Diploid deletion mutants in the S. pombe genome were systematically constructed with targeted mutagenesis at each target ORF. The chromosomal location of the ORFs and their DNA sequence information were obtained from the public fission yeast database at the Welcome Trust Sanger Institute (http://www.genedb.org/genedb/pombe/index.jsp). The deletion cassette module construct contains a selection marker (Fig. 1), tag sequences (molecular barcodes), and the sequences for homologous recombination (Fig 2). The deletion cassette modules were constructed by PCR with well-designed primers. The cassettes were transformed into the S. pombe, SP286 diploid host strain (h+/ h+, ade6-M210/ade6-M216 ura4-D18/ura4-D18 leu1-32/leu1-32). Deletion of the target ORF was screened for by G418 antibiotic selection (Fig 3). If chromosomal integration occurs properly via homologous recombination at the target ORF in a colony, that colony would obtain antibiotic resistance from the KanMX4 selection marker gene.

1) Selection marker KanMX:
 The KanMX module was first developed as a dominant drug resistance marker to Geneticin® (G418) by the Philippsen group. This marker is a heterogonous module that contains Kan transposon Tn903 ORF fromin E. coli, and the TEF promoter and terminator gene in the fungus Ashbya gossypii. The nutrient markers typically used for deletion mutants give a high false rate of integration to target region if the PCR products are used, or the marker has low sequence similarity to the target region. In contrast, the KanMX module gives a low false rate of integration due to its dominant resistance. This feature of KanMX makes the deletion mutant constructed highly efficiently.

2) Tag sequences
 Tag sequences of 20 bp unique to each deletion mutant were generated from a pool of 20,000 oligonucleotides. This pool of oligonucleotides has features of homogenous melting temperatures, no cross-hybridization, no secondary structures, and no similarities to ORFs in S. pombe. A pair of tags to each ORF consists of up-tag and down-tag sequences designed to be distinguished from the mutant pool. This tag system is useful for the identification of mutants affected by specific drug conditions, nutrient starvations, or various stresses. In order to screen drug-sensitive mutants, for example, a deletion mutant pool is treated with a certain drug and drug-sensitive mutants are screened using a proper bioassay. Genes corresponding to each causing mutation can be identified by PCR amplification of the tag sequences unique to each gene and the subsequent microarray.

3) Deletion cassette amplification
 We used the four-round serial PCR or multi-block PCR to amplify the deletion cassette modules for mass construction of deletion mutants. Chromosomal target ORFs were replaced by deletion cassette modules using homologous recombination at the S. pombe homology site. A standard lithium acetate method was used for this purpose.

a. Four-round serial PCR
Step 1: The pFA6a-KanMX4 plasmid was used as a template. Primers included a part of Kan ORF, tag sequences, and universal sequences. The KanMX module fragments combining the PCR primers at both terminals were amplified.
Step 2, 3, 4: Template was a product of the previous step. The target ORF and the homologous region were sequentially extended. The deletion cassette was amplified to contain an 80-base homologous region at both sides.

b. Multi-block PCR
In construction of deletion cassettes using multi-block PCR, as in four-round serial PCR, the pFA6a-KanMX4 plasmid was used as template, and the same primer of step 1 in four-round serial PCR was used. The KanMX module product was joined together with two 350-bp fragments into a deletion cassette, which correspond to the 5' and 3' flanking region of target ORF. Finally, the deletion cassette that has KanMX module, tag sequence, and the 5' and 3' flanking region of target ORF, were amplified and used for homologous recombination.

S. pombe Haploid Deletion Mutant Construction

S. pombe haploid mutants have been constructed from diploid mutants as follows: