Nonpermissive cells infected with {varphi}X174 gene D amber mutants synthesized some sixfold less viral RNA than permissive cells. The decrease was unaffected by increasing the multiplicity of infection and was a consequence of an overall decrease in all viral RNA species. It is suggested that the gene D product may function in replicative form DNA unwinding to expose the template for transcription

Clements, J. Barklie

Sinsheimer, Robert L.

English

Caltech Authors - Main

JOURNAL OF VIROLOGY, Dec. 1974, p. 1630-1632
Copyright 0 1975 American Society for Microbiology
Vol. 14, No. 6
Printed in U.S.A.
Class of OX174 Mutants Relatively Deficient in Synthesis of
Viral RNA
J. BARKLIE CLEMENTS' AND ROBERT L. SINSHEIMER
Division of Biology, California Institute of Technology, Pasadena, California 91109
Received for publication 17 June 1974
Nonpermissive cells infected with OX174 gene D amber mutants synthesized
some sixfold less viral RNA than permissive cells. The decrease was unaffected
by increasing the multiplicity of infection and was a consequence of an overall
decrease in all viral RNA species. It is suggested that the gene D product may
function in replicative form DNA unwinding to expose the template for
transcription.
The bacteriophage OX174 gene D product, a
protein of 14,500 molecular weight, is made in
largest molar amount of any OX gene product in
infected cells (2, 3). The precise function for this
protein is not known, however; mutants in gene
D do not form progeny single-stranded DNA,
but do make a normal amount of double-strand-
ed, replicative-form (RF) DNA (6, 8). The
present study examines the synthesis of OX
mRNA in cells infected with gene D amber
mutants.
Escherichia coli HF4704 (7) was the nonper-
missive host for viral amber mutants, and E.
coli HF4714 was the permissive host. Stocks of
the gene D mutants used (amlO and am42),
obtained from R. M. Benbow, had a reversion
frequency of 3 x 10-5 and were prepared as
described previously (2). Cells were grown to a
density of 5 x 108/ml in 20 ml of TPA medium
(13) containing one-tenth the normal phosphate
concentration and supplemented with a mix-
ture of 20 natural 1-amino acids (2.7 g/liter)
(Nutritional Biochemicals Corp., Cleveland,
Ohio) and 10 ,ug of thymine per ml. Virus was
then added at the multiplicities of infection
described below. Twelve minutes after infec-
tion, cultures were pulsed with [32P ]phosphoric
acid (carrier free) at 100 MCi/ml for 5 min. RNA
was isolated by phenol extraction, and the
proportion of OX mRNA was determined by
hybridizing to denatured XX RF DNA. RNA
samples were fractionated by polyacrylamide
gel electrophoresis and sedimentation in di-
methyl sulfoxide gradients.
Table 1 shows the proportion of XX mRNA in
the total labeled RNA isolated from permissive
and nonpermissive cells infected with gene D
'Present address: Medical Research Council Virology
Unit, Institute of Virology, Church Street, Glasgow Gll 5JR,
Scotland.
mutants. Values obtained with jX am3, a
lysis-deficient gene E mutant (5), are included
for comparison. The amount of OX mRNA
synthesized in nonpermissive cells was reduced
sixfold as compared with the values obtained
from permissive cells. Increasing the multiplic-
ity of infection by 10-fold had no influence upon
this effect. This decrease was not shown with
RNA from cells infected with the gene E mu-
tant, where values with nonpermissive cells
were in fact somewhat higher due to lysis
inhibition. (Likewise, no decrease was observed
in the amount of XX-specific RNA obtained
from nonpermissive cells infected with am16, a
mutant in gene B, which, like gene D, is
required for single-stranded DNA synthesis.)
In Fig. 1, the gel profiles of XX mRNA in
permissive and nonpermissive cells infected
with am42 at a multiplicity of 4 show no obvious
evidence for a specific decrease in any of the
discrete OX mRNAs. This also was true for
amlO infected cells (data not shown). On dena-
turing dimethyl sulfoxide gradients (Fig. 2), the
