Cell, Vol. 50, 379-389,
July 31. 1987, Copyright
0 1987 by Cell Press
An Inducible Mammalian Amber Suppressor:
Propagation of a Poliovirus Mutant
John M. Sedivy, John P. Capone,’
Uttam L RajBhandary,
and Phillip A. Sharp
Center for Cancer Research
and Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
Summary
We describe a general protocol for controlled
gene
amplification,
which allows conditional
expression of
high levels of amber suppressor
activity in monkey
kidney cells, and we demonstrate
its use in the genetic
analysis of animal viruses by the generation
and
propagation
of the first nonsense mutant of poliovirus. A human amber suppressor
tRNASH gene linked
to the SV40 origin of replication
and a second DNA carrying a temperature-sensitive
SV40 large T antigen
gene were cotransfected
into monkey cells. Cell lines
having stably integrated
the DNAs were isolated.
Shifting the cells from the nonpermissive
temperature
to a lower permissive temperature
caused the amplification of the suppressor tRNA gene, which resulted in
suppression
efficiencies
at amber codons of 50%70%, as measured by suppression
of an amber codon
in the E. coli chloramphenicol
acetyltransferase
gene.
A mutant of poliovirus,
in which a serine codon in the
replicase gene was converted to an amber codon, was
efficiently propagated
on the suppressor-positive
cell
lines. The mutant virus reverted to wild-type by a single base change to a serine codon at a frequency of
approximately
2.5 x 10-6, surprisingly
low for a RNA
genome.
Introduction
Isolation of mutations, a cornerstone of all genetic analyses, has not been easy in cultured mammalian cells and
viruses. In particular, the isolation of conditional-lethal
mutations, required for marking essential genes, has met
with considerable difficulty. The most common conditional-lethal system, temperature-sensitivity
(Horowitz and
Leupold, 1951; Campbell,
1961; Edgar and Lielausis,
1964; Cooper, 1964), is limited by the narrow temperature
range in which mammalian cells can be cultured, resulting in the “leakiness” of many mutations. Permissive and
nonpermissive
cell lines have been exploited to isolate
conditional-lethal
host range mutants in animal viruses,
but this approach is restricted to the gene(s) that is complemented by a particular permissive cell line (Benjamin,
1970).
* Present
HamIlton,
address:
Ontarlo
Department
of Biochemistry,
L8N 325. Canada.
McMaster
University,
The second general conditional-lethal
system is based
on suppression
of “nonsense”
mutations by suppressor tRNAs possessing altered anticodons (Benzer and
Champe, 1962; Epstein et al., 1963). In Escherichia coli,
the availability of numerous strains containing a variety of
nonsense suppressors (Celis and Smith, 1979) was crucial for the exploitation of nonsense mutants in the genetic
analysis of bacterial and bacteriophage
genes. Furthermore, the ability to substitute a variety of amino acids at
a single mutant locus by different suppressor tRNAs has
extended the utility of nonsense mutations for the study
of structure-function
relationships in proteins (Miller et al.,
1979). Among eukaryotes, nonsense suppressors have
been well exploited only in yeast (Sherman, 1982) and,
more recently, in the nematode Caenorhabditis
elegans
(Wills et al., 1983; Hodgkin, 1985; Fire, 1986). In mammalian cells, despite several attempts, classical genetic
methods have not yet yielded nonsense-suppressing
cell
lines.
Nonsense suppressors active in mammalian cells have
recently been constructed using site-specific mutagenesis of cloned tRNA genes (Laski et al., 1982; Temple et al.,
1982; Laski et al., 1984; Capone et al., 1985). The biological activity of these suppressor tRNA genes was demonstrated by suppression of termination of nonsense codons
in viral mRNAs (Laski et al., 1982; Young et al., 1983; Laski
et al., 1984) and of nonsense mutations in reporter genes
such as the E. coli chloramphenicol
acetyl transferase
(cat) (Capone et al., 1986). In the latter case, a defined set
of cat gene mutants has been constructed where the serine codon at position 27 has been converted to either an
amber (UAG), ochre (UAA), or opal (UGA) nonsense
codon. Cotransfection of these mutant cat genes with the
appropriate suppressor tRNA gene resulted in suppression efficiencies as high as 50% in several cell types (Capone et al., 1986). Such high suppression efficiencies
have only been obtained in transient systems with high
copy numbers of tRNA genes per cell. Transient systems
are, however, of limited usefulness for the isolation and
propagation of mutations in essential genes. A few permanent cell lines in which suppressor tRNA genes have been
stably integrated into cellular genomes have been isolated, but the levels of suppression have been disappointingly low, approximately 3% (Hudziak et al., 1982; Young
et al., 1983; Ho et al., 1986). It has not been established,
however, whether this level of suppression in mammalian
cells constitutes a biological limit caused by lethality of
suppression.
In this paper, we report a systematic analysis of construction of cell lines containing nonsense suppressor
tRNA genes, and the development of a general method for
induction of high levels of suppressor activity. We demonstrate the utility and scope of this methodology
by the
generation, propagation, and characterization
of the first
nonsense mutant of poliovirus.
Cell
380
pZipNeoSV(SupAm)
Figure
1. Construction
pZipNeo (am//); see Experimental Procedures). The level
of suppression
in a pool of approximately
50 colonies
selected for viability in the presence of low concentrations
of G418 (190 pg/ml) was less than 3%. At higher concentrations of G418, cell viability sharply decreased. It was not
possible to establish cells with higher levels of suppression by selecting for resistance to higher concentrations
of G418.
of Plasmid
Vectors
Plasmid pZipNeoSV(SupAm)
was constructed
by inserting a 900 bp
Sau3A fragment containing the human serine amber suppressor
tRNA
gene from the plasmid pUCtS Su+(Am) (Capone et al., 1986) into the
BamHl site of the retroviral shuttle vector pZipNeoSV(X)l
(Cepko et al.,
1984). The same 900 bp Sau3A fragment was also inserted,
in the
same fashion, in the vector pZipGpt (R. Mulligan, unpublished
data;
see Experimental
Procedures).
Plasmid pLTRtsA58 was constructed
by deleting an internal Xhol fragment,
which contains the neo gene
and the SV40 origin of replication,
from the plasmid pZipSV4OrsA58
(Jat and Sharp, unpublished
data; see Experimental
Procedures).
Bold
arrows indicate the direction of transcription.
Note that the amber suppressor tRNA is transcribed
from its own RNA polymerase
III promoter
contained within the SaudA fragment. Abbreviations:
LTR, long terminal repeat; 5’SS, splice donor site: 3’ SS, splice acceptor site; ori, origin of replication.
