ha 7 e pasty eyes nee whe Ft tn, oy egw tints ”~ ” Bk
In, i, Ledevbore and Joshua Lederheng Deecmber 1. 1953

_
Nw

+

Recent studies of recombinetion in E, coli (17) have led tw the
discovery of a compatibility mechanism (5) » a lysogenic system subject
te genetic centro? (16), and a systen of limited transduction by temperate
phage (22) comparable to that of Salmonella (28), These three phenomena
involve transfer of heritable fectors by infection in contrast to bacterial
mating which invelwes the entire genotype. The clarification, cifferentiation,
ard interrelationships of these mechanisms were emphasized in this in-
vestigation,

I The LYSOGENIC SYSTEM INE. COLI K-12

The relationship of a temperate phage, ds to a specific locus,
Lp, (Latent phage) has already been reported (10). In summary, the prin«
cipal reaction types of bacterial Strains are: sensitive (Lp®) » lysogenic
(Lp*), ‘and the non~lysogenic resistant type, Tmmune-I (Lp™), In crosses
they behave as a system of multiple alleles, linked most closely with
Galj,. This linkage has been confirmed in a Cal* Lp’ x Gal™ Lp® cross
in another Laboratory (27), In addition, the two factors segregated out
of heterozygous diploids in the parentel coupling. This evidence points,

therefore, to a genic determinant regulating the maintenance of \ provirus.
(2)

Prom a mumber of direct and indirect experiments it is known that all

these types adsorb \e A second locus, Lpo> controls resistance or sen~
sitivity to «2, a virulent Anutant, and is situated in the Mal,--S region
of the chromosome. As Lipo” strains cannot adsorb d, they are therefore
not subject to any consequences whose initial reaction requires adsorption;
Lpo does not interfere with the maintenance of AX previously established

in Lp” strains, The genotype Lp®Lp.° is consequently indistinguishable
from Lp"Lpy* types with respect to lytic effect of Ae Cross-reactions of
\with d-2 antiserum have been observed,

New Data on Immune-1: The status of the various isolates of immne-1
strains has been reported, and the interpretation of their constitution with
respect to prophage had been reserved pending evidence of a “eryptolysogenic"
phage that normally fails to mature to give rise to lytic virus, The
segregation pattern of Gal"Lp* /Gal), Lp" diploids, also heterozygous for
Mtl and Maly {table 7 } is identical with similar Lp’ /up® results, The
hypothesis that Lp* types may carry a non-reproducing prophage is supported
by experiments in which a low titer of was recovered by U-V induction of

at least one (22), Lp” types are also subject to transduction, and the

results of these studies will be deferred to that section.
(3)

incidental Vavient Types: No sew evidence buaring on the probler on
the “semllysogenic® strain (10) can be presented, Tests to determine
whether hostemodified A.was carried (section III) were negative.

An intermediate host reaction, semiregistant to both Xana 2,
comparable to the one in Shigella varadysenteriae (26) and the ¥4P allele
of K~12 (112) has been clarified, Standard . suspensions have a reduced
efficiency of plating (cop) on this mtant such that the plaques produced
are reduced in size and number, and also show a reduced efficiency of
transduction, The mtants have been successfully lysogenized, but are
still seniresistant to j-2. The protocols for cresses which establish
a mutation at a new Lp3 locus not linked to Lpo-Mal or Lp, ~ Gal, and
conferring partial resistance to), are presented in table 13,

Mechanism of infection; Mutation and Selection vs. Induction: Breeding
experinents and diploid segregations reveal only the chromosomal determi-
nant of lysogenicity. The facility of the change Lp? to Lp” encourages
the possibility that A directly induces (rather than selects) Lp’ among
the mmerous survivors of exposure to phage, the following types of

evidence would be useful in elucidating the primary infection process:

(1) identification of a “prelysogenic” genotype in the absence of phage
Cy

would encourage the sutation hypothesis, Tt would be cheracterized as
an apparent immune-1 that would be converted to a stable lysogenic after
treatment with). (2) a careful study of the dynamics of infection, in«
cluding the isolation of clonal pedigrees of single cells exposed to r
which engender lysogenics, A pure lysogenic pedigree would favor the
induction hypothesis.

Attempts to identify the prelysogenic genotype in K-12, and hybrids
of K~12 and other crossable lines have been unsuccessful, Preliminary
experiments of the infection process (10) have disclosed lysogenic colonies
contaminated with sensitive cells and free phage long after initial con-
tact with). These mixed clones have since been confirmed in K-12 (18)
and Salmonella (14,201,233). The pogsibility that spontaneous alteration
of the bacteria predisposing to a lysogenic decision plays some role in
the recovery of lysogenics is thus not yet excluded. However, the simplest
conception remains that the genetic elements of the phage are directly
incorporated in, or attached to the bacterial chromosome as we have been
able to find no indication of an extra-muclear inheritance of lysogenicity.

