Cell, Vol. 27, 299-308,
`
`December
`
`1981
`
`(Part 1). Copyright
`
`0 1981 by MIT
`
`Expression
`Enhanced
`
`Is
`of a j&Globin Gene
`by Remote SV40 DNA Sequences
`
`Rusconi
`
`and
`
`Sandro
`
`Julian Banerji,
`Walter Schaffner
`lnstitut
`fur Molekularbiologie
`Universitat
`Zurich
`Honggerberg,
`CH-8093
`
`II
`
`Zurich, Switzerland
`
`Summary
`
`of a
`expression
`transient
`the
`studied
`We have
`intro-
`/31 gene
`after
`its
`cloned
`rabbit
`hemoglobin
`duction
`into HeLa cells. Two and one-half
`days after
`transfection
`using
`the
`calcium
`phosphate
`tech-
`nique, we extracted
`RNA
`from
`the entire
`cell popu-
`lation
`and analyzed
`it by
`the Sl nuclease
`hybridi-
`zation
`assay.
`Transcripts
`were
`barely
`detectable
`when &globin
`gene-plasmid
`recombinants
`were
`used. However,
`200
`times more &globin
`gene
`tran-
`scripts were
`found when
`the &lobin
`gene
`recom-
`binants
`also contained
`SV40 DNA, and 90% of these
`transcripts
`(about
`1000
`per cell) had
`the same 5’
`end as authentic
`rabbit
`globin mRNA.
`In the
`latter
`case, abundant
`production
`of @-globin
`protein
`was
`readily
`detected
`in a fraction
`of transfected
`cells by
`immunofluorescent
`staining.
`Enhancement
`of glo-
`bin gene
`expression
`was dependent
`on SV40
`se-
`quences
`acting
`in cis, but
`independent
`of
`the viral
`origin
`of DNA
`replication.
`The enhancing
`activity
`was associated
`with
`the 72 bp repeated
`sequence
`element
`located
`at
`the beginning
`of
`the viral
`late
`gene
`region.
`Viral DNA
`fragments
`containing
`the
`transcriptional
`enhancer
`element
`could act in either
`orientation
`at many
`positions,
`including
`1400
`bp
`upstream
`or 3300
`bp downstream
`from
`the
`tran-
`scription
`initiation
`site of the
`rabbit P-globin
`gene.
`These
`studies
`define
`a class of DNA elements
`with
`a mode
`of action
`that
`has not been
`heretofore
`described.
`The activation
`of genes
`by specific
`en-
`hancer
`elements
`seems
`to be a widespread
`mech-
`anism
`that may be used
`for
`the
`regulation
`of gene
`expression.
`
`Introduction
`
`of
`transcription
`for
`requirements
`The DNA sequence
`II in vivo are
`by RNA polymerase
`eucaryotic
`genes
`and Birnstiel,
`studied
`(Grosschedl
`being
`intensively
`1980,
`1981;
`Benoist
`and Chambon,
`1980a,
`1980b;
`Gluzman
`et al., 1980; Dierks et al., 1981a,
`1981 b;
`Faye et al., 1981; Gruss et al., 1981; Guarente
`and
`Ptashne,
`1981; Mellon et al., 1981). Most of the DNA
`important
`for efficient
`transcription
`in vivo seems
`to
`be present
`within
`some 100 bp upstream
`from
`the
`transcription
`initiation
`site. For at least some genes,
`sequences
`far more
`than 100 bp upstream
`from
`the
`initiation
`site were
`found
`to influence
`transcription
`in
`vivo. Grosschedl
`and Birnstiel
`(1980b)
`identified
`a
`“modulator”
`of
`transcription,
`a segment
`of DNA
`in
`
`its
`for
`is required
`that
`gene
`front of an H2A histone
`of the early genes
`efficient
`transcription.
