Difference between revisions of "CommunityW303.html"

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* Diploid W303-based strains that harbor the ''rad5-535'' allele have a much higher rate of ''MET15'' (standard name [http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=Met15 ''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 ([http://labs.fhcrc.org/gottschling/Yeast%20Protocols/lore.html 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 [http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=rdn1 ''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 ([http://www.bs.jhmi.edu/MBG/boekelab/Resources/Met15/Met15updt.html Met15 Update], Carla Connelly and Jef D. Boeke, unpublished observation, and personal communication; Stephan Bärtsch, in August 2013).
 
* Diploid W303-based strains that harbor the ''rad5-535'' allele have a much higher rate of ''MET15'' (standard name [http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=Met15 ''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 ([http://labs.fhcrc.org/gottschling/Yeast%20Protocols/lore.html 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 [http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=rdn1 ''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 ([http://www.bs.jhmi.edu/MBG/boekelab/Resources/Met15/Met15updt.html 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 ([http://www.yeastgenome.org/cgi-bin/reference/reference.pl?dbid=S000128822 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 ([http://www.yeastgenome.org/cgi-bin/reference/reference.pl?dbid=S000086919 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 [http://sift.bii.a-star.edu.sg/ SIFT] to affect protein function with a score of 0.00 (max. score 1.00).
+
* 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 ([http://www.yeastgenome.org/cgi-bin/reference/reference.pl?dbid=S000128822 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 ([http://www.yeastgenome.org/cgi-bin/reference/reference.pl?dbid=S000086919 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 [http://sift.bii.a-star.edu.sg/ SIFT] to affect protein function with a score of 0.00 (?) (max. score 1.00). [http://genetics.bwh.harvard.edu/pph2/ 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 ''Mnl''I, as the mutation creates a ''Mnl''I site.
 
* To assay for its presence in any W303 derivative strain, one can do a PCR and digest the products with ''Mnl''I, as the mutation creates a ''Mnl''I site.

Revision as of 07:04, 9 August 2013

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).
  • 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).

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

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)