Proc. Nat. Ad. Sci. USA
Vol. 69, No. 10, pp. 2904-2909, October 1972

Biochemical Method for Inserting New Genetic Information into DNA of
Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda
Phage Genes and the Galactose Operon of Escherichia coli

(molecular hybrids/DNA joining/viral transformation/genetic transfer)

DAVID A. JACKSON*, ROBERT H. SYMONSt, AND PAUL BERG

Department of Biochemistry, Stanford University Medical Center, Stanford, California 94305

Conli-ibuted by Paul Berg, July 31, 1972

ABSTRACT   We have developed methods for covalently
joining duplex DNA molecules to one another and have
used these techniques to construct circular dimers of
SVM DNA and to insert a DNA segment containing lambda
phage genes and the galactose operon of E. coli into SV40
DNA. The method involves: (a) converting circular
SV40 DNA to a linear form, (b) adding single-stranded
homodeoxypolymerie extensions of defined oomposition
and length to the 3' ends of one of the DNA strands with
the enzyme terminal deoxynucleotidyl transferase (c)
adding complementary homodeoxypolymeric extensions
to the other DNA strand, (d) annealing the two DNA mole-
cules to form a circular duplex structure, and (e) filling
the gaps and sealing nicks in this structure with E. coli
DNA polymerase and DNA ligase to form a covalently
closed-circular DNA molecule.

Our goal is to develop a method by which new, functionally
defined segments of genetic information can be introduced into
mammalian cells. It is known that the DNA of the trans-
forming virus SV40 can enter into a stable, heritable, and
presumably covalent association with the genomes of various
mammalian cells (1, 2). Since purified SV40 DNA can also
transform cells (although with reduced efficiency), it seemed
possible that SV40 DNA molecules, into which a segment of
functionally defined, nonviral DNA had been covalently
integrated, could serve as vectors to transport and stabilize
these nonviral DNA sequences in the cell genome. Ac-
cordingly, we have developed biochemical techniques that are
generally applicable for joining covalently any two DNA
molecules.1 Using these techniques, we have constructed
circular dimers of SV40 DNA; moreover, a DNA segment
containing A phage genes and the galactose operon of Esche-
richia coli has been covalently integrated into the circular
SV40 DNA molecuIe. Such hybrid DNA molecules and others
like them can be tested for their capacity to transduce foreign
DNA sequences into mammalian cells, and can be used to
determine whether these new nonviral genes can be expressed
in a novel environment.

o Present address: Department of Microbiology, University of
Michigan Medical Center, Ann Arbor, Mich. 48104.
t Present address, Department of Biochemistry, University of
Adelaide, Adelaide, South Australia, 5001 Australia.
1 Drs. Peter Lobban and A. D. Kaiser of this department have
performed experiments similar to ours and have obtained similar
results using bacteriophage P22 DNA (Lobban, P. and Kaiser,
A. D., in preparation).

MATERIALS AND METHODS

DNA. (a) Covalently closed-circular duplex SV40 DNA
[SV4O(I)] (labeled with ['HIdT, 5 X 10' cpm/pg), free from
SV40 linear or oligomeric molecules [but Containing 3-5%
of nicked double-stranded circles-SV40(II)] was purified
from SV40-infected CV-1 cells (Jackson, D., & Berg, P., in
preparation), (b) Closed-circular duplex Xdugal DNA labeled
with ["]dT (2.5 X 10' cpm/pg), was isolated from an E. coli
strain containing this DNA as an autonomously replicating
plasmid (see ref. 3) by equilibrium sedimentation in CsCl-
ethidium bromide gradients (4) after lysis of the cells with de-
tergent. A more detailed characterization of this DNA will
be published later. Present information indicates that the
Xdugal (Xdu-1.20) DNA is a circular dimer containing tandem
duplications of a sequence of several A phage genes (including
CI, 0, and P) joined to the entire galactose operon of E. coli
(Berg, D., Mertz, J., &Jackson, D., in preparation). DNA con-
centrations are given as molecular concentrations.

