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Identification of two lysine tRNA cistrons in Bacillus subtilis by hybridization of lysyl-tRNA with DNA
Vol. 43, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IDENTIFICATION OF TWOLYSINE tRNA CISTRONSIN BACILLUSSUBTILIS BY HYBRIDIZATIONOF LYSYL-tRNA WITH DNA Ronald Chuang, Toshio Yamakawa,and Roy H. Doi Department of Biochemistry and Biophysics University of California Davis, California 95616
Received February 2, 1971 Summary. Two lysyl-tRNA species were obtained from Bacillus subtilis by benzoylated-DEAE-cellulose column chromatography. They were found to hybridize in an additive fashion to 2. subtilis DNA. These results indicate that the base sequences of these two lysine tRNA species differ sufficiently to prevent the saturation of both genetic sites by either and indicate that there are at least two cistrons for lysine tRNA. Many examples of multiple reported (1,2).
Two general assumptions are that the tRNA species either
are products of different transcribed
tRNA species for an amino acid have been
genes or are different
modified forms of a tRNA
from one gene. In a sequence study it was found that two
serine tRNA.. from brewer's yeast differed the presence of two serine tRNA genes. that gene duplication
by 3 base changes (3) indicating In this particular
case it appears
had occurred followed by three mutations resulting
two serine tRNAs which were still
very similar
in structure
in
and function.
In previous studies of the lysine tRNA species of Bacillus subtilis during sporulation,
it was reported that two peaks of lysyl-tRNA
resolved by chromatographic columns (4-6). tRNA patterns and functions of -.B subtilis tRNA-DNAhybridization
cells
(7), we have found from
to DNAin an additive
of analysis allows one to distinguish sufficiently
In a continuing study of lysine
studies that two lysine tRNA species separated by
column chromatography hybridized
differ
were
two ieoaccepting
fashion.
This method
tRNA species which
in base sequence to prevent the saturation
of both
genetic sites by either. MATERIALSANDMETHODS Organism and medium: & subtilis
W23 was used as the source of tRNA, 710
BIOCHEMICAL
Vol. 43, No. 4, 1971
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and DNA. Cells were grown to a density of 5 x lo8
aminoacyl synthetase,
cells per ml in Penassay medium (Difco) at 37" C with vigorous shaking. Preparation of lysyl-tBNA:
The tBNA was prepared from cells as The aminoacyl synthetase was
described by von Ehrenstein and Lipmann (8). prepared generally
as described by Zubay (9) followed by chromatography
through Sephadex 675 and DSAE-sephadexA-50 to a stage where no RNaseactivity could be detected (Chuang and Doi, manuscript in preparation).
The reaction
mixture for the preparation
experiments
of lysyl-tBNA
contained in pmoles: 50, Tris-HCl buffer, ATP; 0.5, glutathione;
for the hybridization
pH 8.0; 2.5, MgC12; 5,
100-500 pg, tBNA; 58 pg, partially
purified
KCl;
0.5,
synthetase;
50 pg, bovine serum albumin; and 25 pCi 3H-lysine (50 Ci/mmole); in a total The reaction mixture was incubated at 37' C for 12 min.
volume of 0.20 ml.
The reaction was stopped by adding 0.2 ml of phenol saturated with 0.1 M sodium acetate buffer,
pH 5.
method (8) and the final buffer,
The lysyl-tl7NA was extracted
precipitate
was dissolved in 0.05 M sodium acetate
pH 5.0.
Column chromatography:
The benzoylated diethylaminoethyl-cellulose
(B-DEAE) column was prepared according to Gillam --et al. treatment of lysyl-tBNA cellulose
by the phenol
(10).
and chromatography of oligonucleotides
The Tl RNase on DEAE-
columns were performed by the method described by Ishida and
Miura (11). DNA-RNAhybridization: as described by Gillespie
DNA-RNAhybridization
was carried out essentially
and Spiegelman (12) and Weiss --et al.
denatured DNA (200 pg) was fixed onto nitrocellulose incubated in a total RNA, 2XSSCbuffer as indicated
filters
(13).
Alkaline
(27 mm) and
volume of 1.5 ml with 50%formamide, 1 mg/ml yeast pH 5.0, and 3H-lysyl-tBNA
(0.3 M NaCl + 0.03 M Na citrate),
for 3 hr at 30" C.
