Ib

©

of the traneduction clone. The galactose positive reversions of these
segregants are stable.

A charactersitic HFT culture has been obtained for each
galactose uagt negative as well as for wild type. These cultures
were isolated initially by making lysates of random segregants
from heterogenic transductions and assaying the lysates or the
appropriate cells. This method is laborious and inefficient. To
assist in the isolation a nore rapid method was devised. Random
segregantZ colonies were picked to small volumes of water or broth
and a samples of each suspension were then spotted on an EMB galactose

plate spread with cells suitable for the detection of the HFT

culture desired. The plate upmkked was given a small doee of UV

(about 10-20 seconds at 50 cm froma Sterilamp) and incubated for 24 hours.
At the end of this time HFT cultures were usually detected by the

raised welt of galactose positive growth where lambda produced by

the induction and lysis of the HIT culture had transformed bacteria

of the background film of growth. _

The incidence of HFT galactose negative emkutezx cultures
ig not high. Of 67 segregants tested, 7 wére found to be capable of
HFT lysates, The true frequency might be higher than this, since gurified
segreganfs were examined and there was opportunity to pick .FT
segregants from originally HFT clones.

Cultureskux giving HFT lyeates that are pure for a particular

galactose negative allele are suitable for allelffin teste of unknown

gelactose negative cultures by the cross brush method.
@
v
Experiments with lysates giving a high frequency of transduction
Although the HFT lysates have not yet been obtained with

phage titers comparable to. Mrs lysates the titers have been sufficient
for transforming a large fraction of a cell population exposed to them.
The largest fraction of transformation observed thus far has been 12.5.
percent of exposed cells, but in most experimexts the fraction has been
between l and 5 percent.

_ The use of HFT lysates has permitted the study of several problems
not attackable with NT lysates. One of these is the relationship of trans-
duction to lysogenigation with the phage lambda. Another problem is that
of the interaction of Gal, and Gal,. Both of these problems will be
dealt with in the next sections. With pre lysates, transduction was
experimentally feasible ag X only whan a galactose phenotype is
generated that can be sdlected from a galactose negative background.

HFT lysates, permit the detection of galactose negative segregants from
transductionsg clones derived from galactose positive recipant cells.
Traneductions in this sense have facilitated further studies of the

interaction of the galactose loci with the Lp locus,
“8

The relationship of lysogenization to transduction

| By exposing cultures of Lp” celle to HIT lysates, diluting,
and then plating on galactose medium to obtain isolated colonies it is
possible to study the behavior of individual cells with regard to suet
transduction and lysogenization activities. Table if shows the results
of an experiment in which 1.1 percent of a cell population was transformed
after exposure to a HFT lysate. The second portion of table 14 gives
the phage reactions of the galactose positive (transductions) and
galactose negative colonies derived from cells exposed to the HIT lysate.
All of the transductions were lysogenised or converted to the Lpe state

while the non-transformed colonies were either phage sensitive or

4

contaminated with phage.
carries

These results suggest that lambda f@ the transducing activity.
could be argued

However, under the experimental conditions employed it ia=pesebbte that

the transductions are the resultz of the action of two entities. The

would
first, which,actg upon the cells and makes them "potential" transductions,

and the second, lambda, which in the process of lysogenizing the cells,
would sometimes > opi & , so many phage tontacts to res uit m
convert@ them to actual transductions. In order for transduction} +o-he——
° ?

A
( V3 Q 3%)
obeenrcd=at- | hypothesis, the "potentiating" agent would have
preseat “th about —— -: Overs .
to be tie-opder-of ten-fold te excess ef, lanbaa./' Et might be-argwed that
because Gir kukke theoxperinent sarecorted—in—tarvke=24) onty-ubeet—ere-
thist of=mthe—leambaameell—eentecte=became t rerniuctions=thet the ratio
- of the "petentiatingll_sgemt -to iembia was wet bth. ebe-would-not—necessarity

pe

 

  

meré =<H6..o0 nd B prs Le—@ Sd aT oe ensic pt
ed ; [/Pteebmality
potentiaikty=te-aetuettey,s |The observatéen-of linear mp of number of trane~
ductions to amount of HFT lysate at high dilution} (10 -10 j/mkees  ’ -
such au overwhelming excess of AVY Lacessory te a3 -to“wake tw fggethesee
the intervention aira tector ing ME addition, lanbds wiehty iupeobeble. ”

cv) uteu o b le
 
  
 

At these dicot “at dilutions the wed fabs ty that a wed would

both lactivities would at atud, proximate the cana) ejof ae

The juteraction of Gal, and Gel, [ fosituns-ebteck)). Ay.