pattern of distribution of RNA counts hybridiza-
ble to OX RF DNA was similar with RNA
obtained from permissive and nonpermissive
cells. This result indicates the absence of inter-
nal nicks in RNA made under nonpermissive
conditions.
The decrease in the amount of XX mRNA in
cells infected with gene D mutants is reflected
in a reduction of the amount of virus-specific
proteins (2). A decrease in the amount of virus
mRNA in cells infected with gene A mutants of
the related phage S13 has been observed (11).
However, in this case, the amount of S13 mRNA
was increased by raising the multiplicity of
infection, a result attributed to an increase in
the amount of RF template, as gene A mutants
are unable to replicate RF DNA (14).
1630
0
x
-o
.-c
C-)
a-
'E
cn
-
x
a)
V-
-0
-t-
a-
Fraction No Froction No.
FIG. 1. Gel electrophoresis of OX mRNA. RNA samples (25 to 50 Mg) were coelectrophoresed with 3H-labeled
E. coli rRNA and 3H-labeled Sindbis-specific RNA on 2%o polyacrylamide, 0.4% agarose gels at 6 mA/gel for 4 h
at 4 C. Gels were cut into 1-mm segments, and slices were agitated in a horizontal shaker in vials containing 0.4
ml of 50%o (vol/vol) formamide-6 x SSC (pH 7.0) at 37 C for 48 h. After elution, a 20-,uliter sample was taken for
assay of radioactivity, then a blank filter, followed by an RFDNA filter was added. Vials were incubated at 37 C
for 30 h, and filters were processed as described in Table 1. (a) RNA from permissive cells infected with am42.
(b) RNA from nonpermissive cells infected with am42. Arrows indicate the positions of 16S and 23S E. coli
rRNA's and 26S and 49S Sindbis-specific RNAs.
23S 163S (a) (b)
23S 16S
15-
10
4-c
5 N\
0 10 20 30 40 0 l0 20 30 40
Fraction No Fraction No.
FIG. 2. Sedimentation of XX mRNA on dimethyl sulfoxide gradients. RNA samples were cosedimented with
3H-labeled E. coli rRNA in deuterated dimethyl sulfoxide gradients (12). Gradients were centrifuged at 64,000
rpm for 1I to 12 h at 27 C in a Spinco SW65 rotor. After counting a 10-Mliter sample of each collected fraction,
OX mRNA was located by hybridization to RF DNA in 40% (vol/vol) dimethyl sulfoxide-2 x SSC (pH 7.0) at
37 C for 50 h. Arrows indicate the position of 16S and 23S E. coli rRNA's. Sedimentation is from right to left. (a)
RNA from permissive cells infected with am42. (b) RNA from nonpermissive cells infected with am42.
1631
NOTES
TABLE 1. Proportion of OX mRNA in permissive and
non-permissive cellsa
Host strains
Phage strains
HF4704 (Su ) HF4714 (Su )
XX amlO (D) 1.62 ± 0.6 (4) 8.32 ± 1.1 (4)
1.37 ± 0.3 (40) 7.63 ± 0.8 (40)
OX am42 (D) 1.17 i 0.8 (4) 7.16 ± 0.5 (4)
1.03 ± 0.6 (40) 7.70 ± 0.9 (40)
OX am3 (E) 14.55 ± 2.1 (4) 9.15 ± 0.7 (4)
16.32 ± 2.7 (40) 10.27 ± 1.4 (40)
a The amount of OX hybridizable RNA is expressed
as a percentage of total labeled RNA. Each value is
the mean of six separate estimations with errors
expressed as extreme fluctuations from the mean.
Figures in parentheses show the multiplicity of infec-
tion. Radioactive pulses were terminated by addition
of a sodium azide (0.5 M) and potassium cyanide
(0.03 M) mixture to final concentrations of 0.05 M and
0.003 M, respectively. Cultures were immediately
frozen by immersion in a dry ice-methanol bath. The
cell pellets were suspended in 6 ml of Tris-NaCl-
EDTA buffer (0.05 M Tris, 0.1 M NaCl, 0.01 M
EDTA, pH 7.5) containing 0.2 mg of lysozyme per ml.
These suspensions were maintained at 22 C for 3 min,
then sodium dodecyl sulfate was added to 0.5%. The
lysates were extracted twice at 22 C with phenol
equilibrated with Tris-NaCl-EDTA plus 0.5% sodium
dodecyl sulfate. RNA was precipitated twice from 10
ml of Tris-NaCl-EDTA with 3 volumes of 95% ethanol
at -- 20 C. The precipitates were then suspended in 1
ml of 0.1 M sodium acetate, 2.5 mM magnesium sul-
fate (pH 5.0) containing 25 ,ug of deoxyribonuclease I
(electrophoretically purified; Worthington Biochemi-
cals Corp., Freehold, N.J.). Af'ter incubation at 22 C
for 30 min, the samples were extracted with phenol,
the RNA was reprecipitated twice, and samples were
stored in ethanol at -20 C. More than 95'% of the
product was soluble in trichloroacetic acid after
treatment with 0.3 N KOH (16 h, 37 C) or with 20 Ag
of RNAse A per ml (1 h in 0.1 x SSC). RNA samples
(0.5 to 5 Mg of total RNA) in 0.4 ml of 6 x SSC were
hybridized to 10-pg lots of denatured, DNA (1 to 2 Mg
was sufficient to hybridize all the XX mRNA) on
11-mm nitrocellulose filters at 66 C for 30 h in the
presence of blank control filters. After incubation,
filters were washed twice in 10 ml of 2 x SSC, and
then treated with 1 ml of pancreatic ribonuclease (25
Mg of 2 x SSC per ml (pH 5.0), preheated to 84 C for
10 min to eliminate DNase) for 40 min at 22 C. After
two washes with 2 x SSC, the dried filters were
counted in a toluene-based scintillation fluid. Counts
on blank filters were subtracted from values of the
appropriate RF filter.
In terms of molecular weight, and molar
amount synthesized, the OX gene D product
seems analogous to the gene 5 product of the
filamentous phages, e.g., (fl). The gene 5 prod-
uct has been characterized; it binds tightly to
single-stranded DNA and denatures double-
stranded DNA at physiological temperatures (1,
9, 10). The reduction in the amount of OX
mRNA synthesized in infections with gene D
mutants might be ascribed to a role for the gene
product in RF unwinding, so as to expose the
complementary DNA strand which is the
template for RNA synthesis (4).
This research was supported in part by Public Health
Service grant GM13554 from the National Institute of Gen-
eral Medical Sciences.
LITERATURE CITED
1. Alberts, B., L. Frey, and H. Delius. 1972. Isolation and
characterization of gene 5 protein of filamentous bacte-
rial viruses. J. Mol. Biol. 68:139-152.
2. Benbow, R. M., R. F. Mavol, J. A. Picchi, and R. L.
Sinsheimer. 1972. Direction of translation and size of
bacteriophage kX174 cistrons. J. Virol. 10:99-114.
3. Godson, G. N. 1971. Characterization and synthesis of
OX174 proteins in ultraviolet-irradiated and unir-
radiated cells. J. Mol. Biol. 57:541-553.
4. Hayashi, M. N., M. Hayashi, and S. Spiegelman. 1963.
Restriction of in vivo genetic transcription to one of the
complementary strands of DNA. Proc. Nat. Acad. Sci.
U.S.A. 50:664-672.
5. Hutchison, C. A., III, and R. L. Sinsheimer. 1966. The
process of infection with bacteriophage kX174. X.
Mutations in a OX lysis gene. J. Mol. Biol. 18:429-447.
6. Iwaya, M., and D. T. Denhardt. 1971. The mechanism of
replication of OX174 single-stranded DNA. II. The role
of viral proteins. J. Mol. Biol. 57:159-175.
7. Lindqvist, B. J., and R. L. Sinsheimer. 1967. Process of
infection with bacteriophage kX174. XIV. Studies on
macromolecular synthesis during infection with a lysis-
defective mutant. J. Mol. Biol. 28:87-94.
8. Lindqvist, B. H., and R. L. Sinsheimer. 1967. The process
of infection with bacteriophage kX174. XV. Bacterio-
phage DNA synthesis in abortive infections with a set
of conditional lethal mutants. J. Mol. Biol. 30:69-80.
9. Oey, J. L., and R. Knippers. 1972. Properties of the
isolated gene 5 protein of bacteriophage fd. J. Mol.
Biol. 68:125-138.
10. Pratt, D., P. Laws, and J. Griffith. 1974. Complex of
bacteriophage M13 single-stranded DNA and gene 5
protein. J. Mol. Biol. 82:425-439.
11. Puga, A., and I. Tessman. 1973. Mechanism of transcrip-
tion of bacteriophage S13. I. Dependence of messenger
RNA synthesis on amount and configuration of DNA.
J. Mol. Biol. 75:83-97.
12. Sedat, J. W., A. Lyon, and R. L. Sinsheimer. 1969.
Purification of Escherichia coli pulse-labeled RNA by
benzoylated DEAE-cellulose chromatography. J. Mol.
Biol. 44:415-434.
13. Sinsheimer. R. L., B. Starman. C. Nagler, and S.
Guthrie. 1962. The process of infection with bacterio-
phage <X174. I. Evidence for a "replicative form." J.
Mol. Biol. 4:142-160.
14. Tessman, E. S. 1966. Mutants of bacteriophage S13
blocked in infectious DNA synthesis. J. Mol. Biol.
17:218-236.
1632 J. VIROL.