The filters
were washed with ZXSSC, treated
with BNaseT1 (2.5 Dg/ml) for 30 min at room temperature, washed again with ZXSSC, dried, E.
coli
and then counted in a scintillation
DNAor without
counter.
any DNAwere used as controls.
711
The filters
with
Vol. 43, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS When tRNA obtained from log phase cells of z. subtilis
was charged with
3 H-lysine and chromatographed through a B-DEAE-cellulose column, two peaks of 31i-lysyl-tRNA were obtained as illustrated
in Figure 1.
The fractions
in these peaks were pooled, concentrated by flash evaporation,
deacylated
by treatment at pH 8.8, and recharged with 3H-lysine illustrating were both functionally
active
tRNA fractions.
that they
Furthermore when the first
or second peak was passed through a B-DEAE-cellulose column a second time, it eluted from the column in the original peaks 1 (fractions
position.
130-150) and 2 (fractions
The lysine tRNA from
186-220) are designated as
lysine tRNAl and lysine tRNA2 through the rest of this paper.
6
FRACTION
Figure 1.
NUMBER
Elution profile of 3H-lysyl-tRNA from a B-DEAE-cellulose column. The open and closed CirCles represent A260 and Counts per minute, respectively. The squares indicate NaCl molarity for the elution gradient.
In a preliminary
test to determine whether the two lysine tRNA species
were different
in sequence, lysine tRNAl and lysine tRNA2 were charged with 14 3H-lysine and C-lysine, respectively, mixed together, and then digested 712
BIOCHEMICAL
Vol. 43, No. 4, 1971
with Tl BNase; the resulting
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lysyl-oligonucleotides
were chromatographed
through a DEAE-cellulose column to determine whether the two lysine tBNAs differed
in sequence from the first
guanine residue from the 3'terminus.
The results showed that lysyl-oligonucleotides
(with a chain length of 3
bases) from lysine tENAl and lysine tENA eluted in exactly These results in contrast tRNA (14) indicated
the sameposition.
to the previous results with g. subtilis
that the lysine tRNA species had identical
valine
T1 RNase
susceptible sequences near the amino acid accepting terminus. To test whether lysine tENAl and lysine tENA contained different sequences in the rest of the molecule, hybridization and lysyl-tBNA2
base
between lysyl-tENA
and DNAwas tested by the method of Weiss -et g.
(13).
Each of the pooled fractions
from the B-DEAE-cellulose column (Fig. 1) was 3 charged separately with 3H-lysine and the amount of H-lysyl-tENAl and of 3Ii-lysyl-tRNA2
which would hybridize
results are shown in Figure 2.
with DNAwas determined.
The ratio
of tENA to DNAabove 0.3 (i.e.
of tENA to 200 ng of DNA) resulted in saturation preparations
under the conditions used.
0
Since the samespecific
activity
of
the almost
counts with both tBNA preparations suggested
20
40 ,,g
Figure 2.
60 ng
of DNAsites for both tBNA
3H-lysine was used during charging of both tBNA preparations, equal number of hybridized
These
60 so of %I-lyryl-tRNA
100
120
Hybridization of 3H-lysyl-tENAl and 3H-lysyl-tRNA2 with 5. subtilis W23 DNA. The open and closed circles represent 3H-lysyl-tBNA1 (760 cpm per ng) and 3H-lysyl-tENA2 (1,170 cpm per ng), respectively. 713
Vol. 43, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that equal amounts of lysyl-tRNA1 14%deacylation
and lysyl-tRNA2 were hybridized.
occurred with control
samples of both tRNA preparations
during the experiment under incubation conditions identical hybridization
Only
of the
to that
reactions.
To determine whether the two lysine tRNA species were hybridizing two distinct
DNAsites,
the additive
nature of hybridization
adding saturating
levels of lysyl-tRNA1 and lysyl-tRNA2
the hybridization
reaction
presented in Table 1. tested first
Table 1.
Exp. No.
mixture.
