Marker-fusion PCR for one-step mutagenesis of essential genes in yeast.

We describe a one-step gene replacement method based on fusion PCR that can be used to mutagenize essential genes at their endogenous locus. Marker-fusion PCR can facilitate transfer of alleles between strains as well as PCR-based techniques, such as site-directed and error-prone PCR mutagenesis, all without cloning or strain constructions. With this method, PCR is used to fuse a mutagenized fragment to an overlapping fragment containing a selectable marker flanked by regions of homology to the target. By transforming yeast with these PCR products, specific mutations are introduced at the endogenous locus through homologous recombination. We tested the 'marker-fusion PCR' method using the budding yeast CDC28 gene and were able to efficiently introduce site-directed mutations and integrate genomic or plasmid-borne mutant alleles. As a further application for this method, we used a spiked oligonucleotide to randomize the coding sequence for a single domain of CDC28 and were able to construct highly mutagenized libraries for this region.     

A yeast gene encoding a putative RNA helicase of the “DEAD”-box family

An unknown open reading frame from Saccharomyces cerevisiae was identified and sequenced. The predicted amino acid sequence shows high homology to the DEAD-box family of proteins. Gene disruption revealed that the gene is not essential for yeast but necessary for normal cell growth.     

XV. Yeast sequencing reports. Nucleotide sequence of the GDS1 gene of Saccharomyces cerevisiae

We have cloned and sequenced the GDS1 gene located on the right arm of chromosome XV of Saccharomyces cerevisiae. The gene codes for a 522 amino acid serine-rich protein with no obvious homology to proteins in the database. GDS1 gene was isolated as the multicopy suppressor of the glycerol-deficient phenotype caused by the nam9-1 mutation in the yeast nuclear gene encoding the mitochondrial ribosomal protein homologous to S4 proteins from various organisms. Disruption-deletion of the GDS1 open reading frame leads to a partial impairment of growth on medium containing glycerol as the carbon source, indicating mitochondrial function of the gene product. The sequence has been deposited in the GenBank data library under Accession Number U18262.     

The 50:50 method for PCR‐based seamless genome editing in yeast

The ability to edit the yeast genome with relative ease has contributed to the organism being a model eukaryote for decades. Most methods for deleting, inserting or altering genomic sequences require transformation with DNA that carries the desired change and a selectable marker. One‐step genome editing methods retain the selectable marker. Seamless genome editing methods require more steps and a marker that can be used for both positive and negative selection, such as URA3. Here we describe the PCR‐based 50:50 method for seamless genome editing, which requires only two primers, one PCR with a URA3 cassette, and a single yeast transformation. Our method is based on pop‐in/pop‐out gene replacement and is amenable to the facile creation of genomic deletions and short insertions or substitutions. We used the 50:50 method to make two conservative loss‐of‐function mutations in MATα1, with results suggesting that the wild‐type gene has a new function outside of that presently known. Copyright © 2013 John Wiley & Sons, Ltd.     

The 5-aminoimidazole ribonucleotide-carboxylase structural gene of the methylotrophic yeast Pichia methanolica: Cloning,sequencing and homology analysis

The ADE1 gene of the yeast Pichia methanolica encodes phosphoribosyl-5-aminoimidazole-carboxylase (AIRC, EC 4.1.1.21), which is involved in purine biosynthesis. The gene was cloned by complementation of an ade2 mutation in Saccharomyces cerevisiae and a 3077 nucleotide DNA fragment was sequenced. The sequence possessed a single open reading frame, corresponding to a 543 amino acid sequence. The sequence of this putative protein has been compared to the proteins of homologous genes from S. cerevisiae, Schizosaccharomyces pombe, Escherichia coli, chicken and man. The analysis revealed remarkable homology between yeast AIRCs, while for other proteins homology was limited to defined regions.     

A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells

A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.     

Molecular cloning and sequence analysis of the Schizosaccharomyces pombe ade10+ gene

We have cloned and sequenced the Schizosaccharomyces pombe ade10 gene encoding 5-phosphoribosyl-4-carboxamide 5-aminoimidazole transformylase inosine monophosphate cyclohydrolase. The sequence has an uninterrupted open reading frame of 1755 nucleotides corresponding to 585 amino acid residues. The deduced amino acid sequence shows a high degree of similarity to the purH gene product of many species, including Saccharomyces cerevisiae, human, chicken and Escherichia coli. Moreover our data indicate that intrachromosomal recombination in Schiz. pombe is enhanced if the ade10 gene product is defective. The sequence has been submitted to the EMBL data library under Accession Number Y16419. © 1998 John Wiley & Sons, Ltd.     

Sticky-end polymerase chain reaction method for systematic gene disruption in Saccharomyces cerevisiae

We describe a new procedure for the generation of plasmids containing a large promoter and terminator region of a gene of interest, useful for gene disruption. In a two-step polymerase chain reaction (PCR), a fragment, corresponding to the terminator and promoter regions separated by a 16 bp sequence containing a rare restriction site (e.g. AscI), is synthesized (T-P fragment). This PCR fragment is cloned in vectors presenting a rare blunt-end cloning site and a yeast marker for selection in Saccharomyces cerevisiae (TRP1, HIS3 and KanMX). The final plasmids are used directly for gene disruption after linearization by the enzyme (e.g. AscI) specific for the rare restriction site. This approach was used to disrupt three open reading frames identified during the sequencing of COS14–1 from chromosome XIV of S. cerevisiae.     

I. Yeast sequencing reports. Isolation and characterization of the LEU2 gene of Hansenula polymorpha

A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli. The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae. Further downstream, there is a partial reading frame with no obvious similarity to known proteins. The LEU2 gene of H. polymorpha cannot complement the leu2 mutation of S. cerevisiae. The sequence has been entered in the EMBL data library under the Accession Number U00889.     