Class of {varphi}X174 Mutants Relatively Deficient in Synthesis of Viral RNA

JOURNAL OF VIROLOGY, Dec. 1974, p. 1630-1632
Copyright 0 1975 American Society for Microbiology
Vol. 14, No. 6
Printed in U.S.A.
Class of OX174 Mutants Relatively Deficient in Synthesis of
Viral RNA
J. BARKLIE CLEMENTS' AND ROBERT L. SINSHEIMER
Division of Biology, California Institute of Technology, Pasadena, California 91109
Received for publication 17 June 1974
Nonpermissive cells infected with OX174 gene D amber mutants synthesized
some sixfold less viral RNA than permissive cells. The decrease was unaffected
by increasing the multiplicity of infection and was a consequence of an overall
decrease in all viral RNA species. It is suggested that the gene D product may
function in replicative form DNA unwinding to expose the template for
transcription.
The bacteriophage OX174 gene D product, a
protein of 14,500 molecular weight, is made in
largest molar amount of any OX gene product in
infected cells (2, 3). The precise function for this
protein is not known, however; mutants in gene
D do not form progeny single-stranded DNA,
but do make a normal amount of double-strand-
ed, replicative-form (RF) DNA (6, 8). The
present study examines the synthesis of OX
mRNA in cells infected with gene D amber
mutants.
Escherichia coli HF4704 (7) was the nonper-
missive host for viral amber mutants, and E.
coli HF4714 was the permissive host. Stocks of
the gene D mutants used (amlO and am42),
obtained from R. M. Benbow, had a reversion
frequency of 3 x 10-5 and were prepared as
described previously (2). Cells were grown to a
density of 5 x 108/ml in 20 ml of TPA medium
(13) containing one-tenth the normal phosphate
concentration and supplemented with a mix-
ture of 20 natural 1-amino acids (2.7 g/liter)
(Nutritional Biochemicals Corp., Cleveland,
Ohio) and 10 ,ug of thymine per ml. Virus was
then added at the multiplicities of infection
described below. Twelve minutes after infec-
tion, cultures were pulsed with [32P ]phosphoric
acid (carrier free) at 100 MCi/ml for 5 min. RNA
was isolated by phenol extraction, and the
proportion of OX mRNA was determined by
hybridizing to denatured XX RF DNA. RNA
samples were fractionated by polyacrylamide
gel electrophoresis and sedimentation in di-
methyl sulfoxide gradients.
Table 1 shows the proportion of XX mRNA in
the total labeled RNA isolated from permissive
and nonpermissive cells infected with gene D
'Present address: Medical Research Council Virology
Unit, Institute of Virology, Church Street, Glasgow Gll 5JR,
Scotland.
mutants. Values obtained with jX am3, a
lysis-deficient gene E mutant (5), are included
for comparison. The amount of OX mRNA
synthesized in nonpermissive cells was reduced
sixfold as compared with the values obtained
from permissive cells. Increasing the multiplic-
ity of infection by 10-fold had no influence upon
this effect. This decrease was not shown with
RNA from cells infected with the gene E mu-
tant, where values with nonpermissive cells
were in fact somewhat higher due to lysis
inhibition. (Likewise, no decrease was observed
in the amount of XX-specific RNA obtained
from nonpermissive cells infected with am16, a
mutant in gene B, which, like gene D, is
required for single-stranded DNA synthesis.)
In Fig. 1, the gel profiles of XX mRNA in
permissive and nonpermissive cells infected
with am42 at a multiplicity of 4 show no obvious
evidence for a specific decrease in any of the
discrete OX mRNAs. This also was true for
amlO infected cells (data not shown). On dena-
turing dimethyl sulfoxide gradients (Fig. 2), the
pattern of distribution of RNA counts hybridiza-
ble to OX RF DNA was similar with RNA
obtained from permissive and nonpermissive
cells. This result indicates the absence of inter-
nal nicks in RNA made under nonpermissive
conditions.
