vrroLocy 61, 517-520 (1973)

Studies of SV40 DNA

VI. Cleavage of SV40 DNA by Restriction Endonuclease from Hemophilus parainfluenzae

Cleavage of SV40 DNA by bacterial re-
striction endonucleases has provided specific
DNA fragments which are proving useful in
analyzing the structure and function of the
viral genome (1-4). Danna and Nathans (J)
have previously reported that restriction
endonuclease from H. influenzae produces 11
fragments from SV40 DNA which are sepa-
rable by gel electrophoresis. To determine
the order of these fragments in the DNA
molecule and to simplify the task of nucleo-
tide sequence analysis of SV40 DNA, it
would be helpful to have overlapping sets of
such fragments, produced by different re-
striction endonucleases. To this end, we have
used an enzyme from H. parainfluenzae,
described by Gromkova and Goodgal (8), to
cleave SV40 DNA, and here report that the
major products of this digestion are three
large fragments which are about 40, 30, and
20%, respectively, of the length of SV40
DNA.

Small plaque SV40, isolated from strain
776 by IX. Takemoto and plaque purified, was
grown in monolayers of BSC-1 cells in mini-
mal Eagle’s medium containing 10% fetal
calf serum, penicillin, and streptomycin. Viral
DNA labeled with #P or [4C]thymidine was
prepared by the method of Hirt (6) followed
by isolation of covalently closed form I DNA
by equilibrium centrifugation in CsCl-ethi-
dium bromide and sedimentation through
neutral sucrose gradients, as detailed else-
where (1). Restriction endonuclease from H.
influenzae was prepared as described by
Smith and Wilcox (7). Restriction endonu-
clease from H. parainfluenzae was prepared
from a strain provided by Dr. 8. Goodgal, to
whom we are grateful for advice on the
growth of this organism. H. parainfluenzae
was grown to late log phase in BBL brain-
heart infusion broth supplemented with 2
ug/ml of NAD, and restriction enzyme was
prepared from the packed frozen cells by the

method developed by Smith and Wilcox for
the purification and assay of the H. influ-
enzae restriction enzyme (7). However, in the
case of the H. parainfluenzae enzyme, most
of the enzyme precipitated from the Biogel
eluate between 37 and 45% saturation with
ammonium sulfate. After application of the
ammonium sulfate fraction to a phospho-
cellulose column and stepwise elution with
0.1 M, 0.2 M, and 0.3 M KCl, enzyme was
found primarily in the 0.8 M@ KCl fraction.
The cluate was then concentrated using a
Diaflo pressure device and stored in 25%
glycerol at 4°C; it has retained activity for 12
months. The enzyme was active on many
bacterial DNAs, including that of H. influ-
enzae, but not on the DNA of H. parainflu-
enzae. DNA from M. lysodeikticus was used
as substrate during enzyme purification.

Digestion of SV40 DNA by restriction
endonuclease from H. influenzae was carried
out as described earlier (1). Digestion of
SV40 DNA by the H. parainfluenzae en-
zyme was carried out at 30°C for 90 min in
13 mM Tris-Cl pH 7.4, 20 mM KCI, 5
mM MgCl, 13 maf s-mercaptoethanol,
3% bovine serum albumin and 0.024 units
of enzyme (63 yg protein) per yg of DNA. (A
unit of enzyme is as defined by Smith and
Wilcox (9), but with A7. lysodeikticus DNA
as substrate.) The ratio of DNA to enzyme
appreciably influenced the extent of the re-
action and was optimized to the ratio just
given. Also critical werc the salt concentra-
tion (Tris-Cl and KCl) and the Mg? con-
centration. Purified enzyme still had slight
exonuclease activity under optimal condi-
tions for endonucleolytic cleavage of SV40
DNA. However, as shown below, distinct
digestion products could be readily isolated
by gel electrophoresis. Further purification
of the enzyme is underway.