The results from two experiments are
to determine the concentration
of lysyl-tRNA1
T
DNAon filter
was tested by
simultaneously to
For each experiment, the lysyl-tRNA
Hybridization
with
preparations were
of tRNA required for saturation
and lysyl-tRNA2
to DNA
1
3H-lysyl-tRNA hybridized tRNAy
1 tRNAys let cpm
la b C
2a
b
C
no DNA g. subtilis
W23
130
no DNA B. subtilis
W23
148
no DNA j& subtilis
W23
303
no DNA E. coli -. subtilis
W23
123
no DNA E. coli 5. subtilis
W23
121
no DNA E. coli z. subtilis
567*
320 339 533
W23 3
213
3H-lys~l-tRNAl (80 up) and H-lysyl-tRNA2 at saturating concentrations (80 ug or 100 ng ) were hybridized with denatured DNA (200 ng), respectively. In Exps. lc and 2c, both aminoacyl-tRNAs were added to the incubation mixture. 714
Vol. 43, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of the DNAbefore testing
for additive
hybridization.
were used for the results
reported in Figure 2 and Table 1.
experiments no aberrant hybridization
The samepreparations
was noted when up to 200 ng of lysyl-
tRNA was added to the reaction mixture,
i.e.
did not result
than the saturation
in greater hybridization
Figure 2.
These results
hybridized
to a given saturation
to the reaction mixture,
illustrate
In other
a large excess of lysyl-tRNA levels noted in
that lysine tRNAl and lysine
tRNA2 each
level and that when they were added together
they hybridized
in an additive
fashion.
DISCUSSION Two significant 1)
conclusions can be reached from these data:
These results indicate
whose base sequences differ two isoaccepting duplication,
that two lysine tRNA genes are present
sufficiently
to allow additive
If the genes were originally
tRNA species.
these results suggest that sufficient
during evolution
to prevent hybridization
locus of the other.
At first
hybridization
with
derived by gene
base changes have occurred
of one tRNA species to the genetic
glance these results are surprising;
however,
a change of only a few bases could lead to unstable heterologous hybrid formation in the usual manner or the base changes could give rise to two tRNA species which are modified quite differently
by the various base-modifying
enzymes with subsequent enhanced loss of stable hybrid formation with the heterologous DNAsequence. 2)
In this particular
distinguish
the origin
case the hybridization
of two isoaccepting
technique was able to
tRNA species.
Although this may
not be the case with other isoaccepting tRNAs, it would be judicious this additive
hybridization
test to other bacterial
systems, in which iso-
accepting tRNAs have been reported to determine their Obviously those isoaccepting may still distinguish
tRNAs which differ
genetic origin.
by only one or a few bases
bind to heterologous sites and this particular their
in this particular
origin.
However, the clear and readily
case indicate
test will
715
not
obtained results
that it is worth the effort
this basic information.
to apply
to acquire
Vol. 43, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS This research was supported by National Science Foundation Grant No. GB-8405 and U. S. Atomic Energy CommissionGrant No. AT(O4-3)-34.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14.
N. Sueoka and T. Kano-Sueoka, Prog. Nucleic Acid Res. & Molec. Biol. l& 23 (1970). G. D. Novelli, Ann. Rev. Biochem. 36, Part II, 449 (1967). H. G. Zachau, D. ktting, and H. Feldman, Angew. Chemie. Intern. Ed. Engl. 2, 422 (1966). R. A. Lazzarini, Proc. Natl. Acad. Sci. U. S. 56, 185 (1966). R. A. Lazzarini and E. Santangelo, J. Bacterial. 94, 125 (1967). B. Vold, J. Bacterial. 102, 711 (1970). B. Goehler and R. H. Doi, Proc. Natl. Acad. Sci. U. S. 56, 1047 (1966). G. Von Ehrenstein and F. Lipmann, Proc. Natl. Acad. Sci. U. S. 47, 941 (1961). G. Zubay, J. Mol. Biol. 4, 347 (1962). I. Gillam, S. Millward, D. Blew, M. von Tigerstrom, E. Wimmer, and G. M. Tener, Biochemistry 2, 3043 (1967). T. Ishida and K. Miura, J. Mol. Biol. 11, 341 (1965). D. Gillespie and S. Spiegelman, J. Mol. Biol. 2, 829 (1965). S. B. Weiss, W.-T. Hsu, J. W. Foft, and N. H. Scherberg, Proc. Natl. Acad. Sci. U. S. 61, 114 (1968). R. H. Doi, I. Kaneko, and R. T. Igarashi, J. Bfol. Chem.243, 945 (1968).
