Download Control of Lysogenization by Phage P22. II. Mutations (clyA) in the c1 Gene that Cause Increased Lysogenization

J. Mol.
Rid.
(1981) 152. 233-245
Control of Lysogenization by Phage P22
II. Mutations (cZyA) in the cl Gene
that Cause Increased Lysogenization
FRED VC’INSTONt AND DAVID
Ikpartmen,t
(Received
BOTSTEIN~
of Biology, Ma,ssarhusetts Institute
Cambridge, Mass. 02139. V.S.A.
10 NovembPr
IYSO, and in revised form
qf Technology
24 April
19X1)
p12 cly mutants
lysogenize
at very high frequency
after infection
of wild-type
Salmonella.
One class of P22 cly mutations,
clyA mutations,
are shown to map
within the P22 cl gene and to increase the activity
of t,he cl gene product during
establishment
of lysogeny.
The P22 clyil mutations
also appear to affect c%repressor gene expression
in cis and to cause a Cro- phenotype,
despite the fart)
that the cZyA mutations
map far downstream (with respect to transcription) of the
end of the l’Z2 cm gene.
1. Introduction
The decision between lysis and lysogeny after infection by the temperate
phages
1’22 and h is subject to influence by several phage and host components.
These
caomponents serve to regulate the level of repressors (the c2 and mnt gene products
of 1’22 and the r1 gene product of /\) that’ are necessary to repress most phage gene
expression, thereby allowing establishment’
of lysogeny.
The immC region of P22 is functionally
and structurally
analogous
to the
immunity
region of coliphage h (Gough & Tokuno, 1975: Winston & Botstein, 1981.
accompanying
paper) and, although repression and immunity
of P22 are mediated
by two immunity
systems, immC and imm/ (Bezdek & Amati, 1968: Botstein et al..
1975: Levine et al., 1975), a P22 strain with imml deleted is able to establish and
maintain
lysogeny normally
(Winston.
1980). Also, h-P22 hybrid phages, which
contain t,he immC region of P22 substituted
for the h immunity
region, are able t,o
&ablish
and maintain
lysogeny
normally
(Gemski et cll., 1972; Botstein
&
Herskowit)z.
1974).
Levine (1957) and Kaiser (1957) demonstrated
for P22 and h, respectively,
that
clear-plaque
mutat’ions in genes other than those coding for the repressor (the cl
and c3 genes of P22 and the cII and cl11 genes of /\) greatly lower the frequency of
establishment
of lysogeny but do not affect the maintenance
of lysogeny.
t Present address: Section of Biochemistry, Molecular
N.Y. 14853. U.S.A.
t To whom reprint
requests
should he addressed.
“33
0022%2836181/300233-13 $02.00/O
and Cell Biology. C!ornell University.
Itham.
0 1981 Academic Press Inc. (London)
Ltd.
P. WINSTON
231
AND
I). HOTRTEIS
Reichardt
& Kaiser (1971) and Echols & Green (1971) proposed that the h ~11
and cl11 gene products are needed to activate transcription
from a promoter, p,,
(defined by cy mutations),
in order to allow high-level
transcription
of the c1 gene
during the early stages of h infection.
Tokuno & Gough (1976) later proposed the
same model for P22. More recent work on /\ (Reichardt.
1975; Jones it (~2.. 1979:
Schmeissner rt nl., 1980) has strongly supported this model. Thus the repressor gene
(r? in P22) can be transcribed
from two promoters : transcription
at, high level from
p,,. dependent upon the P22 cl and c3 gene products, is essential for establishment
(but not, maintenance)
of lysogenp
and transcription
at low level from pRM,
dependent> upon the P22 &-repressor
(=\, Poteettx, personal czommunication)
is
essential for maint,enance
(but not establishment)
of lysogeny.
1’22 cly mutants
were isolated as P22 mutants
that form turbid
playues on
hosts on whicah wild-type
1’22 forms calear plaques (Hong et al..
mutant Salmowlla
1971 : Winston R: Botstrin,
1981) and by selecting for mut,ants that can lpsogenize
under condit,ions
in which wild-type
1’22 lysogenizes
at very low frequency
(LVinston & Botstein,
1981). cly mutants t’hus are opposite in phenotype
to clear
mutants. Hong Pt al. (1971) and Winston dz Botst,ein (1981) also showed that P22 cly
mut’ants do not form plaques and lysogenize at’ abnormally
high frequency
after
infection of wild-type
fJalm~ondla.