XV. Yeast sequencing reports. A gene from Saccharomyces cerevisiae which codes for a protein with significant homology to the bacterial 3-phosphoserine aminotransferase

During the sequencing of the gene GSP2 from Saccharomyces cerevisiae, we have encountered an adjacent open reading frame having strong homology to the 3-phosphoserine aminotransferase (E.C.2.6.1.52) from other organisms. In this report, we present the sequence for this yeast SERC, and evidence that its deletion from the yeast genome leads to serine dependency. The sequence has been deposited in the GenBank data library under Accession Number L20917.     

A 37·5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes,a TCP-1-related gene,an open reading frame similar to the DAL80 gene,and a tRNAArg

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromsome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding γ-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Ccη subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA^Arg3 (AGC) gene. The nucleotide sequence data reported in this paper have been deposited in the EMBL and GenBank databases under the accession number X85021.     

The URA5 gene encoding orotate-phosphoribosyl transferase of the yeast Kluyveromyces lactis: cloning, sequencing and use as a selectable marker.

A pair of degenerate primers was used for amplification and cloning of an internal fragment of the K. lactis URA5 gene. Primers were designed on the basis of highly conserved motifs within protein sequences predicted for URA5 genes from several microorganisms. Using the amplified fragment as a probe, we finally cloned and sequenced a 1.9 kb chromosomal fragment containing the orotate-phosphoribosyltransferase-encoding URA5 gene and an incomplete open reading frame strikingly similar to SEC65 of Saccharomyces cerevisiae and other yeasts, in which the gene encodes a subunit of the signal recognition particle. Uracil-requiring mutants of K. lactis CBS 683 were selected on media containing 5-fluoro-orotic acid and used as recipients in transformation experiments using K. lactis URA5 as the selectable marker, thereby proving functionality of the cloned gene.     

Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes. © 1998 John Wiley & Sons, Ltd.     

Molecular cloning and sequence analysis of Zygosaccharomyces rouxii ADE2 gene encoding a phosphoribosyl-aminoimidazole carboxylase.

The nucleotide sequence of a 2.8 kb fragment containing the ADE2 gene of the osmotolerant yeast Zygosaccharomyces rouxii has been determined. The gene was cloned from a Z. rouxii genomic DNA library by complementation of the Saccharomyces cerevisae ade2 mutant strain. The sequenced DNA fragment contains a 1710 bp open reading frame predicting a protein of 570 amino acids. The deduced amino acid sequence shares a high degree of homology with Ade2p homologues in five other yeast species.     

Sequencing and analysis of 51·6 kilobases on the left arm of chromosome XI from Saccharomyces cerevisiae reveals 23 open reading frames including the FAS1 gene

We have sequenced two segments containing a total of 51·6 kb of the left arm from chromosome XI of Saccharomyces cerevisiae. The first segment of 38·5 kb contains 18 open reading frames (ORFs) of more than 100 amino acid residues. Five ORFs encode known yeast genes, including the fatty acid synthase gene (FAS1). Three new yeast genes were discovered with homologies to non-yeast genes and ten new genes without homologies to any known sequences. The second segment of 13 kb contains five ORFs with two known yeast genes and three unknown genes. The sequences from cosmid pUKG041 were obtained entirely with the walking primer strategy resulting in a very low overall sequence redundancy of 2·8 and an average reading length of 443 bases.     

Sequence Analysis of 203 Kilobases from Saccharomyces cerevisiae Chromosome VII

The nucleotide sequences of five major regions from chromosome VII of Saccharomyces cerevisiae have been determined and analysed. These regions represent 203 kilobases corresponding to approximately one-fifth of the complete yeast chromosome VII. Two fragments originate from the left arm of this chromosome. The first one of about 15·8 kb starts approximately 75 kb from the left telomere and is bordered by the SKI8 chromosomal marker. The second fragment covers the 72·6 kb region between the chromosomal markers CYH2 and ALG2. On the right chromosomal arm three regions, a 70·6 kb region between the MSB2 and the KSS1 chromosomal markers and two smaller regions dominated by the KRE11 marker and another one in the vicinity of the SER2 marker were sequenced. We found a total of 114 open reading frames (ORFs), 13 of which were completely overlapping with larger ORFs running in the opposite direction. A total of 44 yeast genes, the physiological functions of which are known, could be precisely mapped on this chromosome. Of the remaining 57 ORFs, 26 shared sequence homologies with known genes, among which were 13 other S. cerevisiae genes and five genes from other organisms. No homology with any sequence in the databases could be found for 31 ORFs. Furthermore, five Ty elements were found, one of which may not be functional due to a frame shift in its Ty1B amino acid sequence. The five chromosomal regions harboured five potential ARS elements and one sigma element together with eight tRNA genes and two snRNAs, one of which is encoded by an intron of a protein-coding gene. © 1997 by John Wiley & Sons, Ltd.     

X. Yeast sequencing reports. Sequence and function analysis of a 9·74 kb fragment of Saccharomyces cerevisiae chromosome X including the BCK1 gene

In the framework of the European BIOTECH project for sequencing the Saccharomyces cerevisiae genome, we have determined the nucleotide sequence of the cosmid clone 233 provided by F. Galibert (Rennes Cedex, France). We present here 9743 base pairs of sequence derived from the left arm of chromosome X. This sequence reveals three new open reading frames and includes the published sequence (5′ end and open reading frame) of the gene BCK1/SLK1/SSP31 also identified as ORFAA. Deletion mutants of two earlier unknown open reading frames J0840 and J0904 are viable and the open reading frame J0902 is essential for yeast growth. The sequence has been entered in the EMBL data library under accession number X77923.     

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre‐existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C‐rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.