716
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IDENTIFICATION OF TWOLYSINE tRNA CISTRONSIN BACILLUSSUBTILIS BY HYBRIDIZATIONOF LYSYL-tRNA WITH DNA Ronald Chuang, Toshio Yamakawa,and Roy H. Doi Department of Biochemistry and Biophysics University of California Davis, California 95616
Received February 2, 1971 Summary. Two lysyl-tRNA species were obtained from Bacillus subtilis by benzoylated-DEAE-cellulose column chromatography. They were found to hybridize in an additive fashion to 2. subtilis DNA. These results indicate that the base sequences of these two lysine tRNA species differ sufficiently to prevent the saturation of both genetic sites by either and indicate that there are at least two cistrons for lysine tRNA. Many examples of multiple reported (1,2).
Two general assumptions are that the tRNA species either
are products of different transcribed
tRNA species for an amino acid have been
genes or are different
modified forms of a tRNA
from one gene. In a sequence study it was found that two
serine tRNA.. from brewer's yeast differed the presence of two serine tRNA genes. that gene duplication
by 3 base changes (3) indicating In this particular
case it appears
had occurred followed by three mutations resulting
two serine tRNAs which were still
very similar
in structure
in
and function.
In previous studies of the lysine tRNA species of Bacillus subtilis during sporulation,
it was reported that two peaks of lysyl-tRNA
resolved by chromatographic columns (4-6). tRNA patterns and functions of -.B subtilis tRNA-DNAhybridization
cells
(7), we have found from
to DNAin an additive
of analysis allows one to distinguish sufficiently
In a continuing study of lysine
studies that two lysine tRNA species separated by
column chromatography hybridized
differ
were
two ieoaccepting
fashion.
This method
tRNA species which
in base sequence to prevent the saturation
of both
genetic sites by either. MATERIALSANDMETHODS Organism and medium: & subtilis
W23 was used as the source of tRNA, 710
BIOCHEMICAL
Vol. 43, No. 4, 1971
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and DNA. Cells were grown to a density of 5 x lo8
aminoacyl synthetase,
cells per ml in Penassay medium (Difco) at 37" C with vigorous shaking. Preparation of lysyl-tBNA:
The tBNA was prepared from cells as The aminoacyl synthetase was
described by von Ehrenstein and Lipmann (8). prepared generally
as described by Zubay (9) followed by chromatography
through Sephadex 675 and DSAE-sephadexA-50 to a stage where no RNaseactivity could be detected (Chuang and Doi, manuscript in preparation).
The reaction
mixture for the preparation
experiments
of lysyl-tBNA
contained in pmoles: 50, Tris-HCl buffer, ATP; 0.5, glutathione;
for the hybridization
pH 8.0; 2.5, MgC12; 5,
100-500 pg, tBNA; 58 pg, partially
purified
KCl;
0.5,
synthetase;
50 pg, bovine serum albumin; and 25 pCi 3H-lysine (50 Ci/mmole); in a total The reaction mixture was incubated at 37' C for 12 min.
volume of 0.20 ml.
The reaction was stopped by adding 0.2 ml of phenol saturated with 0.1 M sodium acetate buffer,
pH 5.
method (8) and the final buffer,
The lysyl-tl7NA was extracted
precipitate
was dissolved in 0.05 M sodium acetate
pH 5.0.
Column chromatography:
The benzoylated diethylaminoethyl-cellulose
(B-DEAE) column was prepared according to Gillam --et al. treatment of lysyl-tBNA cellulose
by the phenol
(10).
and chromatography of oligonucleotides
The Tl RNase on DEAE-
columns were performed by the method described by Ishida and
Miura (11). DNA-RNAhybridization: as described by Gillespie
DNA-RNAhybridization
was carried out essentially
and Spiegelman (12) and Weiss --et al.
denatured DNA (200 pg) was fixed onto nitrocellulose incubated in a total RNA, 2XSSCbuffer as indicated
filters
(13).
Alkaline
(27 mm) and
volume of 1.5 ml with 50%formamide, 1 mg/ml yeast pH 5.0, and 3H-lysyl-tBNA
(0.3 M NaCl + 0.03 M Na citrate),
for 3 hr at 30" C.