`Transcription
`around
`200 bp
`of SV40 depends
`on DNA sequences
`upstream
`from
`the
`initiation
`sites,
`in a region
`of two
`directly
`repeated
`72 bp sequence motifs
`(Benoist
`and
`Chambon,
`1981; Gruss et al., 1981; M. Fromm and
`P. Berg, personal
`communication).
`We show a 200-fold
`increase
`in the level of correctly
`initiated
`transcripts
`from a rabbit P-globin
`gene when
`it is linked
`to SV40 DNA. The DNA segment
`that was
`found
`to enhance
`the expression
`of the P-globin
`gene,
`for convenience
`referred
`to as
`the enhancer,
`was
`found
`to be associated
`with
`the 72 bp repeated
`se-
`quence motif of SV40 mentioned
`above. Most
`inter-
`estingly,
`the viral
`“enhancer”
`can act over very
`long
`distances,
`and
`independent
`of
`its orientation.
`Thus
`the 72 bp
`repeat
`region
`does not act solely as an
`upstream
`promoter
`component
`of
`the SV40
`early
`genes,
`as could be inferred
`from
`the work of Benoist
`and Chambon
`(1981)
`and Gruss et al. (1981).
`Apart
`from
`its biological
`significance,
`the enhancer
`phenom-
`enon
`can also be exploited
`for
`the construction
`of
`high-level
`expression
`vectors
`for mammalian
`cells.
`
`Results
`
`Gene
`
`of the Rabbit &Globin
`
`Expression
`Transient
`in HeLa Cells
`p-
`hemoglobin
`of a cloned
`We
`tested
`transcription
`et al., 1978;
`rabbit
`(Maniatis
`chain
`gene
`from
`the
`obtained
`from T. Maniatis)
`using a transient
`expres-
`sion assay
`in HeLa
`cells. Subconfluent
`cell mono-
`layers were
`transfected
`with recombinant
`plasmids
`by
`a modification
`of the calcium
`phosphate
`coprecipita-
`tion
`technique
`described
`by Wigler et al. (1978).
`The
`recombinants
`contained
`a 4.7
`kb
`long
`segment
`of
`rabbit chromosomal
`DNA encompassing
`the /31 -globin
`gene
`(Figure
`1 A). RNA was extracted
`from
`the trans-
`fected HeLa cells after 2% days
`(60 hr) and analyzed
`by
`the Sl
`nuclease
`hybridization
`assay
`(Berk
`and
`Sharp,
`1977; Weaver
`and Weissmann,
`1979).
`Effi-
`cient globin
`gene
`transcription
`was observed with
`the
`clone
`pSVK+,
`a recombinant
`in which
`the 4700
`bp
`Kpn
`I fragment with the rabbit P-globin
`gene had been
`inserted
`into
`the Kpn
`I site of SV40 DNA
`in an SV40-
`pBR322
`recombinant
`(Figures
`1 B and 1 C). Most of
`these
`transcripts
`had a 5’ end
`indistinguishable
`from
`that of authentic
`rabbit
`/3-globin mRNA
`(Figures
`28
`and 2C lanes 1 and 2). Transfection
`with
`the plasmid
`p/32 X, a recombinant
`of the same size with
`two globin
`genes but no SV40 DNA (Figure
`1 F), resulted
`in only
`0.5% of the
`level of /I-globin
`gene
`transcripts
`(Figures
`28 and 2C
`lanes 3) as compared
`with
`transfection
`with
`the pSVK+
`clone. By comparing
`the high
`levels
`of
`transcripts
`from pSVK+
`with
`the
`/3-globin mRNA
`standard, we estimated
`that
`there were 1000
`to 1500
`correct globin-gene
`transcripts
`per cell.
`If we consider
`a transfection
`efficiency
`of 10%-l
`5%.
`this means
`that
`
`Novartis Ex. 2009
`Page 1 of 10
`
`

`

`Cell
`300
`
`there were
`cell
`transfected
`successfully
`in every
`Equally high
`5,000
`to 10,000
`globin-gene
`transcripts.
