Bot FoR PUBLICATION March 1, 1953

Final Progress Report, March 1, 1952 ~- February 28, 1953
to the
Atomie Energy Commission
Contracts Eo. AT(11-1)-64 Proj. 10
CYTOGENETIC EFFEOTS QF RADIATIONS OW BACTERTA
Department of Genetics, University of Wisconsin

Project Leader: Joshua Lederberg, Ph.D., Associate Professor of Genetics

This project was oxviginally formulated to study the cytogenetic
Consequences of irradiation of haploid and diploid bacteria. During its
early stages, we encounte*ud an unexpected phenomenon which has appeared
so fundamental to all of the studies in this laboratory that the original
project has been diverted, for the time being. It is intended, ultimately,
to return to the original problem but we do not at present have sufficient
personnel or facilities to carry both. When an occasion suitable for re-
sumption presents itself, it is intended to renew this proposal for research
support from the AEC, but in the interval other support is being sought.

In the previous report it was mentioned that our test organiem, the
bacterium Escherichia coli strain K-12 was lysogenic, carrying a symbiotic
bacteriophage, "lambda." The genetic relationships of lambda and strain
K-12 have heen the subject of other investigations from this laboratory
(Lederberg and Lederberg, 1953). It had been discovered by Lwoff that
one of the modalities of radiation-killing of various bacteria concerns
the activation of eymblotic phages, resulting in the lysis of the host
bacterium. In order to substantiate the analysis of similarities and
differences in radiation responses of haploid and dipletd bacteria it
was essential to study the role of the "Iwoff effect": induction of the
a De

latent phage. A preliminary study showed that, under certain conlitions,
the presence of symbiotic lambda did, in fact, potentiate the sonsitivity
of K-12 bacteria to ultravielet light. A by-product of these investigations
was the production in the induced lysates of high titers of lambda (over
1049 per mi) such as had not previously been attainable by more familiar
nethods.

Concurrent studies in this laboratory (Zinder and Lederberg, 1952)
have shown that another bacterium, Salmonella typhinuriun, displays a
mechanian of genetic transfer quite distinct from the apparently sexual
recombination precess in E, coli K-12, In Salmonella, certain lines of
bacteriophage are capable of transferring individual genetic determinants
from one genetically marked strain to another. Repeated attempts have
been made to demonstrate a ecimilar transduction process in E, coli, either
ag an alternative oxmplanaticn of, or as an suxiliary to the sexual mechanism,
These experiments, involving a variety of cell-free preparations, extracts,
and lystaes had been consistently unsuccessful. The availability of high-
titered stocks of lambda led, however, to a casual re-exemination of the
possibility of a transduction in E, Celi. The first stocks tested included
a nomber which gave negative results, but alao one, known to carry Gal-
and Lac- (galactose and lactose-negetive) mutations. When plated with
Lambda from induced lysates of lysogenic wild type bacteria, the Gal- Lac-
stock was poticed to produce numerous papillae on EMB lactose agar.
Further study ehowed that the papillae were, in fact Galt Lac- (cf.
Lederberg, 1952), and subsequent experiments have been carried out on
EMB galactose agar. Testa of several other mutant stocks of strain K-12
have uncovered no character other than galactose fermentation that is
subject to transduction by lambda, thus sharply differentiating this
system from transduction in Salmonella, which may involve any single
trait, and from recombination in K-12 itself which typically involves
~ 3<

numerous linked factors, en bloc, Previous work hed also shown that
semal recoubination in E, coli K-12 was indifferent to the presence of
lembda in either or both of the parents (Lederberg, Cavalli and Lederberg,
1952). Conversely, it has since been shown that the transduction of Galt
proceadis veadily between soxtally incompatible stvaius (i.e., two cultures
both F-),

To sammarizo, the traneduction of the Gal+ factor is detected by the
augmentation in the mumber of galactose~poai tive paepillee evolced from various
Gal- strains on EMB galactose agar. Unfortunately, most of the available
Gal= cultures are capable of reverting spontaneously, so that control
platings Luvarilably show « mumber of papillae. The apparent augmentation
by lambda was therefore scrutinized with some suspicion that a selective
rather than trausductive effect might be operating, and it was some time
before the balance of the evidence weighed in favor of the latter. In
a typical experiment, control platings might show 50 papillae per plate,
while 109 added lambda particles would ovoke 500 papillae. This effect
Was Consistently observed in replications of the oxperiment, and disappeared
if the lembde was previously boiled. I+ was observed whether the Gal-
indicater culture was sensitive or resistant-lysogenic for lambda, bat
not when the indicator was immune, i.e., did not adsorb the phage.
Finally, lambda secured from Gal- cultures lacked the effect.

