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STUDIES ON AMPLIFICATION AND EXCISION OF THE INTEGRATED
POLYOMAVIRUS GENOME :
A REEVALUATION OF THE INTEGRATION
PATTERN OF POLYOMAVIRUS
By

Li-Jyun Syu

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology

1994

ABSTRACT

STUDIES ON AMPLIFICATION AND EXCISION OF THE INTEGRATED
POLYOMAVIRUS GENOME : '
A REEVALUA’I‘ION OF THE INTEGRATION
PATTERN OF POLYOMAVIRUS

By

Li-Jyun Syu

Gene amplification is one of the genomic instabilities occurring
during tumorgenesis. The unique system of amplification and excision of
the genome of the oncogenic Polyomavirus (Py) following its integration
into the host chromosome provides a valuable model system because the
size of the amplification unit is small (5.3 Kb). The structure of the
primary integrated viral sequences is unstable. Both amplification and
excision of the integrated viral sequences were observed in our study. We
used Py temperature-sensitive mutants which encode a thermolabile
large T-antigen to transform a rat cell line. Using temperature shifts to
manipulate the viral DNA replication in the transformant, we were able to
show that amplification occurring at the original integration site changes
a simple integration pattern into an apparently complex ladder-like

pattern. The rapidity and accuracy of the amplification and excision

suggest a model involving unscheduled DNA overreplication in a single S
phase at the viral integration site. The resulting onion-skin like structure
is mitotically unstable and tends to resolve into more stabilized products
by homologous recombination. The polyoma viral oncoprotein(s) may
have an unrecognized function which converts the host cell into a
permissive state for gene amplification. A future study on dihydrofolate
reductase (DHFR) gene amplification which leads to Methotrexate-
resistance or CAD (encoding a trifunctional enzyme) gene amplification
which leads to PALA-resistance in the Polyoma transformed cell lines
may help to identify the trans- or cis—elements involved in the gene

amplification process.

To my parents

iv

Acknowledgments

I would like to express my deep appreciation to my dissertation
advisor Dr. Michele Fluck, for her encouragement, guidance, and
financial support. -

I thank my committee members, Drs. Jerry B. Dodgson, Ronald J.
Patterson, Richard C. Schwartz, and Steven Triezenberg, who have given
me valuable advice and criticisms.

Appreciation is also expressed to all my colleagues in Dr. Fluck’s
laboratory, for their friendships and valuable communications, which are
not only helpful to my growth in scientific knowledge but also to my
understanding of American culture.

Finally, I would like to thank my beloved family and my two best
friends, Dr. Jiann-Yuarn Wu, and Chih-Chou Chao, for their love and

extensive spiritual supports.

1.1
1.2
1.2.1
1.2.2
1.2.3
1.3
1.4
1.4.1
1.4.2
1.4.3
1.4.4
1.5
1.5.1
1.5.2
1.5.3

TABLE OF CONTENTS

List of Tables ...................................... viii
List of Figures ...................................... ix
Literature Review ................................... 1
Introduction ................................... '. . . . 1
Neoplastic Transformation ............................ 6
The Role of Middle T-antigen in Neoplastic Transformation. . . . 7
The Role of Small T-antigen in Neoplastic Transformation ..... 9
The Role of Large T-antigen in Neoplastic Transformation ..... 10
Viral DNA Replication ................................ 12
ViralDNAIntegration.................. .............. 19
General Aspects of Integration and Integration Patterns ...... 19
Number of Integration Sites ........................... 24
Specificity of Integration Sites ......................... 25

Rearrangements Following Polyomavirus and SV4O Integration 26

Genomic Instability ................................. 28
Gene Amplification .................................. 29
Proposed Mechanisms of Gene Amplification .............. 30
Trans- and Cis-Acting Elements of Gene Amplification ....... 36
Bibliography ...................................... 39

vi

Effects of Amplification and Excision of the Integrated

Polyomavirus Genome on Its Integration Pattern ............ 75
Abstract .......................................... 76
Introduction .............. . ......................... 77
Materials and Methods ............................... 79
Results ........................................... 84
Discussion ........................................ 125
Bibliography ...................................... 135
Studies on Amplification and Excision of the Integrated

Polyomavirus Genome from subclonal cell populations ...... 139
Abstract 140
Introduction ...................................... 14 1
Results .......................................... 142
Conclusion ....................................... 165
Bibliography .................................. .- . . . 168

vii

LIST OF TABLES

Chapter 2 Table 1

Chapter 3

Summary of the Polyomavirus ts-A strain and selection
method used, as well as the production of ladder or free
viral DNA for each transformant isolated ............. 91

Table 1 .
Summary of the number and percentage of subclones
which displayed the distinct band of the ladder pattern
derived from BglII digestion ...................... 144

viii

1 Figure 1
Figure 2

Figure 3

Figure 4

Figure 5

2 Figure 1

Figure 2

Figure 3

Figure 4

Figure 5a

Figure 5b

Figure 5c

Figure 6a

LIST OF FIGURES

Physical map of polyomavirus genome .............. 3
Physical map of the noncoding region of Polyomavirus. 13

Depiction of DNA binding sites for enhancer-specific
cellular factors ................................ 17

Illustration of integration analyses ................ 21

A model for amplification of mammalian DNA involving
unscheduled DNA overreplication and recombination. . 34

A flow chart of the procedure for isolation of
transformants derived from infections with Py

temperature-sensitive mutants ................... 82
Integration patterns of Py wild-type transformants. . . . 86
A model which illustrates the hypothesis ............ 88

The effect of a block in viral DNA replication on the
integration pattern ............................ 92

Analysis of 33°C populations of 1 1 ts-A transformants,
which reveal a potential ladder-like pattern ........ 96

Analysis of 39°C populations of the same 11 ts-A
transformants as in Figure 5a .................. 98

Analysis of 33°C —> 39°C populations of the same #1-
#7 (excluding #5) ts-A transformants as in Figure 5a . . 101

Determination of the state of the viral genome in the
ladder-like pattern ............................ 104

Figure 6b
Figure 7

Figure 8a

Figure 8b

Figure 8c

Figure 9

Figure 10

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6
Figure 7
Figure 8

EtBr staining pattern corresponding to Figure 6a ..... 106
Quantitation of amplification ..................... 109
Kinetics of disappearance of the ladder-like pattern in '
transformant #6 .............................. l 1 1
Reproducibility of the ladder-like pattern in

transformant #6 .............................. 113
Stability of original integration site in transformant #6

and #1 ..................................... 1 16
Analysis of the number of integration sites by using

Bgll or Xbal ................................. 1 19
Analysis of 33°C populations of 11 ts-A transformants,

which do not reveal any ladder-like pattern ......... 122

Analysis of BgllI integration pattern of the subclones
isolated from parental transformant #6 ..... 145

Analysis of the arrangement of integrated viral
sequences of the subclones, which displayed distinct
BglII integration patterns as shown in Figure 1 ...... 147

Analysis of the topology of integrated viral sequences

of the subclones which displayed fourth band of the
parental BglII ladder pattern, in comparison with the

one displaying the base band .................... 150

Analysis of BglII integration pattern of the subclones
isolated from parental transformant #1 ............ 152

Analysis of the arrangement of the integrated viral
sequences in the subclone which displays a base

band and a novel band ......................... 155
Kinetics of appearance of the ladder ............... 158
Kinetics of disappearance of the ladder ............ 160
Stability of the ladder .......................... 163

CHAPTER 1
LITERATURE REVIEW

1 . 1 Introduction

The mouse polyomavirus was first isolated in 1953 1, and later
grouped with the papovavirus family 9. A variety of solid tumors in mice
and other rodents have been shown to be associated with experimental
polyomavirus infections 3, whereas the incidence of tumors in natural
populations is quite low. The host anti-polyomavirus immune response
accounts for the protection in immunocompetent mice 4. Over the past
three decades, extensive studies have been carried out with this DNA-
tumor virus, and results from these studies contribute to the
understanding of human cancers.

Tumorigenesis is a process involving a variety of changes in the
regulatory processes, structure, and metabolism of the cell. Many
oncogenic viruses participate in this evolution. The strategy which
polyomavirus uses is distinct from that of RNA tumor viruses
(Retroviruses). The transforming genes of polyomavirus play essential
roles in virus growth and have no close cellular homologues. By
disturbing host signal transduction pathways and thus providing
constitutive growth-promoting signals to the infected cells, polyomavirus

transforms cells to a tumorigenic state. Studies of the events which occur
1

2

after viral infection of the cells have given many insights into the steps
towards tumorgenesis 5.

Polyomavirus has a circular double-stranded genome, consisting of
5297 base pairs. The genome is organized in a way to achieve maximal
utilization of the coding sequences by differential processing of RNA
transcripts 5. Two primary transcripts are synthesized from early and late
coding regions, respectively, and are separated by a noncoding regulatory -
region approximately of 350 base pairs. Alternative splicing of the early
RNA precursor generates three transcripts coding for the three proteins:
large, middle and small T—antigens 7. Likewise, the late primary
transcript is differentially processed to generate three transcripts which
code for the viral capsid proteins: VPl, VP2 and VP3 8 (Figure 1). The
noncoding regulatory region controls viral DNA replication and
transcription. It contains regulatory elements, including the origin of
viral DNA replication, promoters for early and late gene transcription,
and an enhancer region, which is composed of multiple binding sites for
various cellular transcription factors, such as PEAl (the murine
homologue of APl) and PEAS ( the murine homologue of c-ets) 9'10.

The definition of the roles played by individual viral proteins in
tumorigenesis can be achieved in tissue culture systems. The most
compatible and easily studied counterpart to the natural host for the
polyomavirus is mouse fibroblast cell lines. A productive infection, also
defined as a lytic infection, can take place in the vast majority of mouse
fibroblast cells, which are thus designated as permissive cells. Upon
infection of permissive cells, polyomavirus is able to carry out the

complete growth cycle, with expression of all viral genes, and eventually

Figure 1. Physical map of polyomavirus genome

The top half circle and bottom half circle represent two coding
regions, which code for the early and late transcripts, respectively. The
transcripts are differentially spliced to generate the mRNAs for the T-
antigens or the capsid proteins, as indicated. The jagged lines within
those coding sequences are the introns. Splicing signals are conserved
and put to multiple use. The small T-antigen shares its splice donor
sequence with that of middle T- antigen and its splice acceptor site with
that of large T- antigen. The noncoding region located within these two
coding regions, is indicated as the dash lined sequences on the left side

of the map. This figure is taken from Soeda et al. 8

 

 

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produces infectious progeny viruses from lysed - cells. To evaluate the
impact of viral proteins in neoplastic transformation, cell lines which can
survive the viral infection must be used instead, such as those derived
from rat or hamster. Rat or hamster cells sustain a reduced (abortive)
infection, with little and no support for viral DNA synthesis and late
capsid protein synthesis, respectively “-15. Thus, those cells are
designated as nonperrnissive cells. Permissiveness seems to be
determined by trans-dominant host factors. These may include the host
DNA polymerase a/ DNA primase complex, which interacts directly with
large T-antigen 16-18. Perhaps, their conformational compatibility is
responsible, at least in part, for the perrnissiveness of the host 19.

Upon infection of rat or hamster cells, a transformed phenotype is
displayed. However, most infected cells soon return to the uninfected
state probably due to the loss of the viral genome upon cell division.
Such a transient phenomenon is termed abortive transformation 2°. In a
minor fraction of the population (OJ—5% 91), stable transformation is
observed. Stable transformation requires integration of the viral genome
into the cellular chromosome, such that the viral T-antigens can be
expressed constantly to maintain the phenotypic- characteristics of
transformation 2248. Transformed cells display structural and“
morphological changes 29 (such as disrupted cytoskeletal architecture 30),
loss of contact inhibition, reduced requirement of serum for growth,
anchorage independent growth in soft agar 31, and the ability to form
tumors in syngeneic animals 32.

This thesis project concerns the rearrangements of the viral
genome associated with integration. Before the main theme is

introduced, neoplastic transformation, which is stabilized with the viral

6

integration, and viral DNA replication, which is intimately linked to
rearrangements, will be reviewed. After these discussions, genomic
instability (in particular gene amplification), which usually occurs in

tumorigenic cells, will be reviewed. The proposed mechanisms for gene
I amplification may help in the elucidation of the mechanisms underlying

the rearrangements of integrated viral genome.

1.2 NEOPLASTIC TRANSFORMATION I

In early studies, two classes of nontransforming mutants, hr-t and
ts-a, were isolated and complementation studies with these have defined,
in part, the functional domains of T-antigens involved in neoplastic
transformation 33-36. The hr-t (host range) mutants are transformed-cell-
dependent, meaning that they replicate less efficiently in normal than in
polyoma-transformed mouse cells 3". These mutants are shown’ to lack
both middle and small T-antigens. These mutants do not induce either
abortive or stable transformation, although they produce normal large T—
antigen and integrate, presumably normally, into the cellular genome 27.
The ts—a mutants have a thermolabile large T-antigen. At the
nonpermissive temperature, ts-a mutants. are capable of inducing a
transformed phenotype which lasts a couple generations (abortive
transformation), but are defective in stable transformation. However, ts-a
mutants can transform cells at the permissive temperature, and a
delayed shift to the nonpermissive temperature after two days post
infection does not affect transformation 33. Complementation studies with
these two classes of mutants have assigned the transforming function
(maintenance of the transformed phenotype) to the hr—t function (i.e.

middle plus small T-antigens). Results from the transformation of rat

7

cells with a plasmid encoding single T-antigens further corroborate that
middle T—antigen is necessary and sufficient for transformation 39. The
importance of large T-antigen in transformation is thought to reside in its

role in viral DNA integration 4°.

1.2. 1 The Role of Middle T-Antigen in Neoplastic Transformation

Middle T-antigen (mT) is the dominant transforming oncogene 2740‘ .
42 of polyomavirus. Middle T-antigen is a membrane phosphoprotein of
421 amino acids 49 and acts indirectly to regulate the phosphorylation
events in the cells. Middle T-antigen mediates its function by association
with and activation of cellular non-receptor tyrosine kinases of the Src
family (src 43, fgn 44, and yes 45-46). The activation of tyrosine kinases
consequently elicits a series of downstream events, which transmit
signals from the cell membrane to nuclear transcription factors, 'via both
tyrosine and serine/threonine kinases in the cytoplasm 47-48.

Among the Src family, pp60¢'s"¢ is the major species to form the
complexes with mT. The formation of complexes between pp60¢'8”c and
mT can lead to a 10- to 50-fold increase in c-src kinase activity 49'50. This
interaction occurs with serine-phosphorylated species of mT, whose
abundance is increased by the activity of protein kinase C 50. The
activation of c-src is due in part to interference with the normal negative
regulation of the c-src protein, which involves the phosphorylation of c-
src protein on tyrosine-527 by another cellular enzyme 52. The resulting
activated Src kinase will in turn phosphorylate mT on tyrosine residues,
providing binding sites for proteins with SH2 (src homology) domains.
These include ' the protein Shc 53, phosphatidylinositol 3-kinase (P13-
kinase) 53-63, and, perhaps, phospholipase C gamma (PLCy) 64. MT

8

signalling is transduced and propagated to three different signalling
pathways.

