Volume 4 Number 9 September 1977
Nucleic Acids Research
Bacteriophage T4 RNA ligase: preparation of a physically homogeneous, nuclease-free enzyme
from hyperprodudng infected cells
N. Patrick Higgins, Adam P. Geballe^, Thomas J. Snopek, Akio Sugino"*, and Nicholas R. Cozzarelli
Departments of Biochemistry and of Biophysics and Theoretical Biology, The University of Chicago,
Chicago, I L 60637, USA
Received 27 June 1977
ABSTRACT
Infection of Escherichia coli by a bacteriophage T4 regA, gene 44
double mutant leads to about a 7-fold Increase 1n the amount of RNA ligase
obtained after Infection by wild-type phage. Using cells Infected by the
double mutant, RNA ligase was purified to homogeneity with a 202 yield.
Unlike previous preparations of this enzyme, the ligase is free of contaminating nuclease and is therefore suitable for inter-molecular Ugation of
DNA substrates. In the course of these studies it was discovered that
adenylylation of the enzyme — a step 1n the reaction pathway — markedly
decreased the electrophoretic mobility of RNA Ugase through polyacrylamide
gels containing sodium dodecyl sulfate. This behavior allows identification of RNA Ugase among a mixture of proteins and was used to demonstrate
that virtually all of the purified protein 1s enzymatically active.
INTRODUCTION
Bacteriophage T4 RNA ligase is emerging as a powerful reagent for the
synthesis of nucleic acid polymers. Silber et al. first showed that the
enzyme cyclizes single-stranded oligoribonucleotides. Several groups have
since demonstrated intermolecular joining of RNA and this reaction provides
a high yield synthesis of product having a defined sequence. " Our
finding that DNA is a substrate expands the utility of this versatile
enzyme. With single-stranded substrates, RNA Ugase efficiently produces
circular DNA molecules and catalyzes Intermolecular joining to yield DNADNA, RNA-DNA, and DNA-RNA copolymers. 12 ' 13 Double-stranded DNA is also an
excellent substrate. The oligoribonucleotides rA(pA) 5 , rl(pl) 5 , and rC(pC) 5
can be added to the 5'-term1ni of DNA with either base-paired ends (Haelll
nuclease cleaved DNA) or cohesive ends (EcoRI nuclease cleaved DNA) with
nearly quantitative yields. 14 The analogous attachment of single-stranded
DNA to duplex DNA proceeds but with low yield. 14 Finally, RNA Ugase
markedly promotes the T4 DNA ligase catalyzed joining of duplex DNA substrates at base-paired ends, a reaction useful for cloning specific DNA
© Information Retriaval Limited 1 Falconberg Court London W 1 V 5 F G England
3175
Nucleic Acids Research
segments.16"18
The RNA ligase reaction intermediates have been isolated ' ' ; the
pathway 1s analogous to that of DNA ligase.
In the f i r s t step of the
reaction, RNA ligase reacts with ATP producing an adenylylated enzyme intermediate and PP-j. The second step requires the presence of a 5'-phosphoryl
terminated polynucleotide (donor) and a 3'-hydroxyl terminated polynucleotide (acceptor). AMP 1s transferred from the enzyme to the 5'-phosphoryl
of the donor creating a pyrophosphate linkage. In the last step, free
enzyme condenses the activated donor and acceptor to form a phosphodiester
bond and AMP is released. Although an acceptor is necessary for formation
of the activated donor, the donor molecule does not necessarily react in
the third step of the reaction with the acceptor which stimulated its formation.
We have coined the term "acceptor exchange" for cases where activated intermediate was formed in the presence of one acceptor but ligated
to another acceptor.
RNA ligase plays an essential role in T4 development. We have proven
recently that i t is identical to the t a i l fiber attachment protein of T4,
21
the product of gene 63.
The marked differences in requirements and inhibitors of nucleic acid ligation and tail fiber attachment imply that the
reactions have different mechanisms. It is not known 1f this bifunctional
enzyme plays an additional role in T4 nucleic acid metabolism.
Although the enzyme has been purified to physical homogeneity as deter9 12
mined by sodium dodecyi sulfate (SDS) gel electrophoresis, '
the apparently homogeneous preparations that we and others have used contain a DNA exonuclease that releases 5'-mononucleotides from the 3'-terminus of the substrate.
