Download A programme for the construction of a lambda phage

J. Embryol. exp. Morph. 83, Supplement, 75-88 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
75
A programme for the construction of a lambda
phage
By W. J. BRAMMAR AND C. HADFIELD
Department of Biochemistry and The Leicester Biocentre, Leicester University,
University Road, Leicester LEI 7RH, U. K.
TABLE OF CONTENTS
Summary
Introduction
The lambda genome
The expression of lambda genes on infection
(a) The uncommitted phase
(b) Lytic development
Lysogenic development
The decision between lysis and lysogeny
The lysogenic state
Summary and conclusions
References
SUMMARY
Infection of a sensitive host by the lambdoid coliphages can cause death of the bacterial cell
by lysis or can lead to a lysogenic cell, with the viral DNA stably integrated into the host
chromosome. These alternative responses both require the coordination of several host and
phage functions, and lambda infection follows a well controlled developmental plan. The lytic
and lysogenic pathways of lambdoid infection are reviewed, with emphasis on the variety of
control mechanisms involved in the commitment to a particular pathway.
INTRODUCTION
The lambdoid bacteriophages are a family of viruses which multiply in
Escherichia coli. Infection by a lambdoid phage can either cause death of the host
cell by lysis or can lead to the stable integration of the viral DNA as a prophage
into the host chromosome, with survival of a lysogenic cell.
The lytic and lysogenic responses both require the co-ordinated activities of
several functions, encoded by both viral and host genes. In lytic infection, DNA
replication, genome packaging and cell lysis proceed sequentially. In the
lysogenic mode, the synthesis of the phage integration system and the repressor
protein must be synchronized. In both cases, functions that might interfere with
the chosen pathway must be excluded. Thus the lambdoid phage infection
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W. J. BRAMMAR AND C. HADFIELD
follows a simple, properly co-ordinated developmental plan.
While no one would claim that phage infection provides a valid model system
for the study of development, the present, detailed understanding of the lambda
developmental programme might have some valuable lessons for developmental
biologists working on more complex systems. This review will present a simple
account of the key steps in the lytic and lysogenic pathways, with emphasis on
the control mechanisms governing co-ordination and commitment to a particular
pathway.
THE LAMBDA GENOME
The genome of bacteriophage lambda contains approximately fifty genes,
about half of which are essential for lytic growth. The DNA is packaged into the
head of the mature phage particle as a non-permuted, linear, duplex molecule
with single-stranded, 5' projections of 12 nucleotides at each end. These mutually
cohesive termini assure rapid circularization of the genome following infection.
The genetic and physical map of the lambda chromosome (Fig. 1) shows
marked clustering of genes of related function. The essential genes concerned
with head (A-F) and tail (Z-7) formation and assembly are contiguous within the
left-hand third of the genome. The region of the map between J and att, the
phage attachment site, contains no essential genes (Hendrix, 1971). Genes to the
right of att govern site-specific (int and xis) and generalized (red) recombination
of phage DNA. None of the genes between att and N is essential for lytic growth
of lambda on normal hosts, though they may affect growth on certain mutant
host strains. The product of gene iVis the early regulatory protein that is normally
necessary to activate transcription of most other phage genes. The cl gene codes
for the lambda repressor, the regulatory protein that switches off transcription
of prophage genes in the lysogenic state. The presence of the lambda repressor
makes a A-lysogenic cell immune to superinfection by another lambda phage and
is responsible for the characteristic turbidity of lambda plaques. Lambda carries
an additional regulatory gene, cro, that encodes a repressor-like, DNA-binding
protein. The cro gene-product functions as an antagonist to the c/-product,
acting to prevent lysogenic development and to promote lytic growth (Gussin et
al. 1983). The O and P genes are required for replication of ADNA (Brooks,
1965; Joyner, Isaacs, Echols & Sly, 1966), the Q gene product for activation of
late transcription (Dove, 1966; Couturier, Dambly & Thomas, 1973) and the S,
R and Rz gene products for lysis of host cells (Harris, Mount, Fuerst &
Siminovitch, 1967; Young etal. 1979).