With the use of HFT lysates it has been possible to study the
interaction of Gal, -and Gal,- cells with HFT Gal,- and Gal,- lysates.
respectively. The results from one set of Loteractions is shown in tabie
16, After a preliminary period for the adsorption ef the traneducing
activities the cultures were centrifuged, the superhatant lysate discarded —
and the cells resuspended in broth. The celle were then diluted and plated
on EMB gun galactose medium, No galactose positive colonies were observed
On the mxbipthpetyintd eketés plates made from control unexposed cells
or from lysate treated cells, After 24 hours incubation at 37C two raised,

slightly, ovange Stn colonies were observed in each experiment on the

 
(s)

7 roughened papillate surface. On straiing out they gave rise to positive
colonies, negative colonies that remained negative, and to papillating
galactose negative colonies. |

In each experiment a number of galactose positive colonies
derived from the papillating negative colonies were tuukead picked and
streaked out twice for purification purposes. From the second streaking
galactose negative scgregants vere obtained and classified with regard
to negative allele. In each experiment idiotypic and allotypic segregants
were observed and in one experiment amphitypic segregants were found. The
amphitypic ( Gal,- Gal,-) ségregants were transformed to wild type
phenotype by lysates of wild type cells, and a lysate of , the amphitype
, formed galactose positive transduction clones when applied to Gal,-

reciplent cells. —

The failure to realize a wild type phenotype when the positive

trans-
. alleles are in a trumzposition, and its realization in the cis~position

constitutes a positional effect for these loci. FES
\
The action of HFT lysates on lambda-2 resistant cultures | v°
In the previous atacussioG WY? Jyeates were stated not to
transduce lambda-2 resistant recipieng cells. HFT lysates, on the
other hand, do transduce lambda-2 resistant cells, but at a low
frequency (one per 16° transducing particles), This is presumably
|e eet peta potency of HFT lysates, which helps to uncover any
residual interaction of transducing phage and lambda-2 resistant bacteria,
regardlese of which element had varied. Such variation might then be
either phenotypic (expressivity) or genotypic (mutation) wither in
virus or bacterium.
The interaction of HFT lysates with lambda-2 resistant cells
is illustrated by the following observations. Some mutations to lambda~2
resistance are accompanied by a coincident change to tambaz inability
to ferment maltose ( z. Lederberg, unpublished). Reversions to ability
to fereunt maltose is accompanied by reversion to lambda-2 sensitivity
and vice versa. These two phenotypic effects have never been separated
in crosses and it is presumed that they are the result of a single
mutation.
The transductions of a galactose negative, maltose negative
lambda~2 resistant, lambda sensitive culture obtained by the action of
an HFT lysaté are of two types. Maltose positive and lambda~2 sensitive,
and maltose negative and lambda~2 resistant. The first of thaex a=
types represents the detection by the HFT lysate of reverse mutation
of the lecus in the recipient cells controlling lambda-2 resiséance.
The second type of transduckion in about 95 percent of the casés

is stable for galactose fermentation. Study of the transformability of

galactose negative segregante from the unstable transductionz clones fouud
p
showed ‘them not to be suscepti bites

 
 

oO

¢
to a higher frequency of transduction than the parental mukkuxs maltose
negative lambda~2 resistant enilyire. In th#é¢ cases, at least, there has
not been a mutational change in d recipient cell to a FESES greater”
aptitude for transformation.

