The Lister Institute of Preventive Medicine.

Chairman of the Governing Body :

Sire HENRY H. DALE, 0.M., G.B.E., M.D., F.R.C.P., F.R.S. CHELSEA BRIDGE ROAD,

Hon. Treasurer :
THE RT. HON. VISCOUNT WAVERLEY, P.C., G.C.B., LONDON, S.W.1.
G.C.S.1., G.C.LE., F.R.S.

Director : Telegrams : *‘ Bacteriology, Knights, London."
ProFessor A, A. MILES, C.B.E., M.D., F.R.C.P. Telephone : SLOane 2181.

20th.April 1954.

Professor J.tederberg, Thy AMlr w tA nessy | om. forms,
Department of Genetics Tone, t me _.
University of Wisconsin, bie Ze Gumrre fond po
Wide UeS cAe ? wre seh f-

Ve wi gh tong Ond, A oy the ell preclignen 13

Dear Josh,

Many than’s for your letter of 28 March, also for the
"Progress Report) which arrived about the same time. I have read
both several times, and done some cogitation and re-examination of
records of earlier micro-manipulation éxperiments (one or two new
ones but no new information), hence delay in answering.

Before getting down to hypotheses to account for trails,
there are a few points about your technique I would like to get
straight. (1) When you say e.g. "not earlier than 14th.generation”"
do you mean that you have followed one line of descent, splitting up
progeny every generation (or 2 or 3 generations), or is this an
estimate based on estimated population size. and/or calculated
number of generation times assuming exponential growth ? Or, as in
my date, a bit of both ? If the former you must have worked lite a
slave; I take it your oil chambers are vept at room temperature.
(11) I would be interested to now what method you use for isolating
single motile descendent fromcrowded droplets I take it you were
using real micro-pipettes, not the semi-micro ones you mention in
your "simgle method" note. (I failed to ma’e this work last weer,
but on re-reading now I see had misunderstood it. I willtry
again). (As to re-use of pipettes, I find dipping in botling water
a satisfactory way of re-sterilising a {soft glass) micro-pipette,
@eGe after accidentally sucking up a lot of cells). (141) You say
you use lag phase (SW 666) as recipient. Is this a 37° culture
grown to saturation, 6.g. overnight, and then diluted in fresh bboth
before, or at the same time as, adding phage ? I am specially
interested intrying to get your single motiles giving motile and
non-motile clones at 1Ist.edivision, which I expected to find but so
far without result.
   

Now as to hypetheses to account for kAbortive transduction etc.
Ag to polyteny. I think this too far fetched when it gets to
100-fold or so, and I gather you agree. I think any supplementary
hypothesis such as yours on transfer of sgne products, or mine
expanded below, will account for all the things to account for which
we were each inclined to invoke polyteny|\becomes an unnecessary
postulate. Your hypothesis has some attractive features: I am not
keen on having the phage transfer two different kinds of material,
gene and gene product, but as gene product might be partial gene
replicas piled beside gene I suppose there is not much in this.
But if it is to explain qll abortive motilisation one must postulate
transfer of number of products varyihg from 1 to more than a hundred.

A satisfactory hypothesis must cover the macroscopic observations
as well. The long unbranched trails produced byy for instance,
SW 553, prove that there are some cells which carry a unit conferring
motility (in agar) which is never, or almost never, replicated-and-
partitioned. (a)Analogy with the micro-experiments would lead one to
expect that most cells producing 1 trail wouldrroduce several. But
using SW 541 and lysate of TM 2, in parallel macro and micro
experiments, one finds at least the great majority of macro-trails
arise as single trails,contrary to expectation. (b) I did some counts
on numbérs of colonies itn trails, I forget the strain and have not
notes here, but making correction for "end-error"by comparing countable
colonies in a trail after, say, 12 and 24 hours, it was clear that
number was much greater than number of generation times in 12 hours,
henee one must conclude several generations “phenotypic lag% (Ithink
the wide crowded trails produced by @e8. SW 545 make this clear
without counting). But both my and your experiments give no clear
evidence of lag, after the 15"generationg or so anyway. The only
evidence I had for it ware the anomalous droplets, grown from single
cell isolated after, say, 10 generations, containing, amongst usual
O majority, say a dozen motiles, none of which gave anything but 0
progeny. Obviously one can't get positive evidence of phenotypic
lag unless one watches all progeny for several generations, to exclude
death of gene-bearer etc. as explanation, but lag is certainly much
less frequent than one would expect from masro-experiments, if indeed
it occurs at all. (c) Comparisons between micro and macro experiments
are difficult unless one is comparing same cells. But such few
experiments as I have done (and TI gather you have had same results)
showg that if one picks say, 40 motile cells, puts 20 in droplets
and transfers 20 to gel-agar, there is approx. agreement in proportion
giving swa » (5-10%) but gross discrepancy re trails; most cells
which give "trail equivalents” tn ofl chamber give single colonies
in gel-agar at 37°. | k20°

(d) You have had one cell giving motile glone and "semiclones”.
(I don't get rationale for this term which T take to mean same as
my "trail-equivalent"; the latter I agree begs the question but your
term I find insufficiently self-explanatory). We have had “6fie example
“3

(macro) of a trail terminating in a swarm; this looked fairly
definite.

