I've been working on a positional cloning project for some time now.
In trying to find good candidate genes that might correspond to my
mutant, I did RT-PCR to test whether my candidate might be expressed
at lower levels in my mutant as compared to Ler (the background from
which the mutant was isolated), and I found it was. This was
observed in several experiments. I next sequenced my candidate gene
from the mutant and found the translational start codon had been
mutated. However, since my mutant was from a Ler background, I
decided I better blast the Ler sequence with my mutant sequence, and
found that in Ler, the start codon was mutated also. A second,
independent sequence of the Ler allele confirmed that the start codon
is not present, and there don't seem to be any other ORF nearby that
could alternatively be used.
Has anyone else had an experience like this? If the predicted gene
isn't legitimate, I would have thought it is not expressed, yet
clearly our RT-PCR data indicates otherwise. Does anyone have a
sense for how common it might be that a gene found in Col is not
functional in Ler or vice versa? Any references?