Dept. Genetics,
* University of wisconsin.
October 6, 1948.

Dear Jacques,

Thank you for your reprint on adaptation. I hope that the cultures I sent
you wiii heve reachsou you, end perhaps Leen cf scme use.

Using oenitrophenyl beguiuctuside as a chromorenic substrite, I have been
continuing my work un coli Luctuse. The substrate has proven to be a very useful
tool. Gali free extructs of very high potency have been resdily obtained from
K-12 with the Booth-Green “111 (thanks to Dr. Green, who is on our campus now),
but nearly comnpruble activity cin be gotten out by «ntolysis overhicht under
toluene. Prolonged autolysis leads to diminution of recovery.

two interesting points hive comes uo in relation to the "isolated" anzyne. Firstly,
is a reyuirenent for, or rather 4 strong stiaulution by Na ion. Preparations made up
in phosphate or glucerophosphate buffer are stimluted up to 100% by 4/50 NaCl or
NaS0, . The other alkali metuls and AH, are either inert or inhibitory (competitive?).

A requirenuent for phosphate has been rigorously excluded (unless £ i “5 or less is
already optimil) by tests in barbit:l and in glycerophosphate buffer, and anslyses
for inorganic phosphate. Jeconfiix 1s the inhibition net only by lactose, but by every
reducing sucir I have tried, of the enzyme.(with respect to its activity on the
galietoside). Sugars not utilized by the organism are ejually inhibitory (e..
cellobiose) and it mikes no difference whether the c ils are Jial- or Yol4 for inhi-
bition my mltose., Suercee and trehulose ure inert (the first utilizoble; the

second not). This would seem to susrest that the capacity to bind with the enzyne

and block its function is relatively nonespecific, while tie srecificty is required
to split the substr:te. The preparations show good linear vesponses, extrapplating to
the origin both for tine vs. product formed, and for product forned ve. enzyme cone.,
strongls sunpogrting the unitary character of the enzyae. ahe enzy.e is formed only
dn lactose (or other sulactoside) idupted cells of wild typs, anit sone of the several
senstic tynes of Lac- so far tested shows any signs of lactase production. All of
woich further tends to support the notion of s complex kind of gene enzyme relation-
ship.

vy work on this aasterial hus been somewhat diverted by the disoovery of hetero-
2ygosis in prototrcphs from crosses of certain K-12 stocks. is you know, the prototropl
are typically pureline and are assuzed to have segregated already fro: the previous
aygote. 4 stock hus veen found (spontaneous mutant,in 32-161 2?) whieh in crosses
with stundurd stocks gives Lacy prototrophs whien ure not stable, but when put on
complete, “ELC medina, sesrezate out into Lac- amd Laef types, which are of all
possible recombinition eclusses, includiie the hitherto elusive sultiple cautant class,
(ioe. Bee Tebeiq- ete...) This interesting ohenomencn seeus tu be sssociited with
an cherration in the recion of the dal) iccus, but hasn't beer thoroughly worked out.

snotoer item that may be of Interest to you is « new nethod for isolatine biochenic
mutaits “hich haus worked wery successfully so far, primarily with suimonella. It de-
pends on the fact that penic@ilin (300 CU/ml for coli) is bactericidal only for growir
ceiis. If in irradiated suspension is washed :ind inoculated into a synthetic mediua
with oenicililin and incubuted 4-6 nours, the growing, non-mutant celis are killed
preferentially, amplifying the proportion of mutants among the survivors. amplificatic
of 103 and higher nave been found in reconstruction experinents with coli, and with
Salmonella, runs in which } the colonies fail to grow on minimal on first test are
typical, so far, no evidence of recombination in Salmonella.

Jith best rezardsa .
Yours sincerely,