GENETIC RECOMBINATION IN ESCHERICHIA COLI.

By Joshua Lederberg

A Dissertation Presented to the Faculty of the Graduete
school of Yale University in Candidacy for the

Degree of Doctor of Philosophy

1947
ABSTRACT

Wild type or "prototroph" cells were found in a proportion

ca. Lo76 1n mixed cultures of biochemical mutants of E coli.

 

It could be shown that these did not result from spontameous
bacx-mutation. In addition th wild types, other recombination
types were found suggesting the occurrence of a sexual process
in E. coli. The analysis of the segregation of factors for lac-
tose fermentation and for resistance to bacteriophage led to the
conclusion that a single linkage group was present, on which
eight factors Rave been mapped.

Experiments confirming the linear order of genes, and bipa-
rental inheritanee were performed. It was concluded that E. coli
is normally haploid and that 1t undergoes sexual fusion with in-
mediate reduction-division, during which crossing-over takes
place. The alternative explanation that the apparent recombinations
are due to soluble transforming factors is discussed in detail
and shown to be uneconomical. Due to the rarity of the process
cytological examination was unfeasible. Attempts to reveal recom-
bination in two other strains of E. coli were unsuccessful, as
were attempts to induce aberrations in the chromosomal arrangement

or the ploidy of the species.
ACKNOWLEDGMENTS

The author is pleased to acknowledge the cordial
assistance and cooperation given him by numerous indivi-
duals, only some of whom can pe named. They include:

Dr. M. MeCarty, for a generous sample of desoxyribonuclease.
Drs. S.E.turia and M.Demerec for cultures of bacteriophages.
Dr. E. Witkin for E. coli strain B/r.

pr. R.R.eRoepke for E, coli I-15, and mutants thereof.

Dr. H. Zelle, for single“eell isolations of recombinants.

Dr. K. Mather (John Innes Hort. Inst.) for indispensable
suggestions and criticism of the linkage analysis.

Many other participants of the 1946 Cold Spring Harbor
Symposium on the genetics of microorganisms for their
valuable advice and encouragement.

He is particularly indebted to Prof. F.Jd. Ryan for having
introduced him to the subject of the genetics of microorganisms, ©
ana for having facilitated his association with Prof. 4.1L.
Tatum.

To. Prof. Tatum,this final mention is reserved, as an ina-
dequate measure of the author's indebtedness and gratitude for

guidance, active cohlaboration, encouragement, and physical

support of the investigations herein reported.

The author was a Fellow of the Jane Coffin Childs Memorial
Fund,for Medical Research. “his work was supported by a grant

from the Jane Coffin Childs Memorial Fund.
CONTENTS

Introduction
Review of literature: sexuality
: transformation

: "zygotes"

Material and Methods

Prototroph recombinants, recovery

Results and Conclusions
Spontaneous mutation
Prototrophs
Syntrophisn

Attempts to extract transfor-
ming factors

Nutritional recombinations
Lac, Vy segregations
Crossover theory

Linearity; Ve segregation
Attempts to induce aberrations
Four-strand crossing over
Attempts to induce diploidy

Bi-parental inheritance and
transformation

Other E coli strains
Discussion

BibiLLlographny.

WwW Mme HF F

bacterial variation

7
11

13
14
15

17
19
21
26
29
32
33
35

37
40
4)
De

o.

Index tc Tables

Summary of mutants
A. Spmbois Used.
B. Derivation of mutant stocks.

Biochemical recombination types found in a mixed culture of
Y-24 and Y-46.

Comparisons of v,* segregations with altermative parental
couplings.

The segregation of Lac and Vy-

A. Data of individual experiments. Lac, Vi Scores on
colonies isolated from minimal agar plates in the cross
Y-40 x Y~53.

B. Data of individual experiments. Lac, Vj, scores on
colonies isolated from thiamin-supplemented plates in
the cross Y-40 X Y-53.

Cc. Data of individual experiments. Lac, V, scores on
colonies isolated from minimal agar in the cross Y-64 X
58-Lol.

D. As above. Scores on colonies isolated from thiamin-
supplememted agar.

E. Lac, V, scores on colonies isolated from minimal agar
an the dross Y-87 X Y-10.

F. AS above. Isolates from thiamin-supplemented agar.
G. Tests of allelism of mutations at Lac and Vy loci.

Summary of Taples 4.

Data of Table 5 rearranged to bring corresponding inter-
change classes in the same column.

Relative frequency of various biochemical recombination
classes in the cross B-M-T+L+B, + X B+tM+T-L-B~.

- Segregation of Lac, Vy and Ve.

Pairwise occurrence of recombination in mixtures of three
components.

Figure 1. Phenotypes of combinations of Lac and Vy on EMB-lactose

agar. V,¥ mutations from V,*.
The study of inheritance in bacteria has, for the most part,
been confined to the investigation of mutational changes in the
course of clonal reproduction. With the exception of experiments on
pneumococcus type transformations there have been few studies on the
direct hereditary interaction of one bacterial type with another,

The conception that bacteria have no sexual mode of reproduction is

widely entertained. This paper will be devoted to the presentation

of evidence for the occurrence in a bacterium Escherichia coli, of a
process of gene recombination, from which the existence of a sexual

stage may be inferred.

The genic basis of microbial inheritance does not depend
on the demonstrability of a sexual phase in bacteria. However, more
powerful genetic methods paralleling classical Mendelian analysis
would be availavole ir it were possible tu rollow the inheritance of
characters in the products of a sexual fusion. The few examples of
this approach thus far reported have provided no incontrovertible
evidence for sexual reproduction in bacteria.

The phenomenon of paragglutination in the colon-typhoid-~
dysentery group might be regarded as an instance of bacterial
hybridization, and was so interpreted by Almquist (1924). As re-
ported by numerous authors, paragglutination refers to the develop-
ment of new types which react with antisera for each of two distinct
Strains, when these are grown together in mixed culture (Kuhn and
Ebeling 1916; Salus 1925; Wollman and Wollman 1925). The significance
of these observatians has been attacked by several authors (Breinl
1921, arkwright 1930, and Kauffmann 1941), chiefly on the grounds
that the paragglutination represents a non-specific cross-reactivity
Characteristic ot "rougher" phases ot these organisms. Hansen (1929)

failed to obtain paragglutination in her experiments, In the light
of mure detailed recent information on the antigenic structure of
this group, this problem certainly deserves a critical reinvestigation

Sherman and Wing (1937) have described experiments designed
to detect recombinations of fermentative characters in mixed cultures
of various BE. coli and Aerobacter aerogenes strains. While new com=
binations of biochemical characters were found, similar «types were
found to an equal extent in pure cultures, so that these authors coulda
not infer the occurrence of a sexual fusion. Their experiments are
of the greatest interest, however, since they represent the first
attempt to study this problem in bacteria by genetic methods using
clearcut characters. Gowen and Lincoln (1942) later performed
similar experiments with strains otf Phytomonas stewartii using cul~
tures differing in morphological and pigment characteristics, As in
Sherman and Wing's studies, these authors were unable to differentiate
the new types they found in their mixed cultures fron types which arose
spontaneously in single cultures. For this reason a definite con
clusion could not be drawn from their results.

A discussion of hereditary processes in bacteria must take
into account the extensive work on transformation of pneumococcal
types, first described by Griffith (1928), ana culminating in the
isolation of the transorming principle in chemically characterizable
form by Avery, MacLeod and McCarty (1944). These studies have re-
vealed that under special experimental conditions, a product iso-
lated from a serologically specific, smooth, pneumococcus culture
Will convert cells of a non-specific rough culture to the smooth type
Characteristic of the source of the transforming principle. so far
as is known, such transformations can be performed in only one direc~
tion (rough to smooth) and anly under very special conditions,

Boivin (1947) has reported a similar transformation involving the

Somatic polysaccharide of a strain of E. coli, There have been
reported other instances of varying credibility (Kasarnowsky 1926;
Lommel 1926; Legroux and Genevray 1933; Frobisher and brown 1927;
Burnet 1925; Holtman 1939, Cantacuzéne and Bonciu 1926). These
studies have a direct bearing on recombination experiments, since
transformations of this sort might be responsible for the occurrence
in mixed cultures of some new types which are interpretable as re-
combination types. This will be discussed in more detail later.
kiorphologically unusual forms ol various bacteria have been
described by Mellon (1925) as zygosvores, and been taken to imply
sexual 1usion. smith (1944) and Dienes and Smith (1944) have suggest
that the "Large Bodies" obvserved in some Bacteroides cultures might
represent a sexual phase, and Dienes (1946) has made a similar inter-
pretation of Large Bodies in Proteus. Star-shaped aggregations of

Phytomonas tumefaciens have been studied by Stapp (1942), and more

 

recently by Braun and Elrod (1947) and provoked all of these authors
to the suggestion that the stars represented some sort of sexual
fusion. As Luria (1947) has pointed out, however, "most of the older
material presented in support of the hypothesis of sexuality in
bacteria cannot be used as genetic evidence because of the lack of
information on the exchange or recombination of discrete hereditary
characters in the course of the supposed sexual fusion."

In the avsence ot zygote-segregation methods, the only
techniques availaote for analysing the genetic structure of bacteria
have veen mutation analyses. The bacteriological titerature is full
of reports of bacterial variations, or so-called "dissociations"
referring to the development within previously pure clones of new
and distinctive types. While these are superfictally very similar
to the mutations occurring in higher organisms, many bacteriologists
{e.g., Rahn, 1937) have rejected a mutational interpretation of

Dacterial variation, chiefly on the grounds that the variants are
‘often capable of reverting to the original form. This objection is
without substance in view of the commonplace occurrence of reversion
both in Drosophila, (Timofeef-Ressovsky 1937} and Neurospora, (Ryan and
Lederberg 1946).

Another distinction that is deeply implanted in classical
bacteriology is that the germinal material of bacteria is thought to
be capable of direct adaptive modification in response to deleteri-
ous conditions, whereas this Lamarckistic viewpoint has now been
virtually eradicated from the thinking of students of heredity in
higher furms.

Proof that adaptive changes in vacterial populations are due
to pre-occurring spontaneous mutations and their suosequent selec-
tion by the deleterious environment has not readily been attained.
Luria and Delbruck (1943) have, however, studied the occurrence in
E. coli of phage-resistant variants of which are detected by the
application of a specific bacteriophage (Tl) to a sensitive popu-
lation. On the assumption that the resistant variants develop in
response to the application of the phage, there should be no greater
variation in the number o1 resistant cells which can be elicited
by the phage in similar samples taken from a series of similar
cultures than there woula be in the case of similar samples taken
from the same culture. On the ovher hand, it was predicted that
there would be a wide variation in the numver ut mutants demonstrable
in a serles or separate vut similar cultures it these mutants arise
spontanevusly previous tu the application of the phage, which acts
Simply to demonstrate them. This increased variance arises from
the fact that the occurrence of a mutation early in the growth of
a culture will lead to the occurrence of a large clone ot mutant
descendants, whereas a mutation which fortuitously occurs later in

the growth of the culture will have only a few mutant descendants.
De
Although an exact mathematical solution o1 the problem has not

appeared, the variance found in Luria and Deloruck's experiments

was wuch larger than could be explained on the direct adaptation
hypothesis, and was in good accord with the predictions of an approx~
imate mathematical theory which they developed.

