suppressor cloning problems

[Shin Enomoto]

Tien-Hsien Chang (tchang at magnus.acs.ohio-state.edu) wrote:
: 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:

Is it possible that your mutation is only behaving recessive in a diploid 
and not in a haploid.  I have some puzzling example involving 2 alleles, 
which have distinct mutant phenotypes.  One is a null allele I constructed 
and the other is let's call it allele-1.  In a heterozygous diploid 
null/allele-1, I get the phenotyep of allele-1 as expected.  But in a 
haploid null allele in the chromosome, 
allele-1 on a plasmid, I get the phenotype of the null allele. A similar 
thing maybe happening in your case.  I don't exactly have a good 
explanation of how this could be happening but the practical soultion 
maybe trying to clone your suppressor in a diploid.



: (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.