`levels of correct
`transcripts
`(Figure
`2D) were obtained
`with a derivative
`of
`the p/32x
`plasmid,
`designated
`ppSV(-I/?,
`which had a complete
`copy of SV40 DNA
`inserted
`between
`the two globin genes
`(see
`legend
`to
`Figure
`1 F).
`There
`exists
`
`the
`
`formal
`
`possibility
`
`that
`
`the globin
`
`tran-
`longer
`from
`in HeLa cells was generated
`RNA
`scripts by nucleolytic
`cleavage
`at the cap site. How-
`ever,
`there
`is good evidence
`for eucaryotic
`genes
`in
`general,
`and
`for mammalian
`,&-globin genes
`in partic-
`ular,
`that the 5’ ends of messenger
`RNAs are the sites
`of transcription
`initiation
`(Contreras
`and Fiers, 1981;
`Grosveld
`et al., 1981 a; Hofer
`and Darnell,
`1981),
`findings which are supported
`by in vitro
`transcription
`
`cao --B---+
`
`D
`
`EcoRI
`
`SV-ori
`
`Kpn
`
`I
`
`Kpn I
`
`plasmid-DNA
`c
`SV40-DNA
`
`4
`
`rabbit-DNA
`
`(Maniatis
`arrows:
`
`coding
`DNA. Solid bars:
`line: noncoding
`Solid
`et al., 1978).
`restriction
`sites where SV40
`sequences
`were
`integrated
`and
`
`Gene
`the Rabbit &Globin
`DNAs Containing
`Figure 1. Recombinant
`(A) Schematic
`representation
`of the genomic
`rabbit
`hemoglobin
`fll gene
`sequences.
`Open bars:
`intervening
`sequences
`(IVSl
`and
`IVS2). Vertical
`were
`found
`to enhance
`globin gene expression
`(see
`text).
`long; nucleotide
`is 5243 bp
`The DNA
`recombinants.
`of our
`the construction
`for
`(B) Map of SV40 DNA with some of
`the restriction
`sites used
`to Appendix
`A of Tooze
`(1980).
`fashion
`according
`origin
`in a clockwise
`positions mentioned
`in the
`text are
`the distances
`from
`the replication
`(C) Map of pSVK+;
`the 4.7 kb Kpn
`I globin gene
`fragment
`was cloned
`into
`the single Kpn
`I site
`found
`in the SV40
`late
`region of the previously
`constructed
`recombinant
`plasmid
`pBSV-early
`(Schaffner.
`1980). The globin gene was also cloned
`in the opposite
`orientation
`into pBSV-early,
`yielding
`the recombinant
`pSVK-.
`(D) Map of pi-l
`lfi-;
`the 4.7 kb globin gene
`replication
`(Gluzman
`et al., 1980).
`integrated
`of SV40 containing
`Ill C fragment
`(E) Map of pSVHin+K+;
`the Hind
`Procedures).
`the plasmid
`pJC-1
`(pJC-1
`is a derivative
`of pBR322;
`see Experimental
`1981, where
`and Schaffner,
`(see Rusconi
`pJC-1
`(F) Map of pg2X;
`the 4.7 kb globin gene
`fragment
`was cloned
`as a dimer
`insert
`into the plasmid
`I
`into
`the Kpn
`DNAs of various
`size were
`cloned
`this recombinant
`was also
`referred
`to as pJKd-).
`For
`further
`experiments,
`putative
`“enhancer”
`derivatives,
`the
`site between
`the
`two globin genes;
`some of
`these DNAs were
`inserted
`by means
`of Hind
`Ill linkers
`(see
`text). One of the p/32x
`of a COmplete Kpn
`l-cut SV40 genome
`between
`the
`two globin genes
`such
`that
`the SV40 early
`region
`clone pBSV(-)p,
`was obtained
`by
`insertion
`and
`the P-globin
`genes were
`transcribed
`from opposite
`DNA strands.
