A SCREEN FOR GENES REQUIRED FOR MEIOSIS AND SPORE FORMATION BASED ON WHOLE GENOME EXPRESSION - Web supplement.
The description of the phenotype is our own results. For a description please see the publication.
Information on the individual ORF information pages about ORF size, chromosome number, coordinates and other names was copied from SGD@Stanford.
A PCR based gene-replacement strategy was employed to disrupt the selected ORFs. Primers were designed to anneal to a template plasmid coding for the HIS3MX6 gene (Wach A, Brachat A, Alberti-Segui C, Rebischung C, Philippsen P. Yeast, 1997 Sep 15;13(11):1065-75 ), bearing a 52 bp overhang complementary to the targeted gene. Sequence data for designing primers was obtained from SGD. The disruption cassette was amplified by PCR and transformed into a diploid homothallic yeast strain. HIS prototrophic colonies were checked for homologous recombination by PCR. Yeast heterozygous for the deletion allele was sporulated and tetrads dissected. The HIS prototrophic spore colonies were checked for homozygosity of the deletion allele at the targeted locus by PCR.
The oligonucleotide sequences named up-del and dw-del were used for amplifying the disruption cassette for the given ORF. The 3' 18 bases are identical for all deletion primers used and anneal to the template plasmid containing the HIS3MX6 gene. The given positional information refers to the 3' most base of the variable part of the primer in relation to the start codon of the ORF in question, being the last nucleotide identical in wt and disrupted alleles.
The oligonucleotides named up-ch and dw-ch were used for checking of homologous recombination of HIS prototrophic transformants. The positional information refers to the 5' most base of the primer in relation to the start codon of the genomic sequence of the locus.
Oligonucleotides called up-int and dw-int were used if (due to a very large PCR product of a wt allele) an unabigous distinction between a heterozygous and a homozygous strain was not possible with the normal primerset. Both primers are located within the ORF, up-int in the region coding for the N-terminus going together with up-ch and dw-int at the other end. Positional information refers to the 5' most base in relation to the start codon of the genomic sequence of the gene.
In this section links to SGD@Stanford and YPD@Proteome describing the ORF in question are provided.