CommunityW303.html

Information regarding the provenance of Saccharomyces cerevisiae strain W303

Kindly provided at SGD's request by Rodney Rothstein on March 10, 2005.

The original W303 strain is mutated in rad5-535 (an G to R change at position 535 - See Fan et al. Genetics 142:749, 1996).

• The change is subtle resulting in a phenotype in combination with soh1 (Hannah Klein in the Fan paper), sir mutations--increased mms resistance (David Sinclair, unpublished) and no effect on recombination, UV or X-ray sensitivities (Rothstein lab, unpublished).
• To assay for its presence in any W303 derivative strain, one can do a PCR and digest the products with MnlI, as the mutation creates a MnlI site.
  • The primers to use are:
    • 5'-gcagcaggaccatgtaaacg-3' RAD5-L
    • 5'-aaactcgttactccactgcg-3' RAD5-R
  • Run a 3% agarose gel to see the fragments.
    • In wild type: 182 bp and 155 bp.
    • In rad5-535: 155 bp, 120 bp and 62 bp.
  • The RAD5 wild type derivatives of W303 are W1588.

Some relevant information for W303:

MATa/MATalpha {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi+]

• This strain was made diploid by transforming W301-18A (Rothstein, Meth. Enzymol. 101:202-211, 1983.) with an HO-containing plasmid.
• The diploid was dissected to obtain the isogenic MATa (W303-1A) and MATalpha (W303-1B) strains (Thomas & Rothstein, Cell 56:619-630, 1989).
• The {brackets} in the genotype indicate that these genes are homozygous in the diploid. Each haploid strain has only a single copy the gene.
• The [phi+] element is a non-Mendelian trait that affects the efficiency of amber suppression. Unlike the related element [psi+], this element does not affect ochre suppression.
  • ade2-1 and can1-100 are ochre-suppressible.
  • trp1-1 is amber-suppressible.
  • ura3-1 reverts at very low frequency (2 x 10e-9).
  • Both leu2-3,112 and his3-11,15 do not revert at any measurable frequency.
  • Sequence details for the relevant genes are listed in the table at the bottom of the page.

Brief description of the history of W303:

• Many crosses were made with strains from Rothstein's Ph.D. thesis, W87 derivatives
  • see Genetics 85:35-54, 1977 and Genetics 85:55-64, 1977
  • These are mainly but not exclusively X2180-like (S288C).
• It also got part of its genetic background from Fred Sherman's strains, D311-3A
  • see Genetics 94:871-889, 1980 and Genetics 94:891-898, 1980
• Finally, one of the grandparents of W301-18A, D190-9C, is a real mutt, which Rothstein got from Jack Szostak and about which very little is known.

TABLE. Mutant alleles in W303.

allele	nt position	alteration	aa change
ura3-1	701	gga > gAa	Gly > Glu
trp1-1***	247	gag > Tag	Glu > amber
can1-100	139	aaa > Taa	Lys > ochre
ade2-1	27**	taa > ttG	none
	190	gaa > Taa	Glu > ochre
	301*	aga > Gga	Arg > Gly
	372**	gtt > gtC	none
	1617**	acg > acA	none
his3-11,15	208	G deletion	-1 frameshift
	319	G deletion	-1 frameshift
leu2-3,112	168**	gtc > gtT	none
	206*	gtt > gCt	Val > Ala
	249	G insertion	+1 frameshift
	792	G insertion	+1 frameshift
	897**	gtt > gtC	none
	898*	gac > Aac	Asp > Asn

• extra mutation compared to published wild-type sequence
  ** nucleotide change compared to published wild-type sequence, but amino acid is conserved
  ***info from John McDonald, formerly of the Rothstein lab, Genetics 147:1557-1568 (1997)