Construction of heterozygous diploid deletion mutants
The dataset used was 4914 protein coding genes, based on the annotated nuclear genome sequence (http://www.genedb.org/genedb/pombe 01/04/08). Each gene was deleted using a cassette containing the dominant KanMX gene as a G418 resistant selectable marker (Wach et al., 1994), regions of homology 5' and 3' to the CDS (CoDing Sequence) of interest, and unique uptags and downtags (barcodes) (Figure 1 for details). To generate deletion cassettes two PCR based methods were used, serial extension (s-PCR, Figure 2) and block PCR (b-PCR, Figure 3) (Giaever et al., 2002; Winzeler et al., 1999). For 263 genes (5.4% of the total) deletions could not be constructed using either PCR method and for these an oligo-mediated gene synthesis (GS) method was used (Figure 4). All deletions were constructed in an ade6-M210/ade6-M216, leu1-32/leu1-32, ura4-D18/ura4-D18 h+/h+ strain; a stable nonsporulating diploid, which can be sporulated by conversion to h90 (containing plus and minus information) or by transformation with the plasmid pON177 containing the minus mating type locus (a gift from Olaf Nielsen). Each cassette was transformed into the diploid parent strain replacing one copy of the gene of interest by homologous recombination and the heterozygous deletion diploids were identified by selection for G418 resistance. Deletions were confirmed by PCR and sequencing of the junctions between the deletion cassette and the CDS (Figure 5).
Figure 1: Structure of deletion cassette module
A PCR-based strategy(Ref 1,2) was modified for the construction of genome-wide deletion mutants in fission yeast. The deletion cassette module for gene deletion consists of three parts: a proximal KanMX module, a pair of tag sequences with universal sequencing primer sites, and flanking homologous recombination regions. 1) Drug resistant marker, the kanMX4 gene: The kanamycin resistant KanMX4 gene can be used as a dominant positive selection marker in yeast3, see also ( http://sequence-www.stanford.edu/group/yeast_deletion_project/). KanMX4 confers resistance to geneticin (G418). 2) Tag sequences: To facilitate whole genome analysis, each deletion has been assigned molecular barcodes, a pair of 20-mer unique sequences, which are referred to as the uptag and downtag (For details, see Supplementary Table 1.). These sequences were generated using a BioPERL-based computer program to select the following criteria; melting temperature (Tm)=60°C, no cross-hybridization, no secondary structures, and no similarities to genomic sequence. RNAfold and mfold freeware (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) was used for checking secondary structure and the BLAST program was used for checking similarity with genomic sequence. The concentration of each tag was determined by PCR amplification and subsequent hybridization to a custom-made GeneChip (Affymetrix Co.) of 20-mer oligonucleotide probes complementary to each of the tag sequences. 3) Gene-specific homologous recombination regions: Three different methods have been employed to construct the deletion cassette modules: four-round serial PCR(s-PCR), overlapping block PCR (b-PCR), and oligonucleotide-mediated gene synthesis (GS). For successful homologous recombination in fission yeast flanking regions longer than those used in budding yeast are required4. To do this, a pair of primer sites have been selected from between 500-bp upstream of promoter to 100-bp downstream of ORF or from 100-bp upstream of ORF to 500-bp downstream over the transcriptional stop site. These primers have similar Tm of 60%deg;C and no secondary structures. For certain genes it was difficult to select the optimal primer sites because of a high ratio of A/T in the promoter and terminator regions and this resulted in less than 100% deletion of the open reading frame. For genes that could not be deleted using PCR based methods we constructed the deletion cassette using gene synthesis (GS). The length of flanking regions is 80-bp for s-PCR method, 250~450-bp for b-PCR method, and 250-bp for GS method, respectively. G1, G2, G3, and G4 represent 6-bp gap sequences.
Figure 2: Generation of deletion cassettes using four-round subsequent-PCR method
For the construction of deletion cassettes (see Supplementary Figure 1.) by PCR-based methods, subsequent-PCRs (s-PCR) and block-PCRs (b-PCR, see Supplementary Figure 3.), the KanMX4 module was prepared by PCR amplification using the pFA6a-kanMX4 plasmid as template and a pair of 70-mer primers. Each 70-mer primer contains a 20-mer universal priming sequence (U1 for UPTAG forward or D1 for DNTAG reverse, respectively), a pair of 6-mer gap sequence (G1/G2 or G3/G4), a unique 20-mer TAG sequence, and a 18-mer Kan universal priming sequence homologous to KanMX4 (U2 or D2) see Supplementary Figure. 1. The sequences are as follows; 70-mer UPTAG forward, 5'-CGCTCCCGCCTTACTTCGCA (U1)-tttaaa (G1)-UPTAG (20-mer)-gatatc (G2)-TTAGCTTGCCTCGTCCCC (U2)-3'; 70-mer DNTAG reverse, 5'-TTGCGTTGCGTAGGGGGGAT (D1) -tttaaa (G4)-DNTAG (20-mer)-gatact (G3)-TTTCGACACTGGATGGCG (D2)-3'. Using the KanMX4 module as templates, the second round of PCR was performed using pairs of 50-mer primers containing a 20-mer sequence homologous to the universal priming sequence and a 30-bp for the extension of flanking ends. Using the same procedure, the third round of PCR was performed to extend the flanking ends from 30-bp to 60-bp long. Finally, the fourth round of PCR was performed with a pair of 40-mer primers, resulting in 80-bp long flanking ends. We used s-PCR method to construct the first 1,550 mutants. All primers and Taq Polymerase with proof reading capability were confirmed by Mass-Spectroscopy and supplied by Bioneer Corporation. We have constructed 1,515 mutants using this method.
