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== Information regarding the ''Saccharomyces cerevisiae'' strain W303 ==

''Kindly provided at SGD's request by Rodney Rothstein on March 10, 2005.''
''Last additional information provided by Stephan Bärtsch on April 16, 2018.''

Choose your yeast strain carefully: the RAD5 gene matters [http://www.nature.com/articles/s41580-018-0005-2 Elserafy M. and El-Khamisy SF. (2018)], Nat Rev Mol Cell Biol. 2018 Apr 3. doi: 10.1038/s41580-018-0005-2. [Epub ahead of print]

Suggested citation for this website:
 
Rothstein R (2005) Information regarding the provenance of Saccharomyces 
cerevisiae strain W303 (online) Available at: http://wiki.yeastgenome.org/index.php/CommunityW303.html (Accessed ''<Date accessed>'') 

Bärtsch S (2018) Biological effects of the yeast W303 ''rad5-G535R'' mutation (online) Available at: http://wiki.yeastgenome.org/index.php/CommunityW303.html (Accessed ''<Date accessed>'')


The original W303 strain is mutated in ''RAD5'' ([http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=rad5 ''RAD5''], an G to R change at position 535 (''rad5-G535R'') - See [http://www.yeastgenome.org/reference/S000051944 Fan HY. ''et al.'' (1996)], Genetics 142:749-759. Two homologs of Rad5 have been identified in human cells, the [http://en.wikipedia.org/wiki/HLTF HLTF] and [https://en.wikipedia.org/wiki/SHPRH SHPRH] proteins, showing 39% and 21% similarities to Rad5, [http://www.ncbi.nlm.nih.gov/pubmed/20096653 Unk I., ''et al.'' (2010)]).

* 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combination with ''soh1'' (Hannah Klein in the Fan paper), ''sir'' mutations--increased MMS resistance (David Sinclair, unpublished) and no effect on recombination, UV or X-ray sensitivities (Rothstein lab, unpublished).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: [https://www.yeastgenome.org/reference/S000049982 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 ([https://www.yeastgenome.org/reference/S000071643 Kiakos K. ''et al'' (2002)] suggest as a possibility a role of Rad5 in facilitating transcription-coupled DNA repair, [http://en.wikipedia.org/wiki/Transcription-coupled_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; in July, 2000, [https://www.yeastgenome.org/reference/S000042429 Ulrich HD, and Jentsch S., (2000)] conclude that the Ubc13–Mms2 complex is recruited to the chromatin by Rad5 upon its accumulation in the nucleus in response to DNA damage).

* Probably due to the ''rad5-G535R'' missense mutation in the W303 background strains, the sensitivity of the ''mrc1Δ'' mutant ([http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=mrc1 Mrc1] plays a role in mediating the DNA replication checkpoint, [https://www.yeastgenome.org/reference/S000073790 Osborn AJ, and Elledge SJ (2003)]  to 200 mM HU observed by [https://www.yeastgenome.org/reference/S000134925 Komata M. ''et al.'' (2009)] (viability about 80%) differed from that reported previously by [https://www.yeastgenome.org/reference/S000068879 Alcasabas AA. ''et al.'' (2001)] (viability about 30%). All strains constructed in the [https://www.yeastgenome.org/reference/S000134925 Komata M. ''et al.'' (2009)] study were derived from ''RAD5'' strain BY4741 (MKY0027 ''MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 mrc1Δ::LEU2'', [https://www.yeastgenome.org/reference/S000073860 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'', [https://www.yeastgenome.org/reference/S000051282 Allen JB. ''et al.'' (1994)])