The Effect ofh and F on Crossing Behavior: The presence of r in
one, both, er nolther of the parents of a cross does not influence the

yield of recombinsnts, 4s noted earlier (8) eensitives were not eliminated
as lethal phenotypes, bub the progeny of lysogenic x sensitive included
both parental types, and no others, in ratios dependent on the selected
auxotroph markers, On the other hand, the compatibility factor (F)
determines not only the yield but also the segregation pattern of many
overtly unselected markers, Prototrophs are recovered only when at least
one parent is F; F also seems to direct the elimination of certain chro-
mogomal segments after the formation of the hybrid zygote (15,23). The
important distinctions of F and dare summarized in table 1, These

are emphasized to mitigate any confusion that might arise from the
suggestions that have been recorded elsewhere that A may play a direct
role in sexual recombination as well as to emphasize the distinction be»
tween the x controlled transduction of restricted genetic factors and the
Fecontrolled sexual recombination. The independent transmission of these
factors was demonstrated by the recovery of (1) F*Lp® cells on the one

hand, and F°Lp* on the other, from mixturcs of genetically labelled

FLp® and F*Lp*, and similarly, (2) Lp*F? (but no Lp®F* or Lp*F*) as sur-

vivors from F"Lp® exposed to \econtaining filtrates from F*Lp* cultures,

om
(6)
TI TRANSDUCTION

Celi-free filtrates derived from suitable Salmonella strains were
capable of transferring unit genetic factors to a competent recipient (28).
A wide range of independent markers has been equally subject to transduction.
Additional analysis has shown that the temperate phage of the donor strain
is the vector of the genetic material (16,25), Attempts to detect trans~
duction in K.12 among the survivors in the turbid centers of X plaques
were negative (10); but by using high-titer lysates obtained by U-V
induction (20), a successful transduction was achieved (22), Two striking
contrasts with the Salmonella system were demonstrated: (1) the restriction
to a single genetic character, galactose fermentation, and (2) a striking
instability manifested by mosaic Gal*/Gal” colonies after transduction
despite repeated single colony purification on EMB galactose agar.
The incidence of persistent instability, rarely if ever encountered
in Salmonella (1h), varies with the recipient strain,

Confounding of Transduction with Recombination ?: The conditions
required for transduction are generally precluded in crossing experiments,
Moreover, the unstable mosaic Gal* /Gal” colony characteristic of trans~

duction has not been se far recovered among recombinant progeny, A
(7)

more careful inquiry into the effect of X and Gal segregation was necessary,
however, in view of the transduction phenomenon, since it may provide an
alternativ interpretation of the Gal-Lp cosegregation ratios currently
satisfied by a linkage explanation. Crosses of genetically related parents
differing only in the presence or absence of were therefore studied,
Table 2 demonstrates no significant deviation in the yleld of Gal” re~
conbinants where parents vary only for the Lp marker,

is Transduction a Selection Artefact?: Interaction of genetic
factors on reverse mutation of entirely independent loci have been re-
ported before ( 125). An analysis of the Gal- segregation from the une
stable transduction, the allelic transduction, reported below, as well
as many other types of evidence (22) rule out the interpretation that
the transduction is a selection artefact. The most convincing evidence,
however, has been the development of specific Gal” transductions in Gal*
recipient strains by means of 1 with extraordinary high frequency of
transduction (22), when the d donor was Gea",

Transduction and Fetransfer: Just as lysogenization is independent
of the conversion of F” into F* atrains, the transduction mediated by AL

is unreleted to the F status of either the recipient or the donor cells,
(8)

Crosses of F" x F" by standard technicues are completely sterile, Howe
ever, recombination of two nonallelic Gal” mutants can be indirectly deme
onstrated by transduction, Lysates from Lp’Gai*F” were completely
functional in introducing the Gal” factor to Gal“F” cells. Similarly,
norallelion of tuo Gal"F” strains can be established by the formation
of Gal* in transduction experiments whereas the sexual sterility of the
cross would black cell recombination in Loto.

Crosses of a strain characterized by its enhanced fertility, Hfr,
(18) displayed a Linkage of the Hfr trait to Gal (12). These data were
verified (table 3) for Cal“5, Despite this linkage, efforts to trans-
port the Hfr and Gal* factors simultaneously iato Gal F"Lp® recipient
cells vis \ prepared from Hfr bacteria were unsuccessful, The conversion
of F” to F* by ) filtrates from F’ strains was examined by crossing the
Cal* transduction with F* tester strains and was likewise unsuccessful.
The competence of din transduction therefore continues to be confined
to the Gal cluster.

The Concurrence of Transduction and Lysogenization: Observations

 

 

on the E, coli system, as in Salmonella, are consistent with the hypothesis

that the vector of transduction consists of temperate phege, As a rule,
the teeasduetions isolated from Galthp® nactorir exposed to A are con+
sistently pure, stable lysogenics, despite the persistent instability of
the Gal” tralt; the ensuing Gal- segragants are also lysogenic. Lyso-
genization occurs very much more frequently than transduction, but the
correlation of the two remained to be explored as evidence bearing on
the hypothesis. In the first experiment (table h , part A) transductions
were picked as Gat” papilise and streaked out on EMB galactose agar. A
single Gal” (representing nontransinduced cells) and a single Gai*