`(G) Map of pa366(-)8
`cleaved with Xba
`I: example
`of a linear DNA used
`between
`the globin genes of pfl2X
`by means of Hind
`Ill linkers. Digestion
`with
`the SV40 DNA
`insert.
`(H) Map of pSX@+; a 2.1 kb Bgl II fragment
`Ill site near
`the origin of replication.
`
`fragment
`
`was cloned
`
`into
`
`the Kpn
`
`I site of a mutant SV40 DNA
`
`lacking
`
`58 bp at the origin of
`
`the
`
`/3-globin gene was obtained
`
`from pSVK+
`
`and was cloned
`
`into
`
`transfection.
`for
`of the resulting
`
`inserted
`was
`l fragment
`Ill-Kpn
`The 366 bp SV40 Hind
`clone, pp366(-)p,
`with Xba
`l liberated
`one gtobin gene
`
`with
`
`the globin gene
`
`replacing
`
`the SV40 early
`
`region was
`
`inserted
`
`between
`
`the Bet l site and
`
`the Hind
`
`Novartis Ex. 2009
`Page 2 of 10
`
`

`

`SV40 Enhances
`301
`
`Globin Gene Expression
`
`~POIYA
`cap
`
`mRNA
`
`. . . . . . . .
`
`Sl nuclease
`
`L^-----------_,,rll...,,,..,.,~.,,,,..,
`
`1 denaturation
`
`R
`
`123
`
`c
`
`123
`
`527-
`
`404-
`
`309-
`
`242-
`230-
`
`aR
`
`*
`
`+fl
`
`-43
`
`-it
`
`n
`
`527 -
`
`404-
`
`cfl
`
`309-
`
`&it
`
`of Globin RNA
`5’ Terminus
`of the Correct
`2. Detection
`Figure
`1979).
`(Weaver
`and Weissmann.
`(A) Sl nuclease mapping
`scheme
`For
`this experiment,
`a globin gene clone
`lacking
`the
`first
`intervening
`sequence
`(IVSl
`; see Figure
`1 A; Weber
`et al., 1981) was used as a
`radioactive
`probe
`(for
`further
`details
`see Rusconi
`and Schaffner,
`1981). DNA end-labeled
`at
`the Barn HI site was hybridized
`to unla-
`beled RNA,
`treated with Sl nuclease.
`denatured,
`fractionated
`by gel
`electrophoresis
`and autoradiographed.
`(B and C) Autoradiographs
`of the same gel after 8 hr and 48 hr of
`exposure,
`respectively.
`(Lanes
`1) Hybridization
`to 0.2 ng rabbit
`/3-
`globin mRNA.
`(Lanes
`2) Hybridization
`to RNA
`from 2.5 x 10’ HeLa
`cells
`transfected
`with
`the P-globin-SV40-pBR322
`clone
`pSVK+
`(Figure
`10.
`(Lanes
`3) Hybridization
`to RNA
`from
`the p-globin-gene
`recombinant
`plasmid p/32X, which does not contain SV40 sequences
`(Figure
`1 F). The
`intensity
`difference
`in the major band between
`lanes
`2 and 3 was measured
`to be 200-fold.
`This was done by scanning
`a
`series of autoradiographs
`of different
`exposure
`times
`to minimize
`any
`nonlinear
`relation
`between
`radioactivity
`and blackening
`of the x-ray
`film.
`cells
`lo6 HeLa
`from
`to RNA
`(D) Hybridization
`time was 40 hr.
`Exposure
`clone pbSV(-)p.
`with correct
`fragment
`ct:
`fl: full-length
`input DNA
`(453 nucleotides).
`it:
`from
`the Barn HI site.
`terminus, mapping
`354 nucleotides
`upstream
`incorrect
`terminus.
`about 306 nucleotides
`upstream
`from
`the Barn HI
`site. Numbers
`to the
`left: size and position
`of marker DNA
`fragments
`(pBR322
`digested
`with Hpa
`II: Sutcliffe.