In a similar way, 1t has been shown that a number of "adaptive"
changes in bacteria are the result of spontaneous mutations vcecurring
previous to the application ur the agent used to select them. These
include, in addition to mutation of E, coli to phage resistance as
already discussed, resistance to additional phages, (Demerec and
Fano, 194d), resistance of Staphylococcus to peniciitlin (Demerec,
1945) ana sulronamiaes (Oakberg and Luria, 1947), resistance to
radiation in E. coli (witkin, 1947), and nutritional adaptations of
Clostridium septicum (Ryan, et al 1940) and of mutants of Ee coli
(Ryan and Lederberg, unpublished.) Recently published examples of
presumably direct adaptive mutations either have not excluded entire-
ly the possibility or previous mutation and seisction, as in the
adaptation o1 yeast to pantothenate synthesis, (Lindegren and Raut
1947) or have not full demonstrated the neritable character of the
moditication, as in the adaptation ot E. coli to resistance to c-
chloro-p-anonubenzoic acid. (Stranskov, 1947). One may concluce,
then, that as in the higher organisms, the germinal determinants
or bacteria are not in direct adaptive rapport with the environment.

Additional evidence or the overall similarity of bacterial
genes tu thuse ot other organisms is contained in the experiments
on the induction or phage-resistance mutations by Demerec and
Latarjet (1946). ‘They reported that A-rays induced mutations of
E. coli to Tl-resistance, and that the mutations induced were
linearly proportional to the A-ray dose. ‘The linear type of resvonse

furnishes strong support, on the vasis o1 the "hit-theory” for the
occurrence of a single iocalized determinant in the cell whose in-
activation leads to the mutation.

Finally, by the use or X-rays, and other mutagens, Tatum
(1946) has vutained nutritional mutants o1 E. coli which are in every
way anaiLogous tu such mutants in Neurospora wnere their anaitysis poy
orthodox genetic methous has shown them to be effects on single genes.
Other investigators have produced similar mutations in E. coli
(Roepke, et al, 1944) and uuner vacteria (Burkholder and Giles, 1947;
Gray andTatum 1944). The mutants optained py Tatum have subsequently
been utilized to demonstrate factur recombination, as will ve de-

scribed in this dissertation.
Materials and Methods

The strain selected for these experiments is Escherichia
coli, K-lz. It was originally isolated from human feces a number of
years ago, and has been carried on agar slants at Stanford University
since that time. It was used there as a typical E. coli for demon-
strations in student lavoraturies. It has been sound to ferment
lactose, tu produce inavle, and to be susceptivie to each of the E.
cuvli phages, Tl tu T7, collected by Demerec and Fano (1945). Since
it ferments sucrose very slowly, if at all, it would be classified
as Escherichia coli "cummune". It is moderately motile as deter-
mined by hanging-drop observations, and vy its slow movement through
semi-solid gelatine~-agar,.

Mutant strains of E. coli characterized by specific growth
factor requirements have been obtained after treatment with x-rays,
ultra-violet light and nitrogen-mustard. Such strains have been de-
scribed by Tatum (1945, 1946) and by Roepke (1944). A single nutri-
tional requirement is established at a single mutational step, and
on the vasis of studies on Neurospora is regarded as based on a
change in a single gene. By successive treatments, multiple mutant
strains with several genetically and biochemically independent nutri-
tional requirements have been produced, The strains used in these
experiments are descrived in Table 1. In general, the nutritional
Characteristics ot a strain are ascertained by inoculating media
Consisting of the basal medium plus various supplements; lack of
visible gruwth in the absence ot a given gruwtn factor and optimal
growth in its presence are the criteria fur the determination of
the nutritional requirements of a strain.

A mutant stein can be signified by suffixing a "-" sign
to the initial of the substance in question; e. g. B-Pa-C- refers

to a strain which is deficient in the synthesis ot biotin,
phenylalanine and cystine. On the other hand particular emphasis

can be placed on the ability of a strain to synthesize a particular
qrowth factor by suffixing a "+". Thus B-Pa-C-TfL+B i+ would refer

to a strain deficient in the three factors mentioned above, but capable
of growth in the absence of threonine, leucine, or of thiamin. The
representation of a growth factor requirement by a minus sign is
justified by the a priori consideration that a mutation establishing a
growth factor requirement generally represents the loss of a function,
and by the experimental finding of Beadle and Coonradt (1944) that wild
are dominant to mutant genes in Neurospora heterocaryons. Strains
which are "-" for all growth factors have been called prototrophs

(Ryan and Lederberg, 1946) since this is the nutritional condition

of the parental wild type E. coli strain from which all the mutants
were ultimately derived,

Mutations for resistance to specific bacteriophages or
bacterial viruses have proven to be exceedingly useful. They are
readily obtained as spontaneous mutants by plating a large number of
sensitive bacteria with the particular virus in question; only re-
sistant mutants escape lysis and may be recovered as "secondary"
colonies (Fig. 1). Resistant mutants are readily freed from residual
virus by serial single colony isolation. Resistance to a given
virus may be scored by streaking a loopt'ul of bacteria on an EMB or

nutrient agar plate at right angles to a previous streak of the

virus suspension (Fig. 1).

It has been found, however, that mutations for resistance
to a given virus are not entirely specific, but that resistant
mutants display "cross-resistance", i. e., are also resistant to
other viruses, Demerec and Fano 1945). For example, most Tl-
resistant types are also resistant to TS (For the nomenclature of

the bacterial viruses used in this investigation, and a detailed
account of the cross-resistance patterns of another strain of E. coli
B, see Demerec and Fano (1945). ‘the cross-resistance patterns of
K-lé are sinilar to those of E. coli B with the exception that Tl-

| resistant mutants which are sensitive to T5 are not tryptophaneless,
as have been reported by Anderson (1946) for the corresponding
mutants or E. coli B. In this paper, the designation Vj" will be
used for the more frequent Tl-resistant mutant, which is also re-
sistant to ‘5,

The specificity of fermentation reactions of various
bacterial species is a clear indication that the ability to ferment
certain sugars has a distinct genetic basis. Because they would be
so easily scored on indicator media, mutants unable to ferment
various sugars have been looked for. Particular attention was paid
to the possibility of isolating "lactose-negative" or "Lac-" mutants,
because of the taxonomic significance which has been attached
to this character.

The detection of fermentation mutants is readily accomplished
by the use of indicator media. The medium "ENB-lactose" used in
routine bacteriological work was found to be highly useful. It con-
sists of the following: in G./l., Peptone (or "N-Z-Case") 10, Yeast
Extract 1, lactose 10, agar 15, Eosin Y 0.4, Methylene Blue 0.06,
sodium chloride 5, dipotassium phosphate 2. On this mediun,
colonies of bacteria which can ferment lactose (or any other sugar
added in its place) rapidly turn a deep purple color, while colonies
of non-fermenting organisms remain white or pink but may slowly turn
light blue.

Lac-mutations have been recovered in two instances, Among
15,000 colonies of strain Y-10 (f-L-B,-) obtained by spreading a
culture previously treated with ultraviolet light, on EMB-lactase

agar, a single pink colony was noted. It proved to be the same,
10
nutritionally, as Y-10 and was therefore regarded as a Lac-mtant and
not a contaminant, this stock is labelled Ye5s5. Aronsg 50,900 colonies
of Y-40 (B-N-V|") a single Lac- was recovered following treatment with
nitrogen-mustard (Tatum, 1946) and was designated as Y¥-S37. Tests show-
ing that these independent mitations are probably allelic will be de-
seribed in a later section (see Table 4g). Strains ¥-53 and Y-87 differ
in the rate at which the Lac-character reverts to the Lac¢e condition,
but whether this is due to different allelic states or to differences
at other loci, cannot be definitely asserted.

Prelimary attempts to obtain maltose, mannitol, and
galactose-negative mutants were not successful, presumably because the
populations tested were too small. A glycerol-negative strain has
been obtained, but the wild type ferments this polyalcohol so poorly
to begin with that accurate scoring is difficult; studies in this
character have, therefore, not been pursued.

It is particularly fortunate that resistance-tests can be
conducted on ERB agar, Since this allows the characterization of a
strain with respect to virus-resistance and to lactose-fermentation
with a single streaking (see Fig. 1).

Morphological variation has occasionally been noted
(exceedingly rough or very mucoid colonial form) but is relatively
unsuitable for genetic work because the presumably random choice of
prototroph recombinants may be influenced.

In additon to the ENB agar already described, a number
of other natural or "complete" media have been used. The Difco product
"Penassay Broth" has been used most extensively, and is satisfactory
for the preparation of inocula, except that it must be supplemented
with cystine for the crowth of cystineless organisms, such as strain
Y-24, Uther satisfactory media include a broth consisting of:

beptone 5, glucose 5, yeast extract 3, g./1, as well as Difco Nutrient
Broth, and diverse concoctions containing peptone or casein hydrol--

sates and meat or yeast extract,

OH

tne synthetic or minimal medium contains, in g./l.,: VE, cf

Ct

HHaNOz 1, NagSO, 2, Kohpo, 3, EHeP04 1, glucose 5, aspararfine l.

3

.

MgSOq4 0.1, trace elements (Beadle and Tatum 1945) and CaCdo a traz:

at

To avoid flocculation when used with agar, the glucose ani

NY)
>
fo
8

ab
"3

in solution should be autoclaved separately, and mixed with the cc:
components just before using. Unwashed agar (Difco) is sufficier-_-
free of the growth factors under consideration to be Satisfactory for
many experiments; the use of washed agar, however, is recorimendeé 7r

the cleanest results.

