Dear Josh aud Esther, JO PES

The Pseudomonas cultures were shipped about a week ago, I
epologise for the delay, but upon openbng the envelopes I discovered
that some required re~lyophilizing before they could be shipped.

The little viel of Kinetin included in the package was to heve
been shipped with Bob's yeest cultures to Rubbo, but I forgot to
fnelude it, so seut it with the Pseudomouss instead.

The crosses for the heterozggotes (Lac v, Gal +/~) you
requested sre under wey, thovgh it may take e while to get
suitable diploids,

The problems with the diploids snd their peculisr segregantes
(thoée which appeared to cross with both Lac a& Lac), testers )
May turn out to be due to the testing conditions. It a BT, at
this point thet I have no very good indicator for Lac Thet is,

an Hfr. which will consistently Pr qance papillse when trose-brushed
with ygireins crys e Lec nd 9 with etreing carrying

Lac, © or Lac The hr Mo Lee, almost always has given a
negitive rescttou-~ with W112 or its 1 axtrophic derivatives uader
conditions used for testing segregents from the diploids. This
may be due to: 1) the AUXOS: hic markers, Newton hes date

indiceting W3120 (yg Lac Hfr M-) recombines consistently and
well with F- Lacy prototrophe. He has 260 data regarding auxotrophs,
however, and W112 and its derivatives used in making the diploids

are TLB, . 2) the Medium, Complete medium would probably be the

best to use from the stanipoint that then ideally only the
recombination of Lac markers would determine the reaction. Uufor tumstel,
it is somewhet messy to score becsuge of reversions of Lac a and
relatively wenk fermentation reactious, M-lactmethionine would

be ideal, except for potential variability in recombination due—

to the differentcauxotrophic merkers of the segregants from the 7
diploids. Also, for some reason, on M-lact meth. W3120 seems to
produce even fewers papillae due’ to recombiustion.

I used W3146 ‘as the indicator for Lac, W112 in the results 1
sext you last time. This was due partly to my misunderstanding
newton's date, and partly te the fect that this strain gave. ;
the best reactious with controls. Although the results were not
very stroug or consistent, in geueral, ou M~lactmeth W3146
combined more readily with W112 and its derivatives than with
the Y8? stocks, enabling one to distinguish between them.
ewton, however, has shown that W3146 should recombine with any
Lec, » The supposed restions may agein be due te the difference
in auxotrophic markers,

Croas—-brushes on M—-lectmeth of F™ prototrophs x Hfr M~ to
teet allelism agreed with Newton's data. The aveilable Hfr Lac,
streins have been tested, but with no better luck, I eam now ia
the process of trying to derive a prototroph Hfr with a suitable
lac marker. Meanwhile, the only promisiug approach seems to be
to de everything in duplicate or triplicate ou B-lac and one or
more minimal media.

f;e key~sort aards were just where you seid. Thank you.