(the successfal transduction) were each tested for lysogenicity on an
appropriate Lp® indicator, In experiment B, marked Gal Lp® eells in

the approximate proportions expected from transduction were introduced
with the Gal” and the mixed culture on EMB galactose plates. With the
assumption that both Lp® strains would adsorb and be equally affected
bys a disparity in lysogenizations of the two ensuing Gal* classes

was Looked for. thoreas all of the transduction Gal* were lysogenized,
only up to 70% of the artifically inserted Gal* or of the original Gal”
hed been infected, Both parts of the experiment show a distinct corre=
lation of lysogenization with transduction; the incidence of lysogenization

is almost higher in these than in the control bacteria on the same plates.
(10)
Segregation of lysogenie sensitive has not so far been observed (ep te
500 tests) from these simultaneously transduced and lysogenized recipients.
This evidence argues that Aas the passive vector of genetic material frou
its source strain, This material is injected to the bacterium by the phage,
im Salmonella the transduced genetic factors seem to umergo an immediate
substitution for the homologues in the recipient bacterium, if they are
successful at all, In E, coli K-12, however, an intermediate stage is
perceived where one can detect simultaneously the presence of the original
recipient and the new transduced genetic factors in the same celis by virtue
of their subsequent segregation, The relationship between this replacement
of genetic material and the conversion of virulent (into its prophage
atage ("reduction" 6) has not yet been completely worked out. As will be
| described below, however, these processes have been separated and are
therefore not mitually dependent,
Lysogeni zation of Twmme~l in Transduction Experiments: When iImmune-1
strains such as W-1027 and W192) are exposed to As no evidence of their

lysogenization is ordinarily perceived. However, under conditions where

transductions can be selectively isolated about 5% of these altered bacteria
are also found to have been lysogenived, Repeated serial segregation

of the resulting transductions showed that in some cases, lysogenicity
failed to segregate. In others, lysogenicity and Gal segregate together,
while Im a single instance a lysogenic Gal” segregant was found which con»
tinued to segregate Lyp™ colonies. Sometimes a very weak lysogenicity is
observed ("one~plaque types" in cross-brush tests) , which is completely
lost after a few transfers. Some of these atypical cases are presented
in table 5s and suggest the following alternative interpretaticns:

(1) Lp” cells are genetically lysogenic but carry a modified prophage.
These cells are generally resistant to infection with ), However, A
may be exceptionally introduced simultaneously with the Gal* fragment
and there may displace the avirulent form of the prophage, or when

Lp segregation is observed, both prophages persist together for the

time being. (2) The Lp is a "null" allele, In transduction, Lp*

and Gal* factors are introduced, but the lysogenic Ammune segregation
occurs when Gal segregates, This hypothesis can not account easily for
the Gal“Lp*/" types except by devising a complicated scheme ie
crossingover, (3) Iumunes may or may not be genetically lysogenic.

The production of Lp* Signifies the occurrence of a double transduction

at two leei, Gal and Lp. (a) ordinarily these linked factors would tend
to be Lost as @ bleck in the ensuing segrogation, or (b) a linked trans=
duction coas not operate. By a two-step process, two effective particles
have penetrated; one fragment carries Gal*, the other Lp*, Independent
segregation is permitted and a mechanism requiring the breakage of a 20
factor linked fragment as in (2) is not called for.

In any event, special assumptions must be made on the avidity of
the Ly” locus for pro) to account for the failure of transductions to
Lp® to segregate Ip*/Lp® along with Gal*/Gal”. However, the Lp” may
only block the propagation of hor its reduction to pro~),

Hypothesis (1) accounts for the occurrence of immmnes which can
be induced by U-V (22), The recovery of unstable Lp’ transductions in
non~transinduced Gal- would tend to support hypothesis 3, The most
decisive elucidation of whether transduction displaces a mutant phage
particle with a wild type dor whether a normal Lp* allele is substituted
for a mtant or mill host Lp” gene would be provided by experiments with
geretically distinguishable ) preparations, Lp /Lp® transductions were
prominent wita irradiated J, tending to support hypothesis °,

Irradiation effects: Quantitative agsays of transducing potentiality

of phage preparation are necessarily based om plaqre counts, The survival
(13)

baad

afcer varlous treatments of plagisenrocuicinug particles and transducing
particles are not identical either in Salmonella (28) or Kel? (22),

In fact, it is known from both studies that transducing power may be
increased at some intermediate dosages, A comparison of the effects of
U-V and X-radiation is given in table6, A U-V dese reducing plaque

7
assay from 1/2 x 10°° to 16.9 x 10°

per ml yielded 170 transductions
from an initial titer of 10" / wl. A comparable X~ray dose was found
to be between 150,000 and 200,000 r. No recognizable transductions were
recovered at the latter exposure, Two viewpoints are indicated:

(1) the lytic and transducing principles in Aare separable by their
independent survival, and (2) avirulent J particles are produced but
they are damaged only to the extent of virulence for the host cell,
Conclusive evidence favoring one or the other views of lp’, however,

is not yet at hand, A decisive chemical and genetic separation of the

transducing material from the virus particle has not yet been experi-

mentally achieved, whether or not it is at all theoretically possible.

GENETIC DEFINE TION OF THE GAL LOCI
Recombination: Attention was focused on galactose nonfermenting

mutants because of the colncidence of the first recognized Jesensitive
(Li)
mutant ia Gab”) (518) , and the subsequent observation of linked
segregation of lp and Gal), (10). Gal” mutants have been isolated
directly by inspection of surviving colonies after U-V treatment on EMB
galactose agar and also as non-papillating variants of Lac” mutabile
recovered on EMB lactose agar plates. Interaction of Gal™ and Gal*
on the phenotypic expression and reverse mutation of Lacy and Lacy alleles
have been described (9). Recombination analysis provided the evidence for
a cluster of four linked Gal loci (7). Gal, and Galj, show a very lew
order of crossovers. Preliminary data could only differentiate them
on the basis of behavior in Het crossea} Lp and Gal, are both hemizygous,

while Gal,” /Galy,” heverozygous diploids are readily obtained (table 7 ).

