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Differential transcriptional regulation of sulfur assimilation gene homologues in the Saccharomyces carlsbergensis yeast species hybrid
FEMS Yeast Research 1 (2002) 315^322
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Di¡erential transcriptional regulation of sulfur assimilation gene homologues in the Saccharomyces carlsbergensis yeast species hybrid Pia Francke Johannesen a b
a;1
, JÖrgen Hansen
b;
*
Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Received 23 May 2001 ; received in revised form 11 October 2001; accepted 7 November 2001 First published online 30 November 2001
Abstract The allopolyploid yeast Saccharomyces carlsbergensis appears to be a relatively newly formed species hybrid, and therefore constitutes a good model for studying early steps in hybrid speciation. Using reverse transcription-coupled polymerase chain reaction to monitor derepression of the S. carlsbergensis homologues of the sulfur assimilation genes MET14 and MET2, we found that both homologues of these genes are regulated in the same pathway-specific manner, but surprisingly, with different kinetics, as the genes derived from one of the parent species (the non-Saccharomyces cerevisiae-like) are alleviated from repression much faster than the genes from the other parent (the S. cerevisiae-like). This probably reflects differing physiological adaptation of the parent species, and the finding may contribute to the general understanding of hybrid speciation. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Regulation ; Hybrid; Di¡erential ; Transcriptional ; Homologue; Saccharomyces
1. Introduction The Saccharomyces sensu stricto yeast-species complex appears to contain several hybrid species. The best-studied example is that of the Saccharomyces carlsbergensis (syn. of Saccharomyces pastorianus) lager brewing yeast [1,2], the genome of which appears to be derived partly from an Saccharomyces cerevisiae-like yeast and partly from another, as yet unknown, Saccharomyces yeast. Another example is a cider yeast, which was found to contain nuclear gene sequences from an S. cerevisiae-like yeast and a Saccharomyces bayanus-like yeast, while the mtDNA was similar to that of a newly described Saccharomyces yeast, S. kudriavzevii [3^5]. To further complicate matters, it appears that the S. bayanus-type strain (CBS380) is itself a hybrid containing complements from two or even three genomes [6^8]. It appears, thus, that the genomic constitution of yeasts
* Corresponding author. Tel. : +45 33275376; Fax: +45 33274764. E-mail address : [email protected] (J. Hansen). 1
Present address: Novozymes A/S, Molecular Biotechnology, Fungal Gene Technology, KrogshÖjvej 36, DK-2880 Bagsv×rd, Denmark.
in the Saccharomyces sensu stricto complex is a complicated matter, and that inter-speci¢c hybridisation events may be more involved in the speciation process of these yeasts than was originally believed. While an increasing amount of insight is gained into the genomic structure of these hybrid yeasts, little is known about the transcriptional regulation of hybrid parental gene homologues. In a newly consolidated hybrid, perhaps derived from parental species genetically adapted to di¡erent environmental conditions, the presence of homologous genes for enzymes and transcription factors will comprise a rich base for speciation; some genes will inevitably be silenced due to redundancy, or specialise through mutation or recombination, and, certainly, di¡erential regulation of parental gene homologues will in£uence the actual changes in the genetic makeup of the evolving hybrid. How does the environment of the species hybrid in£uence the actual selection of which gene homologues for a particular function `to go' and which `to stay', i.e. which of such homologues are the more important for survival or ¢tness in a given situation ? S. carlsbergensis is an ideal organism for the study of this subject. This yeast is usually found to contain two divergent types of each gene, of which, one is identical to the corresponding S. cerevisiae gene (the `-CE' type, for S. cerevisiae-like) and one is di¡erent (the `-CA' type,
1567-1356 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 1 3 5 6 ( 0 1 ) 0 0 0 5 0 - 2
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for S. carlsbergensis-speci¢c) [1,9^18]. The two types are usually functionally analogous, and are termed gene homologues (see [2]). As this species hybrid appears to be derived from only two parental yeast species, of which, one is very well described, it o¡ers itself as a relatively simple hybrid speciation model. The fact that two homologues are usually found indicates that S. carlsbergensis is a rather newly formed hybrid, which makes it suitable for studies of the early stages of hybrid speciation. To address the issue of how a yeast hybrid genome is regulated transcriptionally, we studied the MET14 and MET2 homologues from S. carlsbergensis. These sulfur assimilation genes are tightly repressed by organic sulfur [19], e.g. in the form of methionine, and derepression of the genes can be monitored in rather ¢ne resolution by the use of RT-PCR (reverse transcription-coupled polymerase chain reaction). Here we describe the isolation and sequencing of the MET14-CA. For the ¢rst time, the relative expression of homologous genes in the hybrid yeast S. carlsbergensis was studied by means of RT-PCR, and both the MET14-CE/MET14-CA and the MET2-CE/ MET2-CA gene pairs were found to be di¡erentially regulated. 2. Materials and methods 2.1. Strains and media S. carlsbergensis (syn. of S. pastorianus) M204 is a Carlsberg lager production strain, and PFJ445 is a M204 meiotic segregant containing only MET14-CA (P.F. Johannesen, unpublished). Escherichia coli strain DH5K [20] was used for cloning experiments. Media were prepared as described [21,22]. 2.2. DNA manipulations Small-scale plasmid DNA was isolated by alkaline lysis [23], and large-scale using Wizard midiprep columns (Promega Corp., Madison, WI, USA). DNA for sequencing was prepared using the Wizard0 Plus Miniprep DNA Puri¢cation System (Promega Corp., Madison, WI, USA). DNA manipulations were according to enzyme manufacturers (Roche Molecular Biochemicals, Hvidovre, Denmark; Promega Corp., Madison, WI, USA; or New England Biolabs, Inc., Beverly, MA, USA). Genomic DNA for use as PCR template was extracted from 10-ml stationary-phase yeast cultures [24]. 2.3. PCR ampli¢cation of MET14-CA The sequence information from S. cerevisiae was used to design primers which enabled PCR ampli¢cation of the S. carlsbergensis MET14-CA gene. This strategy relied on two assumptions: (i) that the order of genes in the
S. cerevisiae MET14 and S. carlsbergensis MET14-CA regions is conserved, and (ii) that the DNA sequences are su¤ciently related to allow annealing of the S. cerevisiae MET14-derived primers to the DNA of the S. carlsbergensis MET14-CA region. We used S. carlsbergensis PFJ445 genomic DNA as template, as this yeast contains no MET14-CE gene, but genomic DNA from Saccharomyces monacensis (syn. of S. carlsbergensis and S. pastorianus) could also have been used, as this yeast usually contains only the -CA type of any given gene (e.g. [17,25]). Fragment 1, covering an internal part of the MET14 open reading frame (ORF), was ampli¢ed using primers #3 and #4 (5P-GCGGATCCGCACATTCTTCAACCGTCTTCTGGTCGG-3P and 5P-GCTCTAGAATGGCTACTAATATTACT-3P), which were based on the sequence of the S. cerevisiae MET14 gene. This fragment contained an XbaI restriction site at its 5P-end and a BamHI site at its 3P-end, and was inserted into the same sites of plasmid pRS316 [26], generating plasmid pPF46. Likewise, fragment 2, covering the 3P-region of the MET14 gene, was synthesised using the primers #1 and #2 (5P-GCTCTAGAGCATTGGAGTTGGTTATGCG-3P and 5PGCGGATCCCCGACCAGAAGACGGTTGAAGAATGTGC-3P). These two primers were based on the most downstream part of the S. cerevisiae MET14 ORF and the most upstream part of the adjacent MRP17 gene. Fragment 2 was inserted into pRS316 in a similar manner as fragment 1, generating the plasmid pPF37. A fragment (3) including the MET14 promoter region was ampli¢ed employing primers #5 and #6 (5P-GCTCTAGAAGTAATATTAGTAGCCAT-3P and 5P-GCGGATCCTGTTCATGATTTCCGAAC-3P). We were not able to clone fragment 3, but the DNA sequence was obtained by direct sequencing of the PCR fragment, revealing a very low homology to the corresponding region in the S. cerevisiae MET14 gene. AmpliTaq polymerase (Applied Biosystems, Foster City, CA, USA) was used with up to 4 mM MgCl2 and annealing at 45^59³C for these PCR ampli¢cations. Based on the sequence information from fragments 2 and 3, new, sequence-speci¢c primers were designed and used to amplify full-length MET14-CA from genomic DNA of strain PFJ445. Employing the MET14-CA-derived primers #7 and #8 (5P-GCGGATCCGGAGTCGGTACTAAATATC-3P and 5P-GCGGATCCGAAAGGTGGCCTATC-3P) we obtained the PCR fragment 4 of 1148 bp, containing the entire MET14-CA gene region. DNA fragments from three individual PCR pools were inserted into pUC18 [27], thus generating the three MET14-CAcontaining plasmids pPF50, pPF51, and pPF52. For these PCR ampli¢cations, Expand1 High Fidelity PCR System (Roche Molecular Biochemicals, Hvidovre, Denmark) was used, with 2.5 mM MgCl2 , and annealing at 52³C. The inserts of all three clones were sequenced using an array of primers designed successively on the basis of the available sequence information. The three plasmids were found to contain identical inserts.