This nuclease level is small when compared to RNA cyclization
activity but i t represents a major obstacle for synthesis of DNA-DNA polymers because these reactions require very high enzyme levels. We report
here a purification protocol that removes all detectable nuclease activity
from RNA ligase. We also provide conditions for augmenting RNA ligase
production in T4-infected cells such that this enzyme represents as much as
0.5% of the total soluble protein. Finally, we have found that adenylylation of RNA ligase alters its electrophoretic mobility on SDS-containing
polyacrylamide gels.
T4 RNA ligase is a very stable enzyme and is now relatively easy to
purify to homogeneity. I t is already the enzymatic method of choice for
synthesizing small circular DNA and RNA polymers, for joining singlestranded RNA molecules together, and for synthesizing RNA-DNA block copoly3176
Nucleic Acids Research
mers. Furthermore, with T4 DNA and RNA Ugases 1n hand, nearly all possible
combinations of inter- and intramolecular ligations of single- and doublestranded RNA and DNA substrates can be performed.
MATERIALS AND METHODS
Growth of phage-infected bacteria. The T4 phage strains, obtained
from J. wiberg, were SP62 (regA), amN82 (gene 44), amElO (gene 45), and the
constructed double mutants SP62,amN82 and SP62,amE10. 22 Extracts of E^
coli B infected by these phages were prepared for RNA ligase assay as
described except the cells were resuspended at 8 x l O 8 / m l prior to lysozyme treatment. For preparation of infected cells for RNA ligase purification, 80 1 of E. coli B were grown to a density of 10 9 cells/ml at 37° In
medium composed of 33 mM KH 2 P0 4 , 74 mM Na 2 HPO 4 -7H 2 O, 0.5% glucose, 1%
casamino acids, and 0.003% gelatin. After addition of SP62,amN82 phage
at a multiplicity of infection of 5, the culture was incubated for 90 min,
then chilled by addition of ice chips, and cells were harvested by centrifugation.
Nucleic adds. The syntheses of lb'-32P}rl^Q, [3H]dT^g-, and
[3H]poly(r(J) have been described.4'23 [3H]dT7 was prepared using d(pT) 5
as a primer for terminal deoxynucleotidyltransferase in the presence of a
limiting level of [3H]dTTP. A 2-ml reaction mixture containing 100 mM
sodium cacodylate, pH 7.0, 100 mM potassium phosphate, pH 7.0, 1 mM 2mercaptoethanol, 1 mM CoCl 2 , 5 yM [^JdTTP (4xlO 7 cpm/ymole), 25 yM d(pT) 5
(the concentration of nucleic acids in this report is expressed as nucleotide equivalents), and 80 units of enzyme was incubated at 37° for 43 h.
The product was applied to a 2-ml DEAE-Sephadex A-50 column and eluted with
a 50-ml 0.01 M to 1 M linear gradient of triethyl ammonium bicarbonate.
Col El DNA was isolated according to the procedure of Staudenbauer.
Enzymes and proteins. Bacterial alkaline phosphatase (Type III-S),
bovine serum albumin, phosphorylase-b (rabbit muscle), glyceraldehyde-3phosphate dehydrogenase, and creatine phosphokinase were purchased from
Sigma Chemical Co. Terminal deoxynucleotidyltransferase was obtained from
Grand Island Biological Co.
Chromatography. DEAE-cellulose (DE-52), Sephadex G-100 and DEAESephadex A-- 50, and hydroxylapatite were purchased from Whatman Ltd., Pharmacia Fine Chemicals, and Bio-Rad Laboratories, respectively. DNA-agarose
3177
Nucleic Acids Research
was prepared by the method of Schaller et al. 2 5 One gram of calf thymus DNA
(Sigma type V) dissolved in 67 ml of 0.02 M NaOH was mixed with an equal
volume of 8% agarose (Sigma type II) which had been heated to 120° and then
cooled to 50°. The solution was solidified by pouring it onto a chilled
dish. The DNA-agarose was forced through a 60-mesh steel sieve twice,
suspended 1n 10 mM Tr1s-HCl, pH 7.5, 1 mM EDTA, and 0.1 M NaCl, and washed
with the same buffer until the absorbance at 260 nm of the wash buffer was
negligible. A DNA-agarose column was packed and washed successively with
2 column volumes of 20 mM Tris-HCl, pH 7.5, 50 yM ATP, 10 mM 2-mercaptoethanol, and 10$ glycerol, 1 column volume of the same buffer with 0.3 mg/ml
bovine serum albumin, and 1 column volume of buffer without albumin.
