Figure 1.
Transformation for deleting or C-terminal tagging genes in yeast.
(A) To delete a gene, PCR-generated DNA fragments containing a selectable marker (DrugR) flanked by target gene homology (green block, upstream of start codon (ATG), and brown block, downstream of stop codons) are used to transform yeast cells. Recombination between the DNA fragment and the genomic locus deletes the gene and replaces it with the selectable marker. Terminal homologies used in transformation are embedded within especially designed long primers (shown as green and brown arrows) which are used to amplify the transforming DNA fragments from a series of previously described vectors (3,4). Primer design involves selecting the correct regions of homology (length = N basepair) relative to the target gene. For the forward (Fwd) primer, ATG plus N-3 bp upstream of the start codon is selected. For the reverse (Rev) primer, the stop codon and N-3 bp downstream of the gene is selected. For non-coding genes or other features, N bp upstream and downstream of the feature for Fwd and Rev primers is selected, respectively. (B) To add a tag to the C-terminus of a CDS, a Fwd primer (green arrow) containing N bp directly upstream of the stop codon along with Rev primer (depicted in brown as described in A) are used to amplify the fragment shown in this figure by PCR. Homologous recombination integrates the tag (orange) in-frame with the ORF, resulting in a fusion gene. The tag carries its own translation termination codon (shown as two asterisks on top of one another).
Table 1.
List of yeast strains in the database and their sources for genome and annotation files, as well as their command line inputs.
Figure 2.
An example of a PRIMED database (fission yeast CDS).
In all databases, we provide the systematic and common names, chromosome number, start/end coordinates, transcription strand and forward and reverse primer sequences for each feature under consideration. For deletion databases, also the name(s) and total number of overlapping ORFs, which are disrupted by creating the gene deletion, are indicated.