`1978).
`
`transfected
`
`with
`
`the
`
`et al.,
`Grosveld
`et al., 1980;
`(Proudfoot
`studies
`1981). We there-
`and Schibler,
`1981 b; Hagenbiichle
`cell assay we are
`in our HeLa
`fore
`conclude
`that
`observing
`correct
`transcription
`initiation
`at the
`,&glo-
`bin-gene
`cap site.
`In addition
`to the correct
`5’ termi-
`nus, we have also detected
`low
`levels of transcripts
`with an “incorrect”
`5’ end
`in our Sl nuclease
`assay
`
`from
`downstream
`about 48 nucleotides
`that mapped
`terminus.
`Such
`transcripts
`are not a pe-
`the correct
`culiarity
`of our assay:
`rabbit
`/?l-globin
`genes
`from
`different
`sources
`have been
`introduced
`into a variety
`of vertebrate
`ceils, and
`the same
`incorrect
`terminus
`described
`above
`has also been
`observed
`in
`these
`other systems
`(Weld et al., 1979; Dierks et al., 1981 a,
`1981 b; Rusconi
`and Schaffner,
`1981). Most of the
`globin-gene
`transcripts
`in HeLa cells were not only
`correctly
`initiated,
`but also quantitatively
`processed
`to
`mRNA. We monitored
`splicing
`of the
`first
`intervening
`in
`sequence
`by
`the Sl
`nuclease
`assay as outlined
`Figure
`2A, using
`a similar
`end-labeled
`DNA probe,
`which,
`however,
`contained
`the
`first
`intron
`(see also
`Rusconi
`and Schaffner,
`1981).
`All
`transcripts
`were
`found
`to be correctly
`spliced,
`since
`they protected
`the
`probe
`only
`from
`the
`intron-exon
`junction
`to
`the
`la-
`beled end. They were also polyadenylated
`(most of
`them were
`selectively
`bound
`to oligo[dT]-cellulose),
`and were
`translated
`within
`the HeLa cells
`to an abun-
`dant peptide
`that comigrated
`with authentic
`rabbit p
`globin
`in gel electrophoresis
`(data not shown).
`An
`indirect
`immunofluorescence
`assay was also
`used
`to examine
`/3-globin production.
`Sixty hours after
`transfection,
`cells were
`fixed and stained with sets of
`antibodies
`so
`that
`the SV40
`large
`tumor
`antigen
`(T
`antigen)
`and p globin
`could
`be detected.
`Fluores-
`cence microscopy
`indicated
`the presence
`of T antigen
`as a green, nuclear
`fluorescence.
`A red, cytoplasmic
`fluorescence
`indicated
`/3 globin
`(Figure 3). After
`trans-
`fection with pSVK+
`(Figure
`1C) or other
`similar
`re-
`combinants,
`T antigen
`could
`be detected
`in
`lO%-
`35% of the cells, and of those, 2%-l
`0% (0.7%-l
`.4%
`of all cells)
`showed
`globin
`gene
`expression.
`The
`higher
`sensitivity
`for detection
`of T antigen
`over
`/3
`globin
`in this assay may be due
`to a higher
`stability of
`T antigen
`and/or
`to a higher
`antibody
`titer.
`In this
`assay,
`the number
`of
`fluorescent
`cells
`is correlated
`with the level of transcripts,
`but the relation
`is probably
`not
`linear. At high
`levels of P-globin
`gene expression,
`differences
`in transcription
`efficiency may be under-
`estimated with
`the
`immunofluorescence
`assay. How-
`ever,
`immunofluorescence
`was a clear-cut
`indicator
`of enhanced
`globin
`gene expression:
`transfection
`of
`HeLa cells with globin-gene
`plasmids without
`SV40
`sequences
`never yielded
`any cells with specific
`cyto-
`plasmic
`fluorescence
`(although
`low
`levels of globin
`gene
`transcripts
`could be detected
`by the Sl nuclease
`assay; see Figure 2). The
`immunofluorescence
`assay
`was therefore
`used
`for all further
`experiments.
