3 TECK ERK
STANFORD UNIVERSITY
MEDICAL CENTER
PALO ALTO, CALIFORNIA

 

DEPARTMENT OF GENETICS

School of Medicine

Inverness,
9 September 1961

Dear Bruce-~

Esther and: I are taking a few days holiday in Mendocino County, and
are happy to have some time, as this evening, to catch up on our more
personal coreespondence -- as you must know already, we are shamelessly

bad at this.

Thinking back over your visit, I have a sense of very deep regret over
having seen so little of you, and especially of having failed to recapture
that exultant spirit of pIanning and doing experiments with you that I en-
joyed so muchbefore. True, this has been a difficult time, and I was only
just beginning to catch up well enough to enjoy the lab more as you were
leaving, but I can only remark how I could have been so unwise or so inef-
fective as to put first things second. But, believe it or not, this has
changed very much for the better and I have been able to spend more hours
in the lab than elsewhere, and to follow through on my own programs, as
well as vet all the others going on. So I am just terribly sorry that we
were out of phase. One important help on this has been the way that Elliott
Levinthal has taken over the immediate management of the exobiology work,
which is now going along beautifully on the engineering side; also, a
Dr. EX Hochstein (who did bis Ph.@ on the metabolism of nicotine by soil
bacteria with Syd. Rittenberg) is coming in a couple of weeks to direct
the other side of that work. So now, exobiology need no longer take the
disprpportinaate past of my time that it did before. Also, Haruko Nagaishi
is working out to be a splendid assistant, and we are now occupying the
space you had in "Lab I4#.

The Kleins (which means mainly Eva, for the actual{ lab work) spent most
of thdr tame surveying additional markers for linkage to the his-aromatic
complex; there are still some dubious possibilities that have to be rechecked
but we have to conclude that we have no evidence yet of any additional lin-
kage groups. I will be following this up as extensively as possible, and we
are putting in a fair effort into collecting more mutants. We were also in-
terested in the question of "digressive co-transfer", i.e., the frequency
with which dAfinked markkrs, e.g., his,” ind’ --x - - gives a mixed progeny

of +- amd -+ . We had some serious technical difficulties, as yeu may imagine:
the most reliable experiments were done by plating the --x mix on nutréent
agar, then replica-plating the lawn to the test media with careful registra-
tion, and looking for congruencies. These were then confirmed by picking

the , e.g., -+ outgrowths and demonstrating the co-incidence of +- cells.

At a (high)DNA level that gave about 5% of the transformants as ++ (proto-
trophic) "congressive" cotransfers, there was about an equal proportion, in
addition, of the digressive mixtures. These results should relate very direc
tly to some of your findings, wn the single cells -- which I hope you will
soon be able to have in typescript. Marion Schafer had done some earlier
experiments along these lines, but with a different and quite unreliable
technique, and with erratic results.

In the course of these experiments, we had indirect evidence, from the
inefficiemcy of recovery of transformants on replica platings at certain
wekixkagaik inoculum levels that again had to be explained by a disadaantage
or delay of the transformants (or rather, I would believe, of competent
cells). We therefore plated out some --x experiments and found, as we
should have inferred long sinwe, that the transformant clones can be
detected rather efficiently as ammg the small colonies on nutrient
agar. In combination with the selection by penicillin, this now gives

us the most convenient method to study the events immediately after --x,
and we mux are now deep in this. So far, every transformant clone has
contained: the 000 residue also, and we should believe that your isolated
that did not must already have segregates.

There would also be some detail to report, that I cannot convenientiy
summarize as the picture is not yet very simple, on the kinetics of
transformation, as functions of time, concentration and method of r:epaka-
tion of the DNA. There is some suggestion of a lag of about 90 seconds
(interval of expsaure to DNA before DNAse is added) before any transformants
are produced, but we have not succeeded in filtration or temperature shift
experiments in demonstrating any preliminary phase of attachment of %NA
to cells still sensitiye to DNAse. Mei has shown that no reaction occurs
of DNA with cells at 0 C., and the optimum temperature is probably about
40-43, The reaction is also inhibited by azide. Cell competence is
destroyed, without much loss of total viability, by treatment with periodate,
and it looks as if periodate has no effect immediately after DNA uptake,
so I doubt if this is a differential susceptibility of competent cells to
killing by periodate. We have also seen a surprising inactivation of DNA
to rather higher concentrations of periodate, which is reminescent of Mc-
Carty's finding of the effect of ascorbogenic peroxide. It is hot obvious
what the chemival basis of these effects should be.

Perhaps the most exciting findings are just the persistence of some
facts you already know -- Gan has repeated the CsCl fractionation of
markkrs, which correlates quite nicely with the melting points, and Gan and
Gene have quite good evidence of the breakage of the his-aromatics linkave
complex by mechanical stirring of the DNA. It seems certain that the
DNA as now prepared is quite heterogeneous, but we have not been able
to conserve larger pieces (as reflected in linkage values) by more careful
preparation -- as we have been doing in following up the experiments on
the behavior of the DNA in tramsformant cells. I have to say that we
have hot been pushing that as hard as it should be, but I felt these other
questions had to be cleared up first, and we are just now in a much better
position to go back to it. Waadter Bodmer is just getting
underway, after hia preliminary apprenticeship, on the éAzymic inactivation
of DNA, and is getting a good deal of help on this from Bill Razzell at
the Syntex Institute (which is now flourishing, Boris included). Esther
will give you her own account of her experiments, which involved sec much
bookkeeping that we could just now get a clear picture of their implications,

oo i /
en ! /

a aan ae