- Methodology article
- Open Access
A MultiSite GatewayTM vector set for the functional analysis of genes in the model Saccharomyces cerevisiae
- Astrid Nagels Durand1, 2,
- Tessa Moses1, 2, 3, 4,
- Rebecca De Clercq1, 2,
- Alain Goossens†1, 2Email author and
- Laurens Pauwels†1, 2
© Nagels Durand et al.; licensee BioMed Central Ltd. 2012
- Received: 19 March 2012
- Accepted: 17 September 2012
- Published: 20 September 2012
Recombinatorial cloning using the GatewayTM technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in GatewayTM compatible vectors. The MultiSite GatewayTM system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast Saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.
Here, we present a set of three-fragment MultiSite GatewayTM destination vectors that have been developed for gene expression in S. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins.
Our vectors make MultiSite GatewayTM cloning accessible in S. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research.
- Gateway cloning
- Saccharomyces cerevisiae
- Fusion protein
- Epitope tag
- Arabidopsis thaliana
The model organism Saccharomyces cerevisiae has contributed greatly to our current understanding on eukaryotic genes, their products, and their functions. Decades of study have resulted in an extensive knowledge on yeast physiology, genetics, and the molecular functions and interactions of its proteins. Furthermore, this unicellular eukaryotic system is well suited for the study of basic cellular processes which are often conserved in higher eukaryotes. Because of its ease for genetic modification and fast growth, yeast became the system of choice for in vivo protein analyses from other eukaryotes. S. cerevisiae was used, for example, to perform proteome-wide analysis of the human protein-protein interaction networks , to systematically analyze protein-DNA interaction networks of the nematode C. elegans, and to produce high-value bioactive plant secondary metabolites through metabolic engineering approaches .
Single-segment pDEST vectors are available for virtually all commonly used systems, such as Drosophila . Moreover, for plants an extensive repertoire of pDEST for two- and three-segment MultiSite GatewayTM has been established . Three-segment MultiSite GatewayTM destination vectors are also available for Gram-positive bacteria . In S. cerevisiae, single-segment pDEST vectors are available for yeast two-hybrid screens (Invitrogen), and two-segment (promoter::ORF) MultiSite GatewayTM vectors have been described . An extensive set of single-fragment GatewayTM vectors was constructed, allowing N-terminal fusions with four fluorescent tags, and C-terminal fusions with five different fluorescent tags, an affinity tag and an epitope tag under the control of the inducible GAL1 or constitutive GPD promoter . Alternatively, tags that are not present in this vector set can be fused to the gene of interest through 2-step PCR fusion  before performing the BP reaction. However, to our knowledge no three-segment MultiSite GatewayTM pDEST vectors exist for S. cerevisiae to date. As a consequence, a large number of commonly used protein tags already available as pENTR clones are not readily applicable in the organism that - together with E. coli - is the workhorse of molecular biology.
In this paper we present a set of eleven functionally validated three-segment MultiSite GatewayTM pDEST vectors for use in S. cerevisiae. The vector set features the four most commonly used auxotrophic markers for selection of yeast transformants combined with three different replication mechanisms. The presence of the CYC1 terminator in these vectors allows construction of any promoter::ORF:tag combination. In addition, a number of useful entry clones harbouring commonly used yeast promoters and entry clones with protein tags to be used with these pDESTs are presented and validated. These vectors have been appended to our collection of ‘Gateway™ vectors for functional studies’ and can be ordered through the website http://gateway.psb.ugent.be/. Finally, we illustrate the applicability of this vector set by confirming the formation of a ternary complex between the jasmonate ZIM-domain (JAZ), the Novel Interactor of JAZ (NINJA) and the TOPLESS (TPL) proteins, with NINJA acting as a bridging protein. This complex was previously shown to be involved in jasmonate signalling in the model plant Arabidopsis (Arabidopsis thaliana) .