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2.5. Nucleotide sequencing Sequence reactions were performed using PRISM1 AmpliTaq0 FS Dye Terminator Cycle Sequencing kit, and an Applied Biosystems 373A DNA Sequencer (Applied Biosystems, Foster City, CA, USA). 2.6. Derepression experiments S. carlsbergensis M204 was cultured in 250 ml YPD medium to an OD600 of 0.4 under repressing conditions (5 mM additional L-methionine). Cells were collected by ¢ltration, washed once with B medium, resuspended in 250 ml B medium and allowed continued growth. 20-ml culture samples were collected at consecutive time-points, and cells pelleted by centrifugation and frozen on dry ice. RNA was extracted from the cell pellets using the FastRNA Kit-Red (Qbiogene, Carlsbad, CA, USA). 2.7. RT-PCR 2.7.1. RT control calculations To obtain a measure of the accessibility of the homologous RNA substrates for reverse transcription, a calculation of the free energy needed to fold the two MET14 genes using the RNA mfold (version 3.1) and the two MET2 (version 2.3) genes as RNA, was performed (http://bioinfo.math.rpi.edu/~mfold/rna/form1.cgi) [28,29]. For the MET14 genes (standard parameters, 37³C), the free energy needed to fold the most obvious 25 structures ranged from 3147.4 to 3154.3 kcal/mol for MET14-CE and from 3136.3 to 3143.4 for MET14-CA. For the MET2 genes (standard parameters, 60³C), free energies varied from 3130.1 to 3136.6 for MET2-CE and from 3120.8 to 3126.8 for MET2-CA. 2.7.2. PCR control studies Various concentration ratios of plasmids pPF29 (pUC18 containing MET14-CE) and pPF50 (pUC18 containing MET14-CA) were used as PCR template DNA with the primers MET14-RT2 (5P-GGTCTAAGTGCGTCAGG-3P) and MET14-RT4 (5P-AGCACATTCTTCAAC-3P). Products were digested with SacI (cuts the 465-bp MET14-CE fragment into fragments of 213 and 252 bp) or TaqI (cuts the 465-bp MET14-CA fragment into fragments of 229 and 236 bp). The amount of undigested PCR product was determined by densitometry after separation on agarose. 2.7.3. MET14 RT-PCR experiments RT-PCR reactions were performed using the ProSTAR1 HF Single-Tube RT-PCR System (Stratagene Inc., La Jolla, CA, USA). 200 ng of total RNA in a total volume of 50 Wl was heated to 37³C for 15 min (reverse
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transcription), followed by 40 ampli¢cation cycles of 95³C for 1 min, 50³C for 1 min and 68³C for 2 min, and ¢nally, 1 cycle of 68³C for 10 min. Products were digested with SacI, TaqI or SacI+TaqI, and separated on 4% agarose. 2.7.4. MET2 RT-PCR experiments RT-PCR reactions were performed using GeneAmp0 Thermostable rTth Reverse Transcriptase RNA PCR Kit (Applied Biosystems, Foster City, CA, USA). 200 ng of total RNA in a total volume of 100 Wl was heated to 60³C for 15 min (reverse transcription), followed by 40 ampli¢cation cycles of 95³C for 1 min and 60³C for 2 min, and ¢nally, 1 cycle of 60³C for 10 min. The products were digested using EcoRI (cuts the 580-bp MET2-CE fragment into fragments of 211 and 369 bp) and XbaI (cuts the 580bp MET2-CA fragment into fragments of 161 and 419 bp), and separated on a 3% agarose gel (NuSieve0 GTG0 agarose (BioWhittaker Molecular Applications, Rockland, ME, USA)). Gels were photographed using an Image Master0 VDS (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and densitometric measurements were performed using Image Quant0 , version 5.0 (Molecular Dynamics, Sunnyvale, CA, USA). 3. Results 3.1. Cloning and characterisation of the MET14-CA gene By Southern hybridisation we found that S. carlsbergensis contains two versions of the MET14 gene (data not shown), which we term MET14-CE and MET14-CA. Whereas the S. cerevisiae MET14 gene was described previously [30], the DNA sequence of the MET14-CA gene was not known. Since an attempt to isolate this gene by complementation failed, the sequence information from S. cerevisiae was used to design primers that enabled PCR ampli¢cation of MET14-CA from S. carlsbergensis DNA. Three MET14-CA fragments were obtained (see Section 2 for a thorough description of this cloning procedure), together covering the locus and adjacent intergenic regions. A preliminary MET14-CA DNA sequence was determined from these fragments, and speci¢c primers and a proofreading DNA polymerase were subsequently used to amplify a full-length MET14-CA fragment, which was then sequenced (GenBank accession number AY017216). The ORF of MET14-CA has the same size as that of S. cerevisiae MET14 and is 86% identical to this gene, with a 96.5% identity at the amino acid level. This high degree of conservation is in sharp contrast to a very low identity between the promoter regions of the genes. All of these observations are, however, in good agreement with comparable data for other S. carlsbergensis gene homologues described [15^18].
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Fig. 1. Alignment of the S. carlsbergensis MET14-CA and MET14-CE promoter regions. Alignment was performed employing the ClustalW algorithm [36] and the Bioedit programme [37]. Elements discussed in the text are in italics and underlined.
3.2. The MET14-CA and MET14-CE genes contain conserved promoter elements involved in pathway-speci¢c regulation Sequencing parts of the S. carlsbergensis MET14-CE gene (isolated by complementation cloning with an S. carlsbergensis genomic library) showed its promoter region to be basically identical to the same region of S. cerevisiae MET14. Two nucleotides are missing (position ^164 to ^165), as compared to MET14, but these are not in regions believed to be involved in the sulfur assimilation pathway-speci¢c regulation [19], and therefore we expect MET14 and MET14-CE to be regulated identically. The rather low overall identity between the promoter regions of the S. carlsbergensis MET14 homologues is visualised in Fig. 1. However, scrutinising MET14-CA for regulatory elements identi¢ed in the S. cerevisiae MET14 promoter [30], the TCACGTG element, which is involved in binding of the Cbf1p transcription factor to the promoter of MET genes [19,31], could be found as CCACGTG in MET14-CA at position 3209 (as TCACGTG at position 3229 in the S. cerevisiae MET14), and conserved in MET14-CE at 3227. The 13 bp immediately upstream of this sequence are conserved between the homologues, in agreement with the observation that this DNA region is crucial for proper activation of MET genes [32,33]. The AAANTGTG element, involved in derepression and binding of the Met31p and Met32p transcription factors [34], found in S. cerevisiae
Fig. 2. PCR control experiment. Linearised plasmid pPF29 and pPF50 containing MET14-CE and MET14-CA, respectively, were used as templates in di¡erent molar concentration ratios. Each PCR reaction was performed in duplicate. 1: pPF29 1U, 2: pPF50 1U, 3: pPF29 1/2U+pPF50 1/2U, 4: pPF29 3/4U+pPF50 1/4U, 5: pPF29 1/4U+pPF50 3/4U (1U, 1/2U, 1/4U and 3/4U indicate the molar fractions of speci¢c template added). The PCR reactions were performed using oligoprimers MET14-RT2 and MET14-RT4, equal amounts of product were digested with SacI and TaqI, respectively, and the digested samples were analysed on 2% agarose gels. M: 1 kb DNA ladder (Promega Corp., Madison, WI, USA).
