Volume 5 Number 10 October 1978
Nucleic Acids Research
Definition of the boundaries of the origin of DNAreplicationin simian virus 40
Kiranur N.Subramanian and Thomas Shenk+
'Department of Microbiology, University of Illinois at the Medical Center, Chicago IL 60612, and
"•"Department of Microbiology, University of Connecticut Health Center, Fannington, CT 06032, USA
Received 26 June 1978
ABSTRACT
We have determined by use of DNA sequencing techniques the exact location of the deletion in dj_ 892, a viable deletion mutant of Simian virus kO
(SV40) reported to map very near the unique replication origin or SV'+O.
With the help of this localization w e have narrowed down the boundaries of
the replication origin to 85 nucleotides within the sequence of SV'+O.
INTRODUCTION
The replication of Simian Virus (SV40) DNA proceeds bid! rect ional ly,
starting from an unique origin located near the junction between Hind
(11 + 111) fragments A and C at 0.67 map units clockwise from the single
Eco Rl cleavage site on the DNA 1 - 2 .
One of the fragments of SV40 DNA obtained
by cleavage with the Eco RlI restriction endonuclease, called £co Rll-G,
maps between 0.64 and 0.70 map units in the physical map of the DNA 3 and
overlaps the region containing the replication origin.
Subramanian, Dhar and
Weissman have determined the complete nucieotide sequence of the Eco Rll-G
fragment . This sequence contains the following interesting structural features;
a 27-nucleotide long palindromic sequence possessing a perfect two-
fold rotational axis of symmetry; three other shorter palindromes; a 17-long
stretch containing only A and T residues; and a 17-long "true" palindrome.
In addition, three sets of reiterated sequences occurring within Eco Rll-G
and the neighboring small fragment Eco Rll-N have been detected and sequenced
by Subramanian, Reddy and Weissman5.
These repeats occur distal to the Hind
(11 + 111) - A - C junction within nucleotides 191 and 368 (ref. 5 ) . Two
of these are perfect tandem repeats, that are 21 and 55 nucleotides long,
respectively; the third is a short sequence 8 to 10 bases long that is repeated
four times within the two copies of the 21-long repeat and twice more occurring to the left of the 21-long repeat^'5.
1
the 5 ends of the early and late viral RNAs
Eco Rll-G fragment also specifies
and the possibility has been
suggested ^'° that the replication origin itself could also serve as a
© Information Retrieval Limited 1 Falconberg Court London W 1 V 5 F G England
3635
Nucleic Acids Research
promoter for the transcription of the early and/or late viral RNAs.
It would be of interest to define the boundaries of the SV^O replication origin accurately within the sequence of Eco RI I —G . This could be
achieved by analysis of deletion mutations which eliminate sequences on either
side of the replication origin.
We have determined the exact location of the deletion in jdl_ ^92, a
viable mutant originally isolated by Shenk ejt aj_.', and reported to map at
0.68 map unit.
With the help of this localization, we have narrowed the
boundaries of the replication origin to 85 nucleotides within the Eco
RlI-G fragment.
MATERIALS AND METHODS
SV40 wild type strain 830 and viable deletion mutant strain dj_ 892
(ref.7) were used in this study.
Restriction endonucleases Hae 111 and
Al u I were purchased from New England Biolabs, Beverly, Mass. Alkaline phosphatase (BAPF) was purchased from Worth!ngton Biochemicals, Freehold, N.J.
Bacteriophage T4 induced polynucleotide kinase was a gift from Or. Kan
Agarwal of the University of Chicago. 7-32p_ATP of specific radioactivity
1,000 to 2,000 Ci/m mole was purchased from ICN Pharmaceuticals, Irvine,
California.
SV40 wild type and deletion mutant ONAs were digested with the Hae 111
restriction endonculease.
their 5
1
The ONA fragments in the digests were labeled at
32
ends by use of T4 polynucleotide kinase and y- P-ATP as described
earlier,^ and were fractionated by electrophoresis on a *+% polyacrylamide
slab gel.
The fragments were located by autotradiography.
Fragment d\_ F)a
containing the deletion and the corresponding wild type fragment Fia were
cut out from the gel and extracted by methods described earlier^.
The two
labeled 51 ends of each fragment were separated by digestion with the Alul
restriction endonuclease and fractionation of the subfragments by electrophoresis on a 5% polyacrylamide slab gel containing a 12% polyacrylamide
plug.
The subfragments produced were each labeled in only one of their two
5' ends and were sequenced by the method of Maxam and Gilbert.
RESULTS AND DISCUSSION
An autoradiograph of the Hae 111 restriction digest of wild type and d\_
892 DNAs labeled at their 51 ends and fractionated by electrophoresis on a
4% polyacrylamide gel is show in Figure la.
Fragment _dj, Fja is seen to have
moved slightly faster than its wild type counterpart since it is smaller pre-
3636
Nucleic Acids Research
sumbaly due to the deletion that
it contains.
