J. Mol.
Biol.
(1977)
117, 497-513
Polyoma Virus Defective DNAs II. Physical
Map of a Molecule
with Rearranged
and Reiterated
Sequences
P74) ELSEBET
BEVERLY
LUND?,
E. GRIFFIN AND MIKE
FRIED
Imperial Cancer Research Fund P.O. Box 123, Lincoln’s Inn Fields London WC2A 3PX, England (Received
27 January
1977, and in revised form
29 July
1977)
The structure of the polyoma virus defective species D74 (74% the size of fulllength polyoma virus DNA) has been determined and compared with that of polyoma virus A2 DNA. D74 appears to be composed entirely of viral DNA sequences. (No host DNA sequences have been detected.) It is made up of three DNA segments, each about 24,24 and 27% in size. The two 24eh segments appear to be identical and the 27% segment contains one copy of all the sequences found in the 24% fragments as well as a duplication of some of the sequences. When related to the physical map of A2 DNA, each segment is found to be composed of viral sequences from 1 to about 19 map units, 67 to 69 map units and 70 to 72 map units. Three features found in other polyoma virus defective species (Lund et al., 1977) are also present in D74. (1) Sequences from the region around 67 map units arc linked to other (non-contiguous) viral sequences. (2) Sequences at about 72 map units are linked to sequences at 1 map unit. (3) Multiple copies of sequences from around the origin of viral DNA replication are present. From studies on other polyoma defective molecules (Griffin & Fried, 1975; Lund et al., 1977), the origin of DNA replication for polyoma virus has been defined to lie within the sequences from 67 to 72 map units. Since D74 replicates efficiently but lacks the sequences between 69 to 70 map units, the origin of DNA replication appears to be further defined as lying within 67 and 69 map units and/or 70 to 72 map units.
1. Introduction A number
of clonally
isolated
defective
1974) have been purified and analysed in Robberson & Fried, 1974; Griffin & Fried, species
have
been
shown
to contain
only
virus DNAs (Fried, considerable detail (Fried et al., 1974; 1975; Lund et al., 1977). All of these sequences with no detectable host DNA the origin of viral DNA replication. in the amount and arrangement of their
(non-infectious)
viral
polyoma
and to have repeats of the region around They have differed considerably, however, viral sequences. Earlier studies on the defective species (D74) defined in this paper suggested that contained little (if any) homology with polyoma viral DNA sequences (Robberson Fried, 1974). For this reason, a detailed study on D74 has been carried out using combination of methods which include DNA-DNA hybridization, cleavage t Present Chemistry,
address : University 1216 Linden Drive,
of Wisconsin Medical Center, Department 589 Medical Science Building, Madison, Wise.
497
of Physiologioel 53706, U.S.A.
it & a by
498
E.
LUND,
restriction endonucleases, studies have shown that sequences. The structure defective DNAs and the
B.
E.
GRIFFIN
AND
M.
FRIED
and analysis of depurination products. The results of these D74 is composed mainly of rearranged and reiterated viral of D74 is compared with structures determined for other biological implications of the findings are discussed.
2. Materials
and Methods
All procedures for preparation and characterization of wild-type A2 DNA and the defective (D74) DNA species were carried out as described in the preceding papers (Griffin, 1977; Lund etal., 1977) and by Fried (1974). The original notation (Fried, 1974) for the D74 defective virus isolate was changed (Robberson & Fried, 1974). The latter notation is used in this paper.
3. Results (a) Hybridization
analysis
of 1374 DNA
In an initial attempt to determine the DNA sequencespresent in the defective speciesD74, DNA hybridization analyses between the defective DNA and A2 polyoma virus DNA (or fragments thereof) and of mouse DNA were carried out. (i)
Analysis
using A2 HpaII
fragments
as
probes
The data obtained with three of the A2 HpaII fragments are shown in Figure l(a) to (c) ; these are representative of results obtained by this method of analysis. Only two of the polyoma virus DNA fragments, A2 HpaII-2 and 3, showed any increase in the rate of reannealing following the addition of D74 DNA, thus establishing that D74 DNA contains viral DNA sequencesthat correspond to a very limited portion of the polyoma DNA. In the caseof A2 HpaII-2 (Fig. l(a)), the initial rate of reannealing in the presence of D74 DNA (indicated by the broken line) was higher than that observed in the presenceof the homologousA2 DNA. The subsequentpoints obtained in the presence of D74 DNA did not fall on a straight line and the rate of reannealing of the probe appeared to decreasewith increasing time of incubation. Similar kinetics of reassociation were observed in three independent experiments. In the presence of A2 DNA, on the other hand, the points produced a straight line, as expected for a DNA which contains one complete copy of the probe DNA sequences.These results suggest that D74 DNA contains more than one copy per molecule of a certain fraction only of the A2 HpaII-2 DNA sequences. In the caseof A2 HpaII-3 (Fig. l(b)), the increasein the rate of reannealing following the addition of D74 DNA was significant, but much smaller than that observed in the presenceof A2 DNA. The data did not allow the precise amount of A2 HpaII-3 sequencesin D74 DNA to be determined. By similar analyses, DNA sequencesfrom none of the other A2 HpaII fragments could be detected in the defective DNA. As an example, the data obtained with A2 HpaII-4 are shown (Fig. l(c)). Clearly, the addition of D74 DNA did not change the rate of reannealing of this probe. (ii) Analyses with defective specijic HpaII fragments and A2 HpaII-1 as probes In agreement with the finding (above) that D74 DNA contained several copies of certain viral DNA sequences,the complexity of D74 DNA was found to be severalfold lessthan that of A2 polyoma DNA (data not shown).
A REITERATED
POLYOMA
VIRUS
DEFECTlVE
DE.4
4Y!l
(cl /a
l IO
I 20
I 30
I IO Cot
Cot
x IO”
I 20
I 30
(mol
nucleotides
x s/l)
x IO4 (mol
nucleotldes
x s/L)
I
I
IO
20
Jha. 1. (a) to (c) Reannealing of A2 HpaII fragments in the presence of defective and wild-t)ype A2 DNAs. Portions of %‘-labelled A2 HpaII DNA fragments (spec. act. 3 x lo5 aaP cts/min per pg DNA) and unlabelled viral DNAs were denatured and allowed to reanneal at 68’C in 200 ~1 of standard hybridization buffer containing 0.2 M-N&Cl. The rate of reassociation was monitored by 81 nuclease digestion and the results were plotted as described (Sharp et crl., 1974; Lund et r/Z., 1977). The reassociation kinetics of 1.8 x 1Om8 M (mol nucleotides/l) A2 HpczII-2 (a), 1.8 x lWB M-A:! HpaII-3 (b) and 1.1 x lo-* M-AZ HpnII 4 (c) DNAs are indicated by (A). The same concentrations of probe DNAs were also reannealed in the presence of 1.5 x 1Om6 M-/d! (a) or I>74 (c:) DNAs. This concentration of homologous unlabelled A2 DNA corresponds to an la-fold molar excess of A2 HpaII-1 and HpnII-4 DNA sequences and a 14-fold molar excess of A2 HpaII-3 DNA sequences. As compensation for the difference in molecular weight of A2 and D74 DNAs the C,t values for D74 DNA-containing samples have been normalized t,o those of the A2 DNAcontaining samples as described (Lund et al., 1977). (d) and (e) Reannealing of fragments D74 HpaII 24% and A2 HpnII-1 in t,ho presence of AZ?, D74 and whole mouse embryo DNAs. The hybridizations were carried out as described in (a) to (c) using 300.~1 portions containing 0.5 M-NaCl in standard hybridization buffer. The reassociation kinetics of 32P-labelled probe DNA fragments (spec. act. 1 x lo5 32P cts/min per pg DNA) were measured in the presence of 3 x 10m3 NI (mol nucleotides/l) calf thymus DNA (A) for D74 HpaII 24% (d) and for A2 H-p&I-2 (e) with the probes at a concentration of 3.6 x lo-* M. Also shown is the reannealing of the same concentrat,ion of 32P-labelled probe DNAs in the presence of 3 x 10e3 M-calf thymus DNA and 6.7 x lo-’ MD74 DNA (0); 3 x 10m3 M-calf thymus DNA and 7.3 x 10e7 M-M DNA (0); and 3 x 10m3 M-whol? mouse embryo DNA (A). This concentration of unlabelled D74 DNA (d) corresponds to a 12-fold molar excess of D74 HpaII 24% DNA sequences assuming that each molecule of full-length D74 DNA contains 2 copies of the D74 H;oaII 24% fragment. Similarly the concentration of unlabelled 82 viral DNA (e) corresponds to a 7-fold molar excess of A2 HpaII-1 DNA sequences. In the case of A2 DNA the C,t values have been normalized (as described, Lund et nl., 1977) to those of D74 DNA to compensate for the differences in nucleic acid concentration and molecular weight of the viral DNAs. 33
500
E.
LUND,
B.
E.
GRIFFIN
AND
M.