About 95 percent of the txaunductixzsxef maltose negative

lambda-2 resketant transductions have been found Lp*, the remainder Lp? °
The Lp” forms may be stable or segregating for galactose, but dl segregating
clones are Lp’ . Segregation for Getnctose fermentation is usually accompanied
by segregation at Lp. Presumably in th8GE cases there has been variation
in the transducing particles, although it is possible that in the transductions

ending in Lp® clones that an agent distinct from lambda is operating.
+yraud duce clnw
Crossing behavior of the unstobbe_qalectore-peckbive-cnivures

In previous sections it was noted that transduction clones
gave HFT lysates after UV induction. Pee produced phage is
similar tp phage produced by the induction technic it might be expected
that in crosses between transduction clones and gakactose negative cultures,

or between HFT galactose negative cultures and nbn-allelic galactose

0 ae there are several observations whieh: soegent that : ‘transduction ~ ”. secur
“a does not play: an. important pars. in wach niosseas ‘The first oveervation, gation

  

; ae SERIE Tye .
whieh mintnises: 6 aEEatt , ‘Ls that ins cross Detvean an | EST allotypic
segregent and an fatotypic tester, 12,200 prototrophe were exatined vefore

a exlactove positive recombinant was encountered, A second observation is

“trem the compart vou of a cross niece a lysogento unstable traneduction. x

(ompatte 0 of giving HPT Lysates) ey s Ip® ‘lactone negative calture, wah
/: eross e an Ig? tranedustion (1noapedle of giving arr iymstes) ae
Spe sone te galactose negative gal ture, A comparison of thess orosses

_ Bowed no significant dnorease ta ‘the number of galactose positive Golontes

¢ thave .
in the crocs bebteczi ‘the lyscgeaic transduction ant the “senat tive, Apparently

transduction does not confuse (arcey-tanortantsiney the results of crosses.

 
r
The tranamission of galactose heterogenicity in cesses is greatly

influneced by the F SSKIFIE¥ polarity of the cross (table 1. When an _

wuxtubie heterogenic F’ culture is crossed with a noneallelic galactose
negative Lp® F° culture, unstable galactose positive prototrophs are rare,
When the unatable culture ie F’, and crossed with a non-allelic & galactose
negative I Lp*® Ft culture, most of the prototrophs are galactose positive
and unstable. Some of the galactose negative prototrophs in these crcesses

can be explained by galactose negative segregante in the unstable zix

‘sm-., Galactose positive parent clone.

 

of ultraviolet light in aurotrpphia stocks sit table for crossing with
the Gal,~, Gal,- and Gal,~ stocks used above. Of these 17, 1+ were found

'" to be transformed r lysates (to wild typ Five of the 14 were recurrences

n of Gel,~ amt Oats four were epparently dowdie mutations at. Gal, and D:
CW23.07 2
« aly, and the remaining five were new loi ‘transduced by lysates. one of

‘the “three cultures nht transformed wy any. iyeate was axangned urge.
+ me PEARENTLN * Nor

“Tysates of 18 emi transduced Gal;, Oly. ani Gaiz, but/iot Gal. In
reoonbination tests thie culture has given galactose positive Socombinasits

with beth Gal,- and Gal. For exphanation of these remults {t is necessary
OyTs ioe “SE Sher TRAT CAN BE TRANS OUCED
_ to postulate a double change, one ‘ebthent “the penton ef teerstrced

the meme ics

) zoel, ‘and one witht the region, non-allelio to ary of the kuewn lect,

Be
Bae.

    
 

Ss erp

     
  

a which 1n eddition gives an interaction with Gal,~ euch that the hetere- Be ot

Hdlygous combination Ls not phenotypically galactose positive,” onvesogd¥ Pal

| technic ad df icu tty SeemS a mere Ikety ttplauahm .