Shc becomes tyrosine-phosphorylated by mT-bound pp60¢'s"°. As
there is evidence that mT antigen, pp60‘=‘3'c and p2 1°?"s lie in the same
signal transduction pathway 65-66, events downstream from the
phosphorylated Shc have been studied. Results show that an interaction
between She and Grb2 is promoted in the mT/ pp60¢'s'c mediated signal .
transduction pathway 67. As a consequence, the Shc/ Grb2 complex
stimulates p2 1¢'"1s activity, presumably through the mammalian Sos
homologue ‘68-59, which functions as a guanine-nucleotide-exchange
factor to convert Ras-GDP to active Ras-GTP 7°42. Ras then passes
signals down to the c-Raf-l serine/threonine kinase 73. c-Raf-l is also
activated by protein kinase C 73. Protein kinase C is activated via another
mT signalling pathway, which starts with the association of the activated
mT—pp60c'src complex with PLCy 54. Activated PLCy .will, in turn, cleave
certain phospholipids and result in a concomitant increase in diacyl
glycerol (DAG) and inositol 1,4,5-triph03phate (InsP3) 64. Both DAG and
InsP3 are second messengers in the transduction pathway of many
extracellular signals, including a number of different growth factors 74.
DAG is an activator of protein kinase C 75 and InsP3 is an important
regulator of cytosolic free calcium 76.

Two mT signalling pathways, via Shc and PLCy, thus converge to
activate c-Raf-l. c-Raf-l once activated, phosphorylates a second kinase,
known as MAP (mitogen activated protein) kinase kinase 77 or Mek 73,
which controls a third kinase, the MAP kinase 79. The activated MAP
kinase sends signals into the cell nucleus by phosphorylating

transcription factors, including c-jun 80-81. These transcription factors

9

then, in response, activate banks of genes. Events downstream of the
activation of MAP kinase, such as the induction of pp90'3k activity 32 and
the stimulation of transcription factors PEAl and PEA3, have been
observed in mT-antigen-transformed cells 83.85. PEAI and PEA3 in turn
activate the gene expression of collagenase and other genes 35.

The third mT signalling pathway involves the association of
activated mT-pp60c's’c complex with PI3-kinase. The activated PIB-kinase
can convert three kinds of phosphatidylinositol (PI) into phosphorylated
phosphatidylinositols (PI3Ps), which are not cleavable by any known
phospholipase 87-88. Because the metabolic fate and physiological
function of the P13? is still' unknown, the mle of the PI3-kinase in
mediating transformation is not clear.

Aside from the interaction, via pp60°'sr°, with She, PLCy, and PI3-
kinase, mT also associates with protein phosphatase 2A (PP2A) 39. The
precise role of association with PP2A in cellular transformation is less
defined, but possibly involves the alteration of the phosphorylation state
of mT, and, in turn, may thus promote the binding of mT to pp60°'8’° 9°.

The binding site for PP2A is different from that for pp60‘3'src 91.

1 .2.2 The Role of Small T-Antigen in Neoplastic Transformation
Small T-antigen (sT) contains 196 amino acid residues. All except
the carboxyl-terminal 4 amino acids are included in the amino-terminal
half of middle T-antigen. Small T-antigen is not a DNA-binding protein,
and is localized both in the nuclei and in the cytoplasm in a soluble form
”'93. Studies of sT have revealed no unique biochemical properties.
Similarly to mT, sT can interact with PP2A 94, and perhaps affect PP2A

function. Small T-antigen does not contain a transforming function;

10

however, a stimulating activity in cellular growth and in viral DNA
replication has been demonstrated 93535-101. This activity may be mediated

by PP2A.

1 .2.3 The Role of Large T-Antigen in Neoplastic Transformation

Large T-antigen (LT) is a multifunctional nuclear phosphoprotein
that contains 758 amino acids. LT is involved in transcriptional '
regulation, transformation, integration, as well as viral DNA replication
(initiation and elongation).

LT regulates viral transcription in a differential manner. Binding of
LT to sequences near the early promoter blocks binding of RNA
polymerase, thereby suppressing early transcription 102-104. Whereas, in a
more complex way, when the concentration of LT reaches a certain level
during the late phase, late transcription is stimulated 105-106.

In the early phase of infection, LT can also alter the cellular
transcription pattern of those genes expressed in G1/ S border of the cell
cycle 107-109. This alteration subsequently stimulates quiescent cells to
synthesize cellular DNA 110. The activation of cellular replication
machinery thus at the same time facilitates the replication of viral DNA.
The stimulation starts with the binding of the retinoblastoma gene
(known to be a tumor suppressor gene lll)-encoded protein (pRB). In
normal cells, one of the pRB targets is cellular transcription factor E2F.
E2F has been shown to be tightly regulated, in part, by complex
formation with pRB 112, and controls the expression of those genes whose
functions are essential for DNA replication (113-114. Therefore, large T-
antigen has evolved to dissociate E2F by outcompeting one of its

regulators, pRB 115-118. The dissociation, in turn, leads to a dramatic

11

activation of certain E2F-responsive cellular promoters 119. Genes
affected are those coding for c-myc, N-myc, thymidine kinase 12°,
thyrnidylate synthase 121, DNA polymerase-a 122, ‘ ribonucleotide
reductase, and dihydrofolate reductase 114.

These responses, induced by the interaction of LT with pRB, may
account for the irnmortalization ability of LT, which has been called an
"immortalization ' oncogene" 123. Irnmortalization usually occurs in '
primary cells, such as rat embryo fibroblasts, and results. in an infinite
life span. Immortalized cells do not display transformation-like
phenotypic changes except a decreased requirement for serum growth
factors 194498. However, irnmortalization may contribute to the
transformation phenotype in cultured cell lines. Interestingly, the ability
of LT to irnmortalize cells in culture is not essential to its ability to induce
tumors in the animal 129.

LT affects neoplastic transformation by facilitating a stable
association of the viral genome with the cellular chromosome 36, leading
_ to viral integration. Integration is essential for stable transformation,
providing a means to maintain a cell lineage that contains the complete
viral coding sequences for early proteins. The role of LT in the integration
process may be linked mechanistically to its other known role in
initiation of viral DNA replication. Analyses of the topology of integrated
viral genomes, which typically show partial tandem duplications, provide
evidence to support the view of this linkage. In the absence of a
functional LT, the transformation frequency is dramatically decreased by
100-fold and the ensuing transformants are found to contain nontandem
insertions of viral DNA segments shorter than the infecting polyoma

molecule 39. However, the role for LT in integration-transformation is not

12

simply to amplify the viral genome and thus enhance the probability of
its integration 19. It still remains unclear as to how large T-antigen
facilitates integration and regulates the topology of integration. The roles
of LT in viral DNA replication and in viral DNA integration will be

discussed in more detail below.

1.3 VIRAL DNA’REPLICATION

In the nucleus of the host cells, where DNA replication takes place,
the viral genome is complexed in a chromatin structure indistinguishable
from that of host. 10-25% of the genome is histone-free around the origin
of replication 130. All the requirements for initiation of viral DNA
replication are provided by the host replication machinery except for a
viral replication origin and a viral protein (large T-antigen). This model
thus is useful for the understanding of eukaryotic chromosomal DNA
replication. The viral replication origin is composed of a core domain (ori-
core) and an auxiliary domain (a and [3) (Figure 2). The ori-core is the
minimal origin, spanning only 66 base pairs 131 and containing a 15-bp
A—T rich sequence, a 34-bp palindrome, and an imperfect 17 -bp inverted
repeat sequence 132. The ori-core, determining where. replication begins,
is absolutely indispensable. The auxiliary domain determines the
efficiency of replication. Either an a or B element is required for
replication in all permissive cells except for the single-cell stage of the
newly developing mouse embryo 133-137.

Large T-antigen is the initiator, whose sequence-specific DNA
binding, helicase, and ATPase activities are essential to catalyze DNA
replication 138-139. The LT binding sites located within the ori-core have

lower affinity compared to those major ones towards the early side of the

13

Figure 2. Physical map of the noncoding region of polyomavirus

The nucleotide numbers extending from the late-gene side on
the left to the early -gene side on the right, as shown on the bottom of
the top half figure. The landmarks include the enhancer region, a 15 bp
A/T sequence (open box), a 34-bp palindrome (pinweel), a 17 -bp inverted
repeat (-><—), a TATA box (solid bos) and early and late mRNA start sites.
There are six DNA binding sites for large-T antigens, and their binding
affinity is B, C > A > 1, 2, 3. Three cis-acting elements (a, )3, core) make
up the polyoma origin of replication. Shaded areas designate the minium
required sequence for these elements). The four enhancer domains within
the enhancer region are shown in boxes. The DNase hypersensitive

regions (DHFR) are shown in ellipses.

14

 

 

 

 

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15

viral origin 140-143. This organization is different from SV40, in which the
major T-antigen binding sites are superimposed with the replication
origin 14“. This and other slight differences in the structural organization
of the replication origin suggest that the mechanism for initiation of
replication in these two viruses may be distinct 132. However, three
observations have demonstrated that SV40 and polyoma use the same
mechanism for initiation of DNA replication 132. Therefore, the model I
will present below, although being deduced from SV40 system, can be
applied to polyoma DNA replication as well.

As a first step, T-antigen binds as a double hexamer to specific
sequences within the origin “‘5, leading to a local conformational change
of the genome (unwinding) 1"“. This reaction requires ATP, a DNA
topoisomerase, and a single-strand-specific DNA binding protein
(variously termed SSB, replication factor A or replication protein A) 147-
150. In the second step, specific contacts between large T-antigen,
replication protein A and DNA polymerase a/primase position the DNA
polymerase a/primase in an orientation suitable for the ensuing DNA
synthesis events 151. These events begin with the synthesis of short
ribonucleotide primers 152. The high specificity of these protein-protein
contacts is thought to be responsible, at least in part, for the
permissiveness of a host to support polyoma DNA replication. In the
third step, T antigen acts in the presence of ATP as a DNA helicase to
unwind the DNA in its vicinity within the core region 153 and
consequently generates two advancing replication forks, which diverge
from each other. This facilitates the extention of the primers into short
DNA chains by the DNA polymerase a/primase complex. Replication is

bidirectional, semi-discontinuous and continues for several rounds.

16

During replicative chain elongation, T-antigen probably remains in
association with the DNA polymerase a/primase complex 154455.

Differential and reversible phosphorylation at eight or more sites
on large T-antigen can modulate its structure and function 15615". Initial
studies suggest that phosphorylation downmodulates DNA binding 153.
More recent data further suggest that the cellular protein phosphatase
2A (PP2A) 159-151 is responsible for dephosphorylation of several serine .
residues in LT and for stimulation of LT-dependent DNA unwinding and
replication 161. Dephosphorylation ’ of LT seems to stimulate the
cooperative interaction between the two hexamers of LT bound to the
origin. Consequently, the efficiency of DNA unwinding by LT is increased
161. Another cellular protein that regulates the phosphorylation state of
SV40 LT is cdc2 protein kinase 159. cdc2 protein kinase phosphorylates
the threonine-124 residue of LT, resulting in an efficient binding of LT to
SV40 ori. Since the activity of cdc2 protein kinase is cell cycle regulated,
this phosphorylation event may represent a mechanism by which the
virus will sense the dormancy of host replication machinery, thus
preventing abortive initiation and premature template unwinding. It is
consistent with the observation that LT of polyomavirus is more
phosphorylated in growing cells early in infection than in quiescent cells
163.

The auxiliary domain of the replication origin is not only involved
in viral DNA replication but also functions as an enhancer in viral early
gene expression 133435.137454-166. In particular, the sequences
corresponding to the PEAl and PEA3 binding sites (Figure 3) are
important in stimulating both processes 167. The coincidence of

transcriptional elements in the vicinity of the replication origin is not

17

Figure 3. Depiction of DNA binding sites for enhancer-specific cellular
factors

Among the cellular factors which bind to the enhancer region,
PEAl (the murine homolog of API) and PEA3 ( the murine homolog of c-
ets) have drawn a lot attention in our studies on polyomaviral DNA
replication. This figure is summerized from Veldman et al. 135 and

Hendrickson et a1. 10

18

Bell Pvull Pvull
< A enhancer > < B enhancer ‘ >

    
   
 

PEA3 EF-C PEAZ PEA3

   

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19

only observed in polyoma and SV40 but also in papillomaviruses,
adenoviruses, some herpesviruses, and mammalian mitochondrial DNA
168. Therefore, transcription and replication may be linked and involve
common factors. In appropriate circumstances, many transcription
factors, such as c-jun 169470 and the yeast factors GAL4 171, are observed
to activate DNA replication. Thus, transcription factors are emerging to
be the common factors between transcription and replication 172-174.

Recently, transcription factors VP16, GAL4, and p53 have each
been demonstrated to bind selectively to human and yeast replication
factor A (RPA) 175-176. Since RPA interacts with DNA polymerase a ‘177, the
recruitment of polymerase a by T—antigen and PRA in viral DNA synthesis
151 might also be aided by a transcription factor 176. This observation
provides direct evidence that transcription factors activate DNA
replication by facilitating the assembly of initiation complexes at core
origin sequences.

Recently a crucial role for mT in Py DNA replication has been
recognized in our lab. The central hypothesis is that transcription factors
activated by mT signal transduction pathways aid the initiation of Py
DNA replication. In particular, the transcription faCtors APl and c-ets,
via binding to the auxiliary domain of the replication origin, may be the

main ones involved.

1.4 VIRAL DNA INTEGRATION

1.4. 1 General Aspects of Integration and Integration Patterns
Integration of the polyoma viral genome into the cellular

chromosome is an essential step for stable transformation of

nonpermissive rat and hamster cells in tissue culture. Integration

20

provides a means to maintain a cell lineage that contains the complete
viral early coding sequences. Extensive studies have demonstrated that
the integration process involves nonhomologous (illegitimate)
recombination. Evidence for patchy homology, involving sequences
between 2-5 base pairs in length, has also accumulated 178-183. However,
the validity of requirements for patch homology has been questioned 184.
Integration appears to be a random or quasirandom process with respect
to the breakpoints within the viral and as well as the host sequences
293336485. A gross rearrangement, most likely a large deletion or a
translocation 179480486489 of the host sequences flanking the integration
site, appears to accompany the integration of polyomavirus or SV40 in
many cases. No specific or preferred chromosomal site in the host
genome has ever been found by restriction endonuclease mapping or by
direct DNA sequencing of junction fragments 179: 190-191 cloned from
integration sites.