FRIED
Cleavage of D74 DNA with the restriction endonuclease HpaII produces three fragments, 27% (one-molar yield) and 24% (t wo-molar yield) the size of full-length polyoma DNA (see section (b), below and Fig. 2)t. The purified D74 fragments appeared to have a complexity similar to that of polyoma virus A2 HpaII-1 (a fragment composedof non-repetitive sequencesand corresponding to 27.3% of A2 DNA) since under identical conditions of hybridization D74 HpaII 24”,/0 (or D74 HpaII 270/,) reannealed with a rate very similar to that of A2 HpaII-1 (compare Fig. l(d) and (e)). This suggested that the individual D74 HpaII fragments were composed (primarily) of non-repetitive DNA sequences.Furthermore, the reannealing of D74 HpaII 24% (or 27%) in the presenceof A2 DNA showed that most of these sequences appear to be of viral origin, since the reassociationkinetics of the defective fragment(s) was very similar to that of A2 HpaII-1. Addition of the homologousD74 to either of the defective specific HpaII fragments (24% or 27%) resulted in a large increase in the rate of reannealing, which indicated that the defective DNA contained at least two copies of these probe DNA sequences per molecule. This is shown for D74 HpaII 2476 in Figure l(d). In order to determine whether D74 DNA also contained host DNA sequences, D74 HpaII 24% (or 27%) was reannealed in the presenceof mouseembryo DNA in a concentration sufficient to detect repetitive host DNA sequencesin the probe DNA. Since the addition of host DNA did not influence the rate of reamlealing of the defective HpaII fragments (Fig. l(d)) it was concluded that D74 did not contain repetitive host DNA sequences.Using these techniques, however, the presenceof unique host DNA sequencesin the defective molecule could not be ruled out. In summary, the hybridization analysesshow that D74 DNA is composedprimarily (if not entirely) of viral DNA sequences,which in large part are derived from the region of A2 DNA that correspondsto sequencesfound in A2 HpaII-2. Furthermore these viral sequencesappear to be present in more than one copy (two or three) per molecule of D74 DNA. (b) Restriction
enzyme analyses
One of the immediately striking aspectsof the defective molecule D74 was the fact that the cleavage patterns obtained by digestion with either restriction endonucleases HpaII, Hind111 or HhaI were indistinguishable (Fig. 2). Digestion with each endonuclease produced one fragment approximately 24% (two-molar yield) and one fragment approximately 27% (one-molar yield) in size. (1) Both of the Hind111 defective specific products, when cleaved with HpaII, gave small fragments (about 1.7% in size) which were electrophoretically indistinguishable. In addition, D74 Hind111 24% gave a fragment approximately 2259/, and D74 Hind111 27% a fragment approximately 2550/, in size (seeTable 1). (2) Both of the HhaI defective specific products, when cleaved with HpaII, gave fragments (about 95% in size) which were electrophoretically indistinguishable. In addition, D74 HhuI 24% gave a fragment approximately 14.5% and D74 HhaI 27q/, a fragment approximately 17.5% in size (seeTable 1). (3) Both of the Hind111 defective specific products, when cleaved with HhaI, gave fragments (about 11% in size) which were electrophoretically indistinguishable. In t Unless percentage
otherwise indicated, of the A2 polyoma
a11 restriction virus DNA.
endonuclease
fragment
sizes
me expressed
as a
.I
REITERATED
POLYOMA
VIRUS
DEFEL”1’IVE
T)NA
r?o I
D74
A2
\
I
HpaJI
HhaI
BumI
-
HoeD
27% 24%
3.7%
3.0% 5 6 . 2.1%
FIG. 2. Autoradiograms of D74 DNAs (s2P-labelled) after cleavage with restriction endonucleases HJwII, HindIII, HhaI, Hum1 and Hue111 and of A2 DNA cleaved with HpaII. Fragments were separated on slab gels (20 cm x 20 cm) composed either of 0.0% polyacrylamide/0.15% bisacrylamide and 0.5% agarose (HpaII, HindIII, HhaI) or of 5.0% acrylamide/0~25°/0 bisacrylamide (HumI, HuelII). Fragment sizes for the HpaII restriction endonucleases are taken from Griffin et (II. (1974); sizes for the other restriction endonuclease fragments are given as a percentage of whole polyoma A2 DNA and were determined relative to the HpnII sizes.
addition, D74 Hind111 24% gave a fragment approximately 13:/, and D74 HindIII 27”;, a fragment approximately 16% in size (see Table 1). For further analysis, the action of the restriction endonuclease HaeIII on D74 DNA and on defective specific fragments produced by digestion with HpaII, HindIII and HhaI was studied. DigesCon of D74 with HaeIII produced five fragments with sizes approxima.tely 10.2%, 7.9%, 3.7% and 2.1 oh (each in three-molar yield) and 3.00/, (in one-molar yield) ; the latter product was found exclusively in the D74 27y; fragment (see Fig. 2). The 10.2% fragment was electrophoretically indistinguishable from A2 HaeIII-3, a subfragment of A2 HpaII-2 (Griti, 1977). Cleavage of the D74 Hind111 24% and 27 “/o fragments with HaeIII resulted in the dixappearancc of the D74 HaeITI 3.7 76 fragment ; two new fragments were generated
E.
502
LUND,
B.
E.
GRIFFIN
AND
of data obtained
FRIED
1
TABLE
Xummary
M.
from D74 DNA
by cleavage with restriction
endonucleases HpaII D74
HpaII
D74
Hind111
D74
HhaI
D74
Burnt
D74
H&II
24% 27% 24% 27% 24% 27% 14.5% 12.6% 11.5% 10.2% 7.9% 3.7% V.O% 2.1%
Hind111 22+i+ 255+
22.5+ 1.7% 26*5+ 1.7% 14x5+ 9.5% 17.6+9.6% 12+2.5% NC 9+24x NC NC NC NC
=%I
1.7% 1.7%
13+11y0 lS+ll% 10.4+4*1y0 NC 7.4+4.1y0 NC NC 2.2+ 1.5% NC NC
Fragment sizes we expressed as percentages of A2 DNA. NC signifies fragment not cleaved. t Specific to the D74 27% fragments. $ The electrophoretic mobility of this fragment w&s slightly
HhaI
Bum
14.5+9.5y0 17.5$9-5% 13+11y0 1s+11%
12.6+ 9+2.5y0 12*5+12+2.5y0 12.6+ 7.4+4y0 12.6+10.4+4% 11X+ 7.3+5.2% 14*6+ 7.3+6.2%
NC 7.3+6.2% NC NC 7.7+0.2% NC NC NC
retarded
5.2+ 6.0% 7%i+o.4y0 NC NC NC
after
cleavage
(see text).
with sizesabout 2.2% and 1.5% (Table 1). (In A2 DNA, the Hind111 site in HpaII-2 is found in HaeIII-8 about 2.2% away from the juncture with A2 HaeIII-3 (Griffin, 1977).) Cleavage of the D74 HhaI 24% and 27% fragments with Hue111resulted in a size change of the D74 Hue111 7.9% fragment (from approx. 7.9 to 7*70/,) (Table 1). (In A2 DNA, the HhaI site in HpaII-2 is found approximately 93% from the juncture of the A2 HaeIII-3 and 7 (Griffin, 1977).) Cleavage of the D74 HpaII 24% and 27% fragments with Hue111 resulted in the D74 Hue111 2.1% fragment migrating with a mobility characteristic of a slightly larger fragment. This phenomenon is characteristic of A2 fragment HaeIII- 17 (Griffin, 1977), the subfragment of A2 HpaII-3 which lies at the HpaII 3-5 junction (near the origin of viral DNA replication). The assignment of the above cleavage sites to the D74 Hue111fragments is consistent with results obtained by cleavage of D74 DNA with other combinations of endonucleases(Table 1). D74 DNA was further analysed with a number of other restriction endonucleases (data not given). Cleavage with HhaI together with either Hph, MboI, Mb011 or XbaI gave fragments which were electrophoretically indistinguishable from some of the fragments obtained from A2 HpaII-2. This indicated that D74 DNA contained continuous sequencesof A2 DNA from at least 1 to 18.2 map units (XbaI site at 18.2). Moreover, analysis with these restriction enzymes gave results which were consistent with the 3% extra sequencespresent in the D74 27% fragment being derived from sequencesfrom about 1.5 to 4.5 map units in A2 DNA, these 3% sequencesbeing tandemly duplicated in the defective fragment. The endonuclease Bum1 cleaves D74 into three fragments (Fig. 2) with sizes approximately 14.5% (one-molar yield), 12.5% (th ree-molar yield) and 11.5o/o(twomolar yield). The pattern obtained with Bum1 is consistent with there being two
A REITERATED
POLYOMA
VIRUS
DEFECTIVE
DNA
503
cleavage sites each in the D74 24% and 27% fragments. (A2 DNA has four Bum1 cleavage sites, Fried & Griffin, 1977.) The Bum1 cleavage sites in D74 DNA have been located relative to the Hpall cleavage sites by double digestion with these two endonucleases. The Bum1 12.5% fragment (three-molar yield) was not cleaved by HpaII, but the other two Bum1 fragments were cleaved. New products were found with sizes approximately 12%, 9% and 2.5% (Table 1). By a similar analysis, the Bum1 sites were located relative to Hue111 sites in D74 IINS. One Bum1 site was found in the D74 Hue111 10.20/, fragment. This fragment was cleaved to two products with sizes (5~2% and 5.0%) indistinguishable from the products obtained by cleavage of A2 Haelll-3 (a subfragment of Hpall-2) with RumI (Griffin 1977). The second Bum1 site lies very near to one end of the D74 HaelI 17.9% fragment (see Table 1). These data are summarized on the physical maps shown in Figure 3. (c) Depurination analyses Defective D74 DNA (32P-labelled) and the two viral-specific Hpall fragments (27% and 247/o) were chemically depurinated and the pyrimidine products were separated as described by Ling (1972). The fingerprints of these two species were indistinguishable and had many pyrimidine tracts in common with A2 Hpall-2 (Fig. 4). Notably absent from the fingerprints of D74 DNA were mo.st of the large (greater than decamer size) characteristic products of A2 DNA (Griffin, 1977). The only large oligopyrimidine in D74 DNA was one which appeared identical (in size and mobility) to the oligomer derived from A2 Haelll-14’, a subfragment of A2 Hpall-5 (see Fig. 7). The fingerprint of the defective DNA also contained the long run of a subfragment of A2 thymidines (T, or a) known to be a product of A2 HaeIII-17, HpaII-3 (see Fig. 8). Another predominant feature of the D74 fingerprint was that, as a group, the hexamers seemed to be in higher yield than would be expected on a random basis. (Compare, for example, the yield of hexamers with pentamers, Fig. 4.) This is especially true for hexamers with the composition C,T, and CT, and is a characteristic also of the depurination products of A2 Hpall-2. It does not appear to he characteristic of any of the other A2 Hpall fragments (Griffin, 1977). In order to characterize the defective species further, fingerprints of the five D74 HaeIII fragments were made and compared with HaeIII fingerprints from A2 DNA. The results are shown (in part) in Figures 5 to 8. Some aspects of the analysis of each fragment are considered below. (i) D7P HaeIII
10.2%
(three-molar
yield)
(Fig.
