- Email: [email protected]
Identification of a X174 coded protein involved in the shut-off of host dna replication
Vol. 65, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IDENTIFICATION OF A ~X174 C O D E D
PROTEIN I N V O L V E D IN THE
S H U T - O F F OF H O S T D N A REPLICATION Douglas F. Martin and G. Nigel Godson Radiobiology Laboratories, Yale University Medical School N e w Haven, Connecticut 06510 R e c e i v e d May 22,1975 S U M M A R Y : W h e n Escherichia colt is infected with c>X174, the host cell D N A replication ceases i0 - 12 minutes after infection. This shut-off is prevented by the addition of 30~g/ml chloramphenicol indicating a requirement for post-infection protein synthesis. Some amber mutants in ,6X174 cistron A, which codes for two proteins, do not shut off the host D N A replication. Amber mutants in all other #X174 genes shut off host replication. Mutants near the amino-terminal end of cistron A, which still produce the small 35,000 molecular weight cistron A polypeptide, shut off the host DI~A synthesis whereas mutants near the carboxy-termihal end, which do not produce the small A polypeptide do not shut off the host.
INTRODUCTION:
Infection of Escherichia colt by ~X174 results in the shut-off
of host cell D N A replication i0 - 15 minutes after infection ( i, 2 ). Synthesis of host protein and R N A is not affected by ~X174 infection ( i, 3 ). The shut-off of host D N A replication is inhibited by 30~g/ml C A M I ( i, 2 ) indicating a requirement for at least one phage induced protein. This communication reports the finding that some amber mutants in cistron A do not shut off host cell D N A synthesis whereas amber mutants in each of the other eight known Cistrons ( B - H and I ) shut off the host.
Cistron A of ~X174 contains an internal initiator and codes for
two polypeptides whose molecular weights are 60,000 and 35,000 daltons (4). Amber mutants near the amino-terminal end of the gene still synthesize a 35,000 molecular weight polypeptide and mutants near the carboxy-terminal end of the gene synthesize neither of these polypeptides.
The function of the small A
protein is not known, however, this report shows that the lack of the small A protein correlates with the lack of shut-off of host cell D N A synthesis indicating that this protein is involved in the shut-off. 1
CAM = Chloramphenicol
Copyr[ghf © 19 75 b), Academic Press, h~c. All rights o f reproduction in any/brm reserved.
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MATERIALS A N D M E T H O D S : _Bacteria and Bacteriophage: Escherichia coli C strains HF4701 ( u vrA ) ( 6 ) and HF4704 ( thy , tlr derivative of HF4701 ) ( 1 ) were obtained from Dr. P. Howard-Flanders. Phage ~X174 and mutants a m 86, a___m50, a__m 8, a m 8A, a m 18 (all in cistronA ), a m 14 (cistron B), och 12 a m 3 (double mutant; och in cistron C, a m in cistron E ) a___mi0 (cistron D ), a m 3 (cistron E ), a m 6 (cistron ~), a m $5-I0 (cistron F), a m 9 (cistron G ) and a m 23 (cistron H ) were obtained from Dr. Robert L. Sinsheimer. A m 8 is also temperature sensitive in cistron F ( F. Funk, personal communication ). A m 8A and a m 8tr are temperature resistant revertants of a m 8. ~X174 mutants NI4 and Hg0 ( both in cistron A ) were obtained from Dr. Masaki Hayashi. Experimental Procedure: E. coli HF4704 w a s grown in T P G m e d i u m ( 1 ) su~0plemented with 2~g/ml thymidine an~-l~o ~asein hydrolysate, to a density of 1.3 x I0 u Cells/ml and infected at a multiplicity of 3. At the time of infection4 and at 5 minute intervals thereafter, 1.5 ml of cells were pulsed for 2 minutes with [~H] thymidine ( i0 ~ci/ml ). The pulse w a s terminated by pouring the cells over i0 ml crushed ice containing 0.i ml of I M Na azide. The cells were pelleted by centrifugation at 5,000 rpm for 5 minutes in a Sorval SS34 rotor. The cells were resuspended in 1.0 ml of buffer containing 0.10 M Tris.HCl, p H 8.0, 0.01 M E D T A and 1 m g / m l of lysozyme ( Sigma, eggwhite, grade 1 ), incubated at 0 ° C for 5 minutes, and lysed with 50 ~I of 10% Sarkosyl. The D N A was extracted with phenol and precipitated with 2 1/2 volumes of ethanol at -20Oc overnight. The precipitate w a s collected by centrifugation at 9,000 rpm for i0 minutes in a Sorval SS34 rotor and resuspended in 0.2 ml of 0.1 N N a O H . The absorbance at 260 m~ was measured and the samples were layered on a 5 ml 5 - 2 0 % alkaline sucrose density gradient containing 0.01 M Tris .HCI, p H 8.0, 0.001 M EDTA, 0.15 M N a C l a n d 0.3 M N a O H . The gradients were centrifuged for 2 i/2 hours at 45,000 rpm in a B e c k m a n S W 5 0 . 