Integration patterns have been classically deduced by restriction
endonuclease digestion of high molecular weight cellular DNA, followed
by electrophoresis, Southern transfer and hybridization with a labeled
viral probe. As first shown by Botchan et al. for SV40 185 and later by
Birg et al. for Py 2’2 (Figure 4), an estimate of the minimum number of
independent insertions of viral DNA into the genome of transformed cells
is obtained theoretically when using a restriction enzyme which has no
recognition site within the viral genome (i.e., a no-site enzyme) and thus
digests only within surrounding cellular sequences. The existence of
nonintegrated free viral DNA can also be observed as a band
corresponding to the supercoiled genome. Likewise, the topology of

integrated viral sequences is deduced with a appropriate restriction

21

Figure 4. Illustration of integration analyses

Integrated viral genome is expressed as a head-to-tail tandem
arrangement. The cleavage sites for EcoRI (a one-site restriction
endonuclease), BglII and BstEII (no-site restriction endonucleases) are

indicated.

22

 

 

 

 

 

 

 

 

 

BstEII EcoRI BstEll
l Bglll l Bglll 1
Cellular DNA 1 0" TCellular DNA
EcoRl EcoRI
BSIE" ' BSIEII
l 38'" Tandem copies BglII 1
Cellular DNA 1 O" T O" T 0" TCellular DNA

EcoRl

EcoRl

EcoRl EcoRl

23

enzyme which cleaves once within viral sequences (i.e., a one-site
enzyme). In such a digestion of high molecular weight cellular DNA
separated from viral genomes by the Hirt procedure 199, no more than
two viral-host junction fragments can be detected for a single insertion.
However, a genome size band (linear form 5.3 Kbp) is often observed and
taken as evidence for a head-to—tail tandem arrangement of the viral
genome. .

As established by integration analyses of independently
transformed cell lines, the viral genome appears to be inserted at
multiple sites in the host genome, and each site may contain head-to-tail
tandem copies of the viral genome. Rare transformants harbor only one
viral integration site, containing less than one complete copy of the viral
genome ”3336-185: 193-196. Models proposed for generation of the head-to-
tail tandem arrangement of integrated viral genomes have invoked a role
for either viral DNA replication (perhaps, the rolling circle-type) or
interviral recombination 193,196' 198. Results which are consistent with the
former model have been obtained by Basilico and collaborators, who have
shown that formation of tandems requires a functional large T-antigen 39
and the viral origin of replication 199. Based upon the existence of
unintegrated high-molecular-weight species of the viral genomes in
infections of nonpermissive cells with SV40 200, the rolling circle-type
replication has been suggested for the generation of multimeric DNA,
which is assumed to represent the unintegrated precursors of the
transforming viral genomes. However, rolling circles have not yet been
documented in polyomavirus-infected Fisher rat cells. Furthermore,
increased levels of DNA replication do not correlate to increased

transformation frequency 19. On the other hand, a role for interviral

24

recombination in the formation of tandems has been suggested in
studies of transformants derived from mixed infections with two
distinguishable polyoma strains 193301402. First, two parental genomes
(ts-a and hr-t strains) were found cointegrated at a single site in the host
chromosome 196. Later, in a system not selecting for recombination of two
suitably marked input viruses in mixed infections, a very high level (38%)
of interviral recombination was detected in the integrated viral genome
202. However, a closer analysis of the reCombination events suggests the
models involving interviral recombination or DNA viral replication are not
mutually exclusive. '
1.4.2 Number of Integration Sites

It is generally believed that multiple sites for polyomavirus
integration are available in the host. In 80% of the cases reported in the
published literature, an average of over three sites is found in each
transformant ”433537496303. However, it seems that the apparent
number of integration sites is independent of the number of templates
available in infections. No matter how low a multiplicity of infection (M01)
is used in infections with a single viral strain, similar integration
patterns with multiple integration bands were observed in different
transformed cell lines (data from our lab). Furthermore, in contrast to the
general belief, sites for integration seem to be limited in the study of
double integration events in transformants derived from mixed infections
with two distinguishable Py strains at different ratios 204. Thus, possibly,
there is an alternative interpretation for the apparent multiple number of

integration sites revealed in the integration analysis;

25

One study implies that the apparent number of integration sites
correlates to the degree of viral DNA replication in the integrated viral
genome. Thus, the apparent number of integration sites is greatly
reduced in transformed cells derived from infection with a mutant strain,
whose ability to undergo viral DNA synthesis is strongly diminished 195.

There are only two modes of integration pattern with respect to the
topology of the integrated viral genome and the apparent number of .
integration sites. First, if multiple integration sites are apparent in the
integration analysis, each site usually contain head-to-tail tandem copies
of the viral genome. Second, if only one viral integration. site is
demonstrated, the integrated sequences are comprised by less than one
complete copy of the viral genome 224339305. Basilico and collaborators
have shown that the formation of tandems requires a functional large T-
antigen 39 and viral origin of replication 199 (i.e., viral DNA replication).
These correlations may also imply that the apparent number of
integration sites is affected similarly to the formation of tandems by viral

DNA replication.

1.4.3 Specificity of Integration Sites

Analysis of a number of viral-host junctions by restriction
endonuclease mapping and by direct DNA sequencing of cloned junction
fragments has not revealed any sequence-specificity for polyoma or SV40
integration 179:190‘191. This type of analysis may not reveal other levels of
selectivity in integration sites; for example, selective integration in larger
domains of the host chromosome, in which the structural and functional
characteristics of chromatin may determine regional specificity for viral

integration. Some preferential integration sites have been observed with

26

several viruses. To date, adeno-associated virus (AAV) is unique among
eukaryotic viruses in its ability to integrate in a site-specific manner into
the human chromosome 19q 206408. A strong selectivity for retrovirus
integration sites was found by Shih et al., who analyzed many
independent integration sites collected in integration libraries 209. The
basis of this selectivity has been observed to reside in DNase-I

hypersensitive regions 9104“.

1.4.4 Rearrangements Following Polyomavirus and SV40 Integration

The integrated viral DNA is found to be highly unstable.
Spontaneous rearrangements, mostly duplication (amplification) 23.912
and deletion (excision) 181213 events involving homologous recombination
193314-217, are observed very frequently. Prasad et a1. observed that a
spontaneous induction of free viral DNA (i.e., excision) occurs .with low
(on average, 20 to 50 copies per cell) but constant, probability in a small
percentage of the transformed cell populations 14.218.

The free (unintegrated) viral DNA. originates by a mechanism of
coupled amplification and excision of the integrated viral genome. This
phenomenon is best explained by the ”onion skin" model, proposed by
Botchan and Sambrook 905319. In this model, the integrated viral DNA
can undergo multiple rounds of asynchronous replication during a single
cell cycle. The resulting localized onion-skin with amplified sequences
can then serve as a substrate for intramolecular homologous
recombination, which leads to the excision of free viral DNA and the
amplification of tandems of integrated viral DNA. Usually a functional
large T—antigen and replication origin 920, which are prerequisite for viral

DNA replication, as well as redundant sequences in the integrated viral

27

DNA 189-1933 16, which are prerequisite for homologous recombination, are
required for precise excision. Rare excision events occur when there are
no homologous regions in the integrated viral DNA itself. The
recombination between viral and nearby flanking cellular DNA results in
a low level of heterogeneous free DNA molecules 1894933053914”. The
resultant ”cured" cells, without complete early coding regions in their
integrated viral DNA, will become phenotypic revertants 23-24.

The role of large T-antigen in amplification and excision has been
investigated in polyoma transformed eells. Results from Pellegrini et al.
”3 show that in the presence of a functional large-T antigen, tandems of
integrated viral DNAs with defective replication origins are stable and
those with normal origins can amplify in situ and subsequently excise
free viral DNA. The dependence upon the presence of a viral replication
origin suggests that large T-antigen is involved in viral DNA replication to
provide localized substrates for excisional recombination. Whereas,
results from St-Onge et al. suggest that a role of LT, uncoupled with its
role in viral DNA replication 224-296, is to promote homologous
recombination across the tandem repeats, leading to excision.

Constant amplification and excision of the integrated viral DNA
can generate cell-to-cell heterogeneity among the transformed cell
population. Furthermore, because of the instability of integrated viral
DNA, the real number of original integration sites may have been
inaccurately estimated using the integration analysis. The number of
original integration sites may be distinguished by subcloning the
parental cell population and comparing the integration patterns of
subclones with those of the parental cell population. The rationale is that

every cell (i.e., subclone) should inherit parental cell's integration sites.

28

The results of Birg et a1. 22 show that the integration patterns obtained
with BgllI (no-site enzyme) digestion of subclones are related but not
identical to those of parental cells. The relative abundance of different
high-molecular-weight fragments varies; in some subclones, certain
fragments are even missing and novel fragments show up in the pattern.
Therefore, within the parental integration pattern at least some
fragments do not represent the original integrated viral genome but
rather the rearranged ones 22, which can not be distributed equally

within every cell in a transformed cell population.

1 .5 GENOMIC INSTABILITY

Genomic instability has been shown to be a hallmark of
tumorigenesis 227'929. These rearrangements include translocations,
deletions, duplications, and amplifications. Other alterations, . such as
polyploidy changes and chromosomal aberrations (dicentrics, double
minutes, fragments, rings, and chromatid breaks) are also frequently
observed in the process of tumorigenesis. These changes create a
heterogeneous population, allowing selective growth of a more malignant
cell and may be responsible for tumor progression 230. Cellular
protooncogenes, such as c-myc, as well as viral oncogenes, such as SV40
T antigen and adeno ElA, have been found to be able to drive karyotypic
abnormalities in cultured cells 231-236.

In particular, gene amplification seems to be most prevalent
genetic anomaly in metastasizing tumors 237. The occurrence of oncogene
amplification in a variety of human tumors underscores the important
role of gene amplification in carcinogenesis and tumor progression 938-942.

The amplification of the multiple drug resistance (MDR) gene or the

29

dihydrofolate reductase (DHFR) gene also diminishes the benefit of
chemotherapy with anticancer. drugs, such as methotrexate. Among the
amplified oncogenes, c-myc and neu (i.e., HER-2 or c-erbB-2) have been
demonstrated to be common in many primary human adenocarcinomas
arising at numerous sites including breast, ovaries, lung, stomach and
salivary gland 243446. Scattered cases of amplification of N -myc 247-248, L-
myc 249, c-myb 250’ c-Ki-ras 251-252, int.2 253-254, "17123 255, mm 256, and
EGF receptor 257 are also observed in various tumors. It has been
reported that oncogene amplification contributes to tumor progression
and a poor prognosis. Protooncogenes usually have functions in the
mitogenic pathway and are tightly regulated in normal cells; therefore,
the high degree overexpression of particular oncogenes as a result of
DNA amplification in tumors enhances the probability for emergence of

more malignant cells.

1.5. 1 Gene Amplification

Gene amplification is a normal widespread phenomenon in lower
eukaryotes during development 2584“. Rapid increases in gene
expression in particular cells or tissues are achieved by a programmed
dramatic increase in the copy number of certain genes. Examples include
the amplification of ribosomal genes during oogenesis in Xenopus laevis
and Tetrahymena, and the amplification of the chorion genes in
Drosophila.

Developmentally programmed gene amplification in mammals is
unknown. Furthermore, in normal diploid human and rodent cells, gene
amplification is not detectable by a clonogenic assay for resistance to

drugs (frequency = <10'9) 264-265. However, very rare examples of

3O

spontaneous gene amplification have been found in normal cells by
chromosome flow-sorting analysis 956 and Luria-Delbruck fluctuation
analyses 26536" in absence of selection pressure. Therefore, it is reasoned
that gene amplification is a random event within the cell population and
that it takes place spontaneously before the selection process 268-271. The
difficulty in detection is hypothesized to be due to the instability or to the
low copy number of the amplified genes 272.

In contrast, gene amplification oCcurs commonly in cultured cell
lines and in tumors 279475. In most tissue culture cells selected for drug-
resistance, the rate of amplification ranges from 10‘4 to 10'5 events/ per
cell per cell generation 238. Such a rate is much greater than the standard
mutation rate. Moreover, tumorigenic cell lines derived from rat,
hamster, and human, regardless of the type of tumor, exhibit rates of
gene amplification several orders of magnitude greater than their
nontumorigenic counterparts 230376-978. It is likely that a common
mechanism underlies this process and that amplification gives a growth
advantage to tumor cells 979. The presence of mitogenic substances
(hormones or tumor promoters) during selection 279-281 or the presence of
carcinogenic agents, cytotoxic agents or metabolic inhibitors before
selection 274381;?“ can even further enhance the frequency of gene
amplification. Trans-acting factors that stimulate gene amplification thus

may be involved 235.

1.5.2 Proposed Mechanisms of Gene Amplification
The most studied examples of gene amplification in cultured cell
lines are those selected for resistance to cytotoxic drugs, such as the

amplification of the dihydrofolate reductase (DHFR) gene, resulting in

31

methotrexate-resistance 286-287 and that of the CAD gene (encoding an
enzyme with carbamylphosphate synthetase, aspartate transcarbamylase
and dihydro-orotase activities), leading to N-(phosphonacetyl)-L-asparate
(i.e., PALM-resistance 988-289. Both encoded enzymes are important in
nucleotide metabolism 290492, providing substrates for DNA replication.
Numerous analyses of the cytogenetic and molecular characteristics ‘of
amplified sequences have been performed to gain an insight into the
mechanisms of amplification.

The analysis of the amplification end products for a given gene
shows substantial variation in the cytogenetic location, structure, as well
as in the number of amplified genes. Thus, it is thought that the earliest
amplification intermediates are unstable. Amplified regions are always
far larger than the selected gene, ranging in size from about 50 Kb to
more than 10Mb ”5:293. Amplified sequences are often organized as
extrachromosomal structures, or as arrays within chromosomes, the
former appearing to be unstable 293. The extrachromosomal structures
usually consist of paired, acentric chromatin bodies and are referred to
as double minute chromosomes (DMs) 294. DMs can integrate into
chromosomes 295. The chromosomal regions harboring amplified
sequences are designated as expanded chromosomal regions (ECRs) 293.
There is strong evidence that integration of DMs can generate ECRs 296-
297. In contrast, the breakdown of chromosomally amplified arrays either
does not occur, or occurs at frequencies too low to be measured 298-300.
The results also fit very well with the quantitative predictions of a
mathematical model based upon the assumption that the amplification
precursors are DMs and integrate over time 301. Therefore, the examples

of gene amplification in tissue culture systems suggest a process that

32

involves a unidirectional progression from populations dominated by
cells with extrachromosomal elements at an early time to those
dominated by cells with chromosomally amplified regions at later times.
In contrast, the analysis of biopsies from diverse human tumors
harboring amplified sequences reveals that DMs constitute the majority
(95%) of cases and ECRs are detected very rarely 302.

