Date: Fri, 14 Jul 2006 16:22:51 -0400
From: Natalie Kuldell <nkuldell@MIT.EDU>
To: "Thomas D. Fox" <tdf1@cornell.edu>
Cc: nkuldell@mit.edu
Subject: yeast strain request

Hi Tom-
This email is a follow up on the many helpful conversations I've had with you.
The project I'd like to try is this:

1. recode the nuclear HEM1 gene with the mitochondrial genetic code, adding an
HA tag before the stop. As you know, a strain that's deleted for the nuclear
HEM1 depends on d-ala for growth, fails to respire and is a methionine
auxotroph...so lots of good phenotypes to look at (I hope), and since the Hem1
enzyme is normally functioning in the mitochondrial matrix, I'm hopeful it will
function when expressed there.
2. flank the recoded HEM1-HAm with COX3 sequences. I'm planning to put in 100 bp
from upstrm of the ATG and 100 bp downstream of the stop, but please let me know
if you think this is not enough sequence or if its not the right target for
recombination. [and with apologies for a naive question: Does the plasmid I put
this in matter?}
3. Biolistically transfrom DFS160 (MATalpha ade2-101 leu2del ura3-52 arg8::URA3
kar1-1 rho0)  with the HEM1-HAm plasmid and pRS415 then select for Leu+.
4. Test the Leu+ candates for mitochondrial transformation. I was hoping TF236
might do the trick (ino::HIS3 arg8:hisG pet9(op1) ura3-52 lys2 cox3::ARG8-m)
since I think the pet9 mutation will select against the rho0 parent and any
mated cells that fail to grow on SC-arg are presumed to have recombined at the
cox3 region with a transformed DFS160 strain. I hope this is right.
5. Mate the strain I identify as mitochondrially transformed to the hem1
deletion strain I have. The strain I have is MAT A. I would also like to try an
intronless strain described in the Seraphin paper (PNAS, 1987) if you can find
it. I will knock out the nuclear HEM1 gene in this strain if I have some
success with the strain I have.

So if all this makes sense then the strains requested are:
1. DFS160
2. TF236
3. some MAT A Seraphin strain like GF137-13A

In addition, the plasmids from Aimee Dudley's work would also be enormously
helpful if you can send them. They are:
pT18, which expresses the T7 polymerase targeted to mitos from the ADH1 promoter.
pET3-COX2, which has the T7 promoter driving COX2 expression.

Thank you for all your help with this project. I am grateful for all your expert
advice as well as your patience as I get spun up on this interesting work.
Please email me if you have any ideas or questions about what I've written. I
will not be going to the yeast meeting in Princeton at the end of the month but
Fred Winston will be there.

Appreciatively,
Natalie