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Fig. 3. RT-PCR analysis of the ¢rst hour of derepression of MET14-CE and MET14-CA. RT-PCR reactions were performed on total RNA prepared from cell samples taken every 5 min during the ¢rst hour of derepression, and the products were restriction-digested and separated by agarose gel and electrophoresis. Abbreviations : S, SacI; T, TaqI; S+T, SacI+TaqI; ud, undigested RT-PCR product.
MET14 as AAAATGTG at position 3196, can be found in MET14-CA as AAATTGTG at position 3178 and conserved in MET14-CE at 3194. The strong conservation of promoter elements in the two genes in regions with poor overall identity emphasises their importance and strongly suggests that the genes are regulated by the same mechanisms. 3.3. Derepression of MET14-CA and MET2-CA is signi¢cantly faster than derepression of their homologues Derepression of MET14-CA and MET14-CE was chosen as the method to study pathway-dependent gene regulation in the S. carlsbergensis strain M204. The yeast was cultured exponentially under repressing conditions and shifted to sulfur-free medium, and the changes in MET14 transcription were monitored in cell samples taken at intervals. Northern analysis showed that derepression of both MET14-CE and MET14-CA occurs within the ¢rst hour (data not shown). To determine the ratio of expression of the two S. carlsbergensis MET14 genes, RT-PCR was performed. The oligonucleotide primers MET14-RT2 and MET14-RT4 are 100% identical to both MET14 homologues, and the equally long MET14 PCR products obtained can be distinguished by restriction digestion, since SacI cuts only the MET14-CE product, and TaqI cuts only the MET14-CA product. Control experiments with plasmid-borne MET14-CA and MET14-CE as template for the PCR reaction showed that the molar ratio of the genes correlates well with the ratio of products formed (Fig. 2). RT-PCR analysis was performed on total RNA from cells taken every 5 min during the ¢rst hour after the shift
to non-repressing conditions. The restriction-digested PCR products were separated by agarose gel electrophoresis, and the amount of undigested product assessed by densitometry. The derepression experiment and ensuing RTPCR analysis of the 12 RNA samples were performed thrice, and the results are visualised in Fig. 3 (a sample experiment agarose gel) and Fig. 4 (the MET14-CA/-CE transcript ratio). Immediately after the shift to non-repressing conditions, approximately equal amounts of the two MET14 transcripts were present, whereas from then on, the MET14-CA/-CE transcript ratio increased, up to 7, indicating that MET14-CA was derepressed much faster than MET14-CE. After 45 min the MET14-CA/-CE transcript ratio started to decrease. To study if the di¡erential derepression of the two genes also takes place during the usual environmental conditions
Fig. 4. RT-PCR analysis of the ¢rst hour of derepression of MET14-CE and MET14-CA. The MET14-CA/-CE transcript ratio from each timepoint was calculated after densitometric determination of the amount of MET14 PCR product which was not restriction-digested by one or the other restriction enzyme. The standard deviations of the values from the three experiments are shown as bars on the graph columns.
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Fig. 5. RT-PCR analysis of the expression of MET14-CE and MET14-CA during a production- scale lager beer fermentation. RT-PCR reactions were performed on total RNA prepared from cell samples taken daily during a 4500 hl main fermentation (day 1 and days 3^11), the products were restriction-digested and separated by agarose gel electrophoresis as described in Section 2. Abbreviations : S, SacI; T, TaqI; S+T, SacI+TaqI; ud, undigested RT-PCR product.
of brewing yeast, the transcription pattern of MET14-CE and MET14-CA in S. carlsbergensis brewing yeast during real beer fermentation was analysed. RNA isolated from yeast samples taken out from day 1 through day 11 in a 4500 hl production scale fermentation (supplied by K. Olesen and J. Hansen, Carlsberg Research Laboratory) was subjected to RT-PCR analysis and restriction-digested as described above. An almost complete dominance of MET14-CA transcription was seen: MET14-CE transcripts could only be identi¢ed at days 1 and 4, whereas in the remaining fermentation period, only the MET14-CA product was detected (Fig. 5), indicating that under `real' growth conditions, MET14-CA is by far the more important homologue. In order to assess whether the di¡erential regulation of the MET14 genes is unique or perhaps a characteristic of the genes of the methionine biosynthetic pathway, the derepression pattern of the S. carlsbergensis MET2 homologues was also studied. RT-PCR was performed using a set of primers, MET2-C1 and MET2-C2, previously designed to PCR amplify the same 580-bp DNA fragment from MET2-CA and MET2-CE [17]. The RNA samples previously used were employed for the MET2 RT-PCR. The RT-PCR products could be distinguished by restriction digestion with XbaI and EcoRI, which cut MET2-CA and MET2-CE, respectively. The MET2 homologues also seem to be derepressed di¡erentially, with a maximal MET2-CA/-CE transcript ratio of 2.5 after 10 min, but an earlier decline in this ratio was seen, reaching 1 somewhere between 20 and 25 min (Fig. 6). A possible objection to these experiments is that for both the MET14 and MET2 gene homologues, the free energy needed to fold these genes as RNA seems to be lower for the -CA genes at the temperatures used for reverse transcriptions (see the Section 2). While this could
possibly result in a systematic error in the RT-PCR procedure, i.e. a bias towards higher ampli¢cation of the -CA genes, it is highly unlikely that it would result in an increase in the -CA/-CE ratio through time. Therefore, we do not consider these calculations to question our main conclusion, that the -CA types of both pairs of homologues are released from repression before the -CE types. 4. Discussion We were able to demonstrate that two di¡erent pairs of homologous genes from a particular metabolic pathway in the species hybrid S. carlsbergensis are regulated di¡erentially. Our results could indicate a general phenomenon in this hybrid yeast and in hybrid speciation in a broad sense: even though two versions of a gene responsible
Fig. 6. RT-PCR analysis of the ¢rst hour of derepression of MET2-CE and MET2-CA. The MET2-CA/-CE transcript ratio from each timepoint was calculated after densitometric determination of the amount of MET2 PCR product which was not restriction-digested by one or the other restriction enzyme. The standard deviations of the values from the three experiments are shown as bars on the graph columns.
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for a particular biosynthetic step are subject to the same general regulation, they may be regulated slightly di¡erently from one another. One could speculate that the MET genes from the non-S. cerevisiae-like genome contribute the most to survival of this hybrid yeast in environments with frequent sulfur stress, and consequently that if gene redundancy would become an issue later in the evolution of this particular hybrid, the -CA genes would perhaps be the last to go. Our results support recent similar ¢ndings from Northern analysis of the S. carlsbergensis BAP2 genes [35], and indicate that the Saccharomyces sensu stricto species complex constitutes a rich base for further speciation : even though the species are su¤ciently related to allow hybridisation, they are adequately genetically adapted to confer new genetic and phenotypic opportunities to the resulting hybrid yeasts. What, then, may explain such di¡erences in homologue regulation? If a complete set of both the -CA- and -CEencoded MET gene transcription factors are present in this yeast, the reason for the di¡erential regulation may be mechanistic di¡erences between the -CA and -CE system, perhaps re£ecting the putative biological di¡erences between the yeasts that originally donated their genomic content to form the S. carlsbergensis hybrid: while S. cerevisiae has its optimal growth temperature of 28^30³C, the lower optimal growth temperature of 20^25³C for the S. carlsbergensis hybrid is likely due to a low optimal temperature for the unknown -CA parent, and one could imagine that in such a yeast some protein^protein or DNA^protein interactions would be weaker than the equivalent interactions in S. cerevisiae, and that this would result in di¡erent derepression kinetics. In another scenario, some -CA- or -CE-encoded transcription factors are missing, and accordingly, either some -CE transcription factors regulate MET-CA promoters or vice versa. One possibility in this situation would be that a weaker interaction between -CE-encoded negative regulatory factors and the -CA promoters could result in a faster derepression of the -CA genes. In any event, we believe that the described method is excellent for studying di¡erential gene homologue regulation in hybrid yeasts, and that it will become increasingly valuable as more such gene sets are described.