The detection of recombinants is based uvon the inabilit

biochemical mutant bacteria to proliferate in the absence of the->

ed

Specific growth substances. Flatine in mininal agar, therefore, “2:
the effect of a sieve for prototroph cells. To insure asainst conteci-
nation with prototrophs derived by reverse mutation which has beer
noticed at certain loci, it has been desirable to use multiple bic-
chemical mutants as the parvental stocks in recombination studies,
Coincidental reversion at two or more loci is the theoretically ir-
brobable, and experimentally undenonstrable (see below). For exXarmiis,
platings either Bau-T+L4B, + or Bti+T-L+B.- separately into nominal eran
did not lead to the appearance of prototrophs, Belt? +4L4B74%. When,

however, a mixture of these cell types was so "sieved", one prototrrin

Was found for ca. each 10° cells inoculated. These have been assvu--:

to arfise from the recombination of "4" alleles to form the prototrich,
In early experiments, the two multiple mitants were inocui:-:2

together into ea complete medium and allowed to erow in mixed cultur:

before Plating into minimal agar. This method is not entirely satis-

Py

actory because it allows possible selective differentials to alter

4

“e relative frequencies of different recomination classes. <A modéiiisd

ct
was
procedure developed, which will now be descrited in cetail.
The mutant stocks are maintained on "complete" agar slants,

transferred at intervals of 6-S weeks. “hey are in noculated serarately
nto test-tubes containing about 10 ml. of liquid complete medium and
incubated overnicht at 30 0, with gentle shaking. The following morning,
an additional 10 ml. of the same medium is added to each culture, and
the tubes are incubated in the same manner for an additional 5-5 hours.
These cultures contain from 1-4 x 109 cells ver ml. They ere chen
washed in the following manner: the cotton plugs are replaced with
sterile corks which have been kept in 95% alcohol end the alechol

flared off just before using. The cultures are then centrifuged at ca.
2500 r.pem. for 20 minutes, which suffices to pack the cells in the
bottom of the test tubes. The supernatant medium is carefully roured
off, end the tube is rinsed with ca. 10 nl, sterile distilled water, care
being taken not to disturb the pellet. The cells are then resusitenced
in an additional 15-26 ml. sterile water, and recentrifuged. ‘Tre super-
natant wash water is decanted and replaced with an equal volume of fresh
sterile water, in which the cells are suspended. In the meantime,
minimal agar plates are prepared. A bottom layer of ca. 15 ml. minimal
acar is poured into each Petri plate and allowed to solicify. Cell-
suspensions of different mutmt stocks are mixed at this time and
measured quantities (usually ca. 108 ~ 109 cells) eve pipetted onto

the agar surface. At this time also, one may add such growth factor
Supplements as are desired to permit the growth of recombination tytes
other than prototrophs. The cell suspensions are then mixed into a
layer of ca 10 ml. moffen minimal agar (at ca.4&-509 C.) which is

Foured onto the plates, After the asar has hardened, the plates are
incubated at 309 C, for a period of 48 hours, At this time recombi-
nant prototroph colonies will be found distributed throughout the

Flate, many of them et or near the surface and accessible to picking
for further characterizetion.
The procedure may be varied in severel ways. It is imrortent,

Since cultures

0,
Lad

nowever, that the inoculum consist of "vouns" ¢

of £4 hours or older have siven quite inconsistent results. It is Lossi-
1

ple to store the inoculum in distilled water for at least twenty-four
nours without erpreciably affecting the yield, which suggests that the
ing to the recombination process occurs in
the molten or in the solidified agar. This occurrence must, however,

take place within a few hours, since the recombinant prototrophs are

ar

fe
nl

not epprectably slower to arrear than wild tyre cells ina sim

te

pl

~

oO

hystolosical state which are streaked on the surface of th
phy &

Presumably therefore one could increase the yielc of prototrophs by
making conditions more favorable for the free contact or the cells;
as by vacking them together in a centrifuge tube in minimal liduid

UG
medium. However, the complication of proliferation of prototrophs

8

already formed would interfere with the interpretation of such an ex-
periment. l.any physiological factors may interfere with the recombina-
tion vrocess, and, for example, the yield may be reduced markedly by
inoculating too heavily, or by omitting an under-layer of agar into

which, presumably, deleterious retabolic products may diffuse. Instead

t

I

S possible to streak the

fe

of mixing the cells in semisolid agar,
mixture on tne surface of slightly dried minimal agar plates. Under
these conditions, however, the prototroph colonies are likely to be
more heavily contaminated with the residual parental mutant tyves.
For most purposes, however, this conta ation may be ignored,
&S will be shown in a later section. fFrototroph colonies are, then
fished and streaked directly on EMB plates, or otherwise tested, to
Classify them with respect to other factors that; may be segregating.

Experimental Results. - Spontaneous mutations of bacteria in pure

 

Culture were studied as a preliminary to the investigation of recombi-
mation. The overall frequency of rancor piochemical mutations in
é
untreated cultures is less than O.1 (Tatum 1646) althoush semples

totalling not more than 5,C0O cells have been studied so that the pre-

cision of this measurement is coubtful. In view of the low rate and

ons, however,

ps

sporadic, inderendent occurrence of such stbontaneous rUtat
they may be regarded as a negligible factor in this study.
The stontaneous reversion of biochemical rutarnts to prototrovrs
is under continuing study (Ryan and Lederberg) and will be reported
on more fully elsewhere. It has been found that many biochemical
rutants of E, coli, K-12, will revert at a low rate, prototropns being
found in the proportion of 10-7 in 24-hour cultures of single mutants.
Reversions of different factors are, so far as has been yet ascertaired,
entirely indevencent; as vredicted from the low rate of reversion
of the individual factors, in ca. 19770 cells examined no instence was
found where reversion had occurred at toth loci of a double mutant. Suet
a coincicence would have led to the appearance of a prototroph in a
culture inoculated with a double mutant such as T-L-. On the basis of
these considerations, only double and triple mutents have been used in
the study of reccmbination.
The frecuency of spontaneous mutations to virus resistance
has the same low order of magnitude es nutritional reversion (Luria
and Delbruck, 1943). Mutations from resistance (V¥2") to susceptibility
(V,8) nave not been described, owing to the lack of efficient

techniques for the detection of such reversions.

Prototroph Recombination Types: Since coincidental spontaneous reversion

 

at two or more loci does not occur at e sufficiently high rate to be
detected, the presence of prototrophs in mixed cultures of multiple

mutants is evidence for gene recombination. Each mutant is capable
of synthesizing all the growth factors for which it is not deficient;

therefore, different mutants should have "+" alleles for all but the
ILS.
two or three mutant genes that characterize each strain. The segre-
gation of prototrophic alleles of every gene into one cell would
result in a prototrophic cell. It would develop into a visible
colony on minimal medium while other mutant cells would be unable
to proliferate due to the absence in minimal medium of their nutri-
tiona.s requirements, |

When washed samples of mixed cultures of B-M-P+T+rand B+M+P-T-
were plated into minimal medium, about 100 colonies aeveloped for each
pillion (10°) cells inoculated. No colonies appeared after innoculation
of samples from the individual double mutants. One interpretation of
the occurrence of prototrophs, designated as B+M+P+T+, is that the Pe
and T+ genes of B-M-P+T+ and the B+ and M+ genes of BtM+P-T- have
segregated into the same cell. ‘this is a recombination hypothesis;
alternatives will be discussed in the next section.

The possibility must be consicered that the prototrophs do
not consist of some sort of association of the unaltered mutants.

In a classical illustration of nutritional symbiosis, since designated
as syntrophism (Lederberg 1946), Valentine and Rivers (1927) showed

that Hemophilus canis and H, parainfluenzae, which require X and Vv

 

factor respectively, would grow in mixed culture in media lacking
these substances. They concluded that these growth factors,
synthesized by the individual bacteria, were exchanged via the medium.
While there is no good reason to doubt this conclusion, these authors
did not, in fact, conclusively demonstrate that this was the mechanism
of the interaction. It is possible that cells were present in their
mixed cultures which, as a result of gene recombination, required
neither of the two factors. The situation is obscurea by the use
by these authors of serial transfers of large numbers of bacteria.
Syntrophism has been shown to occur with E. coli mutants

(Lederberg 1946) (Lampen et al, 1947). It is not likely, however,
zi

16.

that it plays a significant role in the appearance of prototrophic

Golontes. Washed cells inoculated into minimal medium do not show

OE ees

wes

2 pie

isyntrophism urtil smail quantites of their recuired growth factors

ont

‘are added. In minimal agar plates heavily inoculated with a washed

SEIT gsr

iiixed culture a uniform turbidity does appear which is ascribable to

- “Jimited exchange of factors and subsequent syntrophic growth.

 
 
   
 
  
   

Evidence of several sorts has been obtained for the homogeneity
nd uniqueness or prototrophs isolated from mixed cultures. Most
plenttioue, they are quite stable and attemps to detect the original
fmutants in recombination prototroph cultures by an efficient selective
echni que (Lederberg and Tatum 1946a, 0o,) have been unsuccessful.

‘Massive doses of ultra-violet light, killing all out 10°° of the ceils

the culture, were no more successful in breaking up the supposed

 
  
  
  
  
 
 
 
 
 
  
 
 
   

B ssoctartone. In addition, prototropns obtained from B-M-P+T+Vy> and
aMeP-T-V45 were studied. oth susceptible and resistant cultures
fare obtained, Although one of the parental strains is resistant,

she susceptible cultures were uniformly lysed upon application of the

papplication of the virus, which would be expected, in an association
oe the original mutants, to lyse the susceptible Belf¢e-T-V4 cells

a leave only B-H-P+l+V,*.

A nicotinicless mutant has been obtained by ultra-violet
rradiation of a prototroph derived from P-I- and BeM-, ‘he pro-
otroph in which this mutation occurred coulc have been neither a

éterocaryon nor an association of diverse types, since in either

saSe the absence of nic# genes in the mutant would récuire tne

is coincidence is hiphly improbaole. The microscopic examinaticn

= seeded agar sunvarted the conclusion that the cells of strain
17
K-12 are well dispersed, so that most of the colonies that appear
would be derived from single cells when only a few hundred cells are
inoculated per plate, as was Gone subsequent to the initial isolation
of prototrophs. Single cell isolations from a "recombination proto-
troph" strain have been mace by Dr. HK. Zelle or the National +nstitute
of Health; all of the single-cell cultures tested were of the same
nutritional and virus-resistance type as the culture from which they
were isolated. Finally, the diversity of recombination types de-
scribed below is incompatible with the Uhnypothesis that they result
fron a gimple combination of cells,cr
Transformation, preliminary expts: The evidence just presented points
to the conclusion that the prototrophs are a new type of cell, which
aid not arise by spontaneous changes in a single double-mutant strain.
Gene recombination, which was postulated abwe, is however, not the
only interpretation for the origin of these new types which would fit
the evidence that has been presented. By analogy with the systems
which have been described in prReaumococci (Avery et al, 1944) and other
strains of E. coli (Boivin 1947) one might postulate that genotypicaily
distinct cells interact not through cell fusion, but through the
release of “transforming substances" diffusing through the medium.
such transforming substances would have the property of inducing or
directing mutational changes in the cell receiving them so as to
lead to what appear to be recombination types.

Since the conditions of the recombination experiments require
that any transforming substance be present in the medium, an attempt
was made to modify a nutritional mutant with a culture filtrate from
another mutant. B4M4+P-T-V,§ was grown in YB broth, and samples of le-
and 36- hour cultures were freed of cells by centrifugation and
filtration through an ultra-fine sintered glass filter. The

filtrate was diluted with an equal volume of YB and inoculated with
18
B-M-P4+T#V,". As a control, 5-H-P+T+#V," cells were inoculated with
B4M4+P-T-V, ° into filtrate broth. After the cultures were incubated
for 48 hours, they were analyzed for prototrophs by the methods de-
scribed avove. None were found in the B-li-Pe1+V,° cultures grown in
the presence of BAM+P-T-V4" filtrate, indicating the absence of
an active transforming principle in the medium under these conditions.
On the other hand, the growth in mixed culture of B-M-P¢T¢v," and
B4M4P-T-V,$ cells resulted in the appearance of numerous prototrophs.

‘Additional attempts were mace to determine whether "trans-
forming activity™ could be separated from the living cell. This is
tantamount to replacing one of the parental cell types in a recombina-
tion experiment with an extract prepared from it, or with comparable
materials. Conditions comparable to the plating described on p.l2d
were used, as well as cell extracts prepared by Doivin's method.
(Roivin, 1947).

No activity was found in supernatants of suspensions of Y40
or Y53, together or separately, as tested by plating the supernatants
with ¥40 or with Y53, into minimal medium and looking for prototrophs.
The only manipulation involved here consists or the removal of most
of the bacteria from suspension in miniral liquid medium, in which
they had been allowed to remain for varying periods up to six hours,
"activity" remained in association with the cells, as tested by
plating them with the alternate type. Equally negative results
Characterized attempts to reveal transforming activity in culture
filtrates and cell autolysates prepared, as mentioned, according to
Boivin (1947).