The study of The qalachse neqa hve Culbuves wet tran bw wel by

lysates hos beet gar tly hindeve by di Frc thy 14 da scev ining chick culties

So
> ae aud w hick culfures we ve mee! “ slow posibwe | ,

trul
wees “" celecked,

Tet galachse porthwe Nansduchms are wel reads ly
oe

DI s6USSION
The xaux results presented above can be placed in an orderly
fashion by the following scheme. When lysogenic cells are exposed to
ultraviolet radiation and the prophage is induced to form mature phage,
on rare cccasions a fragment of the bacterial chromosome is included
within a phage particle. When thie particle injects its genetic material
into another bacterial cell, the fragment is also injected and if the

recipient bacterial cell has the proper genetic constitution the presence

+

of thie extra genic material is made obvious. The—frapnent—renains—within
‘ +

 

The allotypic fragment usually persists at cell ditision, so that

segregating clones can be maintained indefinitelyyin mass culture. At
least two additional events are inferred: (1) diploid crossing over leading
to reorganized digenotes. Since these may be hetepogenic or homogenic, a

Four
oper sea strand (or more) stage is implied. (2) seergetion eccurs leading to

ctieter ne heplogencte, the state typical of E. coli. The fate offthe fragment is
unknown, Crossover haplogenotes (amphitypes) have also been isolated and
may represent either a third process, or the first two in sequence ( ef
Pontecorvo, 1954). Since heterogenotes give HFT lysates, the fragment or
a replica of it, is assumed to have a high probability of incorporation
in the phage obtained by UV induction. The low yields suggest a burst of
one phage particle, a reversal of transduction.

| From this description it is evident that the genetic transfer is
intimately associated with the process of lysogenization and lysogenicity.

Concerning the process of lysogenization in K-12 little is known beyond the

fact that cell and phage interact, there is a period of indecision, amd the
Oa

infected bacterium either dies or generates a clone containing lysogenized
cells. Once lysogenicity is established the capacity to produce phage
behaves as a nulcear gene that is closely linked with a number of loci
controlling galactose fermentation. -

The rire step in the scheme is the inclusion of a fragment af
sum within a phage particle. In Salmonella the fragment is a random
, section of the cell's genetic material, but in E. coli K-12, it is m quite
specific, for only a restricted group of loci are transduced by lambda.
Again in contrast to Salmonella, "lytic" lambda is incompetai/widt in
transduction. This may reflect an inherent difference between lytic and
UV induced phage.

In the establishment of lysogenicity the genetic material of
lambda enters the cell and adsociates itself in some way with a specific
region of the bacterial genome, In the induction process it is presumably
‘emerges from its place and starts to mitiply. Transduction could be
gccounted for by some latitude in the separation of the galactose loci

from the prophage linked to them, and their common bnelusion in some
nature phage particles. The close genetic proximity of the galactose loci
would suggest their increased liklihood of gnclusion, but there is no

closely
necessity makquiness§ genes be also spatially close to one another.

: CBT wa VET)
‘There are two ‘types,of culture in which » tranpductng particles

, ere formed and it ts Legitinate to ask whether the two are ‘different
phenonens « or tex nerely quanti tetvely, disterent aspects: of a a single
phenomenon. The evidence for a unitary process is negative in patugg. tat

ies. no difference have voen noted vetween HFT lysates and art lysates, .

YF he Lalita ated vee hens
except possibly the higher incidence of tradetustions, “ath, Lp", react
.

  

with the former, This exception, if it be one, “gould itself de oxplpines

on ‘the basis of quantitative differences between the two lysates.
  

of tue =
ent ta, of transducing particles in cultures giving |

tr

Saysates has not passed beyond the Preliminary stage. The evidence thus

> mest et why
far suggests that ee PTE of the cells yleld transducing particles
The defeveunahin of

“With
ant that the yield per eel), ial pot large. wa regard to athe frequency of
cells oat tting transducing activity it should be noted that cul tures _ ,

started fron a single colony with HP! property may contain #8 much as
hie Se Sacgshm