Enzymes. The circular SV40 and Adogal DNA molecules
were cleaved with the bacterial restriction endonuclease RI
(Yoshimori and Boyer, unpublished; the enzyme was gen-
erously made available to us by these workers). Phage
A-exonuclease (given to us by Peter Lobban) was prepared
according to Little et al. (5), calf-thymus deoxynucleotidyl
terminal transferase (terminal transferase), prepared ac-
cording to Kat0 et al. (6), was generously sent to us by F. N.
Hayes; E. coli DNA polymerase I Fraction VI1 (7) was a gift
of Douglas Brutlag; and E. coli DNA ligase (8) and exo-
nuclease I11 (9) were kindly supplied by Paul Modrich.

Substmtes. [~r-~*P]de~xynucle~~ide triphosphates (specific
activities 5-10 Ci/pmol) were synthesized by the method of
Symons (10). All other reagents were obtained from com-
mercial sources.

Centrifugations. Alkaline sucrose gradients were formed by
diffusion from equal volumes of 5, 10, 15, and 20% sucrose
solutions with 2 mM EDTA containing, respectively, 0.2,
0.4, 0.6, and 0.8 M NaOH, and 0.8, 0.6, 0.4, 0.2 M NaCl.
100-pl samples were run on 3.8-ml gradients in a Beckman
SW56 Ti rotor in a Beckman L265B ultracentrifuge at 4'
and 55,000 rpm for the indicated times. 2- to 10-drop fractions
were collected onto 2.5cm diameter Whatman 3MM discs,
dried without washing, and counted in PPO-dimethyl
POPOP-toluene scintillator in a Nuclear Chicago Mark I1

2904

Proc. Nat. Ad. Sn'. USA 69 (197.2)

Insertion of the Galactose Operon into SV40 DNA

2905

scintillation spectrometer. An overlap of 0.4% of    into
the 'H channel was not corrected for.

CsCl-ethidium bromide equilibrium centn'fugation was per-
formed in a Beckman Type 50 rotor at 4" and 37,000 rpm for
48 hr. SV40 DNA in 10 mM Tris.HC1 tpH 8.1)-1 mM Na
EDTA-10 mM NaCl was adjusted to 1.566 g/ml of CsCl
and 350 pg/ml of ethidium bromide. 30-Drop fractions were
collected and aliquots were precipitated on Whatman GF/C
filters with cold 2 N HCl; the filters were washed and counted.

Electron Microscopy. DNA was spread for .electron micros-
copy by the aqueous method of Davis et al. (11) and photo-
graphed in a Phillips EM 300. Projections of the molecules
were traced on paper and measured with a Keuffel and Esser
map measurer. Plaquepurified SV40(II) DNA was used as
an internal length standard.

Conuersian of SV4O(r) DNA to Unit Length Linear DNA
[SV~O(LRI)] with Rr Endonuclease. ['H]SV40(1) DNA (18.7
nM) in 100 mM Tris.HCI buffer (pH 7.5)-10 mM MgC12-2
mM 2mercaptoethanol was incubated for 30 min at 37'
with an amount of RI previously determined to convert 1.5
times this amount of SV40(I) to linear molecules [SV~O(LRI)];
Na EDTA (30 mM) was added to stop the reaction] and the
DNA was precipitated in 67% ethanol.

Removal of B'-Terminal Re- from SV40(LRr) with X
Exonuclease. ['H]SV.U)(LRI) (15 nM) in 67 mM K-glycinate
(pH 9.5), 4 mM MgCl,, 0.1 mM EDTA was incubated at 0"
with A-exonuclease (20 pg/ml) to yield ['H]SV~O(LRI~XO)
DNA. Release of ['HIdTMP was measured by chromato-
graphmk aliquots of the reaction on polyethyleneimine thin-
layer sheets (Brinkmann) in 0.6 M NHaHCOa and counting
the dTMP spot and the origin (undegraded DNA).

