J. gen. Virol. (1983)i 64, 69-82. Printed in Great Britain
69
Key words: polyoma virus/flanking cell sequences/chromosomal DNA
Integration of Polyoma Virus DNA into Chromosomal DNA in
Transformed Rat Cells Causes Deletion of Flanking Cell Sequences
By A D A N E E R , N A V A B A R A N AND H A I M M A N O R *
Department o f Biology, Technion - Israel Institute o f Technology, Haifa, Israel
(Accepted 5 August 1982)
SUMMARY
In order to find out whether polyoma virus (Py) integration into chromosomes causes
rearrangements in the cell D N A flanking the integration site, we have mapped the
flanking sequences in the inducible LPT line of Py-transformed rat cells and the
corresponding sequences in normal rat fibroblasts, and then compared the two maps.
To carry out this study we have cloned a segment including Py D N A and flanking
sequences in the bacteriophage vector 2gtWES and subcloned the flanking cell D N A in
a bacterial plasmid. We performed a Southern blot analysis of LPT and rat fibroblast
D N A digested with various restriction enzymes and used the cloned flanking cell D N A
and Py D N A as hybridization probes. Autoradiography of the LPT D N A blots
revealed two sets of fragments. One set includes fragments containing both Py and cell
D N A sequences; the second set consists of fragments which contain no virus D N A
sequences, and are identical to the fragments observed in the corresponding normal rat
D N A digests. These data indicate that LPT cells are heterozygous with respect to the
Py inserts. The same data were used to map the flanking sequences in the two types of
cells. A comparison between the two maps revealed that a 3.0 kb cell D N A segment,
which is located next to the unoccupied integration site in the normal rat chromosomes,
has been deleted from the LPT chromosome which carries Py DNA, but not from the
LPT chromosome which does not carry the virus DNA. The implications for
papovavirus integration are discussed.
INTRODUCTION
In cells which are permanently transformed by the small D N A tumour viruses SV40 and
polyoma virus (Py), virus D N A sequences are integrated into chromosomal D N A (Sambrook et
al., 1968; Shani et al., 1972; Manor et al., 1973). Studies of the pattern of integration of these
viruses revealed the following characteristics. (i) Some SV40- and Py-transformed cell lines
contain single virus inserts. Other transformed lines contain t> 2 inserts which map at different
chromosomal sites. (ii) In independently derived transformants the cellular and the viral sites of
integration map at different positions in the two genomes. No specific viral or cellular sequences
occur at the virus-cell junctions. (iii) None of the inserts analysed so far consists of a single
unmodified virus genome. Instead, many inserts include deletions or inversions of virus D N A
sequences. Partial or complete duplications of the virus genome are also prevalent in SV40 and
Py inserts. The duplications are often arranged in a direct tandem repeat (Botchan et al., 1976;
Ketner & Kelly, 1976; Birg et al., 1979; Campo et al., 1979; Lania et al., 1979; Della Valle et al.,
1981; Clayton & Rigby, 1981; Stringer, 1981).
One important aspect of SV40 and Py integration, which has not been thoroughly investigated
as yet, is whether insertion of the virus D N A into chromosomal D N A causes rearrangements of
the flanking cell sequences. This question can be examined by comparing the flanking
sequences in cell lines transformed by these viruses to the corresponding sequences in the normal
cells from which these lines were derived. By making such a comparison, Botchan et al. (1980)
have found that in one SV40-transformed rat cell line, which contains a single SV40 insert, the
sequences flanking the insert have undergone a rearrangement. They have not determined
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0022-1317/83/0000-5147 $02.000 1983 SGM
70
A. NEER, N. BARAN AND H. MANOR
whether the rearrangement involved deletion, inversion or translocation. Recently, Stringer
(1982) has reported a cellular deletion of at least 3.0 kilobase pairs (kb) at the site of SV40
integration in another SV40-transformed rat cell line. In this article we report an attempt to
determine whether Py integration causes structural alterations of cell D N A sequences flanking
the integration site. The systems employed for this study were an inducible line of Pytransformed rat cells designated the LPT line, and normal rat fibroblasts.
The pattern of integration of Py D N A in the LPT line is rather unusual (Mendelsohn et
al.,1982). The Py inserts in LPT cells include a variable number of whole Py genomes arranged
in a direct tandem repeat and hence have variable lengths. However, all inserts map at a single
chromosomal site and are therefore flanked by the same cellular D N A sequences. The Py
sequences found next to the cell-virus D N A joints are also invariable.
To compare the flanking sequences in LPT cells with the corresponding sequences in normal
rat fibroblasts, we carried out a Southern blot analysis of LPT and rat fibroblast D N A digested
with various restriction enzymes (Southern, 1975; Botchan et al., 1976). We used as
hybridization probes Py D N A and cloned flanking cell DNA. This analysis provided maps of
the unoccupied and the occupied integration sites in the two types of cells. A comparison
between the two maps revealed that 3-0 kb of rat cell D N A located next to the Py integration site
have been deleted in the LPT chromosomes which carry Py DNA. This study also showed that
LPT cells are heterozygous with respect to both the virus inserts and the cellular deletion.
METHODS
Cells and virus. A clonal derivative of the LPT line designated clone 1A was used for these studies. The methods
employed for propagating this clone were described previously (Manor & Neer, 1975). Growth of polyoma virus
and purification of Py DNA were also carried out as described before (Manor et al., 1973; Manor & Neer, 1975).
Rat secondary cultures were prepared according to Winocour & Sachs (1960).
Preparation of cell DNA. High molecular weight chromosomal DNA was prepared from LPT cells by a modified
Hirt procedure (Hirt, 1967; Mendelsohn et al., 1982). The method of preparation of D N A from rat secondary
cultures was also described by Mendelsohn et al. (1982).
