nobody at hgmp.mrc.ac.uk
Mon Jan 14 08:20:50 EST 2002
I have dwarf dominant mutant of A. thaliana (we call it
nana), which is mapped to the top of 1 chromosome. I would
like to clone this gene by transposone mutagenesis. I know
that gai gene was cloned by transposone mutagenesis using
the Ac/Ds lines donated by Ian Bancroft and Caroline Dean,
Cambridge Lab, JICPSR, Colney Lane, Norwich, NR4 7UH, UK.
However, my attempts to find these lines on TAIR DB were
failed, but there I have found new, more upgraded, Ac/Ds
lines containing many marker genes in special construction
(Launching pad). Unfortunately, there is only limited
information concerning to principles of exploitating this
system for mutagenesis in A. thaliana. I would like to
know, if I can use these lines for cloning my gene and how
could I do this?
Below I get my suggestions how to use the Launching pad
lines for mutagenesis, and I would be very appreciated if
you could correct my plan and answer to my questions.
1. If I can use only one line with Ds element nearest to my
gene, or I have to use several lines containing Ds elements
in different loci.
2. How can I get the line containing sAc (what is the stock
number of this line?)
3. After excision of Ds from T-DNA, where it can insert -
upstream or downstream of T-DNA insertion ?
4. I should cross my mutant line with Ds-containing T-DNA
line, and in F2 I will select plants homozygous for na and
Ds. Can I detect the presence of Ds-containing T-DNA using
the NPT marker or I have to use the BAR marker?
5. Then, for transactivating the Ds element I should cross
these homozygotes with the line containing sAc construct
and in F2 I will select plants homozygous for na, which are
resistant to the herbicide BASTA (the BAR marker) and
homozygous for sAc. Can I detect the presence the sAc
construct using the visible marker Lc (this marker is
recessive or dominant?)?
6. Then in several generations from self-pollinating of
these plants I should screen for the plants with
restored stem growth, which would connected with the
excision of Ds element from T-DNA and insertion of this Ds
element in my mutant gene. Can I detect this event using
the restoration of ALS gene activity only, or I should use
GUS-marker gene also?
7. Where can I find out the concentrations of selective
agents (Km, the herbicides BASTA and chlorsulfuron?)
Thanks you in advance
Merry Christmas and Happy New Year
PhD student Olga Sklyarova
Department of Genetics
Moscow State University
Trinity2001 at newmail.ru
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