THE EXPRESSION OF LAMBDA GENES ON INFECTION
(a) The uncommitted phase
The infection of an E. coli cell by lambda is initiated when a phage particle
adsorbs, via its tail fibre, to a specific receptor on the cell surface (Schwartz, 1976;
A programme for the construction of a lambda phage
11
early
control
DNA
replication
replies
immunity
mmunity
/
head
tail
lysis
recombination
P
pE
att
ABC
"
"
EFZV
H LK
'• • • " ' i
'
II
II
i
J
II
\
red
Vmf
i
*
._
i i IUI
II
late
control
|
cro
N
_
cI OP
II
i IIU i i
l
9
!
10
!
!
20
!
!
30
1
!
.
i
Pk
PR
I
SR
i im i
40
!
R
!
kb
Fig. 1. Genetic and physical map of bacteriophage lambda. Gene clusters with related
functions are indicated above the brackets. There are ten known genes at the left end
of the map concerned with head formation, and eleven genes from Z to/inclusive that
are required for formation of the tail. The region between / and att, the b region (for
buoyant density changes in CsCl gradients), codes for several proteins that are not
essential for vegetative growth (Hendrix, 1971). The att (attachment) site is the site of
integrative recombination between phage and bacterial DNA, catalysed by the
product of the int gene. The red genes, redA (= exo) and redB (= bet), encode functions that catalyse general phage recombination. The N and Q genes encode the
proteins that activate early and late transcription respectively. The phage repressor is
encoded by the cl gene, while the product of the cro gene is a DN A-binding protein that
acts as an anti-repressor and promotes lytic growth. Promoters are indicated hyp with
a subscript to indicate their role: pi is the promoter for int gene expression (= pmt); PL
the major leftward and p& the major rightward promoter; p^ is the promoter for
establishment of repressor synthesis, often called /?RE , and pu is the promoter for
maintenance of repressor synthesis (=/?RM); P'R is the late promoter. Horizontal
arrows indicate the extents and directions of specific transcripts, with — indicating
significant readthrough beyond a termination signal. The scale is in kilobase-pairs.
Thirion & Hofnung, 1972). The linear phage genome is injected through the cell
membrane into the cytoplasm (Mackay & Bode, 1976), where it rapidly circularizes via its cohesive termini and is covalently sealed by host DNA ligase.
Transcription of lambda genes by the host's RNA polymerase proceeds leftwards from promoter ph through gene N and rightwards from promoter /?R
through the cro gene (Fig. 2). Most of these initial transcripts terminate at *LI and
tei, immediately beyond genes N and cro respectively. The leftward transcript
is translated to yield the N protein and the early rightward transcript produces
the cro protein. Both of these gene products have important regulatory functions
that influence subsequent A development.
The N protein exerts its controlling effect by influencing RNA polymerase to
ignore transcription-termination signals (Adhya, Gottesman & de Crombrugghe, 1974; Franklin, 1974; Segawa & Imamoto, 1974). In the presence of N
protein, transcription initiated at/?L and/?R is elongated through the terminators
tu and JRI and through a series of 'delayed early' genes whose products regulate
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W. J. BRAMMAR AND C. HADFIELD
Fig. 2. Transcription of lambda genes during lytic growth. I: Transcription immediately after infection or induction. In the absence of lambda's Nprotein, leftward
transcription from p^ terminates largely at tu, while rightward transcription ter :
minates inefficiently at fo\, beyond the cro gene. About 20 % of the rightward
transcripts (thin arrow) avoid termination atfaxand proceed through the replication
genes 0 and P to terminate at fe. II: The product of the TV gene influences
subsequent transcription to override termination sites tu, fei and tja.. Leftward
transcription now expresses the recombination (red) genes, while rightward transcription proceeds through the O, P and Q genes, leading to DNA replication and
the activation of late transcription. Ill: The product of the Q gene elongates transcription from/?'R through genes 5, R (and Rz), governing cell-lysis, and through
genes A to J, coding for head and tail proteins.
both the lytic and the lysogenic responses. The infecting phage genome shortly
thereafter becomes committed to one pathway or the other.
The apparent specificity of the N protein for transcripts initiating at /?L and
PR is not a consequence of the promoters themselves, but of nucleotide
sequences, termed ^/-utilization ('nuf) sites, located between the promoters and
A programme for the construction of a lambda phage
79
the first transcription termination signals (Salstrom & Szybalski, 1978; de Crombrugghe, Mudryj, Di Lauro & Gottesman, 1979). The two nut sites, which have
16 out of 17 identical nucleotides, show hyphenated dyad symmetry, consistent
with the formation of a stable stem-loop structure in the RNA or the DNA
(Rosenberg et al. 1978). Though the nut sequences have been shown to be
sufficient to allow the Af-protein to influence RNA polymerase to ignore
subsequent termination sequences (de Crombrugghe et al. 1979), the precise
mechanism by which this is achieved has not been elucidated.