We hare isolated a large number of new P22 rly mut)ants and have found that
they fall int’o at least three different classes (Winst,on & Botstein,
1981 : Winston,
19X0). All 1’2% cly mutants,
however. ahart, the phenotypes
of a greatly elevat)ed
frequency of lysogc>ny (the Cly IvsogenizaGon
phenotype)
and a host range for lyt,ic
growth
limited
t)o the set of &permissive
Snlmouella
hosbs (t)hr (‘1~ growth
phenotype)
(Winston & Botstein.
1981).
In this paper we report results concerning what’ we call 1’22 clyA mutants, which
include some of the P22 cly mutants studied by Hong et al. (1971). Tokuno & Gough
(1977) showed that, some of the mutants
of Hong et al. (1971) overproduce
c2repressor and have a reduced ability t,o synthesize phage DN4. Roberts d ~2. (1976)
showed that the clyd mutants
of Hong rf al. code for an altered
“h” RX4
(analogous
t’o the h oop RNA),
and Hilliker.
Brady and Rosenberg
(cited by
Rosenberg 8: Court, 1979) showed t)hese altered RNAs to be t,he result’ of a base
change at the 3’ end. Our results indicabe that the P22 clyd mutations
are within
the struct,ural
gene for the P22 cl protein.
We suggest, that cly14 mutations
increase the level of activity
of the P22 cl function after infection. In addition. our
resultas suggest t,hat these mutations
may simultaneously
be affecting
cg gene
expression in a more direct fashion. not involving
the ~2 gene product.
2. Materials
and Methods
All materials and met,hods are destarihpd in the accompanying
1981).
paper (Winston & Botstein,
3. Results
(a)
Frrquency of lysogrny
by PC2 clyA
Like all P22 cly mutants,
P22 clyA mutants
frequencies after infection of wild-type
Salmonella.
mu,tanh
lysogenize
at greatly
increased
The frequencies of lysogeny of
P22 clyil
MUTATIOXS
TABLE 1
Freguemy
of lysogeny
of
1’22’ and P22 cly mutants
Multiplicity
@1
Phage
p22+
cZyN3
clyH109
in DB7OOO
of infection
10
67
92
97
1.7
95
81
Phage were infected into the &non-permissive
(wild-type)
host DB70
at the indicated
multiplicities. The frequency of lysogeny was determined as described previously (Winston & Botstrin,
1081). The numbers above indicate the percentage of infected cells that became lysogens.
two P22 clyA mutants are shown in Table 1, where it can be seen that the effect is
most pronounced
at a low multiplirit,y
of infection.
(h) Mapping
of c1y.A mutations
by four-factor
crosses
One basis for the distinction
of P22 clyA mut’ations from other P22 cly mutations
is that the cZy+ allele can be rescued from the deletion prophage in strain DB147.
This is not the case for the other cly mutations
(Winston & Bot,stein, 1981). We also
have mapped more precisely two of the cZyA mutations
by four-factor
crosses. The
results of those crosses (Table 2, Fig. 1) demonstrate
that cZyH109 and clyX3 map
within t,he cl gene, right of cl-t&m37 and left of cl-am214.
These flanking cl-am
mutations
show a classic cl phenot’ype (Levine. 1957) and fa#il to complement
all
st’andard cl alleles.
4
--z--l
PRM
(repressor 1 (
I
cro
(c&B)
1
I
I
CY
(p,RE)
Cl
I
I8
clyA
cl-h726 [&3]
0, I PR
I
I
I
I
bRNA
c,:omm
-
FIG:. I. Fine structure map of the P22 cl gene. All mutations were mapped by 4-factor reciprocal
crosses (see text). The genes O-O, cl and 1X are expressed rightward from the p, promoter, as are the
genes I;! and 24 (not shown, see Susskind AZBotstein. 1978). The r2 gene is normally expressed leftward
from the pRE promoter during establishment of lysogeny and from the pa, promoter in an rst,ablished
Iysogen. The map distances are not to scale.
In order to determine
we crossed each of three
all combinations.
The
recombination
between
whether the clyil mutations
are different from each other.
cZyA mutjations (cZyH109, cZyN3 and clyH102) pairwise in
results (not shown) were that there is no detectable
any pair of cZy=l mutants. The high reversion frequency ot
F.
236
( h3ss
WINSTON
+
-YL/-Lrrjl
+
Reciprocal
cross
y--
a
12-nm
h
0
b
I2-am
ANI)
I).
