* THE ROCKEFELLER INSTITUTE
FOR MEDICAL RESEARCH

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66T STREET AND YORK AVENUE December 20, 1952

NEW YORK 21, N. Y.

Dear Josh,

The reprints finally arrived this morning. I was all set to call W a
for after receipt of your letter yesterday I feared we were ti have even
greater difficulties, I have received to date some fifty requests which with
your 22 and some 20 to be distribtited here will leave about 100, Unfortunately
our departmental secretary is on a two eek vacation and your plan can not be put
into operation. I could send you all of the cards and the seventy-five
reprints to cover them plus the 25 or so that you need or I colud just send
you the cards have you check them and return them to me for distribution.

I shall await your reply before doing anything further with them other than
local distribution.

The new manuscript which you propose has been on my mind for some time now.
It should be written both because of vagueries in the first and the fact
that the hypothesis has now reached a simplified state that is wobthy of
explicit statement. I've enclosed a tentative outline for the paper. It is
in reality nothing more than the enumeration of the various points we can bring
to bear. I shall as soon as possible start writking up the varimous sections
that are already completeand getting the data in useful tables. I too have
the impression that the general consistency of the story has caused me at times to
gloss over certain aspects and move on t o another before securely settling
any particular point. Some battracking may be necessary but it would be
wiser to do it now before my notes become totally uncomprehendable., The material
ska * is in fairly good shape while the } is either incomplete or not yet
done. You may add or subtract any particular section.

As to further work, -- I should be gald to get the thermal inactivation
rate--- on differenti,1 centrifugation the only ‘data available are those
obtained during the course of purification of whagE plate lysates with
alternating cycles of high and low speed centrifugation and the recovery of FA
remaining in constant ratio.to the phage. --- the rate of phage and FA
ma adsorption has not heen done as both can not be measured in a single
experiment.With the AS we could stop the adorption at any particular time
but only phage or FA recovery could be measured in any particular system, Thus the
lytic variant could be used for phage and the parent phage for FA on a phage
sensitive assay system. Probably one of the most informative ways of —‘%
settling the cohort pme problem is an experiment a 1a Hershey. I shall be out
to CSH some time in the near future and shall discuss this with him, --

For the U.V adsorption of the phage I should be able to get a complete curve
as they have a recording spectrophotometer here.

while I think of it-- Since in some respects an MS is written for
a journal how about J. Exp Med as I should be able to swing it from here
and so ease editorial matters etc.

Kad shgerk
1 couldn't help but be amused by the enclosure with your reprint. Hdwards et
al must be pulling their hair out by now. Hope Spicer can do the same for the
somatic antigens. Have you kept a record of the phage to FA ratio of your
various preps on LA-22 as I think this is worthy of inclusion. If you haven't
I would be gag to carry this along. I have a few data on this point thus
PLI-22 = PLT=22/2 =PLT-2277 > PLT=22/547 and is €PLT=22/558 PLT-22/Ed 57.

The extremes of the ratgo growg on different strains or affcecting different
characters has been 10 to 10 , certainly nowhere near the values I'd like,-

wy U.eVe inactivation data are as follows; the phage is inactivated
45 %/minute through three decades and then the curve breaks sharply downward,
the first part seems to have a soft slope rather than be linear; the FA as
assayed on LA-22 Aux to Prot has a slope of 18 %/min. after the activation and
was followed only tHrough one decade.( 9 minutes). Your data are therefore not
too discrepant with mineg as some changes might have occurredtin the output of the
lamp or different conditions of irradiation.

As you probably by now realizexy your 22V and mine are comparable. Sorry

to have been so obfuse and obtuse. The mutant is quite frequent and is about
one percent of the phage secreted by LT~22, lt is not readily seen on LT-2
unless plating conditions are just right but scores beautifully on S. gal
(Didn't I send you soe pictures of it?). It seems to be a typically lytic phage
although I've not yet tested whether U.V. inactivated it still kills. Am now
running its inactivation curve for comparison with the parent. On LA-22 I also
observed what I thought to be a low transducing ability and in fact was concerned
as to whether it or some contaminating reverse mutations were responsible for the
transductions, It seems to adsorb as well as the parental reaching the same
saburation level of about 8/ bacterium but unlike the parental where the
number of transductions is independent of the multiplicity here they are
dependent. That is at dilution,6.1 per to 1 per the line is linear and the phage
FA ratio is 16//1 ,above one the curve starts braeking and bends around with
actually a lower yield at five than one. As I mentioned thirty tested transinductions
all secreted the parental phage not the mutant. If we assume thatxmp the
replacement of the parental phage by the mutant ,results in the death of the
cell a la Bertani the results are straightforward. However I have not been able
to show explicitly the death of any portion of the population so infected.
Then again such death may not result in the loss of a clone and by taking into
consideration the fact that, as ‘you mention, transduced clones are ordinarily

* pure lysogenic and that there is probably ‘a delay in the clonalization of a
transduction only transduced cells where they has been phage replacement can die.
This results in losses of transductions but not of infected clones, I hope I've made
this reasoning clear for it is the same line of reasoning one canlise to explain the
U.V. activation. You may remember we calculated the predicted peak on the basis
that the probability of e transduction was composed of the probability of the
adsorption of the proper particle as a function of dose and the probability that
the phage did not kill as a function of dose. The results fit the observed peak
but necessitated that some 90 % of the cells die which we could not
demonstrate to occur. However 90% of transduced cells could die with no clonal
lossesy demonstrable.

x plas FA go Pn te TL fore) Za mpm Ly sith
a) paply tiekeeim aU
D) Lpeesp ait, Pm ffs Phen ig tr 666
Transduction by lytic variants should also be tested on a synthetis assay
system ( a sensitive made lysogenic for the parental phage) as here ¢he role
of multiplicity should be even clearer due to the larger numbers of particles that z
can be adsorbed. Also to be tested is the role of U.V in such a system.
One experiment with U.V'd 22V , multiplicity slightly less than one, indicates
U.V inactivated phage kills sensitive cells, however a simultaneous experiemmet
with 22 also killed (Used fflose adequate for 22 , Hawentk am now running
killing curve of 22 V). you've not yet run your protection mikx
experiemnt might I suggest you use SW-18S as it is far stabler than any LT-2
derivative I had.

let me wish you and all in the lab a happy New Fear.

Sincerely,

b bro