GOTTSCHLING LAB -- Lab Lore

Lab Lore

This information is intended to serve as a source of unpublished observations about working with S. cerevisiae. 

alpha-aminoadipate (aAA) is associated with two separate phenotypes in yeast. aAA is converted to alpha-aminoadipate semialdehyde (aAAsemiA) by Lys2p/Lys5p alpha-aminoadipate reductase activity, and this is a necessary step in the biosynthesis of lysine. However, if excess aAA is provided in the medium, Lys2p/Lys5p converts this all to aAAsemiA, which accumulates and is toxic to the cells. So cells lacking Lys2p or Lys5p will be resistant to aAA toxicity because they don't accumulate aAAsemiA.
However, cells lacking Lys2p or Lys5p don't convert aAA to aAAsemiA, and this leaves aAA available for a salvage pathway that can break down aAA and use it as a nitrogen source. So cells lacking Lys2p or Lys5p will be able to use aAA as the sole nitrogen source, while wildtype cells cannot.
Apparently, a LYS2/lys2 heterozygote has enough Lys2p/Lys5p reductase activity to convert enough aAA to aAAsemiA to make lysine, so the cells are still prototrophic for lysine. But, it doesn't have enough activity to accumulate toxic levels of aAAsemiA, so LYS2/lys2 heterozygous cells are quite resistant to aAA toxicity. Although they don't have full reductase activity, however, they have enough that they do not leave enough aAA unconverted to aAAsemiA to allow it to be used as the sole nitrogen source.
So LYS2/lys2 cells are haploinsufficient for Lys2p/Lys5p alpha-aminoadipate reductase activity as regards formation of toxic levels of aAAsemiA, but not as regards lysine biosynthesis or utilization of aAA to prevent salvage.
If the medium contains a good nitrogen source (such as ammonia), neither phenotype is useful for selection: cells will take up aAA only when grown in a poor nitrogen source, because of an active repression mechanism in rich nitrogen. Therefore, synthetic media must not contain ammonium sulfate. However, also remember that when using minimal/synthetic media, the cells must be prototrophic for any amino acids that can be used as the sole nitrogen source: supplementing the medium with amino acids that can be converted to nitrogen prevents testing the cells' ability to use aAA for nitrogen. In our hands, media lacking other nitrogen sources can include uracil, adenine, methionine, lysine, tryptophan, leucine, and histidine, and apparently still derepress amino acid uptake, allowing use of the toxicity of aAA-semiA in this medium. Amino acids that can be used as the sole nitrogen source may include: proline, glutamate, glutamine, ornithine, serine, threonine. Amino acids that cannot be used as the sole nitrogen source may include: leucine. This information is hard to find and inconsistent, however. The aforementioned toxicity media containing uracil, adenine, methionine, lysine, tryptophan, leucine, and histidine obviously contains sufficient nitrogen source (there is no other nitrogen in the media) to preclude this medium from use in testing the ability of cells to use aAA as the sole nitrogen source.

The leu2delta0 allele of the designer deletion series strains (Brachmann, et al. Yeast 14:115-132 (1998)) co-segregates with slower growth, probably associated with the tRNA gene that is also affected by the deletion.

The his3delta200 allele of the designer deletion series strains (Brachmann, et al. Yeast 14:115-132 (1998)) deletes the promoter of the adjacent gene, PET56. The resulting pet56 mutant has decreased mitochondrial translation capacity and an increased frequency of spontaneous petites (Sirum-Connoly and Mason, Science 262:1886-9 (1993)). However, this does not itself account entirely for the elevated frequency of spontaneous petites in S288C backgrounds versus wild isolates. Additionally, S288C HIS3 and his3delta200 strains form colonies with "nibbled" borders after long periods of growth, and these peripheral clones of slow-growing, pale cells seem to be petites. The his3delta200 strains have more of a "skirt" of petites around the perimeter of the colony, as opposed to sectors in HIS3.

Silencing in YPH strains is a little stronger at 37°C and a little weaker at 23°C.

The ADE2 gene fragment contains an ARS element and may be difficult to clone.

Despite published and unpublished reports to the contrary, ade2- petites can turn red. This has been observed in diploid strains of both the designer deletion series and W303 background. The color is fainter and more of a pink/orange, but is readily distinguishable, especially on synthetic or minimal media. See images.

Diploid W303-based strains that harbor the rad5-535 allele have a much higher rate of loss of heterozygosity than other backgrounds, and presumably than RAD5+ W303 derivatives. Not surprisingly, they also have a much higher level of extrachromosomal rDNA circles.

Grande cells will sporulate (albeit at low efficiency) in up to 2% raffinose (plus KAc, etc.). However, petites still will not sporulate with this extra carbon source, suggesting that it is not lack of an adequate carbon source that prevents petite sporulation.

Lead (MLA) plates for met15 screening, made according to the recipe, do not support growth well of plated single cells of certain strains. This does not affect replicaplated or patched cells. It may be due to the effect of the lead solution (which is strongly acidic and oxidizing) on the glucose. We have found that decreasing the amount of lead in the media can help: 0.5 mg/ml gives no noticeable growth defect, though fainter color; 0.7 mg/ml has an intermediate effect, with slightly slow growth but relatively strong color development (see image).For plating directly to lead media for single colonies, we routinely use 0.5 - 0.7 mg/ml final.

Lead plates do not age well: plates used, for example, 6 months after being poured do not support good growth of met15 clones, though MET15 clones still grow fine -- this probably means only the cells precipitating the lead are affected. See image.

G418 can be added to lead plates at the same concentration as for YEPD (200 µg/ml) to utilize the KanMX marker. Also, galactose-based lead plates seem to work as well as glucose-based (4-6% galactose tested).

The MET15 ORF present in pRS401 (Brachmann, et al. Yeast. 14:115-132 ) is not fully functional: it does not provide optimal growth on media lacking methionine, and strains with an integrated version of this (or a PCR knockout using this) as their sole source of Met15p grow slowly on -met media and turn brown on MLA. Diploids with this as their only copy (hemizygous) will still give darker/lighter sectors signaling loss of hemizygosity, but if hemizygous diploid strains are grown under Met+ selection, LOH events that duplicate the MET15 gene will be enriched in the population. The plasmid pGC3 (Cost GJ, Boeke JD. Yeast 1996 Aug;12(10):939-41) contains a larger MET15 insert that provides complete Met15p activity at one copy per cell.