1 rotor at 5°C. Fractions were collected from the bottom of the gradient and counted in a toulene based scintilation fluid. I n t r a c e l l u l a r p h a g e - c o d e d p r o t e i n s were l a b e l e d a n d a n a l y z e d on a 7 1/2% p o l y a c r y l a m i d e g e l s l a b a s p r e v i o u s l y d e s c r i b e d ( 7 ) e x c e p t t h a t t h e [14C] a m i n o a c i d m i x t u r e w a s a d d e d a t 5 a n d 20 m i n u t e s a f t e r i n f e c t i o n . Luria broth w a s a d d e d a t 30 m i n u t e s a n d t h e c e l l s were h a r v e s t e d 4 m i n u t e s l a t e r . RESULTS: E. c o l i HF4704 w a s i n f e c t e d w i t h ~X174 a n d s a m p l e s w e r e p u l s e d for 2 m i n u t e s w i t h [3HI t h y m i d i n e at 0, 5, 10, 15 a n d 20 m i n u t e s a f t e r i n f e c t i o n . DNA w a s e x t r a c t e d a n d s e d i m e n t e d i n a l k a l i n e s u c r o s e . of t h e 5 ( A ) ,
l0 ( B ) ,
a n d 15 ( C )
minute p u l s e s .
The
Figure I s h o w s t h e g r a d i e n t s
This d e m o n s t r a t e s
the s h u t off of h o s t c e l l DNA r e p l i c a t i o n . In a s i m i l a r e x p e r i m e n t , a c u l t u r e w a s d i v i d e d i n t o t h r e e p o r t i o n s a n d the s u b c u l t u r e s were t r e a t e d a s f o l l o w s :
l)
i n f e c t e d w i t h am 3, 2)
infected with
am 3 i n t h e p r e s e n c e of 3 0 ~ g / m l CAM, 3) t r e a t e d w i t h 30 ~ g / m l CAM. w e r e p u l s e d a n d t h e DNA e x t r a c t e d a n d s e d i m e n t e d a s b e f o r e .
The c e l l s
The t o t a l c o u n t s i n
e a c h of t h e h o s t c e l l DNA p e a k s i n t h e s e g r a d i e n t s were c a l c u l a t e d a n d n o r m a l i z e d b y the a b s o r b e n c e a t 260 m~ of t h e s a m p l e s .
T h e s e d a t a were p l o t t e d a s p e r c e n t
of the n o r m a l i z e d c o u n t s i n c o r p o r a t e d i n t o h o s t DNA i n the 0 - 2 m i n u t e p u l s e .
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I00
IA. ' i /~
o
I
75[
HosT
'
I
ss
o c.
Fraction
number
Figure I: Alkaline sucrose density gradients of pulsed labeled, infected cells. E. colt H F 4 7 0 4 w a s infected with ~X174 and portions were pulsed with [311] thymidine. The 13NA w a s phenol extracted and sedimented in an alkaline sucrose density gradient as described. ( A ) 5 - 7 minute pulse ; ( B ) i0 - 12 minute pulse; ( C ) 15 - 17 minute pulse.
The results ( figure 2 ) s h o w that the shut-off of host D N A replication is prevented by G A M ,
indicating that post infection protein synthesis is required for this shut-
off activity. Figure 3 shows the results obtained w h e n cells were infected with amber mutants in 7 of the 9 ~X174 cistrons.
The results of the experiment with wild type
phage ( figure 1 ) are included for comparison. in figure 2.
The cistron E results are s h o w n
The shut-off activity is present in each of these mutants.
Figure 4
shows the results obtained by infection with a series of mutants in cistron A. This demonstrates that some, but not all mutants in cistron A lack the shut-off activity.
The results are not s h o w n for a m 8tr and NI4 which did not shut off the
host D N A replication and a m 50 which did.
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BIOCHEMICAND AL BIOPHYSICAL RESEARCH COMMUNICATIONS i
i
i
I
I II
16
i
m
"5O , Os~o75 ~ c
E
oJ I
0 o
1_ 0
I
I 6
A
Minutes afterinfection
21
Figure 2 : Sensitivity of shut off activity to chloramphenicol. A culture of E. coli H F 4 7 0 4 w a s divided into three parts. The first w a s infected with ~X a m 3 ( cistron E ). The second w a s infected with ~X a m 3 and treated with 30~g/ml CAM. The third w a s treated with 30~g/ml C A M . Each portion w a s pulsed with [3HI thymidine and the D N A w a s extracted and sedimented as before. The total number of counts incorporated into host cell D N A w a s calculated from the gradients and normalized against the absorbance at 2 6 0 m ~ of the sample. These data were plotted as percent of the counts incorporated into the 0 - 2 minute pulse. O - O, uninfected plus C A M ; Q - Q, infected without C A M ; /k .... ~ , infected plus C A M .