Results
Establishment
of Permanent
Cell Lines with
Constitutive
Low Levels of Suppression
of Amber Codons
In the initial experiments,
an amber suppressor, constructed from a human serine tRNA gene (Capone et al.,
1985) was linked to several dominant selectable genes
(neo, gpt, and dhfr; see Experimental
Procedures) and
transfected into NIH3T3 cells. An equal number of colonies survived the selection when plasmids containing either Sup+ or Sup- genes were transfected, suggesting
that cells with integrated copies of the Sup+ tRNA gene
were viable. Disappointingly,
none of the cell lines transfected with the Sup+ gene expressed detectable levels of
suppression when assayed by transfection with reporter
plasmids containing a suppressible nonsense mutation in
the cat gene (see Experimental
Procedures). This suggests that the levels of Sup+ tRNA expressed from the
few integrated genes were inadequate to compete effectively against the activity of the cellular translational release factors (Caskey and Campbell, 1979).
In another series of experiments, functional expression
of the amber Sup+ tRNA gene was selected by cotransfection with a plasmid containing a suppressible amber
mutation in the dominant selectable marker, neo (plasmid
Establishment
of Permanent Cell Lines Inducible to
High Levels of Suppression
of Amber Codons
The apparent inability to propagate cell lines constitutively
expressing high levels of amber Sup+ tRNA necessitated
the development of a system where high levels of suppression could be induced for limited periods of time. A controlled and rapid amplification
of the Sup+ tRNA gene
would satisfy these conditions. The amber Sup+ tRNA
gene was linked to the SV40 origin of replication and
cotransfected into the monkey cell line WC-40 along with
the tsA58 allele of the SV40 large T antigen gene (plasmids pZipNeoSV(SupAm)
and pLTRtsA58; see Figure 1).
Cell lines were grown from colonies selected for resistance to G418 (330 pglml) at the nonpermissive temperature (39.5%). A parallel control cotransfection with plasmids pZipNeoSV(X)l
and pLTRtsA58 was also included.
Upon shifting to the permissive temperature (33%) the A
protein (large T antigen) was expected to stimulate initiation of replication at the SV40 origin, resulting in the
amplification of the linked Sup+ tRNA gene, and, consequently, high levels of suppression (Botchan et al., 1979;
Rio et al., 1986).
All cell lines were initially screened, at both 33% and
39.5%, for the presence of the large T antigen by indirect
immunofluorescence
as well as growth and cellular morphology. Strong nuclear fluorescence was observed at
33% but not at 39.5% in three out of ten control cell lines,
and in two out of nine experimental cell lines. The control
cell lines showed no change in morphology after shifting
to 33% and continued to grow at this temperature,
whereas both of the experimental cell lines (designated
BSC4OsupD3
and BSC40supD7;
see Experimental
Procedures for genetic nomenclature) displayed a marked
change in morphology
after several days of culture at
33%. This change in morphology was accompanied by a
complete cessation of growth. Most important, all of these
cell lines behaved normally at 39.5%. After this initial
screen, nine more experimental cell lines were examined,
and three (designated kEG4OsupD2,
BSC4OsupD4, and
BSC4OsupD72) were found to arrest growth and assume
an altered morphology at the permissive temperature.
Characterization
of Permanent Cell Lines Inducible
to High Levels of Suppression
of Amber Codons
Quanfitation
of the Levels of Suppression
Reporter plasmids containing either wild-type, amber, or
ochre mutant car genes (pRSVcar, pRSVcar(am27)
and
pRSVcat(oc27)
respectively), were transfected into cell
lines taken from passage one (see Experimental Procedures), and the transient levels of car activities were determined. The control BSC4OsupOS cell line did not sup-
An Inducible
381
Mammalian
Amber
A
El
sup0
supD7
39.5”
W
W
A
D
C
supD3
33”
39.5”
33”
A
Suppressor
39.5”
33”
WAOWAOWAOWAO
supD2
supD4
supD12
supD3
supD3
33”
33”
33‘
33”/,p3
33”lpll
WA
WA
W
A
WA
W
A
1
0)
Frgure 2. Amber
Suppressor
Activity
of Stable
BSC4OsupD
Cell Lines
Measured
by Suppression
of a Cat Amber
Mutatron
Cell lanes to be tested were transiently
transfected
with reporter plasmids pRSVcat, pRSVcat(am27).
or pRSVcaf(oc27).
Prior to transfection,
all cell
lines were grown at 39.5%; immediately
after transfection
cells were placed at the indicated temperature
and incubated for 72 hr. The assays were
normalized
for both the relative transfection
efficiencies
and the protein content of the extracts.
(A) Sup- controls at 33% and 39.5%.
(B) Induction of amber suppression
by a temperature
shift-down
in cell lines BSC4OsupD3
and BSC4OsupDZ
(C) L.evels of suppression
at 33% in three additional BSC4OsupD
cell lines.
(D) Stability of the inducible suppressor
phenotype
during continuous
passage at 39.5%.
W, wild-type (pRSVcar); A, amber (pRSVcai(am27));
0, ochre (pRSVcaf(oc27));
p3, passage 3; pll, passage
11.
press the cat(am27) mutation at either 33°C or 39.5X
(Figure 2A). In contrast, at 33OC, the cell lines BSC4OsupD3 and BSC40supD7showed
approximately 20%30% levels of suppression (Figure 2B). At 39.WC, the
BSC40supD3
cell line did not suppress while the cell line
BSC40supD7suppressed
at a level of approximately 5%.
This constitutive level of suppression
at 39.YC in the
BSC4OsupD7 cell line may account for its instability (see
below). Neither cell line suppressed the cat(oc27) mutation at either temperature. The levels of suppression at
33OC in three additional cell lines, BSC4OsupD2,
BSC4OsupD4, and BSC-4OsupDlP ranged from <100/o to 70%
(Figure 2C). The cell line BSC4OsupDIP
reproducibly
showed a 500/o-70% level of suppression.
Analysis of Integrated
and Amplified
DNAs
The level of gene amplification
in the cell line BSC4OsupD3, as well as the structure of the transfected DNA
before and after amplification, was analyzed by Southern
blot hybridization
using a fragment of the neo gene as
probe. The results, presented in Figure 3, revealed the following: first, the transfected DNA in cells grown continuously at 39.5”C was present as stably integrated tandem
concatenates of the input plasmid; second, during the first
48 hr following shift-down to 33OC, low molecular weight,
episomal species rapidly accumulated to approximately
2,000-4,000 copies per cell; and third, analysis of the replicating episomes indicated the presence of approximately equal amounts of two species, a unit-length input
pZipNeoSV(SupAm)
plasmid species, and a “one LTR circle” species.