Finally,the addition of desoxyribonuclease, (kindly provided
by Dr. HM. McCarty) in a final concentration of .05 mg./ml. to the
mixing and plating medium had no effect on the numver of protsotrophs

which appeared on "crossing" Y¥40 and Y53.
Additional experiments ana considerations or a purely
genetic character will be described below; at this point in the
experiments, the interpretation of prototrophs as recombination types
resulting from a sexual process was adopted as a working hypothesis,

and further experiments were designed to elucidate it in detail,

Other recombination types.

If prototrophs arise from the segregation into the same cell
of + alleles from its sexual parents, there might, (in a haploié
system such as E. coli might well be thought a priori, to represent,)
be found in the same mixed cultures other combinations involving -
as well as + alleles. itn the first attempts to detect addition segre-
gation types the cultures Ye4, B-Pa-C-T+L+B,+V,8 and Y 46, BePatC+T-L-
By-Vz were used. There were, thus, availaole 7 markers some of which
might be expected to segregate from the others, and give rise to a
variety of recombination types.

Unfortunately, it would not be possible to detect all of the
a? or 128 possible recombination types. Ynily those types could be
detected which would grow in a medium in which both of the parents
would be suppressed, That is, either biotin, phenylalanine or cystine
would have to be omitted to keep Y24 from predominating in the plates,
and either threonine, leucine or thiamin to suppress ¥46. In fact,
it would be preferable to omit at least two factors required by each
parent in order that "contamination" by back-mutants at a single
locus be eliminated, While this restricts the number of recombination
classes that could be isolated, it still leaves a great many. Jour
markers (two ol the - alleles of each parent) would be used up in
order to detect the “protetroph" recombinations, but the other three
woulda be free to segregate in such combinations as the genetic
system determines.

since the V, lo@us has not been used for detecting
recombinants, there are a total or nine nutritional double-requirement
types - B-T-3 B-L-j B-Bj-; Pa-T-; Pa-L3; Pa-by-; C-1-; C-L-3 C-B,- which
could be detected, as well as six single-requirement types and proto-~
trophs. Such types would be found by plating mixtures of Y24 and Y46
into minimal agar containing two supplements such as biotin and
threonine. On this medium, both parents would be suppressed, but the
recombinant (ex hypothesi) types : B-'-3; B=; T-3; and prototrophs
should be able to grow. Colonies isolated from such a plate would
possibly be any of these four classes, and must be classified more
fully.This was acoomplished by fishing them into small tubes of
sterile water, and taking small inocula into a series of tubes of
minimal medium supplemented with threonine, with biotin, with neither
and with both. ‘the inability ot a culture to gruw in the absence of
a growth tactor indicates tne - allele, while the tube containing both
biotin and threonine serves as a control. frototrophs, of course,
will grow on each of these four media; B- only on the media containing
biotin, T- similarly, and B-T- only in the doubey y supplemented tube.
The result of such an experiment is summarized in Table 2,
In order to determine the proportions of the various types, the number
of prototrophs was used as a standard. The total number of prototrophs
obtained from those plates which had supplements allowing the develop-
ment of a given type was compared with the total number of that type
isolated. The relative frequency ot prototrophs and 5 -, for example,
was found using plates supplemented with thiamin, with thiamin ana
biotin, thiamin and phenylalanine, and thiamin and cystine. The growth
requirements of the more interesting "double-requirement" segregants
were checked several times using 10 ml. volumes of medium » and
isolates purified by serial single colony isolation, Vi Was, of

course, also segregating, but was scored only for some of the

thiaminless and prototroph isolates.
As indicated ty the table (Taple £) several different: re-
combination types were found. tor example, considering the factors
B ana By only, it will be recailed that the parental arrangements
are B-by* and Beby- in Yo4 and Y¥46 respectively. In addition to the
parental arrangements in such recombinants as Bi- (B+) and B- (by4),
the non-parental combinations are found in such types as prototrophs
‘(BAB 4...) and B-B,-. However, since these can only be detected in the
uniform recombination class Pa4C+T4L+ the four types mentioned are not
strictly complementary to each other, and comparison of the frequency is
not particularly meaningful, as it would be if the cross were simply
of the form xy x X¥, where xY and Xy should be equally frequent

Beyond tne mere existence or many of these types, it will be
noted that there are considerable differsnces in their relative fre-
quencies. However, too much weight cannot be placed on these dis-
crepancies since, in this experiment, the two parental types are grown
together in mixed culture in "complete" medium for 48 hours before being
washed and plated. Different recombination types formed during growth
in liquid might be subject to selective growth differentials which would
alter their relative frequencies. It was to counter this objection
that the modified procedure described on p.ldwas developed.

Although nutritional requirements are not objectionable as
markers, and are indispensable for the detection of recombinants, it
was considered that such markers as virus resistance and sugar fermen-
tations might be more readily manipulated and scored in large nunbers.
For further study of segregations, therefore, the behavior of the
factors Lac and va have been especially scrutinized, .

the character V1" has been particularly useful because a

Selective procedure exists by means of which it can be introduced
Alo.

mutationally into any desired gene combination. The application of
a "oross"heterozysous at this locus to the demonstration that proto~
trophs are not simply cell-associations has already been mentioned,
In the course of those tests, 10 prototrophs each were isolated from
the crosses BeM-P#T#Vi" x B+l+T-P-V,8, and B-N-P¢T+V,$ x BeM¢P-T-yyT
respectively. In the first case, 8 were Vi? while 2 were vi°. In the
second, "reversed" cross, 3 were Vi" while 7 were v1*. The apparent
reversal of ratios in reversed crosses, in this small sample, suggested
a technique by which the basis of the non-random distribution of re-
combination classes might be examined. The observations were extended,
therefore, to collect more data for this cross and to study other com-
binations as well.

_ the segregation of V4 alleles into prototrophs resulting
from three different sets of mutant combinations in which the parents
were heterozygous for this locus is shown in table oS. It will be noted
immediately that there is a large discrepancy between the frequency
with which prototrophs are Vj" or V,° as a result of the "reversal" of
the parents in which these alleles are introduced. This discrepancy
amounts to a x* = 199 (for three degrees of freedom) when the values
are cumulated. On the other hand comparisons made between the results
of "reversed"crosses, the ratios being similarly reversed, show a

2.

fairly good fit, a cuhulative X” = 9.8 (for three degrees of freedom)

P= .02) being obtained. These tests illustrate the combinatorial opr
Mendelian character of inheritance whereby gametic frequencies are
invariant in respect of any gene substitution applied systematically
to the genic content of an Organism and of the gametes it produces.
(Fisher, 1947) A value of p=.02 for goodness of fit under the
hypothesis of Mendelian behavior is not as reassuring as one would

iike, but may perhaps be ascribed to errors in scuring V, rather

than to a real deviation from the theory. There can, at any rate,
‘be no doubt as to which of the two modes of comparison gives the

  
 

‘potter fit.

Subsequent to the completion of the experiments of Table 3,
"the Lac-mutant stocks Y53 (T-L-B,-Lac-) and Y87 (B-H-V47 Lacs-) were
‘optained as already described. In addition, a vir mutant of Y53, Y64,
'(p-L-By-Lec-V3") was readily obtained. The development of these
Sghooks permitted a more critical experiment similar to those summarized
‘in Table 3, out in which the segregations of two factors from various
parental combinations could be readily studied. in tables 4, the data
‘from a number of individual experiments are recorded, and analysed

‘tor their homogeneity. In tables 5 and 6 the data are summarized,

fand the results of the different crosses are compared much as in

Inasmuch as the segregation of 51 +B, - was also used in this

 

io speriment, a word will have to be said concerning this locus, in

+t was noted in crosses

 

ines as great on thiamin-supplemented as on minimal agar, indicating

 
  
   

B,-/B]+ ratio of about 10;1. Rather than separate the 10% By}
olonies from the total found on thiamin-supplemented plates, the
‘Oxpedient of comparing the segregations ot bac and V in colonies from
‘hininal and from thiamin-supplemented plates was used. In the
latter, Bi- types would be so preponderant that any appreciable de-
viation from the B + segregation frequencies should be noted among
these colonies despite their "contamination" with 10% B it.

2 The significant heterogeneity of the data of Tables 5 and

6 is rather disturbing, out no method has yet been found of avoiding
it, andits extent and character are such as to make very laborious

any attempts in this direction. The nuli hypothesis of table 6 must

 

86 modifica accordingly. +t should read: "that the deviations

Be
256
between the experiments of Table 4 are not greater than can be
counted by their intrinsic heterogeneity.”

An attempt was made to deal with this problem quantitatively
py the variance-ratio method. The X© for the various sets of experi-
mental data are given in the tables. The variance of Table 4a is,
however, very different from that of the others, and none is available
for 4e and 4f as these were the results of single, large experiments.
It has not, therefore, been possible to select a characteristic
variance for the error or heterogeneity of the data by means of which
to test the deviations between the three main experiments. The shift
in the type of the rarest class with change in the parental couplings
is, however, very striking and affords the clearest qualitative veri-
fication of the principle of mechanical recombination.

The conclusions which may be drawn from the analysis are:

Ll. A far vetter agreement with a Mendelian hypothesis than
with a direct comparison of class frequencies in alternated crosseése

2, No difference in the segregations of Lac and V, in the 5,-
as against the Bi+ progeny.

3, Non-random segregation of Lac 3nd of V, with respect to the
nutritional factors, as indicated by deviations from 1:1 ratios of
alleles in the prototrophs.

4, Interaction between Lac and W\ themselves. In this case,
for example, the ratio of Lac x: Lac x' is 1389:817 = 1.7 in the
subclass Va y's but is 828:63 = 13.1 in the subclass Vj y. (Table 6).

In addition, experiments are recorded in these tables (4g)
indicating that the separately obtained Lac- and the vi" mutations

used in the experiments are indeed allelic, i.¢., that no Lact or
ViS segregants, respectively, occur among the progeny of crosses

homozygous for Lac- or for vit.
24
The simplest combinatorial mechanism known is that of random

or independent recombination. since the data do not support this
hypothesis, one is led to try the next most simple, and the one
characteristic of all other organisms studied: the organization of
genes into linkage groups, presumably corresponding to chromosomes.

The first problem which must be solved is: "How many linkage
groups can be identified?" The data already presented are not suffi-
cient; the behavior of the various nutritional markers themselves
must be studied first.

If one of the five markers in the cross B-M-T+L4B,+ x B4lit
{T-L-B,- were independent of the others, we might predict that the
class "x"~+ would be equally frequent with "x"+ in the detectable re-
combination classes where all the other markers were+ . That is to
say, ‘either B-, M-, T-, L- or B,-, depending on which ones were
segregating independently, would occur in a l:l ratio with prototrophs.

To test this hypothesis, mixtures of these parental types
were plated into agar medium supplemented with one of the five growth
factors involved: biotin, methionine, threonine, leucine, or thiamin.
On the biotin-supplemented plates for example, the two recombination
classes: B-M+T4¢L+B,+ and B+M+T+£+B1* are capable of forming colonies.
If B is independent, they should be equal in number; if not, there
should be a discrepancy from a 1:1 segregation. Colonies were,
therefore, picked from such plates and scored by testing them on
liquid mediun for their growth requirements. The results are in
Table 7.