ROTSTEIN
a+
Single
ah
Single
+ b
Single
++
Triple
Single
Triple
Single
Single
+Q
Depending
upon the relative
position
of the inside unselected
reciprocal
cross will require a triple crossover
to generate wild-type
cross second
listed below. the cross is listed first,. the reciprocal
described
in the previous
paper (Winston
C%Botstein.
1981). p.f.u..
tJnselected
a
I)educed
map order
markers
b
1. clyN3
cl-lslOl
cl-t8101
rlyN3
5611740
1u/2093
3.2
0.9
2.
rlyN3
cl-nm214
cl -am214
cl,yN3
O/1422
IO/1324
< oao7
0.08
clyH109
cl -am37
cl-am37
rlyH109
49/1672
1711540
2.9
I.1
4.
rlyHlO9
rf -lslOl
cl-t8101
dy H 109
55/1116
10/948
5.
cl-am59
cl-t.s101
rz-lslOl
cl -am59
38/25 16
o/932
6.
rlyH109
cl-urn26
cl -o/n26
rlyHlO9
61/3150
x/so00
7.
clyHlO9
cl-am214
cl-am214
rZlyH109
O/l686
4911472
-LO96
3.3
X.
cl -om%
rl -am37
cl -nv137
cl -/rm26
98/3240
312834
3.0
0.1
10.
ry27
cl -nrrr37
rz -am37
3/600
ryfi
16/680
0.5
2.4
cl -nrt137
r2 ~tslO1
rf -lslOl
rram37
O/2450
O/2100
< oG4
< 0.05
rl~nm37
cl-am59
cl -am59
cl -am37
l/1746
17/1468
0.1
1.1
3.
Il.
12.
markers,
either the cross or t,he
progeny.
For each pair of crosses
(see above).
Crosses were done as
plaque-forming
units.
P22
clyit
Eli
MI’TATIONS
the clyd mutations
does not allow det’ection of recombination
below one wild-type
progeny
per lo4 total progeny:
nevertheless
one can conclude that the cly.-l
mutations
are. at the least, very close to each other.
(c) Domir~ance
and cis-trans
tests
Dominancae tests were done in two ways: at a high multiplicity
of infection of a
cly-permissive
host, measuring frequency of lysogeny, and at a low multiplicity
of
host. measuring
phage growth.
The low
infection
of a cly-non-permissive
multiplicity
tests (Winston & Botstein,
1981) were done in a way that guarantees
that each cell that yields progeny must have been co-infected by just one of each
parent,.
The results of the high multiplicity
dominance tests (Table 3) show that clyHlo9
is partially
dominant to wild-type
P22 : that is, in the co-infection with elyH109 and
High multiplicity
dominance
tests in IIR7158
Phage
(rif39)
9; I,ysogeny
P22+ +cZyHlO9
p22+
elyH109
9.4t
1.1
19.6
Single parental infections
were done at a multiplicity
of 10 phage per cell and co-infections
at 5 phage
per cell of each parent. When single infections
were done at a multiplicity
equal to half the total of the
co-infections.
the results were the same. The frequency
of lysogeng was measured as described
in the
preceding
paper (Winston
& Botstein,
1981).
t Of 19 prophages
scored, 11 were cly’. 8 were clyH109.
TABLE 4
Lww multiplicity
dominance
5-amH312
R-amH202
cly +
cZyH109
clyHlO9
thy +
C?-OH100
eZyHlO9
&/HI09
UYJHlOO
e1y+
croH 100
cZyH109
Cl?/ +
croHlO0
cZyHlO9
The low multiplicity
low multiplicity
(<
complementing
amber
and the other parent
parent would produce
no more than one of
cly and cro alleles
X-nmH202
alleles.
tests for clyH109
Burst
size
49.3
58.2
0.9
I .5
755
2.4
0.1
dominance
tests are done under conditions
in which each parent was infected at
@I) into the ely-non-permissive
host DB7000
and each parent
carried
a
mutation
in a P22 late gene. In every case. one parent carried a 5-am mutation
carried an a-am mutation.
In this way, only cells that were co-infected
by each
a burst of phage and these co-infected
cells would virtually
always be infected b>
each parent.
are listed by whether
they were on the same genome with the 5-amH312
or the
“38
P. WINSTON
ANI)
L). HOTSTEIN
t’he frequency
of lysogeny
is intermediak
between that, of the c2yH109
infect,ion and the cly+ infection.