5)
This fragment was found to be identical (at least qualitatively) to A2 Haelll-3, a subfragment of A2 Hpall-2. Since these two fragments are also indistinguishable in size and give similar Bum1 products (Table l), D74 Haelll 10.2% and A2 Hue1113 are probably identical. As a striking feature, both species can be seen to contain only one pentapyrimidine (TC,). (ii) D7d HaeIII
7.9%
(three-molar
yield)
(Fzg. 6)
This fragment was found to have sequences similar to those of A2 HaeIII-7 (a subfragment of Hpall-2) and A2 Haelll-15 (a subfragment of Hpall-3). For ever.y oligomer in A2 Haelll-7, a corresponding oligomer could be found in D74 HaeIII 7,R”(,. A superimposition of the fingerprints of A2 Haelll fragments 7 and 15 will
604
E.
LUND,
B.
E.
GRIFFIN
AND
HmdIU
M.
FRIED
D74
0
HhaI HhaI 0
HhaI
(a) HpaD .
3.7%
HPoII
Bum1
‘HaelII
pa I
HueID
:2d-:D74
7.9%
10*2%
Hpall
24%
HPQ~
‘yfA2 (!:2’=,&)+&-8
( .9%)
Hoellt-3
I:-2
(02%)
Hpan
HpUIIHDe~ (b)
FIQ. 3. Physical maps of A2 DNA and D74 DNA. The 92 map is divided into 100 units with the single EcoRI cleavage site at zero map unit. The units shown on the physical map of D74 correspond to A2 map units. The origins (0) of viral DNA replication are indicated on the maps. (a) A2 physical maps. Inner ring, obtained by cleavage with restriction endonuclease HpaII (8 cleavage sites, Griffin et al., 1974). Outer ring, obtained by cleavage with Hue111 (24 cleavage sites, Griffin, 1977). Other cleavage sites are indicated. These maps were modified from Griffin (1977). Proposed D74 physical map, obtained by cleavage with restriction endonuclease HpaII (3 cleavage sites) (Fig. 2). The HpaII cleavage sites are indicated by continuous lines within the circle. Broken lines show where non-contiguous viral sequences have been covalently linked (see text) and the numbers indicate the A2 HpaII fragments from which these sequences are derived. Sequences between 69 and 70 map units in A2 HpaII-3 (near the origin of viral DNA replication) are deleted in D74 (indicated by V); sequences between 67 and 69 and 70 and 71 map units are present. The restriction endonucleases Hind111 and HhaI also cleave D74 DNA into three fragments, as shown. (b) Linear representation of the Hue111 physical map of one of the reiterated segments of D74 DNA (D74 H@lI 24%) and of A2 restriction fragment H@I-2; restriction sites common to homologous regions are indicated ( 2 ).
produce the fingerprint of D74 Hue111 7.9%; that is, all oligomers generated defective fragment can be found in a combination of the two A2 fragments. (iii) D74 HaeIII This
3.7% (three-molar
yield)
(Fig.
from the
7)
fragment was qualitatively very similar to A2 HaeIII-8 (a subfragment of but appeared to be missing the largest oligopyrimidine (greater than
HpaII-2),
A REITERATED D74
DNA
POLY
OMA
VIRUS
DEFECTIVE
A2
Electrophoresis, pH first dimension
HpalI
DNA
505
- 2
3-5
FIG. 4. Autoradiograms showing a comparison of the depurination products of 3ZP-labelled 1)74 DNA and A2 HpaII-2. The pyrimidine tracts were separated in the first dimension by electrophoresis and in the second dimension by homochromatography (see text and Ling, 1972) as indicated. The easily identified oligopyrimidines in D74 DNA which are derived from A2 HneIII fragments 14’ and 17 (see Figs 7 and 8) are indicated as (14’) and T, Dr 8, respectively. The characteristic oligomer present in A2 H&I-2 (which comes from A2 HueIII-8, see Fig. 7), but absent, from D74, is indicated (*). The hexamers on each fingerprint are indicated (VI). The fingerprints of the D74 HpaII 24% and 27% fragments were indistinguishable from that, of t,he whole D74 molecule.
dccamer size) normally found in the latter (Fig. 4). (Some traces of this oligomer could be seen on the fingerprint of the 3.7% fragment, but as it was present in considerably lower than one-molar yield and not found either in the whole D74 molecule or in its HpaII fragments (see Fig. 4) it was thought to arise from a small amount of contaminating HpaII-2 sequences from the helper viral DNA.) Quantitatively there were some other differences between D74 Hue111 3.7 o/oand A2 HaeIII-8, most notably at the hexamer level. For example, T,C, was clearly present in greater yield than other hexamers in the A2 fragment, but this was not the case for the D74 fragment. Moreover, in the defective fragment the very large oligopyrimidine present in A2 H&11-14’ (a subfragment of HpaII-5) is apparent. All the pyrimidine tracts found
A2
HaeIlI
-3
074
pH
Electrophoresis,
FIG.
10.2%
HaelII
5
3.5
D74
HoeIU
3 *0 %
A RElTERATED
POLYOMA
in the defective specific fragment Hue111 fragments 8 and 14’. (iv)
D74 HaeIII
VIRUS
DEFECTIVE
(3.7%) can be accounted
I)XA
for by products
507
from A2
3.0% (olte-molur yield) (Fig. 5)
All of the pyrimidine tracts in the defective fragment can be found in A2 HaelIl-3. The D74 Haelll 3% fragment contains only one pentamer which has the same composition (TC,) as the single pentamer in A2 Huelll-3 (see (i), above). The defective fragment is located between the D74 Hue111 10*2% and 3.7% fragments on t)he physical map of D74 (Fig. 3) and probably contains some sequences from the latter fragment, but these cannot be distinguished in the fingerprint of the defective specific 3.0!/, fragment. (u) D74 HaeIII
2.1% (three-molar yield) (Fig. 8)
This fragment was especially interesting as it was found to contain the oligothymidine tract (T7 ,,p 8) unique to A2 Huelll-17 (a subfragment of Hpall-3) and so must be composed (at least in part) of sequences from A2 Hpall-3. The D74 Haelll fragment, however, is approximately 2*1o/0 in size, whereas the A2 Haelll-17 fragment is only 1.394. Two oligomers, larger than any found in A2 Huelll-17, are present in the defective fragment. They have been shown to be identical (in mobility and size) to a pair of oligomers (octamers or nonamers) in A2 Huelll-14, the Hue111 fragment adjacent to Haelll-17 in A2 Hpall-3 (Griffin, 1977). The pentamer, T,C, in the defective species is also present in A2 Huelll-14. It could be concluded from further examination of A2 Hue111 fragments 14 and 17 that all the oligomers present in D74 HaeIlJ 2.1% could be found in one or the other of the two A2 fragments, but not all the oligomers present in these two A2 fragments were present in the defective specific fragment. D74 Hue111 2.1 y0 appears to represent a fusion of the two A2 Hue111 fragments (14 and 17) with a resulting deletion of some sequences.
4. Discussion By three independent methods of analysis (cleavage with restriction endonucleases, two-dimensional separation of depurination products (fingerprinting) and DNA-DNA hybridization), the structure of the polyoma defective molecule D74 has been deduced (Fig. 3). This molecule contains selected portions of the viral DNA sequences from Hpall-2 (1 to about 19 map units), Hpall-3 (67 to 69 and 70 to 71 map units) and Hpall-5 (71 to 72 map units). It is composed of three DNA segments, each about 24%, 24% and 27% the size of full-length A2 DNA. The two 24% segments appear to be identical, but the 27% segment contains, in addition to the sequences found in the 24y/, segments, 3% of DNA sequences which appear to be derived from Hpall-2, from the region on the physical map which includes the Hue111 3-8 junction (Griffin, 1977). Tandem duplications of viral sequences have been described in other defective
FIG. 5. fragments The latter fragment either A2 pentamer.
Autorediograms showing a comparison of the depurinetion products of 3aP.lebelletl D74 HaeIII 3.0’$& D74 HaeIII 10.2% and A2 HaeIII-3 (a subfragment of H@I-2). 2 fingerprints are qualitatively indistinguishable. The fingerprint of D74 Hue111 3.07; is much simpler, but does not have any oligopyrimidines which are not also present in HaeIII-3 or D74 HaeIII 10.Z”/& All 3 fragments contain TC( (indicated) as t,heir s&l
A2
HaellI
- 7
Electrophoresis, FIG.