During the phase of uncommitted growth, phage DNA replication is activated
by transcription in the vicinity of the unique origin within the O gene (Dove et
al. 1969; Dove, Inokuhi & Stevens, 1971) and by the products of the genes O and
P (Ogawa & Tomizawa, 1968). The early phase of replication, giving monomeric
circular molecules, ceases after a few minutes. At this stage the genome either
stops replicating and enters the lysogenic phase or switches to rolling-circle
replication as it begins the lytic cycle.
(b) Lytic development
During lytic growth, the production of mature phage particles requires the
synthesis of concatemeric phage DNA, composed of covalently joined, tandemly repeated unit copies of the lambda chromosome (Szpirer & Brachet, 1970;
Stahl etal. 1972; Feiss & Margulies, 1973). The rolling-circle mode of replication
is believed to provide the mechanism for the production of the concatemeric
molecules (Eisen, Pereira de Silva & Jacob, 1969; Gilbert & Dressier, 1969),
which are the ideal substrate for DNA encapsidation (Skalka, 1977). The
packaging of lambda DNA into phage heads requires the unit chromosome to be
bounded by cos sites (Emmons, 1974; Feiss & Campbell, 1974); a monomeric
circular lambda chromosome cannot be packaged in vivo (Szpirer & Brachet,
1970).
The onset of rolling-circle replication is facilitated by lambda's gam gene
product, an inhibitor of the host's recB, C-encoded exonuclease V (linger &
Clark, 1972; Unger, Echols & Clark, 1972; Enquist & Skalka, 1973). In the
absence of the gam-product this nuclease attacks both the rolling circles themselves and an intermediate required for their formation, thus inhibiting the
synthesis of maturable phage DNA.
An alternative route to packageable DNA can be provided by recombination
between two monomeric circular molecules to produce a circular dimer containing two cos sites. Both the red systems of the phage and the rec system of the host
can catalyse such recombinational dimerization (Stahl et al. 1972; Enquist &
Skalka, 1973), but the latter does so very inefficiently with wild-type lambda
DNA (Stahl, Crasemann & Stahl, 1975).
While 'delayed early' leftward transcription gives rise to gam gene expression,
similar transcription rightwards facilitates expression of the Q gene. Note that
the expression of the Q gene will necessarily be delayed by some 2-5 min, the
80
W. J. BRAMMAR AND C. HADFIELD
time required for RNA polymerase to reach the gene from its promoter /?R , 7000
base-pairs away. The product of Q activates transcription initiated at P'R to
traverse the late genes, whose products are responsible for DNA encapsidation
and cell lysis. The Q protein acts as an antiterminator, employing a <2-utilization
('qut') site in a manner formally analogous to the N protein, allowing transcripts
initiated atp'R to proceed through the termination sequence, J'R into the large
late gene region (Forbes & Herskowitz, 1982).
Delayed early transcription begins to diminish after about 5 min of infection,
dropping to a very low level by 10 min (Szybalski etal. 1970). This diminution is
caused by the binding of the cro gene-product to the OL and OR operator
sequences, interfering with the initiation of transcription at/?L and/?R (Fig. 3).
The kinetics of action of the croproduct are explained by the observation that the
active form of the DNA-binding protein is a dimer, and that relatively high concentrations are needed to block the RNA polymerase-binding sites at/?L and/?R.
From about 10 min until the end of infection, transcription from/?'R activated
by the Q protein is predominant and expresses all the late genes. The products
of the A and Nul genes aggregate to form the terminase, which binds to the
concatemeric substrate DNA at the cos sites. Several other late gene products
are assembled into a capsid prohead, while others are independently assembled
to form the tail fibre. The prohead attaches to a DNA-terminase complex, and
a unit length of the phage chromosome is taken up until the next terminase
complex is reached. The terminase then cleaves the DNA to yield a mature,
DNA-filled head. The final stage of capsid assembly is the addition of a tail fibre
to the mature phage head.
Coincident with the production of mature virions is the continuing expression
of the lysis genes. About 60min after infection initiated, the accumulated
products of the S, R and Rz genes are sufficient to lyse the host cell, releasing a
burst of progeny phage.