Enzymatic reactions. The standard RNA ligase assay measures the conversion of [ 5 ' - 3 2 P > A 2 Q - to a phosphatase-resistant form.
One unit of RNA
Ugase catalyzes the formation of one nmol of phosphodiester bonds in 30 m1n
at 37°. Two DNase assays were used. The first contained in 40 yl, 50 mM
Tris-HCl, pH 7.5, 10 mM MgCl 2 , 20 mM dithiothreitol, 0.5 mg/ml of bovine
serum albumin, 23 uM [3H]dTj2K, and enzyme. After a 30-min Incubation at
37°, 50 yl of 1.5 mg/ml of salmon testes DNA and 50 pi of 0.07% uranyl
acetate-2.5 M perchloric acid were added, and the soluble radioactivity was
measured. One unit of DNase catalyzes the conversion of 1 nmol of [ 3 H]dT^g
to an add-soluble form in 30 min at 37°. RNase was assayed in the same
manner using [3H]poly(rU) 1n place of [3H]dTyxg. The second DNase assay
used in 20 yl the same conditions except 1 mM ATP was added and [^]dT, was
the substrate. After 1 h at 37° the reaction mixture was spotted onto
Whatman 3MM paper and [3H]dTMP was separated from [ H]dT, by electrophoresis
in 0.05 M citrate buffer, pH 3.5.
Formation of adenylylated RNA ligase and analysis by polyacryiamide gel
electrophoresis. The RNA ligase-AMP complex was formed 1n a reaction mixture (20 yl) containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 5 mM dithiothreitol, 400 yM [14C]ATP (2xlO 7 cpm/ymol), and 1.6 yg of RNA ligase.
After 5 min at 30° the mixture was split into two portions. One was prepared for SDS-polyacrylam1de gel electrophoresis, and the other was incubated for an additional 5 min at 30° with 1.3 mM sodium pyrophosphate to
reverse adenylylation before electrophoresis. 26 Samples were electrophoresed through a 102 polyacrylamide gel containing 0.22 SDS,prepared
?7
according to Laemmli , for 7.5 h at 25 mA. Other experiments used the same
SDS concentration and 15% or 10-17.5S polyacrylamide gradient gels prepared
3178
Nucleic Acids Research
by the procedure of Anderson et al. 2 8 Gels were fixed and stained with
Coomassie blue; channels were cut Into 2.5 mm slices which were counted In
Triton X-100 based scintillation fluid.
RESULTS AND DISCUSSION
Increased RNA ligase activity Induced by regA, DMA defective phage
mutants. To facilitate enzyme purification we sought conditions in which
RNA ligase was abundant In T4 infected cells. We tested combinations of
DNA defective and regA mutants which are known 22 '29 to lead to increased
production of many early enzymes (Fig. 1). After Infection by a gene 45
mutant, RNA ligase activity is higher than that Induced by the regA mutant
but reaches a plateau about 40 min after infection at 37°. The pattern
after Infection by wild-type phage is similar to that after infection by
regA phage (data not shown). In contrast, after Infection by a regA, gene 45
double mutant, ligase activity Increased throughout the 2 h time -interval
20
40
60
80
100
Length of infection, min
120
Figure 1.
Effect of T4 regA and DNA synthesis mutations on RNA ligase
levels. E. coli B was grown at 37° 1n tryptone broth to 4 x l O 8 cells/ml
and infected with either SP62 (regA) phage ( • ), amElO (gene 45) phage ( o ),
or an SP62,amE10 double mutant ( Q ) at a multiplicity of infection of 4.
At the indicated times after infection, extracts were prepared and assayed
for RNA ligase.
3179
Nucleic Acids Research
examined.
The ligase a c t i v i t y was 2-3 tiroes that reached in the gene 45
Infected cells in three separate experiments.