The results of t,he low multiplicity
dominance
test’s (Table 4) also show an
intermediate
result the burst size being intcrrnrdiatr
between that’ of the c!l/H109
and t,he rly+ infett’ions. In this low rnultiplicit~y
experiment,
however, the burst, size
frorn the mixed infect~ion is only some IS-fold below the wild-type
burst size. while
it is some 500-fold greater than the mutant’ burst size.
Since clyH109 shows partial dominance in t’he high multiplicity
dominance tests.
\ve next’ did cis+travvs tests in an att’empt to clucidat,e the mechanism of a&on of
clyH109.
The results of cZyH1099-(.I-nm214
cis--tm~s t,ests (Table 5) show that,
c!yHlOS can only exert it)s effect with a cl+ all&~ irt &s. This result. in conjunct~ion
alter t,he c-1 prot~ein bo
with t,hc mapping. supports the idea that the cly.4 mutations
increase
its level of activity
after infect,ion.
result indicating
a need
cis-bans
test (Table H) g‘IWS an unexpeckd
A cZyH log-r2
for a funct~ional r8 gene ill cis, suggesting that r/yH 109 also affects directly r2 ge~w
expression.
dJ+.
The low multiplicity
significantly
increased
dominance
strain
when
clyHZOY-cl
tests showed that the rlyA mutant
burst was
DR7000 was co-infected wit,h a P22 cl.yy=l mutant
cis-trans
tests irr ZIB7ISS
eZyHlOY+cl-ccnr214
elyHlO0 cl-nm214+P22+
(rif39)
5.5
*:I
c!yH 109
P22 +
elyHlO9 cl~nm214
cl -am214
I-i.3
1.7
1.0
6.4 x 10-X
(‘o-infections werr done at multiplicities of’5 phage per cell of each parent. Single infections w-ere done
at a multiplicity
of 10 phage per cell. The frequenr~ of I?-sogeny was measured as described in the
previous paper.
cl,vHIOS-~$2 cis-trans
fusts:
high
multiplicity
ixfwtion
InfecGng phage
Infections
clyHlO9+c2-am08
cZyHlO9 c2-om08+P22+
20.0
3.9
cZyH 109
P22 +
cZyHlO9 c2-aran
c2-am08
45.5
37
1.0 x 10-Z
2.0x lo-*
were done as described in the legend to Table 5
of DB7158
(rif39)
P22 rly.4
“39
ML’TATIOSS
and wild-type
1’22. We were, t,herrfore, able to ask whether the cZyA mut’ants can
complement
the P22 cZyB (cro-) mutants, which have been shown to be recessive
(Winston 8r Botstein, 1981). The results of that experiment
(Table 4) show that the
cly3 and clyB mutants do not complement.
This result suggests that) the rlyd
mutants
are deficient in expression
of the P22 cro gene: even though the cly.4
mutat,ions map well downstream
of the end of the cro gene.
(e) Pro-
phe?/otypes
of clyA
mutants
Sincbr the cl~y.4 mutant
P22 cZyH109 did not complement
P22 cro mutant,s. cly.4
mutant’s were examined for two other Cro- phenotypes.
First. a defective
P22 lysogen carrying
a clyil prophage was constructed
to
drt’ermine
whether it could become an&immune.
The result (Table 7) was that
Iysogens containing
a cZyH109 prophage. like those containing
a cro- prophagr
(Winston & Botstein,
1981), are not able to become anti-immune.
This indicates
t.hat the cZyH109 mutation
is able to inhibit expression of the cro gene under these
conditions.
The fact that a cl - mutation
does not alter this effect of clyHlO9
shows that this C’lyA phenotype
is cl-independent,
unlike the growth-failure
and
o\-er-l?rsogenization
phenotypes
of the same mutation
(see section (g), below).
Second, we checked the kinetics
of c2-repressor
synthesis
and erf prot,ein
synthesis after infection by t,he rlyB mut’ant, cZyH109, to see whether. like WC
mutatjions,
rly4 mutations
cause failure to shut-off synthesis of these proteins.
Those results (Figs 2 and 3) show the following
t’o be true : first, in the clyH109
inft>chtion. c2-repressor is greatly over-produced
relat’ive to the wild-type
repressor
lcvrls: srcaond. the kinetics of &repressor
synthesis in t’he clyH109 infection differ
from those in the wild-type
infection in that &?-repressor synthesis continues at high
rates lat’e in infection:
and t)hird. t~f protein.
another
P22 early function.
is
rxpressrd
at, higher levels in the cZ?yH109 infection:
its rate of synt,hesis eventualI>
p22+
P22 Ap3lpjrI
p22+
P22 Ap31pjrl
P22+
P22 ApYlpj4
1’22 +
P22 Ap3 1p&l
The rftirienry of plating (e.o.p.) is normalized to the titer on strain IIRT621. All strains wew grown at
W5’C and incubated at 35°C after plating.