Analysis of restriction enzyme digests by the Southern technique. The procedures employed for digestion of D N A
with restriction enzymes, electrophoresis in horizontal agarose gels, blotting, DNA D N A hybridization and
autoradiography were described by Mendelsohn et al. (1982). The lengths of the fragments observed in the blots
were determined by comparing their electrophoretic mobilities to those of known markers which were
electrophoresed in parallel (ibid). The markers used for these experiments were bacteriophage ,l DNA cleaved
with EcoRI, or with both EcoRI and BamHl, and Py DNA cleaved with either HpalI, or with HindlII, or with
EcoRI. DNA hybridization probes were prepared by nick translation (Rigby et al., 1977). The specific
radioactivities of the probes were in the range of 2.5 × l0 s to 5.0 × l0 s ct/min/lag.
Heteroduplex mapping. DNA molecules prepared from phages ,~lptl and 21pt2 were dissolved in a solution
containing 5 0 ~ formamide, 1 mM-EDTA and 10 mM-PIPES pH 7. The final concentration of each type of D N A
was 2 ~tg/ml. The solution was heated for 3 min at 80 °C and quenched in ice. NaC1 was then added to a
concentration of 0.4 M and the mixture was annealed for 30 min at 37 °C. Next, the D N A was spread for
microscopy by the cytochrome c method (Davis et al., 1971). The grids were rotary-shadowed with platinumpalladium and were visualized in the JEM-100B electron microscope. Length measurements were carried out as
described previously (Zuckermann et al., 1980).
Cloning in the plasmid pBR322. Cloning the 0.90 kb fragment into the EcoRI site of the plasmid pBR322 was
carried out as described by Bolivar & Backman (1979).
Construction of a library of L P T celt DNA fragments in bacteriophage 2gtWES. High molecular weight
chromosomal DNA was prepared from LPT cells by the modified Hirt procedure. It was partially cleaved with the
enzyme EcoRI at an enzyme/DNA ratio of 0.12 units/lag DNA for 1 h at 37 °C. The DNA was centrifuged for 16 h
at 20000 rev/min and 10 °C in a 5 to 2 0 ~ sucrose gradient in the SW27 rotor of the Spinco ultracentrifuge.
Fractions sedimenting faster than 20S were collected and pooled. Bacteriophage 2gtWES was grown and purified,
and the phage DNA was extracted, as described by Enquist et al. (1976). The D N A was cleaved with EcoR] into
three fragments (ibid). The two longer fragments which contain the genes needed for lytic growth (the 2gtWES
arms) were purified by agarose gel electrophoresis. These fragments were ligated with the purified LPT cell DNA
fragments and packaged into 2 coats according to Enquist & Sternberg (1979).
Isolation of bacteriophage clones containing Py DNA sequences from the L P T cell DNA library. The procedure
described by Benton & Davis (1977) was used for screening the in vitro packaged phages for recombinants
containing Py DNA sequences. The screening was carried out with no prior amplification of the library. The
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Polyoma integration causes chromosomal deletion
71
phages were seeded on a lawn of the Escherichia coli strain LE392 in 24 x 24 cm square plates (Nunc). Each plate
contained 50 000 to 75 000 plaques. Nitrocellulose filters were brought in direct contact with the phage plaques and
the DNA was later denatured, neutralized and fixed on the filters. Plaques that gave positive signals were picked,
propagated in E. coli LE392 and further analysed.
RESULTS
Isolation and characterization of a recombinant phage containing Py and non-viral DNA from
L P T cells
High molecular weight LPT cell DNA was prepared and partially cleaved with the restriction
enzyme EeoRI. The fragments were cloned into the bacteriophage vector 2gtWES (Enquist et
al., 1976; see Methods). The genomic library generated by this procedure was screened by a
plaque hybridization assay (Benton & Davis, 1977) for recombinant phage clones containing Py
DNA sequences. Three recombinant phages out of 1-5 x 106 that have been screened contained
Py DNA sequences and the DNA of one of them, designated 21pt2, was also found to include
non-viral sequences (Neer, 1981).
Phage 21pt2 was further characterized as follows: one sample of the phage DNA was partially
digested with the enzyme EeoRI, and another sample was digested to completion with this
enzyme. The digests were analysed by gel electrophoresis. Fig. 1 (track 1) shows the
electropherogram of the complete digest. It can be seen that this digest includes two fragments
derived from the LPT DNA insert and the two 2gtWES arms. The lengths of the LPT DNA
fragments are 2-6 kb and 0-9 kb respectively, and the lengths of the 2gtWES arms are 21-9 kb
and 13.9 kb. Track 3 in Fig. 1 presents a blot of this gel hybridized with 32p-labelled Py DNA. It
can be seen that only the 2-6 kb fragment contains Py DNA sequences. Partial cleavage with
EeoRI produced two additional fragments of 6.1 kb and 3.5 kb from the cloned DNA (track 2),
both of which were found by blot analysis to contain Py DNA sequences (track 4). These results
indicate that the length of the cloned segment in phage 21pt2 is 6-1 kb and that it consists of the
0-9 kb fragment inserted between two repeats of the 2.6 kb fragment; only this arrangement can
yield a 3.5 kb fragment upon partial digestion. Further support for this presumed structure was
obtained by heteroduplex analysis. In this study, phage 21pt2 DNA was annealed with DNA of
another recombinant phage clone, designated 21ptl, which contains two whole Py genomes
inserted in a head-to-tail configuration between the two 2gtWES arms (Neer, 1981). The
hybrid molecules were visualized by electron microscopy. Fig. 2(a, b) shows an electron
micrograph and a trace of a typical heteroduplex, and the legend to Fig. 2 presents the average
lengths of various regions of the heteroduplexes analysed in this study. This analysis has shown
that the cloned LPT DNA in phage 21pt2 contains two direct repeats of a continuous 2.15 kb
segment of Py DNA (regions D and G in Fig. 2b) which hybridized with the corresponding
sequences in phage 21ptl. These two Py repeats are separated by a 1.15 kb segment that
remained single-stranded in the heteroduplexes (region F in Fig. 2b), and apparently includes
the 0.9 kb segment which was shown in Fig. 1 to consist of non-viral sequences. In addition,
there is a shorter region of DNA at one end of the 21pt2 insert (region C in Fig. 2b), which is not
homologous to the Py DNA sequences found in the corresponding region of phage 21ptl. The
regions B, E and H in Fig. 2(b) are 21ptl sequences which are absent from 21pt2. The left
boundary of the regions B and C, and the right boundary of the regions G and H correspond to
the EcoRI site on the Py physical map (Griffin et al., 1980). Measurements of distances of the
various regions from these points allow mapping of the Py DNA regions D and G.