Addition of Homopolymeric Ezlen~im~ lo SV~O(LRI~ZO)
with Tenniml Transferase. ['H]Sv40(L~1exo) (50 nM) in 100
mM K-cacodylate (pH 7.0), 8 mM MgClt, 2 mM 2-mercapto-
ethanol, 150 pg/rnl of bovine serum albumin, [a-"P]dNTP
(0.2 mM for dATP, 0.4 mM for dTTP) was incubated with
terminal transferase (30-60 pg/ml) at 37". Addition of
[ '2PIdNMP residues to SV40 DNA was measured by spotting
aliquots of the reaction mixture on DEAEpaper discs
(Whatman DE-81), washing each disc by suction with 50 ml
(each) of 0.3 M NH4-formate (pH 7.8) and 0.25 M NHIHCOa,
and then with 20 ml of ethanol. To determine the proportion
of SV40 linear DNA molecules that had acquired at least one
"functional" (dA), tail, we measured the amount of SV40
DNA ('H counts) that could be bound to a Whatman GF/C
filter (2.4-cm diameter) to which 150 pg of polyuridylic acid
had been fixed (13). 15pl Aliquots of the reaction mixture
were mixed with 5 ml of 0.70 M NaCl-0.07 M Na citrate
(pH 7.0)-20/, Sarkosyl, and filtered at room temperature
through the ply(U) filters, at a flow rate of 3-5 ml/min.
Each filter was washed by rapid suction with 50 ml of the same
buffer at O', dried, and counted. Control experiments showed
that 9%100~o of [aH]oligo(dA)~25 bound to the filters under
these conditions. When the ratio of [a2P]dNMP to ["]]DNA
reached the value equivalent to the desired length of the
extension, the reaction was stopped with EDTA (30 mM) and
2% Sarkosyl. The [3H]SV40(~~exo)-[~ZPfdA or 4T DNA
was purified by neutral sucrose gradient zone sedimentation
to remove unincorporated dNTP, as well as any traces of
SV40(I) or SV40(II) DNA.

Fanalion of Hydrogen-Bw&d Circular DNA Molecules.
[szP]dA and dT DNAs were mixed at concentrations of 0.15
nM each in 0.1 M NaCl-10 mM Tris.HC1 (pH 8.1)-1 mM
EDTA. The mixture waa kept at 51" for 30 min, then cooled
slowly to room temperature.

Formation of Covalently Closed-Circular DNA  Molecules.
After annealing of the DNA, a mixture of the enzymes, sub-
strates, and cofactors needed for closure was added to the
DNA solution and the mixture was incubated at 20" for 3-5
hr. The final concentrations in the reaction mixture were:
20 mM Tris.HCI (pH 8.1), 1 mM EDTA, 6 mM MgC12,
50 pg/ml bovine-serum albumin, 10 rnM NH,Cl, 80 mM NaCl,
0.052 mM DPN, 0.08 mM (each), dATP, dGTP, dCTP,
and dTTP, (0.4 pg/ml) E. coli DNA polymerase I, (15 units/
ml) E. coli ligase, and (0.4 unit/ml) E. coli exonuclease 111.

RESULTS

General approach
Fig. 1 outlines the general approach used to generate circular,
covalently-closed DNA molecules from two separate DNAs.
Since, in the present case, the units to be joined are them-
selves circular, the first step requires conversion of the circular
structures to linear duplexes. This could be achieved by a
double-strand scission at random locations (see Discus&)
or, as we describe in this paper, at a unique site with RI re-
striction endonuclease. Relatively short (W100 nucleotides)
poly(dA) or poly(dT) extensions are added on the 3'-hydroxyl
termini of the linear duplexes with terminal transferase; prior

R, Endonucleaee

on 3'

OP 5'

5' PO 0-
3'H0 a

3' HO

  J-J hExonucleas4
5'PO OH 3'

OP 5

Terminal rmnsferose

       dATP or dTTP
5' PO OH3'

OP5'

3'HO

dT

or

S'PO

3'HO 7 OP5'

n Annealing

OH 3'

Fro. 1. General protocol for producing covalently closed
SV40 dimer circles from SVM(1) DNA.
o The four deoxynucleoside triphosphates and DPN are also
present for the DNA polymerase and ligase reactions, respec-
tively.

2906 Biochemistry: Jackson et al.

-

-12
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-10

-

-30

-8

- 40

-1-6

-
-30

4

- 20

-

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-10

-

Proc. Nat. Acad. Sci. USA 69 (1978)

10,M

0"

9

2s

N

-20

I5

10

0

//#,I,,

FIG. 2.