Stability of Suppressor-Positive
Cell Lines during
Continuous
Culture at 39.5%
The usefulness of the amber-suppressing
cell lines is dependent on their stability. The inducibility of the suppressor phenotype was found to be very stable during extended
passage in culture at the nonpermissive temperature, with
the exception of one cell line (BSC40supD7).
During four
passages from clonal isolation (approximately 20 generations) in the absence of G418, this cell line had lost its cold
sensitivity, resistance to G418, inducibility to amber suppression, and transfected pZipNeoSV(SupAm)
DNA (see
Exoerimental Procedures and Figure 3, lane 6).
The instability of the BSC4OsupD7 cell line was probably caused by its relatively high constitutive level of suppression at 39.5’C (approximately 5%). The rapid loss of
suppressor activity by segregation of the transfected DNA
clearly indicates the presence of a strong negative selection, and agrees with our previous observations that levels
of suppression above 5% are deleterious to cell viability.
In contrast, both BSC4OsupD3
and BSC4OsupD72 cell
lines were stable during passage for many generations at
39.5’X in the absence of G418. For example, Figure 2D
shows that the inducibility of suppressor activity was identical in BSC-40supD3 cells after more than 60 generations
in the absence of G418 selection. This is consistent with
the fact that both the BSC-4OsupD3 and BSC-4OsupD12
Cell
382
A.
h c
E
-g”
rEx”$d::
B.
supD3
~po7
COPY NO. CONTROLS
7% gz’
DAYS AT 33-C
10’
103
102
1
2
3
4
7
8
9
10
11
12
13
123456
C.
cell lines express very low levels of constitutive
sor activity at 39.5% (data not shown).
D.
Cla
Xba
\
Cl.9 Eco
SPh
Sph
14 15
16 17 18
19 20
21 22
Figure 3. Southern Blot Hybridization
Suppressing
Cell Lines
Analysis
suppres-
Suppression
of a Nonsense Mutation in Poliovirus
Construction
of an Amber Mutant of Poliovirus
The availability of inducible amber-suppressor
cell lines
should enable the isolation and propagation of amber mutant viruses. Poliovirus has been well studied; however,
nonsense mutations in this virus have not been isolated
(Koch and Koch, 1985; Bernstein et al., 1985). Propagation of nonsense mutants of poliovirus was expected to be
particularly difficult, since the total viral genome is expressed as a single polypeptide and a high efficiency of
suppression
would probably be essential to generate
adequate levels of viral proteins. A cDNA clone of the
poliovirus RNA genome has previously been characterized (Racaniello and Baltimore, 1981). In the plasmid,
pSV2polio, this cDNA segment was positioned downstream from a eukaryotic promoter such that the transcribed RNA corresponds to the poliovirus genome (Bernstein et al., 1986). Transfection of permissive ceils with this
plasmid generates infectious polio virions by initiation of
a replicative cycle.
To generate a defined nonsense mutant, the serine
codon (position 28) in the poliovirus RNA replicase gene
was replaced with an amber codon by in vitro site-directed
mutagenesis. Mutagenesis was performed by direct replacement of a small region in the pSV2polio plasmid with
a synthetic double-stranded
oligonucleotide
(Figure 4).
The mutant cDNA plasmid pSV2polio(am28)
was trans-
of BSC-4OsupDAmber
Cells were from passage 4, as defined in Experimental
Procedures.
Total DNA, or Hirt supernatant
DNA (Hirt, 1967) was extracted from confluent monolayer
cells either grown continuously
at 39.5%, or grown
to 80% confluency,
shifted down to 33%. and held for the indicated
times. The probe was homologous
to the neo gene, as indicated by the
solid bar in the diagram, and was prepared by the random priming procedure (Feinberg and Vogelstein, 1983) of an agarose gel electrophoresis purified DNA fragment.
All lanes contain BSG4OsupDS
DNA,
except lanes with size markers (Hindlll-digested
1 DNA), plasmid controls (as indicated),
and lane 6. which contains BSC-40supD7
DNA.
(A) Analysis of cells grown continuously
at 39.5%. Digestion with Xbal
(lane 3, which cuts once in each LTR; see diagram) produced a major
band with migration identical to a control digest of pZipNeoSV(SupAm)
plasmid DNA (lane 2, loaded as one copy per genome equivalent).
This indicates that the majority of the transfected
plasmid DNA has integrated in a form that leaves the sequences
encompassed
by the
retroviral vector intact. Comparison
of band intensities relative to the
control lane indicated a copy number per cell of approximately
ten.
Digestion with BstEll (lane 4, cuts pZipNeoSV(SupAm)
plasmid once)
produced a major band with mobility of the unit-length plasmid, suggesting that most of the transfected
DNA has integrated in intact, headto-tail concatenates,
Digestion with BamHl (lane 5, does not cut pZipNeoSV(SupAm)
plasmid) produced predominantly
two slow migrating
bands, consistent
with the concatenates
being integrated at two sites.
(B) Kinetic analysis of inducible amplification.
Total cellular DNA was
extracted
1, 2, 3, and 4 days after shift-down
to 33% digested with
Xbal (lanes lo-13), and compared to samples containing lo’, 103, and
lo4 copies per cell equivalents
of Xbal-cut pZipNeoSV(SupAm)
plasmid DNA (lanes 7-9). Assuming
that all cells in the culture amplified
the neo gene uniformly, a copy number of 2,000-4,000
per cell was observed 48 hr after shift-down.
This level of amplification
then remained
constant for another 48 hr.
(C) Analysis of cells grown at 33°C for four days. Digestion with BstEll
(lane 15) and Sal1 (lane 16), each ofwhich cuts the pZipNeoSV(SupAm)
plasmid once in flanking sequences
(see diagram), produced a single
prominent band with the mobility expected for a unit-length plasmid.
The generation of apparently intact pZipNeoSV(SupAm)
plasmid DNA
during amplification
suggests that homologous
recombination
among
the tandemly integrated species may have occurred
during excision.
Digestion with Clal (lane 17) and Sphl (lane 18), which cut once and
twice, respectively,
in retroviral vector sequences
between the LTRs,
revealed two prominent bands. The mobilities of these bands are consistent with the presence of two episomal species, the larger species
being the intact pZipNeoSV(SupAm)
plasmid, and the smaller species
containing the proviral sequences
between the LTRs and one copy of
the LTR (see smaller circle in diagram). Such a “one LTR circle” could
be generated
by homologous
recombination
between the two tandemly repeated LTRs in pZipNeoSV(SupAm).