A culture of E. coli HF4701 w a s U.V. irradiated and portions were infected with the various cistron A mutants.
The intracellular ~X174 coded proteins were
labeled with [14C] amino acids and electrophoresed on a 7 i/2% polyacrylamide gel. An autoradiograph of the gel is s h o w n in figure 5.
The gel shows that none of
these mutants synthesize the large protein coded for by cistron A, and two mutants, H g 0 and a___m18, also fail to make the small A protein.
A m 8 has been m a p p e d in the
region of the genome that codes for both A proteins ( 8 ) however, a m 8tr and a m 8A, temperature resistant revertants of a m 8,both synthesize the small A protein. Both mutants which lack the small A protein, Hg0 and a m 18, fail to shut-off host replication, shut-off.
However,
indicating that this protein plays a role in the
a m 8tr and NI4 synthesize the small A protein and fail
to shut-off the host D N A replication,
suggesting that the relationship between
the small A protein and the shut-off activity is not direct. DISCUSSION:
Infection of E. coli with ~X174 results in the shut--off of host
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I00
75
= =~ .E
50
25
E 0 0
=::
o.
jf
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50
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\\
\~'\ \\
A ,
6 II 16 21 Minu#es o f t e r infec#ion
Figure 3: Host cell shut ~ff by mutants of ~X174. Cultures of E. coli HF4704 were infected with amber mutants of c~X174 and pulsed with [3HI thymidine. The D N A w a s extracted and sedimented as described and the data w a s calculated and plotted as inthe legend to figure 2. ( A ) O - - O , wildtype ; A - - - A , aml4 (cistronB); [3 ..... [:], och 12, a m 3 (cistrons C and E); • - - • , a m l 0 (cistron D ) . ( B ) ©--O, a__mmSS_I0 (cistron F ); L~ .... ~ , a__m 9 (cistron G)[3 .... [[], a m 23 (cistron H ) ; • •, a m 6 (cistron ~).
cell D N A replication at about i0 - 12 minutes after infection, as has been observed by others ( i, 2, 9 ). The results of Lindqvist and Sinsheimer ( 1 ) and Stone ( 2 ) that this shut-off is prevented by C A M
have been confirmed.
These results are in conflict with the observations of Ishiwa and T e s s m a n ( 9 ) w h o found that the shut-off w a s not prevented by C A M .
As Stone ( 3 ) has
suggested, this discrepency is probably due to Ishiwa and T e s s m a n working at high multiplicities of infection ( i0 p.f.u./ml ). Stone has observed a nonspecific inhibition of all macromolecular synthesis at high multiplicities of infection ( 3 ). This nonspecific inhibition is observed in the presence of C A M . Mutants in each of the 9 k n o w n cistrons of c~X174 have been tested for the ability to shut-off host cell D N A replication.
The only mutants which did
not s h o w the shut-off activity were located in cistron A.
327
This cistron codes for
Vol. 65, No. 1, 1975
BIOCHEMICAl AND BIOPHYSICAl. RESEARCH COMMUNICATIONS
150
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== I00 E 0
5O
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,
, %~'~,~
6
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, 21
Minutes offer infection
26
Figure 4: Host cell shut off by mutants in cistron A. Experiments were performed as described in legend to figure 3. O - ©, H90;/k ..... /k, a m 8A; • a m 8 6 ; • .... • a m 18.
two polypeptides of molecular weights 60 - 65,000 and 35,000 ( 4 ). Both mutants which do not synthesize the small A protein failed to shut-off the host D N A replication,
indicating that this protein has a role in the shut-off.
However,
two other cistron A mutants, NI4 and a m 8tr, which do synthesize a 35,000 molecular weight polypeptide also failed to shut-off the host replication. the relationship is not clear.
However,
Thus,
there is no evidence that the 35,000 molecular
weight polypeptides synthesized by these mutants are active small A proteins. The status of the a m 8 mutation is confused.
This double mutant carries
a temperature sensitive mutation in cistron F as well as the amber in cistron A. T w o independently isolated temperature resistant revertants of a__m 8 ( a m 8A and a m 8tr ) have been used in this study.
Although a m 8 has been m a p p e d in
the region that codes for both A proteins ( 8 ), both of the temperature resistant strains synthesize a 35,000 molecular weight polypeptide.