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[3]
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[5]
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[7]
[8]
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[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements
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Nanna Hansen is acknowledged for valuable technical help and Morten C. Kielland-Brandt for help with the manuscript.
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[18]
References [1] Nilsson-Tillgren, T., Gjermansen, C., Kielland-Brandt, M.C., Petersen, J.G.L. and Holmberg, S. (1981) Genetic di¡erences between Sac-
321
charomyces carlsbergensis and S. cerevisiae. Analysis of chromosome III by single chromosome transfer. Carlsberg Res. Commun. 46, 65^ 76. Kielland-Brandt, M.C., Nilsson-Tillgren, T., Gjermansen, C., Holmberg, S. and Pedersen, M.B. (1995) Genetics of brewing yeast. In: The Yeasts, 2nd Edn. (Wheals, A.E., Rose, A.H. and Harrison, J.S., Eds.), Vol. 6., pp. 223^254. Academic Press, London, UK. Masneuf, I., Hansen, J., Groth, C., Piskur, J. and Dubourdieu, D. (1998) New hybrids between Saccharomyces sensu stricto yeast species found among wine and cider production strains. Appl. Env. Microbiol. 64, 3887^3892. Groth, C., Hansen, J. and Piskur, J. (1999) A natural chimeric yeast containing genetic material from three species. Int. J. Syst. Bacteriol. 49, 1933^1938. Naumov, G.I., James, S.A., Naumova, E.S., Louis, E.J. and Roberts, I.N. (2000) Three new species in the Saccharomyces sensu stricto complex: Saccharomyces cariocanus, Saccharomyces kudriavzevii and Saccharomyces mikatae. Int. J. Syst. Evol. Microbiol. 5, 1931^ 1942. Rainieri, S., Zambonelli, C., Hallsworth, J.E., Pulvirenti, A. and Giudici, P. (1999) Saccharomyces uvarum, a distinct group within Saccharomyces sensu stricto. FEMS Microbiol. Lett. 177, 177^185. Nguyen, H.-V., Lepingle, A. and Gaillardin, C. (2000) Molecular typing demonstrates homogeneity of Saccharomyces uvarum strains and reveals the existence of hybrids between S. uvarum and S. cerevisiae, including the S. bayanus type strain CBS380. Syst. Appl. Microbiol. 23, 71^85. Casaregola, S., Nguyen, H.-V., Lapathitis, G., Kotyk, A. and Gaillardin, C. (2001) Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization. Int. J. Syst. Appl. Microbiol. 51, 1607^1618. Nilsson-Tillgren, T., Gjermansen, C., Holmberg, S., Petersen, J.G.L. and Kielland-Brandt, M.C. (1986) Analysis of chromosome V and the ILV1 gene from Saccharomyces carlsbergensis. Carlsberg Res. Commun. 51, 309^326. Holmberg, S. (1982) Genetic di¡erences between Saccharomyces carlsbergensis and S. cerevisiae II. Restriction endonuclease analysis of genes in chromosome III. Carlsberg Res. Commun. 47, 233^244. Pedersen, M.B. (1985) DNA sequence polymorphism in the genus Saccharomyces, II. Analysis of the genes RDN1, HIS4, LEU2 and Ty transposable elements in Carlsberg, Tuborg and 22 Bavarian brewing strains. Carlsberg Res. Commun. 50, 263^272. Petersen, J.G.L., Nilsson-Tillgren, T., Kielland-Brandt, M.C., Gjermansen, C. and Holmberg, S. (1987) Structural heterozygosis at genes ILV2 and ILV5 in Saccharomyces carlsbergensis. Curr. Genet. 12, 167^174. Casey, G.P. (1986) Molecular and genetic analysis of chromosomes X in Saccharomyces carlsbergensis. Carlsberg Res. Commun. 51, 343^ 362. Casey, G.P. (1986) Cloning and analysis of two alleles of the ILV3 gene from Saccharomyces carlsbergensis. Carlsberg Res. Commun. 51, 327^341. Gjermansen, C. (1991) Comparison of Genes in Saccharomyces cerevisiae and Saccharomyces carlsbergensis. Ph.D. Thesis. University of Copenhagen, Copenhagen. Fujii, T., Nagasawa, N., Iwamatsu, A., Bogaki, T., Tamai, Y. and Hamachi, M. (1994) Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene. Appl. Env. Microbiol. 60, 2786^2792. Hansen, J. and Kielland-Brandt, M.C. (1994) Saccharomyces carlsbergensis contains two functional MET2 alleles similar to homologues from S. cerevisiae and S. monacensis. Gene 140, 33^40. Hansen, J., Cherest, H. and Kielland-Brandt, M.C. (1994) Two divergent MET10 genes, one from Saccharomyces cerevisiae and one from Saccharomyces carlsbergensis, encode the K subunit of sul¢te reductase and specify potential binding sites for FAD and NADPH. J. Bacteriol. 176, 6050^6058.
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[19] Thomas, D. and Surdin-Kerjan, Y. (1997) Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 61, 503^532. [20] Woodcock, D.M., Crowther, P.J., Doherty, J., Je¡erson, S., DeCruz, E., Noyer-Weidner, M., Smith, S.S., Michael, M.Z. and Graham, M.W. (1989) Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 17, 3469^3478. [21] Sherman, F. (1991) Getting started with yeast. In: Methods in Enzymology 194 : Guide to Yeast Genetics and Molecular biology (Guthrie, C. and Fink, G.R., Eds.), pp. 3^21. Academic Press, San Diego, CA. [22] Cherest, H. and Surdin-Kerjan, Y. (1992) Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway. Genetics 130, 51^58. [23] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. [24] Ho¡man, C.S. and Winston, F. (1987) A ten-minute DNA preparation from yeast e¤ciently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267^272. [25] Pedersen, M.B. (1986) DNA sequence polymorphism in the genus Saccharomyces IV. Homoeologous chromosomes III of Saccharomyces bayanus, S. carlsbergensis, and S. uvarum. Carlsberg Res. Commun. 51, 185^202. [26] Sikorski, R.S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strain designed for e¤cient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19^27. [27] Yanisch-Perron, C., Vieria, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33, 103^119. [28] Zuker, M., Mathews, D.H. and Turner, D.H. (1999) Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide In RNA Biochemistry and Biotechnology (Barciszewski,
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
J. and Clark, B.F.C., Eds.), pp. 11^43, NATO ASI Series. Kluwer Academic Publishers BV. Mathews, D.H., Sabina, J., Zuker, M. and Turner, D.H. (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911^940. Korch, C., Mountain, H.A. and Bystro«m, A.S. (1991) Cloning, nucleotide sequence, and regulation of MET14, the gene encoding the APS kinase of Saccharomyces cerevisiae. Mol. Gen. Genet. 229, 96^ 108. Blaiseau, P.-L. and Thomas, D. (1998) Multiple transcriptional activation complexes tether the yeast activator Met4 to DNA. EMBO J. 17, 6327^6336. Thomas, D., Cherest, H. and Surdin-Kerjan, Y. (1989) Elements involved in S-adenosylmethionine-mediated regulation of the Saccharomyces cerevisiae MET25 gene. Mol. Cell. Biol. 9, 3292^3298. Thomas, D., Jacquemin, I. and Surdin-Kerjan, Y. (1992) MET4, a leucine zipper protein, and centromere-binding factor 1 are both required for transcriptional activation of sulfur metabolism in Saccharomyces cerevisiae. Mol. Cell. Biol. 12, 1719^1727. Blaiseau, P.-L., Isnard, A.-D., Surdin-Kerjan, Y. and Thomas, D. (1997) Met31p and Met32p, two related zink ¢nger proteins, are involved in transcriptional regulation of yeast sulfur amino acid metabolism. Mol. Cell. Biol. 17, 3640^3648. Kodama, Y., Omura, F. and Ashikari, T. (2001) Isolation and characterization of a gene speci¢c to lager brewing yeast that encodes a branched-chain amino acid permease. Appl. Env. Microbiol. 67, 3455^3462. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95^98.