     Alkaline sucrose gradient sedimentation of [aHlSV4C-
(L~~exo)-[~~Pl (dA)sa DNA. 0.16 pg of DNA WBS centrifuged for
6.0 hr.

removal of a short sequence (30-50 nucleotides) from the
5'-phosphoryl termini by digestion with X exonuclease facili-
tates the terminal transferase reaction. Linear duplexes con-
taining  (dA), extensions are annealed to the DNA to be
joined containing (dT),, extensions at relatively low concen-
trations. The circular structure formed contains the two
DNAs, held together by two hydrogen-bonded homopolymeric
regions (Fig. 1). Repair of the four gaps is mediated by E.
coli DNA polymerase with the four deoxynucleosidetriphos-
phates, and covalent closure of the ring structure is effected
by E. coli DNA ligase; E. coli exonuclease I11 removes 3'-
phosphoryl residues at any nicks inadvertently introduced
during the manipulations (nicks with 3'-phosphoryl ends
cannot be sealed by ligase).

Principal steps in the procedure

Circular SV4O DNA Can Be Opened to Linear Duplexes by
RI Endonuclease. Digestion of SV40(I) DNA with excess RI
endonuclease yields a product that sediments at 14.5 S in
neutral sucrose gradients and appears as a linear duplex with
the same contour length as SV40(II) DNA when examined by
electron microscopy [(ls); Jackson and Berg, in prepara-
tion; see Table I]. The point of cleavage is at a unique site
on the SV40 DNA, and few if any single-strand breaks are
introduced elsewhere in the molecule (18) ; moreover, the
termini at each end are 5'-phosphoryl, 3'-hydroxyl (Mertz,
J., Davis, R., in preparation). Digestion of plaque-purified
SV40 DNA under our conditions yields about 87% linear
molecules, 10% nicked circles, and 3% residual supercoiled
circles.

Addition of Oligo(dA) OT -(dT) Ezlensians to the 3'-Hydrozyl
Termini of SV4O (LRI). Terminal transferase has been used
to generate deoxyhomopolymeric extensions on the 3'-
hydroxyl termini of DNA (7); once the chain is initiated,
chain propagation is statistical in that each chain grows at
about the same rate (12). Although the length of the exten-
sions can be controlled by variation of either the time of in-
cubation or the amount of substrate, we have varied the time
of incubation to minimize spurious nicking of the DNA by
trace amounts of endonuclease activity in the enzyme prep-
aration; we have so far been unable to remove or selectively
inhibit these nucleases (Jackson and Berg, in preparation).

Incubation of SV40(h1) with terminal transferase and

either dATP or dTTP resulted in appreciable addition of
mononucleotidyl units to the DNA. But, for example, after
addition of 100 residues of dA per end, only a small propor-
tion of the modified SV40 DNA would bind to filter discs
containing poly(U) (13). This result indicated that initiation
of terminal nucleotidyl addition was infrequent with
SV4O(L1), but that once initiated those termini served as
preferential primers for extensive homopolymer synthesis.
Lobban and Kaiser (unpublished) found that P22 phage
DNA became a better primer for homopolymer synthesis
after incubation of the DNA with X exonuclease. This enzyme
removes, successively, deoxymononucleotides from 5'-phos-
phoryl termini of double-stranded DNA (15), thereby render-
ing the 3'-hydroxyl termini single-stranded. We confirmed
their finding with SV40(h1) DNA; after removal of 3&50

Proc. Nat. Ad. Sci. USA 69 (1972)

nucleotides per 5'-end (see Methods), the number of SV40(h1)
molecules that could be bound to poly(U) filters after incuba-
tion with terminal transferase and dATP increased 5 to 6-
fold. Even after separation of the strands of the SV40(LRIexo)-
dA, a substantial proportion of the aH-label in the DNA was
still bound by the poly(U) filter, indicating that both 3'-
hydroxy termini in the duplex DNA can serve as primers.
The weight-average length of the homopolymer extensions
was 50-100 residues per end. Zone sedimentation of ["Il-
SV40(L~1exo)-[~~P](dA)~ (this particular preparation, which
is described in Methods, had on the average, 80 dA residues
per end) in an alkaline sucrose gradient showed that (i) 6&
70% of the SV40 DNA strands are intact, (ii) the [azP](dA),
is covalently attached to the [2H]SV40 DNA, and (iii) the
distribution of oligo(dA) chain lengths attached   the SV40
DNA is narrow, indicating that the deviation from the cal-
culated mean length of 80 is small (Fig. 2). SV40(L~1exo),
having (dT)a extensions, was prepared with [aZP]dTTP and
gave essentially the same results when analyzed as described
above.

