CommunityW303.html

Information regarding the provenance of Saccharomyces cerevisiae strain W303

Kindly provided at SGD's request by Rodney Rothstein on March 10, 2005. Correction provided by Stephan Bärtsch on August 17, 2008.

The original W303 strain is mutated in rad5-535 (RAD5, an G to R change at position 535 - See Fan HY. et al. (1996), Genetics 142:749-759).

• 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in combination with soh1 (Hannah Klein in the Fan paper), sir mutations--increased MMS resistance (David Sinclair, unpublished) and no effect on recombination, UV or X-ray sensitivities (Rothstein lab, unpublished).

• The rad5-G535R missense mutation does not cause any growth defect or gamma-ray sensitive phenotype. However, rad5-G535R strains displayed increased sensitivity to UV light at high doses when compared to wild-type strains, and rad52 rad5-G535R double mutants were more sensitive to UV light when compared to RAD52 rad5-G535R and rad52 RAD5 single mutants. Levels of direct repeat recombination were not affected by the rad-G535R allele in rad1, rad52 or rfa1-D288Y backgrounds. The efficiency of plasmid gap repair (outline of the system: Bärtsch S. et al (2000), Mol. Cell. Biol. Feb.; 20(4): 1194-1205) was not significantly affected by the rad5-G535R allele. Also, the rad5-G535R allele had no effect on the proportion of crossover and non-crossover events independent of whether the DNA donor for gap repair was of chromosomal or plasmid origin. These unpublished findings support the hypothesis that the weak DNA repair phenotype conferred by the rad5-G535R mutation is caused indirectly through interaction either with proteins of the transcription machinery (Kiakos K. et al (2002), suggest as a possibility a role of Rad5 in facilitating transcription-coupled DNA repair, TCR, of DNA minor groove adducts) or with chromatin but not by direct involvement in recombination (Stephan Bärtsch and Naz Erdeniz, April 2000, unpublished).

• Probably due to the rad5-G535R missense mutation in the W303 background strains, the sensitivity of the mrc1Δ mutant (Mrc1 plays a role in mediating the DNA replication checkpoint, Osborn AJ, and Elledge SJ (2003) to 200 mM HU observed by Komata M. et al. (2009) (viability about 80%) differed from that reported previously by Alcasabas AA. et al. (2001) (viability about 30%). All strains constructed in the Komata M. et al. (2009) study were derived from RAD5 strain BY4741 (MKY0027 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 mrc1Δ::LEU2, Katou Y., et al. (2003)). The mrc1-1 Y1121 strain used by Alcasabas AA. et al. is isogenic with the W303-derived Y300 strain (MATa trp1-1 ura3-1 his3- 11,15, leu2-3,112 ade2-1 can1-100, Allen JB. et al. (1994))

• Diploid W303-based strains that harbor the rad5-535 allele have a much higher rate of MET15 (standard name MET17) loss of heterozygosity (LOH, brown colored sectors within a colony on lead nitrate plates) than other backgrounds, such as RAD5 BY derivatives, and presumably than RAD5 W303 derivatives. Not surprisingly, they also have a much higher level of extrachromosomal rDNA circles (Lab Lore; Dan Gottschling, personal communication, and Michael McMurray, unpublishd results; Stephan Bärtsch, in August 2012). In regard to the MET15 locus, the Jef Boeke lab noticed a phenomenon occurring at the locus: frequent loss of heterozygosity (LOH). MET15 is distal to the RDN1 multigene locus on chromosome XII. When the rDNA repeats recombine mitotically, as they are prone to do, heterozygosity can be lost (e.g. mitotic unequal crossing-over). Preliminary estimates are approximately a 3% frequency of LOH (Met15 Update, Carla Connelly and Jef D. Boeke, unpublished observation, and personal communication; Stephan Bärtsch, in August 2013).

• A rad5-G535R strain did not show detectable chronic low dose ultraviolet light (CLUV) sensitivity, whereas the ATPase-deficient rad5-K538A mutant showed a CLUV hypersensitivity similar to that observed in a rad5 deletion mutant (Hishida T. et al. (2008), Nature 457 (7229): 612-615; Hishida T., personal communication; Stephan Bärtsch, in December 2008). The rad5-KT538/539AA (also known as rad5-GAA ) mutant was partially sensitive to UV irradiation and its ionizing radiation sensitivity was comparable to that of a rad5 deletion strain (Chen S. et al. (2005), Nucleic Acids Res.;33(18):5878-5886). Substitutions at position 535 from G to R, at position 538 from K to A, and at position 539 from T to A are predicted by SIFT to affect protein function with a score of 0.00 (?) (max. score 1.00). PolyPhen-2 report for Rad5-G535R: This mutation is predicted to be probably damaging with a score of 1.000 (?) (sensitivity: 0.00; specificity: 1.00).

• 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. RAD5/YLR032W S. cerevisiae Strain Sequence Alignment (Annotation of multiple Saccharomyces cerevisiae strains at the Saccharomyces Genome Database, Engel SR, and Cherry JM., Database, Vol. 2013, Article ID bat012)
    

    ClustalW S. cerevisiae strain amino acid sequence alignments covering W303 rad5-G535R region.

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 RJ. (1983) , 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 BJ., and Rothstein RJ. (1989) , 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:

Insights into ancestry of W303

• Many crosses were made with strains from Rothstein's Ph.D. thesis, W87 derivatives
  • see Rothstein RJ. et al. (1977), Genetics 85:35-54, 1977 and Rothstein RJ. (1977), Genetics 85:55-64, 1977
  • These are mainly but not exclusively X2180-like (S288C, Mortimer RK. and Johnston JR., (1986), Genealogy of principal strains of the Yeast Genetic Stock Center, (1986), Genetics, 113,35-43).
• It also got part of its genetic background from Fred Sherman's strains, D311-3A (Sherman F. et al. (1974))
  • see Rothstein RJ., and Sherman F. (1980), Genetics 94:871-889, 1980 and Rothstein RJ., and Sherman F. (1980), 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. D311-3A contributes to the genetic background of W301-18A ( Rothstein RJ. and Sherman F. (1980a), (1980b)). The strain is related to D190-9C (Szostak, JW. and Blackburn EH, (1982), Wildgruber, R., Thesis (2002), page 18)

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)