dransduction: Transduction tests reinforce standard allelism tests
(table 8); and in fact have tentatively identified several new loci,
now awaiting confirmation by recombination analysis. Whether the
relative yield of Gal* transductions is proportional to the map dis-
tance between Lp and the Gal locus is in question. The results of
large-scale allelism teats made available to date by new techniques

to facilitate crossing are summarized in table 9,
(15)

The instability characteristic of the Gal” transduction results
in the mosaic colony already noted and deserves further comment.
Despite passage through a large number of serial single colonies, Cal-
segregants are almost always thrown off. In transductions from Gal*,
i.e. Gal x Gal”, these Gal” segregants have been identified as alleles
of the lecus of the original recipient strain, both by crossing and further
transduction tests, No other icinds of Gal” have been recovered, On the
other hand, if the donor is a non-allelic Gal“, both donor and recipient
Gal” appear among the segregants from the Gal* transduction (22). For
example, Galo ~~X Gal,” gives galactose~fermenting intermediates,
presumably of the constitution Galp Gal,” /Galy Gal”. The segregants
in all these tests are identified by (1) crossing experiments with Cal,”
and Gal)” testers, (2) deriving \ and subjecting the testers to its
action, and (3) applying Afrom Gal”, Gal,”, Gal)“, ete. The Galy”
Gal)”, a crossover type, has not been conclusively and consistently
established, This double mitant would be identified as one which is
subject to transduction by \ from Gal’ and from any Gal” other than
Galp” or Galy,", and would yield no Gal” recombinants in crosses with

Gal,” and Gal, ~ testers,
(16)

Diploid studies: The preceding evidence points to a chromoscmal
localization of the Lp lysogenicity determinant closely linked to a series
of Gal loci. Evidence for the segregation of a prophage linked to the Gal;,
locus ruled cut the possibility of a random distribution of cytoplasmic
particles in cells carrying A(io). These observations have since been
extended to Galp and Gal), hybrids (ann heterozygous Lp /s). and also
Gal) “Lp /Galy,“Lp™ diploids (table 10), A study of such diploids segregating
cut distinguishable ) types is in preparation, Preliminary evidence also
has been obtained elsewhere from crosses with lysogenic parents, one
carrying a mutant d (or one "doubly lysogenic") the other doubly
sensitive, which yielded Gal/Lp progeny in parental couplings (1),

The mutational. independence of Gal and Lp was also examined in
the doubly homozygous diploid, Comparable @xperiments with the closely-
Lae, and ¥¢ loci have already been reported. Lac’ reversions were selected
in Lac“V6F/Lac"Ve% diploids, The resulting doubly heterozygous diploids
were of two types: Lac*V,F /Lac"V,8 and Lae"V¢" /Lac*V,", and with equal
frequency (11).

A double homozygote Gal"Lp*/Galy Lp”, also segregating a few other

markers, (and unfortunately also Lps) was prepared by stepwise exposure of
(17)
the double heterozygote to U-¥ (Ly) and the isolation of suitable
"reorganized" diploids, The resulting diploid, H~331 was infected
with Me Several Gal, Lp /Gal)"Lps isolations, A to G, were then allowed
to papillate on EMS galactose agar, Independently occurring Gal” were
selected, and the segregation pattern of Lp and Galy of the resulting
double heterozygotes was tested, The incidence of mutation to Gai*
on the Lp* chromogome (coupling phase, or cis configuration) was com=
pared with that on the Lp® chromosome (repulsion phase, or trans-
configuration). The analysis included a single Gal and a single Gal
segregant from a large number of diploids, (pair analysis) and the
examination of many segregants from a single mass diploid culture (random
analysis), From diploid B, 5 cis configurations and 6 trans configurations

(table 11)
were scored, The conclusion from this evidence/is that the condition of
the Lp locus, whether lysogenic or sensitive, has no significant bearing
on which one of the 2 Gal” alleles will mtate to cart, (These pre~
liminary data will be expanded, and also extended to a corresponding
study of diploids first nade heterouygous Gal, “Lp*/Gal,"Lp*, and then

infected with },)
(28)

The above studies provide to lsinds of Ep” /Ly®; Gal” /Gat™ diploids:
\ coupled on the one hand with Gal” (eis) and on the other, with Galy” (trans)
Tf the notbrity ef fron "trans bacteria is confined to non Calo recipient
a chronosonnl bus not melear Imitation to Aspe ificlty is indicated,
ALL Gal” Lu cle ing Gal,” is expected te respond to cis A. A difference in aN
fron thoes diploids whieh are phenotypically identical, and seretically
identical cxcept for the arrengenent of component parts established a
“position effect." oa far, only \tron the trans-type diploid has been
prepered., Ta! hle ous that while Gal” (Gal, “Gat iy, “) cells are subject
to transduction, enly rare Cal," transductiong were recovered, The develos-
ment of an adequate diploid culture to satisfy the nutritional prerequisites
for U-V induction in K-1e (3 ,5) ard an interzediate growth period nece
essarily peraits somes selection for haploid seevegants, The yield of A
obtained very probably includes a limited portion derived fron Cal, Lp’
and Gal, Lp” haploids. The latter eroszover types may account for those
transductions which were found, The date so far allow the tentative con~

clusion of a position effect hypothesis and strengthen the concent of an

intimete velationship of doa Gel at a specific ection site on the

z =

Rt tt

chromosome, Vronsductbions of the double honemreote [6331 and lryeon
(19)

desivatives has apparently been obtained. The analysis is complicated
by the fact that diploid—haploid instability ean be confounded with trans»
duction ingtabllity.
COMPARATIVE GENETICS OF Lp AND Gal IN OTHER LINES