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Di¡erential transcriptional regulation of sulfur assimilation gene homologues in the Saccharomyces carlsbergensis yeast species hybrid Pia Francke Johannesen a b
a;1
, JÖrgen Hansen
b;
*
Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Received 23 May 2001 ; received in revised form 11 October 2001; accepted 7 November 2001 First published online 30 November 2001
Abstract The allopolyploid yeast Saccharomyces carlsbergensis appears to be a relatively newly formed species hybrid, and therefore constitutes a good model for studying early steps in hybrid speciation. Using reverse transcription-coupled polymerase chain reaction to monitor derepression of the S. carlsbergensis homologues of the sulfur assimilation genes MET14 and MET2, we found that both homologues of these genes are regulated in the same pathway-specific manner, but surprisingly, with different kinetics, as the genes derived from one of the parent species (the non-Saccharomyces cerevisiae-like) are alleviated from repression much faster than the genes from the other parent (the S. cerevisiae-like). This probably reflects differing physiological adaptation of the parent species, and the finding may contribute to the general understanding of hybrid speciation. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Regulation ; Hybrid; Di¡erential ; Transcriptional ; Homologue; Saccharomyces
1. Introduction The Saccharomyces sensu stricto yeast-species complex appears to contain several hybrid species. The best-studied example is that of the Saccharomyces carlsbergensis (syn. of Saccharomyces pastorianus) lager brewing yeast [1,2], the genome of which appears to be derived partly from an Saccharomyces cerevisiae-like yeast and partly from another, as yet unknown, Saccharomyces yeast. Another example is a cider yeast, which was found to contain nuclear gene sequences from an S. cerevisiae-like yeast and a Saccharomyces bayanus-like yeast, while the mtDNA was similar to that of a newly described Saccharomyces yeast, S. kudriavzevii [3^5]. To further complicate matters, it appears that the S. bayanus-type strain (CBS380) is itself a hybrid containing complements from two or even three genomes [6^8]. It appears, thus, that the genomic constitution of yeasts
* Corresponding author. Tel. : +45 33275376; Fax: +45 33274764. E-mail address : [email protected] (J. Hansen). 1
Present address: Novozymes A/S, Molecular Biotechnology, Fungal Gene Technology, KrogshÖjvej 36, DK-2880 Bagsv×rd, Denmark.
in the Saccharomyces sensu stricto complex is a complicated matter, and that inter-speci¢c hybridisation events may be more involved in the speciation process of these yeasts than was originally believed. While an increasing amount of insight is gained into the genomic structure of these hybrid yeasts, little is known about the transcriptional regulation of hybrid parental gene homologues. In a newly consolidated hybrid, perhaps derived from parental species genetically adapted to di¡erent environmental conditions, the presence of homologous genes for enzymes and transcription factors will comprise a rich base for speciation; some genes will inevitably be silenced due to redundancy, or specialise through mutation or recombination, and, certainly, di¡erential regulation of parental gene homologues will in£uence the actual changes in the genetic makeup of the evolving hybrid. How does the environment of the species hybrid in£uence the actual selection of which gene homologues for a particular function `to go' and which `to stay', i.e. which of such homologues are the more important for survival or ¢tness in a given situation ? S. carlsbergensis is an ideal organism for the study of this subject. This yeast is usually found to contain two divergent types of each gene, of which, one is identical to the corresponding S. cerevisiae gene (the `-CE' type, for S. cerevisiae-like) and one is di¡erent (the `-CA' type,
1567-1356 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 1 3 5 6 ( 0 1 ) 0 0 0 5 0 - 2
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for S. carlsbergensis-speci¢c) [1,9^18]. The two types are usually functionally analogous, and are termed gene homologues (see [2]). As this species hybrid appears to be derived from only two parental yeast species, of which, one is very well described, it o¡ers itself as a relatively simple hybrid speciation model. The fact that two homologues are usually found indicates that S. carlsbergensis is a rather newly formed hybrid, which makes it suitable for studies of the early stages of hybrid speciation. To address the issue of how a yeast hybrid genome is regulated transcriptionally, we studied the MET14 and MET2 homologues from S. carlsbergensis. These sulfur assimilation genes are tightly repressed by organic sulfur [19], e.g. in the form of methionine, and derepression of the genes can be monitored in rather ¢ne resolution by the use of RT-PCR (reverse transcription-coupled polymerase chain reaction). Here we describe the isolation and sequencing of the MET14-CA. For the ¢rst time, the relative expression of homologous genes in the hybrid yeast S. carlsbergensis was studied by means of RT-PCR, and both the MET14-CE/MET14-CA and the MET2-CE/ MET2-CA gene pairs were found to be di¡erentially regulated. 2. Materials and methods 2.1. Strains and media S. carlsbergensis (syn. of S. pastorianus) M204 is a Carlsberg lager production strain, and PFJ445 is a M204 meiotic segregant containing only MET14-CA (P.F. Johannesen, unpublished). Escherichia coli strain DH5K [20] was used for cloning experiments. Media were prepared as described [21,22]. 2.2. DNA manipulations Small-scale plasmid DNA was isolated by alkaline lysis [23], and large-scale using Wizard midiprep columns (Promega Corp., Madison, WI, USA). DNA for sequencing was prepared using the Wizard0 Plus Miniprep DNA Puri¢cation System (Promega Corp., Madison, WI, USA). DNA manipulations were according to enzyme manufacturers (Roche Molecular Biochemicals, Hvidovre, Denmark; Promega Corp., Madison, WI, USA; or New England Biolabs, Inc., Beverly, MA, USA). Genomic DNA for use as PCR template was extracted from 10-ml stationary-phase yeast cultures [24]. 2.3. PCR ampli¢cation of MET14-CA The sequence information from S. cerevisiae was used to design primers which enabled PCR ampli¢cation of the S. carlsbergensis MET14-CA gene. This strategy relied on two assumptions: (i) that the order of genes in the
S. cerevisiae MET14 and S. carlsbergensis MET14-CA regions is conserved, and (ii) that the DNA sequences are su¤ciently related to allow annealing of the S. cerevisiae MET14-derived primers to the DNA of the S. carlsbergensis MET14-CA region. We used S. carlsbergensis PFJ445 genomic DNA as template, as this yeast contains no MET14-CE gene, but genomic DNA from Saccharomyces monacensis (syn. of S. carlsbergensis and S. pastorianus) could also have been used, as this yeast usually contains only the -CA type of any given gene (e.g. [17,25]). Fragment 1, covering an internal part of the MET14 open reading frame (ORF), was ampli¢ed using primers #3 and #4 (5P-GCGGATCCGCACATTCTTCAACCGTCTTCTGGTCGG-3P and 5P-GCTCTAGAATGGCTACTAATATTACT-3P), which were based on the sequence of the S. cerevisiae MET14 gene. This fragment contained an XbaI restriction site at its 5P-end and a BamHI site at its 3P-end, and was inserted into the same sites of plasmid pRS316 [26], generating plasmid pPF46. Likewise, fragment 2, covering the 3P-region of the MET14 gene, was synthesised using the primers #1 and #2 (5P-GCTCTAGAGCATTGGAGTTGGTTATGCG-3P and 5PGCGGATCCCCGACCAGAAGACGGTTGAAGAATGTGC-3P). These two primers were based on the most downstream part of the S. cerevisiae MET14 ORF and the most upstream part of the adjacent MRP17 gene. Fragment 2 was inserted into pRS316 in a similar manner as fragment 1, generating the plasmid pPF37. A fragment (3) including the MET14 promoter region was ampli¢ed employing primers #5 and #6 (5P-GCTCTAGAAGTAATATTAGTAGCCAT-3P and 5P-GCGGATCCTGTTCATGATTTCCGAAC-3P). We were not able to clone fragment 3, but the DNA sequence was obtained by direct sequencing of the PCR fragment, revealing a very low homology to the corresponding region in the S. cerevisiae MET14 gene. AmpliTaq polymerase (Applied Biosystems, Foster City, CA, USA) was used with up to 4 mM MgCl2 and annealing at 45^59³C for these PCR ampli¢cations. Based on the sequence information from fragments 2 and 3, new, sequence-speci¢c primers were designed and used to amplify full-length MET14-CA from genomic DNA of strain PFJ445. Employing the MET14-CA-derived primers #7 and #8 (5P-GCGGATCCGGAGTCGGTACTAAATATC-3P and 5P-GCGGATCCGAAAGGTGGCCTATC-3P) we obtained the PCR fragment 4 of 1148 bp, containing the entire MET14-CA gene region. DNA fragments from three individual PCR pools were inserted into pUC18 [27], thus generating the three MET14-CAcontaining plasmids pPF50, pPF51, and pPF52. For these PCR ampli¢cations, Expand1 High Fidelity PCR System (Roche Molecular Biochemicals, Hvidovre, Denmark) was used, with 2.5 mM MgCl2 , and annealing at 52³C. The inserts of all three clones were sequenced using an array of primers designed successively on the basis of the available sequence information. The three plasmids were found to contain identical inserts.