Hydrogen-Bonded Circular Molecules Are Formed by An-
nealing s v4O (L RIeX 0) - (dA ) 80 and s v4O (L Rl~~) - (d T) gg To-
gether. When SV40(L~1exo)-(dA)gg and SV40(L~~exo)-(dT)~
were annealed together, 30-6070 of the molecules seen by
electron microscopy were circular dimers; linear monomers,
linear dimers, and more complex branched forms were also
seen. If SV40(L~1exo)-(dA)~ or -(dT)m alone was annealed,
no circles were found. Centrifugation of annealed prepara-
tions in neutral sucrose gradients showed that the bulk of the
SV40 DNA sedimented faster than modified unit-length
linears (as would be expected for circular and linear dimers,
as well as for higher oligomers). Sedimentation in alkaline
gradients, however, showed only unit-length single strands
containing the oligonucleotide tails (as seen in Fig. 2).

Covalently Closed-Circular DNA Molecules Are Formed by
Incubation of Hydrogen-Banded Complexes Wilh DNA Poly-
merase, Ligase, and Exonuclease III. The hydrogen-bonded
complexes described above can be sealed by incubation with
the E. colienzymes DNA polymerase I, ligase, and exonuclease
111, plus their substrates and cofactors. Zone sedimentation
in alkaline sucrose gradients (Fig. 3) shows that 20% of the

AB 2 4 6 8 10 12 14 16 k3 20 22 EA

bottom Fraction number

FIG. 4.

     CsC1-ethidium bromide equilibrium centrifugation
of the products analyzed in Fig. 4. Line A, dA-ended, plus dT-
ended SV40 linears, plus (P+L+III) ("P, 0; 'H, 0); line B, the
same mixture without (P+L+III) (azP, A; 2H, A).

Insertion of the Galactose Operon into SV40 DNA

2907

TABLE 1.  Relative lengths of SV4O and Xdvgd-120

DNA molecules

DNA species

Length f standard
  deviation in
  SV40 units'

Number of
molecules in
sample

1.00
1.00 f 0.03
2.06 f 0.19
4.09 f 0.14
2.00 f 0.04
2.95 f 0.04
2.78 f 0.05

224
108
23
65
163
76
13

*The contour length of plaquepurified SV40(II) DNA is

t Data supplied by J. Morrow.

defined as 1.00 unit.

input   label derived from the oligo(dA) and -(dT) tails
sediments with the aH label present in the SV40 DNA, in the
position expected of a covalently closed-circular SV40 dimer
(70-75 S). About the same amount of labeled DNA bands
in a CsCI-ethidium bromide gradient at a buoyant density
characteristic of covalently closed-circular DNA (Fig. 4).

DNA isolated from the heavy band .of the CsC1-ethidium
bromide gradient contains primarily circular molecules, with
a contour length twice that of SV40(II) DNA (Table 1) when
viewed by electron microscopy. No covalently closed DNA is
formed if either one of the linear precursors is omitted from
the annealing step or if the enzymes are left out of the closure
reaction. We conclude, therefore, that two unit-length linear
SV40 molecules have been joined to form a covalently closed-
circular dimer.
Covalent closure of the hydrogen-bonded SV40 DNA di-
mers is dependent on Mg2+, all four deoxynucleoside triphos-
phates, E. coli DNA polymerase I, and ligase, and is inhibited
by 98% if exonuclease I11 is omitted (Lobban and Kaiser
first observed the need for exonuclease I11 in the joining of
P22 molecules; we confirmed their finding with this system).
Exonuclease I11 is probably needed to remove 3'-phosphate
groups from 3'-phosphoryl, 5'-hydroxyl nicks introduced by
the endonuclease contaminating the terminal transferase
preparation. 3'-phosphoryl groups are potent inhibitors of
E. coli DNA polymerase I (14) and termini having 5'-hydroxyl
groups cannot be sealed by E. coli ligase (8). The 5'-hydroxyl
group can be removed and replaced by a 5'-phosphoryl group
by the 5'- to 3'-exonuclease activity of E. coli DNA poly-
merase I (7).