Hue III
6
pH
7.9
3.5
%
-
A2
A REITERATED
POLYOMA
VIRUS
DEFECTIVE
DNA
509
pal-yome virus species (Griffin & Fried, 1974; Lund et aZ., 1977). The peculiar arrangemcnt of viral sequences found in D74 may explain the previous difficulty of obt,aining a single interpretable heteroduplex structure between D74 and -42 DNA (Wild. unpublished results). The three features described for the polyoma defective related species D76, D91 and D92 (Lund et ul., 1977) are also found in D74. One involves the linkage of sequences from around 1 map unit (in H&II-2) to sequences around 72 map units (in HJXZII-5). A srcond feature is the linkage of sequences from around 67 map units (in A2 Hael I I15. a subfragment of HpaII-3) to viral sequences in HpaII-2. The data available do not, allow for the exact location of the latter sequences.Although they appear to lit in A2 NaeIII-7 (near the H&I 2-6 junction), there is a discrepancy (0.3 to 1’:“) in sizck between the A2 fragments concerned and the D74 HueIII 7.9% fragment. Even if ~22the sequencesfrom A2 HaeIII-15 (1.5%) and 7 (6.0%) (Griffin, 1977) were present in t)he defective fragment, the latter is still larger than the sum of the A2 fragments. The discrepancy in sizes may be merely a reflection of base composition and sequences,since these affect electrophoretic mobilities of DNA fragments (Ziegler it nl., 1952). Alternatively, D74 may contain someadditional sequences(host or viral) not found in t’his region of A2 DNA. ‘I’hc bhird feature is the repetition of viral sequencesfrom around the Hpal I 3-5 junction where the origin of viral DNA replication has been mapped (Griffin cf al.. 197-C;Crawford et al., 1974). But, whereas in other polyoma virus defective species. continuous viral sequencesfrom around 67 to 72 map units have always been present, (Griffin & Pried. 1975; Lund et al., 1977), in D74 about 1% of the DNA sequences from this region are deleted. D74 contains continuous viral sequencesfrom about 67 to 69 map units and from 70 to 72 map units, but sequencesfrom 69 to 70 map units including the Hue111 cleavage site at 69.5 map units (between A2 fragment’s HmITI-14 and 17) and the Burn1 site at 70.2 map units (Griffin, 1977) are absent’. It’ seemsreasonableto conclude that these deleted sequencescannot therefore be t’ssent,ial for viral DNA replication since D74 appearsto replicate efficiently in the presence of a helper virus (assumingthe viral origin of DNA replication is being used). It’ may be that in polyoma DNA the sequencesspecifying the origin of viral DNA replication art: loca,ted in two closely spaced but non-contiguous regions (67 to 69 and 70 to 7% map units) separated by a region of non-essential sequences(69 to 70 map units). It is also possiblethat both persisting regions are concerned with viral DNA replication but, play different r8les. One may be the site at which an enzyme (e.g. DNA polymerase) binds to the DNA and the other the site at which synthesis is initiated. On bhe other hand, only one of these two regions may be involved in DNA replication and the other region may be conserved because it is essential for some other viral function. For example, it could serve as a focus for the formation of the viral capsid. Encapsidation must be essential for the successfulinfection and virus growth of both defrct’ive and non-defective viral DNAs. In any case,the sequenceswhich speci[y the origin of viral DNA replication appear to be further defined by D74 DNA as lying eithrsr between 67 and 69 and/or 70 and 72 map units.
FH:. 6. Alctoradiograms showing a comparison of fragments of D74 HneIII 7.9?; with A2 HaeIII-7 (a Fubfragment of HpaII-3). A hypothetical fingerprint wo~~ltl producr the fingerprint found for D74 Hue111
the depurination products of 32P-labelletl subfragment of H@T-2) and HtceIII-15 (a constructed from these two A2 fragments 7.9%.
A2
Huelil-
8
D74
FIG.
Electrophoresis. I
Huern
pH
3.5
3*7%
A2 HueDI
14’
A
REITERATED
POLYOMA
VIRUS
DEFECTIVE
DNA
611
The merit’s and limitations of the three methods used for analysis of D74 are discussed in the accompanying paper (Lund et al., 1977). The extent of homo1og.y detected hy DNA-DNA reassociation kinetics between D74 and A2 DNAs is very limited and appears to be restricted to certain DNA sequences present in A2 HpaII fragments 2 and 3. There was no detectable “cross” hybridization between D74 DNA and DNA from any of the other six A2 HpaII fragments under similar conditions of rcannealing. The analysis did not however permit an accurate quantitation of the viral DN$ sequences in the defective molecule. As previously discussed (Lund et al., 1977) the hybridization conditions used would not permit short viral DNA sequences, present in “novel” (defective specific) arrangcmerits different from A2 polyoma DNA sequence arrangements, to be detected. Therefore. t,his analysis provides only a minimum estimate of the amount of viral DNA sequences in D74 DNA. The data presented in Figure l(d) and (e) suggest that the defective molecule does not8 cont,ain a large proportion of host DNA sequences, since the A2 polyoma DNA appeared to “cross hybridize” with most (if not all) of the D74HpaII 24%, prohr sequences. In addit’ion. from the analysis of the reassociation kinetics of the defective specific D74 HpalI 24’34 and 27% fragments, it could be concluded that (1) the DNA sequences from t’hese fragments are present in more than one copy per D74 molecule. (2) most of these sequences are of viral origin and (3) none of the defective specific HpaTI fragments contains large stretches of repetitive host (mouse) DNA sequences (see Fig. l(d) and (e)). The restriction endonuclease analysis shows that continuous viral sequences from Ar! HpaII-2 between the Hind111 cleavage site (at about 1 map unit) and the Xhal cleavage site (at about 18 map units; Fried & Griffin, unpublished results) are present in t(he two D74 HpaII 24% fragments. Furthermore, the D74 HpaII 27% fragment, appears to contain all the sequences present in the 2404 fragments plus an insertion (3’);)) of DNA sequences between the D74 HaeIII 3.7% and 10.2% fragments. The extra sequences in the 2774 fragment appear to come from a duplication of the sequences of A2 HpaII-2 from about 1.5 to 4.5 map units. The pyrimidine tract analysis proved ultimately most useful in the assignment of sequences in D74. By identifying unique tracts of pyrimidines as well as comparing oligomrrs produced from D74 and A2 HaeIII fragments, a number of rearrangements, deletions and additions could be detected. These results are summarized in Figure 3. D74 was isolated after five high multiplicity viral passages (Fried, 1974) and shows extensive rearrangements of viral sequences. No evidence has been obtained for the presence of any host DNA in D74 although a small amount of host sequences cannot) definitely be excluded. Why these sequence rearrangements occur and how they might confer select,ive advantage to D74 is not clear. The longest continuous segment of viral sequences in D74 is derived from A2 HpaII-2, these sequences being linked
Fro. 7. Autoradiograms showing a comparison of the depurination products of 3ZP-labelled I)74 fragment 29~111 3.7% with A2 HaeIII-8 (a subfragment of HpaII-2) and A2 H&III-14 (a subfragment, of HpuII-6). D74 Hue111 3.7% has a fingerprint which is qualitatively similar to A2 HaeIJI-8, although it lacks one (*) of the oligomers unique to the latter (see text). Quant,itative difference can be seen among the hexamers (VI). (For example, compare T& with other hexamers in the 2 fingerprints.) In addition, D74 NaeIII 3.7% contains the very large oligomer (14’) found in X2 H~wIII-14’, but not present in A2 HneIII-8.
FIG.
Electrophoresis.
8
pH 3.5
A2
HoeEl
- 17
A
RETTERATED
POLYOMA
VIRUS
DEFECTIVE
D?r’A
613
to sequences from HJXXII-5 or HpaII-3. Sequences from HpaIT-2 are not however found in other reiterated defective species (Griffin & Fried, 1975). Like other defective species (Griffin & Fried, 1975; Lund et al., 1977), D74 retains viral sequences from the region between 67 and 72 map units: hut unlike the other species. it does not retain all of these sequences. Thus, D74 may prove to be very important in understanding the biological r81e of the various parts of the viral genomc, and in particular t.he region around the origin of viral D1L’A replication. REFERENCES Crawford, L. V., Robbins, A. K. & Nicklin, P. M. (1974). J. (Yen. ITirol. 25, 133~ 142. Pried, M. (1974). J. Viral. 13, 939-946. Fried, M. & Griffin, B. E. (1977). In Advances itz Cancer Research (Klein, G. & Weinhousn, S., eds), vol. 24, pp. 67-113, Academic Press, New York. Fried, M., Griffin, B. E., Lund, E. 6t Robberson, D. L. (1974). Cold S’y&g Harbor S~yny. Quad.
Viol.
39,
45-52.
Griffin, B. E. (1977). J. Mol. Biol. 117, 449-473. Griffin, B. E. & Fried, M. (1975). Nature (London) 256, 175-179. Ling, V. (1972). J. Mol. Biol. 64, 87-102. Lund, E., Fried, M. & Griffin, B. E. (1977). J. Mol. Biol. 117, 475.-497. Robberson, D. L. & Fried, M. (1974). hoc. Nat. Acad. Sci., U.S.A. 71, 3497--3501. Sharp, P. A., Pottersson, U. & Sambrook, J. (1974). J. illoh Biol. 86, 709-726. Ziqler, R. S., Salomon, R., Dingman, C. W. & Peacock, A. C. (1972). h’ature New 238, 65-69.
Biol.
FIG. 8. Autoradiogroms showing a comparison of t’he depurination products of 3ZP-labelled D74 fragment HaeIII 2.1% with those of A2 Ha111 fragments 14 and 17 (subfragments of HpnII-3). The oligot,hymidine tract present in both A2 HneIII-17 and D74 Ha111 2.0% is indicated (T7 or 8). The pair of nonemers (or octomers) present in both A2 HaeIII-14 and D74 Ha111 2.1 yo are indicated (14) ; the pentamer T,C in both these species is also indicated.
