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NATIONAL RESEARCH COUNCIL

CANADA

ATOMIG ENERGY PROJECT
Biology Brench CHALK RIVER. ONT.

July llth, 1949,

Dr. Joshua Lederberg,
Department of Genetics,

The University of Wisconsin,
Madison 6, Wisconsine

Dear Joshua,

Many thanks for your letter. It would be difficult
to justify doing a detailed study of duplex prototrophs here as my
main concern at the moment is to establish the mumber of loci in-
volved in mutations to sr and the various forms of sd ( and from sd
to sr, * , and the various intermediates), with a view to interpret-
ing some of the irradiation experiments using these characterse
However, I shall be only too glad to give you whatever relevant in-
formation I come across in my crosses.

My previous letter, I think, indicates all of the
details of the various crossing techniques tried. The technique
which I am now using is to spread washed mixtures of overnight cultures
on the surface of minimal agar plus thiamin, incubate three days, pick
into minimal medium plus thiamin, grow overnight, and streak a 1:10.
dilution on EMS=-Lactose plus thiamin, Parent type contaminants are
eliminated by the EMS and the reduced number of colonies in a streak
makes it easier to spot mixtures.

The first evidence of mixed prototroph colonies was
from the 58-161 x W-677 cross. Some of the streaks on EMB-Lac (plus
phage Tl) were Lact in the absence of phage and Lac™ in its presence,
suggestion a mixture of Lac* v5 and Lac™ vzR. This is of course very
easy to spote In the cross Y=40 x Y=53 one would expect fewer Lac* v5
and in fact mixtures containing it are rare. I shall enclose a table
indicating the sort of mixture we do get from this cross. You will
notice that the frequency of duplex prototroph colonies is somewhat
in the vicinity of 5 percent in these experiments although it has been
higher in others. Also, incidentally, SR seems to be to the left of
MBe
Dr. Lederberg -~2- July 11/49.

I gather from Dr. Ford, who visited C.S.H. on his way
to the Oak Ridge meetings, that the student you mention will be doing
somewhat similar linkage studies on SR and SD while there. In view
of this, I should be grateful if you could have a look at my own
results when I get them and have them in presentable forme Also, I am
taking the liberty of sending the manuscript of my Oak Ridge paper
which outlines my own interests in this regard. Do not hesitate to
let me know if there seems to be excessive duplication of effort as
the linkage studies are of use to me primarily in the interpretation
of old experiments and in the planning of new ones, rather than being
an end in themselves, Also, I am anxious not to tread on anyone's
toese

With regard to the nuclear segregation hypothesis my
main reservation is that we don't know whether there is consistently
more than one nucleus in the resting bacterium, and Robinow suggests
that when the mucleus first becomes stainable after renewed growth
only one is visible. Iagree that the B/1 zero=points are probably
artifacts, and have found the number of them greatly reduced when
large excesses of phage are used.

Incidentally zero-point mutations to B/sr are rare,
(see table 2 of Oak Ridge paper), which suggests that the concentra-
tion of streptomycin which I have used inhibits almost all cell
division. For this reason I think it unlikely that delayed action
of the streptomycin could be responsible for the nearly equal
estimates spontaneous rate obtained using the two Luria and Delbruck
formulas. However, it would take much more work than I have done to
be certain on this point,

Thank you again for the very detailed information in

a
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your letter.

Howard B,. Newcombe

ABNsar