20 = 22°

May 16, 1964
Dear Bruce:

At last a tardy and hasty reply to yours of the 20th ult., which I found
on return from Oak Ridge. Your letter and the discussion there provoked a
review of notes and some new experiments, ail some new reaults with cold
(eee later) were particularly distracting, to delay this reply. I trust you
will have returned from your vacation,(which wag another reason I ddd not

‘reply more urgently). I will try to go over your letter point hy point or I
will nover get oa to all of it. | :

i): my experiments are, of course, various combinations of short and
long intervals of growth ond seperation. Sometimes I let the initial cell
go through about 15 evensrationes before seperating tne descendant motiles,
in others the clones were dissected early or middle or late. I had been trying
to cet the wholes Gestalt before inguiring into details. Chambers at room
temperaaure* occasionally refrigerated (no ozvious harm or change) or incubated
for an interval at 30 or 35 C. 41) Same method as for original isolation
of motiles. A large empty frop is placed and fused with the larga clone, and
this stirred somewhat with the pipette. Motile ceils almost invariably
work their way into the trap drop. (My note on simpl2 method hae nothing
whatever to de with pedigree experiments, but is just an exped&ant for
getting aingte cell cultures for “pure line’ work without uuch fuss).
4414) My startfine materiel consiste of overnignt 37° Penncaey oultures
mixed 1:) with lyaste or diluted lysate in sene broth. My Birwrer:y showe
thet of 11 initiale ylelding ewarne, 4 yey’ gave mixed clones. Of these
4, 2 could be told, and did, give semieionea as well as clones (but only
1 end 2 respectively}. I am convinced that the paucity of nixed progeny
de due siroiv to the pedigreas'usvally starting only at tre 24 er 2A
seneration, and until thie problem can be circunvente®, there may act be
ruch poirt in collecting more deta.

Pel T am pretty well ecresd vito you on dricroretetlens, bet do net put
much weight now on macrogcopic observations, I am convinced that chenotaxia
Plays an wneontrolied rols in toe initiation sf vielbie trails. Some diffusion
of celis in agar ie quite possible, with or without ehenotypig las, but I
agree there is tha ancearanse uf Clustering wig.uithis eis diley ( though
this ie not obvious microscopically).  peS Neo cases here oP wewerm from
trail, Could yours have been svontaneous revervion. Tracy sonnebhorn also
suggested continuing activity of non-replicative gene, which fucen two
hypotheses. Hotchkiss also raised auestion tek shout the nctile cell of
a dividing T cell remaining motile, which has been ay exvericave. This means
either a very prompt response of Dhenotyre tu ponotyne, or oo Adontdfication
or binding of the particle to the Plegeilar avparutie ilsel?. p.%-4: as to
this test (distribution of numbers) my déta are not too wall arrenged for this,
and I have little enough intomaation on tie most wpoctacule: cases of multiple
semiciones; these are so rare that one can imegine almost any malse for
the parcelling out. Certainiy it is irreguiar: one could sever get even a
20:0 or 20:1 elit at the first fission on @ randon Ciuicibution basis, but
this misht mean merely a splintering from the large bundle (oa thet notion).
Here are some sample data on distributions (each parentiegis= 1 initial;
split among subclonas na indicated, i.e., humber of semiclones 4n each):

(8) (7:13) 30:6) (10022) (0:0:0:031:1:1:22394:15) (0,8,6,1) (13(20:11 subsibs
t1:1) (O:Otat least 10: 100) (4:6). Other clones ZA6 dissected early had
only a few. odd semiclones. But the important clues may be in ths missed first
divisions, which wight aleo account for Af/dddt such a disparate distribution
of semiclone yields per apparent initial. I think especially important would
be casea ineluding swarn-pqukkalent progeny. My somment on serum inhibi-
tion was cbrrectly quoted, but my esrums may not have been clean, and this
Was not well controlled. Your findinze noted..
v the instant I au still womewhat distracted ty some writing, but aiso by

.@ conjugations. The trick, learned from Saimoneila, ia to use Hfr motile X
.- non-notile. Pairs, in numbers auffisient for incidence ‘of recomblnation, sre

not too difficult to find, and trap, by virtue of their erratic motion, ‘They
generally look like: | disjoin after l- 2 hours, at room
temperature, with up to “one: ‘Antervening fissione The majority ‘of F- exconjugants
yield recombinants + ‘parent. Main trouble: fumgility of the poirs-- many of them
suffer from handling in transfer and are lixely te die if forei bly saparated too

early. But at best, aaa exoonjugants aurvive . -

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PS. The Oak Ridge meetings were mildly interesting, but rather misdirected:
everyone, incluéing myself, was discussing crossing-over in some molecular terms
or other, and ignoring the certainly mors complex etructure of the chromosomes
(which undergo crossingover) sitogetner. I saw Jim Wateon again for the first
time aince he gvaduated from Iilinois-- he has changed somewhat, but gave an
unexpectedly reserved (in light of rumor) sort of discussion of DYA structure.

he nocessary luplications for replication, mutation, creseing over, etc., are
almost nil, and he was, I think wise, to give a straightforward account emphaszing
tine proiabl J aound at the expense of the obvious. (and prosably deceptive) specula-
tion. Tha DvA molecule Ls far from the chramosome, though the phagelisute are
especially Sompiucd to extrapolate right away. Mostly, bho mooting waa 400 concrete:

‘there 1s a tremondous aneunt of exis sung Anfornats ono G=0 and. 4% would have been

wiser - to asointl ate tiis than aad a Pew. indecisive irague. Sle doteliki ee gave an

accomt of his intoreating linked trencdcstion Gin ~ 3) which seous entirely

sornvinsins, eepo analy after the be okeres 3¢ data. There was still too much emphasis

on DNA molecule _Yathor then geaetic “vaguents-- Lerrah (Puck's lab) 4s a prdord
see he'?l hays no trouble eractionating tha dicfarent DNA'c, but he doean't
» seer ave Sek it yeb, .

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