Concerning Heyes’ paper in Neture:

Have similar experiments been done with one parent lysed with
phage? Of course 1 realize that this is not strictly comparable
“to the strep experiments. M. Vogt was thinking of doing this
but whether she did or not 1 was never «ble to find out.

*

Concerning Vevalli Maccscarro in Nature - i.e., that a hi frequency
of recombination is obtained when the suvernatants of the washed
cultures are mixed (concluded to be due to residuum of non-sedimented
bacteria plus growth factors ellowing plate microcolony formetion):

a. the residujm of non-sedimented cells would probably be
Single cells rather than clumps $which form when 0.9 %
is added to effect the secomd washing) hence there is a
Breater chance of cells of different parentage coming into
contact than in clumps thus giving more recovereble progeny

b. M. Vogt, when she decided not to follow the methods I used to
produce kinetics results, would grow both parent*l strains
in NB to about 10 to the 8th, mix equal volumes, allow ér¢é several
divisionsto occur (grow together one hour), then plate, without
washing, various volumes in minimal medium, the total volume
plated being 1/2 of the total volume of the system which was
then made up by adding fresh NB (in this manner she thought that
the cells in the cross were being held in a steady state as far

as the environmental and cell coniitions were concerned)

Obviously the recovery or vrototrophs will be pronortionelly |
gre*ter, due to curry-over of bro¥th which is not used u”,

when a larger volume of inoculum is vl*ted, due to microcolony
formation and nlate recombination.

In Jooking over Peg Lieb's letter in which she seys she crossed K12
S X W177 I note that she has verformed the cross both ways - by my method
with the result that the protos are provortional to the nroduct of the varent
concentrstions, «nd by the Vogt method where the vrotos are proportionsl to
the sum of the narents vl*ted. Arparently ML doesn't reslize th-t this is
just as it should be and to get product relationshin she should run several
tubes with varying concentrations of parental cells. Growth of course louses
uy the data - one has to anyly the theroem of mesn value end then too there
1s the problem of segregstion end division of the vrototrovhic segregants
with math similar to the mutation and growth of mutant vroblem.
Further concerning Hayes:

(1) specific objection

Since there are no methods nor no data given in the Nature

paper it is difficult to make any criticism but I wonder about the
following possibility: (a) crossing was performed by mixing 58~1618T7
(strep treated) with war? in minimal medium (I suppose) while (»b)
the sterility of the 58-161 St (or the amount of killing) was
determined by plating an aliquot in strep-free agar, and probably
nutrient agar either before or after washing with saline (makesiittle
difference since a bacteriocidal amt of strep can remain attached to
cell and cannot be washed away according to some bact 'physiologists')
since 58-161 is auxotrophic. Now killing may be (and is according to
some work 1 looked into and the bact physiol people) different for
minimal and nutrient media. Thus Hayes is not actually testing the
viability of the 58-161 cells ysed in the cross. Actually the cross
should be run in liquid and plated (a) in minimal for recombs, and
using kinetics expressions, and (b) in enriched (with proper controls
to determine differential killing in minimal and enriched to determine
residual viable 58-161 cells; 2. ww. ¢ (Thea },

yo J

(2) Baldo (Arnold Ravin - of Alcaligenes fame) writes from Paris
where he is working with Harriett Ephrussi-Taylor on "mappingthe
transforming principle (TP)" - see ECR for ET's theoretical p@er
(and that's all it is to me - until I see quant data) - and work ing
out effect of agar, complex pneumo medium, culture phase, etc. (bact
physiology) on frequency of transformation. They have just heard of
Hayes work and think that it explains‘all of Lederberg's results’
("better than L's original hypothesis") and mine ("I,imagine your
kinetic studies are much moreunderstyndable in these terms than in
terms of a complicated sexual proceas"). H's hypothesis being of
course that genetic material is carréd on surface of corpses of
lysed K12 cells). I pointed out that:

(1) One should beware of explaining all phenomena in tems
of one's own specialty - apparently the Paris people
think that the genetic factors are really TP's stuck to
the €ell wall.

(2) Sow does one explain - linkage (remembering that ET's
hypothesis concerning TP interaction is that and only
that), heterozygous diploids, cytology (Baldo saw $he
slides in 49450 of diploids that you had at CU)?

(3) Crosses between K12 lysogenic (W1177) and K12 non-lyso
had been carried out and probably between two non-lyso-
genic auxotrophic substrains (yourself? “sther? I think
I heard something about Marguertite Vogt's doing it too).
as well. If W1177 must accept gametes from some other
strain then one certainly is up a tree in thinking that
lysis is necessary since apparently none will occur
with K12S5 since no lambde is present. (Baldo brought up
explanation of Texas effect in terms of lysis and release
of lambda).