`the
`To determine
`the best conditions
`for assaying
`expression
`of the rabbit P-globin
`gene, we performed
`time-course
`experiments
`with
`two P-globin-SV40-
`plasmid
`recombinant
`clones. One was
`the pSVK+
`clone
`used
`previously;
`the other
`clone,
`~1-1 lb-,
`whose construction
`is outlined
`below, contained
`SV40
`DNA
`from a replication-defective
`mutant. These were
`transfected
`into HeLa cells
`that were
`then assayed
`by
`
`Novartis Ex. 2009
`Page 3 of 10
`
`

`

`Cell
`302
`
`of T Antigen
`
`and of p Globin Monitored
`
`by
`
`Figure 3. Production
`lmmunofluorescence
`the
`with
`transfection
`60 hr after
`fixed with methanol
`HeLa cells were
`1 C). The cells were
`p-globin-SV40-pBR322
`clone
`pSVK+
`(Figure
`for T antigen
`(flu-
`stained
`by means of
`indirect
`immunofluorescence
`orescein;
`green) and for
`,!? globin
`(rhodamine;
`red). By switching
`filters
`we could
`screen
`a given cell
`for T-antigen
`production
`as well as
`for
`/3-globin production.
`cells having
`three
`(A) Cell sample with
`the presence
`of SV40 T antigen.
`cells
`(B) The
`lower
`two of the T-antigen-positive
`for
`rabbit
`/3 globin.
`as
`revealed
`by cytoplasmic
`cence.
`Bar = 50 pm.
`
`fluorescent
`
`nuclei,
`
`indicating
`
`in (A) are also positive
`rhodamine
`fluores-
`
`of both T antigen
`for production
`immunofluorescence
`j?-
`are shown
`in
`results with ~1-11
`and p globin. The
`Table 1. The clone pSVK+
`gave essentially
`the same
`results
`(data not shown).
`It was
`found
`that T-antigen
`and
`/3-globin
`levels were highest at 2 to 2% days after
`transfection
`and
`rapidly
`declined
`thereafter.
`
`Effect
`“Enhancer”
`of the SV40
`Analysis
`in
`acted
`the SV40 sequences
`To determine
`whether
`cis or
`in trans, we cotransfected
`HeLa cells with a
`mixture
`of
`the cloned
`DNAs
`pBSV3x
`(Schaffner,
`of
`1980)
`and p/32X,
`containing
`three
`tandem
`copies
`SV40 DNA and
`two
`tandem
`copies
`of
`the
`,&globin
`gene,
`respectively.
`The cells
`in an 8 X 8 mm
`field
`were analyzed
`by
`immunofluorescence.
`Though
`the
`usual
`number
`of cells
`(4800;
`13%
`of all cells) ex-
`pressed
`T antigen,
`only
`three
`cells were
`/3-globin-
`positive.