Similar results were obtained
with the regA.gene 44 double mutant which was used for enzyme p u r i f i c a t i o n .
Purification of RNA Hqase.
The results of a typical p u r i f i c a t i o n
from 200 g of cells are summarized 1n Table I .
A l l operations were per-
formed at 4 ° .
Step 1:
Preparation of extract.
Two hundred grams of T4 regA,
gene 44 infected E. c o l l B were suspended in 250 ml of 20 mM Tris-HCl, pH
7.5, 0.1 mM EDTA, and 10 mM 2-mercaptoethanol (buffer A).
After passage
through a French pressure cell press at 15,000 p . s . i . , the lysate was
centMfuged at 36,000 x g for 90 min 1n a Beckman Type 21 rotor (Fraction I ,
410 ml).
Step 2:
Streptomycin sulfate preparation.
Nucleic acids were
precipitated from Fraction I by addition of a 5% streptomycin sulfate solut i o n over a period of 1 h to a f i n a l concentration of 1.4%.
the suspension was centrifuged at 10,500xg for 15 m1n.
(505 ml) was added 158 g of solid (NH 4 ) 2 S0 4 .
After 30 min,
To the supernatant
After 30 min of s t i r r i n g the
suspension was centrifuged at 10,500xg for 15 m1n. The pellet was resuspended in 30 ml of 20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.1 M KC1, and 10 mM
2-mercaptoethanol and dialyzed f o r 6 h against 3 changes of 2 1 of this
buffer (Fraction I I ) .
Step 3:
DEAE-cellulose chromatography.
One half of Fraction I I ,
62 ml, was diluted with an equal volume of 20 mM Tris-HCl, pH 7.5, and
0.1 n*1 EDTA and was applied to a DEAE-cellulose column (24.6 cm2 x 22.5 cm)
which had been equilibrated with buffer A.
The column was developed with a
3-1 0.1 M to 0.6 M KC1 gradient containing buffer A.
TABLE I :
Fraction and Step
Purification of T4 RNA ligase
Activity
Protein
4
units (x 10" )
I
II
III
IV
V
VI
HI
mg
Specific Activity
un1ts/mg
Crude extract
54
18.000
Streptomycin sulfate
44
12.000
37
DEAE-cellulose*
36
1,700
210
Sephadex G-10O
30
480
630
Hydroxylapatite I
16
28
5.700
DNA-agarose*
18
29
6.200
Hydroxylapatite I I
11
17
6,500
The data from the separate columns have been coatiined.
3180
RNA ligase eluted at
30
Nucleic Acids Research
about 0.22 H KC1. The column was washed with 1 M KC1 in buffer A, equilibrated with buffer A, and the second half of Fraction II chromatographed.
The fractions from both columns containing RNA ligase were pooled (725 ml)
and the enzyme precipitated by the gradual addition of 283 g of (NH^^SC^.
After centrifugation at 10,500xg for 40 m1n, the pellets were resuspended
in 13 ml of 20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 50 uM ATP, 0.1 M KC1, 10 mM
2-mercaptoethanol, and 102 glycerol (buffer B) and dialyzed against 2 1 of
this buffer for 6 h (Fraction III, 34.5 ml).
Step 4: Sephadex G-100 chromatography. Fraction III was filtered
through a Sephadex G-100 column (17 cm 2 x 57 cm) equilibrated with buffer B.
RNA ligase has a K av of 0.37. Fractions rich in RNA ligase were pooled
(Fraction IV, 134 ml).
Step 5: Hydroxylapatite chromatography. Fraction IV was dialyzed
against 2 1 of 20 mM potassium phosphate, pH 7.4, 50 pM ATP, 10 mM 2-mercaptoethanol, and 10% glycerol (buffer C) for 3 h and applied to a hydroxylapatite column (3.1 cm 2 x 14.3 cm). The enzyme was eluted with a 460-ml linear
0.02 M to 0.25 M potassium phosphate, pH 7.4, gradient containing 50 uM ATP,
10 mM 2-mercaptoethanol, and 10% glycerol. RNA ligase eluted sharply at
80 mM phosphate (Fig. 2). This step led to a nearly ten-fold purification
40
50
60
70
80 90
Froction
100 110
Figure 2. Purification of RNA ligase by hydroxylapatite chromatography.
