Derartment of Bacteriology,
Postgraduate Vedical School,
Ducane Road, LONDON,W.12.

> - 25 April,1952,
Dear Lederberg,
° . _ Id have returned from a pleasant week at Oxford to my
labour of Sysiphus(research on recombination - not writing to you!) and
had intended ‘answering all your recent letters tonight. However, this
morning I receiyed your ALC telling me of Miss Cahn's inability to
reproduce the S” effect and,since my experimental notebooks are here,I
thought I had bether reply to this matter now. a i have abstracted the
relevant date from 8 experiments(+ one other involving an F+ prototroph
but lacking control counts). A general account of my technique is append-
ed at the bottom of. the second sheet. These experiments deal only with
58-161/8*t X W677(aW677/ST matings and are,I think,a reasonable sample of
my total. ‘While,as you will see,the A/S*:A ratio of prototrophs is very
‘*-variable and the A/st prototroph count always markedly lower than that
given by A,I have never failed to obtain prototrophs. with A/S suspensions.
Against this,I have never succeeded,in a lesser but considerable no.of
expts.ynder the same conditions,in obtaining a single prototroph colony
‘from matings involving W677/St,despite a high survival rate in some expts.
and although all matings were on min.agar-+ Bi(no SM) while the majority
of 58-161/S* matings were on. MA + By + SM200,thus continuing the SIM action.
I.was(and am) quite sure of the results themselves and considered that
they, Justified a preliminary communication to "Nature", As’ you say,the
unequivocal pesults of crossing expts.on MA +.SM,as well as the associatior
_ of fertile S” with F+,also.strongly: supvort the results. Nevertheless I
am very interested in your views and Miss Cahn's results. The latter I
©@ cannet understand at the moment and. can only suggest tat she tries~
again, You have made. a most important and valid point about the ~.
possible-effect of cytoplesmic SM dn SY cells in inhibiting development of
an SS o§-gamete as,of course,this would only become a’ significant factor
in F-/S" X F+/S? mates. | This had not occurred to-me,; It is a. very neat
“point & one to which,at..the moment, I can only reply-that I have obtsined
- prototrophs with A/st XB matings(using 1000 ug./m1,S¥) thoughwith less
efficiency tha with B/ST(vide Expt.of 5.10.51,p.2).. I will see if t can
_ obtein some SM-antagonist from Mill Hill.: If-E can I will send you -some,
‘IT had considered the possibility ‘that the drop in recombination rate using
° 4/S* suspensions might. be due to deleterious action of adsorbed SM(? due
to charge) on the penetrability or other function of a "zamete" and had
thought of trying the anti-SM effect of hydroxylamine but decided it was'nt
_ worth the trouble on account of its. cytotoxicity. :
- oo , The fact is that my results with SM(whatever their true
interpretation) and UV(do'nt forget this!),together with the stimulus of
correspondence with you and Cavalli,opened. up--such an. interesting field
that I preferred to rush on with the tanks while the going was good: and
have not yet attempted to consolidate my. rear.- I have no infantry and
must go back and do it myself when the front stabilises a bit! This
applies especially to the, st work which. so obviously requires definition.
- In’ the consolidatéon programme which I- have provisionally planned when I
"can get down to it I have noted the.following points: "=": SO
1. ‘Attempts to increase the effectiveness of SM in sterilisation By
addition of sulphonamide or penicillin, ~ ;
ro Analysis of the fertility of St’suspensions in terms of physiological
. ‘MBConditions of the cells and after UV enhancement. The few experiments I
|. have done strongly sugzést that A/UV cultures subsequently SM-treated show
a marked increase in fertility,though my technique b- liffered from that
used in the previous gt expts. I think T told you before that washings
of agar suspensions in saline show an extremely low recombination rate.
If, however,the suspensions are suspended in broth at 37°, the rate rises
rapidly to a maximum in about an hour. This applies to 56=161 and W677
although the UV effect is restricted to 5¢-161 alone. This same phenomen
also occurs with even young broth cultures but to @ much lesser extent.
It is clearly distinct from the UV effect & may be of importance,especially
when coupled with morphological Study. Results of “xpts.1,2 & 3 also
suggest that,under properly controlled conditions,ager suspensions
incubated in broth and subseguently treated with SM may prove better
material for studying the st phenomenon than young broth cultures. Incident
Q@lly,this fertility enhancement resulting from suspending agar growths in
broth is not associated with increased liberation of lambda as is the UV
effect. oe 7 .
3. Study of the effect of UV on non-lysogenic K-12 mutants,which Lwoff
“has promised to provide. ‘ mo,
. As regards the techniques I have used in the st expts.,