Fig. 3 shows a physical map of phage 21pt2 based on the electron microscope data and on the
EcoRI cleavage pattern presented in Fig. 1. The map has been corroborated by other restriction
enzyme data presented elsewhere (Neer, 1981). The positions of the fragments generated by
the various enzymes are also indicated in Fig. 3. It is to be noted that the 6.1 kb insert in phage
21pt2 does not correspond to any known segment of the LPT chromosomal DNA (see
Discussion). However, it will be shown below that the 0.9 kb segment, whose position in the
physical map of the phage is shown in Fig. 3, represents an authentic cell DNA piece which
maps next to the Py integration site in LPT cells.
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72
A. N E E R , N. B A R A N A N D H. M A N O R
1
2
3
4
Fig. 1. Analysis of EcoRI digestion products of the recombinant bacteriophage 21pt2 DNA. One lag
samples of phage 21pt2 DNA were either partially or completely digested with the enzyme EcoRI. The
complete digest (track 1), and the partial digest (track 2), were electrophoresed for 16 h at 2 V/cm in a
0.7~ agarose gel. The gel was stained with ethidium bromide and visualized by illumination with u.v.
light. Subsequently, the fragments were transferred on to nitrocellulose sheets and the blots were
hybridized with 32p-labelled Py DNA. Tracks 3 and 4 show autoradiograms of the blots prepared from
the gels shown in tracks 1 and 2 respectively. The numbers on the left designate the lengths (in kb) of the
fragments observed. The procedure employed for length determinations is described in Methods.
The 0.9 kb fragment generated by E c o R I cleavage of phage ,Upt2 DNA is identical to
fragments produced by E c o R I digestion of L P T and normal rat DNA
The 0.9 kb fragment was isolated from an EcoRI digest of phage 21pt2 D N A by preparative
agarose gel electrophoresis. The purified fragment was re-electrophoresed in another gel along
with EcoRI digests of LPT cell D N A and normal rat fibroblast D N A . Blots prepared from this
gel were annealed with another sample of the purified 0.9 kb fragment which had been labelled
with 32p. Fig. 4 (tracks 1, 2 and 3) shows autoradiograms of these blots. It can be seen that each
of the blots contains a single band. Furthermore, these bands have identical electrophoretic
mobilities. Track 4 in Fig. 4 shows a blot of an EcoRI digest of L P T D N A annealed with 32p.
labelled Py D N A . It is evident that the Py D N A probe did not hybridize with the L P T D N A
fragment observed in track 2. The fragments with which the Py D N A did hybridize have been
characterized previously by Mendelsohn et al. (1982).
These data indicate that both the LPT and the normal rat genomes contain a 0.9 kb segment
which is identical to the 0-9 kb segment present in phage 21pt2 and is also cleavable with EcoRI.
It should also be noted that the presence of a single band in the L P T and normal rat D N A
digests, rather than multiple bands, is consistent with the notion that the 0.9 kb segment
contains only unique rat D N A sequences. This notion is supported by quantitative assays in
which known amounts of the purified 0.9 kb segment were electrophoresed in parallel with L P T
D N A digests and used to estimate its abundance in the digests. The concentration of the 0.9 kb
segment was found to be less than 10-6 p.g per 10 Ixg of L P T cell D N A (not shown).
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73
P o l y o m a integration causes c h r o m o s o m a l deletion
(b)
or>
f"'"..
'~
L# 0
Fig. 2. Heteroduplex mapping of phage 21pt2 DNA. DNA of phage 21pt2 was annealed with DNA of
phage 21ptl, which contains two whole Py genomes arranged in a direct tandem repeat (see text). The
hybrids were analysed as described in Methods. (a) Electron micrograph of a heteroduplex between
21ptl DNA and 21pt2 DNA. (b) An interpretive trace of the micrograph shown in (a). A designates 2
DNA. The 21ptl insert consists of the regions B + D + E + G + H; the 21pt2 insert consists of the
regions C + D + F + G. As indicated in the interpretive trace, the region across the loop H does not
appear to be single-stranded and was probably created in this molecule by folding of the loop. This
region is absent from most of the other heteroduplexes analysed in this study. The average lengths of the
various regions and the standard deviations were determined in a sample of 16 heteroduplexes and are
as follows : B, 0.93 + 0.19kb;C, 0-37 + 0.16kb;D, 2.19 + 0.16kb;E, 2.87 + 0.39kb; F, 1.26 + 0.18
kb; G, 2.18 + 0.16 kb; H, 1.84 + 0.29 kb. Single-stranded and double-stranded circular DNA
molecules of bacteriophage ~bX174 were used as standards for these length measurements. Bar marker
represents 0.50 kb...-., Single-stranded DNA;
, double-stranded DNA.
