FEDERAL SECURITY AGENCY

U. S. PUBLIC HEALTH SERVICE
c. Hesearch Laboratory,
1l East 69th St., New York 21, N. Yo

April 14, 1950.

Dr. Joshua Lederberg,

Dept. of Genetics,

The University of Wisconsin,
College of Agriculture,
Madison 6, Wisconsin.

Dear Joshua;

Enclosed is a copy of the manuscript that you
suggested I publish. Any comments would be appreciated.

Sincerely,

*

es
“Ee Ey

Bernard D, Davis
BpD/h1 “ eos ;
/ J. Paer. pte” .

ence
Non=filtrability of the Agents
of Genetic Recombination in Escherichia coli

The demonstration of genetic reecmbination in bacteria
(Lederberg, 1947; Tatum and Lederberg, 1947) appears to imply the
existence of a sexual mechanism, although direct evidence of cellular
fusion has not been produced, presumably bec&use of the low frequency
of the phenomenon in the populations studied (ca, 107), Since produc
tion of genetic transformationg by a soluble material has been estab-
lished in pneumocoect (Griffith, 1928; Avery et al., 1944), and
reported in B, coli as well (Boivin, 1947), the possibility of a
similar mechanism must be seriously considered for the recombinations
discovered by Lederberg.

. It is true that the appearance of recombinant progeny involv-
ing multiple factors in various combinations is more difficult to
reconcile with the hypothesis of a soluble agent than were the original
observations (Lederberg and Tatum, 1946) on single genetic factors.
Nevertheless, one cannot arbitrarily exclude the possibility of either
a complex transforming substance involving a number of genetic units,
or a gamete with a full complement of genes but smaller dimensions
than the bacterial cell, Previous failure to produce recombination
with culture filtrates of E, coli tiutammumi(Iederberg, 1947) could be
due to greater instability in this case than in that of the pneumococcuse
It therefore appeared of interest to test further for a filtrable factor,
attempting as far as possible to circumvent deterioration by minimizing

the delay in transport from donor to recipient eell.
-2=

For this purpose a bacterial contraceptive device was
fashioned in the form of a U-tube partitioned by an "ultrafine" .
fritted glass a (corning). . its Ampermeability to beeterta—was
verified-withr the organisms under inve stigation, E. coli K~12 mutants
Y~LO (Threonine Leucine Thiamine’) and 58-161 (Methionine ), kindly
furnished by J. Lederberg, were separately inoculated (1 ml. in 10 ml.
of medium ecntaining 0.5% yeast extract) in the two sides of the tube.
At frequent intervals during cultivation a large portion of the fluid
was alternately forced through the dise from one side to the other by
application of a vacuum. Simultaneously, cultures were made with a
mixture of both inocula in a single tube, as well as with single inocula
in separate tubes. After & hours all 9 eultures were washed and plated
in minimal medium. Conditions of plating were such that at least 90%
of the prototroph colonies obtained from the mixed tube had arisen from
reeombinations occurring in the tube, rather than during. subsequent
incubation of the mixed plate, This fact. had been established by
numerous experiments comparing inocula mixed in the tube with inocula
of identical size mixed in the plate.

The mixed tube gave 58 prototroph colonies in a 0.05 ml.
inoculum; the cultures from each side of the U-tube, and from separate
tubes, gave none in volumes up to 0.5 ml. Similar rasults were obtained
“in another experiment. It is concluded that recombination by means of
a filtrable transforming substance or gamete is unlikely; such a mechanisa
could be reconciled with these observations only if the material were
exceedingly unstable. .

Bernard D. Davis,
U. S. Publie Health Service,
Tuberculosis Research Laboratory,

Cornell University Medical College,
New York 21, N. Ye
References

Avery, 0. T., Macleod, C. M., and McCarty, Me 1944 Studies on
the chemical nature of the substance inducing transformation
of pneumococcal types. Induction of transformation by a
desoxyribonucleic acid fraction isolated from pneumococcus

Type lII. J, Exp. Med. 79, 137-158.

Boivin, A. 1947 Directed mutation in colon bacilli, by an inducing
principle of desoxyribonucleic nature: its meaning for the
general biochemistry of heredity. Cold Spr. Harb., Symp. Quant.
Biol, i2, 7-17.

Griffith, F. 1928 The significance of pneumococcal types.
J. Hygiene, Cambridge, 27, 113-159.

Lederberg, J. 194,79 Gene recombination and linked segregations
in Escherichia coli. Genetics 32, 505-525.

Lederberg, Jo, and Tatum, E. Le 191.6 Gene recombination in
Escherichia coli. Nature, 158, 558.

Tatum, E. Le, and Lederberg, Je 1947 Gene recombination in the
bacterium, Escherichia eoli. J. Bact. 53, 673-684.