April ll, 1955

Dear Bruce:

I am just in the middle of mamscripting, but hasten to answer yours of the
6th. I finally got our 35mm camera setup to work smoothly, anfl am enclosing
a (duplicate) negative which illustrates the plating of a single clone in MOA
(motility gelatin agar, standard) diluted 40% with Penassay, and incubated about
16 hours. You won't be able to count all the colonies in the trails, but the
two largest had about 150, 100 respectively (sic). I think this answers what
you asked for; the result is not at all unusual for these platings, but this
happens to be about my best picture.

However, I do not think this is a very strong argument. It may mean that
each of two subclones was plubicatenate, but you already postulated that the
sib of an E cell might give up to 10 chains, which could amply account for the
trails of this magnitude. So, don't misundegatand me, I do not think to have
critical evidence against two orders of chains. But I hold that it is very dif-
ficult to get decisive evidence for it. Unless you have some reliable technique
for alwagy Binding the FE cell in a clone, you can't be sure that there will be
no more than 1. For a time you held that the platings in MGA identified the unique
E cell, but this is hard to indist upon in view of the effect of diluting the
agar. Even in platings in MGA, of single clones, I would find every transition
between clusters ani trails; an occasional clone that gave more than one trail
(arbitrerily categorized) might be ascribed to accidental variations in the fluidity
mfx of the agar. So I don't tyhnk this line of evidence is crucial, and the two
orders of chains remains, in mbwt my mind, one of a few plausible hypotheses.

Perhaps your pedigree experiments are more decisive, though it is hard to see
how they can distinguash between a usual and invariable disparity in partition
of chains among subclones without « technically unfeasible amount of work. I will
send my own draft by, I hopem the end of the week for your more detailed comment.

As to trails in i, tranduetions, I must have mentioned (or did I) at one time
having seen then in ~ S. abongy —x S. miami (in presence of anti 2, 1,5). This was
almost two years ago, but I have not noticed them again (and it aight be tricky to,
in view of the usual sidgh’ spread of the recipient) nor followed them up. It may
mean that only one antigen is ever expressed at one time, evan in a temporary
heterogenote. You may redall that in the duplication stock (CDC 157), which is

4” Hw?” (sic) only one of the H, loci is expressed at any moment; the same is
b

true for various derivatives, ¢.g. 4, * H°, Hy u,*. The trails may be

uy? frag, / H,* cells in which the fragment is controlling the phase.

Have I sent you the squibs enclosed? Iino is putting som steam again on
the phase variation story.

I'll write again in a few days when I've done whth the as.

Yo rely,
fi
UN pn

oshua Lederberg