combinations of deletion mutant strains covering the THI5 gene family in this strain and we report their phenotypes with respect to HMP biosynthesis. The position of the putative isozymes relative to pyridoxine is also reported, an issue not yet addressed for the Schizosaccharomyces pombe homologue. Further analysis looks at the regulation of each of the four genes (THI5, THI11, THI12 and THI13) and the roles of their products in the anaerobic formation of the precursor.
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METHODS |
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Reaction conditions for degenerate PCR analysis.
The primers SNZfwd, THI5back, THI5fwd and AADfwd (Table 3) were designed to anneal to conserved regions of the SNZ, THI5 and AAD genes in S. cerevisiae. As the SNZ gene family has two almost identical subtelomeric members, the primer was designed to anneal to portions conserved between S. cerevisiae SNZ and Saccharomyces mikatae SNZ (sequence kindly supplied by P. Clifton, University of Washington, USA). Reaction conditions were 94 °C for 2 min, followed by 30 cycles at 94 °C for 1 min, 52 °C for 1 min and 72 °C for 55 s, with a final incubation step at 72 °C for 10 min.
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Identification of THI5, THI11, THI12 and THI13 gene disruptions.
Identification of disrupted loci was carried out using Southern blot hybridization analysis of yeast genomic DNA digested with XhoI (Gene Images; Amersham Pharmacia Biotech). A ClaIXhoI THI5 DNA fragment from plasmid pRH4 was used as a probe. For strains RWY15 and RWY16, disrupted loci were examined by PCR as described in the text using the oligonucleotide primers listed in Table 3.
THI5 copy number survey.
Yeast genomic DNAs were digested with XhoI, PstI and EcoRI. Southern blot analysis used as probes the S. cerevisiae THI5 sequence, obtained on a ClaIXhoI fragment from pRH4, and the Kluyveromyces lactis THI5 sequence, obtained on HindIII fragment from pDW5a.
Slot-blot hybridization of yeast RNA.
All strains were inoculated into Wickerham's glucose medium without thiamin. After overnight incubation, each yeast culture was then inoculated to a final cell density of 3x105 cells ml-1, into the same medium supplemented as indicated with thiamin, HMP or HET. Samples for RNA preparations were harvested at a cell density of 25x107 cells ml-1.
Total yeast RNA was isolated using the rapid phenol/SDS extraction protocol described by Schmitt et al. (1990). Approximately 2 µg RNA was applied to a Hybond-N+ membrane using a Hybri-Slot filtration manifold apparatus (Gibco-BRL). The membranes were then hybridized as described by Church & Gilbert (1984)
using either the pRH4 THI5 probe or a 342 bp ACT1 EcoRIHindIII fragment of pBS-Actin (gift of T. Pillar, Universitats Krankenhaus-Eppendorf, Hamburg, Germany). Both probes were labelled by the incorporation of [
-32P]CTP. The relative emitted radioactivity of positively hybridizing bands was measured using a Molecular Dynamics Phosphorimager. Analysis of the intensities was carried out using IMAGEQUANT software.
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RESULTS |
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Fig. 1 shows the results of these analyses in the form of a simplified physical map. Centromere proximal to all four THI5 genes is a member of the AAD gene family (Delneri et al., 1999a
), originally postulated to encode an aryl-alcohol dehydrogenase. Two of these genes, AAD4 and AAD6, are expressed in response to oxidative stress in a Yap1p-dependent manner (Delneri et al., 1999b
). The chromosomal sequences that are centromere proximal to the AAD genes and adjacent to the four THI5 genes diverge extensively, showing no conservation and the ORFs are not repeated. These AAD genes, therefore, appear to represent the boundary of the subtelomeric repeated DNA.
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The block adjacent to THI11 and THI13 on chromosomes X-R and IV-L contains genes that encode enzymes involved in carbohydrate metabolism: HXT15 and HXT16 encode proteins that resemble hexose transporters and both function in the uptake of mannose, fructose and glucose, but not galactose (Wieczorke et al., 1999); SOR1 and SOR2 appear to encode a putative zinc-containing alcohol dehydrogenase that is homologous to sorbitol dehydrogenase (Gonzalez et al., 2000
); both MPH2 and MPH3 encode high-affinity
-glucoside permeases capable of transporting maltose, maltotriose, methyl
-glucoside and turanose (Day et al., 2002
); the function of YJR157w and its homologous pseudogene sequence on chromosome IV-L is unresolved.
