Hi, I am interested in nucleotide metabolism and performed serial dilution and spotting using ade1 and ade2 knockout yeast strains. it seems like ade1 grows slower than ade2 eventhough they are in the same pathway. Has anyone come across such observation and is there any link between URA3 overexpression and color of these strains? Thanks in advance
Hi there,
If the yeast background is the same and if you grow the strains on the same plate they should grow equally. About the color, it should appear earlier in ade2 strain because the ADE2 substrate is responsible for this. As ADE1 acts immediatly downstream ADE2, ade1 mutant will first accumulate ADE1 substrate and then ADE2 substrate.
About the link with ura phenotype, the following paper should be helpful:
https://pdfs.semanticscholar.org/1a67/1864470acd3e2edf822d27fb77453e9e6c11.pdf
Dear Dr. Dominique Liger
Thank you very much for your response. Both the knockout strains I am working with are BY4741 background. But upon transformation with a cen plasmid (PCM189-URA3 marker), serial dilution and spotting I see that ade1 grows slower than ade2 transformants on SD-URA plates containing hypoxanthine. another observation I have seen is as follows.
I transformed ade1 and ade2 with PCM189 and PCM189 containing yeast AMP deaminase. In my serial dilution and spotting I see that under derepression conditions the ade1 transformant with yeast AMP deaminase showed pink color and the vector control was colorless. We hypothesise that as these strains are purine auxotrophs, hypoxanthine is salvaged by HGPRT and further converted to AMP, but AMP deaminase overexpression depletes AMP levels and removes the inhibition on de novo pathway which leads to accumulation of the metabolite AIR which is responsible for Pink color. But this phenomenon should be common to both ade1 and ade2 strains. And as you said ade2 should give pink color before ade1. But contrastingly we see that ade1 shows this phenotype and ade2 transformants are all white in color.
the strains are genotyped and validated for auxotrophy. Also when streaked on YPD plate ade2 showed pink color before ade1. So we are confused as to what might be happening...
sorry for the long write up...
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