Among the Independently iselated eressable strains of E, coli (12)
the wild type of three lines (28,47, and $1) were sensitive to carried
by ine 1. A fourth, Line 31, threw off rough variants which were all
h sensitive. These strains occurred in nature as F” but could be

+

altered to FP ty grovth with K-12 or suitable derivatives, So far,
ab least ons Gal” mutant is subject to transduction, Preliminary intra~
Line-? evosses established an Lp locus like that of Ku12,' and a Gal~Lp
linkags, Very little mapping work has teen completed among these strain,
and the cuphesis go far in these studies has been ths genetic behavior
of (in outerosses with E-12,

Sensitives of each Line are readily lysogenized by K-12 \ovat
these lysogenics show a reduction of cop on K~12 sensitive indicators,
This system is entirely anolagous to host modification demonstrated for

f2 (19) and ) produced by strain G (2), The terminology established

for these systems will be used to describe the properties of our strains,
Thus Lines 28,31, and h? can be designated as Ke lysogenic or (it sensitive,
Line 1 senusitives are more resistant to he than to type je he can be

. introduced at low rates into A sensitive hosts, but normal rather than

de is recovered, Similarly, normal Ais converted to fe after a single
passage in A* sensitive hosts, The four phenotypes are readily dis
tlnguishable in cross~brush tests as follows:

Reaction with:

 

~S6NnS . t=SONB
Cc B
Exanple Type bacteria bacteria \ he
line 1 lysogenic A * + R R
line h? sensitive B - ~ Ss Ss
line 1 sensitive C - ~ 8 R
line 7 lysocenic D ~ + R R

 

+/- = lysogenic or not; R/S = resistant or sensitive
Two major hypotheses can be tested by intercrossing these types?
I ip controls all reactions: the types A-D are determined at a
single locus.
It inp controls lysogenicity/ sensitivity; another locus, Mp, controls
resistance or sensitivity to Mite
(a) Both and Ne are fixed at Lp in phenotypes A and D,

(b) Ais fixed at Lp in type As )# 49 fined at Mp in type D.
(21)
The consequences of these hypotheses are shown im table 12, The eritical
evosses for Land IT are Ax Band C xD. The only decisive cross for II a
ve. If bd is Ax D, IL b would be favored by the recovery of sensitive
recombinants as well as a novel genotype whose phenotypic'effects are
unpredictable. Since there is a possibility that Lp and Mp are closely
linked a large sample of progeny many be required, One must bear in mind,
in reviewing these intercross data that the prototrophs represent recon
bination ef as yet unmapped mutritional factors, In addition, chromosome
and other irregularities correlated with interstrain hyorids: have not
been analysed,

Effective transductions have been ackleved in these strains, Gal-
in lines h7 end 31 have been used as recipients, for A produced by line
1, 28, 31, and h?, A reduction in the effectiveness of transduction to
line 1 reciplents is parallel with the reduced effectiveness of lyso-
gerization, In general no important differences with the K~12 mechanism
have been demonstrated, Hypothesis II b is doubtful.so far, The dif-
ferentiation of the }+ of different lines is still to be tested, A
single intercross shows no genetic difference so far,

In preparing this report, it has been necessary to make mmerous
references to the unpublished work carried on in this laboratory by

Professor J, Lederberg, Mr. M. L. Morse, and others, under other auspices.
These are cited by ruber to the bibliography,
Tabie 1

Characteristics of F (compatibility factor) and A(virus)

 

 

Criterion F status ) (effects)
(1) Yield of reconbinants Decisive None
(2) Pype of recombinants Decisive None
(3) Trenemisslon to recombinants 100% Segregated according to linkage with

selected mutritional markers; behaves
as a genetic locus,

{1} Transmission by infection Rapid and Results in mixed clones (3).

 

fixed
{¢) Cell-free preparations Not yet Easily filtered.
acconplished
(6) Effect of antiserun Slight if Blocks adsorption
any
(7) Role in Gal.* transduction None Decisive
Table 2

The Effect of Aon % Gal” Progeny Lt

- NO ad”
_ T-L~Th-Gal* parent Res -
M°Gal™ parent x lysogenic = iios:'¢ Immune
F lysogenic 8.0 Mh

immone . 633 6.3
sensitive 667 10.1
Linkage of Gal, Lp, and Hfr

Table

3

W+1895 x W-2208

 

 

Part A: Genotypes recovered! Total
Gal Ip F
+ + + Li; *
+ s + 5
- + ee Oo
+ ga h
- + + 0
Part B: 2 2 2 contingencies
Gal” Gal” =—sdTotal, «Ss P* oF” Total.
Fr 20% 0 20
r 9 Bue ho
ip* 15% 0 15 13% 5 18
Ly® EL oe ho 6 33% 39
Lac* 26% 5 32 a) 31
Lac™ k 26% 30 7 278 3)
Vy Lt 9 10 Le 9 10
v8 28 21s L9 23 20% 13
Xylo" He i 10 je 2 9
Kylo 20 30% 50 16 Fe 23

 

% Parentsl conbination "
1 Selected as Gal* and Gal” prototrophs,
Table hk

Lysogenization in Transduced and Nontransduced Lp®

 