(The explanation for the
absence of a second prominent
band after digestion with BstEll or
Sall. which do not cut the “one LTR circle:’ is that covalently
closed
DNAs do not transfer to the hybridization
membrane
as efficiently as
linear DNAs.)
(D) Demonstration
of the episomal nature of the amplified DNA after
induction. Total DNA and Hirt supernatant
DNAs from parallel cultures
were isolated 24 and 48 hr after shifting to 33°C and digested with
EcoRI. The same number of cell equivalents
of DNA was added to
each lane. The amplified DNAs had identical EcoRl digestion patterns,
and were equally abundant in the total and Hirt supernatant
fractions.
The mobility of the slower band is consistent
with the pattern generated from intact pZipNeoSV(SupAm)
plasmid, while the mobility of the
faster band is that expected for a linear DNA derived from a ‘one LTR
circle” episome (see diagram).
Plasmid diagrams: All enzymes shown in A-D are indicated. The upper diagram corresponds
to the plasmid pZipNeoSV(SupAm).
The
lower diagram corresponds
to a putative segregant
generated
by
reciprocal intramolecular
homologous
recombination
between the tandemly oriented LTRs in pZipNeoSV(SupAm).
An Inducible
363
Mammalian
Amber
Suppressor
LGTL
ACTTGGG
TC
A CGAAAGGTG
S@,o
Figure 4. Construction
of an Amber Mutation in the RNA Replicase
Gene of Poliovirus by In Vitro Site-Directed
Mutagenesis
The mutant was constructed
in an infectious cDNA clone of poliovirus
(plasmid pSV2polio) by direct replacement
of a small fragment of the
RNA replicase gene with a synthetic double-stranded
oligonucleotide,
as described
in Experimental
Procedures.
The stippled bar in the diagram designates
polio cDNA sequences,
and the solid line, vector sequences.
D, Dralll; H3. Hindlll; RI, EcoRI.
fected into BSC-40 (Sup-) or BSC4OsupDS (Sup+) cells
(see Experimental
Procedures).
The transfected cells
were overlayed with liquid medium and incubated at 33%.
A distinct cytopathic effect (CPE) was evident on the
Sup+ cells after approximately 6 days while no CPE was
observed on the Sup- cells even after extended incubation. The liquid stock from the Sup+ cells was harvested,
serially diluted, and plaqued on Sup+ and Sup- cells. At
low dilutions plaques were observed after 36 hr on both
Sup+ and Sup- cells, with no apparent differences in titer.
The size and morphology of these plaques were indistinguishable from those of wild-type poliovirus plated on control dishes. Several plaques were picked from both Sup+
WT
VIRUS
33”
39.5”
AMBER
VIRUS
33”
39.5”
BSC40
sup0
Figure
Amber
5. Plaque Assays
Mutant rep(am26)
Showing the Host Range of the PolIovIrus
on Suppressing
and Nonsuppressing
Cells
Plaque assays were done as described
in Experimental
Procedures.
Plaques were visualized by staining with crystal violet. Times of incubatlon were as follows: wild-type virus at 33%. 44 hr: wild-type virus
at 39.5%. 30 hr, amber virus at both temperatures,
96 hr. The suppressmg cell line used was BSC-4OsupD3.
and Sup- dishes; all isolates had equal titers on Sup+
and Sup- ceils. These rapidly developing plaques were
thus revertants generated during the growth of the amber
mutant virus on Sup+ cells.
When the liquid stock from the transfection of the Sup+
cells was titered at higher dilutions, smaller turbid plaques
were observed after extended incubation (96 hr). Such
plaques were seen only on the Sup+ cells. Several independent isolates were picked and plaque-purified
on
Sup+ cells. All of these isolates displayed the host range
properties expected of a nonsense mutant. A single, twice
plaque-purified
isolate was chosen for further study, and
designated rep(am28). Figure 5 shows a complete host
range plaque assay experiment for the rep(am28) mutant,
including controls for temperature effects and the presence of the SV40 large T antigen. As can be seen, the
rep(am28) mutant produced plaques only on the BSC4OsupD3 cell line, and only at 33%. Thus the presence of
plaques correlated strictly with the presence of the ambersuppression phenotype in the host.
Characterization
of the Amber Mutant
Poliovirus rep(am28)
In addition to the expected biological properties of the mutant virus, namely the host range growth characteristics,
it was necessary to demonstrate physically the presence
of the am28 allele in the mutant genomes. RNA extracted
from purified virions of the rep(am28) mutant, as well as
five independent wild-type revertants of the am28 mutation, were sequenced by the dideoxynucleotide
chain termination method (Sanger et al., 1977) using avian myeloblastosis virus (AMV) reverse transcriptase essentially as
described by Burke and RajBhandary
(1982) (see Experimental Procedures). The strategy, shown in Figure 6,
employed a synthetic oligonucleotide
primer complementary to a viral sequence near the am28 locus. The results
show that the mutant virion RNA has the sequence UAG
at the expected position (Figure 6).
The sequence of the five revertant RNAs showed, surprisingly, that in all cases, the amber codon had mutated
by an A to C transversion to the serine codon UCG. Since
the amber codon (UAG) can mutate by a single base
change to six different amino acid codons, including tyrosine (UAU, UAC), our data indicate that the serine at amino
acid position 28 in the RNA replicase of poliovirus is
functionally constrained.
Growth Characteristics
and Reversion Frequency
of the rep(am28) Mutant Virus
Growth of multi-cycle liquid stocks of amber virus proved
to be difficult because of the rapid outgrowth of wild-type
revertants in a few generations. Both the frequency of
reversion and the relative growth rates of mutant versus
revertant virus probably contributed to this problem.
The frequency of reversion was determnned in the following manner. A single plaque of the rep(am28) mutant
virus was picked, titered, and re-plated on Sup+ cells to
obtain well-isolated
plaques. Numerous plaques were
picked and individually titered on Sup+ and Sup- cells.
Assuming that each mixed plaque represented a single
reversion event, the reversion frequency was calculated to
Cell
384
WILD
TYPE
AMBER
GATC
MUTANT
REVERTANT
GATC
GATC
Figure 6. Sequence
Analysis of Virion RNAs
of Amber Mutant and Revertant
Polio Viruses
Sequencing
of virion RNA was performed
by
the dideoxynucleotide
termination
method, using the specific synthetic DNA oligonucleotide
primer shown in the figure, as described
in Experimental
Procedures.
Vlrion
RNA
” AGCUU~CCCA~~UUL~~ACUAUGUGUUVGAAGGGGUC
TCA
SERINE
CCCCACTTCClTGGTCGTCAffi
Sequencing
Prlmar
1
1
UCG
AGC
SERINE
be approximately
2.5 x 10m6 revertants/amber
plaque
forming units (pfu) (see Experimental Procedures for derivation).