I hope this quells the Paris sentiment that "This is terrific stuff
and I am convinced as is everybody else in Paris that "sexuality" is
an incorrect explanation for bacterial recombination.,?!
Concerning the sf paper - I got your remarks and puzzled them out
on a camping trip over the week-end, by the aid of a flashlight in the
tornado's tail:

(1)

(2)

(3)
(4)

origin of Kl2 s-: It was the stock that Ryan gave me as K12.
Of course it was reisolated severa! times from a single cell.
The use of EMS is OK - both s- and sf will grow on it - the
s- not as rapidly or as luxuriently nor will they reduce Tphenyl
tetrazolium compounds as well. However I am willing to reword
sections of the paper to let the onus of identification fall
upom the s-.
"classical coliform" is Bergey's. Caginess re contam is Fi's
doing. OK by me to cut table II - plating of sf and s-.
temp sensitivé mutant reference can be reworded
and (8) Wide v riations in oxidation rates exist for log phase
and stationary phase cells - but using the log cells the variation
is less and significantly different in sf amd s-4 Of course
Manometric analysis is a necessary evil if cell-free preps are
not used (I had no TPN to measure the reduction of at 240 m
in the Beckmann - malic enzyme and dehydrogenase cre TPN specific
in coli according to Korkes of Ochoa's gang). But, as you say,
it is fruitless in exp lating the nature of the block and doesn't
analyze the position, exactly, of the block, that's the rub. I
doubt if all the reaction steps of the Szent-Gyorgyi scheme have
been worked out - high energy phosphate is known to be generated
but no intertlediates have been isolated that ere phosphorylated.
sof might be a residual original K1l2 - tho! thenoriginal K1z2
went thru several single colony isolations
Thanks for the reprint. I have it, your diploid, and the T md L
J Bact. 53 papers. True, s- ME might have slipped in but it
wasn’t my fault since 2 mutants, prepared prior to my obtaining
the culture,were prepared (A-3 and B-6) and are s-.
s- is lysogenic 1 believe - Feg Lieb tested it for me by
UV lysis and plating on S.
Please «- my original gebiet and you won't leg’ me mention it.
So what if Delbruck's person couhkdn't repeat it? You know
who it was (grapevine says she tried to repeat H,yes stuff and
couldn't). Besides U,lbruck is reported to have been telling
people "Unrortunately we have been able to repeat Nelson's
work" tho' I must check on this from more reliable sources.
Knowing D. I'm sure this is not a misquotation since he would
never say ‘Unfortwainately we have been A£616/%6, unable to repeat
Nelson's work". In any cross I use kinetics methods just to
check that I'm getting crossing.

I'g willing to include the genetics but how to revort it? All it is
is numbers and if you can figure a consistent linkage you're good. If I'd
only known about the replica technique when I did this!

Tye paper is padded considerably - Mitchell want’ it shorter and Ryan

longer (and cagier). ‘Trying to serve several mestergsat once is difficult.
Trouble is when I talk genetics in geneticshorthand Mitch wents it expanded
and when 1 talk chem in chem shorthand Rydn wants it expanded.
Your recent letter just arrived re the Ff. -+here seems to be a
whole new business opening up.
Concerning the light workusee the ff attached experiment. ‘The rates
(K in the kinetics equation) were somewhat lower at Tech than CU so I
decided to see what happened under illumination. The cross is run between
679-680 and Y24 using the standard plate teghique - Sml total volume is
mized on 10 ml of agar-salts in a Petri (no glucose) - «llowed to stand
for a varying length of time for different plates and then 10 ml of 1.5 X
concentrated agar-salts-glucose added. One series was kept in dark and the
other exposed to a battery of fluorescents. The difference is obvious.
Now comes work on the mechanism:
(a) is it the texas effect? probably not since the filter consists
or several layers ot iron glass as well as pyrex and fluorescents
give off little UV. Conversely is the texas effect this? I
don't know.
(b) 4s it infra-red and increase in chissma frequency? Probably
not since Y24 X p gives increase. I1 must test other crosses
and see if linkage values are the same in light end dark
before the final answer. But the filter includes a copper
sulfate filter to take care of IR.
(ci Ig effect upon rate or upoy, saturation level? Upon rate

ee but may also affect sat level, don't know yet since I haven't hit

a cross with saturation yet.

(d) Is effect temp difference? Probably not - 410 run now being made
but dark controls only 1.8 degrees lower (this means 410 of
about 10) in given experiment and in later‘Xable was heated in

dark controls by flame below to give slightly higher temp.

(e) Is effect due to triggering something in cells or must light be
supplied continuously? Don't know -‘must irradiate cells before
mixing to answer this. :

(f) dls effect due to release of something into medium? (Lysis may
be caused by visible but viable count remains the same yet this
is not proof positive since obly ‘syngamable cells may lyse and
wouldn't be detected). Haven't tested culture filtrates yet.

If something is thrown off then comes the biophysics (action
spectrum) and the chemistry - Kuhn, here we come.

So far I've applied only to Merck. USPHS is next if Merck says no
but it would be best to wait until June 15th deadline since this set is
decided just after (and not before) the new USPHS budget is granted July 1.
Tye application states oept. 1, 1952 - thought we could always modigy this.

Sorry to have written a book.
Regards,