Nucleotide sequence and characterization of the Saccharomyces cerevisiae RPL19A gene encoding a homolog of the mammalian ribosomal protein l19

A gene designated RPL19A has been identified in the region downstream from the 3′-end of the Saccharomyces cerevisiae MIS1 gene encoding the mitochondrial C₁-tetrahydrofolate synthase. The gene codes for the yeast ribosomal protein YL19 which exhibits 57·5% identity with the mammalian ribosomal protein L19. RPL19A is one of two functional copies of the YL19 gene located on chromosome II. The disruption of RPL19A has no effect on the growth of the yeast. The RPL19A gene contains an intron located near the 5′-end. The 5′-flanking region contains one similar and one complete UAS_rpg upstream activating sequence. RPL19A was also found to be adjacent to the chromosome II AAC3 gene, encoding the mitochondrial ADP/ATP carrier protein. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^tm/EMBL data bank with the accession number Z36751.     

In vivo cloning by homologous recombination in yeast using a two-plasmid-based system

In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient ‘recombination in vivo’ technique. Two sets of vectors were developed. The first set, called ‘expression vectors’, contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloning in these vectors of a DNA fragment generates a ‘transfer vector’ which is compatible with the second set of E. coli–yeast shuttle vectors. This set of ‘recombination vectors’ contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host. Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 μm replicon. Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E. coli for sequencing or site-directed mutagenesis. The sequence presented (Figure 1a) has been entered in the EMBL data library under Accession Number Z48747.     

Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae

Fluorescent proteins are convenient tools for measuring protein expression levels in the budding yeast Saccharomyces cerevisiae. Co‐expression of proteins from distinct vectors has been seen by fluorescence microscopy; however, the expression of two fluorescent proteins on the same vector would allow for monitoring of linked events. We engineered constructs to allow dicistronic expression of red and green fluorescent proteins and found that expression levels of the proteins correlated with their order in the DNA sequence, with the protein encoded by the 5′‐gene more highly expressed. To increase expression levels of the second gene, we tested four regulatory elements inserted between the two genes: the IRES sequences for the YAP1 and p150 genes, and the promoters for the TEF1 gene from both S. cerevisiae and Ashbya gossypii. We generated constructs encoding the truncated ADH1 promoter driving expression of the red protein, yeast‐enhanced Cherry, followed by a regulatory element driving expression of the green protein, yeast‐enhanced GFP. Three of the four regulatory elements successfully enhanced expression of the second gene in our dicistronic construct. We have developed a method to express two genes simultaneously from one vector. Both genes are codon‐optimized to produce high protein levels in yeast, and the protein products can be visualized by microscopy or flow cytometry. With this method of regulation, the two genes can be driven in a dicistronic manner, with one protein marking cells harbouring the vector and the other protein free to mark any event of interest. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural comparison of the Pichia pastoris alcohol oxidase genes

In methylotrophic yeasts, alcohol oxidase is the first enzyme in the methanol-utilization pathway. The genome of one such yeast, Pichia pastoris, contains two alcohol oxidase genes, AOX1 and AOX2. Sequence analysis indicated that each gene encodes a similar protein of 663 amino acids. The protein-coding regions of the genes were 92% and 97% homologous at the nucleotide and predicted amino acid sequence levels, respectively. In contrast to homology observed within the protein-coding portions of the AOX genes, no homology was found in either the 5′ or 3′ non-coding regions. Although alcohol oxidase is found in peroxisomes of P. pastoris, the AOX amino acid sequences did not contain a peptide sequence similar to the peroxisomal transport sequence found at the C-terminus of some peroxisomally located proteins in higher eukaryotes.     

Ribosomal Protein L9 is the Product of GRC5, a Homolog of the Putative Tumor Suppressor QM in S. cerevisiae

Genes encoding members of the highly conserved QM family have been identified in eukaryotic organisms from yeast to man. Results of previous studies have suggested roles for QM in control of cell growth and proliferation, perhaps as a tumor suppressor, and in energy metabolism. We identified recessive lethal alleles of the Saccharomyces cerevisiae QM homolog GRC5 that increased GCN4 expression when present in multiple copies. These alleles encode truncated forms of the yeast QM protein Grc5p. Using a functional epitope-tagged GRC5 allele, we localized Grc5p to a 60S fraction that contained the large ribosomal subunit. Two-dimensional gel analysis of highly purified yeast ribosomes indicated that Grc5p corresponds to 60S ribosomal protein L9. This identification is consistent with the predicted physical characteristics of eukaryotic QM proteins, the highly biased codon usage of GRC5, and the presence of putative Rap1p-binding sites in the 5′ sequences of the yeast GRC5 gene. © 1997 John Wiley & Sons, Ltd.     