30 percent of cells wlth MFT proper ty “* Mipeaees|
gehe aes a” Doge
The Saataeme of ‘the lysates: of segregating hgerosygous

is
@Alactose posit2ve clones indicates that the fragnent preferentially
fg

Lpoluded within the phage particles, oo - Presumably exchange between
= Cadalse’*"™ gee
fragnent ant intact chromo sone joscury mah that instead of giving lysates

predominately allotypic in character, idiotypic lysates are obtained, The

exchange ia sufficinetly rare, however, that observation remains cbjeative

in nature.
The nature of the association of the fragment with the infective

phage particle is not known. Presumably the material ts within the phage
membrane since it is not attacked by desoxyribomuclease. The availability
- of lysates in which she most of the phage pabticles have activity (HFT
lysates) or have no activity (NFP lysates) suggests that morphological
comparisons might possibly be made via electron micrsocopyof intact BEAES
PRELTGI“S or disrupted phage particles.
The fragment enters the bacterial cell in company with the prophage,
by analogy with 12, probably by the injection process (Hershey and Chase,1952).
The association of the fragment with the prophage in transduction
to lysogenic cells cannot be stated in the absence of phage markers, since
it is not possible to distinguish between the previously carried and the
newly enstered prophage. The carriage of more than a single prophage by

cells of E, coli K-12 has been reported by Appleyard (1954) and it is

 

likely that the transductions of lysogenic recipient cells are also

carrying more than a single prophage.
In only one instance, from more than 250 segregations studied, has segregation
from a transduction of lysogenic cell resulted in a change at Lp. In this
case an idiotypic segregant became Lp®, and this might have been a spontaneous

"mutation",

. In the transductions to Lp® recipient cells the associatéon
between transduckng prophage and fregnent is possibly better seen, These
transductions are of two kinds, Ip” and Lp’. All/segregants from Lp* clones
have been lysogenic. On the other hand, Lp" transduction clones segregate
Ip" /Lp* as well as Galt/Gal-. The incidence of Lp® Gal- idiotypes supports
the notion that these loci are linked.

In considering the rekationship of the fragment to the rest of
the’ genome no specific statements can be made with regard to its perpetuity
in the heterogenic clone. One would depend upon its possession of a
functional centromere, so that it would behave as a small autonomous
chromosome, or the fragment would be attached to the homologous chromosome

attachment
segment, either intersitially or terminally. Either, position presents

difficulties for crossing over, and the fragment as a separate chromosome
#,.20ems more plausible. |
. In the above sections the results have been treated and discussed
‘dn ogeneral way. It is obvious that the study of this transduction system
has only begun and that many experiments and intereéting observations will
be made before the problem is completely understood, It is proposed to investi~
gate lambda transduction further along the following lines.
1. ‘Whether the production of transducing activity in aFT cultures
is related to the interaction of radiation and cells, or is the result of
a matation\ Like event in the cell poputation.
2. The production of transducing particles in HF? lysates.
3. The action of radiation on transducing particles and the possibility

of inducing mtations.
2 @

6. Farther studies on crossing over between fragment and idiotypic

>

loci using additional markers.
7, The relationship between lysogentsation and transduction, ank

between lysogenization and crossing over.

8. Estimation of the gene order of the transduced loci and their xet

relationship to other mapped loci,
9. Study of the biochemical steps controlled by the various loci

gud the fexxeuxtzi fermentation of galactose.

 

4. The detection of other loci within the transduced region.
Syngam ae
5. fhe bahavior of the fragment transduced during | meiosis,
SUMMARY
A cluster of loci in Escherichia coli K-12 was found previously

to control ‘the fermentathon of galactose. Lyeogeni city for the temperate
bacteriophage, lambda, was also found to be closely linked to these loci
in crosses. The phage lamkda now has been fqund to transduce these loci,
as can be readily demonstrated by mixing lysates of galactose positive
cultures with galactose negative cells on a selective medium, EMB
galactose agar. / The transductions
result in clones that are heberogenic, that is, they are diploid
for a small region of chromosome. The small fragment of chromosome
transduced appears to have a functional centromere, and is perpetuated
within the clone even after many single colony isolations, but it may
on some occasions be lost. While in the cline #t has been found to
eresnorer with its hoftologous region, On some Occasions at least,
at a four strand stage. Each of the new phage particles formed in lysates
of hetergenotes has a high probability og containing set—owky—o-deeguent,
wet the fragment iemth.tee—casmemey carried in the heterogenic clone.
A position effect on the expression of two of the transduced loci has
been observed. Dtheterogenotes of Gal, and Gal, are not phenotypically