Located telomere proximal to THI5 and THI12 on chromosomes VI-L and XIV-L is a block that contains members of the SNO and SNZ gene families. Both SNO and SNZ were first identified as genes expressed at the entry to stationary phase (Padilla et al., 1998). Homologues of SNZ from other fungi have since been found to encode pyridoxine biosynthetic enzymes (Ehrenshaft et al., 1999
; Osmani et al., 1999
). Work done in this laboratory has shown that the subtelomeric copies of S. cerevisiae SNO and SNZ exist to provide pyridoxine (or a pyridoxal intermediate) as a precursor for the HMP pathway (L. Marsh, R. Wightman & P. A. Meacock, unpublished data); this has recently been confirmed by other workers (Rodríguez-Navarro et al., 2002
). The determination of a role for the homologues YFL061w and YNL335w awaits the construction of a mutant S. cerevisiae strain containing deletions of both genes.
The COS genes and adjoining ORFs, which are found distally upstream of all four THI5 genes on the other side of the duplicated gene blocks, are members of much larger gene families of unknown function.
Distribution of THI5, as a gene family, among the hemiascomycetes
To date, the existence of THI5 as a gene family has only been observed in the sequenced genome of S. cerevisiae S288C. The homologous gene nmt1 of Schizosaccharomyces pombe and those of filamentous fungi appear to be present only as a single copy per genome. Therefore, to gain information about when and why amplification of the THI5 gene occurred we decided to investigate other related hemiascomycete yeasts. A survey of THI5 copy number was carried out by Southern blot analysis of digested yeast genomic DNAs using a probe that cross-hybridized with all four Saccharomyces cerevisiae genes. Restriction digests using the enzymes XhoI, PstI and EcoRI individually can distinguish each of the genes THI5, THI11, THI12 and THI13 in strain S288C (Fig. 2). Therefore, equivalent hybridizations were carried out on genomic DNAs isolated from a sample of hemiascomycetes classified as Saccharomyces or Kluyveromyces, so allowing an estimate of THI5 copy number based upon the number of positively hybridizing fragments.
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Other laboratory strains of S. cerevisiae were also surveyed for THI5 copy number (data not shown). Both BY4705 and W303a, which are descended from S288C, showed the same four hybridizing fragments as S288C, whilst a pseudohyphal strain, 1278b, gave no signal corresponding to the THI5 gene on chromosome VI. This same strain had previously been shown to lack the adjacent SNZ3 gene (Padilla et al., 1998
), suggesting that the whole of the left subtelomeric portion of this chromosome is absent. A survey of three commercial strains of S. cerevisiae, the CBS 1171 type strain and two strains (NCYC 1681 and NCYC 1324) formerly designated as Saccharomyces uvarum used in the production of ales and lagers (Goodey & Tubb, 1982
), revealed THI5 to be present as a gene family but with fewer copies than S288C (data not shown); from the number of positively hybridizing fragments we deduce that it is likely that these yeasts possess only two copies of THI5.
Other species of yeasts from the Saccharomyces and Kluyveromyces genera were surveyed using the same S. cerevisiae-derived THI5 hybridization probe (Fig. 3). Within the Saccharomyces collection were a group of strains very closely related to S. cerevisiae and termed the Saccharomyces sensu stricto complex, which included Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces pastorianus and three newly identified species, S. mikatae, Saccharomyces kudriavzevii and Saccharomyces cariocanus (Naumov et al., 2000
). Southern analysis showed that all these yeasts possess multiple copies of THI5. Yeasts classified outside of this subgroup, including the Saccharomyces sensu lato species and several Kluyveromyces species, possess either one or no copies of a THI5 homologue. To eliminate the possibility that our failure to detect positively hybridizing signals in some species was because of sequence divergence between the S. cerevisiae-derived probe and the Kluyveromyces genomic DNAs, we also used as probe the THI5 gene of K. lactis; the results obtained with the original S. cerevisiae DNA probe were confirmed. Therefore, we conclude that the existence of THI5 as a gene family is exclusive to those yeasts of the Saccharomyces sensu stricto complex, and some yeasts of both genera contain no THI5 gene at all.