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2.5. Nucleotide sequencing Sequence reactions were performed using PRISM1 AmpliTaq0 FS Dye Terminator Cycle Sequencing kit, and an Applied Biosystems 373A DNA Sequencer (Applied Biosystems, Foster City, CA, USA). 2.6. Derepression experiments S. carlsbergensis M204 was cultured in 250 ml YPD medium to an OD600 of 0.4 under repressing conditions (5 mM additional L-methionine). Cells were collected by ¢ltration, washed once with B medium, resuspended in 250 ml B medium and allowed continued growth. 20-ml culture samples were collected at consecutive time-points, and cells pelleted by centrifugation and frozen on dry ice. RNA was extracted from the cell pellets using the FastRNA Kit-Red (Qbiogene, Carlsbad, CA, USA). 2.7. RT-PCR 2.7.1. RT control calculations To obtain a measure of the accessibility of the homologous RNA substrates for reverse transcription, a calculation of the free energy needed to fold the two MET14 genes using the RNA mfold (version 3.1) and the two MET2 (version 2.3) genes as RNA, was performed (http://bioinfo.math.rpi.edu/~mfold/rna/form1.cgi) [28,29]. For the MET14 genes (standard parameters, 37³C), the free energy needed to fold the most obvious 25 structures ranged from 3147.4 to 3154.3 kcal/mol for MET14-CE and from 3136.3 to 3143.4 for MET14-CA. For the MET2 genes (standard parameters, 60³C), free energies varied from 3130.1 to 3136.6 for MET2-CE and from 3120.8 to 3126.8 for MET2-CA. 2.7.2. PCR control studies Various concentration ratios of plasmids pPF29 (pUC18 containing MET14-CE) and pPF50 (pUC18 containing MET14-CA) were used as PCR template DNA with the primers MET14-RT2 (5P-GGTCTAAGTGCGTCAGG-3P) and MET14-RT4 (5P-AGCACATTCTTCAAC-3P). Products were digested with SacI (cuts the 465-bp MET14-CE fragment into fragments of 213 and 252 bp) or TaqI (cuts the 465-bp MET14-CA fragment into fragments of 229 and 236 bp). The amount of undigested PCR product was determined by densitometry after separation on agarose. 2.7.3. MET14 RT-PCR experiments RT-PCR reactions were performed using the ProSTAR1 HF Single-Tube RT-PCR System (Stratagene Inc., La Jolla, CA, USA). 200 ng of total RNA in a total volume of 50 Wl was heated to 37³C for 15 min (reverse
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transcription), followed by 40 ampli¢cation cycles of 95³C for 1 min, 50³C for 1 min and 68³C for 2 min, and ¢nally, 1 cycle of 68³C for 10 min. Products were digested with SacI, TaqI or SacI+TaqI, and separated on 4% agarose. 2.7.4. MET2 RT-PCR experiments RT-PCR reactions were performed using GeneAmp0 Thermostable rTth Reverse Transcriptase RNA PCR Kit (Applied Biosystems, Foster City, CA, USA). 200 ng of total RNA in a total volume of 100 Wl was heated to 60³C for 15 min (reverse transcription), followed by 40 ampli¢cation cycles of 95³C for 1 min and 60³C for 2 min, and ¢nally, 1 cycle of 60³C for 10 min. The products were digested using EcoRI (cuts the 580-bp MET2-CE fragment into fragments of 211 and 369 bp) and XbaI (cuts the 580bp MET2-CA fragment into fragments of 161 and 419 bp), and separated on a 3% agarose gel (NuSieve0 GTG0 agarose (BioWhittaker Molecular Applications, Rockland, ME, USA)). Gels were photographed using an Image Master0 VDS (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and densitometric measurements were performed using Image Quant0 , version 5.0 (Molecular Dynamics, Sunnyvale, CA, USA). 3. Results 3.1. Cloning and characterisation of the MET14-CA gene By Southern hybridisation we found that S. carlsbergensis contains two versions of the MET14 gene (data not shown), which we term MET14-CE and MET14-CA. Whereas the S. cerevisiae MET14 gene was described previously [30], the DNA sequence of the MET14-CA gene was not known. Since an attempt to isolate this gene by complementation failed, the sequence information from S. cerevisiae was used to design primers that enabled PCR ampli¢cation of MET14-CA from S. carlsbergensis DNA. Three MET14-CA fragments were obtained (see Section 2 for a thorough description of this cloning procedure), together covering the locus and adjacent intergenic regions. A preliminary MET14-CA DNA sequence was determined from these fragments, and speci¢c primers and a proofreading DNA polymerase were subsequently used to amplify a full-length MET14-CA fragment, which was then sequenced (GenBank accession number AY017216). The ORF of MET14-CA has the same size as that of S. cerevisiae MET14 and is 86% identical to this gene, with a 96.5% identity at the amino acid level. This high degree of conservation is in sharp contrast to a very low identity between the promoter regions of the genes. All of these observations are, however, in good agreement with comparable data for other S. carlsbergensis gene homologues described [15^18].
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Fig. 1. Alignment of the S. carlsbergensis MET14-CA and MET14-CE promoter regions. Alignment was performed employing the ClustalW algorithm [36] and the Bioedit programme [37]. Elements discussed in the text are in italics and underlined.
3.2. The MET14-CA and MET14-CE genes contain conserved promoter elements involved in pathway-speci¢c regulation Sequencing parts of the S. carlsbergensis MET14-CE gene (isolated by complementation cloning with an S. carlsbergensis genomic library) showed its promoter region to be basically identical to the same region of S. cerevisiae MET14. Two nucleotides are missing (position ^164 to ^165), as compared to MET14, but these are not in regions believed to be involved in the sulfur assimilation pathway-speci¢c regulation [19], and therefore we expect MET14 and MET14-CE to be regulated identically. The rather low overall identity between the promoter regions of the S. carlsbergensis MET14 homologues is visualised in Fig. 1. However, scrutinising MET14-CA for regulatory elements identi¢ed in the S. cerevisiae MET14 promoter [30], the TCACGTG element, which is involved in binding of the Cbf1p transcription factor to the promoter of MET genes [19,31], could be found as CCACGTG in MET14-CA at position 3209 (as TCACGTG at position 3229 in the S. cerevisiae MET14), and conserved in MET14-CE at 3227. The 13 bp immediately upstream of this sequence are conserved between the homologues, in agreement with the observation that this DNA region is crucial for proper activation of MET genes [32,33]. The AAANTGTG element, involved in derepression and binding of the Met31p and Met32p transcription factors [34], found in S. cerevisiae
Fig. 2. PCR control experiment. Linearised plasmid pPF29 and pPF50 containing MET14-CE and MET14-CA, respectively, were used as templates in di¡erent molar concentration ratios. Each PCR reaction was performed in duplicate. 1: pPF29 1U, 2: pPF50 1U, 3: pPF29 1/2U+pPF50 1/2U, 4: pPF29 3/4U+pPF50 1/4U, 5: pPF29 1/4U+pPF50 3/4U (1U, 1/2U, 1/4U and 3/4U indicate the molar fractions of speci¢c template added). The PCR reactions were performed using oligoprimers MET14-RT2 and MET14-RT4, equal amounts of product were digested with SacI and TaqI, respectively, and the digested samples were analysed on 2% agarose gels. M: 1 kb DNA ladder (Promega Corp., Madison, WI, USA).