Biol.
(1977)
117, 497-513
Polyoma Virus Defective DNAs II. Physical
Map of a Molecule
with Rearranged
and Reiterated
Sequences
P74) ELSEBET
BEVERLY
LUND?,
E. GRIFFIN AND MIKE
FRIED
Imperial Cancer Research Fund P.O. Box 123, Lincoln’s Inn Fields London WC2A 3PX, England (Received
27 January
1977, and in revised form
29 July
1977)
The structure of the polyoma virus defective species D74 (74% the size of fulllength polyoma virus DNA) has been determined and compared with that of polyoma virus A2 DNA. D74 appears to be composed entirely of viral DNA sequences. (No host DNA sequences have been detected.) It is made up of three DNA segments, each about 24,24 and 27% in size. The two 24eh segments appear to be identical and the 27% segment contains one copy of all the sequences found in the 24% fragments as well as a duplication of some of the sequences. When related to the physical map of A2 DNA, each segment is found to be composed of viral sequences from 1 to about 19 map units, 67 to 69 map units and 70 to 72 map units. Three features found in other polyoma virus defective species (Lund et al., 1977) are also present in D74. (1) Sequences from the region around 67 map units arc linked to other (non-contiguous) viral sequences. (2) Sequences at about 72 map units are linked to sequences at 1 map unit. (3) Multiple copies of sequences from around the origin of viral DNA replication are present. From studies on other polyoma defective molecules (Griffin & Fried, 1975; Lund et al., 1977), the origin of DNA replication for polyoma virus has been defined to lie within the sequences from 67 to 72 map units. Since D74 replicates efficiently but lacks the sequences between 69 to 70 map units, the origin of DNA replication appears to be further defined as lying within 67 and 69 map units and/or 70 to 72 map units.
1. Introduction A number
of clonally
isolated
defective
1974) have been purified and analysed in Robberson & Fried, 1974; Griffin & Fried, species
have
been
shown
to contain
only
virus DNAs (Fried, considerable detail (Fried et al., 1974; 1975; Lund et al., 1977). All of these sequences with no detectable host DNA the origin of viral DNA replication. in the amount and arrangement of their
(non-infectious)
viral
polyoma
and to have repeats of the region around They have differed considerably, however, viral sequences. Earlier studies on the defective species (D74) defined in this paper suggested that contained little (if any) homology with polyoma viral DNA sequences (Robberson Fried, 1974). For this reason, a detailed study on D74 has been carried out using combination of methods which include DNA-DNA hybridization, cleavage t Present Chemistry,
address : University 1216 Linden Drive,
of Wisconsin Medical Center, Department 589 Medical Science Building, Madison, Wise.
497
of Physiologioel 53706, U.S.A.
it & a by
498
E.
LUND,
restriction endonucleases, studies have shown that sequences. The structure defective DNAs and the
B.
E.
GRIFFIN
AND
M.
FRIED
and analysis of depurination products. The results of these D74 is composed mainly of rearranged and reiterated viral of D74 is compared with structures determined for other biological implications of the findings are discussed.
2. Materials
and Methods
All procedures for preparation and characterization of wild-type A2 DNA and the defective (D74) DNA species were carried out as described in the preceding papers (Griffin, 1977; Lund etal., 1977) and by Fried (1974). The original notation (Fried, 1974) for the D74 defective virus isolate was changed (Robberson & Fried, 1974). The latter notation is used in this paper.
3. Results (a) Hybridization
analysis
of 1374 DNA
In an initial attempt to determine the DNA sequencespresent in the defective speciesD74, DNA hybridization analyses between the defective DNA and A2 polyoma virus DNA (or fragments thereof) and of mouse DNA were carried out. (i)
Analysis
using A2 HpaII
fragments
as
probes
The data obtained with three of the A2 HpaII fragments are shown in Figure l(a) to (c) ; these are representative of results obtained by this method of analysis. Only two of the polyoma virus DNA fragments, A2 HpaII-2 and 3, showed any increase in the rate of reannealing following the addition of D74 DNA, thus establishing that D74 DNA contains viral DNA sequencesthat correspond to a very limited portion of the polyoma DNA. In the caseof A2 HpaII-2 (Fig. l(a)), the initial rate of reannealing in the presence of D74 DNA (indicated by the broken line) was higher than that observed in the presenceof the homologousA2 DNA. The subsequentpoints obtained in the presence of D74 DNA did not fall on a straight line and the rate of reannealing of the probe appeared to decreasewith increasing time of incubation. Similar kinetics of reassociation were observed in three independent experiments. In the presence of A2 DNA, on the other hand, the points produced a straight line, as expected for a DNA which contains one complete copy of the probe DNA sequences.These results suggest that D74 DNA contains more than one copy per molecule of a certain fraction only of the A2 HpaII-2 DNA sequences. In the caseof A2 HpaII-3 (Fig. l(b)), the increasein the rate of reannealing following the addition of D74 DNA was significant, but much smaller than that observed in the presenceof A2 DNA. The data did not allow the precise amount of A2 HpaII-3 sequencesin D74 DNA to be determined. By similar analyses, DNA sequencesfrom none of the other A2 HpaII fragments could be detected in the defective DNA. As an example, the data obtained with A2 HpaII-4 are shown (Fig. l(c)). Clearly, the addition of D74 DNA did not change the rate of reannealing of this probe. (ii) Analyses with defective specijic HpaII fragments and A2 HpaII-1 as probes In agreement with the finding (above) that D74 DNA contained several copies of certain viral DNA sequences,the complexity of D74 DNA was found to be severalfold lessthan that of A2 polyoma DNA (data not shown).
A REITERATED
POLYOMA
VIRUS
DEFECTlVE
DE.4
4Y!l
(cl /a
l IO
I 20
I 30
I IO Cot
Cot
x IO”
I 20
I 30
(mol
nucleotides
x s/l)
x IO4 (mol
nucleotldes
x s/L)
I
I
IO
20
Jha. 1. (a) to (c) Reannealing of A2 HpaII fragments in the presence of defective and wild-t)ype A2 DNAs. Portions of %‘-labelled A2 HpaII DNA fragments (spec. act. 3 x lo5 aaP cts/min per pg DNA) and unlabelled viral DNAs were denatured and allowed to reanneal at 68’C in 200 ~1 of standard hybridization buffer containing 0.2 M-N&Cl. The rate of reassociation was monitored by 81 nuclease digestion and the results were plotted as described (Sharp et crl., 1974; Lund et r/Z., 1977). The reassociation kinetics of 1.8 x 1Om8 M (mol nucleotides/l) A2 HpczII-2 (a), 1.8 x lWB M-A:! HpaII-3 (b) and 1.1 x lo-* M-AZ HpnII 4 (c) DNAs are indicated by (A). The same concentrations of probe DNAs were also reannealed in the presence of 1.5 x 1Om6 M-/d! (a) or I>74 (c:) DNAs. This concentration of homologous unlabelled A2 DNA corresponds to an la-fold molar excess of A2 HpaII-1 and HpnII-4 DNA sequences and a 14-fold molar excess of A2 HpaII-3 DNA sequences. As compensation for the difference in molecular weight of A2 and D74 DNAs the C,t values for D74 DNA-containing samples have been normalized t,o those of the A2 DNAcontaining samples as described (Lund et al., 1977). (d) and (e) Reannealing of fragments D74 HpaII 24% and A2 HpnII-1 in t,ho presence of AZ?, D74 and whole mouse embryo DNAs. The hybridizations were carried out as described in (a) to (c) using 300.~1 portions containing 0.5 M-NaCl in standard hybridization buffer. The reassociation kinetics of 32P-labelled probe DNA fragments (spec. act. 1 x lo5 32P cts/min per pg DNA) were measured in the presence of 3 x 10m3 NI (mol nucleotides/l) calf thymus DNA (A) for D74 HpaII 24% (d) and for A2 H-p&I-2 (e) with the probes at a concentration of 3.6 x lo-* M. Also shown is the reannealing of the same concentrat,ion of 32P-labelled probe DNAs in the presence of 3 x 10e3 M-calf thymus DNA and 6.7 x lo-’ MD74 DNA (0); 3 x 10m3 M-calf thymus DNA and 7.3 x 10e7 M-M DNA (0); and 3 x 10m3 M-whol? mouse embryo DNA (A). This concentration of unlabelled D74 DNA (d) corresponds to a 12-fold molar excess of D74 HpaII 24% DNA sequences assuming that each molecule of full-length D74 DNA contains 2 copies of the D74 H;oaII 24% fragment. Similarly the concentration of unlabelled 82 viral DNA (e) corresponds to a 7-fold molar excess of A2 HpaII-1 DNA sequences. In the case of A2 DNA the C,t values have been normalized (as described, Lund et nl., 1977) to those of D74 DNA to compensate for the differences in nucleic acid concentration and molecular weight of the viral DNAs. 33
500
E.
LUND,
B.
E.
GRIFFIN
AND
M.