Preparation of the Galactose Operon for Insertion into SV4O
DNA. The galactose operon of E. coli was obtained from a
Xdvgal DNA; Xdvgal is a covalently closed, supercoiled DNA
molecule four times as long as SV40(II) DNA (Table 1). After
complete digestion of Xdvgal DNA with the RI endonuclease,
linear molecules two times the length of SV40(II) DNA are
virtually the exclusive product (Table 1). This population has
a unimodal length distribution by electron microscopy and ap-
pears to be homogeneous by ultracentrifugal criteria (Jackson
and Berg, in preparation). The RI endonuclease seems, there-
fore, to cut Xdvgal circular DNA into two equal length linear
molecules. Since one Rr endonuclease cleavage per Adv mono-
meric unit occurs in the closely related Xdv-ZO4 (Jackson and
Berg, in preparation), it is likely that Xdvgal is cleaved at the

2908 Biochemistry: Jackson et al.

-8

-7

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-5

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Proc. Nut. Ad. Sci. USA 69 (197%)

-8

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4

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20

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I         t ,     I
5 x)    I5 90    25 ABC

bottom Fmstion number

     Alkaline sucrose gradient sedimentation of annealed
[JH]SV40(L~~exo)-[axP] (dA)ao and [aHH]XdugaZ-f%O) (LRI~xo)-
[azP](dT)s~ incubated for 3 hr with and without (P+L+III).
Centrifugation was for 60 min. Line A, dA-ended SV40, plus
dT-ended kdvgal-f$O linears, plus (P+L+III)("P, 0; JH, 0);
line B, dT-ended hdugal-f 20 linears, plus dT-ended SV40 linears,
plus (P+L+III) ("P, A); line C,dA-ended SV40 linears, plus
dT-ended Xdugal-f%O linears, without (P+L+III) ("P, m).

The arrows indicate the position in the gradient of supercoiled
marker DNAs having the indicated multiple of SV40 DNA mo-
lecular size.

FIG. 5.

same sites and, therefore, that each linear piece contains an
intact galactose operon.
The purified Xdugal (h~) DNA was prepared for joining
to SV40 DNA by treatment with X-exonuclease, followed by
terminal transferase and [12P]dTTP, as described for SV40-
(LRI).

Formation of Coualently Closed-Circular DNA Molecules
Containing both SV4O and Xdugal DNA. Annealing of ["I-
SV40(h1exo)- ["PI (dA), with [ aH]lhdugaZ(h~exo)- [
(dT)m, followed by incubation with the enzymes, substrates,
and cofactors needed for closure, produced a species of DNA
(in about 15% yield) that sedimented rapidly in alkaline
sucrose gradients (Fig. 5) and that formed a band in a CsCl-
ethidium bromide gradient at the position expected for co-
valently closed DNA (Fig. 6). The putative hdugal-SV40
circular DNA sediments just ahead of Xdu-1, a supercoiled
circular DNA marker [2.8 times the length of SV40(II)DNA],
and behind Xdugal supercoiled circles [4.1 times SV40(II)DNA]
in the alkaline sucrose gradient. Electron microscopic measure-
ments of the DNA recovered from the dense band of the CsCl-
ethidium bromide gradient showed a mean contour length
for the major species of 2.95 f 0.04 times that of SV4O(II)
DNA (Table 1). Each of these measurements supports the
conclusion that the newly formed, covalently closed-circular
DNA contains one SV40 DNA segment and one Xdugal DNA
monomeric segment.

Omission of the enzymes from the reaction mixture pre-
vents XdugalSV40 DNA formation (Figs. 5 and 6). No co-
valently closed product is detectable (Fig. 5) if Xdugal and
SV40 linear molecules with identical, rather than comple-
mentary, tails are annealed and incubated with the enzymes.
This res& demonstrates directly that the formation of co-
valently closed DNA depends on complementarity of the
homopolymeric tails.
We conclude from the experiments described above that
Xdugal DNA containing the intact galactose operon from E.
coli, together with some phage X genes, has been covalently
inserted into an SV40 genome. These molecules should be
useful for testing whether these bacterial genes can be intro-
duced into a mammalian cell genome and whether they can
be expd there.