Electrophoretic separation of digestion
products of [?P]SV40 DNA was performed

517

Copyright © 1973 by Academic Press, Inc.
All rights of reproduction in any form reserved.
518

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Fic. 1. Radioautogram of electrophoretically
separated fragments of SV40 DNA produced by
restriction endonucleases from H. parainfluenzae
(Hpa-A to D, center column) and H. influenzae
(Hin-A to K, right and left columns). Origin is at
the top. The actual distance of fragment Hpa-A
from the origin was 28 mm. The gel contained
4% acrylamide with 5% cross linking.

in slabs of 3 or 4% polyacrylamide gel (5%
cross linking) measuring 15 X 40 X 0.16 cm
as described earlier (8), after which the slabs
were dried (9) and applied to X-ray film for
radioautography.

Digestion of [?P]SV40 DNA by restriction

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20

Dd

io-)

 

 

 

NUMBER OF MOLECULES

0
105

 

 

 

T qT Tt
40 60 80 100
LENGTH AS % OF Sv40

 

Fic. 2. Histogram of the lengths of Hpa-A,
B, and C as determined by electron microscopic
measurements. Electrophoretically separated
fragments were individually mixed with open
circular SV40 DNA II reference molecules and
spread on parlodion-covered, 200-mesh copper
grids and stained with uranyl acetate (10). Photo-
graphs were made using an AEJ EM6B micro-
scope. Photomicrographs were projected to a
final magnification of 2.1 X 105 and the contour
lengths measured with a Dietzgen map reader.
Lengths are expressed as percentage of the lengths
of open circular DNA on the same grid. Fragments
are on the left, reference circles on the right.

enzyme from H. parainfluenzae resulted in
the appearance of four main DNA frag-
ments scparable by electrophoresis, desig-
nated Hpa-A, Hpa-B, Hpa-C, and Hpa-D
(Fig. 1). In addition, a small amount of
another fragment is often found between
Hpa-B and Hpa-C (Fig. 1), which we believe
is derived from a minor population of SV40
DNA molecules present in some prepara-
tions of DNA. Prolonged incubation or addi-
tion of more enzyme did not affect this digest
pattern, and we, therefore, conclude that the
fragments detected are limit products. Also
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shown in Fig. 1 are reference digests of SV40
DNA made with the H. influenzae restric-
tion enzyme (1).

Molecular weights of the I{pa fragments
have been estimated by the relative amount
of DNA present in each fragment (P radio-
activity), by electron microscopic measure-
ments of length relative to open circular
SV40 DNA molecules, and by electrophorctic
mobility. For #P measurement each frag-
ment was first localized by radioautography
of the wet gel slab and the dissolved frag-
ments directly counted. For length measure-
ments, fragments Hpa-A, B, and C were
separated in 3% polyacrylamide gels, lo-
calized by radioautography, and eluted in
0.015 Af NaCl, 0.0015 47 Na citrate, pH
7.0. Each fragment was then mixed with
open circular SV40 DNA II and samples
mounted for clectron microscopy. The re-
sults of length measurements are presented
in Fig. 2. The size of the Hpa fragments
could also be estimated by electrophoretic
mobility relative to H. influenzae fragments
of known molecular weight (). All of the
molecular weight estimates obtained by the
various methods are summarized in Table 1.
Fragment Hpa-A is about 40% of the length
of 8V40 DNA, Hpa-B is about 30%, and
Hpa-C is about 20%. Since the values ob-
tained by length measurements and relative
yield ure similar, these fragments are each
equimolar with the starting DNA. In the
case of Hpa-D, the amount of DNA present
appears to exceed even a rough estimate of
molecular weight based on electrophoretic
mobility relative to H. influenzae fragments
(1). Therefore, it is likely that Hpa-D is
multiple.

