Hi all,
I've got a problem I don't know what to make of. I transformed some
mutants with a gene on a multicopy plasmid (Yeplac195) and noticed
suppression of the mutant phenotype. Now, I ran out of this particular
multicopy construct so I tried to rescue the plasmid from these mutants
but the plasmids I get don't at all look like what I transformed in
initially. Even the normal URA3 sites don't seem to be there. I must
mention that it was quite difficult to transform in this plasmid from
the beginning resulting in fewer transformants than usual. Another
strange feature is that when I streak these mutants with the plasmid on
5-FOA, they cannot grow while without this plasmid there is no obvious
growth defect. Northern analysis show that the gene in this plasmid is
downregulated in these mutants. The gene is essential in yeast. I am
quite puzzled by this all and don't know what to make out of it! Anyone
out there who can explain this? What is going on?
N. Amjad