Two parameters of virus growth were investigated: the
generation time (eclipse period) and the burst size per cell
(see Experimental Procedures). A single growth cycle of
the rep(am28) mutant virus on BSC4OsupD3
cells was
about three times longer than the wild-type cycle, the
eclipse periods being approximately 15-18 hr and 5-6 hr,
respectively. The burst size per cell was determined to be
approximately
10 pfu for the amber mutant virus and approximately 100 pfu for the wild-type virus. Thus, in addition to growing slower, the amber mutant virus produces
approximately lo-fold less progeny per infected cell than
the wild-type virus.
Induction of Suppressor
Activity in BSC-4OsupD Cells
As evaluated by transfection with a cat amber mutant
gene, the BSC4OsupD72 cell line generated an appreciably higher level of suppression as compared to the BSC4OsupD3
cell line (Figure 2C). The consequences
of this
higher level of suppression were clearly reflected by the
mutant poliovirus generated
size of plaques the rep(am28)
on the BSC4OsupD72
cells. Plaques of equivalent size
(approximately 3 mm in diameter) were formed after 72 hr
on the BSC4OsupD72 cell line, but only after 96 hr on the
BSC40supD3
cell line (wild-type virus produced equivalent size plaques on both cell lines after 36 hr). In addition,
the plaques on the BSC4OsupD3 cell line were more turbid. Microscopic examination revealed that the turbidity
was due to the apparent survival of a fraction of the cells
within a plaque, probably indicating that not all cells in the
population can be uniformly induced to the Sup+ phenotype. In addition, the efficiency of plaque formation of the
rep(am28)
mutant was approximately 3- to 5fold higher on
the BSC4OsupDIP
line.
cell line than on the BSC4OsupD3
cell
Discussion
The efficiency of suppression of termination at a nonsense codon should reflect the competition between the
activity of a Sup+ tRNA, promoting read-through,
and
that of a translational release factor, promoting termination. Little is known about the level of release factor activity in mammalian cells. The steady-state level of a typical
tRNA species in a mammalian cell is thought to be the
product of transcription
from a pool of approximately
lo-40 genes. Thus it was not surprising that introduction
of a few Sup+ tRNA genes per cell, using selection for a
linked dominant marker, yielded cell lines that did not express appreciable
suppressor activity. This combined
with other indications that constitutive suppression levels
above 5% are deleterious to mammalian cell viability
probably explains why it has thus far not been possible to
isolate mammalian cells carrying nonsense suppression
using genetic selection.
We have developed a generally applicable protocol for
controlled, rapid gene amplification in cell culture, which
allows the generation of high levels of suppression. This
protocol yielded cells with suppression
efficiencies as
high as 50%. To control tRNA gene amplification,
the
Sup+ tRNA gene was covalently linked to the SV40 origin
of replication as well as to a selectable marker (neo), and
cotransfected into monkey cells along with a vector encodmutant of the SV40
ing the &A58 temperature-sensitive
large T antigen gene (Tegtmeyer, 1972; DePamphilis and
Bradley, 1986). Cells were selected for G418 resistance at
the nonpermissive
temperature (39.5%) which resulted
An Inducible
385
Mammalian
Amber
Suppressor
in the stable integration of a few copies per cell of each
plasmid. When such cell lines were shifted from the nonpermissive to the permissive temperature (33%), the A
protein stimulated initiation of replication at the SV40 origin, which resulted in the amplification of the linked tRNA
gene to levels of 2,000-4,000 copies per cell. This amplification, as an episomal DNA, was complete within 48 hr,
and occurred in the majority of the cells. Concomitant with
the amplification of the Sup+ tRNA gene, cell division
ceased, but viability for virus growth was retained for at
least two weeks. (While this work was in progress, two amplification systems similar in concept were reported, one
based on SV40 [Portela et al., 19861 and the other on polyoma virus [Kern and Basilica, 19861.)
The levels of suppression achievable with the inducible
cell lines described here should allow genetic analyses
based on conditional-lethal
amber mutations in a variety
of animal viruses. To explore the utility and scope of this
methodology, an amber mutant of poliovirus was isolated.
The amber mutation was introduced into the RNA replicase gene of a cDNA clone of poliovirus by in vitro sitedirected mutagenesis. Amber mutant virus was generated
from the mutated cDNA clone by transfection of Sup+
cells induced to express suppression activity. After plaque
purification, the mutant virus, rep(am28), was shown to
contain the expected amber mutation by direct sequence
analysis of virion RNA. The rep(am28) mutant virus displayed the expected host range: it formed plaques only on
Sup+ cell lines. On the strongest suppressor cell line, the
rep(am28) mutant virus had a 2-fold longer eclipse period
and a lo-fold smaller burst than wild-type virus. Both of
these traits may be due to a reduced rate of RNA replication caused by incomplete suppression of the amber mutation. This effect may be further aggravated by the fact
that mutations in the replicase gene cannot be complemented in trans (Bernstein, 1987).
The mutation rate of poliovirus was previously estimated to be as high as 10m3 to 10m4 per nucleotide per
replication event (Holland et al., 1982). It has been suggested that this high frequency may be important in permitting picornaviruses to evolve during passage between
susceptible hosts. Measurement of a precise mutational
rate for poliovirus has, however, not been possible. Calculation of mutational rates from the frequency of generation
or reversion of temperature-sensitive
mutations is difficult
because of the uncertainty in the number of potential
nucleotide changes involved. Similarly, the relationship
between the frequency of generation of guanidine resistant variants (2 x lo-‘) and mutational frequency is unclear since the resistant phenotype may require multiple
nucleotide changes (Pincus et al., 1987). In general, the
replication of RNA genomes is thought to be much more
error-prone than DNA replication, since the former process does not possess the exonuclease-type
proofreading function typical of the latter process. As an example
of the high mutation rate of RNA genome replication, the
error level in RNA bacteriophage
Q3 has been precisely
measured and is between 10e3 to 10m4 per base (Batschelet et al., 1976).