These observations indicating that lambda could, in fact, transduce
the Galt+ factor were supported by a further study of the induced papillae.
Whereas the papillae arising by spontansons reversion were invariably
stable Galt, most of the papillae on the experimental plates gave rise
to cultures which were unstable Galt, having a sectored appearance on
EMB galactose, and contimuing to split off Gal- types after repeated
single-colony isolations. Ina few instances stable Galt types have

also been noted to arise from the unstable cultures, but tha facts here
-4.
are still not well-established. It is also not clear whether all the
transduction Gal+ are unstable. The number of initially stable Gal+
papillee en experimental plates is generally greater than on controls,
so that it is likely that some of the transductions are initially stable,
while others are unstable. In the absence of further evidence to confirm
the numerical differences this point remains unsettled.

Further experiments have been designed to study the scope of trans-
duction from a genetic viewpoint. Remarkably, there are at least three
or four distinct Gal- mutations, at different loci ("Galj," "Gal," ete.),
each subject to transduction, while no other marker hae been so far.
However, it is quite likely, from the results of crossing experiments,
that these Gal factors form a fairly closely linked cluster, perhaps
comparable to the “pseudoalleles" now so prominent in the genetics of
higher forms. The different Gal- types can be distinguished in two
waye: their interaction in transduction experiments, and their re-
combination to give Galt in crosses. The two criteria, after certain
technical problems were cleared up, have given consistent results. This
has enabled a study to be made of the apparent segregation process in-
Volved in the unstable Gal+ behavior. For example, Galp- Gal,+ —-x
Galgt Gal,- has given unstable galactose-positives which split off
predominantly Galy,-, but also some Galo-. (The symbol --x denotes
transduction by lambda from the left-hand to the right-hand term).

It is concluded from this experiment that the unstable positives have
a genotypic formula somewhat similar to:
Galot Gal,-

 

eeee eeso

Galo- Gal yt

 
-~5-

in which the short lever line represents a smsll "chromosome" fragnent
traneduced by lambda. An important question remaining to be settled is
how such fragments come to be incorporated in the stable genotype of the
cell, e.g, in the establishment of stable Galo” "segregants." The same
question applies to transduction in Salmonella, bat we do not have the
advantages there of the unstable intermediate state, nor of analysis by
soxual, recombination.

Proof that the trensdvueing element in lysates is the phage itself
in afforded by the correlation botween ability to adsorb lanbda, and
susceptibility to transduction among various indicator stocks and similar
rates of absorption of the twe activities on indicator bacteria, In
aidition, there 13 a close correlation between the lysogenization of
sensitive and the transduction of Gal to Gal- cells. However, only
about 1 phage particle per million capable of lysogenizing also tranus-
ducer Gait, Current studies are in progress on a system showing a much
higher efficiency.

Preliminary studies have shown an anomalous offect of ultra-violet
light on the transducing efficiency of lambda. Small doses, which reduce
the plaque-forming titre of a lambda preparation by no more than Log
potentiate the transducing activity by 10-fold. Higher doses will then
ultimately reduce transduction, but uot 30 rapidly as infectivity. No
explanation for this behavior 1s offered at present.

The experinents summarized in this report are principally the work
of Mr. Me L. Moree, in collaboration with E. M. Lederberg and the

under signed.
-~6-

Lederberg, BE. M. 1952 Allelic relationships and reverse mtation
in Escherichia coli. Genetics 373469-483.

Lederberg, E, 4M. and Lederberg, J. 1953 Genetic studies of lyso-
genicity in E, coli. Genstics 38:51=64,

Lederberg, J., Cavalli, L. L., and Lederberg, FE, MH. 1952 Sex
Compatibility in E, coli. Genetica 37:720-730.

Zinger, J. D. and Lederberg, J. 1952 Genetic exchange in
Salmonella. J. Bact. 643:679-699.

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