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Fig. 3. RT-PCR analysis of the ¢rst hour of derepression of MET14-CE and MET14-CA. RT-PCR reactions were performed on total RNA prepared from cell samples taken every 5 min during the ¢rst hour of derepression, and the products were restriction-digested and separated by agarose gel and electrophoresis. Abbreviations : S, SacI; T, TaqI; S+T, SacI+TaqI; ud, undigested RT-PCR product.
MET14 as AAAATGTG at position 3196, can be found in MET14-CA as AAATTGTG at position 3178 and conserved in MET14-CE at 3194. The strong conservation of promoter elements in the two genes in regions with poor overall identity emphasises their importance and strongly suggests that the genes are regulated by the same mechanisms. 3.3. Derepression of MET14-CA and MET2-CA is signi¢cantly faster than derepression of their homologues Derepression of MET14-CA and MET14-CE was chosen as the method to study pathway-dependent gene regulation in the S. carlsbergensis strain M204. The yeast was cultured exponentially under repressing conditions and shifted to sulfur-free medium, and the changes in MET14 transcription were monitored in cell samples taken at intervals. Northern analysis showed that derepression of both MET14-CE and MET14-CA occurs within the ¢rst hour (data not shown). To determine the ratio of expression of the two S. carlsbergensis MET14 genes, RT-PCR was performed. The oligonucleotide primers MET14-RT2 and MET14-RT4 are 100% identical to both MET14 homologues, and the equally long MET14 PCR products obtained can be distinguished by restriction digestion, since SacI cuts only the MET14-CE product, and TaqI cuts only the MET14-CA product. Control experiments with plasmid-borne MET14-CA and MET14-CE as template for the PCR reaction showed that the molar ratio of the genes correlates well with the ratio of products formed (Fig. 2). RT-PCR analysis was performed on total RNA from cells taken every 5 min during the ¢rst hour after the shift
to non-repressing conditions. The restriction-digested PCR products were separated by agarose gel electrophoresis, and the amount of undigested product assessed by densitometry. The derepression experiment and ensuing RTPCR analysis of the 12 RNA samples were performed thrice, and the results are visualised in Fig. 3 (a sample experiment agarose gel) and Fig. 4 (the MET14-CA/-CE transcript ratio). Immediately after the shift to non-repressing conditions, approximately equal amounts of the two MET14 transcripts were present, whereas from then on, the MET14-CA/-CE transcript ratio increased, up to 7, indicating that MET14-CA was derepressed much faster than MET14-CE. After 45 min the MET14-CA/-CE transcript ratio started to decrease. To study if the di¡erential derepression of the two genes also takes place during the usual environmental conditions
Fig. 4. RT-PCR analysis of the ¢rst hour of derepression of MET14-CE and MET14-CA. The MET14-CA/-CE transcript ratio from each timepoint was calculated after densitometric determination of the amount of MET14 PCR product which was not restriction-digested by one or the other restriction enzyme. The standard deviations of the values from the three experiments are shown as bars on the graph columns.
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Fig. 5. RT-PCR analysis of the expression of MET14-CE and MET14-CA during a production- scale lager beer fermentation. RT-PCR reactions were performed on total RNA prepared from cell samples taken daily during a 4500 hl main fermentation (day 1 and days 3^11), the products were restriction-digested and separated by agarose gel electrophoresis as described in Section 2. Abbreviations : S, SacI; T, TaqI; S+T, SacI+TaqI; ud, undigested RT-PCR product.
of brewing yeast, the transcription pattern of MET14-CE and MET14-CA in S. carlsbergensis brewing yeast during real beer fermentation was analysed. RNA isolated from yeast samples taken out from day 1 through day 11 in a 4500 hl production scale fermentation (supplied by K. Olesen and J. Hansen, Carlsberg Research Laboratory) was subjected to RT-PCR analysis and restriction-digested as described above. An almost complete dominance of MET14-CA transcription was seen: MET14-CE transcripts could only be identi¢ed at days 1 and 4, whereas in the remaining fermentation period, only the MET14-CA product was detected (Fig. 5), indicating that under `real' growth conditions, MET14-CA is by far the more important homologue. In order to assess whether the di¡erential regulation of the MET14 genes is unique or perhaps a characteristic of the genes of the methionine biosynthetic pathway, the derepression pattern of the S. carlsbergensis MET2 homologues was also studied. RT-PCR was performed using a set of primers, MET2-C1 and MET2-C2, previously designed to PCR amplify the same 580-bp DNA fragment from MET2-CA and MET2-CE [17]. The RNA samples previously used were employed for the MET2 RT-PCR. The RT-PCR products could be distinguished by restriction digestion with XbaI and EcoRI, which cut MET2-CA and MET2-CE, respectively. The MET2 homologues also seem to be derepressed di¡erentially, with a maximal MET2-CA/-CE transcript ratio of 2.5 after 10 min, but an earlier decline in this ratio was seen, reaching 1 somewhere between 20 and 25 min (Fig. 6). A possible objection to these experiments is that for both the MET14 and MET2 gene homologues, the free energy needed to fold these genes as RNA seems to be lower for the -CA genes at the temperatures used for reverse transcriptions (see the Section 2). While this could
possibly result in a systematic error in the RT-PCR procedure, i.e. a bias towards higher ampli¢cation of the -CA genes, it is highly unlikely that it would result in an increase in the -CA/-CE ratio through time. Therefore, we do not consider these calculations to question our main conclusion, that the -CA types of both pairs of homologues are released from repression before the -CE types. 4. Discussion We were able to demonstrate that two di¡erent pairs of homologous genes from a particular metabolic pathway in the species hybrid S. carlsbergensis are regulated di¡erentially. Our results could indicate a general phenomenon in this hybrid yeast and in hybrid speciation in a broad sense: even though two versions of a gene responsible
Fig. 6. RT-PCR analysis of the ¢rst hour of derepression of MET2-CE and MET2-CA. The MET2-CA/-CE transcript ratio from each timepoint was calculated after densitometric determination of the amount of MET2 PCR product which was not restriction-digested by one or the other restriction enzyme. The standard deviations of the values from the three experiments are shown as bars on the graph columns.
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for a particular biosynthetic step are subject to the same general regulation, they may be regulated slightly di¡erently from one another. One could speculate that the MET genes from the non-S. cerevisiae-like genome contribute the most to survival of this hybrid yeast in environments with frequent sulfur stress, and consequently that if gene redundancy would become an issue later in the evolution of this particular hybrid, the -CA genes would perhaps be the last to go. Our results support recent similar ¢ndings from Northern analysis of the S. carlsbergensis BAP2 genes [35], and indicate that the Saccharomyces sensu stricto species complex constitutes a rich base for further speciation : even though the species are su¤ciently related to allow hybridisation, they are adequately genetically adapted to confer new genetic and phenotypic opportunities to the resulting hybrid yeasts. What, then, may explain such di¡erences in homologue regulation? If a complete set of both the -CA- and -CEencoded MET gene transcription factors are present in this yeast, the reason for the di¡erential regulation may be mechanistic di¡erences between the -CA and -CE system, perhaps re£ecting the putative biological di¡erences between the yeasts that originally donated their genomic content to form the S. carlsbergensis hybrid: while S. cerevisiae has its optimal growth temperature of 28^30³C, the lower optimal growth temperature of 20^25³C for the S. carlsbergensis hybrid is likely due to a low optimal temperature for the unknown -CA parent, and one could imagine that in such a yeast some protein^protein or DNA^protein interactions would be weaker than the equivalent interactions in S. cerevisiae, and that this would result in di¡erent derepression kinetics. In another scenario, some -CA- or -CE-encoded transcription factors are missing, and accordingly, either some -CE transcription factors regulate MET-CA promoters or vice versa. One possibility in this situation would be that a weaker interaction between -CE-encoded negative regulatory factors and the -CA promoters could result in a faster derepression of the -CA genes. In any event, we believe that the described method is excellent for studying di¡erential gene homologue regulation in hybrid yeasts, and that it will become increasingly valuable as more such gene sets are described.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements
[16]
Nanna Hansen is acknowledged for valuable technical help and Morten C. Kielland-Brandt for help with the manuscript.