BamHI.
HpaI
EcoRI
Partial
cleavage
]/r 17"2kb
~ ~.~ r~o #,
,
,,!
~
~
EcoRI
HpalI/EcoRI
HpalI
HindllI/EcoRI
3'5kb
16"3 kb
6'1 kb
3.9 kb
3"5 kb
2-6 kb
0.90 kb
2.6 kb
0.~76 0..16 0.89.0.__3~ kb 0:76 0.16 0.89 0--34'kb
O.,TA~)a~34kb
,1.O Q-~60.sqo-34kb
0-49===kb
//----t100
T
~
I
Cell DNA
Polyoma DNA
Boundary region
J 2 DNA
T ;)11 11 ti Jl :tl\ TI l\
t
i1.0 kb
Fig. 3. Physical map of the recombinant phage 21pt2 based on the EcoRI cleavage pattern shown in
Fig. 1 and on the electron microscope data shown in Fig. 2. It has been corroborated by cleavage of
phage 21pt2 DNA with other enzymes (Neer, 1981). The fragments generated by the various enzymes
are designated as bars drawn above the map, and their sizes (in kb) are also indicated. The boundary
regions designate limits of uncertainty for the positions of the junctions between the 2, Py and cell
DNA. Note that this map is rotated 180° relative to the heteroduplex shown in Fig. 2.
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74
A. NEER, N. BARAN AND H. MANOR
1
234
1
Fig. 4
2
3
4
5
6
7 8
Fig. 5
Fig. 4. Demonstration of the identity of fragments generated by digestion of LPT DNA, normal rat
DNA, and phage 21pt2 DNA, with the enzyme EcoRI. The 0-9 kb fragment was isolated from an EcoRI
digest of phage 21pt2 DNA by preparative agarose gel electrophoresis. A 10-5 ~tg amount of the purified
fragment was re-electrophoresed on a 1~ agarose gel at 2 V/cm for 16 h (track 1). Ten ~tg samples of
LPT cell DNA (tracks 2 and 4), and of normal rat fibroblast DNA (track 3), were digested with EcoRI
and electrophoresed on the same gel. Blots prepared from this gel were hybridized with another sample
of the 0.9 kb fragment which had been labelled with 32p (tracks 1, 2 and 3), or with 32p-labelled Py
DNA (track 4), and were exposed to an X-ray film for autoradiography. The lengths (in kb) of the
fragments observed in these blots are indicated on the left side of the autoradiogram. The 0.9 kb
fragment is indicated by an open arrowhead according to a convention described in the text.
Fig. 5. Analysis of XbaI, BamHl and XbaI/BamHI cleavage products of LPT and normal rat DNA.
Ten ~tg samples of DNA were digested and electrophoresed on a 0.7~ agarose gel. The DNA species
analysed and the enzymes used for the analysis were as follows. Tracks 1 and 2, LPT DNA, XbaI; track
3, normal rat DNA, XbaI; tracks 4 and 5, LPT DNA, BamHI ; track 6, normal rat DNA, BamHI; track
7, LPT DNA, Xbal + BamHI; track 8, normal rat DNA, XbaI + BamHI. Blots prepared from this gel
were hybridized with the recombinant plasmid including the 0.9 kb cell DNA probe (tracks 2, 3, 5, 6, 7
and 8), or with Py DNA (tracks 1 and 4) and exposed to an X-ray film for autoradiography. The open
and closed arrowheads indicate fragments used for mapping the 0-9 kb segment and the region
surrounding this segment in LPT ceils and in the normal rat fibroblasts, as described in the text. The
lengths (in kb) of these and other fragments are indicated on the left side of the photograph.
Evidence that the 0.9 kb segment maps next to the Py integration site in L P T cells
Our n e x t goal was to d e t e r m i n e the position of the 0-9 kb s e g m e n t r e l a t i v e to the Py i n t e g r a t i o n
site in L P T cells. T o this end L P T D N A was digested w i t h restriction e n z y m e s o t h e r t h a n
EcoRI, whose sites o f c l e a v a g e w i t h i n the sequences flanking the Py inserts h a v e b e e n previously
m a p p e d in our laboratory ( M e n d e l s o h n et al., 1982). Southern blots p r e p a r e d f r o m these digests
were hybridized w i t h Py D N A or w i t h a r e c o m b i n a n t p B R 3 2 2 p l a s m i d in w h i c h the 0.9 kb
s e g m e n t was subcloned. A similar analysis of n o r m a l rat D N A was carried out in parallel. Fig. 5
shows blots o f L P T and n o r m a l rat D N A digested w i t h the e n z y m e s XbaI a n d B a m H I t h a t cut
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Polyoma integration causes chromosomal deletion
75
the Py DNA once, or with both enzymes used in succession. Tracks 1 and 4 show blots of the
XbaI and the BamHI digests of LPT DNA that were hybridized with Py DNA. A blot of the
corresponding double-digest has been presented elsewhere (Mendelsohn et al., 1982). These
blots display two linker fragments containing both Py and cell DNA sequences and also whole
linear Py DNA molecules, or the double-digestion products of these molecules. These fragments
have been characterized previously (ibM). In each digest one linker fragment indicated by a
closed arrowhead also hybridized with the 0.9 kb segment in the recombinant plasmid probe, as
shown by the blots presented in tracks 2, 5 and 7 of Fig. 5. These particular linker fragments
include sequences derived from the left end of the Py inserts, and 5-7 kb or 12.2 kb of flanking
cell DNA respectively (Mendelsohn et al., 1982). We conclude that the 0-9 kb segment maps less
than 5-7 + 0.9 = 6.6 kb from the left cell-virus DNAjoint. More precise mapping of the 0.9 kb
segment is presented in another section of Results.