Fraction IV RNA ligase was applied to a hydroxylapatite column (3.1 cm2 x
14.3 cm). The column was developed with a 460-ml, linear 0.02 M to 0.25 M
potassium phosphate, pH 7.4, gradient containing 50 uM ATP, 10 mM 2-mercaptoethanol, and 10% glycerol. Fractions of 4.5 ml were assayed for ONase
(A ) using a [3H]dTy*g- substrate and for RNA ligase ( o ).
3181
Nucleic Acids Research
and removed the bulk of the contaminating nuclease activity (Fig. 2). The
active fractions were pooled, made 50% in glycerol, and stored at -20°
(Fraction V, 40 ml). While Fraction V is sufficiently pure for many uses
of the enzyme, it contains a contaminating DNA exonuclease activity which
was removed by DNA-agarose chromatography.
Step 6: DNA-agarose chromatography. One third of Fraction V was
diluted with 0.25 M potassium phosphate, pH 7.4, to a final phosphate concentration of 0.1 M and applied to a DNA-agarose column (8.5 cm 2 x 14.2 cm)
equilibrated with 20 mM Tris-HCl, pH 7.5, 50 uM ATP, 10 mM 2-mercaptoethanol,
and 10% glycerol. The column was eluted with 240 ml of the same buffer.
Fractions which had no detectable nuclease activity were pooled. The
remainder of Fraction V was treated similarly and active fractions from the
three columns were pooled (Fraction VI, 195 ml). The DNA-agarose column has
been run with twice as much Fraction V with the same results. If potassium
phosphate was not added to Fraction V before application to DNA-agarose, the
ligase and nuclease eluted as overlapping peaks, in that order, from the
column. The phosphate may impede an Interaction between ligase and nuclease
and cause the nuclease to adhere to the column so tightly that it is not
eluted by the buffer used.
Step 7: Hydroxylapatite chromatography II. In order to concentrate
the enzyme and to remove DNA which may have leached off during the DNAagarose step, a second hydroxylapatite column was employed. The conditions
were the same as for Step 5, except Fraction VI was applied to a 2 cm 2 x
10 cm column and the eluting gradient was scaled down to 200 ml. The active
fractions were dialyzed against 20 mM Tris-HCl, pH 7.5, 50 uM ATP, 10 mM
dithiothreitol, and 50X glycerol for 8 h. This final fraction (Fraction VII,
23.4 ml) was stored at -20°.
The RNA Ugase was purified about 225-fold with a yield of 20X. The
enzyme is quite stable and Fractions V, VI, and VII have been stored at
least one year at -20° without loss of activity. For many purposes Fraction
V enzyme which was obtained In about a 30? yield is adequate. At this stage
1n the purification, the enzyme shows one major band after SDS-polyacrylamide gel electrophoresis and has no DNA endonuclease activity and little or
no RNase activity. Fractions VI and VII are nuclease free. There is no
detectable hydrolysis of [3H]poly(rU) in 30 m1n at 37° by 25 ug/ml RNA
ligase and no detectable relaxation of covalently-closed, duplex circular
Col El DNA. The exonuclease activity in Fraction V specifically attacks
single-stranded DNA from the 3'-end. As 1s common for exonucleases, the
3182
Nucleic Acids Research
activity is greater with short substrates and these are particularly useful
for stepwise synthesis of DNA with RNA ligase. A very sensitive nuclease
assay was employed which measured the release of [^HjdTMP from [^HDdT^ with
only the two 3'-term1nal residues labeled. As shown in Fig. 3, there was
no detectable digestion with Fractions VI (7.5 ug/ml) or VII (36 ug/ml) in
1 h. Fraction V (1.4 pg/ml) RNA Ugase nearly quantitatively released the
mononucleotide. Experiments with [5'-32p]dT7 similarly showed no release
of label by Fractions VI or VII enzyme.