Unfortunately, the B-H- parental type was not entirely
suppressed when heavily inoculated into methionine-supplemented plates,
due to a low level of contamination with biotin either or the agar,
the methionine, or the cell suspensions. This resulted in the

crowding out of any recombinants which were formed, so that figures
for the M~-:M+ ratio cannot be given. Fortunately, this data turns
out not to be critical, as noted below.

It will be seen from Tapdle 7 that none of the other markers
have segregated at random, 3B)- being more frequent, and the others
less frequent than prototroph recombinants.

To account for B- being less frequent than B+ in the MpT+L4+b5 +
class, we may postulate that B is linked to one or more of these
markers. Comparing the parental couplings: Bt with Mt, T-,; L-,B,-
we may conclude that it must be with M that B is linked.

Similarly, we note that Bi- is more frequent than B+. The
parental couplings are By - with Be, N+, T-, L-. ‘therefore, By must be
linked with B or with M, or since these are linked in turn, with both
of them. The linkage group: (B, 2B3M) therefore appears, although at
this gtage the relative urder is not determined.

Both L- and T- are rarer than prototrophs. They must therefore
be linked with each other, since the parental couplings are: I+ with

[+(B]+;B-3M-), L& with (B,+3B-M-), T+ respectively. We are not en-
titled to infer that the groups (B-3;B3M) and (T3;L) are or are not in-
dependent of each other since an interchange between them is necessary,
although insuffient, to produce the detectable prototroph class. With
this reservation, the map at Table. 6 © may be written, for these factors.

The linkage relations of Lac and V may be gotten from Table 6.
In the cross (B,-3B+M+) (T-L-)Lac- xX (B,+5B-M-)(T+L+)Lac+, it may be
seen that Lac- is the more frequent, both in the B, -BeM+T4L+ and the
B 4B4+N+L4+L4+ classes, Lac, therefore, is linked with the (By-3B5M3). -
group although probably not closely with B, inasmuch as the 5, ane Lac

segregations do not interact. We may, then write: (B,53B,M;Lac).
26
In the cross (By, -3B4M4) (T-L-)v, X (1 +5B-M-)(T#Lt)V,*, vi" is more
frequent both in By- and in prototrophs. It is linked therefore to
(G,L). However, it is also linked to Lac, in the other linkage group.
There is, therefore, but a single linkage group, of which the only
suitable arrangement is: B.-(B,M)..Lac...V,..(T,L).., the order of

the factors within the parentheses being indeterminate from the present

datae

crossover theory

In an attempt to compute map distances from the available
data, a crossover theory must be used in which,unfortunately, no
correction for chiasma interference can be made. Such interference
conceivably may result in large discrepancies between true and esti-
mated values of map distance, particularly in the region (B,M) to (T,L).

As can be seen from the map, Table 6 » an interchange between
By and (B,M) results in B )4B4M+...; lack of interchange in B,-B+M+...
The ratio between these two types is 8:79. indicating a proportion
of interchange to total of 8/79+8 or 9.2%. With so small a distance,
correction for double or multiple crossovers would be negligible
compared to the experimental error. With the formula developed below:
tanh x = interchange: no interchange, a value of 10.1% would have been
obtained.

The estimation of the distance (B,M) - (T,L) requires a
detailed consideration of multiple crossing over. Absolute values
for the distances (B.M) - Lac; Lac - Vz 3 V,-(TL), or a, b, c,re-
spectively, are not available but only their relative proportions
as given by the ratios of the single-crossover types in Table 6 .

The values of a,b,c can however be estimated from their ratios,
Tag » Ty » Yo and the proportion of the "triple crossover class of
table ct, rq, since the trequency of multiple crossing over will de-

pend on the absolute map distances.
A recovered prototroph chromatid will fall into the classes
a..ed according to the distribution of crossover "breaks" in its
various segments. Since interchange between B,M and T,L is required
to produce a prototroph, only those chromatids with an odd number of
breaks in the region a+b+c will be recovered.

The map distance, x, may be defined as (100 X) the mean
number of crossover "breaks" in a segment. In the absence of inter-
ference, there should be a Poisson distribution of éhromatids with
varying numbers of breaks, the frequencies of 0,1,2,n, breaks being
given by successive terms of the expression? e* (1, x, x°/2!, xM/n!),
In this case, only chromatids representing the odd terms of this
expansion can be considered, their sum veing e"* (sinh x) which is
equivalent to 1 - @7&% . The sum of the even terms is e~* (cosh x)
whence the expression tanh x for the ratio of interchanges to non-
interchanges. It can be shown from the addition formula for tanh

KLtXo that this formulation is equivalent to Haldane's .

addition formula X,9 = *1 + Xo - 2K] Xo

The expression e~*sinh x also applies to the chances of
interchange in any segmental part of xX, Ge&.+ a; b, ce. We have then,
the four expressions following:

(e78sinhaa)(e7>sinn b)(e7°sinh c) = rg = .020
e-X sinh x

(e74sinh a)(e7°cosh b)(e7°cosh c) = Pe = ,264
e7* sinh x

and soa forth ~

x 2 -b =-¢ “ ~
Since e@ “.e ~,e ° = e* appears in both numerator and denominator of

each of these expressions, they can be cancelled out leaving only

the hyperbolic terms:

sinh a. sinh b. sinh c = .O020
sinh x

 
28

sinh a. cosh b. cosh c
sinh x

7
264 hula VEE

cosh a. sinh b. cosh c = .448
sinh x

cosh a. cosh b. sinh c
sinh x

268

While the solution of this system of equations would provide
a theoretically exact solution for a,b,c and x, we are here concerned
primarily with the estimation of x, and this can be more readily
obtained with the help or certain approaimations. In particular, a
may be taken aS Sgx , where Sg 18 Pa/ratlptre » i.6., the fraction
of tne" single" crossover types represented by a. This is not exact
insofar as the proportions of the types a,b,c will not be directly
related to the distances a, b, c in all cases, the contribution of
the triple-crossover, single-interchange, types being as the Srd
powers of the distances, etc., rather than the first power.

The result of this approximation is the equation in one
variable:

sinh s,x . sinh spx . sinh 8¢% = .020 , which can be solved

errs en ee ee ee

by successive approximations. The solution is x = .80, or 80 units
(morgans). a, b, ¢ are then <&lt, 36+, and 22 units respectively.
As a check on the approximation used, the result of sub-

etituting a = » = c = x/3 may be considered, This leads to the

equation:
3 -
sinh x/3 = ,.020, x = .75, which considering
“sinh x”
the crudity of the approximation is in good agreement.

On the other hand the application of the uncorrected formula
(x/3)9= -020 gives the result, still lower, x = .73. This cor-

x
responds to counting single-crossovers only in the a,b,c types, and

taking the three regions as equal in length.
29

The values already cited: 21+, 56+, and ey will be taken
as representing the best available estimate of the three regions.

Interference, by shifting the distribution of crossover types
towards the lower values, would be expected to diminish ra and there-
fore lead to a low estimate of x. However, interference would also
tend to cause a spacing of crossovers, increasing the likelihood that
where there are, for example, three crossovers, one shall be found in
each segment and lead to a "d" type. With a random distribution of
crossovers, there is only about 1 chance in 5 that 6 crossovers will
be distributed l:l:1; by interference this figure could conceivably
be increased to 1/1, which would compensate for a fivefold bias
against triple-crossovers compared to single-crossovers for a given
value of x. Interference has not yet been sufficiently analysed in
other organisms to permit of any more direct evaluation of the extent
to which these effects will cancel each other. It would clearly be
desirable to find other means of estimating these distances, perhaps
py the use of biochemical markers located in the left hand region of
the map. A comparison of the results might give direct information
concerning the possible role of interference.

Linearity

In constructing a map, and calculating distances, it has been
taken for granted that there is in E. coli a system of linear linkage,
such as nas been demonstrated quite conclusively in Drosophila, and
inferred in all higher organisms. What direct evidence may one bring
to bear on this question?

The method which one is forced to employ in hybridizing this
bacteriun introduces certain complications. The classical proot of
linearity is based on the additive character of distances, expressed
in morgans, between loci occurring within the same linkage group.

The determination of map distances is based upon a comparison between
50

parental andinew combinations of linked genes, as determined in the.
progeny of zygotes selected at random. In E. coli, on the other hand,
one is limited to the recovery of that recombination class in which
there has necessarily been an interchange between certain biochemical
loci, in the cases here discussed, between (B,M) and (T,L).

. The data analysed above, concerning the segregations of Lac
and V1 cannot be used for a demonstration of linear order without an
error of circular reasoning. This is shown by the indeterminacy of
interference which affects very vitally the linear additive properties
of adjoining crossover segments.

The bearing of the reversed crosses tabulated in Tables 3a 4
has already been mentioned. They illustrate the combinatorial charac-
ter of the segregation mechanism but do not specify it more closely.
For example, one might postulate that genes of bacteria are embedded
in an n-dimensional matrix, which is ordinarily conserved, but which
occasionally permits of a gene-for-gene interchange. This is equivalent
to the "Konversion" theory once proposed by Winkler (1952) as an
alternative to crossing-over theory in higher organisms, and which has
been revived most recently, in modified form, by Lindegren (1947). The
Konversion theory ismade untenable by evidence for the interaction of
different interchanges, for example, between bac and V as already
cited. The Konversion theory can be made to fit such results only by
making it experimentally indistinguishaple from the classical cross-
ever theory.

The interactions between interchanges also exclude similar
matricial theories where the wnits are perhaps not single genes, but
blocks ot them- for example a multilinear radial arrangement. At
least for the genes involved in such interactions, one is forced toa
conclude that they are in a continuous segment. Other genes, as yet

unstudied, or course might be shown to be placed on branches or
other bizarre modifications of the chromosome, but thus far no need
for such exceptions has arisen.

Additional, perhars more direct support for the linear
order of genes is provided by data on the segregation of Ve summerized
in Table 8. It will be noted that the segregations of Lac, V, and Ve
are congruous between the By- and By + classes (the latter in the sense
discussed on p. ++). In the totals of "prototrophs" isolated from
B-H-T+#L+By ¢Lac+V7"V65 Xx Beli¢T-L-B, ~LaceVy Ves one finds Lac- 78%,
Ver 82%, and v4® 36%, indicating that, as in previous experiments,
Lac ig linked to (B,M) and V, to (T,L). In addition, Vg is linked to
(B,M) somewhat more intensely perhaps than is Lac. Since linkage to
By is already eliminated, one would predict Prom these totals, on the
hypothesis of linearity, that Lac and V, should be linked, with
Ve to the left of Lac.

The data of Table 8 confirm this prediction. The parental
couplings of Lac and V, are -P and #s respectively. Of 137 Lac-, 154
were Vg"; of 39 Lac+, 29 were V,%. The order V, ; Lac, Vz is also
supported, since the four most frequent types are those corresponding
to single-crossovers on this basis, while they would include multi-
ple-crossover classes with any other order.