Evidence that L P T cells are heterozygous with respect to the Py inserts
Tracks 2, 5 and 7 in Fig. 5 also display, in addition to one linker fragment, a second fragment
which is indicated by an open arrowhead. These fragments do not hybridize with Py DNA. On
the other hand, fragments having the same mobilities are present in the corresponding digests of
normal rat DNA (tracks 3, 6 and 8 in Fig. 5). These data indicate that in addition to the
chromosomal site which is occupied by Py DNA, LPT cells also contain a corresponding
unoccupied site. According to this interpretation, the linker fragments in tracks 2, 5 and 7 are
generated from the site occupied by Py DNA and the fragments indicated by open arrowheads
are generated from the corresponding unoccupied site. This interpretation is also supported by
cleavage patterns of other enzymes, as reported in the next section.
The presence of an unoccupied integration site in LPT cells would be accounted for if LPT
cells are heterozygous with respect to the integrated Py DNA. This hypothesis is supported by
the observation that the fragments representing the unoccupied site in the normal rat DNA are
more abundant than the corresponding fragments derived from LPT DNA (compare, for
example, the intensities of the bands indicated by open arrowheads in tracks 2 and 3 of Fig. 5);
in LPT cells only one homologous chromosome contains an unoccupied site, whereas in normal
rat cells the sites present on both homologous chromosomes are not occupied.
Detailed mapping of the 0.9 kb cell D N A segment and of sequences flanking this segment in the
L P T chromosome which carries Py D N A and in the homologous normal rat chromosomes
Figures 6, 7, 8 and 9 present additional blot analyses of various restriction enzyme digests of
LPT and normal rat fibroblast DNA. The blots were hybridized with either Py DNA, or the
purified 0-9 kb cell DNA segment, or the recombinant plasmid including the 0-9 kb segment.
The symbols used in Fig. 5 are also employed in the rest of the blots. Thus, the linker fragments
are indicated by closed arrowheads. The open arrowheads designate fragments which are
observed in both the LPT and the normal rat DNA digests. Other fragments, which hybridize
with Py DNA but do not hybridize with the cell DNA probe, are not marked in these
photographs. These fragments were previously characterized by Mendelsohn et al. (1982).
The data presented in Fig. 4 to 9 were used to map the 0.9 kb segment and the sequences
flanking this segment in LPT Cells and in normal rat fibroblasts. The method is similar to that
used for mapping virus inserts by the Southern technique (see Botchan et al., 1976; Mendelsohn
et al., 1982). The fragments derived from the LPT chromosome which carries Py D N A can only
be arranged in one way consistent with the previously derived physical map of the virus inserts
(Mendelsohn et al., 1982). This unique arrangement was used to determine the position of the 0-9
kb segment in this LPT chromosome relative to that of the virus DNA, as shown in Fig. 10. Fig.
11 shows the physical map of the 0.9 kb segment and flanking sequences in normal rat
fibroblasts, and the unique fragment arrangement which was used to construct this map. As
discussed above, the same fragments are also present in the LPT cell D N A digests. Hence, the
map shown in Fig. 11 also represents the structure of the corresponding region in the LPT
chromosome which does not carry Py DNA.
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76
A. NEER, N. B A R A N A N D H. MANOR
1
2
3
4
5
6
1
Fig. 6
2
3
4
5 6
7
8
9
Fig. 7
Fig. 6. Analysis of Bgll digests of the purified 0.9 kb fragment, and of LPT and normal rat DNA. A
10-5 ktg sample of the 0.9 kb fragment was digested with Bgll and electrophoresed on an agarose gel, as
described in the legend to Fig. 4 (track 6). Another sample of this fragment was similarly
electrophoresed without being digested (track 1). Other samples applied to the same gel were: LPT
DNA (t0 gg) digested with BglI (track 3), or with BglI + EcoRI (track 5), and no~-mal rat fibroblast
DNA (10 gg) digested with BglI (track 2), or with Bgll + EcoRI (track 4). Blotting and hybridization
were carried out as described in Methods. All blots were hybridized with the purified 0-9 kb cell DNA
segment. The numbers on the left designate the lengths (in kb) of the fragments observed.
Fig. 7. Analysis of HindIII, HindIII/EcoRI and HindIII/BglI digests of LPT and normal rat DNA.
Fifteen pig samples of cell DNA were digested with restriction enzymes and electrophoresed for 16 h on
a 2 ~ agarose gel at 2 V/cm. The DNA species analysed and the enzymes used for the analysis were as
follows. Tracks 1 and 2, LPT DNA, HindIII; track 3, normal rat DNA, HindIII; tracks 4 and 5, LPT
DNA, HindIII + EcoRI; track 6, normal rat DNA, HindIII + EcoRI; tracks 7 and 8, LPT DNA,
HindlII + BglI; track 9, normal rat DNA, HindlII + BglI. Blots prepared from this gel were
hybridized with either Py DNA (track 7), or with the Py DNA fragment HpalI1 which extends from
27.0 map units to 53-8 map units on the standard Py physical map (Griffin et al., 1980) (tracks 1 and 4),
or with the purified 0.9 kb cell DNA segment (tracks 2, 3, 5, 6, 8 and 9). The lengths (in kb) of the
fragments indicated by arrowheads, and of several other fragments, are shown on the left side of the
photograph.