The decrease in the amount of XX mRNA in
cells infected with gene D mutants is reflected
in a reduction of the amount of virus-specific
proteins (2). A decrease in the amount of virus
mRNA in cells infected with gene A mutants of
the related phage S13 has been observed (11).
However, in this case, the amount of S13 mRNA
was increased by raising the multiplicity of
infection, a result attributed to an increase in
the amount of RF template, as gene A mutants
are unable to replicate RF DNA (14).
1630
0
x
-o
.-c
C-)
a-
'E
cn
-
x
a)
V
-
-0
-t-
a-
Fraction No Froction No.
FIG. 1. Gel electrophoresis of OX mRNA. RNA samples (25 to 50 Mg) were coelectrophoresed with 3H-labeled
E. coli rRNA and 3H-labeled Sindbis-specific RNA on 2%o polyacrylamide, 0.4% agarose gels at 6 mA/gel for 4 h
at 4 C. Gels were cut into 1-mm segments, and slices were agitated in a horizontal shaker in vials containing 0.4
ml of 50%o (vol/vol) formamide-6 x SSC (pH 7.0) at 37 C for 48 h. After elution, a 20-,uliter sample was taken for
assay of radioactivity, then a blank filter, followed by an RFDNA filter was added. Vials were incubated at 37 C
for 30 h, and filters were processed as described in Table 1. (a) RNA from permissive cells infected with am42.
(b) RNA from nonpermissive cells infected with am42. Arrows indicate the positions of 16S and 23S E. coli
rRNA's and 26S and 49S Sindbis-specific RNAs.
23S 163S (a) (b)
23S 16S
15-
10
4-c
5 N\
0 10 20 30 40 0 l0 20 30 40
Fraction No Fraction No.
FIG. 2. Sedimentation of XX mRNA on dimethyl sulfoxide gradients. RNA samples were cosedimented with
3H-labeled E. coli rRNA in deuterated dimethyl sulfoxide gradients (12). Gradients were centrifuged at 64,000
rpm for 1I to 12 h at 27 C in a Spinco SW65 rotor. After counting a 10-Mliter sample of each collected fraction,
OX mRNA was located by hybridization to RF DNA in 40% (vol/vol) dimethyl sulfoxide-2 x SSC (pH 7.0) at
37 C for 50 h. Arrows indicate the position of 16S and 23S E. coli rRNA's. Sedimentation is from right to left. (a)
RNA from permissive cells infected with am42. (b) RNA from nonpermissive cells infected with am42.
1631
NOTES
TABLE 1. Proportion of OX mRNA in permissive and
non-permissive cellsa
Host strains
Phage strains
HF4704 (Su ) HF4714 (Su )
XX amlO (D) 1.62 ± 0.6 (4) 8.32 ± 1.1 (4)
1.37 ± 0.3 (40) 7.63 ± 0.8 (40)
OX am42 (D) 1.17 i 0.8 (4) 7.16 ± 0.5 (4)
1.03 ± 0.6 (40) 7.70 ± 0.9 (40)
OX am3 (E) 14.55 ± 2.1 (4) 9.15 ± 0.7 (4)
16.32 ± 2.7 (40) 10.27 ± 1.4 (40)
a The amount of OX hybridizable RNA is expressed
as a percentage of total labeled RNA. Each value is
the mean of six separate estimations with errors
expressed as extreme fluctuations from the mean.
Figures in parentheses show the multiplicity of infec-
tion. Radioactive pulses were terminated by addition
of a sodium azide (0.5 M) and potassium cyanide
(0.03 M) mixture to final concentrations of 0.05 M and
0.003 M, respectively. Cultures were immediately
frozen by immersion in a dry ice-methanol bath. The
cell pellets were suspended in 6 ml of Tris-NaCl-
EDTA buffer (0.05 M Tris, 0.1 M NaCl, 0.01 M
EDTA, pH 7.5) containing 0.2 mg of lysozyme per ml.
These suspensions were maintained at 22 C for 3 min,
then sodium dodecyl sulfate was added to 0.5%. The
lysates were extracted twice at 22 C with phenol
equilibrated with Tris-NaCl-EDTA plus 0.5% sodium
dodecyl sulfate. RNA was precipitated twice from 10
ml of Tris-NaCl-EDTA with 3 volumes of 95% ethanol
at -- 20 C. The precipitates were then suspended in 1
ml of 0.1 M sodium acetate, 2.5 mM magnesium sul-
fate (pH 5.0) containing 25 ,ug of deoxyribonuclease I
(electrophoretically purified; Worthington Biochemi-
cals Corp., Freehold, N.J.). Af'ter incubation at 22 C
for 30 min, the samples were extracted with phenol,
the RNA was reprecipitated twice, and samples were
stored in ethanol at -20 C. More than 95'% of the
