suppressor cloning problems

Tien-Hsien Chang tchang at magnus.acs.ohio-state.edu
Mon Feb 26 11:28:14 EST 1996


We have been puzzled by the following observations, and would greatly 
appreciate any valuable input from the netters.

(1) We started out with a cold-sensitive mutant unable to grow at 16oC.  In 
this mutant, an essential gene on the chromosome was nearly completely deleted 
and marked by HIS3.  A conditional cold-sensitive allele was carried on 
pRS314/CEN/TRP1.

(2) We then isolated, via spontaneous reversion, a number of second-site 
suppressors which can now grow happily at 16oC.  A RECESSIVE second-site 
suppressor mutation, which yields no additional growth phenotypes, was chosen 
for cloning by transforming the cells with a YCp-LEU2 genomic library.

(3) It is anticipated that the wild-type suppressor allele (DOMINANT) in the 
genomic library will render the recipient cells cold-sensitive again.  We 
plated out transformants on SD-Leu (or on SD-Leu-Trp) plates and incubated at 
30oC.  Upon replica plating, we found that MORE THAN 40% of colonies failed to 
grow at 16oC.  These colonies were not rho mutants, as they can grow on 
glycerol plates.

(4) The question is why there are such an incredibly high number of candidates?
 How should we go from here?

At the present time, we can only entertain two possibilities:

(a) The suppressor mutation is a by-pass suppressor, rendering the conditional 
allele to be lost freely.  But why a huge number of colonies become 
cold-sensitive upon receiving the YCp clones?

(b) There is a problem of recombination between the pRS314 and YCp-LEU2 
plasmids, deleting the conditional allele, which leads to lethal event.  But 
why this only shows up at such a high frequency upon replica plating at 16oC.



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