Hi Natalie,
	Some quick comments:
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?}

I would use more homology if possible.  It will aid in getting integration and also allow you to use a positive selection to identify the initial transformants: see below.

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.

In principle this would work.  But rather than looking for loss of Arg+ (very tedious) you could set this up to look for restoration of respiratory growth if you had the complete COX3 5'-UTL and some upstream sequence and then looked for the ability of your transformants to restore a tester strain with a cox3 leader mutation to the ability to respire.  This is the strategy that Steele used (using a strain described in ref 10 of that paper (GW22).  Take a look at this.  I'm sorry, but I'm really rushed this week.

I can't remmeber now whether oyu ahve any plasmid wiht the COX3 leader?

Alternatively, you could knock out hem1 in DFS160, transform it and then straight for HEM+ aftern mating.  The problem here is that the hem1 variant of DFS160 iwll probably be sick and may not transform well.

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  This isn't the strain you want.  It has a missense mutation in ARG8m and is Arg-.
3. some MAT A Seraphin strain like GF137-13A

This we have.  I've sent a picture of the card as an attachment.

later,
Tom