Construction of MultiSite GatewayTM vectors
Nomenclature of the S. cerevisiae MultiSite Gateway TM pDEST vectors
Testing the versatility of the system
Another advantage of the MultiSite GatewayTM system is that several different tag sequences are readily available (Figure 2) and can easily be introduced to acquire translational fusions, since the att sites do not disturb the reading frame. For proof-of-concept, we cloned different plant genes (without STOP codon) in a C-terminal translational fusion with either a V5, c-myc, or FLAG-HIS tag. To make these constructs compatible with protein interaction studies in yeast, we additionally fused each epitope tag with a nuclear localization signal (NLS) derived from SV40, thereby creating the NLS-3xV5, NLS-3xc-myc, and NLS-3xFLAG-6xHIS tags, respectively. Such constructs allow avoiding false negative experimental outcomes that result from protein localisation in different cellular compartments, for instance. Tagged plant proteins were expressed in yeast under the control of the constitutive GPD or ADH1 promoters in different vectors from our set. Total protein extracts were obtained and the expressed proteins were visualized through immunoblot analysis (Figure 3B). The (NLS)FLAG-HIS tag is particularly suitable when protein purification under denaturing conditions is needed, while, at the same time, low background detection on immunoblots is desired.
To express C-terminal translational fusions by means of our promoters, tags and destination vectors, entry vectors containing the GOI should be cloned with a start codon and without a stop codon, and the manufacturer’s guidelines regarding the reading frame should be adopted (http://www.invitrogen.com). Organisms for which ORFeome collections contain vectors that match these criteria include several bacterial collections (Brucella melitensis, Pseudomonas aeruginosa, Bacillus anthracis, Francisella tularensis, Helicobacter pylori, Mycobacterium tuberculosis, Rickettsia prowazekii, Staphylococcus aureus, Streptococcus pneumoniae, Vibrio cholerae), human and mouse , plants (Arabidopsis , maize, sorghum, sugarcane, rice ), viruses  and yeasts (S. cerevisiae and Schizosaccharomyces pombe). Caution is advised since some ORFeome collections contain entry clones both with and without stop codon [15, 16]. ORFeome collections that comprise only GOI’s provided with a stop codon, as is the case for some bacterial and viral ORFeomes (Neisseria gonorrhoeae, Sinorhizobium meliloti, Yersinia pestis, and Hepatitis C ) are not compatible with our vectors. Other ORFeomes in which the GOI is cloned without a start and without a stop codon (Caenorhabditis elegans[24, 25], E. coli), are not compatible with our current promoters but can be used for expression of C-terminal translational fusions with our vectors provided the promoter is cloned followed by a start codon. Since this ORFeome list is non-exhaustive and, as described above, different approaches are used when cloning the GOI, the compatibility of available pENTR collections should always be corroborated either in silico before assembling the different sequences into expression vectors or through epitope tag detection in immunoblots. Finally, the destination vector set presented here also allows expression of N-terminal fusions, provided the att sites flanking the different fusion components are adapted accordingly.
Value of the vector set
The value of this vector set was exemplified by a yeast three-hybrid (Y3H) experiment, in which we investigated the formation of the ternary protein complex by the Arabidopsis JAZ3, NINJA, and TPL proteins. We have proposed recently that the adaptor protein NINJA bridges the JAZ proteins to TPL proteins and thereby forms a repressor complex that blocks the cellular programs regulated by the jasmonates, ubiquitous plant hormones that regulate various aspects of plant growth, development, and survival . TPL interacts with the ETHYLENE RESPONSIVE FACTOR–associated amphiphilic repression (EAR) motif that is present in NINJA  but absent in most JAZ proteins, including JAZ3. Some JAZ proteins however, i.e. JAZ5 to JAZ8, contain EAR motifs themselves and are capable of direct interaction with TPL [34–36].
The N-terminal domain of TPL contains the LisE and CTHL domains and was previously shown to be essential for binding the EAR motif in Aux/IAA proteins . Therefore we cloned this part (denominated TPL-N) as a bait protein for Y2H. In agreement with the proposed models, the interaction of TPL-N with NINJA was confirmed but TPL-N could not interact with JAZ3 (Figure 3C).