galactose positive in the trans positiong, but are so,in the cis.
4d

ese

BIBLIOGRAPHY

Alexander, H. E., and G, Leidy 1951
Determination of inherited traits of H. influenzae by desoxyribonucleic

actd fractions isolated from type specific cells
J. Exp. Med. 93, 345-359

Alexander, H. E. and Redman 1453 ‘ Chante ta
, ty af Mening 9c? ch, t .
Therionc ryge weaucl of Ce peafe eniPacks canteoning desonriboruclel
ve -
qed. fh test ved ag 1941- wot
Eppleyard, R. K. 1954
Segregation of new lysogenic types during growth of a doubly
lysogenic strain derived from Escherichia coli K-12

Genetics 39, 440-453

Atchley, W. A. 1951
Cold Spring Harbor Symp. Quant. Biol, XVI, 441
(Discussion of Lederberg et al)

Austrian, BR. 1952
Bacterial transformation reactions

Bact. Rev. 16, 31-50

Boivin, A. 1947
Direcged mutation in colon bacilli by an inducing principle of

desoxyribonucleic nature: its meaning for the general biocemistry

SEXRBERALEZ of heredity
Cold Spring Harbor Symp. Quant. Biol. XII, 7-17

Herehey, A. D. and M. Chase 1952 ‘
Independent functions of viral protein and nucleic acid in growth
of bacteriophage
J. Gen. Physiol. 36, 39-56

Hotchkiss, R. D. 1954
Double marker transformations as evidence of linked factors in

desoxyribonucleate transforming agents
Proce, Nat. Acad. Sci. 40, 55-60

Lederberg, E. 1950
Genetic. control, of mutability in the bacterium Escherichia coli

Ph. D. ShakSmniversity of Wisconsin, Madison, Wisconsin

hederbers, €,. and J, Lederberg 1953
Genetic studies of lysogebicity in Escherichia coli

Genetics 38, 51-64

Lederberg, J. 1947
Genétit-recembination in Escherichia coli
Ph, D. Dissertation, Yale University, New Haven, Conn.

beevber J, 1950
g! Isolation and characterization of biochemical mutants of bacteria

Methods in Medical Research 3, 5-22
The Year Book Publishers,Inc, Chicago, Ill.
Lederberg, Z., E.M.Lederberg, N.D.Zinder and E.R.Lively 1951
Recombinational analysis of bacterial herédity
Cold Spring Harbor Symp. Quant. Biol. XVI, 413-443
Lederberg, J. and E, L. Tatum 1953
Sex in bacteria: genetic studies 1945-1952
Science 118, 169-175

Lederberg, J. 1954
Recombinational ,mechanismse in bacteria

J. Cell, Comp, Physiol. Supplement 1954
( Symposium on genetic recombination, ORNL, April, 1954

Pontecorvo, B, 1955 ; -
Analysis of mipbtic vecambination ih Aspergillus niger

‘J. Genetics 52, 226-237
Stocker, B.A.D.,cMsD.Zinder and J. Lederberg 1953

Transduction of flagellar chafachers in Salmonella
J. Gen. Microb. 9, 410-433

 

Tatum, E. L. and J. Lederberg 1947
Gene recombination in the bacterium Escherichia coli

J. Bact. 53, 673-684

Weigle, J.J. and M. Delbrick 1951
Mutual exclusion between an infecting phage and a carried phage

J. Bact. 62, 301-318

Zinder, N.D. and J. Lederberg 1952
Genetic exchange in Salmonella
J. Bact. G+, 679-699
 

 

Table 16
The transmission of heterogenicity
in crosses
parental elle ; Prototrophic recombinante
Zz ¥F G + & -
Galo=(}) Galy- tp* 1" abcut 6000.
Galye ip’ al,=(2) sue 99

 

* unstable for galactose fermentation, 6 galactose negative
segregants tested were Gal ~

** 25 of 30 examined were unstable for galactose fermentation.
One segregant from each of the 25 was tested, all were Galo~.