Since all host 1,vsogens are deleted for imml because of the ApXlpj~l marker, they arc all sensitive to
P22’. Only those which can become anti-immune
are sensitive
t,o P22 Ap3lpfrl.
240
F.
WINSTOS
AND
11. BOTRTEIX
p22+
5
u
10
5
P22clyHl09
20
25
5
10
15
20
25
c2
c2
erf
erf
u
5
10
15
20
25
5
10
15
20
25
PIG:. 2. Rates of protein
synthesis
after infection
by wild-type
P22 and P22 clyH109.
Samples of
infected
cells
were
pulse-labeled
with
[35S]methionine
and
run
on
sodium
dodecyl
sulfate/polyarrylamide
gels as described
in Materials
and Methods.
Lanes are labeled with the time
(min) at which the I-min pulses were begun. The lane labeled u is an uninfected
sample.
being t’urned down, hut not shut off nearly so efficiently
as in t’he wild-type
infection.
These experiments
demonstrate.
therefore,
that the cZyH109 infection
has the characteristic
C’ro- phenotype
of failure to t,urn off expression
of early
phage genes at’ normal times after infect,ion.
Frrqu,mcy
of lysoyeny
by P22 cl-am814
Phage
su -
supI
SUPIT
SUPF
p22+
cl -am214
clyH109
2.3
102
64.0
3.7
<0.2
97.7
6.0
< 0.2
446
1.8
15.0
48.0
in HU+ hosts
Host
supH
1.7
<O.l
23.1
tyrf:
1.8
-CO%
56.7
.411 infections
were done at low multiplicity
(0% to @l) at 37°C. The amber suppressor
strains listed
above insert amino acids at nonsense codons as follows: supl). serine; supE, glutamine;
supF, tyrosine;
and nupH, leucine; the ochre suppressor tyrU inserts tyrosine
at amber or ochre codons (Winston
etal..
1979).
E
? 5 I- Q-repressor
Time
after
infection
-1
(min)
FIG. 3. Rates of synthesis of erj protein and &repressor after infection by wild-type P“ and I’22
cZyH109. Infections and pulse-labelings were performed as described in Materials and Methods. The
curves indicate the relative rates of synthesis of &repressor
and erf protein. Autoradiograms
we’~‘v
traced and the areas were normalized to the amount of trichloroacetic acid-precipitable
radioactivity
in
each gel slot.
(f) A cl-umber
mutan,t that owr-lyxogenizes
a supF host
In the process of isolating c7 mmants as revertants
of cZyN3, cZyH109 (see below)
and rroH 100 (Winston & Botst’ein, 1981), we isolated five rl amber mutants and
crossed them all against wild-type
1’22 to isolate the cZy+ cl -am single mutants. At
a low multiplicity
of infection, one of these mutants, cl -am214, lysogenizes at very
low frequency in RU- or supD, supE and supH hosts. However,
in a supF host, it
lysogenizes at considerably
greater frequency
than does wild-type
P22 (Table 8).
This result clearly demonstrates
that an alteration
in the primary structure of the
cl protein can increase the frequency of lysogeny by P22. It is interesting
to not’e
t’hat cl -am214 maps at the carboxy end of the cl gene, close to the clyA mutations.
As noted above cl-am214
is. by all criteria, phenotypically
a classic cl mutation
when not’ suppressed.
(a) rlyA
p,~pudo-revertan,ts
and analysis
To understand
the clyA mutants
mutants
and constructed
cly&clear
Winston & Botstein,
1981).
of cly-clear
better we isolated
double mutants
double mutants
second-sit’e clear-plaque
by recombination
(see
“12
F. k%‘INSTON
AND
I). BOTSTEIS
(Year-plaque
mutants
are present, at, high frequency
(about 50%) among cly
revertants.
Among 97 independent
clear-plaque
revertants
of clyN3 and cZyH 109
t’ested by complementation
(Winston & Botstein.
1981). 92 were cl mutants, foun
were c2 mutants. and one was a mutant that is simultaneously
cl - and cy - (as was
found for a rev&ant
of P22 cro- ; W’inxtjon & Botstein.