The molecular amplification unit (amplicon) of the expanded
chromosomal regions is known to be arranged as either direct tandem
repeats 303 or, more commonly, as a mixture of tandem and inverted
repeats 304-306. Extrachromosomal elements containing the CAD and ADA
(encoding adenosine deaminase) amplicons have now been shown to be
circular and to be organized as imperfect inverted repeats 30.7. Due to the
difficulty in isolating and investigating the initial intermediates of
amplification, mechanisms are proposed and evaluated based upon the
types and number of recombination events which would have to occur to
generate the observed amplification end products. The diversity of sizes,
structures, and locations of amplified sequences suggests that a number
of distinct mechanisms can operate in different situations or that a
primary mechanism can predominate and lead to a wide range
amplification structures. Three major groups of mechanisms have been
proposed : the rereplication model (also referred to as onion skin
replication and disproportionate replication) 274,308, the unequal exchange
model 309'310, and models involving acentric elements 297°98'13“.

In the rereplication model 308, the loss of control of DNA replication
within each .cell cycle accounts for the extensive and rapid increases in
gene copy number. ”Onion skin"-like structures are proposed to be

generated by multiple unscheduled initiations of DNA synthesis at a

33

single origin in a single S phase; the structures can create substrates for
recombination and can be resolved into a stable amplified region 205.312-
3". Cases in which this model has been proposed include the
developmentally programmed amplification of chorion genes in
Drosophila 263315315, the amplification of adenine phosphoribosyl
transferase-thymidine kinase genes transfected into mouse cells 313, the
overreplication of integrated SV40 in nonpermissive transformed cells.
following treatment with chemical carCinogens 317-318 or with Herpes
Simplex virus 319390, and the generatiOn of unintegrated circular polyoma
and SV40 genomic DNA from a single integrated linear viral genome 94-905
(Figure 5). The model is flexible enough to account for almost any type of
amplified structure, including extrachromosomal DNA 321.

The unequal exchange model 309 proposes that two misaligned
chromosomes or chromatids can undergo either homologous or
nonhomologous recombination and generate multiple gene copies in one
chromosome. Only one exchange is likely to occur in a single cell cycle,
and thus, the production of multiple gene copies requires numerous
independent recombination events. Support for this model comes from
the studies of the formation of tandem arrays of rRNA genes in
Drosophilia 3” and budding yeast 323-324. The model accounts for head-to-
tail tandem amplified structures but not structures organized as inverted
repeats. For amplification of very large regions of DNA, this model may be
most applicable 285.

The models involving acentric elements include the episome
excision model (1) 293, the double rolling circle model (2) 311, and the
chromosome breakage model (3) 300. In (1), amplification can result

simply from unequal segregation at mitosis. In (2), the first circle is

34

Figure 5. A model for amplification of mammalian DNA involving
unscheduled DNA overreplication and recombination

This picture is taken from Stark and Wahl 975. Bidirectional
replication at an origin generates a bubble that can undergo further
rounds of unscheduled DNA replication. The resulting structure is
mitotically unstable and tends to resolve into more stabilized products by

recombination.

35

 

_ REPLICATION V

sbcdgh.-°tgh

 

 

WSCHEDLLED REPLICATION '.
f"\

MITOTICALLY
UNSTABLE

K\
.@.

 

 

 

 

 

COO!

 

            

36

proposed to be a dimer organized as an inverted repeat. This DNA
molecule undergoes normal replication initiation from one of its two
potential origins to form a partially replicated circle. Homologous
recombination then takes place between one strand of the newly
replicated sequence and that of unreplicated sequence. Further
replication of the product leads to an array of inverted duplications with
multiple copies of the parental DNA. Up to now, no observations in .
mammalian cells support the predictions made according to this model.
In (3), the first step is the introduction of single-strand or double-strand
cleavages within a replication "bubble” which contains the target gene
and one or more adjacent replication origins. The breakage of DNA
results in a replication proficient acentric element and broken centric
chromosomes. Over multiple cell doublings, normal replication followed
by unequal segregation of an acentric element will amplify the target
gene to heterogeneous copies. The broken centric chromosomes may also
become terminal deletions or fuse to other chromosomes, creating
perpetual genomic instability mediated by bridge-breakage fusion cycles '
325. The finding that chromosome breakage is an early or initial event in
amplification of an DHFR gene 30°, implies that chrOmosome breakage

may be an important general mechanism for gene amplification 293.

1.5.3 Trans- and Cis-acting Elements of Gene Amplification
trans-Effects on gene amplification are more readily studied than
cis-effects. Three examples of trans-effects have been demonstrated.
First, various DNA damaging agents and treatments, including
hydroxyurea, aphidicolin, carcinogens, hypoxia, ultraviolet irradiation,

and ionizing radiation, have been shown to induce amplification 975.

37

Secondly, certain tumorigenic cells have higher rates of amplification
than their non-tumorigenic counterparts 230376477. Thirdly, cell lines with
an "amplificator phenotype" that amplify certain genes at an increased
frequency have been isolated 326427 and this phenotype is dominant upon
cell fusion 328. In contrast, using human-human somatic cell hybrids, it
has been demonstrated that the ability to amplify is a recessive genetic
trait 278. This suggests that normal mammalian cells contain a gene or.
genes in a pathway(s) regulating the ability to amplify endogenous genes
and loss or mutation of that gene(s) results in increased genomic
instability. .

The p53 gene, a cell cycle checkpoint regulator that arrests cells in
G1 phase 329-330, is known to be one determinant in a pathway controlling
amplification. Primary human and mouse diploid fibroblasts that have
lost wild-type p53 were shown to amplify a drug resistance gene at a high
frequency 331‘333. However, suppression of gene, amplification is not
coupled to suppression of tumorigenicity and therefore amplification and
tumorigenicity are under independent control 278.

c-myc may be another gene in the pathway regulating the ability to
amplify endogenous genes. The overexpression of transfected c-myc has
been found to stimulate DHFR gene amplification in established rat
embryo fibroblasts 334. Since there is increasing evidence that c-myc is
involved in DNA replication 335-337, Denis, et al. suggest that the c-myc
overexpression may lead to DNA over-replication 334.

There are also cis-acting elements capable of increasing the
frequency of gene amplification. In mammalian cell cultures, the
chromosomal location of transfected CAD genes has been found to affect
the frequency of CAD gene amplification up to 100-fold 338. Consistently,

38

in another study, the cytogenetic outcome and the stability of amplified
CAD genes are ascribed to their flanking sequences and nuclear
environment 339. cis-acting elements may interact with adjacent origins
or function as an autonomous origin of replication: For example, in
Dmsophila, cis-acting sequences have been shown to positively interact
with adjacent origins 34° and influence chorion gene amplification 341-343.
Other cis-acting sequences, possibly acting as recombination-promoting,
elements in gene amplification, have also been isolated. For Mple,
HSAG—l has been shown to promote the amplification of the DHFR
selectable vectors in a cell line derived from Chinese hamster ovary cells
344'345. HSAG-l is a repetitive mammalian genetic element and contains
Alu-like sequences, inverted repeats, and A/T-rich regions 344346. All of
these sequence characteristics have been identified in a "hot spot" for
recombination in the region of the adenylate deaminase gene 305.
Moreover, HSAG-l does not contain regions similar to the simian ori 347.
Thus, the effect of HSAG-l in gene amplification is not due to its function

as an autonomous origin of replication 348.

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CHAPTER 2

EFFECTS OF AMPLIFICATION AND EXCISION OF THE INTEGRATED
POLYOMAVIRUS GENOME ON ITS INTEGRATION PATTERN

Li-Jyun Syu and Michele M. Fluck

Department of Microbiology, Michigan State University,

E. Lansing, MI 48824-1101

75

76

Abstract :

Previous investigations have suggested that in most polyomavirus
(py) transformed cells, the viral genome is integrated at multiple sites in
the cellular genome. When we carried out similar integration site
analyses, but with higher resolution electrophoresis, we observed a
regular ladder-like pattern in multiple clonal cell lines derived frOm
infections with a wild—type strain. To determine the origin of the ladder,
ts—A type mutants (encoding a thermolabile large T-antigen) were used to
transform FR3T3 cells. Using temperature sensitivity to manipulate
competence for viral DNA replication, we were able to show that
replication converts a simple integration pattern (i.e., a single band
resulting from integration at a single site) into an apparently complex one
characterized by multiple bands with an interband distance
corresponding to the size of the Py genome. The amplification unit of
integrated viral DNA is a head-to-tail tandem repeat rather than an
inverted repeat. It is the heterogeneity in the number of head to tail
amplified genomes among different cells of a clonal population that

results in the deceivingly complex integration patterns.

77

Introduction :

The understanding of how a viral genome, such as that of
polyomavirus and simian virus 40, integrates into host chromosomes,
has been pursued mostly by the analysis of the integration pattern of
transformed tissue culture cells 3» 4. In such an analysis, high molecular
weight cellular DNA 28 is digested with an appropriate restriction
endonuclease, 'lfollowed by electrophoresis, Southern transfer and
hybridization with a labelled viral probe. Depending upon the position of
the restriction endonuclease recognition site in the viral genome, an
estimate of the minimum number of independent insertions and the
topology of integrated viral sequences are deduced. The integration
patterns vary from transformant to transformant. Hence it has been
suggested that the integration of Py and SV40 genomes is a random or
quasirandom process, occurring at no particular site in either the
cellular or viral genome. In related literature, most integration analyses
demonstrate apparently multiple integration sites with numbers
averaging over three in each transformant 13347312940. Rare
transformants harbor only one viral integration site. Thus, it is generally
believed that multiple sites are available in the host chromosomes for
integration.

Based upon this general belief, Oh et al. 33 assumed that each
integration event should happen independently in any transformant and
thus integratable viral genomes in the pool should have equal probability
of interaction with random sites available on the host chromosomes. The
expectation would be that within a single transformant, different Py
strains can be integrated at independent sites, with a probability

78

proportional to their ratio in the mixed infection. However, instead, Oh et
al observed a low probability (1 1%) of double integration events in
transformation and established the generality of this phenomenon in a
series of experiments that utilized mixed infections with two
distinguishable Py strains at different ratios. Consequently, it was
hypothesized that the number of sites in the host chromosome available
for integration is actually limited. .

lntriguingly, transformants with apparently multiple integration
sites usually exhibit the topology of a head-to-tail tandem arrangement of
the viral genomes at each site. Whereas, if only one viral integration site
is observed, less than one complete copy of the viral genome usually
comprises the integrated sequences 134.15. The requirement of a
functional large T-antigen 15 and the viral origin of replication 13 in the
formation of tandem repeats indicate an involvement of viral DNA
replication. The parallel existence of apparently multiple integration sites
and their head-to-tail tandem arrangements in integrated viral sequences
thus raises the possibility of a parallel requirement for viral DNA
replication in the emergence of apparent multiple integration sites
observed in the integration analysis. Several results have indeed
supported this correlation 15.28. For example, the majority (approximately
70%) of transformed cells derived from infections with a Py strain RA or
A2-(E / N RA), which contain mutation(s) strongly diminishing their ability
for viral DNA synthesis, are found to have viral integrations at a single
site in the host genome 25. Similarly, in reducing the apparent number of
integration sites, the mutations are also responsible for a diminution in

the incidence of tandem arrangements of the viral genome, although to a

79'

lesser extent 25. However, the involvement of viral DNA replication is not
simply in providing more viral templates for integration 26.

In this study, we reinterpreted the apparent multiple integration
sites revealed by restriction endonuclease analyses and tested whether
the ability of the integrated viral genomes to engage in DNA replication
would affect the apparent number of integration sites. Transformants
were derived from infections of nonpermissive rat cells with large T-
antigen temperature sensitive mutants, whose ability to replicate viral
DNA was manipulated by temperature shifts between permissive and
nonpermissive temperature. High resolution electrophoresis in 0.4%
agarose at 0.8 volt/cm for 45-48 hours was conducted to reveal the
apparent number of integation sites. For all transformants analyzed,
50% showed an integration pattern containing multiple bands organized
as a regular ladder. Moreover, the ladder would disappear and resolve
into a single band when viral DNA replication wasblocked for more than
16 cell generations. We suggest that the apparent multiple integration
sites do not reflect independent integration events. The post-integration
and replication-dependent events convert a simple integration pattern
into a more complex pattern, which misleads its interpretation. We also
demonstrated in this study that the number of integration sites per cell

for the Py genome is restricted to one or two.

Materials and Methods :
Cell lines and cell culture. The FR3T3 cell line was maintained in a
humidified incubator with 5% C02 at 37°C according to standard

culture technique. The medium for cell culture was Dulbecco's modified

80

Eagle's medium (DME) supplemented with 10% calf serum and
penicillin-streptomycin. The Py transformants were grown as monolayers
in the same media and passaged every 3 to 6 days at a 1:10 or 1:20
dilution.

Viruses. Wild type A2 24 and three different ts-A mutants, ts-a, ts-25-E,
and ts-616, were used to transform FRSTS cells. The mutation in ts-a
has been mapped to nucleotide 2193 (i.e., large T-antigen codon 547)
14.35. Mutant ts-25 E has been localized to nucleotide 2883, which is only
11 amino acid residues from the carboxyl terminus of large T-antigen
(i.e., large T-antigen codon 778) 14-35. Mutant ts-616 maps between the
Hha I sites spanning the region from nucleotides 2331 to 2992 39. These
point mutations affect the structural stability and result in a
thermolabile large T-antigen which turns over almost totally during a 4-

hr chase at the non-permissive temperature (39°C) 17.

Collection of Py temperature-sensitive mutants transformants. Prior
to infection, FRSTS cells were grown to near 100% confluency,
synchronized by starvation, and then released from the G0 phase of the
cell cycle by trypsin treatment and serum addition. This procedure. is
known to optimize transformation 9. Then cells were infected at a
multiplicity of infection (M.O.I.) of 5 plaque forming units (pfu) per cell.
Transformants were selected by two independent criteria: anchorage
independence and overgrowth of the monolayer. In the first criterion,
infected cells were plated in soft agar before the first cell division. In both

cases, infected cells were kept at the permissive temperature (33°C) until

81

individual transformed colonies or foci were seen, i.e., after two to three
weeks. Fifteen stably transformed colonies were isolated by growth in
agar. Seven transformants were isolated directly as focal overgrowths on
the monolayer. As soon as detectable, each individual clonal
transformant was split into two cell populations. One was propagated at
39°C, and the other at 33°C. After 12 or 15 days (19 and 12 generations)
for the respective populations, some cells of each population were shifted
to the other temperature for 10 or 6 days (8 or 10 generations). In the

end, four cell populations for each transformant were analyzed (Figure 1).

