Dear Netters
I have a perplexing result from a recent experiment and am hoping your
insights might clear things up. I have knocked out my favorite gene (MFG1)
in haploid cerevisiae using a TRP1 marker and screened for the k-o by
western blot (I know, I should have done a southern!). Following this, I
mated the k-o with the isogenic strain carrying a disruption in another
allele on a different chromosome (MFG2). The marker for this disruption
was URA3. After dissecting tetrads and checking auxotrophies, it became
clear that the TRP1 allele had integrated twice in the mfg1 disrupted
strain. Okay (I thought), maybe the TRP1 allele had integrated both into
the genomic trp1 locus and also into MFG1.
Then I did a southern on the individual tetrads from the mfg1 x mfg2
cross. From this it seems that there are two alleles of MFG1 (ie. I can
pick up both the disrupted and wild-type fragments from a single haploid)
These really don't seem to be diploid under the microscope so I've ruled
out diploid contamination. I have also blasted the sequence and there does
not seem to be any other sequences homologous to MFG1 in the yeast
database. Sooooooo...now I'm thinking there is some chromosomal
rearrangement in my original strain which led to duplication of the region
of DNA encompassing MFG1. Thus, I knock-out only one or the other copy
(giving two fragments on the southern) or both copies (giving me two TRP1
integrations as indicated by tetrad analysis).
My question is this. Is it possible (probable, highly unlikely?) that this
scenario could occur. Could anyone offer a more realistic explanation? Any
suggestions would be greatly appreciated.
Thanks for letting me pick your brain.
Greg