Evidence for a deletion o f a cell DNA segment in the L P T chromosome which carries Py D N A
Fig. 12 s h o w s a g a i n t h e p h y s i c a l m a p o f t h e c h r o m o s o m a l l y a s s o c i a t e d P y D N A a n d f l a n k i n g
cell s e q u e n c e s d r a w n b e l o w t h e m a p o f t h e c o r r e s p o n d i n g r e g i o n in t h e h o m o l o g o u s n o r m a l r a t
c h r o m o s o m e s . T h e t w o m a p s w e r e o r i e n t e d b y p l a c i n g t h e 0-9 k b s e g m e n t a t e q u i v a l e n t
p o s i t i o n s . It c a n be s e e n t h a t t h e left p o r t i o n s o f t h e m a p s , u p to a n d i n c l u d i n g t h e 0.9 k b
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Polyoma integration causes chromosomal deletion
1
2
3
Fig. 8
4
5
6
1
2
77
3
4
5
Fig. 9
Fig. 8. Analysis of BgllI/BamHI, BgllI/XbaI and BgllI/EcoRl digests of LPT and normal rat DNA.
Ten p.g samples of cell DNA were digested with restriction enzymes and analysed by electrophoresis,
blotting, hybridization and autoradiography, as described in the legend to Fig. 4. The recombinant
plasmid containing the 0.9 kb cell DNA segment was used as a hybridization probe. The DNA species
analysed and the enzymes used for the analysis were as follows. Track 1, LPT DNA, BgllI + BamHI;
track 2, normal rat DNA, BgtlI ÷ BamHI; track 3, LPT DNA, BgllI + XbaI; track 4, normal rat
DNA, Bglll + XbaI; track 5, LPT DNA, BgllI + EcoRI; track 6, normal rat DNA, BgllI + EcoRI.
The lengths (in kb) of the fragments indicated by arrowheads are shown on the left side of the
photograph.
Fig. 9. Analysis of HpalI, HpalI/BglII, HpalI/EcoRI, HindlII and HindlII/BgllI digests of normal rat
DNA. Fifteen gg samples of normal rat fibroblast DNA were digested with restriction enzymes, and
were analysed by the Southern technique, as described in the legend to Fig. 7, except that all blots were
hybridized with the 0.9 kb cell DNA probe. The enzymes used for digestion were as follows. Track 1,
HindlII; track 2, HindlII ÷ Bglll; track 3, HpalI; track 4, HpaII + BgllI; track 5, HpalI + EcoRl.
The length (in kb) of the fragments indicated by arrowheads are shown on the left side of the
photograph.
segment, are identical. Beyond this region to the right, there are in the top m a p two restriction
e n z y m e sites, those of BgllI and BglI, that are missing f r o m the b o t t o m m a p . R e c e n t l y , we found
t h a t a n o t h e r restriction e n z y m e site (a KpnI site), w h i c h m a p s b e t w e e n the BglI a n d BglII sites in
the n o r m a l rat c h r o m o s o m e s , is missing f r o m the L P T c h r o m o s o m e w h i c h carries Py D N A (not
s h o w n in Fig. 12). Thus, it appears that a D N A s e g m e n t i n c l u d i n g these three sites, a n d
i n d i c a t e d on the top map, has been deleted f r o m the region flanking the Py D N A on the right.
T h e distance b e t w e e n the BglI and the BgllI sites, 3.1 kb, is. a m i n i m a l e s t i m a t e o f the length o f
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78
A. NEER, N. BARAN AND H. MANOR
XhoI
Bglll
13,1 kb
8.6 kb
12-2 kb
-~i
BamHl ~/r
,:l
Xbal/BamHl ~ , q
XbaI/Bglll
BamHI/BgllI
BglII/EcoRI
BamHI/EcoRI
Xbal
BamHI/Bgll
Xbal/BglI
BglI/BglI!
HindllI/BglI
Hpall/EcoRl
Hpall
HCndllI/EcoRI
Hindll]
EcoRI/BglI
BglI
EcoRI
13-2 kb
8.~ kb
6.5 kb
3.3 kb
0.90 kb
0.90 kb
0.77 kb
1.3 kb
10.77 kb
2,1 kb
,0.77 kb
2.1 kb
0.66 kb 9"7 kb
0.63 kb.0q26 kb
0.90 kb
1.0kb
O-79 k~
1-32 kb
1.28 kb
.0"68 kl?
0.77 kb
0.90 k
2-1 kb
b
0.90 kb segment
,r . . . . .
q
/lt
,3
L_..... "1'1J4550 6'0 70 80 9'0 160 1'0 2'0 3'0 4()45 [/
~
Polyoma virus D N A
'~" Boundary region
~'
Cell DNA
I 1.0 kb I
Fig. 10. Map position of the 0.9 kb segment relative to that of the Py inserts in LPT cells. The Py inserts
and flanking sequences have been mapped by Mendelsohn et al. (1982). The black bars drawn above
the map indicate the positions of linker fragments which hybridize with both Py D N A and the 0.9 kb
cell D N A segment. The empty bars indicate fragments which hybridize with the 0.9 kb segment and do
not hybridize with Py DNA. The 3.4 kb EcoRI fragment hybridizes only with Py D N A and is indicated
by a hatched bar. The map position of the 0.9 kb segment, shown here as a rectangle, was deduced from
this unique fragment arrangement, Most of the fragments were observed in the blots presented in Fig. 4
to 9. The HpalI cleavage patterns were presented in the paper by Mendelsohn et al. (1982) and the XhoI
data are included in the thesis of A. Neer (1981). The boundary regions designate the uncertainty limits
of the positions of the junctions between the viral and the cell DNA.
the deletion. Beyond the deleted segment the chromosomal sequences are again homologous in
the two maps, as indicated by the presence of similar patterns of restriction enzyme sites.