Integration analysis. Total cellular DNA was isolated from clonal
populations of transformed cells as previously described 31. 10 pg of DNA
from each sample was digested with an appropriate restriction
endonuclease at a concentration of 2-3U/ug for 6 to 18 hrs at the
appropriate temperature. The digested DNA was then precipitated,
resuspended and separated by agarose gel electrophoresis. For the
analysis of samples digested with restriction endonucleases that do not
cut the viral genomes (no-site enzymes), electrophoresis in 0.4% agarose
was used to achieve high resolution. The thickness of gels was 3.5 mm.
Electrophoresis was carried out at 0.8 volt/ cm for 45-48 hours in Tris-
acetate/EDTA electrophoresis buffer with circulation. The DNA was then
denatured in 0.5M NaOH/ 1.5M NaCl and transferred in 0.25M
NaOH/ 1.5 M NaCl to a Hybond—nylon membrane. The membrane was
then dried and exposed to 254 nm ultraviolet irradiation for 2 min to fix
the DNA. Hybridization was performed in 10% dextran sulfate/5X
ser/sx Denhardt's solution/0.5% (w/v) SDS at 65°C for 48-72 hours,

82

Figure 1. A flow chart of the procedure for isolation of transformants
derived from infections with Py temperature-sensitive mutants

12 days at 39°C is about the time for 19 cell generations; 6
days at 39°C is about the time for 10 cell generations; 15 days at 33°C is
about the time for 12 cell generations; 10 days at 33°C is about the time

for 8 cell generations.

83

 

| FR3T3 Cells |

l

I infected with a ts-a, c-2515 or ts-616 Py mutants J

 

 

 

 

I incubated at 33 0C for three weeks I

I

I transformants selected I

A

 

 

 

 

 

 

 

 

 

 

 

 

 

I half focus I per transformant I I half focus / per transformant I
I incubated at 39°C for 12 days I I incubated at 33 0C for 15 days I
| DNA harvested (39°C) DNA harvested (33° C) |

 

 

\ /

I some cells shifted to the other temperature

/ \r

I incubated at 39 °C for6 days | | incubated at 33°C for 10 days |

IDNA harvested (33 °C+39 °C)| IDNA harvested (39°C->33 °C)|

 

 

 

 

 

 

84

using 2-4 X 106 cpm of the labeled polyomavirus genomic probes per ml
of hybridization solution (0.1 ml/cm2). The probes were synthesized
using a random multiprime DNA labeling kit (Amersham Corp.) with
[32pl-dCTP and followed by column purification. The specific activity of
the probes was 2-4 x 109 cpm/pg. After hybridization, the filter was
washed and then autoradiographed. For each transformant, the two
populations permissive for viral DNA replication and the two other
populations nonpermissive for viral DNA replication were analyzed on the
same gel, but their resulting DNAs were transferred to two separate
Hybond-nylon membranes. Subsequently, hybridization was performed
with the same batch and same amount of viral probes. Most
autoradiographs were obtained with the same length of exposure time
(two weeks).

Dot blot amplification analysis: 5 ug DNA samples were applied to a
Hybond-nylon membrane, using a Schleicher 85 Schuell dot blot
apparatus. The DNA on the membrane was denatured, neutralized, fixed
and hybridized as described above. The blot was stripped with 0.1% SDS
and rehybridized with the mouse immunoglobulin uj gene as a DNA
internal control. The intensity of individul dots was measured directly by

a B-scanner (Ambis).

Results :
Integration patterns of Py wild-type transformants. To examine the
number of viral integration sites in polyoma transformants, a high

resolution integration analysis was carried out with a series of

85

independent Py wild-type transformants that were derived following
infection of FR3T3 Fischer rat cells. To reveal the number of integration
sites, cellular DNA was digested with an enzyme that does not cut the
viral genome. BglII was chosen because it generates the smallest possible
fragments. High resolution electrophoresis, followed by blotting and
hybridization, was carried out as described in materials and methods.
Only one of 12 transformants displayed a single band representing the
integrated genome. In contrast, a ladder-like pattern was obtained with
11 transformants (Figure 2). The ladders consisted of a base band with a
size ranging between 8 and 15 Kbp, a series of regularly spaced bands
(as many as ten) and a broad unresolved band at the gel entry limit. A
band corresponding to unintegrated supercoiled genomic viral DNA was
also observed. The distance between bands was approMately 5 Kb, i.e.,
about the size of the Py genome, and the band intensity decreased with
the increasing size of the bands. The consistency of the interband
distance within each individual ladder and between all ladders strongly
suggests that each band within a specific ladder does not represent a
unique and random integration site, but rather that the bands of a given
ladder are related to each other and generated by amplification.
Specifically, we propose that in a given transformant, the viral genome is
integrated in a single chromosomal site, and that each member of a
clonal cell population contains a different copy number of the viral

genome, which is generated by DNA replication (Figure 3).

The effect of blocking viral DNA replication on the integration

pattern. In order to test the proposed model, an experiment involving

86

Figure 2. Integration patterns of Py wild-type transformants

Py wild-type transformants were derived from infections. of
FR3T3 cells with a Py wild-type strain. Cells were propagated at 37°C. 10
ug of total cellular DNA were digested with restriction endonuclease BglII,
electrophoresed on a 0.4% agarose gel, transferred to a Hybond-nylon
membrane, and hybridized with polyoma genomic probes. A ladder-like
pattern was observed in 1 1 out of 12 transformants. Free viral DNA was
also apparent. Lane #9 displayed a pattern with a single band or a very
faint ladder, and no free viral DNA was seen. DNA molecular-weight size

marker is indicated at left.

87

>
b
K
4.
9

6.6 Kb >

Free viral DNA >

 

44Kb>

88

Figure 3. A model which illustrates the hypothesis

Polyoma viral genome is integrated in a single chromosomal
site. The topology is assumed as a partial head-to—tail tandem
arrangement. In a clonal cell population, each cell member contains a
different copy number of the viral genome in its integrated viral DNA
sequences. The cleavage sites for EcoRl, BglIl and BstEII in the integrated

viral genome and host genome are indicated.

89

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90

manipulation of viral DNA replication was designed; large T-antigen
temperature sensitive mutants were used. FRSTS transformants were
derived at the permissive temperature. For each transformant, four cell
populations were derived as described in material and methods and in
figure 1. One population was kept at the nonpermissive temperature
(39°C population), one kept at the permissive temperature (33°C
population), one was shifted after 12 generations at the permissive
temperature to the nonpermissive temperature for 10 generations (33°C
-9 39°C shift-up population), and one was shifted after 19 generations at
the nonpermissive temperature to the permissive temperature for 8
generations (39° —) 33° shift-down population). This procedure was
carried out for twenty-two independent transformants derived with three
different temperature sensitive mutants. Seven were derived as foci
overgrowing the monolayer and fifteen derived as anchorage-independent
colonies (see table 1). Total cellular DNA was isolated and the integration
pattern was determined by analysis with BglII (a no-site enzyme for Py).
The results for one set of four such populations from one specific
transformant (transformant #6; see table 1) are shown in figure 4. In the
39°C population, a single band of approximately 9 Kb was observed,
representing a single integration site. In contrast, in the 33°C
population, the apparent integration pattern was extremely complex,
with a ladder of bands spanning from 9 Kb to the gel entry limit. In the
gel entry limit and in the gel, broad bands which were unable to be
resolved or enter the gel represented even higher molecular-weight
integration products of the viral genome. A high intensity band

corresponding to supercoiled genomic viral DNA was also observed, due

Table 1. Summary of the Polyomavirus ts-A strain and selection
method used, as well as the production of ladder or free viral

DNA for each transformant isolated

 

Transformant #

lowdown-heater...

Strain
ts 25E
ts 616
ts 25E
ts 616
ts 25E
ts 25E
ts 616
ts 616
ts-a

ts25E
ts 25E
ts-a

ts 616
ts 25E
ts 25E
ts 25E
ts25E
ts 616
ts25E
ts 25E
ts 25E
ts 25E

Selection '1

>>>>>>w>>>~o>nnn~o>a>>>>

Ladder

yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
no

no,

no
no
no
no
no

Free viral DNA

yes
yes 1’
yes
yes
yes
yes
yes 0
yes ‘1
yes
yes
yes °
yes
yes
no
no
no
no
yes
no
no
yes
no

 

a: Two selection methods were applied in this study.

A: anchorage-independent growth colony

F: foci overgrowth of the monolayer
The size of free viral DNA is 4.3 Kb.
The size of free viral DNA is 5.0 Kb.
The size of free viral DNA is 3.6 Kb.
The size of free viral DNA is 4.3 Kb.

9.9.9.9?

92

Figure 4. The effect of a block in viral DNA replication on the integration
pattern

Transformant #6 was derived from an infection with a Py ts-
25E mutant and isolated as an anchorage-independent colony. Four
populations of transformant #6 were propagated at temperatures
indicated. 10 pg of total cellular DNA were digested with BglII and
analyzed as described in figure 2. The two populations permissive'for
viral DNA replication (39°C, 33°C —> 39°C) and the two populations
nonpermissive for viral DNA replication (33°C, 39°C —> 33°C) were
analyzed in the same gel. DNA was transferred onto two separate
Hybond-nylon membranes and hybridized with the same batch and same
amount of viral genomic probes. The detection of high molecular-weight
bands required two weeks exposure to X-ray films, reflecting low
representation of cells which reveal those bands in the population. The
arrow indicated between 6.6 Kb and 4.4 Kb of the DNA molecular-weight

size marker is the position of free viral DNA.

93

39 33
Temp (00) 39 T 33 T
33 39

23Kb> ‘

36Kb>

44Kb>

13Kb>
ZOKb>

 

94

to viral DNA replication in this p0pulation. The dark background,
masking the bands, may represent the heterogeneous migration (and
topolog) of viral DNA replication intermediates, because it disappeared
once the population was shifted to 39°C for 10 generations. However, in
the shift-up population, a residual ladder, composed of about 8 bands
with a base band identical to the only major band seen in the 39°C
population, was" still displayed. The band corresponding to unintegrated-
supercoiled genomic viral DNA was eliminated. The interband distance
was approximately the size of the Py genome (i.e., 5.3 Kb), which
suggests that the difference in composition of every band is an integral
copy number of the Py genomes. Possibly, replication events are followed
by recombination, generating a head-to-tail tandem arrangement of the
viral genome. at a specific integration site. Furthermore, the durability of
the ladder after shifting to the nonpermissive temperature, suggests that
the bands in the ladder represent more stabe structures than the viral
DNA replication intermediates, including the unintegrated viral genomes.
When the 39°C population was shifted to 33°C, the complex pattern,
very similar to the pattern seen in cells maintained at 33°C, was
generated. This result supports the model proposed, in which a pattern
with a single band can convert into a complex pattern with bands
comprised of multimeric viral genomes simply by viral DNA replication.
In the 33°C and 39°C —+ 33°C populations, an additional band was
observed between the base band and the second band. We hypothesize
that this band represents the unintegrated viral genome, such as the
concatenated form, because this band disappeared after cells were

incubated under conditions nonpermissive for viral DNA replication.

95

To examine the generality of this observation, 22 transformants
were analysed. In the 33°C populations (Figure 5a), 11 out of 22
transformants displayed an intense to moderate signal which did not
necessarily resolve into a pattern. The level of amplification varied from
transformant to transformant, with #1 being the highest and #11 the
lowest. There was a correlation between the amount of supercoiled free
viral DNA, the intensity of signal remaining in the loading well and of the
broad band in the gel entry limit, as well as the intensity of the
hybridization in the entire lane. In the transformants displaying high
degrees of amplification (#1 - 5), any potential pattern was masked. In
contrast, starting with transformant #6 (i.e., the transformant shown in
Figure 4), a decrease in the amount of free viral DNA was observed; one
strong major band was observed in addition to a few fainter bands of
higher molecular-weight. Identical major bands were observed in the
pattern of corresponding 39°C populations, which were never exposed to
the permissive temperature (Figure Sb). In these patterns of 39°C
population, residual ladders were also observed in addition to the major
band in 10 transformants, except #6, whose results have been presented
above. The band corresponding to free genomic viral DNA was not
displayed. The band of about 8 Kb shown in every transformant could be
a unknown contaminant, since the same band was never seen in other
analyses of the same samples. There were several possibilities to account
for the emergence of residual ladders. This will be addressed in
discussion. .

The ladder-like hybridization pattern was manifested more clearly

in 33° —9 39°C shift-up populations. The masking darkness, which

96

Figure 5a. Analysis of 33°C populations of 11 ts-A transformants, which
reveal a potential ladder-like pattern

Transformants were derived from infections with a ts-A Py
mutant and selected as either foci overgrowth over the monolayer or
anchorage-independent colonies. Four populations propagated at
different temperature conditions for each transformant were harvested as
described in the Materials and Methods. 10 ug of total cellular DNA were
digested with BglII and analyzed as described in Figure 2. The loading
order of each transformant is in parallel with the order of their
amplification potential, which was reflected in the darkness of individual
lanes. The arrow indicated between 6.6 Kb and 4.4 Kb of DNA molecular-

weight size marker is the position of free viral DNA.

9.4 Kb >

6.6 Kb >

4.4 Kb >

97

Temperature : 33°C

«34567891011

 

c--—~-

98

Figure 5b. Analysis of 39°C populations of the same 11 ts-A
transformants as in Figure 5a

10 ug of total cellular DNA were digested with BgIII and
analyzed as described in Figure 2.

99

Temperature : 39°C

1234567891011

32,? a:
n “ tm .I‘,
I C '

23 Kb>'

9.4 Kb> '

6.6 Kb >

 

4.4 Kb > -

100

represented heterogeneous replication intermediates, was eliminated
after cells were incubated for 10 cell generations at the nonpermissive
temperature (Figure 5c). In each ladder, the major band (i.e., base band)
is the same size as saw for the original integration site, suggesting that
the majority of the population contains the original integrated viral
genome. The intensity of the band in the ladder decreased in inverse
proportion to its size, suggesting that the cells with higher molecular-
weight integrated viral genomes are more rare in the population. The
correlation between molar yield of individual amplified tandem repeats
varied from transformant to transformant, reflecting that the probability
for amplification is different from transformant to transformant. Since
the band of higher molecular-weight contained more copies of head-to-
tail tandems of the viral genome in the same integration site, the
frequency of cells containing a specific size of integrated viral genome
should be derived from the ratio of : (band intensity/ sum of each band
intensity) / (the corresponding copy number of the viral genome) (e.g.,
assuming the base band contains one copy). The strong signal in the
broad band of gel entry limit suggests that multiple species of even larger
head-to-tail tandems are resolved by amplification.

Twenty-two transformants were isolated using three different
temperature sensitive mutants and two selection methods as described
in material and methods. However, the appearance of ladders did not
correlate with a specific mutant or selection method as summarized in
table 1. Transformants which did not display a ladder-like pattern, but

instead gave a single band, will be described below.

101

Figure 5c. Analysis of 33°C -—> 39°C populations of the same #1 - #7
(excluding #5) ts-A transformants as in Figure 5a

10 pg of total cellular DNA were digested with BglII and
analyzed as described in Figure 2.