Also, one of these
revertants, a m 8A, shuts off the host, but the other does not.
Stone also
observed shut-off of host cell replication with a m 8 ( 2 ). Undoubtly,
he used
a temperature resistant revertant also, since the phage w a s grown at 37°C. The m e c h a n i s m by which ~X174 shuts off the host D N A replication is u n k n o w n and very little is k n o w n about the properties of the small A protein.
328
It
Vol. 65, No. 1, 1975
x
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~O
0
~
~
"~"
z
c~ x
~ ~
~ ~
A
F
N A,
Figure 5 : 7 I/2% polyacrylamide gel of polypeptides synthesized in UV-irradiated E. coli HF4701 infected with ~X174 and cistron A mutants. Preparation and electrophoresis of samples was as described in methods.
has been shown to bind strongly to D N A cellulose columns ( 5 ). ~X174 virus particles contain one
copy of a 30,000 molecular weight polypeptide ( 6 ) which
coelectrophoreses with the small A protein ( 4 ). W h e n vaccinia virus infects L cells, a single-stranded D N A specific endonuclease is released from the virus core in the nucleus concemitant with the inhibition of host cell D N A synthesis ( I0 ). It is possible that the small A protein interacts with the host D N A at the single-stranded regions of the replication forks, perhaps degrading the single-strands.
This would explain the reduction in size of E. coli D N A
observed in ~X174 infected cells ( Martin and Godson, unpublished results ). The large cistron A protein is known to have endonucleolytic activity ( II ), but whether this activity resides in the portion of the molecule that is c o m m o n to
329
Vol. 65, No. 1, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
b o t h large and s m a l l p r o t e i n s is not k n o w n .
A l t e r n a t i v e l y , the s h u t - o f f a c t i v i t y
may be due to i n t e r a c t i o n s with a n o t h e r p r o t e i n i n v o l v e d in h o s t DNA r e p l i c a t i o n . It is i n t e r e s t i n g to note t h a t the r e p l i c a t i o n of d o u b l e s t r a n d e d ~X174 r e p l i c a t i v e form DNA c e a s e s at the s a m e time t h a t h o s t r e p l i c a t i o n is shu~-off ( 12 ).
The s h u t - o f f of v i r a l d o u b l e s t r a n d e d DNA r e p l i c a t i o n , w h i c h r e s e m b l e s
E. c o l i DNA r e p l i c a t i o n , at l e a s t s u p e r f i c i a l l y ,
is a l s o p r e v e n t e d b y CAM ( 13, 14 ).
It is quite p o s s i b l e t h a t the s h u t - o f f of h o s t c e l l DNA r e p l i c a t i o n is a s i d e p r o d u c t of a m e c h a n i s m d e s i g n e d to s h u t - o f f v i r a l d o u b l e - s t r a n d e d DNA r e p l i c a t i o n .
ACKNOWLEDGEMENTS: technical assistance.
W e wish to thank Ms. Pamela Svec for excellent This work was supported by the Unites States Public
Health Service Grant AI-I1633-01.
REFERENCES:
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. tl. 12. 13. 14.
L i n d q v i s t , B. a n d R. L. S i n s h e i m e r ( 1967 ) J. M o l . Biol. 29_8: 8 7 - 9 4 . S t o n e , A. B. ( 1970 ) Viroloqy 4_2_2:171-181. Stone, A. B. ( 1 9 7 0 ) J, M o l . Biol. 4__Z.7:2 1 5 - 2 2 9 . L i n n e y , E. a n d M. H a y a s h i ( 1 9 7 4 ) Nature 249" 3 4 5 - 3 4 8 . L i n n e y , E . , M. N. H a y a s h i , and M. H a y a s h i ( 1 9 7 2 ) Virology 50" 3 8 1 - 3 8 7 . G o d s o n , G. N. ( 1 9 7 1 ) ~. M o l . Biol. 57: 5 4 1 - 5 5 3 . G o d s o n , G. N. ( 1 9 7 4 ) Virology 58: 2 7 2 - 2 8 9 . Benbow, R. M . , C . A. H u t c h i s o n III, I . D. F a b r i c a n t , and R. L. S i n s h e i m e r ( 1971 ) ~. Virol. 7: 5 4 9 - 5 5 8 . I s h i w a , H. a n d I. T e s s m a n ( 1 9 7 3 ) J. M o l . Biol. 37: 4 6 7 - 4 7 4 . Pogo, B. G. T . , a n d S. D a l e s ( 1974 ) Viroloqy 5_.88"3 7 7 - 3 8 6 . H e n e r y , T. J. and R. Knippers ( 1 9 7 4 ) Proc. N a t . A c a d . S c i . U. S. A. 71: 1549-1553. I w a y a , M. a n d D. T. D e n h a r d t ( 1971 ) ~. M o l . Biol. 5._/.7:159-175. T e s s m a n , E. S. ( 1 9 6 6 ) J. M o l . Biol. 17: 2 1 8 - 2 3 6 . H u t c h i n s o n , C . A . , III, a n d R. L. S i n s h e i m e r ( 1 9 6 6 ) ] . M o l . Biol. 18: 4 2 9 - 4 4 7 .