The effect of adenylylation of RNA ligase on its electrophoretic mobility in the presence of SDS. The purification of E. coll DNA ligase by
Modrich, Anraku, and Lehman^ capitalized on the different affinity for
phosphocellulose of adenylylated and free enzyme. In analogous experiments
directed toward freeing RNA ligase from the contaminating nuclease we prepared a large amount of adenylylated enzyme and discovered that it and free
enzyme have different electrophoretic mobilities in the presence of SDS. As
shown in Fig. 4, preincubation of RNA ligase with 400 pM [14C]ATP for 5 min
at 30° caused 80? of the enzyme to migrate slower (lane 2) than untreated
enzyme (lane 1). The ^ 4 C label is exclusively associated with the slower
15
Fraction
25
Figure 3. Exonuclease activity in RNA ligase purification fractions. The
nuclease assays contained a [3H]dT7 substrate and 1.4 ug/ml of Fraction V
( o ) , 7.5 ug/ml of Fraction VI ( A ) , 36 pg/ml of Fraction VII ( D ) , or no
enzyme ( • ). After 1 h at 37° the reaction products were resolved by paper
electrophoresis using a 0.05 M sodium citrate, pH 3.5 buffer. The radioactivity in 1-cm strips was measured and the position of an Internal dTMP
reference 1s shown.
3183
Nucleic Acids Research
on.
1
eg
2 3
1
t
B
'
'_H2O
X
10
8
6
E
Q.
-4
u
II
-2
I
O
a>
(X
3
6
9
Distance Migrated, cm
12
Figure 4. SDS-polyacrylamide gel electrophoresis of RNA ligase and RNA
ligase adenylate. Fraction VI Ugase was adenylylated with [14c]ATP and
adenylylation was subsequently reversed by the addition of sodium pyrophosphate as described in Materials and Methods. Samples were removed and subjected to electrophoresis through a 102 polyacrylamide gel in the presence
of 0.22 SDS prepared according to Laemml1.27 Panel A is a photograph of the
Coomassie blue stained gel: lane 1, enzyme before addition of ATP; lane 2,
after 5 min at 30° with ATP; lane 3, after 5 min reversal of adenylylation.
Lanes 2 (•) and 3 ( o ) were then cut into 2.5ranslices, solubilized, and
monitored for 14c. The profiles are shown in B. ([14c]AMP not covalently
bound to protein 1s washed out of the gel during fixation and staining.)
The arrows indicate the positions of the stained RNA ligase bands. The
molecular weight standards shown (•) are phosphorylase-b, bovine serum
albumin, glyceraldehyde-3-phosphate dehydrogenase, and creatine phosphokinase in order of increasing mobility.
3184
Nucleic Acids Research
form. Another experiment In which the enzyme bound ^ 4 C label was carefully
measured showed that the stoichiometry of AMP to RNA Ugase was 1. This
alteration of electrophoretic mobility is reversible. Addition of 1.3 mM
PP} to the adenylylated enzyme for 5 min releases the label from the ligase
and all protein migrates with the untreated control (lane 3). The lower
electrophoretic mobility of adenylylated RNA ligase has been observed with
3 different SDS-polyacrylamide gel electrophoresis systems: 10% gels
prepared according to Laemmli, 23 and 152 and 10-17.5% gradient gels prepared
by the method of Anderson et al.
This result was unexpected since the molecular weight of adenylylated
enzyme is only 350 more than free enzyme yet the alteration in electrophoretic mobility is that expected for an increase in molecular weight of 4000.
Changes 1n apparent molecular weight have also been observed after reduction, maleylation, and removal of sialic acid residues from various proteins.
The conclusion from these results is that changes 1n electrophoretic mobility 1n the presence of SDS cannot necessarily be interpreted
1n terms of alteration of protein molecular weight. However, this behavior
provides a tool for Identification of RNA Ugase 1n a mixture of proteins
of the same subunit size. Moreover, the high percentage of RNA ligase which
can be adenylylated shows that the purified protein contains little if any
Inactive enzyme.
ACKNOWLEDGMENTS
This work was supported by NIH grant GM-22729. N.P.H. was supported
by a NIH fellowship (GM-7190) and A.S. received travel funds from the Naito
Foundation.
FOOTNOTES AND REFERENCES
1. This is fifth in a series of publications on RNA ligase; the fourth
1s Ref. 21.