Commonly used Y2H vectors such as pGADT7 and pGBKT7 (Clontech) are designed such that the bait and prey fusion proteins are targeted to the same subcellular compartment (i.e. nucleus) and are equipped with an epitope tag, HA and c-myc, respectively, allowing easy confirmation of expression through immunoblot. To verify whether NINJA can connect EAR-lacking JAZs with TPL, as previously proposed , we performed a Y3H assay in which we expressed NINJA, under control of a constitutive promoter (pGPD), as a bridging protein. Hereby we used the MultiSite GatewayTM vector pMG426 (Table 1) that carries the URA3 auxotrophic marker that is often still available in yeast strains used for Y2H. As a C-terminal tag we used the NLS-FLAG-HIS tag. Only when NINJA is co-expressed, yeast growth was observed on selective –His medium, indicating that interaction between JAZ3 and TPL requires the involvement of NINJA (Figure 3C-D).
We have successfully constructed a set of three-segment MultiSite GatewayTM destination vectors for S. cerevisiae. Our findings make high-throughput recombinatorial cloning of multiple genetic segments in one single reaction accessible in one of the most widely used experimental model systems in molecular biology. The availability of different auxotrophic markers in this vector set, together with the large amount of existing compatible building blocks for MultiSite GatewayTM cloning already available in several research groups, creates a versatile utility for these vectors. In addition, we have cloned two constitutive and one inducible yeast promoter in appropriate pENTR vectors and constructed three novel epitope tags, each including a NLS, which are suitable for interaction studies in yeast.
The usefulness of the MultiSite GatewayTM vectors was demonstrated in a Y3H assay with which we corroborated the hypothesis that NINJA connects the JAZ proteins with the co-repressor TPL. This trimeric complex mediates repression of jasmonate responsive genes in the absence of the hormone .
Implementation of the vector set presented in this article, together with the cloning of more promoters and (epitope) tags according to personal experimental needs, will facilitate gene functional studies and contribute to the high-throughput versatile expression of heterologous (plant) proteins in yeast.
Strains and growth conditions
The E. coli strains used were either the ccdB resistant strain DB3.1 (Invitrogen) or the ccdB sensitive strain DH5α. Both were grown at 37°C in LB broth medium with appropriate antibiotics. Several different commonly used yeast lab-strains were grown at 30°C in synthetic defined medium (Clontech) lacking the appropriate amino acids.
MultiSite GatewayTM cloning and yeast transformation
MultiSite LR reactions were performed in 10 μL total volume containing 10 fmoles of each entry vector, 20 fmoles of destination vector, and 2 μL LR II ClonaseTM Plus (Invitrogen). The reaction was incubated overnight at 25°C. After proteinase treatment, the mix was transformed into E. coli DH5α. Colonies that grew on selective medium were picked and the insert was sequenced using M13 forward and reverse primers (Additional file 1). To maintain the reading frame, necessary for expression of translational fusions, MultiSite GatewayTM cloning was carried out according to the manufacturer’s guidelines (http://www.invitrogen.com). Note that in order to produce C-terminal translational fusions, the ORFs used should be without STOP codon. A convenient method to obtain simultaneously clones of ORFs with and without STOP codon has been described .
Competent yeast cells were transformed using the LiAc/SS carrier DNA/PEG method .
The attB4 and attB1 sites were introduced in the primers used for promoter amplification (Additional file 1). A PCR was performed using Phusion High-Fidelity PCR Kit (Thermo Fisher Scientific) on 50 ng of pBEVY-A, pBEVY-GL , and pGAD424 (Clontech) as template for the GPD, GAL, and ADH1 promoters, respectively. PCR products were purified with the GeneJET Gel Extraction kit (Fermentas). BP reactions were performed in a total volume of 5 μl containing 1 μl enzyme, 300 ng pDONR P4-P1R (Invitrogen), and 30 ng of PCR product. Incubation and subsequent treatments were the same as those for MultiSite LR reactions.