330
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IDENTIFICATION OF A ~X174 C O D E D
PROTEIN I N V O L V E D IN THE
S H U T - O F F OF H O S T D N A REPLICATION Douglas F. Martin and G. Nigel Godson Radiobiology Laboratories, Yale University Medical School N e w Haven, Connecticut 06510 R e c e i v e d May 22,1975 S U M M A R Y : W h e n Escherichia colt is infected with c>X174, the host cell D N A replication ceases i0 - 12 minutes after infection. This shut-off is prevented by the addition of 30~g/ml chloramphenicol indicating a requirement for post-infection protein synthesis. Some amber mutants in ,6X174 cistron A, which codes for two proteins, do not shut off the host D N A replication. Amber mutants in all other #X174 genes shut off host replication. Mutants near the amino-terminal end of cistron A, which still produce the small 35,000 molecular weight cistron A polypeptide, shut off the host DI~A synthesis whereas mutants near the carboxy-termihal end, which do not produce the small A polypeptide do not shut off the host.
INTRODUCTION:
Infection of Escherichia colt by ~X174 results in the shut-off
of host cell D N A replication i0 - 15 minutes after infection ( i, 2 ). Synthesis of host protein and R N A is not affected by ~X174 infection ( i, 3 ). The shut-off of host D N A replication is inhibited by 30~g/ml C A M I ( i, 2 ) indicating a requirement for at least one phage induced protein. This communication reports the finding that some amber mutants in cistron A do not shut off host cell D N A synthesis whereas amber mutants in each of the other eight known Cistrons ( B - H and I ) shut off the host.
Cistron A of ~X174 contains an internal initiator and codes for
two polypeptides whose molecular weights are 60,000 and 35,000 daltons (4). Amber mutants near the amino-terminal end of the gene still synthesize a 35,000 molecular weight polypeptide and mutants near the carboxy-terminal end of the gene synthesize neither of these polypeptides.
The function of the small A
protein is not known, however, this report shows that the lack of the small A protein correlates with the lack of shut-off of host cell D N A synthesis indicating that this protein is involved in the shut-off. 1
CAM = Chloramphenicol
Copyr[ghf © 19 75 b), Academic Press, h~c. All rights o f reproduction in any/brm reserved.
323
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MATERIALS A N D M E T H O D S : _Bacteria and Bacteriophage: Escherichia coli C strains HF4701 ( u vrA ) ( 6 ) and HF4704 ( thy , tlr derivative of HF4701 ) ( 1 ) were obtained from Dr. P. Howard-Flanders. Phage ~X174 and mutants a m 86, a___m50, a__m 8, a m 8A, a m 18 (all in cistronA ), a m 14 (cistron B), och 12 a m 3 (double mutant; och in cistron C, a m in cistron E ) a___mi0 (cistron D ), a m 3 (cistron E ), a m 6 (cistron ~), a m $5-I0 (cistron F), a m 9 (cistron G ) and a m 23 (cistron H ) were obtained from Dr. Robert L. Sinsheimer. A m 8 is also temperature sensitive in cistron F ( F. Funk, personal communication ). A m 8A and a m 8tr are temperature resistant revertants of a m 8. ~X174 mutants NI4 and Hg0 ( both in cistron A ) were obtained from Dr. Masaki Hayashi. Experimental Procedure: E. coli HF4704 w a s grown in T P G m e d i u m ( 1 ) su~0plemented with 2~g/ml thymidine an~-l~o ~asein hydrolysate, to a density of 1.3 x I0 u Cells/ml and infected at a multiplicity of 3. At the time of infection4 and at 5 minute intervals thereafter, 1.5 ml of cells were pulsed for 2 minutes with [~H] thymidine ( i0 ~ci/ml ). The pulse w a s terminated by pouring the cells over i0 ml crushed ice containing 0.i ml of I M Na azide. The cells were pelleted by centrifugation at 5,000 rpm for 5 minutes in a Sorval SS34 rotor. The cells were resuspended in 1.0 ml of buffer containing 0.10 M Tris.HCl, p H 8.0, 0.01 M E D T A and 1 m g / m l of lysozyme ( Sigma, eggwhite, grade 1 ), incubated at 0 ° C for 5 minutes, and lysed with 50 ~I of 10% Sarkosyl. The D N A was extracted with phenol and precipitated with 2 1/2 volumes of ethanol at -20Oc overnight. The precipitate w a s collected by centrifugation at 9,000 rpm for i0 minutes in a Sorval SS34 rotor and resuspended in 0.2 ml of 0.1 N N a O H . The absorbance at 260 m~ was measured and the samples were layered on a 5 ml 5 - 2 0 % alkaline sucrose density gradient containing 0.01 M Tris .HCI, p H 8.0, 0.001 M EDTA, 0.15 M N a C l a n d 0.3 M N a O H . The gradients were centrifuged for 2 i/2 hours at 45,000 rpm in a B e c k m a n S W 5 0 . 1 rotor at 5°C. Fractions were collected from the bottom of the gradient and counted in a toulene based scintilation fluid. I n t r a c e l l u l a r p h a g e - c o d e d p r o t e i n s were l a b e l e d a n d a n a l y z e d on a 7 1/2% p o l y a c r y l a m i d e g e l s l a b a s p r e v i o u s l y d e s c r i b e d ( 7 ) e x c e p t t h a t t h e [14C] a m i n o a c i d m i x t u r e w a s a d d e d a t 5 a n d 20 m i n u t e s a f t e r i n f e c t i o n . Luria broth w a s a d d e d a t 30 m i n u t e s a n d t h e c e l l s were h a r v e s t e d 4 m i n u t e s l a t e r . RESULTS: E. c o l i HF4704 w a s i n f e c t e d w i t h ~X174 a n d s a m p l e s w e r e p u l s e d for 2 m i n u t e s w i t h [3HI t h y m i d i n e at 0, 5, 10, 15 a n d 20 m i n u t e s a f t e r i n f e c t i o n . DNA w a s e x t r a c t e d a n d s e d i m e n t e d i n a l k a l i n e s u c r o s e . of t h e 5 ( A ) ,
l0 ( B ) ,
a n d 15 ( C )
minute p u l s e s .
The
Figure I s h o w s t h e g r a d i e n t s
This d e m o n s t r a t e s
the s h u t off of h o s t c e l l DNA r e p l i c a t i o n . In a s i m i l a r e x p e r i m e n t , a c u l t u r e w a s d i v i d e d i n t o t h r e e p o r t i o n s a n d the s u b c u l t u r e s were t r e a t e d a s f o l l o w s :
l)
i n f e c t e d w i t h am 3, 2)
infected with
am 3 i n t h e p r e s e n c e of 3 0 ~ g / m l CAM, 3) t r e a t e d w i t h 30 ~ g / m l CAM. w e r e p u l s e d a n d t h e DNA e x t r a c t e d a n d s e d i m e n t e d a s b e f o r e .
The c e l l s
The t o t a l c o u n t s i n
e a c h of t h e h o s t c e l l DNA p e a k s i n t h e s e g r a d i e n t s were c a l c u l a t e d a n d n o r m a l i z e d b y the a b s o r b e n c e a t 260 m~ of t h e s a m p l e s .
T h e s e d a t a were p l o t t e d a s p e r c e n t
of the n o r m a l i z e d c o u n t s i n c o r p o r a t e d i n t o h o s t DNA i n the 0 - 2 m i n u t e p u l s e .
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I00
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Figure I: Alkaline sucrose density gradients of pulsed labeled, infected cells. E. colt H F 4 7 0 4 w a s infected with ~X174 and portions were pulsed with [311] thymidine. The 13NA w a s phenol extracted and sedimented in an alkaline sucrose density gradient as described. ( A ) 5 - 7 minute pulse ; ( B ) i0 - 12 minute pulse; ( C ) 15 - 17 minute pulse.
The results ( figure 2 ) s h o w that the shut-off of host D N A replication is prevented by G A M ,
indicating that post infection protein synthesis is required for this shut-
off activity. Figure 3 shows the results obtained w h e n cells were infected with amber mutants in 7 of the 9 ~X174 cistrons.
The results of the experiment with wild type
phage ( figure 1 ) are included for comparison. in figure 2.
The cistron E results are s h o w n
The shut-off activity is present in each of these mutants.
Figure 4
shows the results obtained by infection with a series of mutants in cistron A. This demonstrates that some, but not all mutants in cistron A lack the shut-off activity.
The results are not s h o w n for a m 8tr and NI4 which did not shut off the
host D N A replication and a m 50 which did.