To account for these observations without postulating totally
irregular replication of the“super-numerary gene’, I propose the
following scheme: Phage transfers gene, which is either incorporated
(or replicdd) in continuity into Chromosome, to give clone: or
accepted into some other situation. This might be either as side-~-
branch of chromosome, or in cytoplasm. In the latter case, one must
postulate that the gene decays in some way so as to (nearly always )
prevent later incorporation. In either case the cell is an "Rt cell,
capable of forming a trail in agar-gel. (Alternatively the transferred
particle which gives an abortive was pre-determined to do so before it
entered cell, e.g. was defective in "matching -up" SrOuUpS, or was a
primary gene product, probably partial replica).

The E cell, 1.6. cell containing Fla* gene in abnormal situathn ,
manufactures flagella via a series presumably including “primary
gene products", E cell presumably contains wild-type amount of these
products, including flagella, hence can spread in semi-solid at 37°,
If any of the products are “particulate” effective in dose of 1
particle, and not consumed in producing their effects, and are
divided at random when the cell divides, then when an EF cell divides
it will produce an E cell, and a "7" cell, which will produce n T
cells in its progeny, where 2n is the number of particles present in
the F cell, Possible candidates for the role of non-replicating
weakemotility-producing "particles" would be the primary product,
@e&-. partial replica, of the Flat gene; and the flagella, or their
"basal granules themselves. On this hypothesis, the macro trail
marks the path of the E cell, which owing to a ""dose-effect" of the
particles is highly motile through semi-solid medium and keeps going
till it dies. The super numerary colonies of trail, at first
attributed to phenotypic lag, ane really "second-order" trails ending
because single particle though it confers broth motility does not
suffice (for long anyway) in semi-solid, This accounts for failure
of macro-trails to split or arise in groups, despite micro ffndings.
The trail to clone examples (your micro, my macro) indicate that the
particle which mares a cell an E cell does very rarely later become
incorporated in Chromosome, 1.6. it is a gene or partial gene, which
particle of T cell need not be. When T particle is reduced to one
there might be no carry-over effect (of necessity if the T particle
is a flagellum. Hence absence (in general anyway) of lag in later
generations in micro experiments. The failure of most single motile
cells (hand picked) to produce trails in semt-solid would indicate
they were mostly T cells, not E3 in the only experiments I have done,
the time of isolation was so late that this might well have been so.
Gok ae ciety tong, Tank Tits ptth hbo ton 1 laa a, Wee tanlton PU pombe)

, This theory makes on prediction which can be checked on

existing data. Droplets inoculated with 1 cell which contain man
motile cells must have been tnoculated with an E cell; therefore Te
 

the clone produced by the original picked cell }/has been sub-divided
into its components at, say, the 8 cell stage,/no more than one of the
sub-clones should contain many motiles, (many{particles per mature

E cell). If n may be as large as 7, then my data fit; but this is
rather a high value and anyway there are not enough data to test the
ideae I shall be interested to hear whether your data contain any
exceptions.

As to further experiments, the following seem worth doing,

if possible (a). Further tests to see why micro and macro experi ments
disagree as to early branching etc. Temperature may be relevant, but
one macro experiment on_semi-solid agar (no gelatin) at 23° give same
‘Vind of result as at 37°, both as to singleness of trails, and failure
of most hand=-picred motiles to initiate trails. As to the latter, it
would be nice to Sompare trail counts on gel-agar and number ofmotiles
produced under microscope, but I don't see how to measure latter.

(bo) If the T particle is a flagellum or basal granule then T cells
should differ in electron microscope from wild-type. (They don't seem
to show the excessive wobbling motion one gets with a vibrio, but a |
peritrichous organism with 1 flagellum would be a new&unpredictable
object). I have grids and ar-angements to get them looted at, all I
need to do is to find how to deposit single cells on them. (C) Further
pedigrees, if possible splitting up abortive clone at eachcell division
up to 16 or 32 cell stage, to get max. value for n on my theory.

(a) re-examine some long trail prodtisears, counting colony increase per
generation time, for an independent estimate of n (or of mean phenotypic
lag). Nothing else occurs to me at the moment. I suppose we had

better each work away at what presents itself, and compare results
(and theories )from time to time. The thing has proved surprisingly more
complex than seemed likely when I left Madison.

One further point; most of my micro-manipulation experiments
were done with SW 541, and lysate of TM 23; a few with lysogenic
derivative of SW 541 gave similar results. ‘

Thats all on abortives. We are pressing on with attempt to
map; if the argument is sound, the order must be (544) - (28) - (543)-~
Hi- (966) - (553), but 544 4s an obstinate devil anyway, and 553 not
much better at times. I agree quantitative data needed.

Yours sincerely, Lnx 4 Ente Hien wm
Of rok wed, Cry b Fors,

Bestocrer.,
Jon Ay Yr Frgues Apne Pal tem my prmter/on
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