(1) contrel platings showed the ratiox of (+)/(~-) in this
culture was 109/57

(2) control platings showed the ratio of (+)/({-) in this
culture was 115/13

25

Gok” bp® GoLt Let 324
} 757 (aprox)

Gar t te Gol. ig? 107 (ppror.)
Table 1

Principal cultures

 

 

Wisconsin
Stock Number Ge mtype*
W518 rou Lac,- Gal,- Lp®

+ +
W750 FPF M- Lac,- Galj- Lp
W811 FY Lac,- Gal,- Lp’

+

w902 F- T-I-B)~ Hal,- Gal,- Lp

+
W1210 F M- Lac,~ Gal,- Lp’
W1436 F’ 2-L-B,- Lac,- Gal,~ Lp* s°
W1924 FT Mu Lac,- Gal,- Lp™
W2175 Fr’ Gal,- Lp*
W2279 F* M-Lac,~ Gal,- Lp®

+
W2281 F M- Lac,~ Gal,- Lp®

 

*Genotypic symbols reger to the following characters,
(1) Compatibility status, F

(2) Nutritional requirements; M, methionine; T, threonine;
L, leucine; By» thiamin

{3) Fermentation reactions; Lac~, lactose negative; Gal-, galactose
negative; Mal-, maltose negative

(4) Phage reaction; Lp”, lambda sensitive; Lp’, lambda lysogenic;
Lp, lambda resistant, but not overtly lysogenic.

(5) Drug resistance; S, streptomycin
Table 2

Recombination between the various
ig? Galactose loci

Ww wo

 

 

 

Minimum Number of Percent Galactose
Gross Prototrophic Recombinants Fermenting Recombinants
FiGal,- X F Gal,- (1) 1500 0.13
(2) 6517 0.06 *
(3) 3603 0.03 “
11620 0.06 —
¥’ Gal,- xX ¥ Gal,- 4588 0.13 ¥
FY Galy- xX F Gal,- 2654 0.23
FY Gat, = W750 .
E- Gx2, - W150 (aevalhm phenocopy)

Fo God,- Wier
Ft Gely” = weil wsig  wit3e
Table 3

on

/48)

\

Observations on lambda lysate transductions

 

Locus Number of experiments

1. Leci not transduced
Lacy
(serine or glycine)
Leucine

Methionine

\
we y Streptomzcin

Xylose

Proline

4

Ww r- WwW ~

ro

9

l(lytic lambda)

2

1L

Cultures involved

w112
W1678

W1736 ,W1436"
58-161 ,W811 ,W1821 W518”
wi821°

w51st

W1692,W1920 ,w2062°
42062"

W2331 ,W23478

W2071

23079

 

2, Loci transduced

Gal,
{

We ate
As Gal,
( gs . Gal,

Gal¢

(Footnotes table 3 continued)

f- lytté’ lambda grown on M~ culture

g- lysate of prototrophic HFT Gal
h- lysate of prototrophic HF? Gal

2.
2

culture
culture

W750, W2279 ,W2280 ,W2373
W1210 ,W2175 ,W2281

W2297
W518, W811 ,W1821 ,W1436 ,W1924

w2070

oe ey
Ww)
Table 4

The interaction of lysates ani cells of
falactose negative cultures

 

Recipient has ‘ay Galy- Gal,~ Wild
‘as pH aes Bet
2 : hel Art

Gal fr (2) e196 43 -
(2) 2 2 - - 40S

Galo- = (1) Us 52 lL 43 -
(2) 20 - 10 - 356

Gal,- (1) 89 - 202 - -
(3) 59 85 - - 417

(3) 4? - - 30 394

 

*“ The no added lysate plate which represents the number of
spontaneous reversions occuring on the plate. The remaining figures are
the mmbers of papillae occuring on the plates per 0.1 ml of lysate ,

added,
Table 5

Restoration by reverse mation of the ability
So transduce previously non-transducible lock

 