A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences

Expression/shuttle vectors for the yeast Saccharomyces cerevisiae have usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number in Escherichia coli, and all have the yeast TRP1 gene, and the 2 μm origin including REP3 sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximal EcoRI site and a unique promoter distal XhoI site, allowing for directional cloning and expression of ‘ZAP’-type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters, ADH2, PGK, GAL10 and SV40 were compared for their relative activity, both in E. coli and in yeast. All yeast promoters showed substantial activity in E. coli with ADH2 showing the highest activity. ADH2 also was well-regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site-directed mutagenesis.     

X. Yeast sequencing reports. Sequence and function analysis of a 9·46 kb fragment of Saccharomyces cerevisiae chromosome X

In the framework of the European yeast genome sequencing project, we have determined the nucleotide sequence of the cosmid clone 233 provided by F. Galibert (Rennes Cedex, France). We present here 9464 base pairs of this cosmid located on the left arm of Saccharomyces cerevisiae chromosome X. This sequence contains two new open reading frames and includes the published sequences of the RADH gene (also identified as SRS2/HPR5) and the 3′-end of the gene BCK1/SLK1/SSP31. Deletion mutants of the two unknown genes J0909 and J0911 are viable. The sequence has been deposited in the EMBL data library under accession number X77087.     

Vectors for Cu2+-inducible production of glutathione S-transferase-fusion proteins for single-step purification from yeast

We describe six new yeast episomal vectors which encode glutathione S-transferase (GST) affinity tags. These allow for the production of GST-fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione-Sepharose permits convenient purification of the fusion protein from a yeast lysate. The presence of a protease cleavage site facilitates subsequent removal of the GST tag. The expression and single-step purification of both GST and a functional GST-metallothionein fusion from yeast are shown as an example of the application of these vectors.     

Investigation of two yeast genes encoding putative isoenzymes of phosphoglycerate mutase

Our previous data indicated that GPM1 encodes the only functional phosphoglycerate mutase in yeast. However, in the course of the yeast genome sequencing project, two homologous sequences, designated GPM2 and GPM3, were detected. They have been further investigated in this work. Key residues in the deduced amino acid sequence, shown to be involved in catalysis for Gpm1 (i.e. His8, Arg59, His181) are conserved in both enzymes. Overexpression of the genes under control of their own promoters in a gpm1 deletion mutant did not complement for any of the phenotypes. This could in part be attributed to a lack of expression due to their weak promoters. Higher level expression under the control of the yeast PFK2 promoter partially complemented the gpm1 defects, without restoring detectable enzymatic activity. Nevertheless, deletion of either GPM2 or GPM3, or the two deletions in concert, did not produce any obvious lesions for growth on a variety of different carbon sources, nor did they change the levels of key intermediary metabolites. We conclude that both genes evolved from duplication events and that they probably constitute non-functional homologues in yeast.     

YHp as a highly stable,hyper-copy,hyper-expression plasmid constructed using a full 2-μm circle sequence in cir0 strains of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2-μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir⁰ strain as a host for constructing an entire 2-μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2-μm plasmid contains two long inverted repeats, which is problematic for the amplification by polymerase chain reaction. Therefore, we amplified it by dividing into two fragments, each containing a single repeat together with an overlapping sequence for homologous recombination. TDH3 promoter-driven yEmRFP (TDH3p-yEmRFP) and the URA3 were used as a reporter gene and a selection marker, respectively, and inserted at the 3′ end of the RAF1 gene on the 2-μm plasmid. The three fragments were combined and used for the transformation of sake yeast cir⁰ ura3^- strain. The resulting transformant colonies showed a red or purple coloration, which was significantly stronger than that of the cells transformed with YEp-TDH3p-yEmRFP. The 2-μm transformants were cultured in YPD medium and observed by fluorescence microscopy. Almost all cells showed strong fluorescence, suggesting that the plasmid was preserved during nonselective culture conditions. The constructed plasmid maintained a high copy state similar to that of the natural 2-μm plasmid, and the red fluorescent protein expression was 54 fold compared with the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.     

XIV. Yeast sequencing reports. A 43·5 kb segment of yeast chromosome XIV,which contains MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1, predicts an adenosine deaminase gene and 14 new open reading frames

A 43,481 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae was sequenced. A gene for tRNA^phe and 23 non-overlapping open reading frames (ORFs) were identified, seven of which correspond to known yeast genes: MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1. One ORF may correspond to the yet unindentified yeast adenosine deaminase gene. Among the 15 other ORFs, four exhibit known signatures, which include a protein tyrosine phosphatase, a cytoskeleton-associated protein and two ATP-binding proteins, four have similarities with putative proteins of yeast or proteins from other organisms and seven exibit no significant similarity with amino acid sequences described in data banks. One ORF is identical to yeast expressed sequence tags (EST) and therefore corresponds to an expressed gene. Six ORFs present similarities to human dbESTs, thus identifying motifs conserved during evolution. Nine ORFs are putative transmembrane proteins. In addition, one overlapping and three antisense ORFs, which are not likely to be functional, were detected. The sequence has been deposited in the EMBL data bank under Accession Number Z46843.     

