We are working on the S.cerevisiae bHLH-ZIP protein CPF1 (Centromere and
Promoter Factor). CPF1 binds DNA at both centromeres and in lots of promoters
including those of various MET genes. When you delete the CPF1 gene you get
methionine auxotrophy. However, CPF1 does not act as a simple activator at
these sites. We, in fact, cannot find a site at which CPF1 actually affects
transcription in any major way. In our attempts to look at this problem we
have tried various ways of suppressing the Met- phenotype of cpf1- yeast.
These turn out to include:
1. Secondary mutations in SPT21, SIN3, RPD3 and CCR4.
2. Over expression of PHO4, and mutations in PHO80 or PHO84.
3. When we lower the concentration of all the amino acids in our minus Met
media (for instance 0.5X standard Guthrie and Fink mix).
Okay, are you still with me? :-)
The weird one is point 3. When we use a 0.5X amino acid mix but omit either
aspartate or threonine we get our Met- phenotype back again.
So, the question (finally), is: Does anybody know of a link between the MET
system, the PHO system and the Asp and Thr pathways (other than "they
are all in yeast and involve metabolism! :-))? Or, does anyone have an
explanation for point 3?
Thanks for wading through this....
Microbiology Unit, Dept.Biochemistry, University of Oxford.