Comparison of the electrophore-
tic mobilities of other Hae 11) fragments of known size
shows that .dj. F|a
is about 25 nucleotides shorter than the wild type fragment
F^a.
The dl F)a and F]a fragments were extracted from the gel, redigested
with the Alul
restriction endonuclease to separate the two labeled 5' ends
in each fragment and the two subfragments produced were fractionated by
electrophoresis on a 5% polyacryiamide gel as shown in Figure lb.
The small-
er of the two subfragments of d}_ Fja had the same mobility as its counterpart derived from the wild type F.a fragment whereas the larger subfragment
of d\_ F|a moved faster than the larger subfragment of Fja showing that it
contained the deletion
in the d\_ 892 mutant.
This subfragment, cal led dl
Fja-1 was labeled only at its Hae 11.1 end and was sequenced by the procedure
a
An autoradfograph of the sequencing gel is shown in
of Maxam and Gilbert.
Figure 2.
The sequence
is read from bottom to top.
The sequence of the
corresponding wild type fragment determined earlier
The portion of the sequence missing
the two sequences.
is presented
in dj_ 892 can be determined by comparing
The Hae 111 end of d\_ Fja labeled
in this study corre-
sponds to position 160 in the sequence of the Eco Rll-G fragment
this region.3
186.
in Figure 3.
spanning
The sequence of dj_ Fja reads like wild type through
The next 19 nucleotides of wild type SVtO ONA are missing
position
in d\_ 8 9 2 .
After this gap, the sequence picks up from position 206 and reads like
the wild type sequence.
Definition of boundaries for the SV^O replication origin
The deletion mutant d_l_ 892 was chosen for this study since it maps
closest to the replication origin.
It is followed by other viable deletion
mutants mapping distal to position 205 (Fig.3) in an area between map units
0.68 and 0.74 of the genome.?
It is clear from results presented above that
dl 892 lacks a segment of 19 nucleotides
205 in the Eco Rll-G sequence, both
DNA.
(located between positions
187 and
inclusive) present in the wild type SV'tO
In lieu of the fact that segments of ONA occurring distal
to position
186 could be deleted without affecting the replication of the viral
DNA
(and,
indeed, the ability of the virus to grow without a helper), one of the boundaries for the replication origin
located distal to the Hind
junction could be placed at position
(11 + 111) -A-C
186.
The other boundary for the replication origin can be placed by use of
data obtained by Lai and Nathans^ and Cole £t aj_
.
ucted a deletion mutant called d\_ 1002 lacking Hind
mapping between 0.430 and 0.655 map units.
Lai and Nathans^ constr(II + 111) -A fragment
Cole £t aj_
have constructed
3637
Nucleic Acids Research
41892
v!'-- •*»
NT
WT
-A-
'km
3638
892
Nucleic Acids Research
another deletion mutant called dj_ 1209 that lacks the Hae 111 -E fragment
located between 0.590 and 0.660 map units.
Both these mutants are
in their early functions but can replicate very well
impaired
in presence of a helper
virus, suggesting that their origin of DNA replication
is intact.
These
deletion mutants were created by infection of BSC-1 cells with partial digestion products of SV40 ONA lacking only the fragment
Hind
(11+111)
-A or Hae III -E.
in question, viz,
The absence of just these fragments and
no other ONA sequences from the genome has been confirmed by
7
analysis of the isolated deletion mutant DNAs- '
.
restriction
These results
indicate
that sequences to the left of map position 0.660 are not needed for initiation of SV40 DNA replication.
Therefore, the other boundary of the replica-
tion origin is placed at map position 0.660 corresponding to the Hae III
restriction site at nucleotide
ing to
102 in the Eco RI I-G sequence
.
Thus, accord-
the present estimate, the SV'tO replication origin consists of a
maximum of 85 nucleotides
located between positions
quence of the Eco Rll-G fragment derived earlier
This sequence
102 and 185 in the se-
and
is shown
in Figure k.
includes the 27-nucleotide long perfect palindrome, the 17-
long "true" palindrome
(containing a single imperfection
in its sequence),
the 17-long A-T rich sequence and the 15-long imperfect palindrome, all
shown to be present
in the Eco Rll-G sequence
.
As discussed earlier,
the palindrome s might be the recognition signals for proteins taking part
in the initiation of DNA replication and the A-T rich sequence might be required for unwinding of the DNA strands at this
location.
Nature of the sequence missing in dl 892
The deletion
in ^J_ 892, a viable deletion mutant, affects two copies of
a short reiterated sequence,
(A)TGGG(G)CGGA,
located closest to the repli-
cation origin; one of these is removed in full and the other in part.
However,
it may be noted that two more copies of this repeat are present within the
two copies of a 21-long tandem repeat that follows**'!*.
copies
repeat.
In addition, two
of a similar sequence, GGGGCGGGA, are also present within the 21-long
It appears reasonable that removal of two of the copies of a certain
reiterated sequence which
is otherwise adequately represented within the genome
does not affect the viable nature of the virus.