New vectors for simple and streamlined CRISPR–Cas9 genome editing in Saccharomyces cerevisiae

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double‐strand breaks. Targeting of the DNA break is typically controlled by a single‐guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR–Cas9 technology can be used for efficient, marker‐free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time‐consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9–sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter‐selection using 5‐fluoro‐orotic acid (5‐FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR–Cas9 technology in yeast genome editing. Vectors pT040, pJH001, pML104 and pML107 have been deposited at Addgene ( www.addgene.org ). Copyright © 2015 John Wiley & Sons, Ltd.     

Expression of GFP using Pichia pastoris vectors with zeocin or G‐418 sulphate as the primary selectable marker

Pichia pastoris is a popular host organism for expressing heterologous proteins, and various expression vectors for this yeast are currently available. Recently, vectors containing novel dominant antibiotic resistance markers have become a strong and developing field of research for this methylotropic yeast strain. We have developed new P. pastoris expression vectors, the pPICKanMX6 and pPICKanMX6α series. These vectors were constructed by replacing the zeocin resistance gene of the pPICZA, B, C and pPICZαA, B and C vectors with the Tn903 kan^R marker from pFA6a KanMX6, which confers G‐418 sulphate resistance in P. pastoris. The limits of antibiotic resistance in two transformant yeast strains were investigated, and the selection marker was shown to be stably retained. To demonstrate their usefulness, a gene encoding hexa‐histidine‐tagged green fluorescent protein (GFPH6) was cloned into one of the new vectors and GFP expression examined in P. pastoris cells. The protein expression levels using the pPICKanMX6B vector were comparable with that using the original plasmid, based on zeocin resistance as seen by yeast cell fluorescence. Moreover, GFPH6 was able to be isolated by immobilized metal ion affinity chromatography (IMAC) from lysates of both yeast strains. A model reporter construct has been used to demonstrate successful recombinant protein expression and its subsequent purification using these new vectors. Corresponding vectors can now also be engineered with foreign gene expression under the control of various different promoters, to increase the flexibility of P. pastoris as a cellular factory for heterologous protein production. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of episomal vectors carrying a nourseothricin‐resistance marker for use in minimal media for Schizosaccharomyces pombe

In the post‐genomic era, an immediate challenge is to assign biological functions to novel proteins encoded by the genome. This challenge requires the use of a simple organism as a genetic tool and a range of new high‐throughput techniques. Schizosacchromyces pombe is a powerful model organism used to investigate disease‐related genes and provides useful tools for the functional analysis of heterologous genes. To expand the current array of experimental tools, we constructed two series of Sz. pombe expression vectors, i.e. general and Gateway vectors, containing nourseothricin‐resistance markers. Vectors carrying nourseothricin‐resistance markers possess advantages in that they do not limit the parental strains with auxotrophic mutations with respect to availability for use in clone selection and can be used together with vectors carrying nutrient markers in minimal media. We modified the pSLF173, pSLF273 and pSLF373 vectors carrying a triple haemagglutinin epitope (3×HA) and an Ura4 marker. The vectors described here contain the nmt1 promoter with three different episomal expression strengths for proteins fused with 3×HA, EGFP or DsRed at the N‐terminus. These vectors represent an important contribution to the genome‐wide investigation of multiple heterologous genes and for functional and genetic analysis of novel human genes. Copyright © 2013 John Wiley & Sons, Ltd.     

The human c‐fos and TNFα AU‐rich elements show different effects on mRNA abundance and protein expression depending on the reporter in the yeast Pichia pastoris

AU‐rich elements (AREs) are located in the 3′ untranslated region (3′ UTR) of their host genes and tightly regulate mRNA degradation and expression. Examples for this kind of regulation are the human proto‐oncogene c‐fos and the cytokine TNFα. Despite large effort in this field, the exact mechanism of ARE‐mediated mRNA turnover remains unclear. In this work we analysed the effects of c‐fos‐ and TNFα AREs on mRNA abundance and protein expression of selected human cDNAs in the yeast Pichia pastoris. This yeast is exceedingly well known for its excellent protein production capacity; however, ARE‐like mechanisms have not been studied in this yeast to date. Interestingly, we observed both stabilizing and destabilizing effects of the c‐fos ARE, whereas the TNFα ARE has a destabilizing or expression‐reducing function in all tested cDNAs. Based on this observation, we introduced a number of single‐point mutations upstream of the introduced c‐fos ARE into the 3′ UTR of a single cDNA in order to demonstrate the importance of ARE‐flanking sequences for their own regulation. In conclusion, we illustrate that the analysis of ARE‐mediated effects on mRNA abundance and protein expression of a reporter depends on the sequence of the reporter itself as well as the ARE‐surrounding sequences within the 3′ UTR. For this reason, we question whether already established reporter constructs in other cellular systems display the true type of regulation of the tested AREs for its original host gene. Finally, we propose that AREs should be analysed in their native sequence context. Copyright © 2009 John Wiley & Sons, Ltd.