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Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2242-2246, June 1975
Cloning, Isolation, and Characterization of Replication Regions of Complex
Plasmid Genomes
(DNA/restriction endonuclease/incompatibility/R-plasmids/heteroduplex analysis)
KENNETH TIMMIS, FELIPE CABELLO, AND STANLEY N. COHEN
Department of Medicine, Stanford University School of Medicine, Stanford, California 94305
Communicated by Allan Campbell, April 4, 1975
ABSTRACT EcoRI^ endonuclease-generated^ DNA frag- ments carrying replication regions of the F'lac and R6- plasmids have been cloned and isolated, using as a selec- tion vehicle a nonreplicating ampicillin-resistance DNA fragment derived from a Staphylococcus aureus plasmid. Heteroduplex analysis of the constructed plasmid chimeras and the parent replicons has localized the cloned R6- replication region to a DNA segment between kilobase pair coordinates 1.0 and 88.0 on the R6-5 map. Physical proximity between the plasmid replication functions^ and the locus governing plasmid^ incompatibility^ has^ been shown for both parent replicons. The cloning^ method reported appears to be^ generally^ applicable^ for^ the identi- fication and isolation of replication regions^ of a^ variety^ of complex genomes.
Rapid progress has taken place recently in^ the study^ of DNA
synthesis involving small replicons^ such as^ simian virus^ 40 (1),
the bacteriophages M13 and 4X174 (1, 2), and the colicin El
plasmid (Col El) of Escherichia coli (3, 4). In contrast, bio-
chemical investigations^ of^ the^ replication^ of large^ replicons
such as the E. coli chromosome, the sex plasmid F, and some
antibiotic resistance (R) plasmids have been hindered by the
genetic complexity and structural fragility of these DNA
molecules. The recent demonstration that recombinant plas-
mids are capable of containing and utilizing at least two dis-
tinct sets of replication functions (5) has made evident still
other potential difficulties in investigating the replication of
large genomes.
In this report, we describe the isolation and^ characteriza-
tion of replication regions of the E.^ coli plasmids R6-5^ and
F'lac using an EcoRI-generated fragment of^ a^ Staphylococcus
aureus plasmid as a^ selection vehicle.^ The^ staphylococcal
plasmid DNA fragment, which^ carries^ genetic information^ for
penicillin-ampicillin (Ap)^ resistance,^ and^ which^ apparently
lacks an origin of replication in its original host (R. P. Novick,
personal communication), is^ capable^ of^ propagation^ in^ E. coli
only when linked^ to^ another^ EcoRI DNA^ fragment^ carrying
functions required for replication in this bacterial host (ref.
6 and A. C. Y. Chang and S. N. Cohen, unpublished data).
Genetic and molecular investigations of the cloned E.^ coli
plasmid replication region fragments demonstrate a^ physical
proximity between plasmid replication origins, replication
genes, and^ incompatibility determinants.
MATERIALS AND^ METHODS
Escherichia coli K-12 strain C600 (7) and nalidixic-acid^ re-
sistant (nalr) mutants of strains^ CRT46 (8)^ and^ CR34^ (9)
have been described. Plasmid R6-5 (10) expresses^ resistance
to chloramphenicol (Cm), kanamycin (Km), streptomycin
(Sm), and sulfonamide (Su). The pSC102 plasmid was con-
structed by in vivo ligation of EcoRI-treated R6-5 DNA, con-
sists of three EcoRI-generated fragments^ of^ R6-5, and^ ex-
presses Km^ and Su^ resistance (11). Plasmid pSC101 codes^ for resistance to^ tetracycline^ (Tc)^ (9).^ Plasmid^ pSC113^ was
constructed in vitro^ (6)^ and^ contains^ the^ entire^ pSC101^ plas-
mid plus two EcoRI-endonuclease-generated fragments of
the S. aureus penicillinase plasmid pI258 (12); it codes for
resistance to penicillin-ampicillin and Tc. Plasmids R100-
and R192-F7 (refs. 13 and 14, kindly provided by K. Hardy)
are derepressed fertility mutants of R100 and R192, and ex-
press resistance to Tc, Cm, Sm, and Su. F'lac is^ the classic
Paris F' plasmid and was obtained in strain DF109 (= bromo-
deoxyuridine-resistant isolate of DF87, ref.^ 15) from^ D.^ Frei-
felder via R. P. Silver.