FRIED
Cleavage of D74 DNA with the restriction endonuclease HpaII produces three fragments, 27% (one-molar yield) and 24% (t wo-molar yield) the size of full-length polyoma DNA (see section (b), below and Fig. 2)t. The purified D74 fragments appeared to have a complexity similar to that of polyoma virus A2 HpaII-1 (a fragment composedof non-repetitive sequencesand corresponding to 27.3% of A2 DNA) since under identical conditions of hybridization D74 HpaII 24”,/0 (or D74 HpaII 270/,) reannealed with a rate very similar to that of A2 HpaII-1 (compare Fig. l(d) and (e)). This suggested that the individual D74 HpaII fragments were composed (primarily) of non-repetitive DNA sequences.Furthermore, the reannealing of D74 HpaII 24% (or 27%) in the presenceof A2 DNA showed that most of these sequences appear to be of viral origin, since the reassociationkinetics of the defective fragment(s) was very similar to that of A2 HpaII-1. Addition of the homologousD74 to either of the defective specific HpaII fragments (24% or 27%) resulted in a large increase in the rate of reannealing, which indicated that the defective DNA contained at least two copies of these probe DNA sequences per molecule. This is shown for D74 HpaII 2476 in Figure l(d). In order to determine whether D74 DNA also contained host DNA sequences, D74 HpaII 24% (or 27%) was reannealed in the presenceof mouseembryo DNA in a concentration sufficient to detect repetitive host DNA sequencesin the probe DNA. Since the addition of host DNA did not influence the rate of reamlealing of the defective HpaII fragments (Fig. l(d)) it was concluded that D74 did not contain repetitive host DNA sequences.Using these techniques, however, the presenceof unique host DNA sequencesin the defective molecule could not be ruled out. In summary, the hybridization analysesshow that D74 DNA is composedprimarily (if not entirely) of viral DNA sequences,which in large part are derived from the region of A2 DNA that correspondsto sequencesfound in A2 HpaII-2. Furthermore these viral sequencesappear to be present in more than one copy (two or three) per molecule of D74 DNA. (b) Restriction
enzyme analyses
One of the immediately striking aspectsof the defective molecule D74 was the fact that the cleavage patterns obtained by digestion with either restriction endonucleases HpaII, Hind111 or HhaI were indistinguishable (Fig. 2). Digestion with each endonuclease produced one fragment approximately 24% (two-molar yield) and one fragment approximately 27% (one-molar yield) in size. (1) Both of the Hind111 defective specific products, when cleaved with HpaII, gave small fragments (about 1.7% in size) which were electrophoretically indistinguishable. In addition, D74 Hind111 24% gave a fragment approximately 2259/, and D74 Hind111 27% a fragment approximately 2550/, in size (seeTable 1). (2) Both of the HhaI defective specific products, when cleaved with HpaII, gave fragments (about 95% in size) which were electrophoretically indistinguishable. In addition, D74 HhuI 24% gave a fragment approximately 14.5% and D74 HhaI 27q/, a fragment approximately 17.5% in size (seeTable 1). (3) Both of the Hind111 defective specific products, when cleaved with HhaI, gave fragments (about 11% in size) which were electrophoretically indistinguishable. In t Unless percentage
otherwise indicated, of the A2 polyoma
a11 restriction virus DNA.
endonuclease
fragment
sizes
me expressed
as a
.I
REITERATED
POLYOMA
VIRUS
DEFEL”1’IVE
T)NA
r?o I
D74
A2
\
I
HpaJI
HhaI
BumI
-
HoeD
27% 24%
3.7%
3.0% 5 6 . 2.1%
FIG. 2. Autoradiograms of D74 DNAs (s2P-labelled) after cleavage with restriction endonucleases HJwII, HindIII, HhaI, Hum1 and Hue111 and of A2 DNA cleaved with HpaII. Fragments were separated on slab gels (20 cm x 20 cm) composed either of 0.0% polyacrylamide/0.15% bisacrylamide and 0.5% agarose (HpaII, HindIII, HhaI) or of 5.0% acrylamide/0~25°/0 bisacrylamide (HumI, HuelII). Fragment sizes for the HpaII restriction endonucleases are taken from Griffin et (II. (1974); sizes for the other restriction endonuclease fragments are given as a percentage of whole polyoma A2 DNA and were determined relative to the HpnII sizes.
addition, D74 Hind111 24% gave a fragment approximately 13:/, and D74 HindIII 27”;, a fragment approximately 16% in size (see Table 1). For further analysis, the action of the restriction endonuclease HaeIII on D74 DNA and on defective specific fragments produced by digestion with HpaII, HindIII and HhaI was studied. DigesCon of D74 with HaeIII produced five fragments with sizes approxima.tely 10.2%, 7.9%, 3.7% and 2.1 oh (each in three-molar yield) and 3.00/, (in one-molar yield) ; the latter product was found exclusively in the D74 27y; fragment (see Fig. 2). The 10.2% fragment was electrophoretically indistinguishable from A2 HaeIII-3, a subfragment of A2 HpaII-2 (Griti, 1977). Cleavage of the D74 Hind111 24% and 27 “/o fragments with HaeIII resulted in the dixappearancc of the D74 HaeITI 3.7 76 fragment ; two new fragments were generated
E.
502
LUND,
B.
E.
GRIFFIN
AND
of data obtained
FRIED
1
TABLE
Xummary
M.
from D74 DNA
by cleavage with restriction
endonucleases HpaII D74
HpaII
D74
Hind111
D74
HhaI
D74
Burnt
D74
H&II
24% 27% 24% 27% 24% 27% 14.5% 12.6% 11.5% 10.2% 7.9% 3.7% V.O% 2.1%
Hind111 22+i+ 255+
22.5+ 1.7% 26*5+ 1.7% 14x5+ 9.5% 17.6+9.6% 12+2.5% NC 9+24x NC NC NC NC
=%I
1.7% 1.7%
13+11y0 lS+ll% 10.4+4*1y0 NC 7.4+4.1y0 NC NC 2.2+ 1.5% NC NC
Fragment sizes we expressed as percentages of A2 DNA. NC signifies fragment not cleaved. t Specific to the D74 27% fragments. $ The electrophoretic mobility of this fragment w&s slightly
HhaI
Bum
14.5+9.5y0 17.5$9-5% 13+11y0 1s+11%
12.6+ 9+2.5y0 12*5+12+2.5y0 12.6+ 7.4+4y0 12.6+10.4+4% 11X+ 7.3+5.2% 14*6+ 7.3+6.2%
NC 7.3+6.2% NC NC 7.7+0.2% NC NC NC
retarded
5.2+ 6.0% 7%i+o.4y0 NC NC NC
after
cleavage
(see text).
with sizesabout 2.2% and 1.5% (Table 1). (In A2 DNA, the Hind111 site in HpaII-2 is found in HaeIII-8 about 2.2% away from the juncture with A2 HaeIII-3 (Griffin, 1977).) Cleavage of the D74 HhaI 24% and 27% fragments with Hue111resulted in a size change of the D74 Hue111 7.9% fragment (from approx. 7.9 to 7*70/,) (Table 1). (In A2 DNA, the HhaI site in HpaII-2 is found approximately 93% from the juncture of the A2 HaeIII-3 and 7 (Griffin, 1977).) Cleavage of the D74 HpaII 24% and 27% fragments with Hue111 resulted in the D74 Hue111 2.1% fragment migrating with a mobility characteristic of a slightly larger fragment. This phenomenon is characteristic of A2 fragment HaeIII- 17 (Griffin, 1977), the subfragment of A2 HpaII-3 which lies at the HpaII 3-5 junction (near the origin of viral DNA replication). The assignment of the above cleavage sites to the D74 Hue111fragments is consistent with results obtained by cleavage of D74 DNA with other combinations of endonucleases(Table 1). D74 DNA was further analysed with a number of other restriction endonucleases (data not given). Cleavage with HhaI together with either Hph, MboI, Mb011 or XbaI gave fragments which were electrophoretically indistinguishable from some of the fragments obtained from A2 HpaII-2. This indicated that D74 DNA contained continuous sequencesof A2 DNA from at least 1 to 18.2 map units (XbaI site at 18.2). Moreover, analysis with these restriction enzymes gave results which were consistent with the 3% extra sequencespresent in the D74 27% fragment being derived from sequencesfrom about 1.5 to 4.5 map units in A2 DNA, these 3% sequencesbeing tandemly duplicated in the defective fragment. The endonuclease Bum1 cleaves D74 into three fragments (Fig. 2) with sizes approximately 14.5% (one-molar yield), 12.5% (th ree-molar yield) and 11.5o/o(twomolar yield). The pattern obtained with Bum1 is consistent with there being two
A REITERATED
POLYOMA
VIRUS
DEFECTIVE
DNA
503
cleavage sites each in the D74 24% and 27% fragments. (A2 DNA has four Bum1 cleavage sites, Fried & Griffin, 1977.) The Bum1 cleavage sites in D74 DNA have been located relative to the Hpall cleavage sites by double digestion with these two endonucleases. The Bum1 12.5% fragment (three-molar yield) was not cleaved by HpaII, but the other two Bum1 fragments were cleaved. New products were found with sizes approximately 12%, 9% and 2.5% (Table 1). By a similar analysis, the Bum1 sites were located relative to Hue111 sites in D74 IINS. One Bum1 site was found in the D74 Hue111 10.20/, fragment. This fragment was cleaved to two products with sizes (5~2% and 5.0%) indistinguishable from the products obtained by cleavage of A2 Haelll-3 (a subfragment of Hpall-2) with RumI (Griffin 1977). The second Bum1 site lies very near to one end of the D74 HaelI 17.9% fragment (see Table 1). These data are summarized on the physical maps shown in Figure 3. (c) Depurination analyses Defective D74 DNA (32P-labelled) and the two viral-specific Hpall fragments (27% and 247/o) were chemically depurinated and the pyrimidine products were separated as described by Ling (1972). The fingerprints of these two species were indistinguishable and had many pyrimidine tracts in common with A2 Hpall-2 (Fig. 4). Notably absent from the fingerprints of D74 DNA were mo.st of the large (greater than decamer size) characteristic products of A2 DNA (Griffin, 1977). The only large oligopyrimidine in D74 DNA was one which appeared identical (in size and mobility) to the oligomer derived from A2 Haelll-14’, a subfragment of A2 Hpall-5 (see Fig. 7). The fingerprint of the defective DNA also contained the long run of a subfragment of A2 thymidines (T, or a) known to be a product of A2 HaeIII-17, HpaII-3 (see Fig. 8). Another predominant feature of the D74 fingerprint was that, as a group, the hexamers seemed to be in higher yield than would be expected on a random basis. (Compare, for example, the yield of hexamers with pentamers, Fig. 4.) This is especially true for hexamers with the composition C,T, and CT, and is a characteristic also of the depurination products of A2 Hpall-2. It does not appear to he characteristic of any of the other A2 Hpall fragments (Griffin, 1977). In order to characterize the defective species further, fingerprints of the five D74 HaeIII fragments were made and compared with HaeIII fingerprints from A2 DNA. The results are shown (in part) in Figures 5 to 8. Some aspects of the analysis of each fragment are considered below. (i) D7P HaeIII
10.2%
(three-molar
yield)
(Fig.
