yeast complementation

[Yogesh K Sharma]

Hi yeast experts:

I am trying to clone plant gene homologs by complementation of yeast mutants
sensitive to certain drugs. I am using Arabidopsis (plant) expression library
in lambda YES vector ( Elledge and Davis). It is a Ycp vector and has gal
inducible promoter driven expression in yeast S cerevisiae. The vector has URA 
selection.My yeast mutant has TRP insertion, mutating the gene.

 I  finally selected 9 total yeast transformants on triple selection of the
ura, trp and the drug.  My problems are as follows:

1    a. I observe weird recombinations on transformation of XL-1-Blue cells
subsequent to plasmid isolation from yeast transformants. This is such a
problem that I end up getting only one clone from E coli with the right size 
and restriction pattern of the insert  and sometimes even nothing after 50-60 
minipreps.There has been no such recombinational problem with only vector or 
vector with fatty acid desaturase clone, used as controls.  Does it mean that 
plant genes that is complementing the yeast mutant is prone to 
deletions/recombinations? Is this gene
product toxic to yeast.

2    a.  Next, when I try to backtransform the yeast mutant with the plasmid
isolated from E.Coli (with the right diagnostic restriction pattern for the
vector) that was transformed with plasmid DNA isolated from complemented yeast, 
I have more problems.  Back transformation with only one out of nine clones 
gave drug resistant phenotype.  Furthermore, I could repeat this only twice.
Rest of the attempts (twice) all the backtransformants were nontolerant to the
drug.
     b    Besides even on each of the two succesful  back transformations of
yeast with DNA isolated from E Coli, I had two types of clones: red and white.
The white ones were resistant to drug.and had the  size and restriction pattern 
of insert that was similar to the plasmid used to back transform.  But the red
ones were sensitive to drug and had sometimes deleted and sometimes apparently 
right size and restriction pattern.of insert

3. What are the other ways (other than back transformation) of showing that the
drug tolerance that I see in the complemented yeast mutant is due to the
plasmid containing plant gene.
.4   I am unable to cure the yeast transformants even after 6 generations on
liquid and two on solid YPD media


5. The expression of my right clones are not inducible by galactose i.e. I see
no increase in growth rate or resistance to drug in galactose rather than
glucose as sugar source.
I work in a plant mol.biol. lab and have little yeast experience.  I will
sincerely appreciate, if some yeast expert can give me some insight in to the
problems I am facing.

Thanks
Praveen Sharma