Dear Chlamy folks,
After insertional mutagenesis of CC-425 using ARG7 and isolating some
interesting mutants, i have a problem that seems to be pretty common.
In testing cosegregation of Arg+ and the mutant phenotype, only about
50% of my mutants appear to be tagged by the ARG7 plasmid, and of course
the ones i'm most interested in are among the "other" 50%.
One possible explanation for the "untagged" mutants is that they are due
to insertion of fragments of the ARG7 plasmid. All the repetitive
sequence in the ARG7 gene makes it difficult to test this possibility,
but i tried using the vector (pBluescript) part of the plasmid as a
probe on Southerns of the untagged mutants. In one mutant that seemed to
have only 1 expressed insertion locus according to segregation of Arg+
and Arg- in a cross, there were multiple pBluescript bands present, some
of which did not cosegregate with Arg+. Unfortunately, none of them
cosegregated with the mutant phenotype either.
Another explanation might be that some mutants are spontaneous mutants
caused by insertion of a transposon (that maybe was activated by the
stress of the ARG7 transformation protocol?). At least one of my
mutants does seem to revert at a frequency that is consistent with this
possibility.
So i was wondering:
1. Does anyone out there have evidence for any "untagged" mutants being
due to transposon insertions?
2. Has anyone assembled a collection of probes for all the various
Chlamy transposons that could be used to screen for polymorphisms on
Southerns?
3. How many transposable element families have been identified in
Chlamy? I've seen mention of Gulliver, Tcr1,2, and 3, TOC1 and 2, and
Pioneer1.
Thanks in advance for any help/information you can provide. I'll try to
post a summary of any replies i receive.
Kris Niyogi
Carnegie Institution of Washington
Dept. of Plant Biology
290 Panama St.
Stanford, CA 94305
niyogi at andrew.stanford.edu