Since Lac and Va are sepregating, the totals for the four
combinations of these two factors can be compared with those of
_ previous experiments. A 4x2 table comparison with the corresponding
cross, Table 5, row 1, gives Ye? 19.6 for three degrees of freedom,
While this would pe an exceedingly poor agreement if a normal dis-
tribution obtained, an analysis of variance by means of the variance
ratio shows that p = .O05 that the discrepancy can be accounted
for in terms af the variance of the replicated experiments.

The first 3 factors tested, 5,M,T,L,B,,Lac, Vy, and Vg

have been shown to belong to the same linkage group. It is, there-
Se
fore, extremely likely that there is but one linkage group in E. coli,
the chances that another of the sames magnitude exists being 2-7, or
-CO8, There is no cytological evidence to suggest more than one
chromosome in E. coli. No other genetically investigated organism
has so few linkage-groups. Cytologically, the nearest analogue is
perhaps the compound chromosome of Ascaris megalocephalus v. univalens,
en = 2.

Attempts to Induce Aberrations

Using a chromosomal theory as a working hypothesis, it was
hoped that some verfication could be found by the study of types in
which the normal order of genes was disturbed. Since there is only
one chromosome (from the genetic evidence), the only types of re-
arrangements would be changes, leading to a series of inversion tyres.
It was thought that such types might be detected by genetical pro-
cedures, by virtue of their effect on crossing over. In particular,
the occurrence of an inversion in the region BL - (B,M) would be ex-
pected to have the effect of eliminating the recombination classes
involving interchanges in this region. In the cross B-M-T+L+EBy+ x
BeM4T-L-B,- this would be equivalent to the suppression of prototroph
recombinants; B,- types, however, would be recoverable, and allow the
investigation of the extent of the changes.

Preliminary attempts to find such aberration types have,
to date, been unsuccessful. The procedure was as follows:

Following treatment with nitrogen mustard or 20,000 r of
x-rays, cells of Y-40 and of Y-53 were incubated separately for 24
hours, to allow the separation of cells or nuclei that might have
been associated at the time of treatment. The cultures were then
streaked out on nutrient agar plates. Single colonies of Y-40
were picked and streaked across a nutrient agar plate. Streaks of

similarly treated Y-535 colonies were made from the opposite direction,
556
so that in the center of the plate, cells of the two types were
mixed, treated colony by treated colony. The occurrence of colonies
which would not interact to produce prototrophs, as detected by
plating into minimal medium, would be an indicator that the combina-
tion was heterogeneous for an aberration. Since in these experiments,
both "parents" were exposed to treatment, each plating was equivalent
to the testing of two chromosomes, for the occurrence of an aberration.
No marked variation in the yield of prototrophs was noted in tests
involving lel mustard and 28- K-ray-treated chromosomes. This can
scarcely be regarded as an adequate sample in view of the stringent
selection imposed by the technique, which might be expected to
eliminate any aberration types which are even slightly less vigorous
then the normal. This consideration is especially relevant in view
of the "hemizygous" condition of any aberrations in the probably
haploid vegetative cells. These studies will be continued.

How Many Segregants per Zygote?

In the experiments detailed in this paper, recombinants
were obtained from different cell types vhich were first exposed to
each other in an agar medium. Therefore, each prototroph recombinant
colony seen by the experimenter marks the site of formation of a
zygote. The question may immediately be raised whether there are
at that site other recomination classes which, by virtue of their
biochemical deficiencies, may not proliferate within the prototroph
colony on the minimal selective medium. This is equivalert to in-
quiring whether there is but a single viable product of meiosis
(as in megasporogenesis in many higher plants) or more than one,
as in ascomycetes. The solution to this problem would be of
special interest in relation to the possible occurrence of four-
strand crossing over. In addition, if an appreciable proportion

of prototroph coloniés consisted of two distinct segregation types,
34.
4¢ would be necessary to isolate these types for the collection
of segregation data.

There are at least three ways in which a zygote might yield
more than one haploia recombinant. Firstly, the zygote might be
capable of proliferation in the diplophase (or sporophyte), leading
to the occurrence of several diploid cells, each of which might under-
go meiosis independently, and by chance yield several segregation
types. Secondly, a single zygote might produce, after meiosis, in
addition to the prototroph, the complementary multiple mutant class.
Thirdly, in a system of four-strand crossing-over, there might be two
supplementary prototroph recombinants differing in the segregation
of factors such as Lac and Vy for which the diploid was heteray gous.

Obviously, the proper investigation of these possibilities
requires that one stringently avoid contamination of one colony with
another. For this reason, the cell-suspensions used were diluted so
as to yield only about 5-10 recombination colonies per plate.

Crosses were mace between Y-40 and Y-sd (B-M-T4+L4By,4Lac4Vy"
x Btl+?-L-B, -Lac~V, ©) on B,- containing minimal agar medium. As
already noted, about 90% of the colonies from such a cross are
BtH+T4+L+B,-. The theoretical complementary class would be B-M-'-L-B, +.
Because of its nutritional deficiencies, it could not be expected
to proliferate on the minimal medium even had it been produced after
meiosis. The possibility remains, however, that a few cells of
this constitution might still be present among the 10® or so B,-
cells of the predominant type ina colony. By plating such colonies
into medium lacking By but containing biotin, methionine, threonine
and leucine, the B,- cells would be suppressed, while the postulated
multiple mutant type coulda form colonies and be recovered.

The experiment just described was carried out, testing 5¢

colonies for their content of other cell types. In general, a
thininless colony could be shown to contain from 10-100 cells capable
ot forming colonies on the 6,l,T yL medium. However, in each case
investigated these have been shown to be indistinguishable from the
y-40 parental B-N-type, and must be presumed to arise from a sur-
prisingly low degree of contamination of the colony with these cells
fron the heavily seeded plate. A few colonies were found which could
be characterized as reversions from b,- to Bit These experiments
are, then, incjusive with respect to the occurrence of complimentary
geneotypes in the same colony. With appropriate stocks, not as yet
available, it should eventually be possible to menipulate the situation
so that the complementary type could be recovered selectively, ex-
cluding both parents and the dominant recombination class.

A search for supplementary types was conducted with the
game crosses, except that colonies appearing on By, ager were streaked
out directly on EMB-lactose agar to determine whether any of them
were heterogeneous for this factor. In some cases, a number of
4solated colonies from each FMB-test plate were then also tested for
homogeneity with respect to T1l- resistance. About 90 colonies were so
tested; only 1 colony was found containing both Lact and Lac- cells.
It is impossible to be certain that, with this low frequency, the
single colony which was picked actually was derived from two distinct
zygotes. These experiments cannot be considered as bearing critically
/ on the question of the occurrence of two- oP four-strand crossing
over because of the absence or information concerning the viability
of more than one meiotic product.
Diploidy.

The segregation of characters observed between prototroph
recombinants strongly suggests naploid condition of E. coli, with re-
duction immediately following the zygotic fusion. If this condition

could be modified so as to yield stable diploid variants, which,
36

using with haploid cells, might yield zygotes showing tri-somic

egregation, many questions concerning centromere relations, and the

umber of strands as melosis, as well as the dominance or recessive-

ess of particular characters, coulda be studied. Because of the

4fficulties of cytological examination, a genetic test was divised

hich, it was hoped, would detect stable diploid variants.
In 1913, Penfold, described a peculiar response of KE. coll

o sodium chloroacetate. Wild type strains appeared to be inhibited

yy this agent, but gave rise to resistant mutants, which appear as papill-

1e or button-like projections from the inhibited growth on mutrient

igar containing 0.1% sodium chloroacetate. The resistant mutants were

yeculiar. insofar as they lacked the capacity to form gas from glucose,

atthough abundant acid was formed.

These findings were contirmed with E. coli, K-12. In addition,

it was found that while the resistant mutant, Cla™, could rorm gas

from formate, it could not from pyruvate. The presently accepted

scheme for the formation of gas from carbohydrates by E. coli involves

the splitting of pyruvate to acetate, or similar Co fraction, and

formate. The formate is then decomposed to CO, +H, by the "formic .

hydrogenlyase” complex. Since the capacity to form gas from formate

is intact, while that from pyruvate is impaired, it may be assumed
that there is a correlation between Cla® and the enzymatic splitting

of pyruvate. While other interpretations are perhaps not ruled out,

this was adopted as the most likely explanation, and is the basis

for what follows.

on a priori ground, and from the work of Seadle and Coon-

radt on Neurospora heterocaryons. it is likely that the ability

to perform a reaction wiil generaliy be dominant to the inability.

Ola’ therefore should be dominant in a diploid heterozygote to Cla?

with respect to enzymatic function. If the correlation between
S7

enzymatic function and resistance is maintained, Cla® will also be
dominant with respect to resistance, 1.¢., the combination Cla*%/cla™
will still be sensitive to chloroacetate. Starting from the diploidised
wild type, Cla®/cia*, it will require two mutations to produce the
resistant type ClaT/cia™. On this basis, a diploidised E. coli should
yield mutations to the phenotype of resistance to chloroacetate only
with extreme infrequency compared to the normal haploid form. Since
ane need merely streak out cells of a suspected diploid type on
chloroacetate agar, and record the development of papillae, this
working hypothesis provides a possible tool for the detection of diploids.
Unfortunately, none have yet been found among some dozens of tests of
camphor- or acenaphthene~ treated material. This matter was discussed
primarily to illustrate a possibly very fruittul line for further re-
search, particularly from the point of view of the possibility of
cytogenetical correlations.
Transformation. Experiments designed to extract transforming factors
from cells of E. coli were mentioned in an earlier section. The fail-
ure or such experiments igs in line with the genetical properties of
the recombination system, linkage, etc., but cannot be regarded as
conclusive for the exclusion of aiffusible transforming factors. The
methods used may have been too delicate to extract appreciable
quantities or too rough to preserve what was extracted. There is,
however, a further type of genetic experiment which bears on the
possibility of transformation via soluble substances.

A glance at Tavle 7 shows that some "aultiply-transformed"
classes are more frequent than those involving changes of but one
or two loci of one of the parents, Un the transformation hypothesis,
this might be interpreted in terms of the non-uniform susceptibility
of different cells to transformation, so that wherever it takes

place at all, it is likely to affect several genes. Under these
o&8

conditions, one would anticipate that susceptible cells might be
influenced simultaneously by a mixture of transforming factors.

On the transformation hypothesis for the exchanges described in this
paper, it would be equivalent to predicting tri-parental recombina-
tions in mixtures of three genetic cell-types.

A sexual mechanism has rather different consequence. Among
other sexual organisms, biparental inheritance is the ruls, barring
the dubiously relevant exception of certain multi poric embryo-sac
types in the angiosperms. In mixtures of three genetic types, only
those types of zygotes may be inferred which result from pairwise
fusions of cells. Zygote formation is so infrequent that the coin-
cidence of successive fusions or the segregants of a Type 1 ¥ Typee
zygote with a Type 3 gamete has a negligible likelihood,

This critical point of difference was subjected to experi-~_
mental test in the following way. The same biochemical parents were
used as before, namely B-M-~-, and T-L-B,-. Lac and V, alleles were dis-
tributed among these parents in various combinations. For example,
the types BelM-Lac-VyF ; T-L-B,-bac-V,8; and T-L-B, -Lac+V,* were used.
Suspensions of these types were prepared and all three mixed together
in a manner analogous to that already described for normal, pairwise
crosses, From such combinations, prototrophs or B,-, otner factors
prototrophic, could be produced only by recombination between the
B-M-parent and one or the other of the T-L-B)- parents, By biparental
inheritance, in this case, only three otf the possible combinationsof
Lac ana V could appear among the prototrophs: Lac-V,"5 Lac-V,8;
Lac+V,™, If a ménage a trois were permissiole, however, the fourth
type Lac+V,5 should be found also.