102

Temperature : 33°C —9 39°C

<23 Kb
23 Kb>
.4 <94Kb
<6.6Kb
<44Kb
&4Kb>

6.6 Kb >

 

103

State of the viral genome in the ladder-er pattern. The
characteristics of the ladder strongly support the hypothesis that bands
are generated by viral DNA replication. To further examine the state of
the amplified viral genome, DNA from 39°C and 33°C -+ 39°C shift-up
populations of transformants #1 and #6 were analyzed with two no-site
enzymes, individually or together (Figure 6a). In the undigested DNA
from the shift-up population, all viral sequences comigrated with high
molecular-weight cellular DNA and no ladder-like species were produced.
Bglll and BstEII digests produced distinctive ladder patterns. In double
digests, the Bglll pattern appeared to be dominant. From the EtBr
staining pattern (Figure 6b), it is clear that the cellular DNA was cleaved
more frequently by Bglll than by BstEII and, from the double digests,
that both enzymes were active. The interband distance did not change
and the overall ladder was shifted up or down. Similar digestions were
also performed with cell lines #2, #3, #4, and similar results were
obtained (data not shown), except that no dominant BglII pattern was
obtained. Overall these results suggest that the ladder is generated by
integrated viral genomes rather than unintegrated viral multimers. In
addition, these results exclude the possibility of multiple independent
integration sites, and support the hopothesis that the ladder represents
multimeric forms of the integrated viral genomes originating in the same

chromosomal integration site.

Quantitation of amplification. Two transformants were chosen to
assess the degree of amplification : #1, i.e., the most capable of

amplification, and #6, moderately amplified. Comparisons between the

104

Figure 6a. Determination of the state of the viral genome in the ladder-
like pattern

Transformants #6 and #1 were chosen to be further studied.
10 pg of total cellular DNA were digested with Bglll, or BstEII, or with
both and analyzed as described in Figure 2. The undigested DNA loaded

in lane 1 in each transformant had been sheared slightly by vortexing.

105

Cell line 1

39

Cell line 6

9

39
T 3ST

33

39
BstEll

39

3 39 39

39

T39T 39

T
33
BstEll + Bglll

T39T39t39

Temp (0C)

33
BstEll + Bglll

33
U0 Bglll

33

33
SSE"

33
UD Bglll

33

 

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106

Figure 6b. EtBr staining pattern corresponding to the gel shown in Figure

6a

107

Cell line 6 Cell line]
39 39 39 39 39 39 39 39
t39t39139t 1‘391391391
33 33 33 33 33 33 33 33
up Bglll BstEll BstElI+Bglll uo Bglll BstEll BstEll+Bglll

 

 

 

 

 

23 Kb>

9.4Kb>

6-6Kb>

4-4 Kb>

108

set of four populations were carried out by DNA dot blot. Amplification
was normalized using the immunoglobulin pj gene (Figure 7). In
transformant #1, a 600-fold amplification of the viral genome was
observed following shift-down (i.e., comparing 39°C and 39°C —) 33°C
populations) over a time during which approximately 8 cell generations
occurred. In transformant #6, a 50-fold increase was observed in. a

similar comparison.

Stability of the original integration site. The kinetics of resolution of
the ladder to the original integration site were determined in cell line #6
(Figure 8a). The unintegrated viral genome completely disappeared after
three days of temperature shift-up. Its disappearance seems much faster
than that of any integrated viral genome. The diminution of the ladder
occurred in a step-wise fashion, starting with bands of high-molecular-
weight. The emergence of a homogeneous cell population containing the
unamplified genome at the original integration site (i.e., base band) takes
more than ten days (about 16 doubling times) of temperature shift-up.
The results suggest that without viral DNA replication, continual excision
takes place, leading to the most stabe structure.

The stability of the original integration site was examined with Cell
line #6 by shifting the temperature up and down at various time points
to manipulate the amplification and excision of integrated viral DNA
(Figure 8b). The reproducibility of the amplification and excision patterns
was demonstrated. In a comparison of the ladder generated during the
first three weeks incubation at 33°C with that generated during a

subsequent three weeks incubation at 33°C following an interim two

109

Figure 7. Quantitation of amplification

DNA blots of 5 pg uncleaved cellular DNA probed with whole
Py genome (A) and with the mouse immunoglobulin pj gene as a DNA
internal control are shown. The amplification at 33°C was quantitated
as 60 fold in transformant #6 and 950-fold in transformant #1 using the

39°C level as the basal level.

110

 

 

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111

Figure 8a. Kinetics of disappearance of the ladder-like pattern in
transformant #6

Cells were propagated at 33°C first then were shifted up to
39°C. During the temperature shift-up stage, cells from three time points
were isolated. 10 pg of total cellular DNA were digested with Bglll and

analyzed the same as described in Figure 2.

112

Temp (co) 33 33—939

Days 12 3 710

 

23 Kb>

9.4 Kb >

6.6 Kb >

Free Viral DNA >

 

113

Figure 8b. Reproducibility of the ladder-like pattern in transformant #6

39°C population was shifted down and up for given times .as
noted. Cells were isolated from the four temperature regimes.10 pg of
total cellular DNA were digested with Bglll and analyzed the same as
described in Figure 2.

114

(a) (b) (c)
33 39 33
Temp (0C) T 1‘ ‘l
39 (a) (b) (e)

Weeks 2 3 2 3

6.6 Kb >

Free Viral DNA >

 

115

weeks at 39°C, identical patterns were obtained. Similarly, the bands
observed during the interim two weeks at 39°C were remnants of the
ladder. No novel rearranged fragments were obtained. Collectively, the
results suggest that a very accurate amplification and excision of
integrated viral genomes occurs via homologous reciprocal
recombination. O
To further examine the stability of the original integration site,-
39°C p0pulations of transformant #1 and #6 were incubated at 39°C for
up to three months. Cell populations were analyzed at four time points.
At each time point, the same single band was observed (Figure 8c). From
these results it appears that ladders resolve into a single band upon long

cultivation at 39°C and that the original integration site is very stable.

Further examination of the number of integration sites by analyses
with a one-site or a two-site restriction endonuclease. Theoretically
two flanking bands should be obtained for each independent integration
site with a one or more-site restriction endonuclease digestion. We have
hypothesized that at least some bands in the ladder represent multimeric
forms of the integrated viral genome at the same integration site.
Therefore, we expected that those multimeric forms of the integrated viral
genome would show the same flanking sequences. Two populations from
cell line #6 were chosen to deduce the number of integration sites with
this point of view. In the 39°C population and the 33°C —> 39°C
population, a single band and a ladder were obtained, respectively, upon
digestion with Bglll (Figure 4). However, both populations showed simple

and identical flanking-region patterns upon digestion with Bgll, a one-

116

Figure 8c. Stability of original integration site in transformant #6 and #1
Cells were isolated from four time points of long-term

incubation at 39°C. 10 pg of total cellular DNA were digested with Bglll

and analyzed the same as described in Figure 2, except that the agarose

gel in electrophoresis was 0.7% instead of 0.4%.

117

Cell line 6. 1.

Temp (OC) 39 39

 

Days 31 65 80 92 17 33 67 94

23 Kb >1
----

9'4“”? a---

36Kb>

4.4 Kb >

118

site enzyme, or with XbaI, a two-site enzyme that cleaves at very closely
spaced sites (Figure 9). The tandem repeat-derived band (5.3 Kb)
observed in the 39°C population upon digestion with Bgll indicated that
the partial repeat of the original integrated viral genome centered around
the Bgll site. Whereas, the partial repeat did not encompass the Xbal
site, since no tandem repeat-derived band was observed. The appearance
of a tandem repeat-derived band in the 33°C —+ 39°C population upon-
digestion with Xbal indicated that the original integrated viral DNA had
generated more tandem repeats and created additional viral Xbal sites at
a time when the cells were incubated at 33°C. In Xbal digests, in
addition to tandem repeat-derived band, only two flanking region-derived
bands were displayed. In Bgll digests, only one flanking region-derived
band was detected; apparently the length of linked viral sequences at the
other viral-cellular junction was not sufficient to be detectably hybridized
by the viral probes. Overall, these results support our hypothesis that all
the bands in the ladder represent multimeric forms of integrated viral
DNA genomes at the same chromosomal site. The difference between
individual forms resides in the copy number of tandem repeats rather
than in the viral-cellular junctions.

For every transformant with a ladder pattern in digests with Bglll,
a similar analysis with the one-site enzyme EcoRI was carried out to
further examine the number of integration sites. Most commonly, two
flanking bands were obtained in the 39°C populations. However, in five
transformants (#2, #3, #4, #7, #8), one or two very faint bands were
observed in addition to two major flanking bands. In the 33°C —+ 39°C

population of those five transformants, identical patterns with the same

119

Figure 9. Analysis of the number of integration sites by using Bgll or
Xbal

10 pg of total cellular DNA from 39°C or 33°C —> 39°C
populations of transformant #6 were digested with Bgll (a one-site
enzyme) or Xbal (a two-site enzyme that cleaves at very closely spaced
sites) and analyzed. A 0.7% agarose gel was used in electrophoresis.

After electrophoresis, the gel was treated as decribed in Figure 2.

120

39 39
Temp (0o) 39 T 39 T
33 33

Bgll Xbal

 

23-Kb >

ale>
6.6Kb> ..
“U o

44Kb>

Z3Kb>
20Kb>

 

121

major flanking region-derived bands and the same very faint bands were
observed, except that the intensity of the faint bands was not the same.
The faint bands may not represent the flanking sequences of a second
integration site because of the inconsistency in intensity. However, they
may represent rearranged novel fragments in a very small subpopulation.
In summary, the examination of the number of integration sites with a
one-site enzyme for all the transformants with a ladder pattern suggests
that the apparent multiple bands do not represent multiple independent
integration sites and that the actual number of integration. sites is

restricted to one (or, less likely, two).

Analyses of the transformants without ladder integration patterns.
Eleven out of 22 transformants displayed the BglII pattern of a single
band even in the 33°C population as well as in other populations (Figure
10). According to previous data, we infer that the lack of a ladder-like
pattern reflects a deficiency either in viral DNA replication or homologous
recombination in these transformants.

A viral replication origin, including binding sites for large T-
antigen, and a functional large T-antigen are requirements for efficient
viral DNA repliciation in competent host cells. By restriction mapping
with BclI and Bgll, the fragment around the Py enhancer region of these
11 transformants was confirmed to contain the viral replication origin
(data not shown). By restriction mapping with MspI (a seven nucleotide
recognition site enzyme), seven transformants were shown to lack the
coding sequences for the carboxyl terminus of large T-antigen (data not

shown). However, four transformants (i.e., #12, #13, #18, #21) contained

122

Figure 10. Analysis of 33°C populations of 11 ts-A transformants, which
do not reveal any ladder-like pattern

Transformants were derived from infections with a ts-A Py
mutant and selected as either foci overgrowth over the monolayer or
anchorage-independent colonies. 10 ug of total cellular DNA from 33°C
populations was digested with Bglll and was analyzed as described in

Figure 2.

123

Temperature : 33°C

12 13 14 15 16 17 18 19 20 21 22

23 Kb>

 

4.4 Kb >

124

the complete coding sequences for large T-antigen. Their encoded large T-
antigen was functional in viral DNA replication, as evidenced in two
analyses. First, by the plasmid replication assay, in which a plasmid
containing the Py replication origin was transfected into an individual
transformant, these four transformants were shown to replicate
transfected plasmids (data not shown). Second, a small amount of free
viral DNA was observed in the Bglll pattern in the 33°C populations,

suggesting a functional large T-antigen.

Topology analyses: As we suggested previously, homologous
recombination necessarily occurs to resolve the amplified structures.
Therefore, a redundant sequence in the integrated viral DNA may be
prerequisite. The t0pology analyses presented below are in support of the
redundant sequence being one of the requirements in the formation of a
ladder-like pattern. Restriction maps of integrated ‘viral DNAs with Mspl
and a variety of one-cut enzymes were derived for individual 39°C
populations of 22 transformants. In those 11 transformants capable of
amplification, partial tandems (i.e., redundant sequences) were detected
(data not shown). At least one complete copy of coding sequences for
large, middle, small T-antigens was retained. Among these
transformants, seven transformants contained two copies of the viral
replication origin in their integrated viral genome. Whether two viral
replication origins have any advantage in amplification was not
investigated in this study. No complete copy of a viral genome was
detected in the other 11 transformants incapable of amplification (data

not shown). Overall, the results of a series of restriction analyses suggest

125

that the generation of amplified multimeric forms of integrated viral
genomes requires a viral replication origin and a directly repeated

sequence in the integrated viral genome.

Discussion :

In this study, we reinterpreted the integration pattern of apparent
multiple band,” which are usually revealed by integration analyses of
independent transformed cell lines when their isolated DNA is digested
with a no-site enzyme. The integration pattern obtained in this study
with 22 ts-A transformants, showed that 50% of the transformants after
propagation under conditions permissive for large T—antigen function
displayed apparent multiple bands of integration; these bands were
organized as a very regular ladder-like pattern when a higher resolution
electrophoresis was used in Southern blot analyses. In the ladder-like
pattern, bands were composed of multimeric forms of integrated viral
sequences originating from the same integration site. The multimeric
forms of integrated viral sequencesonly differ in the copy number of
tandem repeats, but contain the same flanking region sequences. The
varying number of tandem repeats were possibly generated by a process
of continuous amplification and excision of the integrated viral genome;
thus, the amplification and excision converted a transformed cell
population into one containing heterogeneous but related integrated viral
genomes. The data showed that the number of integration events
available on host chromosomes in each transformation event of rat cells

by polyomavirus was actually limited to one or two.

126

The primary integrated viral genome is usually destabilized when a
viral replication origin 34, a functional large-T antigen 1 (both required in
viral DNA replication) and a partial or full tandem arrangement 11 are
present in the integrated viral sequences. The destabilization usually
results in a high rate of excision or amplification of integrated viral
genomes 235.1%“ without disturbing the flanking sequences in the
integration site 113. Excision is defined as the removal of an integral
number of viral (genomes from the integration site and has given rise to
the production of free (unintegrated) viral genomes in a fraction of the
cell population at any given time 42. However, the accompanying
amplification, defined as the acquisition of new tandem repeats has only
been documented in a few derivative cell lines and the observed increase
is only one or two copies of tandems 10,11. Such a small extent of
amplification is indicated by one or two extra fragments of higher
molecular-weight in a no-site enzyme-derived integration pattern
obtained with derivative cell lines propagated under conditions
permissive for viral DNA replication. Botchan and Sambrook 5'36 have
ascribed the mechanism to the in situ unscheduled overreplication of the
integrated viral genome, followed by homologous recombination.)

In this study, we used ts-A mutants to transform the FR3T3 cells.
For ts-A mutants, elevated temperatures (39°C) can destabilize large T—
antigen, resulting in a cessation of viral DNA replication 19, as well as a
block in virus-mediated cell transformation 20. The single point mutation
in each ts-A mutant is not localized in the domains of Rb (the protein of
the retinoblastoma gene) binding, of immortalization, nor of nuclear

localization 1435. Other properties of the multifunctional large T-antigen

127

are not necessarily influenced by elevated temperature, including the
recombination-promoting activity, which is dissociated from DNA
replication 37.