The procedures used for^ conjugal transfer^ (16)^ and^ trans-
formation of plasmid DNA^ (17),^ radioactive^ labeling^ and^ iso-
lation of plasmids (18), sucrose and CsCl gradient centrifuga-
tion (5), construction of hybrid plasmid DNA molecules by
means of the EcoRI restriction endonuclease (11), and agarose
gel electrophoresis (5) have been described. Plasmid hetero-
duplex analysis procedures have been described by Sharp et^ al.
(19). The EcoRI restriction endonuclease was^ purified from
E. coli strain RY-13 according to Greene et^ al.^ (20) through
the phosphocellulose chromatography step. E. coli DNA^ ligase
was generously provided by S.^ Panasenko, P.^ Modrich,^ and
I. R. Lehman.
RESULTS
Isolation of the Selection Vehicle. Chang and^ Cohen^ re-
cently described the in vitro construction^ of^ a^ Tc^ and^ Ap
resistance plasmid chimera, pSC1 13, that^ contains^ the^ entire
pSC101 plasmid replicon plus two EcoRI-generated frag-
ments of the penicillin-ampicillin resistance^ staphylococcal
plasmid pI258 (6). Cleavage^ of^ the^ pSC113^ plasmid^ chimera
with the EcoRI endonuclease, ligation of the resulting frag-
ments, and transformation of E. coli with the^ ligated mixture
yielded another plasmid (pSC122) that^ also^ expressed resis-
tance to both Tc and Ap, but which^ contained^ only one^ of^ the
two EcoRI-generated fragments of^ Staphylococcus plasmid
DNA originally present in pSC113 (Fig. 1A). This^ Ap frag-
ment, which^ is^ the^ larger^ of^ the^ two^ staphylococcal^ EcoRI
fragments contained in pSC113, has^ a^ buoyant density in^ CsCl
of 1.692 g/cm3 (Table 1). Because of^ the^ substantial^ difference
in the buoyant density of the^ Ap fragment and^ the^ buoyant
density of the pSC101 plasmid (p =^ 1.710^ g/cm3) the^ two
DNA species can be separated easily by preparative cen-
trifugation of EcoRI-cleaved^ pSC122 plasmid DNA^ in^ CsCl
gradients (Fig. 1B).
Abbreviations: Ap, ampicillin-penicillin; Cm, chloramphenicol; Km, kanamycin; Sm, streptomycin; Su, sulfonamide; Tc,^ tetra- cycline; CCC, covalently closed circular; kb, kilobase; M.W., molecular weight.
Cloning of^ Replication Regions 2243
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FIG. 1. Isolation of staphylococcal (^) Ap-resistance DNA. (A) Construction of (^) pSC122 plasmid. One (^) microgram of (^) pSC DNA in (^50) ,u of a solution of 20 mM Tris.HCl pH 8.0, 1 mM Na2EDTA, 10 mM^ MgCl2 was^ digested^ for^30 min^ with^1 uA^ of EcoRI endonuclease prepared as previously described (5). The nuclease was then inactivated by incubation for 5 min at 600 and the DNA mixture (^) was ligated by addition of (^) (NH4)2SO4 (to 10 mM), NAD^ (to 100 uAM), bovine^ serum^ albumin (to 100 ug/ml), and 5 units of DNA (^) ligase. After (^) incubation at 140 overnight the mixture was used to transform cells (^) of E. coli K12 (^) C600. Cova- lently closed circular (CCC) plasmid DNAs prepared from transformant clones that (^) expressed Ap-resistance were (^) analyzed by electrophoresis of their EcoRI digestion products through an 0.8% agarose gel in Tris-borate buffer (5). One Ap-resistance plasmid, designation pSC122, contained only one of the two original staphylococcal EcoRI endonuclease cleavage products of the parent pSC113 plasmid. Fig. 1A is a photograph of an ethidium bromide-stained agarose gel containing the following EcoRI endonuclease-cleaved plasmids: 1, pSC101; 2, pSC113; 3, pSC122; and^ 4, Ap DNA^ fragment isolated as described in 1B below. (B) Isolation of Ap fragment DNA by buoyant density cen-
trifugation. Thirty-three micrograms of [3H]thymidine-labeled
pSC122 CCC-DNA^ (specific activity =^ 2.5^ X 103 cpm/pg) were cleaved with EcoRI (^) enzyme as described (^) above, dialyzed 4 hr against a solution of 10 mM Tris HCl, pH 8.0, 1 mM EDTA (TE buffer) to remove NP40 detergent present in the enzyme preparation, and then mixed with TE buffer to give a final volume of 8 ml. Solid CsCl was added (refractive index =^ 1.3985) and the solution (^) was centrifuged in a (^) bovine serum (^) albumin-coated centrifuge tube in a 50 Ti rotor at 36,000 rpm for 60^ hr^ at 200. Fractions were collected from a hole pierced in the bottom of the tube and the (^) radioactivity (0) in an (^) aliquot of (^) each fraction was measured (^) and the refractive index (^) was determined. Fractions indicated by the shaded area were pooled and dialyzed ex- haustively against TE buffer. This material was analyzed by agarose gel electrophoresis (Fig. 1A). About 10 jg of^ Ap fragment DNA was recovered.