Epitope-tag design and immunoblot analysis
Synthetic DNA encoding NLS-3xFLAG-6xHIS, NLS-3xV5, and NLS-3xc-myc flanked by attB2R-attB3 sites were designed in Vector NTI® (Invitrogen) and ordered from GenScript as clones in the pUC57 vector. These tags were introduced into pDONR P2R-P3 through a BP reaction. The resulting entry vectors were transformed into E. coli and sequence verified.
Total yeast protein extracts were obtained as described  and concentration quantified using the Bio-Rad Protein Assay (Bio-Rad). Samples were combined with 5x Laemmli loading buffer and denatured for 10 min at 95°C. Subsequently, 30 μg total protein was loaded on a 4–15% Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad) and transferred to a PVDF membrane using the Trans-Blot Turbo transfer system (Bio-Rad). Detection was performed using the following primary antibodies: anti-FLAG (Sigma), anti-c-myc-HRP (Invitrogen), anti-HA (Roche), and anti-V5 (Sigma).
Yeast two- and three-hybrid
The primers were designed to clone the ORF corresponding to TPL-N with and without STOP codon (Additional file 1) . The entry clones pEN-L4-GPD-R1, pEN-R2-NLS-3xFLAG-6xHis-L3, and pEN-L1-NINJA-L2 were recombined by MultiSite GatewayTM LR reaction with pMG426 as destination vector. Construction of the pGADT7- and pGBKT7-clones, and the Y2H and Y3H were carried out as described  except that transformed yeast cells (strain PJ69-4a) were selected on SD-Ura-Trp-Leu.
We thank Mansour Karimi for useful discussion. This work was supported by the Research Foundation-Flanders through the projects GA13111N and G005312N, and a postdoctoral fellowship to LP. This work was supported by the European FP7 project SmartCell (222716).
- Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N: Towards a proteome-scale map of the human protein-protein interaction network. Nature. 2005, 437: 1173-1178.View ArticlePubMedGoogle Scholar
- Deplancke B, Mukhopadhyay A, Ao W, Elewa AM, Grove CA, Martinez NJ, Sequerra R, Doucette-Stamm L, Reece-Hoyes JS, Hope IA: A gene-centered C. elegans protein-DNA interaction network. Cell. 2006, 125: 1193-1205.View ArticlePubMedGoogle Scholar
- Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J: Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature. 2006, 440: 940-943.View ArticlePubMedGoogle Scholar
- Hartley JL, Temple GF, Brasch MA: DNA cloning using in vitro site-specific recombination. Genome Res. 2000, 10: 1788-1795.View ArticlePubMedPubMed CentralGoogle Scholar
- Karimi M, Bleys A, Vanderhaeghen R, Hilson P: Building blocks for plant gene assembly. Plant Physiol. 2007, 145: 1183-1191.View ArticlePubMedPubMed CentralGoogle Scholar
- Petersen LK, Stowers RS: A gateway multisite recombination cloning toolkit. PLoS One. 2011, 6: e24531-View ArticlePubMedPubMed CentralGoogle Scholar
- Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, Parant JM, Yost HJ, Kanki JP, Chien CB: The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Developmental dynamics: an official publication of the American Association of Anatomists. 2007, 236: 3088-3099.View ArticleGoogle Scholar
- Benhamed M, Martin-Magniette ML, Taconnat L, Bitton F, Servet C, De Clercq R, De Meyer B, Buysschaert C, Rombauts S, Villarroel R: Genome-scale Arabidopsis promoter array identifies targets of the histone acetyltransferase GCN5. The Plant journal: for cell and molecular biology. 2008, 56: 493-504.View ArticleGoogle Scholar
