BAIT RETURN PROJECT

We ask you to send us your LexA fusion contructions for a project
described below.  Ideally, we would like to receive both the bait
plasmid DNA and a derivative of yeast strain RFY206/pSH18-34 into
which you have introduced the bait plasmid.

RFY206/pSH18-34 is URA3+ his3-.  We enclose this strain with the
standard kit (see requesting materials).  You should maintain
selection for the pSH18-34 plasmid (a hypersensitive LexAop-lacZ
reporter) by first streaking the strain onto Ura- glu plates and then
growing in Ura-glu liquid culture prior to transformation with your
HIS3+ bait plasmid.

The more information about your LexA fusions that you can provide, the
more useful it will be to us.  If you can't manage the transformation
we would be very grateful for the bait DNA.

You can send the strain and plasmid to:

Russ Finley
Department of Molecular Biology
Massachusetts General Hospital
50 Blossom Street
Boston, Massachusetts 02114
USA

What is this about

We are isolating sets of proteins involved in cell cycle decisions.
Most of these are new, and we hope to use the interaction trap to gain
a clue to the function of some of them, by identifying interactions
between these proteins of unknown function and known bait proteins.

These baits do not need to be involved in cell cycle decisions: any
interaction may be informative.  If, for example we find that one of
our new proteins interacts with known cytoskeletal components, or with
a component of the replication apparatus, we may gain a clue to its
function.

Although this approach is of unproven value, we are quite excited
about it, and we hope that if it works for our new cell cycle
regulators it might be generalizable to newly identified proteins that
may affect other cellular processes.

To this end, Russ Finley has devised simple plate mating conditions by
which a putative interactor can be quickly tested against a very large
number (100s or 1000s) of baits contained in RFY206/pSH18-34.

We are asking people who are using the interaction trap, if they are
willing, to send us back their bait proteins so that we can test them
against our unknowns.  If we do get a hit, we will tell you (e.g. that
our unknown #17, that has the EQRX motif, interacts with your protein
phosphatase), and perhaps we will both learn something.  We will not
distribute your bait or your RFY206 derivative to others without your
permission.

What we ask you to do
      
If you are willing to go along with this scheme, we ask for three
things back.

1) Your bait plasmid(s) DNA.

2) A derivative of RFY206/pSH18-34 into which the bait plasmid has
been introduced.

3) A description of it

What we would like to know about the bait

1) Name of the protein fused to LexA.  Reference for it.  Residues of
the protein that are present.

2) Other significant facts about the bait.  Was it designed so as to
include any motifs or structures of conserved function?  Was it
designed so as not to contain any such motifs?

3) Structure of the bait plasmid.  Is it derived from pEG202, our
later vector?  Into what site(s) on the plasmid was the gene encoding
the fusion protein introduced?  What is the sequence of the junction
between LexA and the fusion partner?  What codon terminates the coding
sequence of the fusion protein?  What do you know about the length,
restriction map, and sequence (if available) of the inserted DNA 3' to
its coding sequence.

4) (If available) sequence or inferred sequence of the DNA fragment
introduced into bait vectors.  Sequence files gladly accepted.

Conclusion

Note that this is an experimental strategy of so far unproven value.
However, we are all pretty optimistic about it.  Thank you for your
help.

Roger Brent

617 726 5925
617 726 6893 (fax)
brent@opal.mgh.harvard.edu

September, 1993