1981). Among the pseudo
revertants,
those with a cl mutation
grew well on a cly-non-permissive
host,
indicating
strong suppression
of the clyA growth
phenotype.
However.
those
I.)seudo-revertarlts
with a c2 mutation
grew poorly on a cly-non-permissive
host,.
The low frequency
of c% pseudo-revertants
and their poor growth suggest that c%
mut,ations are poor suppressors of clyA mutations.
Constjruction
of rZyA -clear double mut*ants generally
confirmed
the pseudo
revertant’ analysis: clyA cl double mut,ants grew well on &non-permissive
hosts.
while cly c2 double mutants grew poorly on the same hosts.
That suppression is definitely
due to a cl mutation
is demonstrated
by the fact
that the clyH109 cl Pn~m26 and c2yH 109 cl ~nmdl4 double mut,ants grow well on a
sl/- host but neither grows well on a strpF host. which suppresses both of t,hose cl
a’rn mutations
well.
The clyH109 &-am08
double mutant grows poorly on a su cZy-non-permissive
host but grows better on a supE c&non-permissive
host. which weakly suppresses
the c%-am mutation.
This is similar
t)o anot,her Cro-phenotype
(the Tro
phenotype:
Eiscn rt 01.. 1975: Ueorgion et al.. 1979).
4. Discussion
The 1’22 cly.-l mutations.
which map wit,hin the P22 cl gene, increase the
frequency
of lysogerly by P22 and cause failure to grow on wild-type
Salmonella
hosts. From the data we present,ed. 1’22 cZyA mutations
appear to have t,wo effects
that result in the Cly phenotype.
First, and foremost. they seem t’o alter the P22 cl
protein to increase the quality
and/or quantity
of its activity.
Second. they also
somehow affect c2 gene expression in cis. These two effects combine to cause the
Cly phenotype,
which for clyA mutants as well as for clyR (CTO-) mutants includes
a (Ire- phenotype.
(a) Evidence
that clgA
mutationjs
alter thr cl protein
Evidence that the clyA mutations
alter t,he ~1 protein comes from several results.
First, the clyA growth defect and high frequency of lysogenization
after infection
of a wild-type
host require a functional
cl gene in cis. The cis effect could mean
either that the rlyA mutations
are affecting expression of the cl gene or t’hat they
are affecting the structure of the cl protein itself. The second possibility
seems more
likely since the rlyA mutations
map within the cl gene.
Second, a known P22 cl -amber mutant, P22 cl -am214 has the Cly phenotype of
high frequency
of lysogeny.
but only after infection
of a supF host (an amber
suppressor
that inserts tyrosine
at, UAC codons: Winston
et al., 1979). This
demonstrates
that
the primary
structure
of the cl protein
is critical
in
establishment
of lysogeny. When a serine or glutamine
is present at the amber site
P22 cly.4
213
MVTATIONS
affected by the cl-am214
mutation,
the frequency
of lysogenization
is virtually
zero: when a tyrosine is present at the same site in t’he cl protein, lysogeny is
established
much more frequently
than in a wild-type
P22 infect’ion. Additionally.
Thus, it seems clear
the cl -am214 mmation maps very close to the clyA mutations.
than an alteration
in the amino acid sequence at the carboxyl end of the cl prot’ein
itself can increase the frequency of lysogeny by P22.
Third, the partial trans-dominance
of the clyA mutants suggests a change in a
diffusible product. The reason for the incompleteness
of the dominance is not clear
P22 cZyH109, is only
from the experiments
done so far. A P22 clyA mutant,
partially
dominant to a P22 cl -amber mutant (data not shown). This suggests that
t’he partial dominance
is not the result of either competition
for a site of action
between mutant and wild-type
cl proteins or of a mixed multimer of mutant and
wild-type
cl proteins, but is somehow intrinsic to mixed infection.
The manner in which the clyA mutations affect the quality and/or quantity
of cl
activity
cannot be determined
from our experiments.
An attractive
analogy can
be drawn with the h can1 mutation
(Jones & Herskowitz,
1978), which has been
shown to increase the stability
of the h cI1 protein (Epp. 1978). However,
the X
ran 1 mutation
is at the amino end of the h ~11 gene, the opposite end from the P22
c1,y.q mutat,ions in the analogous P22 cl gene.
(11)Evidence for a cis effect of clyA rwtations
on c2 expression
Evidence that the clyA mutations
also affect a site which affects c;! expression in
cis comes primarily
from two results.
First, the cly.-l mutation
H109 requires a functional
c2 gene ilr cis to exert, its full
phenotype.