The reversion frequency of the amber mutant rep(am28)
virus has been measured as a single nucleotide change
that can generate wild-type virus. Five revertant viruses
have been sequenced
and each was found to have
reverted by mutation of the amber codon, UAG, to the serine codon, UCG. The frequency of this nucleotide change
was approximately 2.5 x 10m6. This represents an upper
limit as any inefficiency in the titering of mutant virus
would decrease the value. Since only one of the three possible nucleotide changes seems to generate revertant virus, the mutational rate at this locus is 7.51 x 10m6 (approximately 10m5), a rate one to two orders of magnitude
lower than the above mentioned values. An upper limit for
the mutational rate of poliovirus has recently been estimated to be 2 x lo+ by the sequencing of the VP1 gene
of randomly isolated virus stocks (Parvin et al., 1986). Approximately 100,000 nucleotides in total were sequenced
without detection of a mutation. This estimate is qualified
by the limitation that only spontaneous variants that replicate efficiently would have been included in the pool that
was sequenced (Domingo et al., 1978). In conclusion, the
mutational rate at one locus in poliovirus has been measured and is significantly lower than previous estimates.
Whether this rate is typical of other loci awaits the isolation
of more nonsense mutants.
As mentioned above, all the wild-type revertants of the
rep(am28) mutant encoded a serine in place of the amber
codon. This was surprising in that an amber codon can
mutate by a single base change to six ditferent amino
acids, including tyrosine (UAU, UAC). Since it is unlikely
that the limited variety of revertants reflects constraints on
the RNA structure of the genome, the RNA replicase of
poliovirus is probably functionally constrained to have a
serine at position 28. This conclusion is also supported by
the finding that RNA replicases of five related picornaviruses: polio, rhino, coxsackie, endomyelocarditis,
and
foot-and-mouth disease contain either a serine or a threonine at this position, which is adjacent to an invariant proline at position 27, as well as several other invariant
residues in the vicinity (Figure 7). The overall homology
between the five proteins varied from 76% (polio/rhino) to
28% (polio/foot-and-mouth
disease), with three distinct
domains of homology apparent (the first is shown in Figure 7). Since threonine is considered a more conservative
replacement of serine than tyrosine, the analysis of the
revertants suggests that an aliphatic hydroxyl group in a
certain stereochemical environment is required at this position in the enzyme.
The genetic analysis of many DNA and RNA animal
viruses depends crucially on the isolation and characterization of mutants. For most viruses, the suppression system developed here should be adequate for the propagation of nonsense mutants. Poliovirus was selected as a
difficult example since its genome is expressed as a single polyprotein from which all virion and catalytic proteins
are processed. The efficiency of suppression of a nonsense codon in this polyprotein was expected to be critical
for virus reproduction. Nonsense mutants in genes encoding catalytic and structural proteins of other viruses
Cell
386
30
50
RH1:
* .
PIINAPSKTKLEPSZWHYVFEGVKEPAVLTKNDPRLKTD
I
I II II I II I IIIIII
NPVNTATKSKLHPSVFYDVFPGDKEPAVLSDNDPRLEVK
cox :
PVINTPSKTKLEPSVFHQVFVGNKEPAVLRSGDPRLKAN
FMD:
RVL.VMRKTKLXC?TVAHGVFNPEFGPAALSNWPRLNEG
EMC:
PRIHVPRKTALRPTVARQWQPAYAPAVLSKFDPRTEAD
POL :
II
I
III1
II
I IIIIII
liii
I I I
ii
II
I
IIII
II
II
III
I IIII
II
IIIIII
K
Figure
teins
7. Homology
IIIII
I II
I III
LP
Alignment
I
II
IIII
VF
PA L
of Picornavirus
Kaufman, unpublished
data); (4) pZipSV4OtsA58,
which is identical to
plasmid pZipSV40 (Jat et al., 1986), except that it contains the tsA.58
mutation (Tegtmeyer, 1972) in the large T antigen coding sequence (Jat
and Sharp, unpublished
data); (5) the cat nonsense
suppression
reporter plasmids pRSVcat, pRSVcaf(am27)
and pRSVcat(oc27)
(Capone et al., 1986); (6) pUCtS Su+(Am), a clone of the human serine
amber suppressor
tRNA gene (Capone et al., 1985). The retroviral vectors pZipNeoSV(X)l
and pZipGpt were used to leave open a future option of utilizing infection for the introduction
of the Sup+ tRNA gene
into cells, although this method has not been investigated
to date.
IIII
III
DPR
RNA Replicase
Pro-
The protein sequences
of five picornaviruses,
poliovirus (POL), rhinovirus @HI), coxsackievirus
(COX), foot-and-mouth
disease virus (FMD),
and endomyefocarditis
virus (EMC). were compared
pairwise using
the program ALIGN, as described
by the Protein Identification
Resource (PIR) report ALI-1284, National Biomedical
Research Foundation, Georgetown
University
Medical Center. A penalty of six was assigned to a break, 25 random runs were performed
for each pair, and
the mutation data matrix of Dayhoff et al. (1979) was used. The amino
acid numbering
order is based on the poliovirus protein, and amino
acids 15-52 are shown. The asterisk indicates Ser28, whose codon
was changed to an amber codon in the rep(am28) mutant.
should not be more difficult to grow than poliovirus mutants. The high mutation frequency of poliovirus also
makes it a difficult test. This mutation rate is approximately two orders of magnitude greater than that of a typical DNA replication system where replication errors are
continuously edited. The mutational rate of poliovirus is,
however, probably typical of other viruses with RNA genomes, including retroviruses.
Experimental
Procedures
Genetic
Nomenclature
The standard nomenclature
(Demerec
et al., 1966) as now employed
for E. coli (Bachmann,
1983) has been adopted. In accordance
with
the E. coli convention,
the designation
supD is used to indicate a serine inserting amber suppressing
tRNA. The supD gene used in all experiments
presented
here was derived by site-specific
mutagenesis
from a human serine tRNA gene (Capone et al., 1985).
Strains,
Cell Lines, and Plasmids
All plasmid cloning and large scale
DNA preparation
F-afaaD139A(am
galU ga/K hsr-
was in strain JS4
hsm+ strA mcA7,
whrch is strain MC1061 (Casadaban
and Cohen, 1980) made Ret- by
Pl cotransduction
with sr/:TnlCJ from strain SY925, followed by removal
of the TnlO by Pl transduction
to Sri+ (strain SY925 was a gift from M.
Syvanen).
The mammalian
cell lines used were NIH3T3 (Todaro and
Green, 1963; obtained from N. Hopkins);
LMtk- (Hudziak et al., 1982);
and BSC-40 (Brockman
and Nathans, 1974; obtained from R. Condit).