Isolation of Replication Regions of the R6-5 and F'lac Plas- mids. Aliquots (1.5,ug) of Ap fragment DNA purified from the
FIG. 2. Analysis of R6-5-Ap and F'lac-Ap hybrid plasmids by agarose gel electrophoresis. CCC^ plasmid DNA^ preparations were cleaved to completion with EcoRI endonuclease and sub- jected to electrophoresis through 0.8% agarose slab gels as indicated in Fig. 1. Fragments of all plasmids are numbered from top to bottom in the gels, as was described previously (11). (A) 1, R6-5 + pSC135; 2, pSC135; 3, R6-5; 4, PSC136; 5, Ap fragment; 6, pSC139; 7, pSC102; 8, R6-5 + pSC102. (B) 1, pSC138; 2, pSC137; 3, F'lac; 4, pSC140; 5, Ap fragment; 6, pSC141. The^ pSC140 and pSC141 plasmids contain one or^ more other (^) fragments of F'lac in (^) addition to the replication region fragments.
pSC122 plasmid were separately mixed with equal amounts of
EcoRI-cleaved R6-5 or F'lac plasmid DNA and ligation and
transformation were carried out as described in Materials and
Methods and Fig. 1. Covalently closed circular (CCC) plas-
mid DNA samples isolated from 10 separate Ap-resistant
clones obtained by transformation with the R6-5 ligation
mixture were treated with EcoRI endonuclease and examined
by agarose gel electrophoresis; several representative plas-
mids are shown in Fig. 2A. All plasmid DNA preparations
had in common a single EcoRI fragment of the R6-5 plasmid
in addition to the Ap fragment; one of the clones (pSC136)
contained a second R6-5 DNA fragment (fragment XI, ref.
11) which was not essential for replication of the chimera.
Because fragments II and III of R6-5 have almost identical
mobilities in agarose gels, electrophoresis of a mixture of
EcoRI-cleaved pSC135 and R6-5 DNA (Fig. 2A-1) was car-
ried out to identify the R6-5 DNA fragment that enables
replication of the Ap fragment in E. coli. Mobility measure-
ments, using the other R6-5 bands present in gel 1 of Fig. 2A
as internal standards, identified the band having increased
fluorescence intensity as EcoRI fragment II of R6-5. A^ similar
co-electrophoresis experiment indicated that R6-5 fragment
II, and not the slightly smaller fragment III as was previously
believed (11), is contained also in the pSC102 plasmid (Fig.
2A-8). Parallel experiments indicated that a single EcoRI
fragment of F'lac (fragment VI of the parent plasmid) was
common to all of the F'lac-Ap plasmid chimeras isolated (Fig.
2B); several of the chimeras contained various other F'lac
Proc. Nat. Acad. Sci. USA (^72) (1975)
Cloning of^ Replication Regions 2245
63.9 kb from the ends of EcoRI fragment II in the complete
R6-5 plasmid and is situated 1.7 kb and 7 kb from the ends
of this fragment in pSC102. Thus, formation of the pSC
plasmid necessarily occurred by intracellular ligation of
separate DNA fragments, and not simply by in vivo recir-
culation of a single DNA segment containing the three EcoRI
fragments. Direct examination of the pSC102/R6-5 hetero-
duplex (data not shown) confirmed this interpretation.