Further evidence for the existence of the deletion, and additional estimations of the length of
the deleted segment, were obtained by comparing the sizes of the fragments produced by
cleavage of the rat fibroblast D N A with the enzymes XbaI, BamHI, HindlII and XhoI, to the
distances between the corresponding sites in the LPT chromosome containing the virus D N A .
Thus, Xba] cleavage of the normal rat D N A generates one fragment of 13.7 kb which hybridizes
with the 0-9 kb segment. The distance between the two corresponding XbaI sites in the
chromosome containing the smallest Py insert is equal to the sum of the two XbaI linker
fragments, that is 12.2 + 4-3 = 16.5 kb (Mendelsohn et al., 1982). Subtracting from this number
the length of the Py insert, estimated as 5.9 kb, we obtain 16-5 - 5.9 = 10.6 kb. The difference
between the two figures, 13.7 - 10-6 = 3.1 kb, is an estimate of the length of the deletion.
Similar calculations based on the cleavage patterns of the enzymes HindlII, XhoI and BamHI
yield estimates of 2.9 kb, 3.7 kb and 2.4 kb respectively. The average length of the deletion and
the standard error, calculated from these five estimates, is 3.0 + 0-4 kb.
DISCUSSION
The study of the D N A sequences flanking the Py inserts in LPT cells, and of the
corresponding sequences in untransformed rat fibroblasts, was begun by attempts to clone the
flanking sequences in the bacteriophage ;.gtWES. These attempts led to isolation of the
recombinant phage 21pt2 which has been mapped and found to contain a cell D N A piece
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79
Polyoma integration causes chromosomal deletion
XhoI
XbaI
c:==q/
BamHl ~_//
//
BamH1/XbaI ,=:~/
BamHl/BgllI
Xbal/Bglll
BamHI/BglI
Xbal/BglI
BamHI/EcoRI
Xbal/EcoRl
Bgln/EcoRI
BgllI/Bgll
BgllI/HpalI
Bglll/Hindlll
BgllI
HindlII/Bgll
HindlII/EcoRl
HindlII
HpalI/EcoRI
HpaIl
EcoRI/Bgll
Bgll
EcoRI
10-9 kb
13-7kb
16-9 kb
13.2 kb
2-4 kb
2.4 kb
0.77 kb
0.77 kb
0.90 kb
0-90 kb
0.90 kb
0.77 kb 0-53 kb
,0.90 kb 0.6~ kb
1.32 kb
1.2 kb
2.4 kb
0.66 kb
0.79 kb
1-32 kb
0-63 kb 0..,_26kb
0:90 kb
0"~¢ kb
0-77 kb
0.90 kb
//~=~
3.6 kb
6-9 kb
4-2 kb
3.6 kb
0.90 kb segment
~
Cell D N A
~ 1.0 kb~
Fig. 11. Physical m a p of the 0.9 kb segment and the sequences flanking this segment in normal rat
fibroblasts. This m a p was deduced from the unique fragment arrangement drawn above it. Most of the
data used here are presented in Fig. 4 to 9. The XhoI cleavage patterns are included in the thesis of A.
Neer (1981).
t
0.90 kb segment
i~!_1~4550 607080 90 l?010 20 30 40 45ff~
~=
Cell D N A
n m m Polyoma D N A
Boundary region
J Deleted D N A
1.0 kb
t
t
Fig. 12. Comparison between the physical m a p of the sequences flanking Py inserts in L P T ceils and
the physical m a p of the corresponding region in normal rat chromosomes. The two m a p s shown in Fig.
10 and 11 are shown here again. The m a p s are oriented relative to the 0.9 kb segment. The region which
has been deleted from the L P T chromosome carrying the Py D N A , and hence is absent from the bottom
map, is designated as 'deleted D N A ' in the top map. The boundary regions designate the uncertainty
limits of the ends of the deletion and of the Py insert. The positions of the restriction sites on the right
side of the bottom m a p have been deduced on the basis of data presented by Mendelsohn et al. (1982).
The segmented arrows in the top m a p designate restriction sites in the normal rat D N A , whose presence
was predicted but could not be demonstrated by using the 0.9 kb segment as a hybridization probe.
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80
A. N E E R , N. BARAN AND H. M A N O R
inserted between two direct repeats of a Py DNA segment. A continuous portion of this cloned
DNA, consisting of the cell DNA piece and one Py repeat, is co-linear with an authentic LPT
chromosomal segment which includes the left cell-Py DNA joint (compare Fig. 3 and 10).
However, the presence of the second Py repeat and the positions of the E c o R I sites in the cloned
Py DNA show that phage 21pt2 could not have been simply generated by in vitro ligation of a
chromosomal DNA fragment with the 2gtWES arms. Rather, it may have been derived by in
vivo recombination from other recombinant phages during growth of the latter in E. coll.
We have subsequently prepared from phage 21pt2 a DNA fragment containing just cellular
sequences and subcloned this fragment into the plasmid pBR322. We used the purified
fragment, the recombinant plasmid and Py DNA as hybridization probes in a Southern blot
analysis of LPT and normal rat fibroblast DNA digested with various restriction enzymes. This
analysis has led to two significant findings. First, it was shown that LPT cells contain, in
addition to the chromosomal locus into which Py DNA had been inserted, an equivalent site
which remained unoccupied and is therefore identical to the corresponding site in the normal rat
cells. Presumably, during the infection which led to cell transformation, Py DNA has integrated
into one chromosome. The other homologous chromosome remained unmodified and has been
propagated ever since in all LPT cells along with the chromosome which carries the Py inserts.