Mahoney serotype
1 poliovirus was propagated
from a single plaque,
derived from a cDNA-containing
plasmid as described (Racaniello and
Baltimore, 1981). The following plasmids were used: (1) the retroviral
shuttle vector pZipNeoSV(X)l
(Cepko et al., 1984); (2) the retroviral
shuttle vector pZipGpt which is identical to pZipNeoSV(X)l,
except that
the E. coli guanine phoshoribosyl
transferase
(gpt) gene (Mulligan and
Berg, 1980) has been substituted for the neo gene, and the SV40 origin
of replication
has been deleted (R. Mulligan, unpublished
data); (3)
pR7A, a vector containing
a methotrexate
resistant mouse dihydrofolate reductase
(dhfr) cDNA, which is identical to plasmid pCVSVL
(Kaufman and Sharp, 1982) except that it contains two tandem copies
of the SV40 Avall-D fragment, and that it contains a mutation in the dhfrcoding sequence,
introduced
by site-directed
mutagenesis,
which
changes the leucine codon at position 22 to an arginine codon (R.
leu)7697A/acX74
Assay of cat Activity
Extracts were harvested
and assayed
as previously
described
(Capone et al., 1986). Assays were normalized
both for transfection
efficiency and protein content of the extracts.
Levels of suppression
are
shown expressed
as percentages
of the activity obtained from the wildtype cat gene, which was always transfected
in parallel on duplicate
dishes. For the cotransfection
controls, human growth hormone levels
rn the medium were measured
by a solid-phase
two-site radioimmunoassay
kit according
to the manufacturers
recommendations
(Hybritech
Inc.).
Site-Specific
Mutagenesis
pSV2polio(am28).
The mutagenic
oligonucleotides
5’AGCTTGAAC
CCTAGGC’TTTCCACTAT,
S-GTGGAAAGCATCGGGTTCA,
and the
sequencmg
primer oligonucleotide
5’-GGACTGCTGGTTCCTTCACCCC were synthesized
and purified as previously described
(Capone
et al., 1986). The two oligonucleotides
are complementary,
and, after
annealing
produce a duplex with Hindlll and Dralll cohesive
ends.
This double-stranded
oligonucleotide
was cloned by a three-part
ligation with gel-purified
EcoRI-Dralll
and EcoRI-Hindlll
fragments of
pSV2polio, as diagrammed
in Figure 4. Individual E. coli transformants
were screened
for the presence
of the extra Avrll site resulting from
the three base change from serine (AGT) to amber (TAG). The efficiency of mutagenesis
was essentially 100% (six out of six tested). The
sequence spanning the entire replaced region was verified directly by
dideoxynucleotide
sequencing
of one individual
isolate (data not
shown).
pZipNeo(Aml1).
The 2 kb BarnHI-EcoRI
fragment from pZipNeoSV(X)l, containing
the neo gene, was subcloned
into M13mp8, and
mutagenesis
was performed
using the oligonucleotide
B’-ACGCAGGTTAGCCGGCCGCT
by the gapped duplex procedure,
as described
by Capone et al., 1986. The introduced amber mutation creates a new
Nael site. After verification,
the neo(am77) mutation was reconstructed
into the pZipNeoSV(X)i
vector.
Transfection
and Plasmid DNAs into Mammalian
Cell Lines
Transfections
were done by the calcium phosphate
coprecipitation
method of Graham and van der Eb (1973) as modified by Parker and
Stark (1979) and contained 10 kg of DNA per 100 mm dish; carrier DNA
wes not used, and the glycerol shock was omitted. For stable transfections a chloroquine
diphosphate
shock of 50 Kg/ml for 3-4 hr (NIH3T3
cells) or 100 uglml for 4-6 hr (BSC-40 cells) was included (Luthman
and Magnusson,
1983). Cotransfections
were done at a 1O:l mass ratio
of nonselected
vs. selected DNAs. G418 concentrations
during selection of stable cell lines are expressed
as ug of active drug. Gpt selection medium was as described
(Mulligan and Berg, 1981), except that
glycine was omitted and the concentrations
of mycophenolic
acid and
methotrexate
were changed to 10 pg/ml and 0.25 NM, respectively.
For
transient transfections,
the efficiency
of transfection
was determined
by the inclusion of 1 pg of plasmid pXGH5, which contains the human
growth hormone gene (Selden et al., 1986) in all transfections.
Standard
Culture Conditions
of Suppressing
Cell Lines
After the initial screening,
the cell lines BSC-4OsupD3,
BSC-4OsupD7,
and BSC-4OsupO5,
were thawed from original stocks, and reselected
for resistance to G418 (290 vglml) for five generations
at 39.5%. Afterwards, these cell lines were passaged
at a dilution of I:50 every 5-6
days in the absence of G418. Utmost care was taken to minimize exposure to temperatures
below 39.5% during all handling procedures.
Cells for all subsequent
experiments
were withdrawn
from this standard regimen, as required.
An Inducible
387
Mammalian
Amber
Suppressor
Quantitative
Growth
Properties
of Suppressing
Cell Lines
The growth curves at 39.5% of the cell lines BSC-40, BSC4OsupO5,
BSC4OsupD3,
and BSC4OsupD7
at passage 4. as defined by the
standard regimen, above, were identical (data not shown). This was not
the case at 33%. where the cell line BSC4OsupD3
showed a coldsensitive phenotype, while the other three lines grew at essentially the
same rate. As discussed
in the text, these results showed that the cell
line BSC4OsupD7
had lost its cold sensitive phenotype by passage 4.
The total culture of BSC4OsupD3
cells arrested growth after shifting
to 33OC. suggesting
that the majority of cells were induced for expression of suppressor
activity.
Stability
of the 6418 Resistance
Phenotype
in Suppressing
Cell Lines
The efficiency
of plating (EOP) in the presence and absence of G418
was determined
to compare
the stabilities
of the cell lines BSC4OsupD3 and BSC40supD7
in maintaining
the neo gene, which is
linked to the Sup+ tRNA gene. Cells were withdrawn
from the 150
passaging regrmen in the absence of G418 (as defined above) at passage 4, trypsinized,
serially diluted, and plated with and without G418
selection at 39.5%. Dishes were stained after two weeks of incubation
and counted. A BSC40neo
cell line was included as a stable control.
This line had previously
been generated by transfection
with the plasmid pZipNeoSV(X)l.
The EOP ratios (-G418/+G418;
330 Kg/ml) were
as follows: BSC40neo,
3; BSC4OsupD3
at passage
4, 10; BSC4OsupD3 at passage
10, 10; BSC4OsupD7
at passage 4, 100; BSC4OsupD7 at passage 10. >104. The BSC4OsupD3
ceil line thus appears to be stable when cultured at 39.5%. This was not the case for
the BSC4OsupD7
cell fine, which segregated
the G418 resistance
phenotype.
The BSC4OsupD7
cell line originally (passage
1) expressed a constitutive
5% level of suppression
at 39.5% (Figure 2). Not
surprisingly,
suppressor
activity was not detected after passage 10
(data not shown).