Second, the RNA sequencing of the clyA mutation
cZyN3 (Hilliker
et al., cited by
Rosenberg & Court, 1979) shows that the G to A base change it causes at the 3’ end
of t,he P22 0 RNA is in a potential
“stem-and-loop”
secondary structure such t,hat
the base change disrupts
a G.C base-pair
in the potential
stem. Such stem
structures have been implicated
in transcription
termination
in prokaryotes
(for a
review. ser Rosenberg & Court, 1979) and mutations
disrupting
base-pairing
in
these structures have been shown to decrease transcription
termination
(Stauffer et
01.. 197X: Kosenberg et al., 1978). This suggest’s that the cis effect of the P22 fly.4
mutat,ions might be to affect termination
of the P22 h RNA and to allow it to act. as
a leader for cg mRh’A (Roberts et u,l., 1976; Rosenberg $ Court), 1979).
This t,ermination
model could also explain the phenotype
of P22 rl -am214,
which over-lysogenizes
a supF cby-non-permissive
host but has a weaker (‘1s
phenotype,
as it still plates at normal efficiency on such a host. For P22 cl-amdl4.
perhaps only an altered cl protein is made and t,he mutation
has no cis effect on CY
expression as do the cZy=1 mutations.
A role for the b RNA in a normal wild-type
P22 infection, if any, is not demanded
by this model. Evidence from work wit)h X (Jones et aZ.. 1979) virtually
rules out any
major role for the analogous A oop RNA in normal establishment
of lysogeny. There
is some evidence, however, that mutations
in t,he analogous region at or near the
carboxyl end of the /\ cII gene in A can affect establishment
of lysogeny (Honigman
244
F. WINSTOS
ANI)
U. BOTSTEIPZ
rt al., 1975). Thus we are left with the possibility
that the small leftward
RNA in
both phages can result in altered
lysogenization
rates, but only in mutant,s
(Oppenheim,
1977).
(c)
Helatimship
of
rlyA
7n7Ltafion.s
to
pro yena ux:pressio?i
Another
principal
result regarding
the clyA mutants
is their apparent
Crop
phenotype:
(1) they prevent
t,he establishment
of the anti-immune
stat’e when
present in appropriat,ely
defect,ive prophages;
(2) t,hey fail to complement
cZyR
(cro-) mutants:
(3) they fail to turn off at least some P22 early gene expression:
and (4) they fail to grow in the total absence of &repressor
(the Tro phenotype).
The Cro- phenotypes
of the clyA mutants could be explained
by postulating
that
somehow clyil mutations
inhibit expression of the cro gene during an infection. One
attractive
direct mechanism for the inhibition
of wo gene expression might be t>he
blocking of branscription
of CTOfrom p, due to abnormally
high levels of leftward
transcription
from pRE (or, even, from the unterminated
b RNA) in a P22 &J/I
infection
(Ward k Murray,
1979). Unfortunately,
this mechanism
cannot easily
ac*count, for the cl independence
of t,his phenotype.
We cannot rule out, some posttranscript,ional
event in affecting cis expression such as is postulated
for the effect
of 62 inhibit’ion
of in/ expression in phage h (Guarneros & Galindos. 1979). We also
cannot rule out that cw protein normally
acts at a site t’hat is altered by clyd
mutations,
although there is no evidence for this. The cZ+yA mutations,
t’herefore.
although they map in a different place t,han the rl.yR mutations,
appear t,o mimic so
closely cZyH (cro-) mutations
that we must, consider seriously some direct way in
from a position far begond the carboxyl
which expression of cro can be controlled
end coding sequence for 0-0.
In conclusion, our result,s show that the clyrl mutations
increase establishment
of
lysogeny in two distinct ways : first t,hey alter t,he cl protein to increase the quality
and/or quantity
of its activity
; and second, they affect’ r8 expression in cis. possibly
by allowing t,he P22 h RNA to act, as a leader for c2 mRKA. The cZyA mutations
result in a (“ro- phenotype.
These phenomena point once again to the c*omplexitjy
of the regulatory
interactions
controlling
the decision bet>ween lysis and 1ysogeIlJ
and demand in particular
an explanation
for how a gene can be contjrolled
directly
from
a site
well
beyond
its 3’ end.
\Z’r thank Carl Falro and Doug Koshland
for many helpful discussions.
We also thank
Pamela Oppenheimer
for help in preparing
t,his manuscript.
