so UNIVERSITY OF LONDON

POSTGRADUATE MEDICAL SCHOOL
OF LONDON , .

POSGRADMED CHISK LONDON . DUCANE ROAD
Telephone Department of Bacteriology.. LONDON, W.12

SHEpherds Bush 1260 (4 lines) 22 Mareh,19 54
9 *

Dear Joshua,
. Your long letter reached me when I returned to work
. ¢ today. Thanks for your offer to present any comment I might like
y to make at the Oak Ridge symposium. I appreciate this,but there is
, really no point of my own that I want to make . I really have no
x new significant data which I would be prepared to stand over, though
‘ the kinetics work using the T phages which I started at Pasadena
4 has offered me several new lines which I think may be productive.

F poboiu

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x.
. I will tell you briefly my ideas absut this first. The last para.
Y of your letter suggests to me that you may already have a consider—
able inkling of these ideas from the rather meagre clues I gave youl!
X\ First let me say that I have an open mind on the question of when
@ the eliminations occur and fully appreciate the significance of
--your recent experiments on the metatypic hets. and the segregation
‘ of isolated zygotes. Nevertheless,if you do'nt mind,I would like
to keep this important point open since my ideas were born in an
‘ attempt to devise an experiment to clear it up. I like to satisfy
i myself about things in my own way! The fertility of S°F+ Xx S*F-
Na crosses on S-MA suggested either that S®*° dominance was not expressed
iS in the transient zygote(as opposed to your hets.) or else that
} the s©° allele of the F+ parent was not present in the xygote.
This was confirmed by the fact that neither r; nor T, had any effect
on zygotes in the system Hfr.13°.T¢° X E.T, 70-7, 6n the assumpt—
ion that the zygote is complete but thet dominance relationships are
not phenotypically expressed in it,the situation would be that
g while the Hfr parent could be destroyed by phage to which it was
sensitive,the Hfr genome within the diploid zygotes would be
immune until segregation. If,therefore,the parents are mixed in
aerated. broth for fy hr.so that zygotes are formed,the Hfr parent
then destroyed by phage,the residue treated with anti-phage serum
and washed,and then plated,the products of zygote sergegation
retrospectively identified by replica plating should show the Hfr
parental type in a proportion of colonies. I realise the technical
difficulties involved but I think,from my experience with the
system,that these can be overcome. Single cell isolation would .
greatly improve this experiment but I have not yet had any experi-
@ ence with this technique. I also intend to extend this method

 

 
to an investigation of, the peculiarities of Lac, inheritance..
In my Hfr.Lac+ X W677.¥F- cross,Lact+ is consistently inherited by
no more than 20-25% of prototrophs. Without going into details of
possible theories to explain this low frequency,I thought it @
possible that it might be due to "elimination" of the Lac+ locus
from a proportion of prototrophs. lac, and Ve appear to be rather
closely linked so that any aberration in the inheritance of Lac due
to its situation on the chromosome( rather than due to suppressors
&¢e.) should also involve Vg. This seems to be the case. If,in the
system Hfr.Lac+.V¢§ x Be. Lac~.V¢", the zygotes are all complete for
LacVé6(as I anticipate),then these loci should segregate in the
usual ratio after treatment with Tg followed by its neutralisation
as described above. Should this prove not to be the case,then
wither Lac+V¢5 was not represented in those zygotes saved after

T, treatment uxxeisexthexextorxsxXwe re xxE present eix but xthexdeninrmee
ue from which one might suspect that the dominance of Vg was in
fact expressed in these zygotes in which it appeared. This would
in turn suggest that the sparing of zygotes in the Tz experiments
was due to absence of the F+ or Hfr Vz alleles from’ them. A11
this is a very long shot but I intend to try it. Some time back
_< was toying with the idea that recombination might be only an
extended transduction phenomenon,the fairly regular segregation
rations being due the frequency with which bits of chromosome of
different length were brought over to the F— parent and "replaced"
the homologous F- bit,rather than to crossing-over. I was finding
the eliminations,and the anomalies in the TL-V)-Lac segnent(Rothfels)
and the unexplained interactions too much to swallow in the face of
the similarities of the K-12 system to Salmonella transmuctionie®
the taxonomic relationship, the donorrecipient set-up,the involv
ment of a transmissible agent in the donor strain,the evidence for
"breaks". These phage experiments were evolved with this at the
back of my mind,but your recent experiments have shaken me. I
would like to tell you more about some nice experiments(not so far
successful) to prove fusion by the transferrence of vegetative
lambda (after induction) to a /A2.#on-lysogenic F- but feel I should
. get on to more definite things.