2. On leave from Duke University School of Medicine, Durham, North Carolina 27710.
3. Present address: National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina 27709.
4. Silber, R., Malathi, V.G., and Hurwitz, J. (1972) Proc. Natl. Acad.
Sci. USA 69, 3009-3013
5. Walker, G.C., Uhlenbeck, O.C., Bedows, E., and Gumport, R.I. (1975)
Proc. Natl. Acad. Sc1. USA 72, 122-126
6. Kaufmann, G., and Littauer, U.Z. (1974) Proc. Natl. Acad. Sci. USA
71, 3741-3745
7. Kaufmann, G., and Kallenbach, N.R. (1975) Nature 254, 452-454
8. Linne\ T., flberg, B., and PhilUpson, L. (1974) Eur. J. Biochem. 42,
157-162
318S
Nucleic Acids Research
9. Last, J.A., and Anderson, W.F. (1976) Arch. Biochem. Biophys. 174,
167-176
10. Sninsky, J.J., Last, J.A., and Gilham, P.T. (1976) Nuc. A d d s Res. 3,
3157-3166
11. Uhlenbeck, O.C., and Cameron, V. (1977) Nuc. Acids Res. 4, 85-98
12. Snopek, T.J., Sugino, A., Agarwal, K.L., and CozzarelH, N.R. (1976)
Biochem. Biophys. Res. Commun. 68, 417-424
13. Sugino, A., Snopek, T.J., and Cozzarelli, N.R. (1977) J. Biol.Chem.
252, 1732-1738
14. Sugino, A., Higgins, N.P., Snopek, T.J., Geballe, A.P., and Cozzarelli,
N.R., in M1crob1ology-1977, Schlessinger, D., Ed. (in press)
15. Sugino, A., Goodman, H.M., Heyneker, H.L., Shine, J., Boyer, H.W., and
Cozzarelli, N.R. (1977) J. B1ol. Chem. 252, 3987-3994
16. Heyneker, H., Shine, J., Goodman, H.M., Boyer, H.W., Rosenberg, J.,
Dickerson, R.E., Narang, S.A., Itakura, K., Lin, S., and R1ggs, A.D.
(1976) Nature 263, 748-752
17. Marians, K.J., Wu, R., Stawinsky, J., Hozumi, T., and Narang, S.A.
(1976) Nature 263, 744-748
18. Backman, K., Ptashne, M., and Gilbert, W. (1976) Proc. Natl. Acad.
Sci. USA 73, 4174-4178
19. Ohtsuka, E., N1sh1kawa, S., Sugiura, M., and Ikehara, M. (1976) Nuc.
Adds Res. 3, 1613-1623
20. Lehman, I.R. (1974) Science 186, 790-797
21. Snopek, T.J., Wood, W.B., Conley, M.P., Chen, P., and Cozzarelli, N.R.
(1977) Proc. Natl. Acad. Sci. USA (in press)
22. Wiberg, J.S., Mendelson, S., Warner, V., Hercules, K., Aldrich, C ,
and Munro, J.L. (1973) J. Virol. 12, 775-792
23. Low, R.L., Rashbaum, S.A., and Cozzarelli, N.R. (1976) J. Biol. Chem.
251, 1311-1325
24. Staudenbauer, W.L. (1976) Molec. Gen. Genet. 145, 273-280
25. Schaller, H., NUsslein, C , Bonhoeffer, F.J., Kurz, C , and Nietschmann, I. (1972) Eur. J. Biochem. 26, 474-481
26. Cranston, J.W., Silber, R., Malathi, V.G., and Hurwitz, J. (1974) J.
Biol. Chem. 249, 7447-7456
27. Laemmli, U.K. (1970) Nature 227, 680-685
28. Anderson, C.W., Baum, P.R., and Gesteland, R.F. (1973) J. Virol. 12,
241-252
29. Karam, J.D., and Bowles, M.G. (1974) J. Virol. 13, 428-438
30. Modrich, P., Anraku, Y., and Lehman, I.R. (1973) J. Biol. Chem. 248,
7495-7501
31. Tung, J., and Knight, C.A. (1971) Biochem. Biophys. Res. Commun. 42,
1117-1121
32. Griffith, I.P. (1972) Biochem. J. 126, 553-560
33. Segrest, J.P., Jackson, R.L., Andrews, E.P., and Marchesi, V.T. (1971)
Biochem. Biophys. Res. Commun. 44, 390-395
3186