Part At Gal* and Gal” from single papillae

 

 

 

 

 

 

 

Gal” /dal”
Pair type Number Gai Ip® Gai” Lp*
Lp*/ip* 13 Gai* Lp? 2 3
8
Lp*/Lp 15
Lp®/Lp* 3 Gal* Lp* 17 13
LpS/ips 2
Lp* /up® 2
% Gal* sensitive 15.2
% Gal- sensitive 7.2
Part Br Lysogenization of transduced and inserted Gal”
3 Av, Ho, Gal” vocovered
Lp” strains Control ‘Treated types in mixture No. tested % lysogenic
Gal Lact 109 92 Gn*Lac* (inserte) 16 68.5
Cal“Lac” ‘Lhe 432 Gal“Lac™ (origins) ho 72.5
Mixtureisnt 106.5 W9 Gai."Lae~ (transductions) 103 100,
109.)

+ Spontaneous reversions per 10° anoculum
aut: 108 Gal~Lac~ and 109 Gal*Lac*
Table 5

Transductions to Gal)” Inmmune~Is Segregation Patterns
Exp, 385% Strain 192): 27 Gal”

 

Number lysogenic Number semilysogenic Number nonlysogenic
(lys) . “ semi) on (non)
#2 ho é
Colony generation . 7 1
Lal” non 139 Gal* ys +h Gat” iys 18 mi 1 oat semi = 1. Gall" semi ar", |
L (Gai* Vy and Gal
/ and Gal”) \ - 2 Gal” lys non
ob 2 Gal” non
Ir Gal” non t Gal”lys : Gal" non Gal“lys . 2 Gait 2 Gel 49 bait 19 Ge1* non
lys non . non
TIr Gal*tys Gal“non h3 lys 3 non 1 semi
Iv lys non
v PAN non non
VI lye ion

fap. Y3L: Strain 2110: 38 Gal*: 28 non, 1 eemi (#23), and 9 lys

 

Segregation patterns ali Gal” lys, all Gal™ non: 2

of lys ail Gal* lys, all Gal= lys: 5
all Gal* lys, Gal” lys and non 2
both Gal* and Gai” non: #23
Table

curvivel and Transduction wlth Irradiated ny

 

Xray” ( x 10° r)

 

 

 

 

os Uniweatod 4 -

No phase phage Ua" 50 ico 8-150 200

Ay, plaques/ui x10? 9 427,000 16.9 1,667 3,975 377 100
& survival ~ 100 «000013. 3208 = 33 04297 0.008
Lp® bacteria

Yo. Gal papillae 20 1,000 170 250 85 320 30
~ 4 u G45 100 3h 25 17 6 6
tp bacterLa

Wo. Gal” papillae 39 60 ~ 135 lis 31 20
oe # " 65 100 225 191.7 5.2 3.3

 

 

20 minutes, sterileup

103 w/min, ab 250 K.V., courtesy A, Novick, Radiobiology Inst.,

YJ, of Chicago,
Table 7

Segregation of Cal, Lo,... diploids

 

 

A. He32y Bo He325
Segregation of Loo, By, not Segregation of Vg, Mtl, Lpa, By not
tabulated, tabulated,
Galg- Calot Lp Mal Syl M T,2. Galy~ Galy+

 

1 47 + + em 2 ho

‘

 

0 1 + “6 = 0 0
o 1 PF of mF é 0
Oo 0 ; * ~ ee 6 1
1 0 tot Fe 4 0 0
2 9 + ow RF 0 0
25 S Boom » f 13

Go tw ‘SO
So oS © Q 2 4
2
+
>
:
~
~j
o 09 89 09 80 FF 9

Z S = + + + 0
2 s t - + + 3
0 5S = ~~ mS 12

 

50 50 Total tested SL

2

 
Table 8
AlIsLic Specificity of the Gal « \Transduetion at the
Gel 1, Gel 2, and Gal. b 1ocd.

 

 

 

 

d= donor bacteria Recipient cells
Gal 1. Gal 2 Gal Le 2th+ 1+2-))+ 1+2+h-

+ + + + + +

~ + + ~ + +

al ~— + + ~ +
diploids:

2 % + %
ra data (21) (300)
(trans)
+ + + Lp*
z = ie (cis) No data

 

* Gal + papillae per 10” dL
Table 9

Summary of Current Allelism Tests

 

Totalitt No, 7
Exp. No, Gal” type FF" parent F* parent progeny Gal” Maxim, % Galt

 

 

 

 

 

535% xh W~750 Lot Wa223) Lp® 5000 17 0.3
563% 2000 15 0.75
Saige exh W-1210 Lp* W..223) Lp® 6000 25 Ook
563% 1600 WD 0,68
580 21,00 8 0.3
535 hx 3 WeS18 Lp® We2315 Lp* 807 6 O.7h
582 hx? WeG18 LpS W.2215 Lp? 5000 0 0
6700 5 0.06
583 1x? We2291 Lp® We583 Lp* 7603 _— 2 0,026

 

* Ay Gal* recombinants in these experiments are Lp®,
svEstimated total,
Type of cross
(a) (Het)

1. Hat diploids
(b) (Het)
2, Lacl- x Lae)- (a)

(b)

3, Haploid x auxoe (a)
trophic diploid

(b)

but Gal 1 does not.