This interpretation is consistent with the observation that integration of Py and SV40 DNA into
cellular genomes is a very low-probability event and does not involve a specific recognition of
any particular chromosomal site (Botchan et al., 1976; Ketner & Kelly, 1976; Birg et al., 1979;
Lania et al., 1979). Thus, the probability of simultaneous integration of the Py DNA into two
equivalent sites in two homologous chromosomes could be as small as the probability of
simultaneous integration of Py DNA into two unrelated sites, that is the product of the
probabilities of integration into each of the two sites. It should be noted in this connection that
determinations of frequencies of reversion from the transformed phenotype of SV40transformed rat cells have indicated that these cells are haploid with respect to the virus insert
(Steinberg et al., 1978). Recently, SV40-transformed rat cells were found by a Southern blot
analysis to be heterozygous with respect to the SV40 inserts (Stringer, 1982).
The second significant finding reported in this paper is that a chromosomal DNA segment of
about 3.0 kb is missing from the region flanking the Py inserts in LPT ceils. The proximity of the
deletion to the virus inserts indicates that there is a causal relationship between the integration
and the deletion events. This view is supported by the observation that the deleted segment is
present not only in the corresponding normal rat chromosomes, but also in the homologous LPT
chromosome which does not carry a Py insert. Thus, the deletion could not have been caused by
an inherent instability of this particular chromosomal site in LPT cells. Rather, it appears to be
directly related to the integration of virus DNA at this site and to have resulted from an
interaction which operated only in cis.
The causal relationship between the deletion and the integration events does not necessarily
imply that the two events happened simultaneously; recent studies have indicated that virus
integration patterns in Py- and SV40-transformed cell lines are unstable. Rearrangements of
integrated Py DNA sequences were found to occur at a rather high rate in a number of Py
transformants including the LPT line. These rearrangements, which include deletions and
duplications of virus sequences, apparently take place by recombination between homologous
segments of the virus DNA (Basilico et al., 1979; Colantuoni et al., 1980; Chartrand et al., 1981 ;
Mendelsohn et al., 1982; Mendelsohn, 1981). Hiscott et al. (1980) and Sager et al. (1981) have
observed rearrangements of integrated virus DNA sequences in subclones of SV40-transformed
mouse cell lines. Sager et al. (1981) have reported that duplications of segments including both
SV40 DNA and flanking cellular sequences also occur in these cells. In view of these findings, it
is possible that the deletion occurred in LPT cells after the initial integration event. It could have
been generated, for example, by a single crossover between a site within the virus insert and a
site within the flanking cell DNA. However, so far, rearrangements affecting flanking host
sequences have not been reported in Py-transformed cells.
On the other hand, the deletion could have occurred during the initial integration event. This
possibility would be in line with current ideas on integration of papovavirus DNA. The ideas are
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Polyoma integration causes chromosomal deletion
81
Polyoma D N A
X4
Recombination
Cellular D N A
Fig. 13. Double-crossover model for integration of Py D N A into chromosomal D N A . The virus
precursor, of which only a part is shown here, includes a tandem array of Py monomers. Each monomer
is bound by two arrowheads. The cellular D N A segment located between the two crossover points is
replaced by the virus segment located between these two points.
based on studies mentioned in Introduction which showed that in many SV40 and Py
transformants, including the LPT line, the virus inserts contain partial or complete tandem
duplications of the virus genome. It has also been observed that none of the inserts analysed so
far consists of a single virus genome. Thus, it appears unlikely that the immediate precursor to
the chromosomally associated virus D N A is a circular monomer and that the latter is integrated
into the cell D N A by a single crossover event. Instead, it has been suggested that most
papovavirus inserts originate from a precursor consisting of tandemly joined virus genomes and
that it is integrated into the cell D N A by a mechanism involving double-crossover (Topp et al.,
1980). The precursor might be a rolling circle intermediate in virus D N A replication (Topp et
al., 1980; Della VaUe et al., 1981), or an oligomer of tandemly joined molecules of virus D N A
which is synthesized by recombination of free virus monomers (Chia & Rigby, 1981). As Fig. 13
shows, such a mechanism would generate an insert containing duplications of virus D N A and a
cellular deletion. In the case of the LPT line the insert would be longer than the deletion, as
indicated in Fig. 13 (see also Fig. 12). If this mechanism is general, then deletions should be
found next to sites of integration in other Py- and SV40-transformed cell lines. As mentioned in
Introduction, at least in one SV40-transformed rat cell line that has been characterized by an
approach similar to ours, a deletion occurred at the site of SV40 integration (Stringer, 1982).
In conclusion, we have demonstrated in this article that a 3-0 kb deletion of cellular D N A
occurred next to the Py inserts in LPT cells. We have also shown that LPT cells are heterozygous
with respect to both the inserts and the deletion. Our data indicate that the deletion was caused
by the integration event, but the relationship between these two events has not as yet been
clearly defined. We have discussed two alternative ways for deletion formation.
This work was supported by grants from the United States-Israel Binational Science Foundation and the
Leukemia Research Foundation. We thank M. Zabeau, V. Pirrota and L. Melli for their advice and help during
the initial phase of the project. We also thank M. Zuckermann for helping us to carry out the heteroduplex
analysis, J. Kuhn for helpful discussions and Y. Bot for able technical assistance. H. Manor was supported by an
E M B O short-term fellowship during a 2 month visit at the European Molecular Biology Laboratory in Heidelberg,
West Germany, where the cloning was begun.
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