DISCUSSION

The methods described in this report for the covalent join-
ing of two SV40 molecules and for the insertion of a segment
of DNA containing the galactose operon of E. coli into SV40
are general and offer an approach for covalently joining any
two DNA molecules together. With the exception of the for-
tuitous property of the RI endonuclease, which creates con-
venient linear DNA precursors, none of the techniques used
depends upon any unique property of SV40 and/or the Xdugal
DNA. By the use of known enzymes and only minor modi-
fications of the methods described here, it should be possible
to join DNA molecules even if they have the wrong combina-
tion of hydroxyl and phosphoryl groups at their termini. By
judicious use of generally available enzymes, even DNA
duplexes with protruding 5'- or 3'-ends can be modified to
become suitable substrates for the joining reaction.
One important feature of this method, which is different
from some other techniques that can be used to join unrelated
DNA molecules to one another (16, 19), is that here the join-
ing is directed by the homopolymeric tails on the DNA. In
our protocol, molecule A and molecule B can only be joined
to each other; all AA and BB intermolecular joinings and all
A and B intramolecular joinings (circularizations) are pre-
vented. The yield of the desired product is thus increased,
and subsequent purification problems are greatly reduced.

"SI

12c I I I/

2 Pt

Proc. Nat. Ad. Sei. USA 69 (1972)

For some purposes, however, it may be desirable to insert
Xdugal or other DNA molecules at other specific, or even ran-
dom, locations in the SV40 genome. Other specific placements
could be accomplished if other endonucleases could be found
that cleave the SV40 circular DNA specifically. Since pan-
creatic DNase in the presence of Mn2+ produces randomly
located, double-strand scissions (17) of SV40 circular DNA
(Jackson and Berg, in preparation), it should be possible to
insert a DNA segment at a large number of positions in the
SV40 genome.
Although the Xdugal DNA segment is integrated at the same
location in each SV40 DNA molecule, it should be emphasized
that the orientation of the two DNA segments to each other
is probably not identical. This follows from the fact that each
of the two strands of a dudex can be joined to either of the two

strands of the other duplex (e.g., TfF or Tfg). What

- --

possible consequences this fact has on the genetic expression
of these segments remains to be seen.
We have no information concerning the biological activities
of the SV40 dimer or the Xdugal-SV40 DNAs, but appropri-
ate experiments are in progress. It is clear, however, that the
location of the RI break in the SV40 genome will be crucial
in determining the biological potential of these molecules;
preliminary evidence suggests that the break occurs in the
late genes of SV40 (Morrow, Kelly, Berg, and Lewis, in prep-
aration.
A further feature of these molecules that may bear on their
usefulness is the (dA.dT), tracts that join the two DNA seg-
ments. They could be helpful (as a physical or genetic marker)
or a hindrance (by making the molecule more sensitive to
degradation) for their potential use as a transducer.
The Xdugal-SV40 DNA produced in these experiments is,
in effect, a trivalent biological reagent. It contains the genetic
information to code for most of the functions of SV40, all of
the functions of the E. coli galactose operon, and those func-
tions of the A bacteriophage required for autonomous repli-
cation of circular DNA molecules in E. coli. Each of these

The symbols W and C refer to one or the other complementary
strands of a DNA duplex, and the "connectors" indicate how the
strands can be joined in the closed-circular duplex.

Insertion of the Galactose Operon into SV40 DNA

2909

sets of functions has a wide range of potential uses in studying
the molecular biology of SV40 and the mammalian cells with
which this virus interacts.

We are grateful to Peter Lobban for many helpful discussions.
D. A. J. was a Basic Science Fellow of the National Cystic
Fibrosis Research Foundation; R. H. S. was on study leave from
the Department of Biochemistry, University of Adelaide, Aus-
tralia and was supported in part by a grant from the USPHS.
This research was supported by Grant GM-13235 from the
USPHS and Grant VC-23A from the American Cancer Society.

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