Figure 3: Overlapping block-PCR(b-PCR) method
In this method, 250~450-bp flanking regions were prepared by two separate PCR-amplifications using 51-mer and 25-mer primers and chromosomal DNA as templates. The 25-mer primer contains a sequence homologous a region flanking of the gene of interest and each 51-mer primer contains the 6-mer gap sequence (G1 or G4), the 20-mer complementary to the universal priming sequence (U1 or D1), and 25-mer sequence homologous to a flanking region of the gene of interest. These primers were used to amplify UP and DN blocks for homologous recombination as shown in step 2 and were then joined together with the common KanMX4 module (from step 1) by block PCR via the 26-bp overlapping sequences (6-mer gap + 20-mer universal priming sequences) using a pair of 25-mer outmost gene-specific primers. We have constructed 3,058 mutants using this method. Click here for an example
Figure 4: Oligonucleotide-mediated gene synthesis method
In some cases, conventional PCR-based methods have been tried but either failed or it was not possible to design the correct PCR primer sets. In these cases an oligonucleotide-mediated GS method has been used to make deletion cassette modules. We designed the oligonucleotide sets in three parts: 5'-linker-5'-Block, KanMX4 core module, and 3'-Block-3'-linker. The 70-bp up- and down-tags were assigned to 5'- and 3'-Blocks instead of the KanMX4 module (see PCR-based methods). Block2Oligo was used to design 20~40-mer oligonucleotide sequences with Tm of 51~58°C. The Batch_Block2Oligo program was used for joining the three blocks together via overlapping oligonucleotides located in junction parts (see step 2 in Supplementary Figure 3). The 5' termini were phosphorylated by T4 polynucleotide kinase and ligated by 40 cycles of ligation-chain reactions (LCR) using thermostable Tfi DNA ligase (Bioneer). The whole cassette was then amplified through two rounds of PCR to generate sufficient DNA for transformation. This PCR step used 46-bp universal, artificial linkers in flanking regions of the deletion, which were then eliminated by treatment with Bsa I before transformation. The linker sequences are as follows; 46-bp 5' linker, 5'-TGCAATGCGTTGGGCAACAA (for first PCR) -TTACGCTTGTGCAAACGCCA (for second PCR)-GGTCTC (Bsa I)-3'; 46-bp 3' linker, 5'-GAGACC (Bsa I)-TTTTAATGCGCGCCCTTGCA (for first PCR)-TCATAACGCGTTTGCGGCAT (for second PCR)-3'. So far, 263 mutants have been constructed by this method.
Figure 5: Verification of deletion mutants by colony PCR method
Colony PCR was used to verify integration of the deletion cassette at the correct locus, using genomic DNA from G418-resistant colonies as a template and with two gene-specific primers (cp5 and cp3) and four pairs of primers from KanMX module: 5'-CGTCTGTGAGGGGAGCGTTT-3' (CPN1), 5'-GATGTGAGAACTGTATCCTAGCAAG-3' (CPN10), 5'-TGATTTTGATGACGAGCGTAAT-3'(CPC1), 5'-GGCTGGCCTGTTGAACAAGTCTGGA-3' (CPC3). The cp5 primer is located from 400 to 600-bp upstream of the start codon, whereas the cp3 primer from 400 to 600-bp downstream of the stop codon. For successful deletions, PCR reactions with a pair of primers, the cp5-CPN1 (or CPN10) and the cp3-CPC1 (or CPC3), gave a PCR product of the correct size. We then used dideoxy sequencing of the PCR product from each successful deletion mutant to confirm the sequences of up- and down-tags as well as across the junctions to accurately define the region deleted. If the deletion was not successful and no PCR product was generate we used the cp5i and cp3i primers inside the CDS to confirm that the gene coding sequence was still present. All the confirmed S. pombe deletion mutants have kept as frozen glycerol stocks.
References1. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387-391 (2002).
2. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901-906 (1999).