5)
This fragment was found to be identical (at least qualitatively) to A2 Haelll-3, a subfragment of A2 Hpall-2. Since these two fragments are also indistinguishable in size and give similar Bum1 products (Table l), D74 Haelll 10.2% and A2 Hue1113 are probably identical. As a striking feature, both species can be seen to contain only one pentapyrimidine (TC,). (ii) D7d HaeIII
7.9%
(three-molar
yield)
(Fzg. 6)
This fragment was found to have sequences similar to those of A2 HaeIII-7 (a subfragment of Hpall-2) and A2 Haelll-15 (a subfragment of Hpall-3). For ever.y oligomer in A2 Haelll-7, a corresponding oligomer could be found in D74 HaeIII 7,R”(,. A superimposition of the fingerprints of A2 Haelll fragments 7 and 15 will
604
E.
LUND,
B.
E.
GRIFFIN
AND
HmdIU
M.
FRIED
D74
0
HhaI HhaI 0
HhaI
(a) HpaD .
3.7%
HPoII
Bum1
‘HaelII
pa I
HueID
:2d-:D74
7.9%
10*2%
Hpall
24%
HPQ~
‘yfA2 (!:2’=,&)+&-8
( .9%)
Hoellt-3
I:-2
(02%)
Hpan
HpUIIHDe~ (b)
FIQ. 3. Physical maps of A2 DNA and D74 DNA. The 92 map is divided into 100 units with the single EcoRI cleavage site at zero map unit. The units shown on the physical map of D74 correspond to A2 map units. The origins (0) of viral DNA replication are indicated on the maps. (a) A2 physical maps. Inner ring, obtained by cleavage with restriction endonuclease HpaII (8 cleavage sites, Griffin et al., 1974). Outer ring, obtained by cleavage with Hue111 (24 cleavage sites, Griffin, 1977). Other cleavage sites are indicated. These maps were modified from Griffin (1977). Proposed D74 physical map, obtained by cleavage with restriction endonuclease HpaII (3 cleavage sites) (Fig. 2). The HpaII cleavage sites are indicated by continuous lines within the circle. Broken lines show where non-contiguous viral sequences have been covalently linked (see text) and the numbers indicate the A2 HpaII fragments from which these sequences are derived. Sequences between 69 and 70 map units in A2 HpaII-3 (near the origin of viral DNA replication) are deleted in D74 (indicated by V); sequences between 67 and 69 and 70 and 71 map units are present. The restriction endonucleases Hind111 and HhaI also cleave D74 DNA into three fragments, as shown. (b) Linear representation of the Hue111 physical map of one of the reiterated segments of D74 DNA (D74 H@lI 24%) and of A2 restriction fragment H@I-2; restriction sites common to homologous regions are indicated ( 2 ).
produce the fingerprint of D74 Hue111 7.9%; that is, all oligomers generated defective fragment can be found in a combination of the two A2 fragments. (iii) D74 HaeIII This
3.7% (three-molar
yield)
(Fig.
from the
7)
fragment was qualitatively very similar to A2 HaeIII-8 (a subfragment of but appeared to be missing the largest oligopyrimidine (greater than
HpaII-2),
A REITERATED D74
DNA
POLY
OMA
VIRUS
DEFECTIVE
A2
Electrophoresis, pH first dimension
HpalI
DNA
505
- 2
3-5
FIG. 4. Autoradiograms showing a comparison of the depurination products of 3ZP-labelled 1)74 DNA and A2 HpaII-2. The pyrimidine tracts were separated in the first dimension by electrophoresis and in the second dimension by homochromatography (see text and Ling, 1972) as indicated. The easily identified oligopyrimidines in D74 DNA which are derived from A2 HneIII fragments 14’ and 17 (see Figs 7 and 8) are indicated as (14’) and T, Dr 8, respectively. The characteristic oligomer present in A2 H&I-2 (which comes from A2 HueIII-8, see Fig. 7), but absent, from D74, is indicated (*). The hexamers on each fingerprint are indicated (VI). The fingerprints of the D74 HpaII 24% and 27% fragments were indistinguishable from that, of t,he whole D74 molecule.
dccamer size) normally found in the latter (Fig. 4). (Some traces of this oligomer could be seen on the fingerprint of the 3.7% fragment, but as it was present in considerably lower than one-molar yield and not found either in the whole D74 molecule or in its HpaII fragments (see Fig. 4) it was thought to arise from a small amount of contaminating HpaII-2 sequences from the helper viral DNA.) Quantitatively there were some other differences between D74 Hue111 3.7 o/oand A2 HaeIII-8, most notably at the hexamer level. For example, T,C, was clearly present in greater yield than other hexamers in the A2 fragment, but this was not the case for the D74 fragment. Moreover, in the defective fragment the very large oligopyrimidine present in A2 H&11-14’ (a subfragment of HpaII-5) is apparent. All the pyrimidine tracts found
A2
HaeIlI
-3
074
pH
Electrophoresis,
FIG.
10.2%
HaelII
5
3.5
D74
HoeIU
3 *0 %
A RElTERATED
POLYOMA
in the defective specific fragment Hue111 fragments 8 and 14’. (iv)
D74 HaeIII
VIRUS
DEFECTIVE
(3.7%) can be accounted
I)XA
for by products
507
from A2
3.0% (olte-molur yield) (Fig. 5)
All of the pyrimidine tracts in the defective fragment can be found in A2 HaelIl-3. The D74 Haelll 3% fragment contains only one pentamer which has the same composition (TC,) as the single pentamer in A2 Huelll-3 (see (i), above). The defective fragment is located between the D74 Hue111 10*2% and 3.7% fragments on t)he physical map of D74 (Fig. 3) and probably contains some sequences from the latter fragment, but these cannot be distinguished in the fingerprint of the defective specific 3.0!/, fragment. (u) D74 HaeIII
2.1% (three-molar yield) (Fig. 8)
This fragment was especially interesting as it was found to contain the oligothymidine tract (T7 ,,p 8) unique to A2 Huelll-17 (a subfragment of Hpall-3) and so must be composed (at least in part) of sequences from A2 Hpall-3. The D74 Haelll fragment, however, is approximately 2*1o/0 in size, whereas the A2 Haelll-17 fragment is only 1.394. Two oligomers, larger than any found in A2 Huelll-17, are present in the defective fragment. They have been shown to be identical (in mobility and size) to a pair of oligomers (octamers or nonamers) in A2 Huelll-14, the Hue111 fragment adjacent to Haelll-17 in A2 Hpall-3 (Griffin, 1977). The pentamer, T,C, in the defective species is also present in A2 Huelll-14. It could be concluded from further examination of A2 Hue111 fragments 14 and 17 that all the oligomers present in D74 HaeIlJ 2.1% could be found in one or the other of the two A2 fragments, but not all the oligomers present in these two A2 fragments were present in the defective specific fragment. D74 Hue111 2.1 y0 appears to represent a fusion of the two A2 Hue111 fragments (14 and 17) with a resulting deletion of some sequences.
4. Discussion By three independent methods of analysis (cleavage with restriction endonucleases, two-dimensional separation of depurination products (fingerprinting) and DNA-DNA hybridization), the structure of the polyoma defective molecule D74 has been deduced (Fig. 3). This molecule contains selected portions of the viral DNA sequences from Hpall-2 (1 to about 19 map units), Hpall-3 (67 to 69 and 70 to 71 map units) and Hpall-5 (71 to 72 map units). It is composed of three DNA segments, each about 24%, 24% and 27% the size of full-length A2 DNA. The two 24% segments appear to be identical, but the 27% segment contains, in addition to the sequences found in the 24y/, segments, 3% of DNA sequences which appear to be derived from Hpall-2, from the region on the physical map which includes the Hue111 3-8 junction (Griffin, 1977). Tandem duplications of viral sequences have been described in other defective
FIG. 5. fragments The latter fragment either A2 pentamer.
Autorediograms showing a comparison of the depurinetion products of 3aP.lebelletl D74 HaeIII 3.0’$& D74 HaeIII 10.2% and A2 HaeIII-3 (a subfragment of H@I-2). 2 fingerprints are qualitatively indistinguishable. The fingerprint of D74 Hue111 3.07; is much simpler, but does not have any oligopyrimidines which are not also present in HaeIII-3 or D74 HaeIII 10.Z”/& All 3 fragments contain TC( (indicated) as t,heir s&l
A2
HaellI
- 7
Electrophoresis, FIG.