LYSOGENIC DEVELOPMENT
The establishment of the lysogenic state involves the coordinated expression
of two key genes, the cl gene that encodes the phage repressor and the int gene,
whose product catalyses the site-specific integration event. Transcription of cl
and int is synchronized by the action of a positive effector, the ell gene-product,
which stimulates the binding of RNA polymerase to the separate promoters, pre
and /?int (Ho & Rosenberg, 1982) (Fig. 4). The c//-product is metabolically
unstable, due to the action of the host's HflA protein, but is protected by the
lambda clll product (Hoyt et al. 1982). The controlling ell and cIII genes are
both expressed mainly by delayed early transcription from/?R and /?L during the
uncommitted phase of phage development.
The c/-encoded repressor is the effector of the switch into lysogenic development to the exclusion of lytic growth. The protein binds to the phage DNA at the
A programme for the construction of a lambda phage
81
OL and OR operators, with initial preference for regions OLI and ORI . These
operator sub-sites are located within the RNA polymerase-binding sites and the
bound repressor prevents further transcription from initiating atpL andpR. The
repressor, in blocking transcription of the N, gam and red genes from /?L and the
O, P and Q genes frompR, effectively stops phage DNA replication, late protein
synthesis and host cell lysis.
The repressed, circular phage DNA is integrated into the host chromosome by
a site-specific recombination event catalysed by the int-pvotein (integrase). The
int gene is transcribed from pint, under the influence of ell protein, and from /?L ,
driven by the action of Af-protein. However, an interesting post-transcriptional
mechanism, 'retroregulation', ensures that the latter transcription is nonproductive during phage infection (Fig. 5). This phenomenon depends on a exacting regulatory element, sib, located downstream from int, beyond the phage
attachment site (Mascarenhas, Kelley & Campbell, 1981; Guarneros, Montanez, Hernandez & Court, 1982). The sib sequence displays extensive dyad
symmetry, enabling the elongated RNA transcript to form a hairpin structure
that is believed to be a preferential substrate for RNAse III. The action of this
enzyme on the RNA could create a 3' end, processive 3' to 5' degradation from
which destroys the adjacent int region of the transcript (Guarneros et al. 1982).
Transcription from the c//-dependent promoter, pint, terminates at a rhodependent terminator, tmx, located within the sib region (Schmeissner, Court,
McKenney & Rosenberg, 1981), and fails to complete the RNAse Ill-sensitive
(pcro\
A
/
att
N PL cl cro OP Q SR
Fig. 3. The control of lambda transcription in the lytic phase. The product of the cro
gene represses transcription from /?L and /?R , moderating expression of the early and
delayed-early (Af-dependent) genes, and from p™, blocking synthesis of the
repressor. The Q gene product causes transcription fromp'R to avoid termination
and thus to express the late genes.
J
att int
Pint
N cl cro pre ell OP QSR
Fig. 4. Coordinate expression of int and cl leading to lysogeny. The two promoters
Pint and PRE , governing transcription of the int and cl genes respectively, are both
sites for activation of transcription by the ell gene-product, pell.
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W. J. BRAMMAR AND C. HADFIELD
+
. . .
.
lysogenic infection
product
£
1 pell
sib att int xis
= -
D ,w
F
—
Pi
RNAselll
sib att int xis
lytic infection
nv
=========================
;A
•.
G££> PJ2I
pN
prophage induction
——•"
•—~
rms+p_m£
Fig. 5. Retro-regulation of int gene expression. During infection, the cll geneproduct activates promoter p\ (-pmt), within the xis gene, to give expression of int.
The transcript terminates beyond att, and is translated to give the m/-product. During
a lytic infection, transcription from/?L is activated by TV-protein, proceeding through
termination signals to transcribe both xis and int. The transcript proceeds beyond the
sib site, where RNAse III initiates a nucleolytic attack that leads to processive
elimination of the int gene transcript. Potentially deleterious integration of a
chromosome committed to the lytic cycle is avoided. On prophage induction, transcription from/?L can express both xis and int, since the sib site is no longer adjacent
to the int gene. The site-specific recombination event at the att site that gives the
integrated prophage places the sib site at the opposite end of the prophage map. The
products of both int and xis are required for efficient excision of phage DNA from
the prophage state.
structure. This regulatory mechanism ensures that only those int transcripts
initiated at/?jnt are effective as integrase messenger, and the requirement for cll
protein ensures coordination with repressor synthesis.