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i
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Figure 2 : Sensitivity of shut off activity to chloramphenicol. A culture of E. coli H F 4 7 0 4 w a s divided into three parts. The first w a s infected with ~X a m 3 ( cistron E ). The second w a s infected with ~X a m 3 and treated with 30~g/ml CAM. The third w a s treated with 30~g/ml C A M . Each portion w a s pulsed with [3HI thymidine and the D N A w a s extracted and sedimented as before. The total number of counts incorporated into host cell D N A w a s calculated from the gradients and normalized against the absorbance at 2 6 0 m ~ of the sample. These data were plotted as percent of the counts incorporated into the 0 - 2 minute pulse. O - O, uninfected plus C A M ; Q - Q, infected without C A M ; /k .... ~ , infected plus C A M .
A culture of E. coli HF4701 w a s U.V. irradiated and portions were infected with the various cistron A mutants.
The intracellular ~X174 coded proteins were
labeled with [14C] amino acids and electrophoresed on a 7 i/2% polyacrylamide gel. An autoradiograph of the gel is s h o w n in figure 5.
The gel shows that none of
these mutants synthesize the large protein coded for by cistron A, and two mutants, H g 0 and a___m18, also fail to make the small A protein.
A m 8 has been m a p p e d in the
region of the genome that codes for both A proteins ( 8 ) however, a m 8tr and a m 8A, temperature resistant revertants of a m 8,both synthesize the small A protein. Both mutants which lack the small A protein, Hg0 and a m 18, fail to shut-off host replication, shut-off.
However,
indicating that this protein plays a role in the
a m 8tr and NI4 synthesize the small A protein and fail
to shut-off the host D N A replication,
suggesting that the relationship between
the small A protein and the shut-off activity is not direct. DISCUSSION:
Infection of E. coli with ~X174 results in the shut--off of host
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Figure 3: Host cell shut ~ff by mutants of ~X174. Cultures of E. coli HF4704 were infected with amber mutants of c~X174 and pulsed with [3HI thymidine. The D N A w a s extracted and sedimented as described and the data w a s calculated and plotted as inthe legend to figure 2. ( A ) O - - O , wildtype ; A - - - A , aml4 (cistronB); [3 ..... [:], och 12, a m 3 (cistrons C and E); • - - • , a m l 0 (cistron D ) . ( B ) ©--O, a__mmSS_I0 (cistron F ); L~ .... ~ , a__m 9 (cistron G)[3 .... [[], a m 23 (cistron H ) ; • •, a m 6 (cistron ~).
cell D N A replication at about i0 - 12 minutes after infection, as has been observed by others ( i, 2, 9 ). The results of Lindqvist and Sinsheimer ( 1 ) and Stone ( 2 ) that this shut-off is prevented by C A M
have been confirmed.
These results are in conflict with the observations of Ishiwa and T e s s m a n ( 9 ) w h o found that the shut-off w a s not prevented by C A M .
As Stone ( 3 ) has
suggested, this discrepency is probably due to Ishiwa and T e s s m a n working at high multiplicities of infection ( i0 p.f.u./ml ). Stone has observed a nonspecific inhibition of all macromolecular synthesis at high multiplicities of infection ( 3 ). This nonspecific inhibition is observed in the presence of C A M . Mutants in each of the 9 k n o w n cistrons of c~X174 have been tested for the ability to shut-off host cell D N A replication.
The only mutants which did
not s h o w the shut-off activity were located in cistron A.
327
This cistron codes for
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Figure 4: Host cell shut off by mutants in cistron A. Experiments were performed as described in legend to figure 3. O - ©, H90;/k ..... /k, a m 8A; • a m 8 6 ; • .... • a m 18.
two polypeptides of molecular weights 60 - 65,000 and 35,000 ( 4 ). Both mutants which do not synthesize the small A protein failed to shut-off the host D N A replication,
indicating that this protein has a role in the shut-off.
However,
two other cistron A mutants, NI4 and a m 8tr, which do synthesize a 35,000 molecular weight polypeptide also failed to shut-off the host replication. the relationship is not clear.
However,
Thus,
there is no evidence that the 35,000 molecular
weight polypeptides synthesized by these mutants are active small A proteins. The status of the a m 8 mutation is confused.
This double mutant carries
a temperature sensitive mutation in cistron F as well as the amber in cistron A. T w o independently isolated temperature resistant revertants of a__m 8 ( a m 8A and a m 8tr ) have been used in this study.
Although a m 8 has been m a p p e d in
the region that codes for both A proteins ( 8 ), both of the temperature resistant strains synthesize a 35,000 molecular weight polypeptide.
Also, one of these
revertants, a m 8A, shuts off the host, but the other does not.
Stone also
observed shut-off of host cell replication with a m 8 ( 2 ). Undoubtly,
he used
a temperature resistant revertant also, since the phage w a s grown at 37°C. The m e c h a n i s m by which ~X174 shuts off the host D N A replication is u n k n o w n and very little is k n o w n about the properties of the small A protein.