- Van Leene J, Witters E, Inzé D, De Jaeger G: Boosting tandem affinity purification of plant protein complexes. Trends in plant science. 2008, 13: 517-520.View ArticlePubMedGoogle Scholar
- Van Leene J, Stals H, Eeckhout D, Persiau G, Van De Slijke E, Van Isterdael G, De Clercq A, Bonnet E, Laukens K, Remmerie N: A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana. Molecular & cellular proteomics: MCP. 2007, 6: 1226-1238.View ArticlePubMedGoogle Scholar
- Burckstummer T, Bennett KL, Preradovic A, Schutze G, Hantschel O, Superti-Furga G, Bauch A: An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nature methods. 2006, 3: 1013-1019.View ArticlePubMedGoogle Scholar
- Dricot A, Rual JF, Lamesch P, Bertin N, Dupuy D, Hao T, Lambert C, Hallez R, Delroisse JM, Vandenhaute J: Generation of the Brucella melitensis ORFeome version 1.1. Genome Res. 2004, 14: 2201-2206.View ArticlePubMedPubMed CentralGoogle Scholar
- Labaer J, Qiu Q, Anumanthan A, Mar W, Zuo D, Murthy TV, Taycher H, Halleck A, Hainsworth E, Lory S, Brizuela L: The Pseudomonas aeruginosa PA01 gene collection. Genome Res. 2004, 14: 2190-2200.View ArticlePubMedPubMed CentralGoogle Scholar
- Pathogen functional genomics resource center.http://pfgrc.jcvi.org/,
- The ORFeome collaboration.http://www.orfeomecollaboration.org/,
- ATOME 1 and ATOME 2.http://www-urgv.versailles.inra.fr/atome/index.htm,
- Yilmaz A, Nishiyama MY, Fuentes BG, Souza GM, Janies D, Gray J, Grotewold E: GRASSIUS: a platform for comparative regulatory genomics across the grasses. Plant physiology. 2009, 149: 171-180.View ArticlePubMedPubMed CentralGoogle Scholar
- von Brunn A, Teepe C, Simpson JC, Pepperkok R, Friedel CC, Zimmer R, Roberts R, Baric R, Haas J: Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome. PloS one. 2007, 2: e459-View ArticlePubMedPubMed CentralGoogle Scholar
- Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H: Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes & development. 2005, 19: 2816-2826.View ArticleGoogle Scholar
- Matsuyama A, Arai R, Yashiroda Y, Shirai A, Kamata A, Sekido S, Kobayashi Y, Hashimoto A, Hamamoto M, Hiraoka Y: ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nature biotechnology. 2006, 24: 841-847.View ArticlePubMedGoogle Scholar
- Brettin T, Altherr MR, Du Y, Mason RM, Friedrich A, Potter L, Langford C, Keller TJ, Jens J, Howie H: Expression capable library for studies of Neisseria gonorrhoeae, version 1.0. BMC microbiology. 2005, 5: 50-View ArticlePubMedPubMed CentralGoogle Scholar
- Schroeder BK, House BL, Mortimer MW, Yurgel SN, Maloney SC, Ward KL, Kahn ML: Development of a functional genomics platform for Sinorhizobium meliloti: construction of an ORFeome. Applied and environmental microbiology. 2005, 71: 5858-5864.View ArticlePubMedPubMed CentralGoogle Scholar
- de Chassey B, Navratil V, Tafforeau L, Hiet MS, Aublin-Gex A, Agaugue S, Meiffren G, Pradezynski F, Faria BF, Chantier T: Hepatitis C virus infection protein network. Molecular systems biology. 2008, 4: 230-View ArticlePubMedPubMed CentralGoogle Scholar
- Lamesch P, Milstein S, Hao T, Rosenberg J, Li N, Sequerra R, Bosak S, Doucette-Stamm L, Vandenhaute J, Hill DE, Vidal M: C. elegans ORFeome version 3.1: increasing the coverage of ORFeome resources with improved gene predictions. Genome research. 2004, 14: 2064-2069.View ArticlePubMedPubMed CentralGoogle Scholar
- Reboul J, Vaglio P, Rual JF, Lamesch P, Martinez M, Armstrong CM, Li S, Jacotot L, Bertin N, Janky R: C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nature genetics. 2003, 34: 35-41.View ArticlePubMedGoogle Scholar