Twenty-two ts-A transformants, four populations of which were
propagated under multiple temperature conditions, were isolated and
analyzed in detail. In results of integration analyses with a no-site
enzyme, 50% of the transformants showed more complicated patterns
when grown at 33°C than when grown at 39°C. The intensity of the
background hybridization which masked any possible pattern in 33°C
populations (Figure 5a) contrasted with a clear background and easily
recongizable patterns observed in wild-type Py transformants (Figure 2).
The hybridization background resulted from intermediates of very active
viral DNA replication. Hacker et al. have suggested that at 33°C, large T-
antigen and polymerase a/DNA primase complexes are more stable than
those at 37°C, accounting, in part, for greater viral DNA synthesis 26. In
one transformant (#6), a single band representing the original integration
site was obtained at 39°C, in contrast to the multiple bands of a ladder
obtained at 330C (Figure 4). The interband distance was approximately
the size of the Py genome and the intensity of bands decreased with
increasing size. An examination of the state of viral genomes in the
ladder (Figure 6) suggests that the multiple bands originated from
integrated rather than from unintegrated viral genomes. Furthermore,
the consistency of the ladder-like pattern obtained with different no-site
enzymes suggests that the multiple bands do not differ in the flanking
sequences associated with multiple sites of integration, but rather the

copy number of tandem repeats. In particular, the dominance of the Bglll

128

pattern in double digests was explicable only if the multiple bands
contain the same flanking sequences and both Bglll sites are located
within both BstEII sites. The probability for both Bglll sites to be located
within both BstEII sites in up to 10 different flanking sequences would be
very low. The real number of independent integration sites was further
examined by a series of Southern blot analyses with one-site enzymes .to
deduce the number of different viral-host junctions (Figure 9). These
results suggest that the viral-host junctions are preserved between the
apparent multiple bands in the ladder. The absence of any novel viral-
host junctions and the creation of full tandems around the Xbal site
suggest that the difference between the various bands in the ladder
resides in the integral copy number of Py genomes. In conclusion, the
multiple bands in the ladder should represent multimeric forms of the
integrated viral genomes at the same chromosomal site. The size
difference between the multiple forms is about the ‘size of the py genome;
thus the generation of full tandems implies that the amplification "unit”
is likely to be a head-to-tail tandem rather than an inverted duplication.
Inverted duplications have been suggested to be a general feature in one
model of gene amplification and have been demonstrated to be associated
with a number of independent amplifications of DNA containing the Myc
gene (16-50 copies/ per cell) and of DNA containing the CAD gene (30-200
copies/ per cell), which encodes a trifunctional enzyme catalyzing
reactions of the pyrimidine biosynthesis pathways 18.

The amplification of the integrated viral genomes, thus creating
extra tandem repeats, is dependent upon.viral DNA replication. However,

a residual ladder was observed in 39°C populations of 10 transformants.

129

The residual ladder was another line of evidence for the rapid
amplification of the integrated genomes. The transformants were isolated
as foci or colonies, which were developed after three weeks incubation of
the infected cells at 33°C. The exact time point for Py integration has not
been documented yet; whereas, it has been suggested to be as early as
within 20 hours after infection. Therefore, the amplification of the
integrated genOmes may have been initiated at a very early stage. The
39°C populations were isolated after incubation at 39°C for 19 cell
generations, which was not enough time to collapse the ladder as
suggested from the kinetic study (Figure 8a, 8b). Similarly, a residual
ladder was observed in 33°C —> 39°C populations of 11 transformants.
These populations were isolated after incubation at 39°C for 10 cell
generations, which was shorter than the time for isolation of 39°C
populations. Thus, not surprisingly, the residual ladder, a remnant of the
ladder generated at 33°C, was still observed. From’ the pattern, it is also
suggested that 10 cell generations is sufficient to dilute out the
unintegrated viral genomes which were generated from the excision of
the amplified integrated viral genomes. A less likely alternative
explanation is that, when cells were propagated at 39°C, the leakiness of
ts-A mutants, occasionally reported 21, or the unavoidable temperature
variations during cell passage, may be responsible for the partial
amplification of the integrated viral DNA.

The degree of amplification in transformants #6 and #1 was 50-fold
and 600-fold, respectively, in contrast to the very small degree of
amplification (an increase of one to two copies) found in the results of

Colantuoni et al. 10,11, Moreover, the profound ladder-like pattern

130

containing up to 10 bands has not been well recognized in previous
studies. The unresolved signals left in the well slot and gel entry limit
represent species of even higher molecular-weight amplified integrated
viral genomes. In our study, the generation of the ladder-like pattern as
an indication of extensive amplification and excision was a general
phenomenon rather than a specific case. We think the lack of- its
detection in previous studies is due to, at least in part, the lower
resolution electrophoresis, a less sensitive hybridization system, and
fewer transformants systematically studied to reveal an extensive
amplification event. In our analyses, it usually takes probes of 2-4 X 106
cpm/ ml hybridization buffer and two weeks exposure for a clear
autoradiograph. Moreover, there are requirements, which I will discuss
below, for amplification to occur.

Temperature shift does not always give rise to a simple correlation
between the necessity of LT-ag and the generation of multiple forms of
amplified integrated viral DNA as we had expected. However, as
discussed above, the ladder appears to derive from a single original
integration site and involve viral DNA replication. Data from the 11
transformants which display only one band in the no-site enzyme
integration analysis, further support the notion that the available sites
for Py integration are very limited. The reason for this restriction of
integration sites is not yet understood. The composition of integrated
viral genomes of every transformant has been analyzed in detail by a
series of restriction mappings. The difference between the ones
containing multiple forms and those containing only one form of

integrated viral genome resides in three general categories of factors.

131

First is whether the coding sequences are complete for a functional large
T-antigen; second is whether the integrated viral genome contains
tandem repetitive sequences. The third requirement is the viral
replication origin, which includes the binding sites for large T-antigen.
However, the viral replication origin exists in all integrated genomes of 22
transformants. In every one of those 11 transformants which have
ladder-like patterns, the coding sequences for large T—antigen are
complete and the partial tandem repeats exist; in some of them, two
replication origins are included in the original integrated viral genome. In
contrast, in each of the 11 transformants which have one-band patterns,
the complete viral genome is not observed. However, four transformants
contain a functional large T-antigen, as evidenced by the capacity to
support the replication of plasmids which contain the py replication
origin. Therefore, a functional large T-antigen is not the only requirement
for amplification, but also the viral repeats, which are a prerequisite for
homologous recombination.

Gene amplification of cellular oncogenes is one mechanism of
oncogene-activation and a hallmark of tumorigenesis in mammalian
cells. The amplification of drug resistance genes has also been
extensively studied and used as a model system. Two general structures
are usually observed in amplified sequences occurring in mammalian
DNA. Those are extrachromosomal structures such as double minute
chromosomes (DMs) ‘2 and expanded chromosomal regions (ECRs) 41
harboring either direct tandem repeats or a mixture of tandem and
inverted repeats. There is strong evidence that integration of DMs can

generate ECRs 3339. However, in either polyoma or SV40 transformed

132

cells, which provide another model system for amplification and excision,
no example of reintegration of free viral genOmes into the original
integration sites has ever been documented. When SV40 transformed
cells are superinfected with SV40, the DNA of the superinfecting virus
was found to be integrated into other chromosomal sites 5, as evidenced
by the novel new bands in the integration pattern. .
In our study, the reintegration of excised free viral genomes seems
highly unlikely. First, no detectable new integration site has been found
(Figure 8b,8c). However, the accuracy of this point has been reevaluated
in another study using subcloning to separate individual cells containing
different integration sites. Second, the site-specific reintegration into the
original integration site is even more unlikely, not only because of the
very low frequency for integration with such a low amount of free viral
genomes but also because of the generally accepted randomness of the
integration process. In Figure 5a, a much smaller intensity increase in
the supercoiled form of viral genomes compared to the intensity increase
of integrated viral genomes was observed. Most of the free viral genomes
arose from the continual excision from the integrated viral genomes, and
thus the excised free viral genome can continue to replicate as an extra
element only to a small extent. Therefore, the high degree of amplification
of head-to-tail tandems cannot be explained completely as reintegration
of free viral genomes; reintegration may only account for a very small
part of amplification. An alternative mechanism for the generation of
amplification and excision is unequal sister chromatid exchange (SCE).
Brown and Basilico 3 have observed elevated SCE frequencies in polyoma

transformed cells in the presence of large T-antigen. However,

133

theoretically, in one generation only one SCE can occur; thus it is
difficult to apply this model to the extensive amplification and excision in
our study, which showed in one transformant a 600-fold increase in the
Py signal within 8 cell generations. Besides, SCE cannot account for the
production of free viral genomes. The most appropriate mechanism
therefore is unscheduled DNA overreplication in one single S phase
followed by homologous recombination.

In mammalian chromosomes, the DNA replication of a given gene
is temporally regulated according to the transcriptional order of that
given gene in the cell cycle 235”. Therefore, individual replication origins
possibly have differential affinities for rate-limiting cellular replication
factors, and may be vulnerable to overreplication prior to mitosis to
different extents. Since the primary integration of Py or SV40 is usually
accompanied by a large deletion of cellular DNA and profound alterations
of the original integration structure, the chromatin structure,
methylation pattern, and transcriptional activity of flanking sequences
may consequently be changed 7. These changes may influence the
normal controls that prevent reinitation of the replicon in a single cycle.
Direct evidence has demonstrated that by chromosomal rearrangement,
a replication origin of dihydrofolate reductase can be activated,
presumably by deleting negative regulatory elements or by creating a
more favorable chromosomal milieu for initiation 30. DePamphilis
suggests that Py virus is capable of initiating multiple rounds of
replication per S phase by escaping the restriction(s) which applies to the
host cellular origin 16. If there are any trans-acting factors induced in

this process, a similar amplification of cellular genes may be expected. A

134

future study of dihydrofolate reductase (DHFR) gene amplification which
leads to Methotrexate-resistance or CAD (encoding for a trifunctional
enzyme) gene amplification which leads to PALA-resistance in Polyoma
transformed cell lines may help to identify the trans- or even the cis-

elements involved in the gene amplification process.

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mutations. J. Virol. 24:142-150.

CHAPTER 3

STUDIES ON AMPLIFICATION AND EXCISION OF THE INTEGRATED
POLYOMAVIRUS GENOME FROM SUBCLONAL CELL POPULATIONS

Li-Jyun Syu and Michele M. Fluck

Department of Microbiology, Michigan State University,

E. Lansing, MI 48824-1101

139

140

Abstract :

We recently demonstrated that continued amplification and
excision of the polyomavirus (Py) genome at a single integration site
changes a simple, single integration pattern into an apparently complex
ladder-like pattern. We have now extended our studies to subclonal cell
populations derived from two parental transformants, one of which has
been shown to be highly amplified in its integrated viral sequences and
the other, moderately amplified. Most of the subclones, which were
isolated under the nonpermissive condition for further amplification of
the viral genome, contain the original size of the integrated viral
sequences. It appears that during the propagation of subclones, the
excision of the Py genome from the integrated viral sequences continues
and that these excision events occurs via homologous recombination.
When the subclone, containing the original arrangement of the integrated
viral sequences, was incubated under permissive conditions, a
heterogeneity in the size of integrated viral sequences was generated in
the cell population. When cellular DNA was digested with an enzyme
which does not cleave the viral genome and analyzed via Southern blot
hybridization, the heterogeneity in the size of integrated viral sequences
resulted in a ladder-like pattern, which is identical to that of its parental
transformant. We have also shown from kinetic studies that the
generation of unintegrated viral genomes was faster than the
amplification of integrated viral sequences. The amplification and
excision of the integrated viral sequences occur in a rapid and precise
but step-wise manner with an increase or decrease of a unit length of the

Py genome at the original integration site.

141

Introduction:

In previous studies we demonstrated that the amplification and
excision of integrated Py sequences affected the interpretation of the
number of integration sites in integration analyses. We have proposed
that in most Py transformed cells, the multiple bands apparently
corresponding to multiple integration sites indeed represent various
forms of the integrated viral sequences generated at a single
chromosomal site. We have demonstrated that in transformants derived
from infections with a ts-A type Py mutant (encoding a thermolabile large
T-antigen) (5,6,9) that those various forms of integrated viral sequences
can be resolved into a single form and later be regenerated under
conditions in which the viral large T-antigen was either nonfunctional or
functional, respectively, in promoting viral DNA replication. Th'e various
forms of integrated viral sequences can be displayed as a ladder-like
pattern when the cellular DNA is digested with an enzyme which does
not cleave the viral genome (a no-site enzyme for the Py genome) and
analyzed by Southern blotting. The size difference between the distinct
bands in the ladder-like pattern is about the size of the Py genome. These
findings suggest that there exists a heterogeneity within a transformed
cell population regarding the copies of head-to-tail tandem repeats in the
integrated viral sequences. The heterogeneity is achieved by continual
amplification and excision of the integrated viral sequences at the
original integration site. To further address the mechanism underlying
the generation of heterogeneity, a subcloning strategy was adopted to

separate individual cells either by end dilution of the parental

142

transformant in individual wells or by isolating cell clones from soft agar.
Subclones were isolated at the nonpermissive temperature (39°C) to
prevent further rearrangements of the integrated viral sequences. In this
report, we present the integration analysis for these subclones and the
kinetics of the appearance and disappearance of the ladder-like pattern
from a subclone containing the original size of the integrated viral

SCQUCIICCS.