The only region of homology seen in fifteen pSC135/pSC
heteroduplexes (e.g., Fig. 3D) is the 6.4 kb Ap fragment,
indicating that the EcoRI replication region fragments of
R6-5 and F'lac^ contain dissimilar base^ sequences. This is
consistent with the observed differences in replication-associ-
ated properties shown by F'lac and R6-5 (copy number,
acridine orange sensitivity, compatibility; Table 1 and ref.
22). The F'lac/pSC138 heteroduplex (Fig. 3E) shows a region
of homology 7.8 kb in length, which corresponds to the size
estimated by gel electrophoresis for the EcoRI replication
region fragment cloned from the F'lac plasmid (M.W.
5 X 106).
Compatibility Studies. Although incompatibility between
bacterial plasmids has been considered to be a property of
plasmid replication (23), direct evidence for a structural
interrelationship between these separate functions is lacking.
Using constructed Ap resistance plasmid chimeras containing
the replication regions of R6-5 or F'lac, we have investigated
compatibility of the plasmid chimeras with those plasmids
from which their replication functions have been derived
(Table 2). Incompatibility was studied by transformation
of the R6-5-Ap plasmid chimera pSC135, or the F'lac-Ap
plasmid chimera pSC138 into bacteria containing the parent
or a plasmid related to it, and was measured by the frequency
of expression of genes carried by the incoming plasmid in the
presence or in the absence of selection for determinants car- ried by the resident plasmid.
The pSC122 plasmid, which provided the Ap fragment for
these plasmid chimeras, is seen to be compatible with F'lac,
R6-5, R100-1, R192-F7, and pSC102 plasmids, and hence the
Ap fragment does not contribute to incompatibility. In con-
trast, the R6-5-Ap plasmid pSC135 is incompatible with
pSC102, R6-5, and other large plasmids (R100-1, R192-F7)
(Table 2) of the same incompatibility group as R6-5 (FII, ref.
22, and N. Datta, personal communication). The lowest incom-
patibility ratios (about 40) were observed between pSC135 (or
pSC102) and the R6-5, R100-1, and R192-F7 plasmids, which
appear to contain more than one set of replication functions (11, 24, 25). The greatest incompatibility ratio (about 800)
was observed between pSC135 and pSC102. The pSC
plasmid, which contains a replication region from F'lac, is
entirely compatible with plasmid R6-5 and its derivative
pSC102, consistent with the absence of nucleotide sequence
homology in the replication regions of the R6-5-Ap and F'lac-
Ap chimeras (Fig. 3D). As expected, pSC138 is incompatible
with the F'lac plasmid.
Measurements of incompatibility, which used a standard
conjugation method (16) and which employed nonconjugative
plasmids as resident replicons, yielded data which supported
the interpretations derived from Table 2. In the absence of
continued selection for antibiotic resistance determinants
carried by both plasmids, rapid segregation occurred in in-
stances where a high incompatibility index was observed.
However, in both transformation and conjugation experi-
ments, detectable segregation of or recombination between
('Sl1b REP^ jIR1^ (ISl)a^ IIR2{Sl)b 2.2^ I= 1.3 1.0^ I^ /MEE 88.0^ 55.0//\ 44.1 28.0 27.2^ I 2.2 1. 0/98.5 24.1 21. __ RTF - (^) r-DETERMINANT
FIG. 3. Heteroduplex analysis of R6-5-Ap and F'lac-Ap
hybrid plasmids. Standard procedures for plasmid DNA hetero- duplex analysis and electron microscopy were followed (19).
OX174 single-stranded^ and^ PM2^ duplex^ DNA^ were^ used^ as
internal standards for molecular length measurements (19, 27). The bar in each part of the figure represents 1 kb. Arrows indicate the junctions of single-strand (SS) and double-stranded (D) regions of the heteroduplexes. (A) Heteroduplex between^ pSC and R6-5^ plasmids. The^ region of homology is 11.5 kb in length and is^ located between^ 1.0^ and^ 88.0^ kb^ on^ the standard^ R6- physical map [shown in (B); see refs. 28 and 29]. A large single- stranded substitution loop (SS2) containing the two inverted repeats IR1 and IR2 represents the R6-5 segment absent in the pSC135 plasmid. The smaller substitution loop (SS1) represents the (^) Ap-DNA fragment contribution to pSC135. (B) Map of R6-5 showing the location of the cloned replication region. (C) The pSC135/pSC102 heteroduplex. The nonhomologous regions
comprising the Ap-DNA fragment (SS3) and the segment of
pSC102 (SS4) containing IR2 are indicated. (D) pSC135/pSC heteroduplex. The duplex region represents the Ap DNA frag- ment common to both plasmids. The replication region frag- ments of^ R6-5 (SS5) and F/lac(SS6) are indicated. (E) pSC138/ F'lac (^) heteroduplex. The region of homology is (^) 7.8 kb, which corresponds to^ the^ length of^ EcoRI^ fragment VI^ of^ F'lac.^ The single-strand substitution^ loops SS7^ and^ SS8^ are^ Ap DNA fragments and the nonhomologous segment of F'lac, respec- tively.
