Hello, My name is Nathan Blewett, I am a Postbac student at the LMG/NICHD,
working with the fission yeast S. pombe. I have transformed the tRNA
modifying enzyme MiaA which is expressed under the nmt1 promoter into a
pombe strain which contains the suppressor tRNA allele mSERuga; this strain
is red due to anti-suppression caused by the gene maf1 being deleted.
Upon MiaA transformation, I see two dramatically different levels of
suppression, the majority of which is anti-suppressed (red) at the level of
the parent strain. However, about 5-10% of the colonies are efficiently
suppressed. Empty vector gave only red colonies. I have repeated this and
get the same result, I subsequently isolated these suppressed colonies, grew
them out in YES rich media to allow for loss of any plasmid present, plated
for single colonies, and streaked these onto limiting media(adenine) and
they became anti-suppressed, at the same level as the parent strain once
again; indicating that the plasmid present was increasing suppression in a
portion of my transformants.
My question is, has anyone gotten transformation results, on the same plate,
which give varying levels of suppression? There is the possibility that
there is alternate AUG selection occurring with MiaA, and this may be
contributing to the varying suppression level? Any input would be
appreciated.