product was soluble in trichloroacetic acid after
treatment with 0.3 N KOH (16 h, 37 C) or with 20 Ag
of RNAse A per ml (1 h in 0.1 x SSC). RNA samples
(0.5 to 5 Mg of total RNA) in 0.4 ml of 6 x SSC were
hybridized to 10-pg lots of denatured, DNA (1 to 2 Mg
was sufficient to hybridize all the XX mRNA) on
11-mm nitrocellulose filters at 66 C for 30 h in the
presence of blank control filters. After incubation,
filters were washed twice in 10 ml of 2 x SSC, and
then treated with 1 ml of pancreatic ribonuclease (25
Mg of 2 x SSC per ml (pH 5.0), preheated to 84 C for
10 min to eliminate DNase) for 40 min at 22 C. After
two washes with 2 x SSC, the dried filters were
counted in a toluene-based scintillation fluid. Counts
on blank filters were subtracted from values of the
appropriate RF filter.
In terms of molecular weight, and molar
amount synthesized, the OX gene D product
seems analogous to the gene 5 product of the
filamentous phages, e.g., (fl). The gene 5 prod-
uct has been characterized; it binds tightly to
single-stranded DNA and denatures double-
stranded DNA at physiological temperatures (1,
9, 10). The reduction in the amount of OX
mRNA synthesized in infections with gene D
mutants might be ascribed to a role for the gene
product in RF unwinding, so as to expose the
complementary DNA strand which is the
template for RNA synthesis (4).
This research was supported in part by Public Health
Service grant GM13554 from the National Institute of Gen-
eral Medical Sciences.
LITERATURE CITED
1. Alberts, B., L. Frey, and H. Delius. 1972. Isolation and
characterization of gene 5 protein of filamentous bacte-
rial viruses. J. Mol. Biol. 68:139-152.
2. Benbow, R. M., R. F. Mavol, J. A. Picchi, and R. L.
Sinsheimer. 1972. Direction of translation and size of
bacteriophage kX174 cistrons. J. Virol. 10:99-114.
3. Godson, G. N. 1971. Characterization and synthesis of
OX174 proteins in ultraviolet-irradiated and unir-
radiated cells. J. Mol. Biol. 57:541-553.
4. Hayashi, M. N., M. Hayashi, and S. Spiegelman. 1963.
Restriction of in vivo genetic transcription to one of the
complementary strands of DNA. Proc. Nat. Acad. Sci.
U.S.A. 50:664-672.
5. Hutchison, C. A., III, and R. L. Sinsheimer. 1966. The
process of infection with bacteriophage kX174. X.
Mutations in a OX lysis gene. J. Mol. Biol. 18:429-447.
6. Iwaya, M., and D. T. Denhardt. 1971. The mechanism of
replication of OX174 single-stranded DNA. II. The role
of viral proteins. J. Mol. Biol. 57:159-175.
7. Lindqvist, B. J., and R. L. Sinsheimer. 1967. Process of
infection with bacteriophage kX174. XIV. Studies on
macromolecular synthesis during infection with a lysis-
defective mutant. J. Mol. Biol. 28:87-94.
8. Lindqvist, B. H., and R. L. Sinsheimer. 1967. The process
of infection with bacteriophage kX174. XV. Bacterio-
phage DNA synthesis in abortive infections with a set
of conditional lethal mutants. J. Mol. Biol. 30:69-80.
9. Oey, J. L., and R. Knippers. 1972. Properties of the
isolated gene 5 protein of bacteriophage fd. J. Mol.
Biol. 68:125-138.
10. Pratt, D., P. Laws, and J. Griffith. 1974. Complex of
bacteriophage M13 single-stranded DNA and gene 5
protein. J. Mol. Biol. 82:425-439.
11. Puga, A., and I. Tessman. 1973. Mechanism of transcrip-
tion of bacteriophage S13. I. Dependence of messenger
RNA synthesis on amount and configuration of DNA.
J. Mol. Biol. 75:83-97.
12. Sedat, J. W., A. Lyon, and R. L. Sinsheimer. 1969.
Purification of Escherichia coli pulse-labeled RNA by
benzoylated DEAE-cellulose chromatography. J. Mol.
Biol. 44:415-434.
13. Sinsheimer. R. L., B. Starman. C. Nagler, and S.
Guthrie. 1962. The process of infection with bacterio-
phage <X174. I. Evidence for a "replicative form." J.
Mol. Biol. 4:142-160.
14. Tessman, E. S. 1966. Mutants of bacteriophage S13
blocked in infectious DNA synthesis. J. Mol. Biol.
17:218-236.
1632 J. VIROL.