By using different combinations or alleles, the experiment
may be varied so that a different class becomes the exceptional in

each case. In Table 9, the results of four experiments, so constructed
59
that a different class should be lacking on the basis of biparental
inheritance in each experiment, are set down. It will be seen that
no exceptional types appeared in a total of 628 tests. It may be con-
eluded that genetic factors from different cells are not freely
miscible, as would be demanded by the simplest versions of transfor-
mations. On the other nand, gene recombination is restricted in
any inetance to exchanges of genetic material between two cell types.

The results of this experiment are also a check on spontaneous
mutation as the source of what have been claimed to be recombinations.
On the spontaneous mutation hypothesis there should be no discrimina-
tion against the exceptional types which were not found in these 6x-
periments.

The genetics of bacterial transformations is still in an
exceedingly primitive state, and there is no information concerning the
occurrence of interactions in bona fide transforming systems, If
transformation is to account for the results of the present experiments
4t will have to fulfill the following conditions: a) non-independence
of factors, simulating a linkage group; b) potential capacity of
carrying all the genetic factors of the donor in a single parcel;

c) immiscibility of parcels derived from aifferent cells. From a
genetic point of view, such a transforming factor would be indistin-
guishable from a gamete, and its definition would be based on chemic al
properties only. It will be recalled, however, that Muller (1947)

has interpreted the pneumococcus transformation in similar terms:
"still viable bacterial chromosomes oP parts of chromosomes floating
free in the mediunm....these have penetrated the capsuleless bacteria
and, in part at teast, taken root there, perhaps after having under-~
zone a kind of crossing-over with the chromosome of the host.”

Further genetic work on transforming systems will be required

to substantiate this interpretation, particularly in view af the
40
relatively low molecular weight, 600,000, which has peen ascribed to
the pneumococcus factors. (Avery et al. 1944).

Other E. coli strains
In an attempt to find how generally the ability the recombine
49 distributed among bacteria, studies were made on two other strains

ot E. coli. These were designated B/r and L-15. B/r, obtained through

we

 

 

the courtesy of Dr. E. Witkin, is a radiation-resistant mutant of
strain B. Hoth » and B/r have been used extensively in studies on
mutation from phage-sensitivity to phage-resistance. L-15 is a strain
used by Roepke, Libby and Jones (1944) for the production of blochemical
wutations. A variety of biochemical mutants of L-15 were obtained through
the courtesy of Dr. Roepke. Double mutants of B/r, arzinine-methio-
nineless and histidine-p-sminobenzoreless.., were obtained from ultra-
violet treated material by previously aescribed techniques for the
tsolation of mutants, Lederberg and Tatum, (1946a) All combinations of
the T-L-B,- mutant, Y55, of K-12, and of the mutants of B/r and of 4-15
were made and plated into minimal medium as already described, In no
case was there any suggestion of the formation of prototropnhs within
L-15 or B/r mutants, between them, or with K-12. +t is recognized that
the conditions for recombination in those strains may differ from K-iz,
or that there mea be genetic conditions of mating type. ft was esti-
mated that recombination would have been detected had it occurred with
a frequency of not less than 107° of that found in K-12 mutants,
Serious cytological studies seeking to identify the zygote
in K-12 have not been attempted in view of the futility of attempts
to characterize and verify so rare an occurrence. The burden of this
investigation has been the verification of the recombination of genes
in a bacterium, and the elucidation of some of its genetic properties.
The way is open for considerable further work, using recombination as

a tool of genetic analysis, and to the more detailed picturization of
41

process in space and time.
Discussion

Although a transformational interpretation of these experiments
has not been excluded beyond any shadow of ceubt, it makes little
difference for most purposes whether one adheres to invisible zygotes
as against unextractable transforming factors. The techniques de-
scribed should be useful in either case toward the solution of genetic
problems in bacteria. Many of these are discussed in the Cold Spring
Harbour Symposium for Quantitative Biology, Volume 11, 1946, which
deals with the genetics of microorganisms. These problems include
the genetic nature of phenotypically complex mutations, the verification
Or reverse mutation as the bvasis of genotypic reversion, the site or
interaction of certain mutations involving glycolytic enzymes, the
genetic basis of antigenic variations, the verifivation of the "one-to-
one" theory of the relationship between genes and enzymes in bacteria,
and in general, any instance where it is required to test the allelism
or two or more genetic variations,

Genetic recombination has, of course, a far broader meaning in
biology than as a laboratary tool. The recombination of mutations is
a source of variation that may be of crucial importance in the evolu-
tion of new “adaptive peaks". While this statement, in its general
terms is indubitable (see Dobzhansky 1941) recombination can only
effect the reshuffling of preexistent mutations, Concerning the natural
historical ai gnificance of the latter for bacteria, we are in a state
o1 woeful ignorauce, so that we are hardly in a position to discuss
the significauce o: bacterial recombination in concrete terms. To this
must be added the caution that geritic combination was found, luckily,

alfboyah
in one E. coli strain, and not in two others which were studied.
42

It way be wondered at that the apparent recombination rate is
so low. It will be recalled that about 1u7© of the cells insSoculated
ati the cross B-M-T+L+B)+ X B+M+T-L-By- showed interchange in the
regiou (B,M) - (f,L). Since the estimated map distance is 8U
units this 1s also the correct order of wagnitude of the fusion
process. However, this is possibly not to be ascraped to any
sexual imperfections of the colon bacterium, but to the method of
enumeration. It seems likely that an anal gous comparison of the number
ot somatic and generative cells in a nigner plant, or the ratio
of perithecia to total nuclei in a fruiting culture of Neurospora
would not give very different ratios. It 1s also possible that the
optimal conditions ror zygote fommation and germination have
not yet been achieved ahd that by special procedures the rate of
fusion may be wcelerated to the level where there might be some
hope of trapping 1t 1n the field of the microscope.

Since the mutants used in the recombination experiments were de-
rived trom the same wild-type strain, there can be no question of
genotypic mating type determination. The failure of two E. coli
strains to exhibit recombination might conceivably be ascribed
to genetic heterithallism, such as has caused many fungi to be
classified as "imperfect". The chances of finding the appropriate

mates are of course very slim, but should not be entirely overlooked.
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bacteria; with some light on fermentative variability
in the coli-aerogenes group. J, Bact. 35: 315-dé2l.

smith, W. E. 1944. Observations indicating a sexual mode of repro-

duction in a common pacterium (pacteroides funduliformis)

 

gt. Bact. 47: 417-418.

stapp, C. 1942. Der Pflanzenkrebs und sein Erreger Pseudomonas
tumefaciens. Xl. Zytologische Untersuchungen des
pakeriellen Erregers. Qo, Bakt. (IIT) 105: 1-14.

strandskov, #. 1947. Inihibition of methionine synthesis in
Escherichia coli by 2.chloro-4-aminobenzoic acid. J, Bact.
53: 555-560.

Tatum, E. L. 1946. X-ray induced mutant strains of !scherichia coli.
proc, Net. Acad. Scie 31: 215-219.

Tatun, E. Le 1946. Induced biochemical mutations in bacteria. Cold
spring Harbor, Symposia on Guatitative Pioclogy 11* 278-84.

Timoféef-Ressovsky, N. We 1937. Experimentelle vwtationsforschung in

der Verexrbungslchre. Steinkopf: Beesden.
glentins, #. U. Gs £7, Further observaticns

concerning growth requirements of hemophilic bacilli.
J. Exp. ied, 45: 993-1.

linkler, H. 1952. Konversions-heorie und Austausch-Thorie. 5» iol.

Zbl. 52: 163-169.
jitkin, E. M. 1946. Inherited difference in sensitivity to radiation

in Escherichia coli. rroc. “at. Acad. Sci. Sei 59-68.
follman, E. and E. Wollman. 1925. Sur la transmission "oarahéréditaire"

de caractéres chez les Bactéries. C. R. Soc. Biol. 95*

1568-1569.

Crratuure

References snraderrtenth, omtled from toot

Dienes. L. 14H6. Com pley reproductive processes th bacteria.

CAD bor. York. Symp. duant (ict MH, 38 -SO,

Jurdegrere, C.C. and Raut C. lauq. A direct relations lup betareer
parltlamacte oncwtoalion and the tome aepewssd) Ae mdues

Mer production f partstlurake synthroy mg "nmulsnta” 22
aptad Gan. the. (ot. Nard. 34:85 - 73.
Table LA

Symbols used for various mutations

1. Nutritional requirements. The allele for requirement ,i.e.,lack

of synthesis of a substance is "-"; the alternative, independence,
is "+", ae 2 ee
B bviotin L leucine Pa phenylalanine
B, thiamin $M _ methionine. |g” threonine
¢ cystine P proline _ | .

2¢.Sugar fermentation. The ability to ferment is "+"; the alterna-
tive,inability, is "-".

Lactose Lac
3, Bacteriophage resistance. Resistance is designated by the super-
seript "r"; sensitivity by tet,
Vy reaction to bacteriophage Tl

Ve reaction to bacteriophage 16.
Table 1B

A Summary of the Mutants Used

 

Strain No. Genotype
K-12
58 B-

58-161 B-M-

58-278 B-Pa-

Y-24 B-Pa-C-

679 T-
679-680 T-L-

Y-10 T-L-By-

¥-46 @-L-B,-V¥,*
Y-53 1-L-B,-Lac-
Y-64 ?-L-B,-Lac-V;"
Y-40 B-M-V;"

Y-87 B-M-V,7Lac-
Y-25 B-Pa-C-V,"
679-183 T-P-

¥-94 T-L-B,-Lac-Vo

Origin

Genotype

prototroph. Original wild strain.

K-12

58

538
58-278
K-12
679
679-680
Y-10
Y-10

“Y-53

58-161
Y-40
Y-24
679
¥553

B+
B-M+

B-Pat

B-Pa-C+

T+

T-L+
T-L-B,+
T-L-By-V¥15
{T-L-B,-Lact

T-I-B,-Lae-¥15

s.
B-M-V

"] ?

B-M-V. "Lact:
B-Pa-C-V,5

Q-P+

_ P-L-By-Lac-V6"

Agent

X-ray
X-ray
X-ray

UerVe

E-ray

| X-ray

X-ray
selection

UerVe

' gelection

‘selection

N-mustard
selection
X-ray

selection
Table 2.
| Biochemical recombination types found in a mixed culture
of Y-24 and Y-46.
B-Pa-C-T+L+B,+V, 5 x B+PatC+T-L-B,-Vy*.

pype:* No. No. of

Ratio
BPacTLB, isolated prototrophs
-- -+++ £In excess: parental type
++ +2 -- In excess: parental type
++ +e e+ 86 : ; . os --
to eete) 56 37 | 0.97"
++ +--+ 2 31 0.06
++ +e 4+ 4 55 0.07
-+ +444 5 56. «0.09
$+- tees 1 52 - 0.02
++ = +44 21 19 — 0.05
-- +++ 2 41 0.05
++ ++-- 3 16 0.19
-+ +et- 3 28 . 0.11
-+ +- ++ isolated in a different run.
-+ ++-4 isolated in a different run.