In the prophage of a A-lysogenic cell the sib site has been removed from
proximity to the int gene by the integrative recombination event involving the
phage attachment site. On induction of a A-lysogenic cell the adjacent int and xis
genes, whose products are both required to bring about excision of the prophage
DNA, are coordinately expressed via transcription from/?LThe alternative modes of expressing int during infection and induction thus
ensures that the appropriate protein is produced to drive the recombination
event in the required direction.
Host proteins are also important in modifying the lysogenic response. The E.
coli HflA protein decreases the amount of active c//-product in the infected cell,
but is antagonized by the phage c///-product. The synthesis of the HflA protein
itself is repressed by the host cyclic AMP-activated catabolite repression system
(Belfort & Wulff, 1974). The integrative host factor (IHF), a dimeric protein
comprised of the products of the E. coli him A and himD genes (Miller & Nash,
A programme for the construction of a lambda phage
83
1981; Nash & Robertson, 1981) has a dual function in lysogenic development.
It is a DNA-binding protein that is essential for integrative recombination (Nash
& Robertson, 1981) and promotes the synthesis of the phage ell product (Hoyut
etal. 1982).
Overall, a single regulatory protein, the ell gene-product, coordinates the
lysogenic response. Not only does the ell protein synchronize the expression of
the cl and int genes, but it also acts as the receptor protein for transmission of
environmental signals via the host IHF and HflA control proteins.
THE DECISION BETWEEN LYSIS AND LYSOGENY
Within the phage-infected cell, an individual lambda genome can enter either
the lytic or the lysogenic pathway. Although these two pathways have their initial
steps in common, a decision between them is made at an early stage of phage
development.
The particular pathway that ensues depends upon whether the cro-protein or
the repressor wins the competition for the operator sites, particularly OR . The
cro-product is able to lock the genome into the lytic pathway, inhibiting the
expression of ell and clll and ultimately preventing repressor synthesis. The
repressor ensures the lysogenic response by preventing expression of cro itself,
and also of the O, P, and Q genes whose products are required for DNA replication and late gene expression.
The delicately balanced competition between repressor and cro protein is
influenced by several factors, the level of ell protein being a crucial one. The ell
product is required to activate transcription of the cl gene from /?RE , and its
activity is influenced by several host- and phage-coded products. When the
activity of the c//-product is high, the lysogenic response is favoured; when it is
low, the lytic response is enhanced. Thus, for example, carbon-starved cells,
which have reduced levels of HflA protein, favour activity of the ell protein and
show elevated frequencies of lysogenization (Belfort & Wulff, 1974).
THE LYSOGENIC STATE
In the lysogenic cell, the prophage state is maintained by the binding of
repressor to the operator sites, OL and OR , blocking transcription from the two
major early promoters, /?L and /?R .
Each of the operators OL and OR contains three repressor-binding sites
(Ptashne et al. 1976) (Fig. 6). These are similar in sequence and contain axes of
two-fold hyphenated symmetry. The three sites in OR overlap both/?R, the major
early rightward promoter, and /?RM , the promoter from which the cl gene is
transcribed in the prophage. At low concentrations, repressor binds preferentially, as a dimer, at ORI: such binding potentiates subsequent binding of repressor
84
W. J. BRAMMAR AND C. HADFIELD
to OR2 (Johnson, Meyer &Ptashne, 1979). Binding to 0R3 shows reduced affinity
and is only achieved at high concentrations of repressor.
The binding of repressor dimers at om and ORJ. stimulates the binding of the
E. coli RNA polymerase to/?™, by providing extra contact sites at this position,
thereby enhancing transcription of the cl gene (Reichardt & Kaiser, 1971;
Meyer, Maurer & Ptashne, 1980). The repressor, in blocking transcription from
PR , is also acting to stimulate its own synthesis (Fig. 7). At high intracellular
concentrations repressor will bind to OR3 and inhibit its own synthesis, thus
providing an effective homeostatic mechanism.