Characterization and synthesis of OX174 proteins in ultraviolet-irradiated and unirradiated cells.

Direction of translation and size of bacteriophage kX174 cistrons.

Isolation and characterization of gene 5 protein of filamentous bacterial viruses.

Mechanism of transcription of bacteriophage S13. I. Dependence of messenger RNA synthesis on amount and configuration of DNA.

Mutants of bacteriophage S13 blocked in infectious DNA synthesis.

Process of infection with bacteriophage kX174. XIV. Studies on macromolecular synthesis during infection with a lysisdefective mutant.

Properties of the isolated gene 5 protein of bacteriophage fd.

Purification of Escherichia coli pulse-labeled RNA by benzoylated DEAE-cellulose chromatography.

Restriction of in vivo genetic transcription to one of the complementary strands of DNA.

The mechanism of replication of OX174 single-stranded DNA. II. The role of viral proteins.

The process of infection with bacteriophage I. Evidence for a &quot;replicative form.&quot;

The process of infection with bacteriophage kX174. X. Mutations in a OX lysis gene.

The process of infection with bacteriophage kX174. XV. Bacteriophage DNA synthesis in abortive infections with a set of conditional lethal mutants.

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Class of {varphi}X174 Mutants Relatively Deficient in Synthesis of Viral RNA

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