Induction
of the Suppressor
Phenotype
Several procedures
were tested for their efficiency of induction of the
amber-suppressing
phenotype, as judged by the size of plaques of the
poke rep(am28) mutant virus. The best protocol was as follows: Sup’
ceils, grown continuously
at 39.5%. were split from a confluent 10 cm
dash into four 6 cm dashes for the BSC4OsupD3
cell line, or into three
6 cm dshes for the BSC40supD12
cell line. These dishes were placed
immediately at 33%. Incubated for 24 hr, re-fed with fresh medium, incubated further for 24 hr at 33OC, and finally infected with virus.
Preparation
of Virus Stocks
Weld-type and amber poliovrrus plaques typically contained about IO5
and lo4 pfu, respectively.
Stocks of wild-type poliovirus were made by
Infecting BSC-40 monolayers
at a multiplicity of infection (moi) of approximately
0.01, overlaying
wrth liquid medium, and rncubatmg
for
several days until all cells displayed CPE. Stocks of amber mutant
polrovrrus
were grown on BSC-4OsupD72
cells. Individual plaques
were first tested for the presence
of revertant pfu’s by plaque assays
on BSC-40 cells. Plaques that contained no detectable revertants were
pooled and concentrated
by ultracentrifugation
(Beckman
50Ti rotor,
45,000 rpm at 20°C for 1 hr), and resuspended
in a small volume of
PBS. BSC-4OsupD72
monolayers
were infected at an mar of 0.2-0.5,
then treated as described
above for the wild-type virus.
The eclrpse periods were determined
by infecting induced BSC4OsupD3 cells at an mar of approximately
0.05 with either rep(am28)
or wild-type vrrus, and samphng the medium at hourly intervals. The
burst srzes were determined
by infecting induced BSC4OsupD3
cells
at an mar of 0.2 After 18 hr for the mutant virus, or 6 hr for the wild-type
virus, both the medium and the cells were harvested,
the latter were
lysed by freezing and thawing, and the two fractions ware pooled and
htered
Since vrnons m a plaque are concentrated
in a very small volume
by the agar overlay, Infection of neighboring
calls is very efficrent.
Thus, the growth rate of a plaque would be primarily determrned by the
generatlon time of the vrrus, and less by the burst size. In liqurd growth,
however, re-Infection
IS limited by the large dilution factor, and the burst
size can be expected to have profound effects on the overall growth
rate. The resultant exaggerated
growth advantage caused the rapid accumulation of revertant viruses in stocks of intact vrrus when passaged
!n mass culture. For this reason, it has proven difficult to grow the
rep(am28) mutant vrrus to high titers.
Determination
of the Reversion
Frequency
Well isolated amber plaques were picked after 3, 4, and 5 days of incubation (nine plaques for each day), and individually
titered on BSC4OsupD3 and BSC-40 cells. Four plaques out of the 27 contained wildtype revertants (one each from days 3 and 4. and two from day 5). The
individual plaque titers ranged from 3 x lo3 pfulplaque to 3.7 x IO4
pfu/plaque; the average value was 1.8 x IO4 pfulplaque, and the total
number of amber pfu in the 27 plaques was 4.8 x 10s. A Poisson distribution analysis gave 95% probability of recovering
one revertant in
1.3 x lo5 pfu. Using a value of 3 as the EOP ratio of the rep(am28)
mutant virus on the BSC-4OsupD72
vs. the BSC4OsupD3
cell line, this
value becomes 1 in 3.9 x 105. or 2.5 x lo-‘% The true reversion frequency IS probably even lower, since the rep(am28) mutant virus may
have a higher pfulvirion ratio than the wild-type virus. An indication of
this comes from the sequencing
experiments,
where approximately
5-fold less pfu equivalents
of rep(am28) than wild-type virus produced
almost identical signals.
Preparation
of Virion RNA for Sequencing
Cells and medium were harvested,
pooled, frozen, and thawed three
times to lyse the cells, cleared of cellular debris by low-speed centrifugation, and made up to 2 mM EDTA and 0.5% (w/v) SDS. Virions were
concentrated
by ultracentrifugation
as above and resuspended
in TNE
(50 mM Tris [pfi 7.51, 0.1 M NaCI, 0.1 mM EDTA) with 0.5% SDS to a
concentration
of approximately
5 x 10’ pfulml. RNA was extracted
with an equal volume of TNE equilibrated phenol, followed by one extraction with phenol/chloroform
(II), and one extractton with chloroform. RNA was precipitated
with 3 volumes of ethanol in the presence
of 0.3 M NaCl and 100 frglml glycogen
as carrier
Sequence
Analysis
of Virion RNAs
The vrrion RNA sequence
in the vicinity of the mutagemzed
site was
determined by the dideoxynucleotide
triphosphate
termination method
of Sanger and Coulson (1977) using AMV reverse transcriptase
(BioRad) and a specific oligonucleotide
primer (see above) essentially as
described
by Burke and RajBhandary
(1982). Each sequencing
experiment contained
approximately
lo7 pfu equivalents
of wild-type
or
revertant virion RNAs, or 2 x 1Oa pfu equivalents
of virion RNA from
the amber mutant, and 65 frCi of [s*P]-dATP (split equally among the
four sequencing
reactions).
Acknowledgments
We gratefully acknowledge
Dr. Parmjit Jat for his interest, encouragement, and help In this project. We thank Dr. H G Khorana for making
the DNA synthesizer
available for our use, and Drs Michael Nassal,
Tatsushi Mogi, and Tom Sakmar for help with oligonucleotide
synthesis. We also thank Dr. David Baltimore for the clone of the poliovirus
cDNA, Dr. Marie Chow for help with the RNA sequencing,
Drs. Richard
Condit and Harris Bernstein for many stimulating discussions,
William
Gilbert for help with the computer
analysrs, and M. Srafaca for her
careful preparation
of the manuscript.
This work was supported
by
grants GM17151 from the National Institutes of Health and NPl14 from
the American Cancer Society to U. L. R., and by NIH grant GM32467,
National Science Foundation grants DCB-8502718, ancl CDR-8500003,
and partially NCI Cancer Center Core grant P30CAi4051
to P A. S.
J. P C. is the recipient of a centennial fellowshfp and J. M. S. is the recrprent of a postdoctoral
fellowship from the Medrcal Research Council
of Canada.
The costs of publication of this article were defrayed In part by the
payment
of page charges.
This article must therefore
be hereby
marked “advertisement”
in accordance
with 18 U.S C Sectron 1734
solely to rndrcate this fact.
Received
March
20, 1987; revised
May 15. 1987
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