The filters
were washed with ZXSSC, treated
with BNaseT1 (2.5 Dg/ml) for 30 min at room temperature, washed again with ZXSSC, dried, E.
coli
and then counted in a scintillation
DNAor without
counter.
any DNAwere used as controls.
711
The filters
with
Vol. 43, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS When tRNA obtained from log phase cells of z. subtilis
was charged with
3 H-lysine and chromatographed through a B-DEAE-cellulose column, two peaks of 31i-lysyl-tRNA were obtained as illustrated
in Figure 1.
The fractions
in these peaks were pooled, concentrated by flash evaporation,
deacylated
by treatment at pH 8.8, and recharged with 3H-lysine illustrating were both functionally
active
tRNA fractions.
that they
Furthermore when the first
or second peak was passed through a B-DEAE-cellulose column a second time, it eluted from the column in the original peaks 1 (fractions
position.
130-150) and 2 (fractions
The lysine tRNA from
186-220) are designated as
lysine tRNAl and lysine tRNA2 through the rest of this paper.
6
FRACTION
Figure 1.
NUMBER
Elution profile of 3H-lysyl-tRNA from a B-DEAE-cellulose column. The open and closed CirCles represent A260 and Counts per minute, respectively. The squares indicate NaCl molarity for the elution gradient.
In a preliminary
test to determine whether the two lysine tRNA species
were different
in sequence, lysine tRNAl and lysine tRNA2 were charged with 14 3H-lysine and C-lysine, respectively, mixed together, and then digested 712
BIOCHEMICAL
Vol. 43, No. 4, 1971
with Tl BNase; the resulting
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lysyl-oligonucleotides
were chromatographed
through a DEAE-cellulose column to determine whether the two lysine tBNAs differed
in sequence from the first
guanine residue from the 3'terminus.
The results showed that lysyl-oligonucleotides
(with a chain length of 3
bases) from lysine tENAl and lysine tENA eluted in exactly These results in contrast tRNA (14) indicated
the sameposition.
to the previous results with g. subtilis
that the lysine tRNA species had identical
valine
T1 RNase
susceptible sequences near the amino acid accepting terminus. To test whether lysine tENAl and lysine tENA contained different sequences in the rest of the molecule, hybridization and lysyl-tBNA2
base
between lysyl-tENA
and DNAwas tested by the method of Weiss -et g.
(13).
Each of the pooled fractions
from the B-DEAE-cellulose column (Fig. 1) was 3 charged separately with 3H-lysine and the amount of H-lysyl-tENAl and of 3Ii-lysyl-tRNA2
which would hybridize
results are shown in Figure 2.
with DNAwas determined.
The ratio
of tENA to DNAabove 0.3 (i.e.
of tENA to 200 ng of DNA) resulted in saturation preparations
under the conditions used.
0
Since the samespecific
activity
of
the almost
counts with both tBNA preparations suggested
20
40 ,,g
Figure 2.
60 ng
of DNAsites for both tBNA
3H-lysine was used during charging of both tBNA preparations, equal number of hybridized
These
60 so of %I-lyryl-tRNA
100
120
Hybridization of 3H-lysyl-tENAl and 3H-lysyl-tRNA2 with 5. subtilis W23 DNA. The open and closed circles represent 3H-lysyl-tBNA1 (760 cpm per ng) and 3H-lysyl-tENA2 (1,170 cpm per ng), respectively. 713
Vol. 43, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that equal amounts of lysyl-tRNA1 14%deacylation
and lysyl-tRNA2 were hybridized.
occurred with control
samples of both tRNA preparations
during the experiment under incubation conditions identical hybridization
Only
of the
to that
reactions.
To determine whether the two lysine tRNA species were hybridizing two distinct
DNAsites,
the additive
nature of hybridization
adding saturating
levels of lysyl-tRNA1 and lysyl-tRNA2
the hybridization
reaction
presented in Table 1. tested first
Table 1.
Exp. No.
mixture.
