P.S. I forgot to say that Hfr

behaves like F+ in relation to

SM. Some of the Hfr prototrophs

from the E oon ce 8 Her are

@rosses were S”. am ge ng

very involved with teaching and routine work and will not be able to
do much

more with K-12.

Department of Bacteriology.
9th. Mareh,1953.

My dear Jim,

I have some news for you which certainly confirms the
separateness of the TL-Az-Lac chromosome and also throws some light on
Hfr behaviour though I am not clear yet as to what it all means.

I will try to put it in a nutshell and supply details when next I see
you. You remember that I got an Hfr strain arising spontaneously in
a 58-161/sp/F+ culture,and was checking on its behaviour. Among other
things I found that its fertility was not increased by UV(unlike that
of the parent F+ strain) and that it formed an Fe phenocopy to the
same extent as the F+ strain. By crossing the F- phenocopy(Lac+Mal+s*)
with W677/F+ on SMe-minimal agar I obtained prototrophs which were Lac+
MaltSr,in the formation of which I assumed Hfr had acted as acceptor
so that these prototrophs should have been Hfr, “hen,however,I tested
them against 58-161/F-= they all behaved as F+. I then tested two Lac-
Mal+S" prototrophs obtained from the same cross against Y-10( TLB, -Lac+)
and both showed Hfr behaviour,though again only F+ behaviour was
obtained against 58/Fe. It looked as if Hfr behaviour was only assoc-
lated with the TL$Lac chromosome,normal F+ behaviour bsing shown when
M was selected for, To check this I put up tvo experiments:
1. A Laet+b,- "Hfr" prototroph was crossed with an F- phenocopy of
“=705(Lac-N=), a, on MA + M + Lae ... Lac selected for;

b. on MA + Gleuose ... M selected for,
Cross a. showed Hfr behaviour & cross B. normal F+ behaviour.
a. Auxotrophic 58/Hfr was crossed with “677 on 3 media:

a. MA + B] ..e TL selected for ...... Hfr behaviour.

be MA + TLB] + Lac ... Lac selected for ...... Hfr behaviour,

c. MA alone ... By selected for as well as TL ..... instead of
the prototroph count being reduced to approx.1/10-1/20 as in the usual
F+ X Fe cross,it was reduced to 1/1000.

It therefore seems clear that Hfr is only associated
with the TL-Lac chromosome and not with B,-M. This fits the segregation
data for Hfr X W677/F- crosses. I have now scored 300 prototrophs for
Lac,Mal,Az,SM & B]. The Lac & Az crossovers are normal,but there were
no SM or B, crossovers at all,and only one Mal(and this might have been
a mutation) ,as against the usual 2-10%,

I then crossed one of my Lac-Hfr prototrophs, rxkh
before and after UV,mwkkm on MA + B, + Lac with Y-10(Lac+TLBy- ..,.' TL
selected for) and with 58/Fe(Lac+M= ... M s@kected for). ‘Ith TL

a
selection there was Hfr behaviour as before and no UV enhancement;
with M selettion there was F+ behaviour and a normal degree of UV
enhancement. Moreover, treating the Hfr prototroph with F+ antiserum
had no effect on the the Hfr recombination rate but markedly reduced
recombination when M was sélected.

And now comes the crux of the matter. As you know,
in the Hfr X F- cross none of the prototrophs are either F+ or Hfr.
I expected to find that,when selection was made for a chromosome showing
normal F+ behaviour,the prototrophs would be F+. Thus the "bound form"
of F+ in Hfr would be revealed though why it is not transduced by Hfr
strains would still be a mystery. In fact,about 5-10% of prototrophs
from two distinct Hfr X F- crosses,in which B, or M were selected for,
wre Hfr, im An equivalent number of other prototrophs showed only
a few prototroph colonies when mated with 1677/F- or 53/F=- below the
expected number for an F+ ¥ F- cross; in these latter cases I have not
been back to the initial prototroph culture to check it but I guess this
was only a temporary F+ carriage(such as is occasionally found sith
filtretes) and that they would be F- on subculture. A second important
point is this. As you know,when By as well as TL are selected for in
the usual 58/S'/F+ X W677/Fe cross on VA alone,the number of SM & Mal
crossovers is less than 10%. ‘hen the same cross is made using 58/ST/tfr,
selec'ion being made for By as well as TL,however,the following ratios
were obtained: Val-S5... 69%

Males*®... 0 Lac crossovers = 29%...normal.

Mal-ST... 13)= 31% sv. Only 30 prototrophs examined,

Wale+st... 18)
As I mentioned,in the same control cross where only TL were selected,
there were no S? prototrophs among 300 tested.
Again,in the cross "Lac-ByHfr prototroph" X 58/F- on MA + B, + Lae(i.e.
M selected),the following SW & By ratios were obtained — 50 prototrophs
tested: SSBit+ eee 22%

s"Bl- oes AOR

S By+ eee % >

rate = 38% ST. The prototroph Hfr parent

SUBy~ ++ +308) was S™,of course.

I think this work clearly shows the separateness of the
TLeAzg~Lac linkage group. I think it also lends weight to my vector
theory. If F+ is a single cytonlasmic factor which determines ability
to conjugate but can be itself transmitted by fusion without conjugation,
then in Hfr strains why is it that recombination involving one or two
linkage groups only occurs in 1/1000 cells that must be assumed to have
conjugated normally with respect to another linkage group? One could
postulate a different F+ agent for each linkege group,each with its
own potentialities but this does not explain why a recombinoent arising
from the transmission of only one linkage group,or F- strains converted
to F+ without recombination,are capable of showing recombination affect-
ing all the linkage groups(perhaps this has'nt been adequately tested).
It seems to me more plausible to suppose that Hfr is an altered F+ agent,
in a new relationship with the cell such that every Hfr agent liberated
is associated with the TL linkage group,but only a small proportion
hitched to the other linkage groups(perhaps both together) -—a proportion
that can be increased by UV. I have decided to write up the whole story
in detail for C.S.Hi. but will clearly have to present a much more
condensed & dogmatic account to the meeting itself,if only for semantic
reasons. I will shortly let you have a sketch of the paper. I hope to
come to Cambridge about Mar.23 to have some electron microphotographs
done. I am sending a copy of this to Delbruck & Cavalli. Yours, ~