 

celle (int Reversilo lone uyeate Reversion
Gai, M4 = Gal, (1) 0 eves
Gala~ Gal,” (2) 10 96
Gals” (2) 6 552
Gal,,- Gat,” (5) 39 204
Gal,” (8) ' 25 291

 

*number of papillae per plate, 0.1 ml of lysate pleted,
 

Table 6

The necessity of of lambda adsorption
for transduction n¢

 

 

bbe?
a Plate, Addubun-

Recipient | ’ i Oipptiee
Cells (L ___ Hone Wild type Lu Ses
Gal)- 8 1 426"

r | 1 2
Galp- 4 20 356

r Ww 14
Gal,,- 8 / 89 296

¥ «50 57

 

*xumber of papillae per plate, 0.1 ml of lysate plated
at sc ROned-+- Darmeabet | nr fearbda -2 Nroltict.
pone dt wt dane atu lok da oy Aomubde -¥
 

 

 

 

 

_ Bable fe
The actiln'ot Ivtteally aom
lambda,
Plabe obbctur
Experiment Recipient Lp Rial:
cells Allele ___ None Lytic lambda Place titer
228 Gal,- + 3 2" 2.4 x 1020
Galo~ + 9 8
Gal, - 8 9 8
239 Gal, + 2 o 2.4 x 10°C
Galy- + 6 2
Gal,- 8 13 8
254 Gal,- 8 - 6e* 2.4 x 10°4
Gal,~ + - Bae
Gal,- 8 - | ore
Galy- 8 - 6
Gal), + - 39%
280 © Gal,- + 0 Que 6 X 107
Gals- + 1 266
Gal,- + 14 10**

 

*Papillae per plate, o.1 ml lysate plated. Lysate prepared by
growing Gal,~ lambda (UV inducticn) on a gelactose fermenting culture.

®*Thege papillae picked and streaked on EB galactose medium
and found stable for galactose fermentation,
Table 8

The specific activity of lysates of the
transduction clones

 

 

Recipient  Transéucing Titers +
Cell lysate fransductions on Lp assay cells P/T*
Plaques Gal,- Galo- Gal,,-
Gal, - wild type 5.8 x 108 2.4x106 1.8x 107 1.3 x 107 32
Gal,- Gal,~ 7.22109 1.2x10° 1.0 x 108 - 60
Gal,- Gal ** 2? x10° 1.8x10° 63x10 = -
Gal ,- Gal,- 6.2.x 108 4.3x107 1.5 x 108 - 4D
Gal,,- Gal;- 11.5 x108 5.0x107 7.5 x10° 74x10? 8 2
Galy- Gal,- 9 7.9 x 10? 2.5210? 2,8 x 10° “ 29

 

* Ratio of plaques to transductions, the maximum transductign
titer observed is used for this estimate. Usual ratio P/f is about 10 -

*® A second isolation,
The occurrence of

q
Table TS

 

stable transductions

 

 

 

Recipisnt Lys2tea
cells mfg Wild
type Galy- Gal - Gal =_
ceo  * o/c 0 7 PC Cc if %8C C TF
Galj-Ip® 38. 2 34 - = = U/l 2 NL 30/2 1 29
Lp’ 46/1 1 2 - - = S/1 1 4 27/1212 27
Lp* 143/1 1 42 - - - 9/1 1 0 - = =
Galo- Lp® 46/0 0 15 214/00 27 = = = 98/0 0 &
upt 208/17 17 21 83/4414 61 - == 79/1 4 52
Ip’ 23/4 4 6 65/2 2 0 - = = S65 5 0
Bal,- Lp* 835/19 19 383 «= 72/29 29 72 492/11 11 20 - - -
Lp? 573/41 42133 (96/51. 5196 ee - --
Lp” 320/31 31 127 - = = 238/31 31 50 ~ - -

 

* Papillae transduction plate/ papillae control plate. T =

transduction plate, 0 =

control plate

** Corrected for sample taken, stable obs. X Papillae transd, plate

galactose fermenting papillae.

sample size’
With the exception of the T/C column, numbers given are number of stable