The results from two experiments are
to determine the concentration
of lysyl-tRNA1
T
DNAon filter
was tested by
simultaneously to
For each experiment, the lysyl-tRNA
Hybridization
with
preparations were
of tRNA required for saturation
and lysyl-tRNA2
to DNA
1
3H-lysyl-tRNA hybridized tRNAy
1 tRNAys let cpm
la b C
2a
b
C
no DNA g. subtilis
W23
130
no DNA B. subtilis
W23
148
no DNA j& subtilis
W23
303
no DNA E. coli -. subtilis
W23
123
no DNA E. coli 5. subtilis
W23
121
no DNA E. coli z. subtilis
567*
320 339 533
W23 3
213
3H-lys~l-tRNAl (80 up) and H-lysyl-tRNA2 at saturating concentrations (80 ug or 100 ng ) were hybridized with denatured DNA (200 ng), respectively. In Exps. lc and 2c, both aminoacyl-tRNAs were added to the incubation mixture. 714
Vol. 43, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of the DNAbefore testing
for additive
hybridization.
were used for the results
reported in Figure 2 and Table 1.
experiments no aberrant hybridization
The samepreparations
was noted when up to 200 ng of lysyl-
tRNA was added to the reaction mixture,
i.e.
did not result
than the saturation
in greater hybridization
Figure 2.
These results
hybridized
to a given saturation
to the reaction mixture,
illustrate
In other
a large excess of lysyl-tRNA levels noted in
that lysine tRNAl and lysine
tRNA2 each
level and that when they were added together
they hybridized
in an additive
fashion.
DISCUSSION Two significant 1)
conclusions can be reached from these data:
These results indicate
whose base sequences differ two isoaccepting duplication,
that two lysine tRNA genes are present
sufficiently
to allow additive
If the genes were originally
tRNA species.
these results suggest that sufficient
during evolution
to prevent hybridization
locus of the other.
At first
hybridization
with
derived by gene
base changes have occurred
of one tRNA species to the genetic
glance these results are surprising;
however,
a change of only a few bases could lead to unstable heterologous hybrid formation in the usual manner or the base changes could give rise to two tRNA species which are modified quite differently
by the various base-modifying
enzymes with subsequent enhanced loss of stable hybrid formation with the heterologous DNAsequence. 2)
In this particular
distinguish
the origin
case the hybridization
of two isoaccepting
technique was able to
tRNA species.
Although this may
not be the case with other isoaccepting tRNAs, it would be judicious this additive
hybridization
test to other bacterial
systems, in which iso-
accepting tRNAs have been reported to determine their Obviously those isoaccepting may still distinguish
tRNAs which differ
genetic origin.
by only one or a few bases
bind to heterologous sites and this particular their
in this particular
origin.
However, the clear and readily
case indicate
test will
715
not
obtained results
that it is worth the effort
this basic information.
to apply
to acquire
Vol. 43, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS This research was supported by National Science Foundation Grant No. GB-8405 and U. S. Atomic Energy CommissionGrant No. AT(O4-3)-34.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14.
N. Sueoka and T. Kano-Sueoka, Prog. Nucleic Acid Res. & Molec. Biol. l& 23 (1970). G. D. Novelli, Ann. Rev. Biochem. 36, Part II, 449 (1967). H. G. Zachau, D. ktting, and H. Feldman, Angew. Chemie. Intern. Ed. Engl. 2, 422 (1966). R. A. Lazzarini, Proc. Natl. Acad. Sci. U. S. 56, 185 (1966). R. A. Lazzarini and E. Santangelo, J. Bacterial. 94, 125 (1967). B. Vold, J. Bacterial. 102, 711 (1970). B. Goehler and R. H. Doi, Proc. Natl. Acad. Sci. U. S. 56, 1047 (1966). G. Von Ehrenstein and F. Lipmann, Proc. Natl. Acad. Sci. U. S. 47, 941 (1961). G. Zubay, J. Mol. Biol. 4, 347 (1962). I. Gillam, S. Millward, D. Blew, M. von Tigerstrom, E. Wimmer, and G. M. Tener, Biochemistry 2, 3043 (1967). T. Ishida and K. Miura, J. Mol. Biol. 11, 341 (1965). D. Gillespie and S. Spiegelman, J. Mol. Biol. 2, 829 (1965). S. B. Weiss, W.-T. Hsu, J. W. Foft, and N. H. Scherberg, Proc. Natl. Acad. Sci. U. S. 61, 114 (1968). R. H. Doi, I. Kaneko, and R. T. Igarashi, J. Bfol. Chem.243, 945 (1968).
716