* Diploid W303-based strains that harbor the ''rad5-535'' allele have a much higher rate of ''MET15'' (standard name [http://www.yeastgenome.org/locus/S000004294/overview ''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://research.fhcrc.org/gottschling/en/protocols/yeast-protocols/lab-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 ([https://www.yeastgenome.org/reference/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 ([https://www.yeastgenome.org/reference/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 (?); amino acids with probabilities < 0.05 are predicted to be deleterious. With an alternative, [http://provean.jcvi.org/index.php PROVEAN], the variant G535R results in a PROVEAN score of -7.850 (?); variants with a score equal to or below -2.5 are considered deleterious. [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.
** 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''<nowiki>: 155 bp, 120 bp and 62 bp.</nowiki>
** The ''RAD5'' wild type derivatives of W303 are W1588. [http://www.yeastgenome.org/cgi-bin/FUNGI/alignment.pl?locus=RAD5&submit=Submit&rm=display_result ''RAD5/YLR032W S. cerevisiae'' Strain Sequence Alignment]  ([http://www.ncbi.nlm.nih.gov/pubmed/23487186, Annotation of multiple ''Saccharomyces cerevisiae'' strains at the Saccharomyces Genome Database], Engel SR, and Cherry JM., Database, Vol. 2013, Article ID bat012) [[File:Rad5_aa_yeast_strain_SeqAlign-tiff.jpg|thumb|alt=Rad5.|ClustalW ''S. cerevisiae'' strain amino acid sequence alignments covering W303 ''rad5-G535R'' region.]] 
** [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499129/ Matheson K. ''et al.''  (2017)] provide with a Whole-Genome Sequence and Variant Analysis of W303 high-quality, annotated genome sequences for comparative analyses and genome-wide studies. Interestingly, as they and R. Rothstein (personal communication with the authors) report, W303 was selected, besides to have a high transformation efficiency, to have a higher sporulation efficiency than S288C (Gerke et al. 2006; Hong et al. 2008). 

=== Some relevant information for W303: ===

''MAT''a/''MAT''alpha {''leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15''} [''phi+'']

* This strain was made diploid by transforming W301-18A ([https://www.yeastgenome.org/reference/S000080983 Rothstein RJ. (1983)] , Meth. Enzymol. 101:202-211, 1983.) with an ''HO''-containing plasmid.
* The diploid was dissected to obtain the isogenic ''MAT''a (W303-1A) and ''MAT''alpha (W303-1B) strains ([https://www.yeastgenome.org/reference/S000049504 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: ===

*  [https://www.yeastgenome.org/reference/S000151108 Insights into ancestry of W303]

* Many crosses were made with strains from Rothstein's Ph.D. thesis, W87 derivatives
** see [https://www.yeastgenome.org/reference/S000040025 Rothstein RJ. ''et al.'' (1977)], Genetics 85:35-54, 1977 and [http://www.genetics.org/content/85/1/55.long Rothstein RJ. (1977)], Genetics 85:55-64, 1977
** These are mainly but not exclusively X2180-like (S288C,  [http://www.ncbi.nlm.nih.gov/pubmed/3519363 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 ([http://www.ncbi.nlm.nih.gov/pubmed/4367877 Sherman F. ''et al.'' (1974)])
** see [https://www.yeastgenome.org/reference/S000057232 Rothstein RJ., and Sherman F. (1980)], Genetics 94:871-889, 1980 and [https://www.yeastgenome.org/reference/S000049256 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 (  [http://www.ncbi.nlm.nih.gov/pubmed/6254831 Rothstein RJ. and Sherman F. (1980a)], [http://www.ncbi.nlm.nih.gov/pubmed/6254832 (1980b)]). The strain is related to D190-9C ([http://www.ncbi.nlm.nih.gov/pubmed/?term=Szostak%2C+J.W.+1982 Szostak, JW. and Blackburn EH, (1982)], [http://mediatum.ub.tum.de/doc/603343/603343.pdf Wildgruber, R., Thesis (2002), page 17 and 18])

=== TABLE. Mutant alleles in W303. ===

{| border="1" cellspacing="4" cellpadding="2"
| allele
| nt position
| alteration
| aa change
|-
| ''ura3-1''
| 701
| gga &gt; gAa
| Gly &gt; Glu
|-
| ''trp1-1''<nowiki>***</nowiki>
| 247
| gag &gt; Tag
| Glu &gt; amber
|-
| ''can1-100''
| 139
| aaa &gt; Taa
| Lys &gt; ochre
|-
| ''ade2-1''
| 27**
| tta &gt; ttG
| none
|-
|
| 190
| gaa &gt; Taa
| Glu &gt; ochre
|-
|
| 301*
| aga &gt; Gga
| Arg &gt; Gly
|-
|
| 372**
| gtt &gt; gtC
| none
|-
|
| 1617**
| acg &gt; acA
| none
|-
| ''his3-11,15''
| 208
| G deletion
| -1 frameshift
|-
|
| 319
| G deletion
| -1 frameshift
|-
| ''leu2-3,112''
| 168**
| gtc &gt; gtT
| none
|-
|
| 206*
| gtt &gt; gCt
| Val &gt; Ala
|-
|
| 249
| G insertion
| +1 frameshift
|-
|
| 792
| G insertion
| +1 frameshift
|-
|
| 897**
| gtt &gt; gtC
| none
|-
|
| 898*
| gac &gt; Aac
| Asp &gt; Asn
|}

<pre>* 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)</pre>