- Rajagopala SV, Yamamoto N, Zweifel AE, Nakamichi T, Huang HK, Mendez-Rios JD, Franca-Koh J, Boorgula MP, Fujita K, Suzuki K: The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology. BMC genomics. 2010, 11: 470-View ArticlePubMedPubMed CentralGoogle Scholar
- Akbari OS, Oliver D, Eyer K, Pai CY: An Entry/Gateway cloning system for general expression of genes with molecular tags in Drosophila melanogaster. BMC cell biology. 2009, 10: 8-View ArticlePubMedPubMed CentralGoogle Scholar
- Karimi M, Depicker A, Hilson P: Recombinational cloning with plant gateway vectors. Plant physiology. 2007, 145: 1144-1154.View ArticlePubMedPubMed CentralGoogle Scholar
- Perehinec TM, Qazi SN, Gaddipati SR, Salisbury V, Rees CE, Hill PJ: Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria. BMC molecular biology. 2007, 8: 80-View ArticlePubMedPubMed CentralGoogle Scholar
- Cheo DL, Titus SA, Byrd DRN, Hartley JL, Temple GF, Brasch MA: Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones. Genome research. 2004, 14: 2111-2120.View ArticlePubMedPubMed CentralGoogle Scholar
- Alberti S, Gitler AD, Lindquist S: A suite of gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast. 2007, 24: 913-919.View ArticlePubMedPubMed CentralGoogle Scholar
- Atanassov II, Etchells JP, Turner SR: A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps. Plant methods. 2009, 5: 14-View ArticlePubMedPubMed CentralGoogle Scholar
- Pauwels L, Barbero GF, Geerinck J, Tilleman S, Grunewald W, Perez AC, Chico JM, Vanden Bossche R, Sewell J, Gil E: NINJA connects the co-repressor TOPLESS to jasmonate signalling. Nature. 2010, 464: 788-791.View ArticlePubMedPubMed CentralGoogle Scholar
- Causier B, Ashworth M, Guo W, Davies B: The TOPLESS interactome: a framework for gene repression in Arabidopsis. Plant physiology. 2012, 158: 423-438.View ArticlePubMedPubMed CentralGoogle Scholar
- Pauwels L, Goossens A: The JAZ proteins: a crucial interface in the Jasmonate signaling cascade. The Plant cell. 2011, 23: 3089-3100.View ArticlePubMedPubMed CentralGoogle Scholar
- Shyu C, Figueroa P, Depew CL, Cooke TF, Sheard LB, Moreno JE, Katsir L, Zheng N, Browse J, Howe GA: JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis. The Plant cell. 2012, 24: 536-550.View ArticlePubMedPubMed CentralGoogle Scholar
- Szemenyei H, Hannon M, Long JA: TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science. 2008, 319: 1384-1386.View ArticlePubMedGoogle Scholar
- Underwood BA, Vanderhaeghen R, Whitford R, Town CD, Hilson P: Simultaneous high-throughput recombinational cloning of open reading frames in closed and open configurations. Plant biotechnology journal. 2006, 4: 317-324.View ArticlePubMedGoogle Scholar
- Gietz RD, Schiestl RH: High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature protocols. 2007, 2: 31-34.View ArticlePubMedGoogle Scholar
- Miller CA, Martinat MA, Hyman LE: Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expressionvectors with bi-directional promoters for use in Saccharomyces cerevisiae. Nucleic acids research. 1998, 26: 3577-3583.View ArticlePubMedPubMed CentralGoogle Scholar
- Hampton RY, Rine J: Regulated degradation of HMG-CoA reductase, an integral membrane protein of the endoplasmic reticulum, in yeast. The Journal of cell biology. 1994, 125: 299-312.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.