Figure I: Autoradiograph of polyacrylamide gel electrophoresis of a)
Hae 111 fragments of wild type (WT) and dl_ 892 DNAs labeled at their
5 1 ends, and b) Alu I digests of 5 1 end labeled Hae 111-F.a fragments
of wild type and dl_ 892 DNAs. The arrow in b) denotes the larger
Alu I subfragment of dj_ F.a containing the deletion in d\_ 8 9 2 .
3639
Nucleic Acids Research
Figure 2:
Sequence analysis of the
larger Alu
I subfragment of dl Fja
by the technique of Maxam and
The channels of C>T, T>C,
Gilbert".
3*A,
and A>G contain bands corresponding to
fragments arising out of chemical
cleavage at predominantly C,T,G, or A
residues respectively.
The right
hand side contains the sequence of dj_
F)a reading from bottom to top.
The arrow denotes the location where
the sequence changes from wild type
to mutant.
-t-tr
T
A
A
ft
3640
Nucleic Acids Research
51
170
C-C-T-C-T-G-C-A-T-A-A-A-T180
A-A-A-A-A-A-A-A-T-T-A-G190
f
I - C |l AA -- GG -- CC -- C C-!A-!A-I-G-G-C-C-
L
200
V"
r
C - C - G - A I G - A •IA-T-GJG- G - C 210
3'
G - G - A ^ A - C V T - G - G - G - C
Figure 3: The sequence of the portion of wild type SV40 DNA spanning
the deletion in d]_ 892. This sequence is taken f{jom the sequence of
Eco FUI-G fragment of SVUO DNA determined earlier . The sequence
TJeTeted in dl_ 8§2 as determined In this study is shown within brackets.
The boxes denote
a short reiterated sequence present in this part of
SV40 DNA « 5 .
102
110
•[- C-C-T-C-C-A-A-A;A-A-A-G-C-C-T-C-ci
I
...i
I
-(- G-G-A-G-G-T-T-T JT-T-T-C-G-G-A-G-G-f
120
130
T-C-AjC-T-A-C-T-T-C-TjG-G-A-A-T-A-G
A-G-T-lG-A-T-G-A-A-G-A-jC-C-T-T-A-T-C
140
150
C-T- C-A-G-A-G-G-C-C-G-A-G-G-CG-AG-T-C-T-C-C-G-G-C-T-C-C-G160
G-G-C-C-T-C-G-G-C-C-T-C-T-G
C -C -G-G-A-G-C -C -G-G-A-G-A-C
170
;$p
3-
:A-T-A-A-A+T-A-A-A-AJA-A-A-A-TTT-ATG-T-C-
i
i
:
!• •!• • •
:T-A-T-T-T-fA-T-T-T-T-JT-T-T-T-A^A-TTC-A-G••
5'
Figure k: The sequence of 85 nucleotfdes presently assigned to the SV^O
replication origin. This sequencers a part of the sequence of the Eco
Rll-G fragment determined earlier . Boxes drawn with solid lines
denote palindromes; those drawn with dashed lines or dots denote the
true palindrome or the A-T rich sequence respectively.
3641
Nucleic Acids Research
Acknow1edqment
This research was supported by grants from the University of Illinois
(GRSG and Campus Research Board) and the American Cancer Society (#NP-248)
to K.N.S. and by a grant from the National Cancer Institute (CA-19151) to
T,S. Preliminary characterization of d\_ 892 by transcription to RNA and
fingerprint analysis (not reported here) was done while K.N.S. was at Yale
University in the laboratory of S.M. Weissman. This work was carried out
during the tenure of an Established Investigatorship (to T.S.) of the
American Heart Association.
References
1. Fareed, G.C. Garon, C , and Salzman, N. (1972). J. Virol, 10, 484-491.
2. Oanna, K., and Nathans, D. (1972). Proc. Nat. Acad. Sci. USA, 69.
3097-3100.
3. Subramanian, K.N. Pan, J., Zain, B.S. and Weissman, S.M. (1974).
Nucl. Acids Res., }_ 727-752.
4. Subramanian, K.N., Ohar, R., and Weissman, S.M. (1977). J. Biol.
Chem., 252. 355-367.
5. Subramanian, K.N., Reddy, V.B., and Weissman, S.M. (1977). Cell, Jj)
497-507.
6. Dhar, R., Subramanian, K.N. Pan, J., and Weissman, S.M. (1977). J.
Biol. Chem., 7^1_, 368-376.
7. Shenk, T.E., Carbon, J., and Berg, P. (1976). J. Virol., 28, 664-671.
8. Maxam, A.M. and Gilbert, W. (1977). Proc. Nat. Acad. Sci. USA, 7_4,
560-564.
9. Lai, C.J., and Nathans, D. (1974). J. Mol. Biol., 89., 179-193.
10. Cole, C , Landers, T., Goff, S., Manteui 1-Brutlag, S., and Berg P.
(1977). J. Virol., 24, 277-294.
3642