It is clear from the results presented that
the number of sites in SV40 DNA susceptible
to the H. parainfluenzae restriction enzyme is
fewer than the number susceptible to the H.
influenzae enzyme. The two sets of fragments
thus have extensive overlaps and have,
therefore, proved useful in mapping the en-
zyme cleavage sites (Danna, Sack, and
Nathans, submitted for publication). In ad-
dition, the large fragments of SV40 DNA
produced by incomplete or complete diges-
tion with the H. parainfluenzae enzyme
should contain intact genes or operons and

519
TABLE 1

Motecuuar Wercuts or Hpa FRAGMENTS
(as &% or SV40 DNA)

 

 

Apa Electron 2P TDis- Electro-
fragment microscopy” tribution’ —_ phoretic
mobility?

A 36.4 + 5.3 39 2 (34)

B 28.9 + 5.2 32.8 (31)

Cc 18.5 + 3.2 21.1 21

D 6.9 (<4)

 

Electron microscopic length measurements
are taken from Fig. 2 and are percentage of the
length of open circular SV40 DNA + 1 standard
deviation.

®To estimate the molecular weights by dis-
tribution of #P in the various fragments, 0.35 ug
of |@P)SV40 DNA 1 containing 53,400 cpm was
digested to completion in 35 ul of the standard
reaction mixture and subjected to electrophoresis
in the same slab gel after division into three equal
portions. After electrophoresis (150 V [30 mA]
for 18 hr at room temperature) the wet gel was
covered with Saran Wrap and exposed to film for
24 hr; the film was then developed and used to
locate the radioactive areas on the wet gel. Frag-
ments of the gel containing the radioactivity were
then cut out and placed in scintillation vials con-
taining 0.2 ml of 30% H.0, and dissolved at 75°C.
After dissolution of the gel fragments, 5 ml of
Triton-toluene fluor was udded and the samples
were counted. The results are expressed as per-
centage of total radioactivity present in each
fragment. Each value is the average of three
electropherograms.

© To estimate molecular weight from electro-
phoretic mobility, H. influenzae fragments were
used as standards (/). Since no standards were
available above 22.5% or below 4% of the length
of SV40 DNA, only the value for fragment Hpa
C could be accurately determined by this method.

may, therefore, show biological activity, thus
helping to localize SV40 genes.

ACKNOWLEDGMENTS

We thank T. J. Kelly, Jr. and K. J. Danna for
their help in some of the experiments reported.
This research has been supported by grants
from the U. 8. Public Health Service (CA 11895)
and from the Whitehall Foundation. George H.
Sack is supported by Training Grant GM 00624.

REFERENCES

1. Danna, K. J, and Naruans, D., Proc. Nat.
Acad. Sci. USA 68, 2913-2917 (1971).
520 SHORT COMMUNICATIONS

2, Natuans, D., and Danna, K. J., Nature 9. Rem, M. &., and Bieteskr, R. L., Anal.

(London) New Biol. 236, 200-202 (1972). Biochem, 22, 374-381 (1968).
3. NaTHans, D., and DANA, K.J., J. Mol. 10. Davis, R. W., Sron, M., and Davipson, N.,
Biol. 64, 515-518 (1972). In ‘‘Methods in Enzymology” (S. P. Colo-

4. Danna, K. J., and Natuans, D., Proce. Nat.
Acad. Sci. USA 69, 3097-3100 (1972).

§. Gromkova, R., and Goopaat, 8. H., J. Bac-
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wick and N. O. Kaplan, eds.), Vol. 21, pp.
413-428, Academic Press, New York, 1971.

5. Hirt, B., J. Mol. Biol. 26, 365-369 (1967). George H, Sack, Jr
7. Smitu, H.O., and Wiucox, K. W., J. Mol. Biol. DanieEL NaTHANS
51, 379-391 (1970). Department of Microbiology

8 peWacuTer, R., and Fiers, W., In ‘‘Methods The Johns Hopkins University
in Enzymology” (8S. P. Colowick and N.O. School of Medicine
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demic Press, New York, 1971. Accepted November 18, 1972