Hue III
6
pH
7.9
3.5
%
-
A2
A REITERATED
POLYOMA
VIRUS
DEFECTIVE
DNA
509
pal-yome virus species (Griffin & Fried, 1974; Lund et aZ., 1977). The peculiar arrangemcnt of viral sequences found in D74 may explain the previous difficulty of obt,aining a single interpretable heteroduplex structure between D74 and -42 DNA (Wild. unpublished results). The three features described for the polyoma defective related species D76, D91 and D92 (Lund et ul., 1977) are also found in D74. One involves the linkage of sequences from around 1 map unit (in H&II-2) to sequences around 72 map units (in HJXZII-5). A srcond feature is the linkage of sequences from around 67 map units (in A2 Hael I I15. a subfragment of HpaII-3) to viral sequences in HpaII-2. The data available do not, allow for the exact location of the latter sequences.Although they appear to lit in A2 NaeIII-7 (near the H&I 2-6 junction), there is a discrepancy (0.3 to 1’:“) in sizck between the A2 fragments concerned and the D74 HueIII 7.9% fragment. Even if ~22the sequencesfrom A2 HaeIII-15 (1.5%) and 7 (6.0%) (Griffin, 1977) were present in t)he defective fragment, the latter is still larger than the sum of the A2 fragments. The discrepancy in sizes may be merely a reflection of base composition and sequences,since these affect electrophoretic mobilities of DNA fragments (Ziegler it nl., 1952). Alternatively, D74 may contain someadditional sequences(host or viral) not found in t’his region of A2 DNA. ‘I’hc bhird feature is the repetition of viral sequencesfrom around the Hpal I 3-5 junction where the origin of viral DNA replication has been mapped (Griffin cf al.. 197-C;Crawford et al., 1974). But, whereas in other polyoma virus defective species. continuous viral sequencesfrom around 67 to 72 map units have always been present, (Griffin & Pried. 1975; Lund et al., 1977), in D74 about 1% of the DNA sequences from this region are deleted. D74 contains continuous viral sequencesfrom about 67 to 69 map units and from 70 to 72 map units, but sequencesfrom 69 to 70 map units including the Hue111 cleavage site at 69.5 map units (between A2 fragment’s HmITI-14 and 17) and the Burn1 site at 70.2 map units (Griffin, 1977) are absent’. It’ seemsreasonableto conclude that these deleted sequencescannot therefore be t’ssent,ial for viral DNA replication since D74 appearsto replicate efficiently in the presence of a helper virus (assumingthe viral origin of DNA replication is being used). It’ may be that in polyoma DNA the sequencesspecifying the origin of viral DNA replication art: loca,ted in two closely spaced but non-contiguous regions (67 to 69 and 70 to 7% map units) separated by a region of non-essential sequences(69 to 70 map units). It is also possiblethat both persisting regions are concerned with viral DNA replication but, play different r8les. One may be the site at which an enzyme (e.g. DNA polymerase) binds to the DNA and the other the site at which synthesis is initiated. On bhe other hand, only one of these two regions may be involved in DNA replication and the other region may be conserved because it is essential for some other viral function. For example, it could serve as a focus for the formation of the viral capsid. Encapsidation must be essential for the successfulinfection and virus growth of both defrct’ive and non-defective viral DNAs. In any case,the sequenceswhich speci[y the origin of viral DNA replication appear to be further defined by D74 DNA as lying eithrsr between 67 and 69 and/or 70 and 72 map units.
FH:. 6. Alctoradiograms showing a comparison of fragments of D74 HneIII 7.9?; with A2 HaeIII-7 (a Fubfragment of HpaII-3). A hypothetical fingerprint wo~~ltl producr the fingerprint found for D74 Hue111
the depurination products of 32P-labelletl subfragment of H@T-2) and HtceIII-15 (a constructed from these two A2 fragments 7.9%.
A2
Huelil-
8
D74
FIG.
Electrophoresis. I
Huern
pH
3.5
3*7%
A2 HueDI
14’
A
REITERATED
POLYOMA
VIRUS
DEFECTIVE
DNA
611
The merit’s and limitations of the three methods used for analysis of D74 are discussed in the accompanying paper (Lund et al., 1977). The extent of homo1og.y detected hy DNA-DNA reassociation kinetics between D74 and A2 DNAs is very limited and appears to be restricted to certain DNA sequences present in A2 HpaII fragments 2 and 3. There was no detectable “cross” hybridization between D74 DNA and DNA from any of the other six A2 HpaII fragments under similar conditions of rcannealing. The analysis did not however permit an accurate quantitation of the viral DN$ sequences in the defective molecule. As previously discussed (Lund et al., 1977) the hybridization conditions used would not permit short viral DNA sequences, present in “novel” (defective specific) arrangcmerits different from A2 polyoma DNA sequence arrangements, to be detected. Therefore. t,his analysis provides only a minimum estimate of the amount of viral DNA sequences in D74 DNA. The data presented in Figure l(d) and (e) suggest that the defective molecule does not8 cont,ain a large proportion of host DNA sequences, since the A2 polyoma DNA appeared to “cross hybridize” with most (if not all) of the D74HpaII 24%, prohr sequences. In addit’ion. from the analysis of the reassociation kinetics of the defective specific D74 HpalI 24’34 and 27% fragments, it could be concluded that (1) the DNA sequences from t’hese fragments are present in more than one copy per D74 molecule. (2) most of these sequences are of viral origin and (3) none of the defective specific HpaTI fragments contains large stretches of repetitive host (mouse) DNA sequences (see Fig. l(d) and (e)). The restriction endonuclease analysis shows that continuous viral sequences from Ar! HpaII-2 between the Hind111 cleavage site (at about 1 map unit) and the Xhal cleavage site (at about 18 map units; Fried & Griffin, unpublished results) are present in t(he two D74 HpaII 24% fragments. Furthermore, the D74 HpaII 27% fragment, appears to contain all the sequences present in the 2404 fragments plus an insertion (3’);)) of DNA sequences between the D74 HaeIII 3.7% and 10.2% fragments. The extra sequences in the 2774 fragment appear to come from a duplication of the sequences of A2 HpaII-2 from about 1.5 to 4.5 map units. The pyrimidine tract analysis proved ultimately most useful in the assignment of sequences in D74. By identifying unique tracts of pyrimidines as well as comparing oligomrrs produced from D74 and A2 HaeIII fragments, a number of rearrangements, deletions and additions could be detected. These results are summarized in Figure 3. D74 was isolated after five high multiplicity viral passages (Fried, 1974) and shows extensive rearrangements of viral sequences. No evidence has been obtained for the presence of any host DNA in D74 although a small amount of host sequences cannot) definitely be excluded. Why these sequence rearrangements occur and how they might confer select,ive advantage to D74 is not clear. The longest continuous segment of viral sequences in D74 is derived from A2 HpaII-2, these sequences being linked
Fro. 7. Autoradiograms showing a comparison of the depurination products of 3ZP-labelled I)74 fragment 29~111 3.7% with A2 HaeIII-8 (a subfragment of HpaII-2) and A2 H&III-14 (a subfragment, of HpuII-6). D74 Hue111 3.7% has a fingerprint which is qualitatively similar to A2 HaeIJI-8, although it lacks one (*) of the oligomers unique to the latter (see text). Quant,itative difference can be seen among the hexamers (VI). (For example, compare T& with other hexamers in the 2 fingerprints.) In addition, D74 NaeIII 3.7% contains the very large oligomer (14’) found in X2 H~wIII-14’, but not present in A2 HneIII-8.
FIG.
Electrophoresis.
8
pH 3.5
A2
HoeEl
- 17
A
RETTERATED
POLYOMA
VIRUS
DEFECTIVE
D?r’A
613
to sequences from HJXXII-5 or HpaII-3. Sequences from HpaIT-2 are not however found in other reiterated defective species (Griffin & Fried, 1975). Like other defective species (Griffin & Fried, 1975; Lund et al., 1977), D74 retains viral sequences from the region between 67 and 72 map units: hut unlike the other species. it does not retain all of these sequences. Thus, D74 may prove to be very important in understanding the biological r81e of the various parts of the viral genomc, and in particular t.he region around the origin of viral D1L’A replication. REFERENCES Crawford, L. V., Robbins, A. K. & Nicklin, P. M. (1974). J. (Yen. ITirol. 25, 133~ 142. Pried, M. (1974). J. Viral. 13, 939-946. Fried, M. & Griffin, B. E. (1977). In Advances itz Cancer Research (Klein, G. & Weinhousn, S., eds), vol. 24, pp. 67-113, Academic Press, New York. Fried, M., Griffin, B. E., Lund, E. 6t Robberson, D. L. (1974). Cold S’y&g Harbor S~yny. Quad.
Viol.
39,
45-52.
Griffin, B. E. (1977). J. Mol. Biol. 117, 449-473. Griffin, B. E. & Fried, M. (1975). Nature (London) 256, 175-179. Ling, V. (1972). J. Mol. Biol. 64, 87-102. Lund, E., Fried, M. & Griffin, B. E. (1977). J. Mol. Biol. 117, 475.-497. Robberson, D. L. & Fried, M. (1974). hoc. Nat. Acad. Sci., U.S.A. 71, 3497--3501. Sharp, P. A., Pottersson, U. & Sambrook, J. (1974). J. illoh Biol. 86, 709-726. Ziqler, R. S., Salomon, R., Dingman, C. W. & Peacock, A. C. (1972). h’ature New 238, 65-69.
Biol.
FIG. 8. Autoradiogroms showing a comparison of t’he depurination products of 3ZP-labelled D74 fragment HaeIII 2.1% with those of A2 Ha111 fragments 14 and 17 (subfragments of HpnII-3). The oligot,hymidine tract present in both A2 HneIII-17 and D74 Ha111 2.0% is indicated (T7 or 8). The pair of nonemers (or octomers) present in both A2 HaeIII-14 and D74 Ha111 2.1 yo are indicated (14) ; the pentamer T,C in both these species is also indicated.