* these figures do not include tests of the V; character. Of
49 prototrophs tested, 20 (41%) were resistant. of 20 thiamin-

less tested, 7 (35%) were Vy".
Table 3.

Comparisons of v,* segregations with alternative parental

couplings.
Parents Prototrophs isolated
B-Pa-C-T¢p4  BePatC+T+P- vr vi" iy x" (1) x®(2)
V4rx v,° 76 6 92
3 r 101 7.9"
Vi X Vi 30 107 22
BePa-C-T+LFB1t BePatC+l-L-B)-
vy" x v4" 80 23 «77
5 7 65 1.1
Vy x Va 53 133 28
B-M-T+P+ B+M+T-P-
V,7X v,$8 49 8 86
i 1 32 1.8
Vix v," 5 19 21

X*(1) refers to a comparison of the classes directly: Le Ces to
a @x 2 table of the data as they stand.

x” (2) refers to 4 comparison in which the gametic output or v1
4s inverted in the same manner as was done by changing

the parental coupling. In both cases, n = 1 for each tapole

2
The cumulative X3 are 198 and 10.8 respectively.
Table 4a e

Data of individual experiments. Lac, V, scores on colonies
isolated from minimal agar plates in the cross: Y40 x Y53

B-M-T+L+B,+tLactV," x BtM+T-L-B,-Lac-V,$
Class recovered: Beli+TeLeby...

ixp. # Lac-v._~ Lac-V, * Lacev,* Lac+vV, *

1
l. ol 11 25 4
Je 28 el él O
36 el 18 11 0
1. 189 44 129 3
de 52 39 59 2
36 17 10 9 QO
, 355 " 80 35 0
3 20 ee 10 3
i. o7 25 16 1
O. <0 135 15 ze
-le 26 9 11 3
~Le 19 LO 11 2
De ev 12 50 1
-4e ev 13 135 L
Oe 335 28 14 O
otal 602 505 587 Le
% 45.8 25-1 29.4 1.7

. 2. ,
Homogeneity: X° = 101., n= 42, p« ,001
Table 4 bd.

Data of individual experiments. ac, V, scores on colonies
tsolated from thiamin-supplemented plates in the cross

y 40 x Y55.
B-H-T+L+B,+bac¢¥," x B+l+T-L-by-Lac-V)§&
Class recovered: BeM+l+L+B,- ca. 10% yr.

Exp. Lac-V,P  Lac-V,§ LaceV, Lac#V48

 

le 30 21 L? 1
2. 73 "46 50 4
3. er 12 30 1
4. 59 34 31 3
Be 16 12 13 0
6. 21 19 5 O
7. 18 12_ 13 1
Total 244 156 159 10
% 42.9 27.4 27.9 1.8

(

Homogeneity: X"2 16.9 n= 16, p =

ev

Homogeneity or Bit totals with B,- totals (Tables 4a & 4 b)

 

 

 

 

 

602 303 387 22. «1814
244 156 159 10 | 569
B46 459 546 32 | 1683
2
XB = 4.4

With a normal distribution of x*, p would be 0.2. In view of

the heterogeneity of the 5,t data however, this result is

artificially low.
Table 4 Ce

Data or individual experiments. Lac, V scores on colonies
,solated from mininal agar in the cross: Y 64 x 58-161.

BeM-T +L+B,+Lac 4v,8 as Beli¢T-L-B, -bac -V4*

Class recovered: BelieT-L=By-.-+ ee

EXP. Lac-V_" Lac-¥y" LactV," LacrVv;° Sum
i. ov 55 5 19 L116
ee 42 53 1 29 119
Oe 8 9 1 2 20
4 13 16 1 9 39
5. 8 12 1 8 89

votal 108 145 9 61 $235
% 3004 45.0 2.3 18.9

Homogeneity: Xen 5.6 na le. p = .95-
Table 4d.

As above, scores on colonies isolated from thiamin-supplemented

agar
Class recovered! p4el+T+L¢B, - + cas. 10% Bit
1. 109 125 8 70 312
2. 20 21 1 9 51
3, 4 5 0 1 10
Yotal 133-181 9 80 375
bs 35.6 40.5 204 21.4
Homogeneity: X27 .65 n=- 3, ps -85 (expts. 1 & 2 only!

Comparison of 4c and 4a tatals (Bl + with B-)

 

4a LES 151 9 80 375
4¢e 108 145 9 61 O20
241 196 18 141 696
Table 4e

Lac, Va scores on colonies isolated from minimal agar in
the cross: Y87 % Y1O. -.
B-h-'T+L+By+Lac-V\{"7 x BEF T-L-B,-Lac+V, §

Class recovered Beli+T+L+By tees

Single Experiment .

~ Pr GO ue 8 ° iL s m
Lac Va Lac V4 Lac+rV, acdV, Total
28 6 46 o7 117
% C02 5.1 39-9 51.6
Taple 4F

As above. Isolates from thiamin agar.
Class recovered’ BelieT+L4b,- + 10% Bit
102 7 201 91 401

J 2504 1.7 50.1 2247

Comparison of 4e and 4f (B,+ with Bi-

28. «6 46 37 » 117
loz 7 201 91 | 401

347 128 | oL&

| oad
CH
oO
je
G
to
nS
Table 4g

Tests of allelism of mutations at hac and V loci.

1. Test of allelism of bac- in stocks Y535 end Y87.
Belitl-L-B,-bLac- x Beh L+L4b, -Lacr. 134 prototrophs

isolated: all bac-.

2. Test of allelism of V,* in stocks ¥40 and Y-46
BeM-T4+L4B,+bactV," x BeMet-L-B,-Lac-Vy"

161 prototrophs isolated: all v,”

128 Lace 3; 35 Lact.
Table 6
summary of Tables 4,

The segregation of Lac and V into "orototrophs" derived from
various crosses. Scores of B44 and By- classes have
been pooled in view of theif nomogetieity-

 

Parents Recoibinants: BaleT+Le+B 2
peMeVaL4B+ | Bele -L-By- LaceV,F bae-V1* LactV,r Lac+V,* total
A. LaceV,? Lac-V,8 846 459 546 32 1883

1 1 j
% 44.9 24.4 29.0 1.7
r
B. Lac-V,§ Lac-V1 241 296 18 141 696
4 34.6 42.5 2.6 20.3
C. Lac-V4* Lac-V1§ 130 13 247 128 518
% 25.1 2.5 47.7 24.7

35097.

Thesx4 contingency tables of able 5 gives the following x*

Value (for 6 degrees of freedom.)

x2 = 77745
ab

cd

ef

Table 6.
The data of Tawle 5. are rearranged so as to bring corresponding
interchange classes in the same column.

B-M-T+I4B,+Lac x V,Z XX B+M+TsL~B,-Lam x! vz!

Parents Class recovered: B+M+T+L+...
x y x! y! xty' xy! xy xty Total
+ 3 - s 546 846 459 32 1883
(29.) (44) (24) (2)
+s - ¢ 141 296 241 18 696
(20) (42) (35) (3)
- r + s 130 247 128 13 518
(25) (48) (25) (2)
Totals 817 138y 828 |. 63 3097
% 26.4 44.8 26.8 2.0.
3 270 : .458 : .273 ---
a D c

x2 = 40.7, n= 6. No true value of p can be calculated
because of the inconsistent variance of the different popu-
lations. Compare, however, with the value of X° = 777 of
Table 5. |

The figures in parentheses are the percent contribution of
each class to the row total. "s" are the proportions of the

single interchanges according to the map:

 

ou bh . c
7 + +f x x 7
ee u y > +
g, (BM) Lae v, (TL)
Table V

RELATIVE FREQUENCY OF VARTOUS BIOCHEMICAL RECOMBINATION CLASSES
TN THE CROSS ,

BeM-T+L9B,* B44 T-L-By- *

 

 

 

 

 

 

 

 

 

——

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

From plates Number of Recombination classes found
supplemented colonies 2
with tested Type Number Type Number Ratio X
f '
Biotin 70 B- 10 Be 60 0.17 |ss
Threonine 46 T- 9 Tt 37 0.24 | 1”
Leucine 56 L- 5 Le 51 0.096 | 58
Thianin 87 Bi- 79 Bat 8 9,88 E

 

 

 

 

 

 

 

 

 

 

 

 

3 Gells of the parental types were mixed and plated into agar
supplemented with the growth factor indicated. Yn this medium,

the two recombination classes indicated on each line of the table
could form colonies. Contrasting alleles only are specified;

other loci, unless otherwise specified, have the "+" configuration.
rhe X" for the ratio of single piochemicall aeficient types 5
types to prototrophs is calculated for a comparison with the 1:1
expectation of a random segregation. As can be seen from the

x" values, the probability that the deviations are due solely to

chance is, in each case, less than .OOl.
Table 8

SEGREGATION Of Lac, Va and Ve

wag 5., 7r
B--THL4B,sLactV, "V8 x Belle T-L-By-bac- Vy Ve

1

 

 

 

 

 

 

 

 

 

 

 

Bel-TsLe- Lac: - - -* - + + * 4 To-
Vii Yr Ss rs r s Yr 8 tal
Ve: r or s s r Yr s 8&8
20 eBit 2416 1 Oo 2@ 1 10 €@ 56
ee eB 52 42 €& 0 6 1 16 1 120
Total 76 58 3 0 8 2 26 8 176
% 43 351.7 0 4.61.1 151.7
Crossover e f cde cdf dad def c ced
region
B (Be eHeeVae hace. Vy ee Te eh ci
13 b oc Cg e f
+ - - § + r + +
- r + - 3 - -

“This map is not intended to represent the map distances,

merely the linear order.
Table 9

Pairwise Occurrence of Recombination in Mixtures of Three Components

 

 

 

 

 

 

Parental Types Recombinant Frototrophs
B-lh-T+L+B5 + _ BeH+T-L-By - . Balit'+LeB, - or By+ |
ac-v_ -V.% Lacey, aV "
7 z Lac V5 Lac vi Lac<V. Lac+V,s Lot
LactV, + = s
Lac-V.2 173 4 4 O eee
i Lac-V, *
;
Lacty_© ;
Lac+v,§ ’ 1 0 136 37 40 el:
i Lac-v_§
— -- : i oo 7”
Lac+V, © 5
Lac-Vj 65 48 O 25 13€
Lac-V_P
Pe - 7 ne es
Lac+v_* 5 16 O 7 28 51
i Lac+V,

 

 

Totaleccessvrevsvvcevees 628.
 

Caption for Fig. 1.

Fig. 1. The phenotypes of the four combinations of Lac and V are illus
trated. In order they are: LactV," : Lac+¥,°; Lac-V,*; Lac-V;%.

An EMB-lactose agar plated was first streaked vertically with the
virus Tl. Subsequently, each of the bacteria was streaked, from left
to cight, perpendicularly across the virus streak. sfter 16 hours in-
cubation, both the Tac and Vz phenotypes are well developed. Developin
in the zone where Lac-V;* has been lysed can be seen two colonies of

resistant mutants: Lac-¥,*.