suppressor cloning problems
shin at biosci.cbs.umn.edu
Tue Feb 27 21:30:48 EST 1996
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
: (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.
More information about the Yeast