plasmids, as^ determined by examination of^ CCC-DNA, did
not occur in cells carrying compatible plasmid replicons.
DISCUSSION
The Ap-resistance EcoRI staphylococcal plasmid DNA frag-
ment used in these experiments has particular advantages
as a probe and vehicle for selection and cloning of replication
regions in E. coli. Because its buoyant density is substan-
tially different from the buoyant density of pSC101 DNA, the
fragment can be prepared in large quantities by CsCl den-
sity gradient equilibrium centrifugation of the EcoRI-cleaved
composite plasmid pSC122. The fragment can be separated
Proc. Nat. Acad. Sci. USA (^72) (1975)
2246 Biochemistry: Timmis et al.
from cloned E. coli plasmid replication region fragments by the same procedure, thus permitting the isolation of large amounts of replication region DNA for physical character- ization and in vitro studies. While the level of ampicillin resistance expressed by the staphylococcal DNA fragment in
E. coli (minimum inhibitory concentration, 200 ,g/ml) is
substantially lower than the (^) levels achieved by common
penicillin-ampicillin resistance plasmids indigenous to and
widely distributed among E. coli (Cabello and Cohen, in
preparation), it is sufficient for the selection of replication re-
gions as described here. Although earlier results, and those
presented in Table 2, suggest that at least two separate
replication regions are located on R6-5 and related plasmids
(11, 24, 25), all of the plasmid chimeras cloned in these studies
by the use of the Ap fragment selection vehicle contain a
unique EcoRI fragment. This finding suggests that essential
components of other R6-5 replication region(s) may be dis-
tributed on separate EcoRI fragments of the R6-5 plasmid.
The use^ of^ other restriction endonucleases for cloning replica-
tion regions of R6-5 should permit investigation of this pos-
sibility.
The demonstration that a 5.2 megadalton fragment of F'lac
DNA and a 7.6 megadalton fragment of the R6-5 plasmid
carry all of the functions required for replication of a DNA
fragment in E. coli indicates that the replication origin and
the replication genes of the F'lac and R6-5 plasmids are clus-
tered together in a small region of the genomes of these plas-
mids; similar clustering of replication origin and replication
genes has been observed in the genome of the bacteriophage X
The ability to clone specific segments of complex genomes
that carry genetic information for particular biological func-
tions such as DNA replication appears to be highly useful for
genetic and biochemical^ investigations of such functions.
The current experiments report the selective cloning and
study of^ EcoRI-generated replication region fragments of
two large plasmid genomes, R6-5 and F'lac. The methods
described are (^) potentially applicable for the isolation of DNA segments containing the (^) replication origin and/or genes of
any complex replicon capable of^ functioning in^ bacteria^ and
may be useful in the study of chromosome replication. The sequestration of replication functions onto small plasmid DNA molecules should facilitate in vivo and in vitro investiga- tions of gene products involved in replication. With appro- priate modification, the^ procedure describe1^ may also^ permit the isolation of^ particular DNA^ segments specifying functions involved in the (^) conjugal transfer of (^) plasmids.
These studies were supported by National Institute of Allergy
and Infectious Diseases Grant Al^ 08619, National^ Science
Foundation Grant GB-30581, and American Cancer^ Society
Grant VC-139. K.T. is the^ recipient of^ a^ postdoctoral fellowship
from the Helen Hay Whitney Foundation. We thank J. Zabielski
for technical assistance during part of this investigation.
1. Kasamatsu, H. & Vinograd, J. (1974) Annu. Rev. Biochem.
2. Schekman, R., Weiner, A. & Kornberg, A. (1974) Science
186, 987-993.