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By using a PCR approach based on degenerate primers, gene order conservation between THI5 and its adjacent genes was investigated in the sensu stricto strains S. cerevisiae CBS 1171, S. bayanus, S. pastorianus, S. paradoxus and S. mikatae in comparison to S. cerevisiae S288C. The primers were designed to amplify the intergenic regions between SNZTHI5/12 and THI5/12AAD by annealing to conserved sequences within the two genes. All strains gave SNZTHI5/12 products equal in size to that derived from S288C (Fig. 4), implying a conservation of gene order. In the case of the THI5/12AAD analysis, all except S. bayanus gave products of similar size. The S. bayanus exception might be a consequence of DNA polymorphisms within the primer-binding region or it could be due to this particular yeast having no AAD gene downstream of THI5/12. Even so, the detection of amplified DNA from these two gene intervals in these strains implies synteny between the chromosomes of the sensu stricto group. Further evidence of a common genome environment around the THI5/12 genes of the sensu stricto group is documented within the Synteny Viewer on the Saccharomyces Genome Database web site (http://genome-www.stanford.edu/Saccharomyces/), part of which has been published as a survey of several Saccharomyces species belonging to this complex (Cliften et al., 2001
).
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Mutants with deleted alleles were detected by Southern blot hybridization using the THI5 cDNA as probe. Strain BY4705 gives rise to four positively hybridizing XhoI fragments of different sizes; these are 14·0 kb for THI11, 10·2 kb for THI12, 7·0 kb for THI13 and 3·8 kb for THI5. Thus, after each round of gene disruption, transformants were scored for the presence/absence of each of the four genes (data not shown). A genealogical tree (Fig. 5) describes the routes to the construction of all the mutant strains. The genotype of the RWY15 strain containing deletions of all four members was confirmed by a PCR approach. This made use of primers, four marker-gene-specific and one common, that amplified the DNA between each of the individual integrated disruption cassettes and a conserved region of the four promoters (Fig. 6
). A second quadruple mutant strain, RWY16, was constructed using the kanMX4 cassette, in order to allow for the stable maintenance of URA3+ vectors.
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Regulation of THI5, THI11, THI12 and THI13 by thiamin and its precursors
Slot-blot RNA hybridization was used to investigate the expression of each of the THI5 genes in response to exogenous thiamin and its precursors. Total RNA was isolated from BY4705 and each of the triple mutants (RWY11RWY14) after growth to late exponential phase in Wickerham's medium supplemented with thiamin, HET and/or HMP. Equal amounts of each RNA sample were applied to two membranes that were hybridized, one with a THI5 probe and the other with an ACT1 probe. The intensity of each THI5-hybridizing RNA signal was recorded after normalization for loading differences using the intensity levels of the ACT1 signals (Table 5). For each yeast strain, the intensities of the hybridization signals have been expressed as a percentage relative to the RNA sample isolated from medium without added thiamin. The actual hybridization signals, as measured on a Phosphorimager, from strains grown in thiamin-deficient medium are shown (Table 5
).
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Differences in transcript levels between the four genes were seen for samples from medium supplemented with only HMP. The THI11 (RWY12) and THI13 (RWY11) genes both exhibited the low levels of transcription described above, indicating that these genes were almost fully repressed by the presence of HMP. In contrast, THI5 (RWY13) and THI12 (RWY14) yielded transcript levels that were approximately 20 % of their maximum and so were not fully repressed by HMP (Table 5). These differences in patterns of gene expression mirror the type of promoters that are found upstream of these genes. Both THI11 and THI13 possess 100 % DNA sequence identity for approximately 1 kb upstream of their ATG translation start site but only 60 % identity to the equivalent region upstream of THI5 and THI12. Within the 1 kb sequence upstream of THI5 and THI12, there are differences at just eight nucleotide positions. As the Thi5p isozymes have been shown to be involved in HMP biosynthesis, it was surprising to see a noticeable effect of HET upon mRNA levels of members, most notably THI13. This suggests that the regulatory circuits between HET and HMP biosynthesis are linked.