This work was supported
by
grant,s to D.B. from the National
Institutes
of Health (GM18973 and GM21253). F. W. was
supported
by a training grant to the Department
of Biology from the National
Institutes
of
Hralth
(GM07287).
REFERENCES
Bezdek, M. & Amati, P. (1968). J’iroloyy, 36, 701-703.
Botstein,
D. & Herskowitz,
I. (1974). Natz~r (London), 251, 584-589.
Botstein,
D., Lew, K. K., ,Jarvik, V. & Swanson, (‘. A. ,Jr (1975). J. Mol. Biol. 91, 439-462.
Echols, H. & Green, L. (1971). Proc. Nat. Acad. Sri.. I’.S.il. 68, 2190-2194.
P22 clyA
MITTATIOSS
Eisrn,
L’13
H.. Georgiou, M., Georgopoulos,
(‘. I’., Selzer, S.. Gussin, G. & Herskowitz.
I. (1975).
I’irology,
68, 26G-269.
Epp, C. (1978). Ph.D. dissertation,
University
of Toronto.
(irmski,
I’. ,Jr, Baron, L. S. & Yamamoto.
IV’. (1972). Pror. Sat. Acad. A’ci., 1TS.A. 69.31 IO3114.
Gorgiou.
M., (+eorgopoulos,
C. P. & Eisen, H. (1979). J’irology, 94, 38-54.
Gough, M. 8r Tokuno, S.-I. (1975). Mol. Grn. Grnet. 138, 71-79.
(iuarneros.
U. & Galindos, ,J. M. (1979). Virology, 95, 119-126.
Hong, *J.-S.. Smith, G. R. & Ames, B. N. (1971). Proc. Xat. =Icad. Sri., L~.S.A. 68,2X58-2262.
Honipman,
A., Oppenheim,
A. & Oppenheim , A. B. (1975). Mol. Grrc. Gem-t. 138, 85-I Il.
.Jones. M. 0. & Herskowitz,
I. (1978). l’irology.
88, 199-212.
,Jones, M. O., Fiscsher. R., Herskowitz.
I. & Evhols, H. (1979). Proc. Sat. ilead. Sci.. I ~.A”.;I
76. 150~154.
Kaiser. A. I). (1957). J’irology, 3, 4%-(il.
L(+nta, M. (1957). J’irology, 3, 22-41.
Levine, Jl., Truesdell.
S.. RamakrishlLan.
T. & Bronson, M. .J. (1975). .I. ,Vol. Riol. 91, 421
438.
Oppenheim.
A. (1977). J. Mol. Biol. 111, 83-89.
Reirhardt.
L. (1975). J. Mol. BioZ. 93, 267-288.
Reichardt,
L. 8: Kaiser, A. D. (1971). P rot. AVat. Acad. h-i., T,T.S.A. 68, 2185-2189.
Roberts. .I. W.. Roberts,
C. W.. Hilliker.
S. & Botstein,
D. (1976). In RNA Polymeraxr
(Losick, R. $ Chamberlin,
M., eds), pp. 707-718. Cold Spring Harbor Laboratory,
Cold
Spring Harbor,
New York.
Kosenberg,
M. 8: Court, D. (1979). dnnu. Rev. &net. 13, 319-353.
Rosenberg, 31.. Court, D., Shimatake,
H.. Brady, C. & Wulff, D. L. (1978). Nature (Londo~l),
272. 414- 423.
Schmeissner,
U., Court, D., Shimatake,
H. 8: Rosenberg,
M. (1980). Proc. A’at. Acad. Sci..
l’.S..-l. 77, 3191-3195.
Stauffer. (:. V., Xurawski.
G. & Yanofsky,
C. (1978). Proc. Sat. Acad. Sci., I~.A”.A 75, 4833
4837.
Susskind. M. M. & Botstein,
D. (1978). Microbial.
h’ee. 42, 385-413.
Tokuno.
S. 8i (iough, M. (1976). Mol. Gun. Genet. 144, 19%-204.
Tokuno.
S. Ns.Gough, M. (1977). J. J’irol. 21, 956-964.
Ward, D. F. & Murray,
N. E. (1979). J. Mol. Bill. 133, 249-266.
Winston,
F. (1980). Ph.D. dissertation,
Mass. Institute
of Technology.
Cambridge,
MA.
Winston,
F. & Botstein,
D. (1981). J. Mol. RioZ. 152, 209-232.
\Vinston,
V.. Botstein,
D. 8r Miller. .J. H. (1979). J. Racteriol. 137, 433439.
Edited
by M. Gottecsman