- Sakakibara, Y. & Tomizawa, J.-I. (1974) Proc. (^) Nat. Acad. Sci. USA 71, 802-806.
- Lovett, M. A., Katz, L. & Helinski, D. R. (1974) Nature 251, 337-340.
- Timmis, K., Cabello, F. & Cohen, S. N. (1974) Proc. Nat. Acad. Sci. USA 71, 4556-4560.
- Chang, A. C. Y. & Cohen, S. N. (1974) Proc. Nat. Acad. Sci. USA 71, 1030-1034.
- Bachman, B. J. (1972) Bacteriol. Rev. 36, (^) 525-557.
- Hirota, Y., Mordoh, J. & Jacob, F. (1970) J. Mol. Biol. 53, 369-387.
- Cohen, S. N. & Chang, A. (^) C. Y. (1973) Proc. Nat. Acad. Sci. USA 70, 1293-1297.
- Silver, R. P. & Cohen, S. N. (1972) J. Bacteriol. 110, 1082-
- Cohen, S. N., Chang, A. C. Y., Boyer, H. W. & Helling, R. B. (1973) Proc. Nat. Acad. Sci. USA 70, 3240-3244.
- Peyru, G., Wexler, L. F. & Novick, R. P. (1969) J. Bacteriol. 98, 215-221.
- Nishimura, Y., Ishibashi, M., Meynell, E. & Hirota, Y. (1967) J. Gen. Microbiol. 49, 89-98.
- Meynell, E. (^) & Cooke, M. (^) (1969) Genet. Res. 14, 309-313.
- Freifelder, D. R. & Freifelder, D. (1968) J. (^) Mol. Biol. 32, 15-23.
- Timmis, K. (1972) J. Bacteriol. 109, 12-20.
- Cohen, S. N., Chang, A. C. Y. & Hsu, L. (^) (1972) Proc. Nat. Acad. Sci. USA 69, 2110-2114.
- Clewell, D. B. & Helinski, D. R. (1969) Proc. Nat. Acad. Sci. USA 62, 1159-1166.
- Sharp, P. A., Hsu, M.-T., Ohtsubo, E. & Davidson, N. (1972) J. Mol. Biol. 71, 471-497.
- Greene, P. J., Betlach, M. D., Goodman, H. M. & Boyer, H. W. (1974) in Methods in Molecular Biology, ed. Wickner, R. B. (Marcel Dekker, Inc. New York), Vol. 9, pp. 87-
- Sharp, P.^ A., Cohen, S. N.^ & Davidson, N.^ (1973) J. Mol. Biol. 75, 235-255.
- Willetts, N.^ (1972) A^ nau.^ Rev. Genet.^ 6, 257-268.
- Jacob, F., Brenner, S. &^ Cuzin, F.^ (1963) Cold Spring Harbor Symp. Quant. Biol. 28, 329-348.
- Yoshikawa, M. (1974) J. Bacteriol. (^) 118, 1123-1131.
- Rownd, R. H., Perlman, D. & Goto, N. (^) (1974) in Micro- biology-1974, ed. Schlessinger, D. (American Society for Microbiology), pp. 76-94.
- Stevens, W. F., Adhya, S. & Szybalski, W. (1970) in The
Bacteriophage Lambda, ed. Hershey, A.^ D.^ (Cold Spring
Harbor Laboratory, Cold^ Spring Harbor, N.Y.), pp. 515-
Espejo, R.^ T., Canelo, E.^ S. &^ Sinsheimer, R.^ L.^ (1969) Proc. Nat. Acad. Sci. USA (^) 63, 1164-1168.
28. Hu, S., Ohtsubo, E., Davidson, N. & Saedler, H. (1975) J.
Bacteriol., 122, 764-775.
29. Ptashne, K. & Cohen, S. N. (1975) J. Bacteriol., 122, 776-
Cooper, S. & Helmstetter, C. (1968) J. Mol. Biol. 31, 519-
31. Nishimura, Y., Caro, L., Berg, C.^ M.^ &^ Hirota, Y.^ (1971)
J. Mol. Biol.^ 55, 441-456.
- Hirota, Y.^ (1960) Proc. Nat. Acad.^ Sci.^ USA^ 46, 57-60.
Proc. Nat. Acad. Sci. USA (^72) (1975)
