July 23, 1946.

Dear Francis:

The culture labelled '679-680 double reversion’ which you sent me some
days ago has been scrutinized with the following results:

After preliminary tests indicated that I should expect to find what I
did, I scraped most of the inoculum from the agar slant you sent, and inoculated
complete medium with it, incubated with shaking and plated ca. 10”
cells into minimal, leucine, and threonine agar. There were no prototrophs,
Nor dic any colonies appear on the threonine agar. In leucine, about 107
colonies appeared. The culture is thus predominantly the original double
mutant with a few (167° of the population) threonine-reverted members,

I cannot account for the original appearance of the colony. It is possible
that it represented a contaminant which was heterotrophic, and supported

by the other cells syntrophically, and has since died out, or that selection
plays an important role in the mixed populations containing mutant and
reverted cells. It will be important to compare ratios before and efter
growth of prepared mixtures of mitants and revertants,

Checking up on the triples that 1 sent you earlier (Y9 and Y10) the
following comments are apropos: Y, which is classified as Threonine-leucine-
methionine does respond slightly te methiohine, but much more markedly to
some unidentified yeast extract component, which we hope to track down.

Y1O ~ threonine-leucine-thiamin is what it purports to be except that
there is a delayed growth in the absence of threonine, probably ang adaptation,
which I shall look into. I have gotten a Houble mutant in B/r (Arginine-

methionine) by the detection method, which is working very beautifully in
that strain.

No new biochemival recombination types yet, mostly because I've been waiting
té@ use twontriples together, and Y9 + Y 24 (B¢C) did not work. However, from
BeJ—C-P+T+ and 3+9+C+P-T-R (resistant to T-1) have gotten B-R end P- sens.
which are recombination types, and support fusion vs. transformation.

Am working also to improve the 'mating' medium, and have found a hundred
fold increase in proportion of prototrophs using « beef extract broth, or
a supplement of ecid hydrolysed E. coli . Other veriations (pH, sugar, salt,
temperature, shaking) hed little or no effect. This looks like a good icad,

It's raining here now so I would have no immeddate envy of your position
but I hope you're having an invigorating vacation, so that there will be as
productive a year next yezr as 1 think the lest one was. If possible I will
try to get up there towards the end of August,

Best regards to Bett; und L.S.

Sincerely,

Josh.