Table 10

Behavior of Gal. and Lp in Lac +/~ Diploids

F (TL th)
+ «
- >
+ o
+ +
+ +
+ «
= +

+

+

Wf ef +f +/u -/* + +/o

Parents
Lac, Lac), Galy Galj, Lp

+ + + + +
a + + = 8
+ + + + +
~ + - + +
+ ~~ + + +
oe + + - 8
+ - + + +

same, except M~ parent is Lp*

1/ In Het crosses, Lp does not segregate. Gal 1 and Gal );

Diploid progeny

Gal Lp

+/ +/> or ~/o V 3/
+/> or -/e not. segregating
Mostly +/+ Mostly +/e 2/
Mostly «/* Mostly 6/ 2/

+/o Gal* Lp* / Gal-Lp (linked) 3/
8

Gait Lp” / Gal-Lp” (linked)

3 two closely linked loci also differ: Gal ) segregates,

2f Diploids resulting from delayed disjunction revealed by heterozygotes of two Lac pseudoalleles show no segregation

of Gal or Lp.

3f The only successful demonstration of heterozygosity of Gal and Lp,

L/ Aeration phenocopy.

5/ +/+ indicates purity for +, whether hemizygous or homozygous.

Reversal of F status reverses the polarity of the Gal, Lp segregation.
Table 12

Segregation Patterns of Cal Reversions in Gal,“Lp"/Gal,“Lp* Diploids

 

 

 

2 2
Diploid Total + ~ Galt - + ~ Inferred
number segre= cal cal = Gal bat bal type of
gants Lp’ ip? Lp” Lp® Lp, Lpp® Lp.” Lpo® Mal Mal” Mal* Mal~ diploid
AL 161 76 6 3 76 LS 0 39 0 1 53 17 36 cis
Bi 121 2 6 6 i 52 8 60 1 38 22 61 0 trans
B 2 73 0 Lo kl 0 32 7 3L 0 33 7 33 0 trans
Ba 76 62 A 2 10 65 0 57 5 65 0 lh =:18 cis
G1 Lg a 2 am oO 23 1 oh 0 9 15 ak; 0 trans
El 60 30 0 3 27 26 4 oh 6 30 6) 16 86 cis
BE? oh Oo 12@ #322 =40 12 0 12 0 6 6 12 0 trans
E3 23 12 0 0 12 12 0 1 0 12 0 3 8 cis
Fu 66 32 1 2 31 31 2 30 3 32 1 a 4612 cis
Poo ho 20 0 i 19 20 0 20 0 20 0 7 #13 cis
r3 23 12 0 oO il 12 0 16 1 12 0 3 8 cis
Fh 18 a 0 1 6 10 3 0 7 at 0 7 0 cis

 
Genetic Determination of Host Modification:

Table 12

line

i

lines 28, 31, h7

 

Hypothesis I
Lp locus with

Genotypes Under

Hypothesis Ila
fixed at Lp,

Hypothesis Tib
fixed at Lp in line l,

 

 

 

 

 

 

 

 

alleles modified by Mp at Mp in other lines
Phenotypes Symbol Lp Lp Mp Lp Mp
lysogenic A + + r + r
sensitive B git 8 8 8 8
sensitive C Ss 8 r 8 r
lysogenic D +t + 8 8 +
AXB None c, D c, D
BXC Nore None None
cCxD None A, B A, B
AXD None None B ami Lp’Mp*
EXPTL. RESULTS: Lines crossed Type A C D Gal char.
Expt. No. Lx 28 A Gal” x B O 16 1 0 +
ig 18 0 0 -
CGal™ xD 0 oO 3h +
2 18 3 7
418 Lx 31 AGalm™ xB 3 43 26 1 No record
420 AGal“~xB kh 22 28 = 12 Gal* only
4,23 AGal“ xB 8 2 1 37 +
0 1 0 0 ~
423 CxDGal* 8 1 3 0 (and 28 Lpo”)
B or ©
Why CGal-xD 9 9 a9 oO mostly Gal”
502 BGai”xC oO 15 13 0 +
c. 8613 —l 68 0 -
bh3 31 x 32 BxA 0 2 0 1
468 Lx h7 Ax B Galm 51 0 0 6 +
ee ee ee :
2 al” x 7 1 9 +
al hl 0 0 2 -
528 BxC @Gal- 0 13 17 0 +
0 8 ah 0 =
52 C Gal” xD 3 2 2 a +
2 2 28 0 -
523 AGal” xD 8 0 Oo 52 +
37 0 0 19 ~

 

F” parent underlined,
Table 13
Genetic Control of the Semiresistant Phenotypess

Nonlysogenic (W-21)7) and Lysogenic (W-2172)

 

 

 

 

 

 

 

 

 

