A PCR-generated (Baudin 1993; Wach 1994) deletion strategy was used to systematically replace each yeast open reading frame from its start- to stop- codon with a KanMX module and two unique 20mer molecular bar codes. The presence of the tags can be detected via hybridization to a high-density oligonucleotide array, enabling growth phenotypes of individual strains to be analyzed in parallel.
Nearly all ORFs larger than 100 codons were disrupted; highly similar ORFs were not attempted (~3%). Four different Yeast Knock-Out (YKO.'s) collections have been generated: haploids of both mating types, homozygous diploids for non-essential genes, and heterozygous diploids, which contain the essential and non essential ORFs.
Approximately 5% of the yeast genome was not knocked out for various reasons. ORFs that were not unique due to gene duplication or contained regions of high sequence similarity were not attempted. Additionally, a number of ORFs were unsuccessfully deleted for unknown reasons; ~62% of these ORFs have no known biological function. For a list of these ORFs, click here.
The YKO collection of gene disruption mutants is an unparalleded resource for the scientific community
Each deletion "cassette" was constructed using two sequential
In the second PCR reaction, two ORF specific 45-mer oligonucleotides (UP_45 and DOWN_45) are used to extend the ORF specific homology to 45 bp, increasing the targeting specificity during mitotic recombination of the gene disruption cassette.
The U1/D1 and U2/D2 sequences being common to all the deletion strains are used to amplify the 5' and 3' "molecular bar-codes" respectively via PCR for subsequent identification and analysis. The presence of the two tags (UPTAG and DNTAG) increases the quality of the hybridization data from the oligonucleotide arrays by adding redundancy. Early in the project one tag (UPTAG) was used exclusively; approximately 3.2 % of the strains harbor only one unique UPTAG sequence.
A second attempt was made to delete ORFs that were not successfully deleted in the first attempt. The success rates for Round 1 and 2 deletion attempts were 92% and 74% respectively. Genes not successfully deleted after the second round were attempted again using an additional pair of longer (63 mer) primers (UP_90 and DOWN _90) that extended the sequence flanking the ORF to be deleted to 90 bp. About 10% of the collection was attempted using such primers; the success rate for these disruptions (rounds 3 and 4) was >97%.
During construction of the YKO collection, two
problems had to be avoided.
Second, a substantial fraction of the haploid deletion mutants were found to carry a wild-type copy of the gene to be deleted, in addition to the correct deletion mutation as confirmed by the appropriate PCR tests. Such cases are likely due to aneuploidy: duplication of all or part of the chromosome. They comprised about 1% of the heterozygous KanMX-containing primary transformants (estimated from a sample of about 1300 mutants), and upon identification through PCR they were discarded. (Hughes, 2000; B. Dujon, personal communication; our observations,described).
The "A" and "D" primers were positioned 200-400 bp from the start and
stop codons of the gene, respectively. The "B" and "C" primers were located
within the coding region of the ORF and, when used with the A or D primers,
gave product sizes between 250-1000 bp. The "KanB"
(5'-CTGCAGCGAGGAGCCGTAAT-3') and "KanC"
(5'-TGATTTTGATGACGAGCGTAAT-3') primers are internal to the KanMX4 module.
For haploid or homozygous isolates, the junctions of the disruption were verified by amplification of genomic DNA using primers "A" and "KanB" and primers "KanC" and "D". Deletion of the ORF was verified by the absence of a PCR product using primers "A" with "B" and "C" with "D". In the case of heterozygous strains a successful deletion was indicated by the additional appearance of a wildtype-sized PCR product in reactions 3, 4 and 5. Finally, each deletion mutant was checked for a PCR product of the proper size using the primers flanking the gene.
In addition, each strain background was checked for the appropriate
auxotrophic markers and mating capabilities.
Deletion strains were grown individually at 30°C with 0.8ml liquid YPAD in 96-well microtiter plates with shaking (Dot Scientific, U.S.A.); each well contained one 3.5 mm glass bead to facilitate mixing. The cells were grown to stationary phase, then diluted and grown to mid-log-phase (at least six generations). Cells were fixed by the addition of formaldehyde to a final concentration of 3.7%, incubated for one hour at 30°C, washed with PBS, resuspended in PBS, and examined by phase-contrast and differential interference contrast microscopy.
The mutants were scored using a scale of 1-4 (see table and legend
below) and grouped into seven classes: elongated, round, small, large,
football-shaped, clumpy and other. "Other," being defined
as a deletion mutant having 3 or more distinct phenotypes. Cells that
exhibited a "chain" or "branched" phenotype were
also catergorized as "other."
Morphological Screen Results