HMP production during anaerobiosis
It has been suggested by Tanaka et al. (2000) that S. cerevisiae uses an alternative route for the formation of HMP when propagated under anaerobic conditions. Moreover, this anaerobic pathway does not proceed via pyridoxine. With this in mind, the effects of aerobic and anaerobic conditions on the growth of S. cerevisiae strains S288C, BY4705 and RWY15 were investigated with regard to thiamin biosynthesis. The strains were inoculated, in duplicate, onto Wickerham's glucose agar medium supplemented with or without thiamin, or with HMP or HET replacing the vitamin. Both sets of plates were incubated at the same temperature with one set placed under anaerobic conditions and the other under aerobic conditions; growth was monitored after 7 days incubation (Fig. 8
). On medium with added thiamin, all three strains displayed similar growth in both aerobic and anaerobic conditions (Fig. 8a
). Without added thiamin and in an aerobic environment, the quadruple mutant strain RWY15 exhibited the expected minimal growth due to the deficiencies in HMP formation described previously (Fig. 8b
). Normal growth was seen for the other strains. However, when incubated on the same medium in an anaerobic environment, all three strains showed the same severely restricted growth as seen for aerobic RWY15. This phenotype was not rescued by supplementation of the medium with HET (Fig. 8c
). Instead, normal anaerobic growth of all strains was restored by HMP (Fig. 8d
). All the triple mutants (RWY11RWY14) exhibited identical growth to each other and to BY4705 under the conditions tested (data not shown). Taken together, these data confirm that under anaerobic conditions even strains that contain functional THI5 family genes only use this alternative pathway to produce the HMP for thiamin, and imply that the Thi5p-catalysed pathway includes an obligatory oxidative step that cannot be accomplished under anaerobic conditions.
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DISCUSSION |
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Through the application of a PCR-mediated disruption method, S. cerevisiae strains that cover all 15 combinations of deletions of the THI5 gene family have been constructed. Included in this set of deletants is a subset of strains that possess just one of the four members (RWY11RWY14) plus strains that contain deletions of all the genes (RWY15, RWY16). Each subset of deletion mutants, although differing in genotype, has the same overall phenotype (e.g. triple mutants are all TRP+ HIS+ LEU+). This will be useful for carrying out effective competition experiments in a single culture.
Deletion of all four THI5 genes in a single strain resulted in severely retarded growth on medium lacking thiamin. Vigorous growth of this strain was dependent upon the presence of thiamin or its precursor HMP but not pyridoxine, showing that all isozymes are involved in the production of HMP from pyridoxine. The presence of any one of the genes THI5, THI11, THI12 or THI13 conferred apparently normal thiamin prototrophic growth, showing that this gene family is functionally redundant for HMP formation.
Fig. 9 shows how these genes might feature in HMP formation from the metabolite 5-phosphoribosyl pyrophosphate. The only other known enzymes in this pathway are the hydroxymethylpyrimidine phosphate kinases Thi20p and Thi21p (Llorente et al., 1999
), and the glutamine amidotransferase activity catalysed by Ade4p (Mantsala & Zalkin, 1984
). As Ade4p is feedback-inhibited by the products of purine biosynthesis, another glutamine amidotransferase must exist to produce phosphoribosylamine for pyridoxine and thiamin biosynthesis. The SNO and SNZ genes, which are also represented by multicopy gene families with some members positioned adjacent to THI5 gene family members, are potential candidates for this role since multiple null mutants display pyridoxine auxotrophy (L. Marsh, R. Wightman & P. A. Meacock, unpublished data; Rodríguez-Navarro et al., 2002
).
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The existence of two aerobic biosynthetic pathways is consistent with the observations of Grue-Sorenson et al. (1986), who examined the incorporation of [14C]formate into the pyrimidine ring of thiamin and found the radiolabel to be present at two sites, C-2 and C-4, within the HMP moiety. Moreover, the radiolabel was not uniformly distributed between the two atoms; 70 % was located at C-4 whilst only 20 % was located at C-2. This observation can only be explained by the presence of two independent pathways where [14C]formate enters C-4 by a more efficient route and C-2 by a less efficient route. The C-4 route represents the pathway that occurs via pyridoxal/THI5 as described in Fig. 9
.