[17]
[18]
References [1] Nilsson-Tillgren, T., Gjermansen, C., Kielland-Brandt, M.C., Petersen, J.G.L. and Holmberg, S. (1981) Genetic di¡erences between Sac-
321
charomyces carlsbergensis and S. cerevisiae. Analysis of chromosome III by single chromosome transfer. Carlsberg Res. Commun. 46, 65^ 76. Kielland-Brandt, M.C., Nilsson-Tillgren, T., Gjermansen, C., Holmberg, S. and Pedersen, M.B. (1995) Genetics of brewing yeast. In: The Yeasts, 2nd Edn. (Wheals, A.E., Rose, A.H. and Harrison, J.S., Eds.), Vol. 6., pp. 223^254. Academic Press, London, UK. Masneuf, I., Hansen, J., Groth, C., Piskur, J. and Dubourdieu, D. (1998) New hybrids between Saccharomyces sensu stricto yeast species found among wine and cider production strains. Appl. Env. Microbiol. 64, 3887^3892. Groth, C., Hansen, J. and Piskur, J. (1999) A natural chimeric yeast containing genetic material from three species. Int. J. Syst. Bacteriol. 49, 1933^1938. Naumov, G.I., James, S.A., Naumova, E.S., Louis, E.J. and Roberts, I.N. (2000) Three new species in the Saccharomyces sensu stricto complex: Saccharomyces cariocanus, Saccharomyces kudriavzevii and Saccharomyces mikatae. Int. J. Syst. Evol. Microbiol. 5, 1931^ 1942. Rainieri, S., Zambonelli, C., Hallsworth, J.E., Pulvirenti, A. and Giudici, P. (1999) Saccharomyces uvarum, a distinct group within Saccharomyces sensu stricto. FEMS Microbiol. Lett. 177, 177^185. Nguyen, H.-V., Lepingle, A. and Gaillardin, C. (2000) Molecular typing demonstrates homogeneity of Saccharomyces uvarum strains and reveals the existence of hybrids between S. uvarum and S. cerevisiae, including the S. bayanus type strain CBS380. Syst. Appl. Microbiol. 23, 71^85. Casaregola, S., Nguyen, H.-V., Lapathitis, G., Kotyk, A. and Gaillardin, C. (2001) Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization. Int. J. Syst. Appl. Microbiol. 51, 1607^1618. Nilsson-Tillgren, T., Gjermansen, C., Holmberg, S., Petersen, J.G.L. and Kielland-Brandt, M.C. (1986) Analysis of chromosome V and the ILV1 gene from Saccharomyces carlsbergensis. Carlsberg Res. Commun. 51, 309^326. Holmberg, S. (1982) Genetic di¡erences between Saccharomyces carlsbergensis and S. cerevisiae II. Restriction endonuclease analysis of genes in chromosome III. Carlsberg Res. Commun. 47, 233^244. Pedersen, M.B. (1985) DNA sequence polymorphism in the genus Saccharomyces, II. Analysis of the genes RDN1, HIS4, LEU2 and Ty transposable elements in Carlsberg, Tuborg and 22 Bavarian brewing strains. Carlsberg Res. Commun. 50, 263^272. Petersen, J.G.L., Nilsson-Tillgren, T., Kielland-Brandt, M.C., Gjermansen, C. and Holmberg, S. (1987) Structural heterozygosis at genes ILV2 and ILV5 in Saccharomyces carlsbergensis. Curr. Genet. 12, 167^174. Casey, G.P. (1986) Molecular and genetic analysis of chromosomes X in Saccharomyces carlsbergensis. Carlsberg Res. Commun. 51, 343^ 362. Casey, G.P. (1986) Cloning and analysis of two alleles of the ILV3 gene from Saccharomyces carlsbergensis. Carlsberg Res. Commun. 51, 327^341. Gjermansen, C. (1991) Comparison of Genes in Saccharomyces cerevisiae and Saccharomyces carlsbergensis. Ph.D. Thesis. University of Copenhagen, Copenhagen. Fujii, T., Nagasawa, N., Iwamatsu, A., Bogaki, T., Tamai, Y. and Hamachi, M. (1994) Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene. Appl. Env. Microbiol. 60, 2786^2792. Hansen, J. and Kielland-Brandt, M.C. (1994) Saccharomyces carlsbergensis contains two functional MET2 alleles similar to homologues from S. cerevisiae and S. monacensis. Gene 140, 33^40. Hansen, J., Cherest, H. and Kielland-Brandt, M.C. (1994) Two divergent MET10 genes, one from Saccharomyces cerevisiae and one from Saccharomyces carlsbergensis, encode the K subunit of sul¢te reductase and specify potential binding sites for FAD and NADPH. J. Bacteriol. 176, 6050^6058.
FEMSYR 1439 7-3-02
322
P.F. Johannesen, J. Hansen / FEMS Yeast Research 1 (2002) 315^322
[19] Thomas, D. and Surdin-Kerjan, Y. (1997) Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 61, 503^532. [20] Woodcock, D.M., Crowther, P.J., Doherty, J., Je¡erson, S., DeCruz, E., Noyer-Weidner, M., Smith, S.S., Michael, M.Z. and Graham, M.W. (1989) Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 17, 3469^3478. [21] Sherman, F. (1991) Getting started with yeast. In: Methods in Enzymology 194 : Guide to Yeast Genetics and Molecular biology (Guthrie, C. and Fink, G.R., Eds.), pp. 3^21. Academic Press, San Diego, CA. [22] Cherest, H. and Surdin-Kerjan, Y. (1992) Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway. Genetics 130, 51^58. [23] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. [24] Ho¡man, C.S. and Winston, F. (1987) A ten-minute DNA preparation from yeast e¤ciently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267^272. [25] Pedersen, M.B. (1986) DNA sequence polymorphism in the genus Saccharomyces IV. Homoeologous chromosomes III of Saccharomyces bayanus, S. carlsbergensis, and S. uvarum. Carlsberg Res. Commun. 51, 185^202. [26] Sikorski, R.S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strain designed for e¤cient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19^27. [27] Yanisch-Perron, C., Vieria, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33, 103^119. [28] Zuker, M., Mathews, D.H. and Turner, D.H. (1999) Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide In RNA Biochemistry and Biotechnology (Barciszewski,
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
J. and Clark, B.F.C., Eds.), pp. 11^43, NATO ASI Series. Kluwer Academic Publishers BV. Mathews, D.H., Sabina, J., Zuker, M. and Turner, D.H. (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911^940. Korch, C., Mountain, H.A. and Bystro«m, A.S. (1991) Cloning, nucleotide sequence, and regulation of MET14, the gene encoding the APS kinase of Saccharomyces cerevisiae. Mol. Gen. Genet. 229, 96^ 108. Blaiseau, P.-L. and Thomas, D. (1998) Multiple transcriptional activation complexes tether the yeast activator Met4 to DNA. EMBO J. 17, 6327^6336. Thomas, D., Cherest, H. and Surdin-Kerjan, Y. (1989) Elements involved in S-adenosylmethionine-mediated regulation of the Saccharomyces cerevisiae MET25 gene. Mol. Cell. Biol. 9, 3292^3298. Thomas, D., Jacquemin, I. and Surdin-Kerjan, Y. (1992) MET4, a leucine zipper protein, and centromere-binding factor 1 are both required for transcriptional activation of sulfur metabolism in Saccharomyces cerevisiae. Mol. Cell. Biol. 12, 1719^1727. Blaiseau, P.-L., Isnard, A.-D., Surdin-Kerjan, Y. and Thomas, D. (1997) Met31p and Met32p, two related zink ¢nger proteins, are involved in transcriptional regulation of yeast sulfur amino acid metabolism. Mol. Cell. Biol. 17, 3640^3648. Kodama, Y., Omura, F. and Ashikari, T. (2001) Isolation and characterization of a gene speci¢c to lager brewing yeast that encodes a branched-chain amino acid permease. Appl. Env. Microbiol. 67, 3455^3462. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95^98.
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