Results:
Isolation of subclones and analysis of their integration patterns

Two Py ts-A transformants, one of which has been shown in
previous studies to be highly amplified in its integrated viral sequences
(transformant #1) and the other one, moderately amplified (transformant
#6), were chosen to be subcloned in order to separate individual cells in
the population. Three subcloning experiments were conducted with both
transformants. Among these three, two subcloning experiments used the
cell populations grown at 33°C (conditions permissive for viral DNA
replication) for 12 cell generations. Under these conditions, the stabilized
structures as well as intermediates relating to with any amplification
could be detected. One subcloning experiment used the 33°C —> 39°C
cell populations, in which the cells had been first propagated under
condition permissive for 12 cell generations and then shifted to
conditions nonpermissive for viral DNA replication for 10 cell generations
to allow dilution of any intermediates of amplification of the viral genome.
All subclones were isolated at 39°C to prevent any further

rearrangements of the integrated viral sequences. In total, 36 subclones

143

were isolated from transformant #1, and 58 subclones were isolated from
transformant #6 (Table 1). When genomic DNAs of the subclones were
analyzed by Southern hybridization using a no-site enzyme for the Py
genome (Bglll), 60% displayed a single band corresponding to the size of
integrated viral sequences without any amplification (the basse band). In
three subclones from transformant #6, a single band comigrating withor
very close to the second, or third band of a ladder-like pattern was
displayed (Figure 1). All subclones with a band corresponding to the base
band and the subclones with a band corresponding to the second band
were shown to contain the same flanking sequences when the cellular
DNA was digested with either BclI or Bgll (Figure 2). Both Bell and Bgll
are enzymes which cleave the Py genome once. Thus, the difference in
size between the base and second bands of the ladder corresponds to one
copy of the Py genome. However, the subclone with a band comigrating
with or very close to the third band of the ladder displayed one additional
2 Kb Bell and one additional Bgll fragment in addition to flanking
fragments identical to those shown in the parental transformant and the
subclones with either the base or second hand of the ladder. Moreover,
following EcoRI digestion, a 9.5 Kb fragment was observed instead of the
5.3 Kb tandem band when the pattern was compared to that of the
parental transformant. This suggests that the integrated viral sequences
of this subclone contain an internal 2 Kb duplication, which includes the
Bgll and BclI sites but excludes the EcoRI site. The subclone displaying
the band with a size very close to the fourth band in the parental ladder
of transformant #1 was observed to contain a tandem repeat of the

region containing either Bgll or BclI sites, which was created due to the

144

Table 1. Summary of the number and percentage of subclones which
displayed the distinct band of the ladder pattern derived from

Bglll digestion

transformant #1 transformant #6 percentage

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Subclones 36 58

base band only ($1) 18 38 60%
second band only (SQ) O 2 . 2%
third band only (83) O 1 1%
fourth band only (S4) 1 O 1%
base plus second bands 2 8 11%
(31+ 82)

second plus third bands 1 O 1%
($2 + $3)

faint ladder O 3 3%
ladder (Sladder) 7 2 10%
internal deletion or 5 2 7%
rearrangement (Sn)

(band with novel size)

reintegration (81 + Sn) 2 2 4%

(base band plus novel band)

 

 

145

Figure 1. Analysis of Bglll integration pattern of the subclones isolated
from parental transformant #6

A Southern blot of BglII integration pattern of distinct
subclones, probed with Py whole genome, is shown. The integrated viral
sequences of parental transformant #6, containing ts-A strain (encoding
a thermolabile large T-antigen) of Py genome, have been shown to be
moderately amplified at permissive temperature. Subclones have been
isolated mainly by end dilution of the parental transformant in individual
wells and propagation at nonpermissive temperature to prevent further
rearrangements. The starting number of cells in each well after dilution
was monitored by the visible number of colonies observed at a stage as
early as possible. Only the wells containing a homogeneous cell
population originating from a single cell were randomly selected for
further studies.

Total cellular DNA was extracted from cells as previously
described. 10 ug of DNA from each sample were digested and analyzed
by Southern blot analysis. The electrophoresis of Bglll cleaved DNA was
done with 0.4% agarose gel. The labels for each subclone are abbreviated

as defined in Table 1.

 

5'31“.“
"I

82 Sm

Sl+2

Sl+n

 

51a

146

 

147

Figure 2. Analysis of the arrangement of integrated viral sequences of
the subclones, which displayed distinct Bglll integration patterns .as
shown in Figure 1

Southern blots of Bell, Bgll and EcoRI restriction patterns of
distinct subclones, probed with Py whole genome, are shown. BclI, Bgll
and EcoRI all cleave once in the Py genome. The labels for each subclone
are abbreviated as defined in Table 1. P shown in the first lane of each
restriction pattern represents the parent, as a control. The parent used is
the 39°C population, which has been incubated at 39°C for 19
generations. There was no detection of any amplification of the integrated
viral sequences in that cell population. The electrophoresis was done

with 0.7 % instead of 0.4 % agarose gel.

148

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nxmdv

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2m 2m a

:5

N+~m mm

NW Dfim

:om

 

2m a

149

amplification and did not exist in the integrated viral sequences of the
subclones displaying the base band (Figure 3). However, the Bgll and Bell
tandem repeats were not contributed by four copies of the Py genome in
its integrated viral DNA sequences. The one or two additional bands
shown in the Bell or Bgll pattern, respectively, may be evidence of an
internal duplication or rearrangement besides amplification. -
Aside from the 64% of the subclones which displayed a single band
of the ladder, 12% of the subclones displayed two bands corresponding
to either base and second bands or second and third bands (Table l). The
intensity of these two bands varied. The data suggest that within a cell
population, the dynamic changes between integrated viral sequences
from higher to lower molecular-weight occur constantly, even at 39°C.
The ladder-like pattern seen in the subclones (Figure 1, Figure 4) may
also be explained as a heterogeneity generated from excision 'of the Py
genome from a highly amplified species of integrated viral sequences
which were contained in the single cell when propagation of the
subclones was started within an individual well. This interpretation
appears to be the best explanation rather than the one which attributes
the heterogeneity to the amplification from a single species of integrated
viral sequences (i.e. a single cell), since at 39°C there was no viral DNA
replication unless the mutation of large T-antigen was leaky.
Consistently, all the subclones containing the ladder patterns were
derived from the parental transformants which had been incubated at
33°C for one month and contained highly amplified species of integrated

viral sequences.

150

Figure 3. Analysis of the topology of integrated viral sequences of the
subclones which displayed fourth band of the parental Bglll ladder
pattern, in comparison with the one displaying the base band

Southern blots of Bglll, Bell, and Bgll restriction patterns,
probed with Py whole genome, are shown. The subclones shown in this
figure have been isolated from parental transformant #1, whose
integrated viral sequences have been shown to be highly amplified in the
condition permissive for viral DNA replication. The labels for each
subclone are abbreviated as defined in Table l. P shown in the first lane
of each pattern represents the parent, as a control. The parent was the
33°C —> 39°C population, which has been incubated at 33°C for 12
generations first, then shifted to 39°C for 10 genetations. There was
heterogeneity in the size of the integrated viral sequences in the parental

cell population.

151

Bglll Bcll Bgll

P S1 54 p 51.1 S1b 34 p 31:. Slb S4

 

23Kb>

 

53Kb>
95Kb>

44Kb> ‘

 

152

Figure 4. Analysis of Bglll integration pattern of the subclones isolated
from parental transformant #1

A Southern blot of Bglll integration pattern, probed with Py
whole genome, is shown. Parental ladder derived from 33°C —> 39°C
population is also shown. The labels for each subclone are abbreviated as

defined in Table 1.

23Kb >

9.4Kb>

153

Sladder Snl

Sn2 Sn3 Sn4 p

 

154

7% of the subclones displayed a single band of novel size (Figure
4). Because of the lack of detailed restriction maps, it is not known
whether they contain flanking fragments identical to those of their
parental transformants. Thus, the novel size may be due to an internal
deletion, internal amplification or rearrangement of the integrated viral
sequences. There were more subclones displaying more than one band, a
ladder pattern, or a band with novel size among the subclones derived
from transformant #1 than transformant #6. This probably indicated that
transformant #1 contained more highly amplified species of integrated
viral sequences. The heterogeneity of viral species within that cell
population was more complex than that found in subclones of
transformant #6.

4% of the subclones displayed a band of novel size in addition to a
band with the size of the base band (Figure 1). The intensity of the novel
band was either equal or much weaker than that of the base hand. These
subclones also displayed novel flanking sequences in addition to the
flanking sequences identical to their parental transformant (Figure 5).
Thus, these subclones may reflect a reintegration event into a new
integration site, although an internal amplification may result in a
similar alteration of the pattern. Nonetheless, these data suggest that the

maximal occurrence of reintegration is 4%.

Kinetics of appearance and disappearance of high molecular-weight
amplified species of integrated viral sequences
One subclone, displaying the band corresponding to the base band

of the parental ladder of transformant #1, was selected to determine the

155

Figure 5. Analysis of the arrangement of the integrated viral sequences
in the subclone which displays a base band and a novel band

Southern blots of Bgll and BamHI restriction patterns, probed
with Py whole genome, are shown. Bgll and BamHI cleave once and
twice, respectively, in the Py genome. S1+n shows a novel band in both

restriction patterns.

156

nove|>

53Kb>

 

 

157

kinetics of appearance and disappearance of high molecular-weight
amplified species of integrated viral sequences, following temperature
switches between the permissive (33°C) and nonpermissive (39°C)
conditions for viral DNA replication (Figure 6, Figure 7). Figure 6 shows
that the amplification of the viral genome was initiated within the first
cell generation at 33°C. The first indication of amplification was the
appearance of a band corresponding to the size of unintegrated viral
DNA. This suggests that the generation of unintegrated viral DNA is more
rapid than the appearance of amplified species of integrated viral
sequences. The intensity of unintegrated viral DNA increased as a
function of the time of incubation at 33°C from day 1 to day 9 until an
equilibrium was reached. The distribution of amplified species of
integrated viral sequences also followed a similar tendency; band
intensity was shifted from the lower to the higher molecular-weight
bands as a function of the time of incubation. These results suggest that
in the population, the number of cells containing the original size of the
integrated viral sequences decreases; cells gradually contain more and
more copies of the Py genome in their integrated viral sequences due to
the amplification. Within 5 days, which represents 4 generations, cells
containing five copies of the Py genome at the integration site appeared.
These results support the in situ replication model proposed by Botchan
and Sambrook (2,10) as the most likely mechanism for the generation of
the amplified species of integrated viral sequences.

After incubation at 33°C for 9 days, the cells displayed
heterogeneity in the size of integrated viral sequences at the same

integration site. The heterogeneity was displayed as a band-loss

158

Figure 6. Kinetics of appearance of the ladder

A Southern blot of Bglll-cleaved DNA from subclone S1. is
shown. Cells from the subclone population were incubated at permissive
temperature and DNA was collected every day up to day 9. The
amplification of viral DNA was initiated within the first cell generation in
the permissive condition. The 5.3 Kb band of viral genome size was
generated in parallel with bands corresponding to the amplified species
of integrated viral sequences. The molecular weight of amplified viral

DNA increased gradually along the time course.

159

1.81

33

Temp (OC)

 

23456789

1

0

Days

>
A
N
D

Ira

Free V

 

160

Figure 7. Kinetics of disappearance of the ladder

A Southern blot of Bglll-cleaved DNA is shown. The population
of cells cultivated at 33°C for 9 days was shifted to 390C and DNA was
harvested every other day. The disappearance of highly amplified species
of integrated viral sequences was quite rapid. Within 15 days, only the

band of original size of integrated viral sequences was left.

 

161
1.81

Temp (co) 33 33 —> 39

Days 91358101215
“9"” "'

1 'i— ’
(- L.

i

 

 
  
 

23 Kb>,

9.4 Kb >

6.6 Kb >

Free Viral DNA >

4.4 Kb >

162

hybridization pattern of high intensity (Figure 7 ). After incubation at
39°C for three days, a clear ladder pattern was regenerated. Bands of
intermediate sizes probably representing replication intermediates
disappeared much faster than bands corresponding to the integrated
viral sequences containing amplification with integral copies of the Py
genome. The complete disappearance of the unintegrated viral genomes
also took place faster than the complete disappearance of the ladder
pattern. The simplification of the ladder pattern occurred in a step-wise
fashion from the higher to the lower molecular-weight species and it took
more than 24 generations to reach the most simple pattern, a single
band. The most stable species correspond to the original structure of the
integrated viral sequences, containing only a partial tandem repeat of the
Py genome. The removal of integral copies of the Py genome must occur
in a very precise manner involving homologous recombination. In a single
cell generation, only one homologous recombination can possibly occur.
Thus, the amplified species of the integrated viral sequences can contain,

at a minimum, 24 integral copies of the Py genome.

Stability of the ladder

The subclone displaying the band corresponding to the base band
of the parental ladder of transformant #1, was expanded at 33°C for 65
days (Figure 8). The heterogeneity in the cell population reached a steady
state and displayed a complete ladder at 10 days incubation. The steady
state was maintained up to 65 days. The ladder patterns observed in
samples Collected from each time point were exactly the same. It appears

that an equilibrium was established between amplification and excision

163

Figure 8. Stability of the ladder

A Southern blot of Bglll-cleaved DNA from a long term 33°C
kinetics experiment is shown. A steady state in the ladder-like pattern
was reached before 10 days of growth at 33°C (see Figure 6) and was

maintained stably through 65 days.

164

1.81

Temp (0C) 33 33 —> 39

 

Days 0 1o 17 34 47 58 65 12

9.4 Kb >

6.6 Kb >

Free Viral DNA >

 

4.4Kb>" .

165

of the integrated viral sequences at 33°C. These kinds of dynamic
changes within the cell population further support the involvement of
homologous recombination in this process. The alteration in the size of
integrated viral sequences follows a precise fashion, with either an
increase or decrease in the copy number of the Py genome. Once the
temperature was shifted to 39°C, all of the highly amplified species. of
integrated viral sequences disappeared, suggesting that at 39°C, the

excision of integrated viral sequences is the only process which occurs.

Conclusion:

In this study, we have dissected the heterogeneity in banding
patterns of Py seqquences occurring at the original integration site due to
the amplification and excision of the integrated viral sequences within a
transformed cell population. Before initiating the subcloning
experiments, we had thought it should be possible to obtain mostly
subclones which contained different amplified species of the integrated
viral sequences; each one of them would correspond to the individual
band of the ladder-like pattern. The results of our previous studies have
shown that all the amplified species of the integrated viral sequences are
stabilized structures with head-to-tail tandem repeats of the Py genome.
We thus expected to isolate the stabilized structures, if the isolation
procedure was performed at the nonpermissive temperature to prevent
further rearrangements of the integrated viral sequences. However, 60%
of the subclones were observed to contain the original size of the
integrated viral sequences. This result did not indicate that in the

parental transformant, 60% of the cell population contained the original

166

size of the integrated viral sequences without any heterogeneity. Rather,
it suggests that the excision took place predominantly at 39°C and all
the amplified species have undergone dynamic changes which result in
formation of the most stable structure of the original size of the
integrated viral sequences.

The in situ amplification event is controled by large T-antigen
because of its ‘1 required prerequisite .role in viral DNA replication.
However, relative to amplification, the excision of the Py genome from the
amplified species of integrated viral sequences is relatively large T-
antigen independent. According to St-Onge and Bastin (11), the
recombination-promoting activity of large T-antigen is dissociated from
its function in DNA replication. Therefore, the elevated temperature may
inactivate the function of large T-antigen in viral DNA replication but not
its function in homologous recombination. Moreover, the Py genome has
been shown to be highly recombinogenic (4). Thus, possibly, even without
the involvement of large T-antigen, the Py genome may have a high
probability to recombine with its amplified copies within homologous
regions.

We also demonstrate that the heterogeneity in the integrated viral
sequences can be regenerated from the subclone which contains the
most stable structure of integrated viral sequences. The most stable
structure of integrated viral sequences is the one with the minimal size of
the partial tandem repeat of the Py genome without any amplification. In
the kinetics study, we show that the generation or dilution of the
unintegrated viral genome is much faster than that of amplified species

of integrated viral sequences. The amplification and extision of the

167

integrated viral genome occurs in a very rapid, precise, and step-wise
manner with an increase or decrease of a unit length of Py genome at the
original integration site.

Overall, an insight into the mechanism underlying the
amplification and excision of the integrated viral sequences has been
obtained. This may indicate that this is a valuable model system in
understanding gene amplification, which is a normal widespread"
phenomenon in lower eukaryotes (3,7) during development, and it occurs

in a variety of human tumors (e.g. myc, ras, neu) (1,8).

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