History of Sigma

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The History of Sigma1278b and notes on other Sigma1278b derivative sets

These notes have kindly been provided by the Fink lab, collected Nov. 1998 by Cora Styles

Per Ljungdahl was doing a mutant screen, looking for mutants that would grow on high concentrations of histidine (30mM). These were dubbed SHR (Super-high Histidine Resistant). One was called SHR3 and we realized that this mutant with unknown function had been found several times in previous mutant screens by others. Carlos Gimeno was assigned to gather these other mutants and see if they had the same histidine phenotype as Per's shr3.

One of these mutants was among The Fink lab Foreigner strains, F35 MATa aap. This strain was very old - more than 20 years - and had been saved in a variety of ways - continuous subculturing on fresh YPD, and dessication on silica gel. On 30mM histidine it grew, but displayed a mold-like phenotype, ie. pseudohyphal growth. A number of people in the lab claimed this "wasn't yeast" and Carlos should get rid of it.

Carlos was not so easily convinced. He found that F35 didn't mate, but it did sporulate, and the clincher was when he showed the ascospores could mate with S288C strains. We deduced in retrospect that somewhere in its checkered storage history the strain first had diploidized, and then a MAT switch occurred so it now was a/x. Before we had glycerol/-70C storage, diploidization was not uncommon, something I had to watch out for in maintaining our stocks. We surmised that these diploids that spontaneously form must have a competitive advantage over the haploid in conditions where stocks are kept on YPD slants and re-passaged on new medium from time to time.

The mutant aap was in the Sigma1278b background. This wild-type lab strain is different from S288C with respect to its GAP1 permease response to ammonia. It is avoided by researchers who want to do quantitative biological studies because the haploid daughters don't regularly separate from their mothers. It is "clumpy" and further, flocculent, that is, unrelated cells also tend to cling to each other. Flocculent groups can be separated by sonication, but vegetative offspring require zymolyase treatment for separation.

To expand our repertoire of Sigma1278b strains, we asked Marjorie Brandriss to send us her ura3-52 congenic strain which she had produced by the classical alternative to cloning, namely by crossing a foreign strain carrying ura3-52 by Sigma1278b, then taking a ura3-52 offspring and crossing it again back to the Sigma1278b parent for 10 generations. The thought is that over 99% of the foreign strain's genes will be replaced by Sigma1278b genes.

There are undesirable mutants that could be predicted to hide in such a procedure, and Nature, who shares her bounty with us, provided some. They were recessive mutations in sporulation and germination (sgl=red1), which silently passed along through the heterozygous diploids. When Carlos crossed the ura3-52 Sigma strain of Brandriss by Sigma1278b, sporulation and germination were fine, but when he crossed those offspring together and sporulated them, he ran into all kinds of trouble. The ascospores of tetrads disintegrated and formed a sloppy mess within the diploid cell wall. After repeated crosses and throwing out the bad stuff, we got rid of that.

We saved our favorite MATa and Mat x (alpha) ura3-52 strains, 10480-5C and -5D. Haoping Liu blasted these repeatedly to get single MATa and Matx hisG insertion - disruption mutants for leu2, his3 and trp1. These we intercrossed to build triple and quadruple marked strains. Then we discovered the presence of a spore germination lethal (RED1) in 10480-5C (MATa). The diploid red1/red1 forms perfect ascospores, but none will germinate. Our carefully built collection was riddled with this gene. A high school student, Rupa Mukerjee, switched our four MATx (alpha) strains (RED1+) to MATa and we got rid of the previous red1 MATa set. Cora made diploids and dissected crosses to test for the presence of this mutation. A strain bearing all four markers that was RED1+ MATa was identified. Cora crossed it by the original wild type Sigma1278b to produce our 10560 series of ascospores. These strains are not isogenic. They are congenic.

We also learned that shr3 is defective in processing a family of amino acid permeases, including proline. Unable to take up much proline, the sole nitrogen source provided in the original experiment, the shr3 diploid strains form pseudohyphae essentially in response to nitrogen starvation. SHR3+ diploids (wild type) will likewise form pseudohyphae simply on minimal medium with very low nitrogen concentration. (Some people omit the nitrogen (ammonium sulfate) altogether.

One cannot observe pseudohyphal growth in diploids which require leucine or tryptophan supplementation, because these amino acids can serve as nitrogen sources. The minimal medium can be supplemented with uracil or histidine, however, because these cannot be used as nitrogen sources by S. cerevisiae. Despite this fact, the pseudohyphal growth achieved is poorer than if these additions were absent, i.e. prototrophs on unsupplemented SLAD make the best pseudohyphae.

In Joe Heitman's lab, Mike Lorenz started with Sigma1278b itself and blasted out ura3 and built other mutations by transformation. See Legacies donated by Lorenz, L6621 & ff.

Microbia's Sigma 2000 series starts not with Sigma1278b but a Fink derivative, a MATa prototroph, 10560-5A These are in our Foreigners collection.

Lorenz reports that the activity of the MEP3 gene differs between Fink's and his set. Steffen Rupp found no function in the Fink strain(s), but Mike found a phenotype in his strain(s). Lorenz reports another undescribed difference between the two.

Todd Milne reports that the "dig" phenotype (haploid adhesion to YPD medium) is different for Ura+ vs Ura-. Ura+ cultures wash off more readily than Ura-. Differences are not seen for our other most-used auxotrophies: TRP, HIS, LEU.

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