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.