I was very surprised to hear that Tom Nelson had found no
substantial difference in behaviour between our two Hfr strains,
There is something funny here. I may be fond of speculation but
I am a rather careful and self-critical technician and tend to be
cautious about my statements of fact. I have checked and rechecked
my Hfr strain and am now quite certain of the facts set forth in
my CSH paper about it. In addition to these I have examined a
series of 300 prototrophs for Mal(total 600),Xyl,Mtl,ara and found
the Hfr allele absent from all save one. I have told you that the
S allele has been completely absent. from 500. 300 prototrophs
on MA + By were all B]- although a control cross using the parent
F+ strain showed about 8% Bi+. You do not say to what extent
addition of B, to MA increased the yield in Tom's experiments.

I have had a lot of difficulty scoring for B] and,to be f fiI
got these results I would doubt my scoring ahd repeat in an he

controlled experiment using very small inocula. I find I cannot
use replica plating for Bj scoring. I was particularly struck b

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2

“the difference between the two strains in connection with the fool-

proof(!) marker S. Cavalli,in the MGB and in a typescript of his
Rome talk,says that his strain shows Hfr behaviour when S is the
selective marker. This is very definitely not the case with mine.
Moreover,recently I have tested my strain in recombination with

an arginineless,tryptophaneless F~ strain of Coli "C"(8ritish N.C,
123). The Tr locus is very closely linked to(or identical with)

the WY," locus in this strain. The Hfr X C& cross showa Hfr
behaviour on MA + Arg (when selection is made for the Hfr locus Tr+
which probably lies on the TI4Lac "chromosome") but Nfr behaviour
on MA + Tr. On the other hand,approximately the same number of
prototrophs arise in the equivalent P+ X CF- crosses.I honestly feel
there is an important difference between our strains. Both are
derivatives of 58-161,so0 that a comparative test of F+ and the two
Hfr strains in crosses with W677,on MA + B,,scoring for S,lac,and
the other sugars by replica plating should be decisive. Perhaps you
and Tom Nelson would consider this. If you do not find a decisive
diffemence,then there must be some peculiar selective difference
between our minimal medium or else my strain has changed; I could
send you a checked single colony isolation of my strain for recheck.
I am sure that neither you nor Tom Nelson will mind my suggesting
that the results of scoring for B) may be unduly high. I found that
when inocula. were large,considerable growth of B,- prototrophs arose

_ on my MA and that the reversion rate to B+ tended to be considerable.

I therefore went to the trouble of making a dilute suspension of the
purified prototroph in saline and transferring from this to the MA
with a straight wire. Anyway I woud appreciate it if you would
check:on your results,though there is no hurry about this from my
point of view.

As regards the kinetics experiments,I was confused by the
fact that Marguerite Vogt had found(unpublished) that mixing F+ and
F- strains in buffered saline lead to a large increase in the number
of recombinants,although a nutritional supplement was thereafter
required for maximal recombination. She was following up Nelson's,
original work and you have probably; seen the typescript. I failed
to substantiate the occurrence of any increase in saline with either
F+ or Hfr. I will compare the rate of zygote formation in saline,
minimal medium and broth using my phage technique.

I did not make a copy of my last letter to you and cannot

remember just what I said. I do not remember mentioning any experi-

ments on the separation of F from the cells. I did'nt even attempt
this in the short time I had at Caltech. The only relevant thing I
have heard is that F+ cells broken up by HF sound appear to have
yielded prototrophs,though this has not yet been properly confirmed.
This is: being done at Urbana. I think that the question of the nature

of F is perhaps the most interesting aspect of K-12 genetics and

would like to get on to it. As regards my experiment No.1,the term
“reciprocal segregant" connoted only those Hpr markers which appear
in recombinants formed at high frequency. I fully agree that if
your recent work really implies what it appears to imply,and is
applicable to my Hfr strain,then the idea of a deficient contribution

to the zygote from the Hfr parent is no longer tenable. But I would

 
 

mb ook
like to hol3 on to this idea for a little longer.

It will probably be some time before I settle down to ~
experimental work again as I have a lot of teaching and routine

work ahead of me. However,as soon as I have any worthwhile results

I will let you know. I would much appreciate a typescript of you

Oak Ridge talk should you have one. available. Incidentally,in the
report you sent me,it was-not. clear whether you had found more
than one recombinant type ‘segregating from the zygote.

Please give my kind regards” to Nelson and Morse if he is still
with yous With: every: good wish to ad and yourself,

Yours ‘sincerely, “

 

 

at

joe BT ag gem

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