328
It
Vol. 65, No. 1, 1975
x
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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Figure 5 : 7 I/2% polyacrylamide gel of polypeptides synthesized in UV-irradiated E. coli HF4701 infected with ~X174 and cistron A mutants. Preparation and electrophoresis of samples was as described in methods.
has been shown to bind strongly to D N A cellulose columns ( 5 ). ~X174 virus particles contain one
copy of a 30,000 molecular weight polypeptide ( 6 ) which
coelectrophoreses with the small A protein ( 4 ). W h e n vaccinia virus infects L cells, a single-stranded D N A specific endonuclease is released from the virus core in the nucleus concemitant with the inhibition of host cell D N A synthesis ( I0 ). It is possible that the small A protein interacts with the host D N A at the single-stranded regions of the replication forks, perhaps degrading the single-strands.
This would explain the reduction in size of E. coli D N A
observed in ~X174 infected cells ( Martin and Godson, unpublished results ). The large cistron A protein is known to have endonucleolytic activity ( II ), but whether this activity resides in the portion of the molecule that is c o m m o n to
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b o t h large and s m a l l p r o t e i n s is not k n o w n .
A l t e r n a t i v e l y , the s h u t - o f f a c t i v i t y
may be due to i n t e r a c t i o n s with a n o t h e r p r o t e i n i n v o l v e d in h o s t DNA r e p l i c a t i o n . It is i n t e r e s t i n g to note t h a t the r e p l i c a t i o n of d o u b l e s t r a n d e d ~X174 r e p l i c a t i v e form DNA c e a s e s at the s a m e time t h a t h o s t r e p l i c a t i o n is shu~-off ( 12 ).
The s h u t - o f f of v i r a l d o u b l e s t r a n d e d DNA r e p l i c a t i o n , w h i c h r e s e m b l e s
E. c o l i DNA r e p l i c a t i o n , at l e a s t s u p e r f i c i a l l y ,
is a l s o p r e v e n t e d b y CAM ( 13, 14 ).
It is quite p o s s i b l e t h a t the s h u t - o f f of h o s t c e l l DNA r e p l i c a t i o n is a s i d e p r o d u c t of a m e c h a n i s m d e s i g n e d to s h u t - o f f v i r a l d o u b l e - s t r a n d e d DNA r e p l i c a t i o n .
ACKNOWLEDGEMENTS: technical assistance.
W e wish to thank Ms. Pamela Svec for excellent This work was supported by the Unites States Public
Health Service Grant AI-I1633-01.
REFERENCES:
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. tl. 12. 13. 14.
L i n d q v i s t , B. a n d R. L. S i n s h e i m e r ( 1967 ) J. M o l . Biol. 29_8: 8 7 - 9 4 . S t o n e , A. B. ( 1970 ) Viroloqy 4_2_2:171-181. Stone, A. B. ( 1 9 7 0 ) J, M o l . Biol. 4__Z.7:2 1 5 - 2 2 9 . L i n n e y , E. a n d M. H a y a s h i ( 1 9 7 4 ) Nature 249" 3 4 5 - 3 4 8 . L i n n e y , E . , M. N. H a y a s h i , and M. H a y a s h i ( 1 9 7 2 ) Virology 50" 3 8 1 - 3 8 7 . G o d s o n , G. N. ( 1 9 7 1 ) ~. M o l . Biol. 57: 5 4 1 - 5 5 3 . G o d s o n , G. N. ( 1 9 7 4 ) Virology 58: 2 7 2 - 2 8 9 . Benbow, R. M . , C . A. H u t c h i s o n III, I . D. F a b r i c a n t , and R. L. S i n s h e i m e r ( 1971 ) ~. Virol. 7: 5 4 9 - 5 5 8 . I s h i w a , H. a n d I. T e s s m a n ( 1 9 7 3 ) J. M o l . Biol. 37: 4 6 7 - 4 7 4 . Pogo, B. G. T . , a n d S. D a l e s ( 1974 ) Viroloqy 5_.88"3 7 7 - 3 8 6 . H e n e r y , T. J. and R. Knippers ( 1 9 7 4 ) Proc. N a t . A c a d . S c i . U. S. A. 71: 1549-1553. I w a y a , M. a n d D. T. D e n h a r d t ( 1971 ) ~. M o l . Biol. 5._/.7:159-175. T e s s m a n , E. S. ( 1 9 6 6 ) J. M o l . Biol. 17: 2 1 8 - 2 3 6 . H u t c h i n s o n , C . A . , III, a n d R. L. S i n s h e i m e r ( 1 9 6 6 ) ] . M o l . Biol. 18: 4 2 9 - 4 4 7 .
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