I began by using 24 hr.agar washings suspended in NB + SM to a final cone.
of 1000 pg./ml. for 2 hrs.at, 37°. This appeared to give good prototroph
counts but "sterility" was poor. I therefore aimed at producing sterility
(as judged by no growth from the St inoculum X 2 on NA after 48-72 hrs.
at 379) & found that this was best. achieved by Reatememed the regimen used
in expts.5,6,7,8. The intermediate log phase culture was designed to.
eliminate "persisters"(temporarily' dormant cells from the overnight culture
ft thought that if the 2nd.log phase culture was grown for more than 4 hrs.
before adding SM,allowing: for another division or so some cells might have
‘entered the stationary phase and: become more resistant.to SM. You will
observe that this regimen,while quite effective in producing "sterility",
appeared(usually but not always)to have a markedly adverse effect on the
fertility of St suspensions.’ This may be due(as you wish to imply,I guW@es)
to the fact that most of the cells wee now really sterilised. On the
other hand the shaking may have promoted alteration of F+—F- phenocopy,
which I could interpret as a relative failure of "gameté" extrusion (is
this good 'sciencemanship?).,- An alternative explanation which I previously
entertained to account-for the low fertility of S® suspensions was -this.
When untreated heterologous mutant suspensions are mixed and spread on
-minimal agar a certain amount of growth (& physiological function) goes
. on. Thus,living cells constitute a dynamic population so that cells which

had not extruded their genes("gamete") on first mixing could still do so.

a On the other hand SMV-treatment "freezes" the population so that only those

cells which had exposed their "gamétes" at the time of initiation of
treatment would: be effective in recombination. .
I feel that this problem of S* suspensions is so intimate-

_ ly associated with other more recent findings that it (& certainly its
' interpretation). cannot: be separated from the problem as a whole and its
solution. I therefore propose,in my paper which will be published in the
same issue of .the JGM as yours & Cavallits,to stick to my theory, This
has served me well in prediction & -I feel | hat it. may well present a facet
of the truth. Moreover,I feel. that the most productive- results usually
arise: from two or more persons attacking a problem from different perspect—
ives. Even if my views are completely wrong,as they may be,they will have
served a useful purpose in being provocative, As regards the morphological
Be oe pags, You, know Mrs .Klieneberger-Nobel is working on this in London

am nelping her as much as I ean. I have supplied her with F+ &F-.
Strains of 58-161.& W677 which may prove useful in’ ‘separating morphol dqwcal

- distinction ; the’ ! S
factors. associated with ‘the sex process from those due ta nutritional

 
Oo

On
“itoin the next two days I will send you a copy of my paper to the san
& this will give you,I hope,a lucid acount of my theory. I agree with
you about semantics and would Like to point out that,in this paper,l
have used the word "transformation" in its common connotation and with
no esoteric implications. The word "transduction" does have rather a
specific meaning and,since time precluied any semantic discussion,I picked
on "transformation" to express the F+—F- alteretion. T am sorry this
paper has been so long but I have been held up by the photographic dent.
Thanks for your remarks about the persistent diploids,whicr I think I
understand,& about the possibility of the segmental eliminations. I am
not qualified to criticise or diseuss such views from you or Cavalli but
it does strike me that fitting the facts into an orthodox genetical
pieture is involving increasing complications and that Occam's razor may
prove a doudle-edged weapon! I think we both have open minds about this
problem and to attempt to describe and explain what we observe in terms
of a context we already know and understand is not a failing but inevit-
able. Incidentally I have told Cavalli that I have no desire to establish
any priority(which I do not have in fact) over the F+ business and,there-
fore,do not intend to publish an abstract of my SGM talk in the next
number of the journal. Your "Genetics" paper will,I hope,come out first
and then our synchronous publications in the JGM.

Yours sincerely,
 

 

 

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METHODS. :

SM-treatment - =xpts.1,2,3. Ovérnight nutrient dgar(NA)culture suspended in
small vol.saline—pdiluted in X 20 vol.nutrient broth at 37°9(NB37) containing
SM1LOOOpg./m1. —» 37° 2-5 hrs.—» washed X 3 & renusperjded in appropriate small
vol.saline-buffer.

Expts.5,6,7,8. 0.1 ml.overnight NB culture(ex Dorset egg at
4OC,) —> 5 ml. NB37— > mechanically shaken 37° 2 hrs. — >» 0.5-1.0 ml.— 50 ml.
NB37 —> 2-4 hrs.379—» SM added to 1000(rarely 2000 )pg./ml. —» shaken 37°
4-18 hrs. —»wash X 3 as before.

(The reason for all this is hidden in the letter).

Control untreated suspensions were always an aliquot of the same NA suspensicr
of NB culture,identically treated except for refrigeration at 4° instead of
SM-treatment.
Heterologous mating suspensions were prepared in an identical way to the
control untreated suspensions.

Control Countg. Immediately before mating,appropriate dilutions of each

suspension were made & one standard loop vol.(0.0055 ml.) spread on NA(in

ob 370° or triplicate). Counts of S* suspensions were made after 48-72 hrs.

at 37°,

Mating. Equal vols.suspension mixed & one standard loop vol.spread on surface
min.agar(+ By:with or without SM200 according to expt.)in (usually)small
m.diam.plates(duplicate,triplicate or quadruplicate).

Total counts quoted = (No.organisms of each suspension in mixture actually
plated on min.agar)X 2, Prototroph counts usually read 40-48 hrs.370,