Why then should the Saccharomyces sensu stricto group require the THI5 gene in multiple copies? Presumably, there must be either a strong metabolic need to produce large amounts of the pathway end-product, ThdP, or one of the intermediates, or the proteins themselves fulfil other functions needed when cells are depleted of thiamin; expression of these genes is repressed in thiamin-replete conditions. This group of yeasts are identified by their petite-positive character and ability to ferment a variety of sugars, all of which are metabolized via glycolysis to pyruvate and thence either by anaerobic fermentation to ethanol or by aerobic metabolism to produce biomass.
It is well documented that multiple gene copies allow for rapid and abundant synthesis of their cognate RNAs and proteins; classic examples are the genes encoding rRNAs and proteins, and histone proteins in various organisms. Consistent with this, examination of THI5 mRNA levels in the triple mutant S. cerevisiae strains showed that no single gene is able to generate the maximal level of expression seen in the wild-type cell possessing all four genes. Additionally, multiple gene copies facilitate evolution through the functional divergence and differential gene expression of the individual members, in this case with respect to the production of the thiamin precursor HMP. The subtle differences that we have observed in the growth patterns of mutant strains (Fig. 7) and in expression levels of individual genes (Table 5
) suggest that different family members might function with differing efficiencies under different growth conditions.
If the reason for multiple copies of THI5 is to respond to a high ThdP requirement then what is causing this in the sensu stricto yeasts? It is interesting to note that a great many of the amplified genes that are found near telomeres, including those in one of the duplicated THI5 blocks, encode proteins involved in sugar uptake and metabolism (e.g. HXT, SUC, MAL, MEL, MPH, ERR, FSP). These amplifications probably confer rapid uptake mechanisms leading to a greater glycolytic flux to pyruvate. The fate of pyruvate, towards either fermentation products or respiratory metabolism, requires ThdP-dependent reactions catalysed by pyruvate decarboxylase (Pdc) and pyruvate dehydrogenase (Pdh) respectively. It is perhaps relevant in this context to note that the commercial production of pyruvate makes use of thiamin auxotrophic yeasts (Li et al., 2001).
It is curious that the enzyme activities of thiamin biosynthesis encoded by multigene families are those of the HMP and pyridoxine branch (THI5, SNO, SNZ, THI20), whereas enzymes of the HET branch (THI4) and those involved in condensation of the precursors (THI6) and the downstream steps (THI80) are encoded by genes that are only present in single copy. A priori there should be no requirement for HMP in larger quantities than HET since thiamin is synthesized by the condensation of these precursors in equimolar quantities. If HMP is itself an intermediate in another metabolic pathway then this could explain the need for a greater HMP pool relative to HET. However, data from this study seem to contradict this model since the HMP auxotrophy of the quadruple mutant RWY15 can be rescued completely by the addition of just thiamin to the growth medium.
Alternatively, the selection pressure might be the intracellular pyridoxine pool. The depletion of pyridoxine or a derivative is known to occur by three processes: as an enzyme cofactor in amino acid biosynthesis, as a HMP and thiamin precursor, and as a free radical scavenging activity as shown by Ehrenshaft et al. (1999) and Osmani et al. (1999)
. The extra production of pyridoxine for HMP formation has been met through the amplification of the SNO and SNZ genes and by their regulation by ThdP. Co-amplification of THI5 in the same gene block would provide extra Thi5p to ensure that this extra pyridoxine (or intermediate) is channelled into thiamin biosynthesis for metabolism of sugar growth substrates, rather than being expended completely in free radical breakdown.
A solution to this somewhat paradoxical and enigmatic situation will only come from physiological studies of normal and mutant yeast cells growing in a carefully controlled situation, such as in chemostat cultures.
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ACKNOWLEDGEMENTS |
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Received 19 December 2002;
revised 4 February 2003;
accepted 6 March 2003.
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