Part I
Hypothesis I Hypothesis II
A new allele at Lp: A 3rd locus, Lp3, is involved:
Phenotype symbol Lp, Lp9 Example Lp. Lpo Lp3
A + 3 Type lysogenic + s 8
B + r Immune-2 lysogenic + r 8
Cc + P We2172 mtant + s p
D s s Type sensitive 8 8 8
E s r Imemne-2 s r 8
F s Dd We21h7 mutant 8 8 )
BxfF Yields: B, F, BE, © progeny Yields B, F, E, C, A, D
Cx " "
Results: BxfF No, of Progeny Cx
A B C D BE YY A B c D E F
Mal* 5 1. i 1 0 1 22 2 1 26 0 1
Mal.” o 58 0 0 1 0 0 0 0 0 59 0
Part II linkage of Lp3 to Lpy--Gal and Lpo--Mal ?
No. of Progeny
Parents Mal* Lpj® = Mal” bp)” = Mal” Ipy® Mal” Lp,"
F Mal” x B Mal™ \ 56 1 58
C Mai* x E Mal” 27 25 59 0
Mal* Lpo® Mal” Ip. = Mal” Lpg® = Mall” Upp”
F Mal* x B Mal™ 59 1 0 59
C Mai* x E Mal” 51 2 0 59
Mal* Lp3° Mai* Lp? Mal” Lp3” Mal” Lp3P
F Mal* x B Mal” 57 3 59
C Mal* x E Mal” 50 2 50 0
C Gal* x D Gal” Gai* Lp;* Gal* Lp}8 Gal~ Lp* Gal~ Lp®
60 0 0 2
Gal* Lp,§ Gal* LpsP Gal~ Lp,f Gal~ Lp5P
37 23 37 26

 

The above data are consistent with the hypothesis that an Lp3 locus separable
from Lp and Lp modifies the reaction to )-1 and \-2. This locus is not
linked to Lp,--Gal or Lpo~-Mal.
REFERENCES
1. Appleyard, R. K, 195) Segregation of lambda lysogenicity during bacterial roe
combination in E, coli EL2, Ccld Sprirz Harbor Symp. Quant. Blol. 19.
In Press.
2, Bertani, G. and J, J, Weigle. 1953 Host controlled variations in bacterial
viruses, J, Bact. 65: 113-121,
3. Borek, FE, 1952 Factors controlling aptitude and phage development in a

‘lysogenic Escherichia coli K-12, Bichim. et Biophys. Acta 8: 211-215,

ences eee

 

h. Cavalli-Sforza, L. L, and J. L, Jinks. 1953 Observations on the genetic and
mating system of E, coli K-12, Abstr. 9th International Congress of
Genetics, Bellagio.

5. Gots, J, S. and Gd. R. Bunt, Jr. 1953 Amino acid requirements for the
maturation of tacteriophage in lysogenic Escherichia coli. d. Bact. 66:
353+36)..

6. Jacob, Fy. A. lwcff, A, Siminovitch and E, Wollman. 1953 Definitions de
quelques termes relatifs a la lysogenie, Ann. Inst. Pasteur 8h: 222-22).

7. Lederberg, E. M, 1950 Genetic control of mutability in the bacterium
Escherichia coli. Ph.D. Thesis, University of Wisconsin.

8, Lederbery, E, M, 1951 Lysorenicity in E, coli K~12, Genetics 36: 560
(abstruct).

9, Lederberg, E. M, 1952 Allelic relationships and reverse mitation in
Escherichia coli. Genetica 37: 1,69~1,83.

10. Lederbery, E, M. and J, Lederberg. 1953 Genetic studies of lysogenicity
in Escherichia coli. Cenetics 38: 51-6.

11, Lederberg, J. 1951 Genetic atudies with bacteria, In? Genetics in the
20th Century, MacMillen: New York. pp. 263-289.

12, Lederberyz, J. 1952 Cell genetics and hereditary symbiosis, Physiol.
Rev. 322 102130.

1h Lederberg, J. Unpublished,

15 Lederberg, J., L, L, Cavalli, and E, M, Lederberg, 1952. Sex compatibility

in Escherichia coli, Genetics 37: 720-730.
16,

17.

16,

19,

226
234.

233.
25.
26,

27.4

REPERENCAS continued

Lederberg, 3, and P, R, Edwards, 1953 Serotypic recombination in Salmonella.
J. Inemmol, Tl: 232-20.

Lederberg, J., E. M, Lederberg, N. D. Zinder, and E, R, Lively, 1951
Recombination analysis of bacterial heredity. Cold Spring Harbor Symp,
Quant. Biol. 16: )13+h33,

Lieb, H, 1953 The establishment of lysogenicity in Escherichia coli.

J. Bact, 65262-6512,

Luria, S. E, and M, L, Human, 1952 A non~hereditary, host-induced vari«
ation of bacterial viruses, J, Bact. 6h: 557-569,

Imoff, Av, Le Siminovitch, and N. Kjeldganrd. 1950 Induction de production
de phage dans une bacterie lysogene, Ana. Inst, Pasteur 79: 815-858,
Kaplan, A, S. and A. lwoff, 1953 Factors affecting the lysogenization of
Salmonella typhimurium, VI Int'l Congress of Microbiology II:203.

Abstract No. 55.

Morse, HM, Ly Unpublished.

Nelson, T. C, ani J, Lederberg, Unpublished.

Parry, W. 2. and J, Edwards, 1953 The induction of lysogenesis in
Salmonella typhimurium, J, Gen. Microbiol. 9: 32-349.

Skaar, P, D, Unpublished,

Stocker, B, A. D,, N, D, Zinder, and J, Lederberg, 1953 Transduction of
flagellar characters in Salmonella, J, Gen, Microbiol. (In press).

Wahl, R. and L, Blum-Emerique, 1952 Les bacte ies semi-resistantes au
bacteriophage, Ann, Inst. Pacteur 82: 29«l3,

Wollman, E, 1953 Sur le determinisme genetique de le lysogenic, Amn,
Inst. Pasteur 8h: 281-293.

Zinder, N, D, and J, Lederberg, 1952 Genetic exchange in Salmonella,

J, Bact. 6h: 679-699.