THE ROCKEFELLER INSTITUTE FOR MEDICAL RESEARCH

66TH STREET AND YORK AVENUE
NEW YORK @21,N_Y.

August 26,1953

Dear Joshua:

Congratulations and best wishes on your award. No one deserved it MOTres
Had I known I really would have made some effort to come to the meetings.

ky part of the bacterial genetics course went very well and the data we
obtained on the Lac, vi segregations agreed beautifully with the published data.
The teaching of the coli story forced me to sit down and seriously review tim it
in doing so the appeal of the Hayes! hypothesis became apparent. In the simplest
of ways it encompasses almost all of the complexity of coli recombination. Without
it things tend to be sanewhat chaotic and explanations of the findings not at all
concrete. One of the difficulties 1 found was that I'd been equating mating
frequency with recombination frequency within a small factor, Is it possible that
matings occur as Hfr or better in all cases, and that iek it is in the region of
producing successful and viable recombinants that the strain differences lie.? That is
F~ X F- produces no matings, F= X F+ many matings but only a few viable recombinants,
etc. ‘his would be one way of explaning Hayes' peculiar Hfr strain, Are there any
data other than fram the heterozygotes( which as you may know are not too kindly
looked upon in some quarters) that bear on the timing of the elimination. I was
aware of the het data and had to sit on my hands at CSH to keep from presenting
them but the het itself is an anomaly and to explain one anomaly with another
can oftentimes be a compounding of errors, 4mough of coli and let me tell you of some
of the new things in transduction,

Before I go any further I suppose that you've seen the paper by Baron in the
Proc Soc. I wish he had consulted either of us prior to its submission. is
misquote on the preparation of FA and that uncomprehendible stem dose-response
curve has created some confurgsion.From thmat paper alone one could quite easily draw
the conclusion thaat the phage had nothing to do with the transduction,

While at CSH, Garen,as an able tator,and I played with the doxearmhes PLT-~22V

and the following information is now available. High titer 22V can be obtained by
growing LT-2 to a litile better than 10°/ ml, centrifuging and resuspending in fresh
broth giving a burst size of some 500 and about 1 phage. Shaking with chloroform
removes most of the bacterial debris and also makes the solution sterile for
further manipulation, Three alternating cyclesxiouckhexipinexx of centrifugation
yields very pure phage ( 28,000 g in the Spinco sufficient),

The phage was hydrolysed and its NA composition determined. Unfortunatley
it has the usual bases in equimolar composition, about 1/3 the amount of T2 which
is about right for its e A prio it seems unlikely that a temperate phage
would have differe se composition\fran its bacterial host.

We also determined the transfer of phosphorus fram the bacterium to the
phage progeny and found it to be on the order of 20% which is too hich for ‘eerie
there to be any specificity involved and just about kills any hopes of being able
to identify the transducing part. Another experiment with labeled material bearsn, this
point. The transducing material id turned over in each phage generation while with
T2 about 30 4 of the phosphorus label is transferred to the progeny phage.
Phage was labeled in two ways 1) hot bacteria with cold phage and cold medium 2)

cold bacteria and phage and hot medium. The former should have the label in large
molecule DNA prior to its incorporation into the phage progeny,the latter comes direct
from the medium, The transfer from these batches of phage to their progeny was
measured on the hope that one would turn over and the other wouldn't. Within the
limits of the sensitivity of the method, which is quite poor, both transferred

30 % to their offspring. Obviously the bacterial contribution to the phage is too

ix large and non-specific and overshadows the probably small amount of transducing
material,

There are of course several further things that might be done to aid in the
localization and characterization of the transducing part. The most xm promising
being some experiments whereby sufficient hot phosphorus is incorporated such that
the disintegrations lead to a measurable loss of viability of transduction and
plaque formation depending upon the methed of incorporation. This , if it works » may

not provide any more information thman the U.V. data but one can never be sure in
biology.

All in all there is almost as much information about PLT-82 as there is

for T2, It certainly is a lovely phage to work with and in some respects even more
interesting than T2 considering the diversity of systems that one can study witht

it. I have enough mutants now to begin a recombination analysis and test the

randomness of FA distribution in both growing and recombining (detectibly that #s)
phage populations, I have been kicking around the notion that ‘hier there may not

be an isurtky identity of the particles that produce transduction and those that
produce plaques. By incorporating smmgk enough anti-phage serum in the selective
warm agar it may be possible to show that transduced cells from sensitive populations
become lysogenic for the mostpart by plate infection,

I hope this finds you all well. Busy I'm sure you are. ‘wish you'd come Zast
one of these dgys and give us a chance to repay your hospitality. I was terribly
shocked to hear of the death of Dr. Husicins.I guess he died of a broken heart.

If you should see DTB tell him to drop us a line about his his new offspring etc.
We're expecting another one next spring. A bit more planned than the first,

Best regards to everybody,
Sincerely,

bard