When the DNA of a A-lysogenic cell is damaged, for example by ultraviolet
irradiation, thymine starvation or a chemical carcinogen, the prophage is induced into the lytic cycle. The induction process is triggered by the proteolytic
cleavage of the repressor, catalysed by the activated product of the cellular recA
gene (Roberts, Roberts & Craig, 1978; Craig & Roberts, 1981). Destruction of
the repressor leads first to transcription from/?R , with production of the cro gene
product, and slightly later to transcription from pL. (Repressor binds more
tightly at OL than at OR .) The cro-protein, also active as a dimer, has high affinity
for the sites within OR , though in the order ORS > OR2 = ORI . Binding of croprotein to OR3 blocks access of RNA polymerase to /?RM , eliminating the maintenance mode of repressor synthesis (Fig. 7). This action of the era-product
effectively prevents recovery of repressor synthesis and locks the induced
prophage into the lytic cycle.
PL
f
att
4
int
xis
red
cIII
NOL
PRM
«.
4
rex
,
^
,
cl
OR cro
I
PRM
ell 0 P
1I
Q
PR
J
Fig. 6. The sites controlling early lambda transcription. Transcription of lambda's
early genes is initiated at p^ and /?R and is controlled by the interaction of repressor
with the operator-sites, OL and OR . Transcription of cl can be initiated at pie, the
establishment promoter, or p^, the maintenance promoter. Transcripts are indicated by dashed lines. The expanded diagrams show the three repressor-binding
sites within OL and OR and their spatial relationships with /?L , p™ and pR. The cro
gene-product also binds at OL and OR to reduce transcription from /?L , PRM and pn.
A programme for the construction of a lambda phage
85
repressor
-M
induction
pcro
cl
Fig. 7. The lysogenic state and prophage induction. In the lysogenic state the
repressor dimers bind cooperatively at ORI and OR2 , blocking /?R and activating /?RM
by providing extra contact'sites for the interaction of RNA polymerase with /?RM .
Following induction, involving proteolytic inactivation of repressor by the host's
recA gene-product, polymerase gains access topR to transcribe the cro gene. The cro
gene-product binds as a dimer to om and prevents expression of cl from /?RM •
SUMMARY AND CONCLUSIONS
Lambda has evolved an effective system for monitoring the environment in the
infected cell and using this information in the simple decision between lysis and
lysogeny. The alternative responses branch from a common pathway, the
ultimate direction being governed by the products of the cro and cl genes. These
two DNA-binding proteins compete for the same region of the phage DNA and
repress each other's synthesis. The balance between the cl and cro products is
modulated by the ell protein, whose activity is sensitive to the cell's metabolic
state via its interaction with the host HflA gene-product.
Lambda's relatively simple control circuits reveal great variety in the mechanisms by which gene expression is controlled. Genes can have alternative modes
of expression; control proteins can affect either initiation or termination of
86
W. J. BRAMMAR AND C. HADFIELD
transcription and can act at multiple sites to coordinate the expression of
separate genes, and the same protein can be both an activator and a repressor
of transcription. It is generally the case that regulatory interactions between
competing pathways are reinforced, to prevent possible interference, and that
many regulatory proteins are metabolically unstable, allowing rapid switching
should circumstances change. It will be interesting to see how many of these
themes are rediscovered in examining the mechanisms of determination and
commitment in more complex developmental systems.
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BELFORT, M. & WULFF, D. L. (1974). The roles of the cIII gene and the Escherichia coli
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BROOKS, K. (1965). Studies on the physiological genetics of some suppressor-sensitive mutants
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CRAIG, N. L. & ROBERTS, J. W. (1981). Function of nucleoside triphosphate and polynucleotide in Escherichia coli recA protein-directed cleavage of phage A repressor. /. biol. Chem.
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ENQUIST, L. & SKALKA, A. (1973). Replication of bacteriophage A DNA dependent on the
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FEISS, M. & CAMPBELL, A. (1974). Duplication of the bacteriophage lambda cohesive end site:
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FORBES, D. & HERSKOWTTZ, I. (1982). Polarity suppression by the Q gene product of phage
lambda. /. molec. Biol. 160, 549-569.
FRANKLIN, N. C. (1974). Altered reading of genetic signals fused to the N operon of bacteriophage: Genetic evidence for the modification of polymerase by the protein product of
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GILBERT, W. & DRESSLER, D. (1969). DNA replication: the rolling circle model. Cold Spring
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G. C , MONTANEZ, C , HERNANDEZ, T. & COURT, D. (1982). Posttranscriptional
control of bacteriophage A int gene expression from a site distal to the gene. Proc. natn.
Acad. Sci., U.S.A. 79, 238-242.
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