`In a parallel
`transfection
`the @globin-SV40
`recombinant
`pflSV(-)p
`gave
`rise
`to 4200 T-antigen-
`expressing
`and 466 globin-expressing
`cells
`in an area
`of equal
`size (Table
`1). If the SV40
`sequences
`were
`acting
`in trans, one would
`expect
`about 450
`rather
`than 3 globin-expressing
`cells
`in the mixing
`experi-
`ment, since
`cells
`that are
`“competent”
`to express
`transiently
`one kind of DNA
`from a mixture
`of
`two
`transfected
`DNAs are found
`to express
`the other DNA
`also
`(see
`footnote
`to Table
`2). The same
`“compe-
`
`Table 1. Production
`Cells
`
`of T Antigen
`
`and p Globin
`
`in Transfected
`
`HeLa
`
`Total Cells
`(8 x 8 mm)”
`
`Cells Positive
`for T Antigen
`
`Cells Positive
`for
`/3 Globin
`
`Days after Trans-
`fection with
`Clone ~7-1 7p-
`
`2
`
`Y/z
`
`3’/2
`
`5’/2
`
`8
`
`Transfecting
`
`DNA
`
`PflSV( - )fl
`pBSV3 x , p/32X
`
`PSVBX
`
`P82 x
`
`33,000
`
`32,000
`
`29,000
`
`35,000
`
`31,000
`
`41 .oqo
`
`38,000
`
`37,000
`
`41,000
`
`12,000
`
`(35%)
`
`242
`
`(0.7%)
`
`11,000
`
`(34%)
`
`265
`
`(0.8%)
`
`4.900
`
`(17%)
`
`53 (0.2%)
`
`2.500
`
`(7%)
`
`11 @.03%)
`
`800
`
`(2%)
`
`0
`
`4,200
`
`(10%)
`
`466
`
`(1 .l%)
`
`4,800
`
`(13%)
`
`5,200
`
`(14%)
`
`0
`
`3 (0.008%)
`
`0
`
`0
`
`in an 8 X 8 mm area was extrapolated
`of cells
`total number
`* The
`of 0.145 mm* each
`(400-fold
`magnifica-
`areas
`counting
`three
`from
`that
`is, a total number of 150
`to 300 cells was counted.
`Similarly,
`tion);
`the number of T-antigen-positive
`cells was extrapolated
`from counting
`7 to 12 areas of 0.145 mm* each. The number
`of globin-positive
`cells
`was always
`counted
`in the whole 8 x 8 mm
`field.
`
`been well docu-
`has already
`phenomenon
`tence”
`in cell-transformation
`experiments
`(Wigler
`et
`mented
`al., 1979). The three globin-positive
`cells
`in our mixing
`experiment
`described
`above
`(Table
`1) are
`likely
`to be
`the
`result of
`intracellular
`recombination
`between
`the
`transfected
`plasmids,
`since
`unrelated
`mixed DNAs
`used
`for cell
`transformation
`can be
`linked
`together
`within
`the recipient
`cell eventually
`to form
`large DNA
`entities
`(Pellicer
`et al., 1980; Perucho
`et al., 1980).
`We next wanted
`to determine
`if enhanced
`expres-
`sion of
`the globin
`gene was a copy-number
`effect
`This
`resulting
`from
`the
`activity
`of an SV40
`replicon.
`possibility
`did
`not
`appear
`very
`likely,
`since
`the
`repli-
`cation
`SV40
`is
`severely
`inhibited
`by
`cis-acting
`of
`plasmid
`sequences
`(Lusky and Botchan,
`1981).
`In all
`our experiments
`such plasmid
`sequences
`were pres-
`ent whenever
`a complete
`SV40
`replicon was
`linked
`to
`the P-globin
`gene, and analysis
`of pSVK+
`DNA
`from
`HeLa cells by Southern
`blot hybridization
`transfected
`of
`(Southern,
`1975)
`did not
`indicate
`any
`replication
`we
`this
`recombinant
`(data not shown).
`In addition,
`tested viral DNAs
`that either
`lacked a functional
`origin
`of replication
`or did not contain
`coding
`sequences
`for
`T antigen,
`both
`of which
`are
`required
`for
`SV40
`repli-
`cation
`(Tegtmeyer
`and
`Ozer,
`1971;
`Gluzman
`et
`al.,
`1980). Cloned mutants of SV40 with
`deletions
`of 9 bp
`and of 58 bp at
`the origin
`of DNA
`replication
`were
`provided
`by Y. Gluzman
`(Gluzman
`et al., 1980;
`see
`Figures
`4d
`and
`4e).
`Both
`mutants,
`after
`transfection
`into
`CVl
`monkey
`cells,
`produce
`normal
`amounts
`functional
`T antigen
`but are unable
`to replicate.
`
`of
`The
`
`Novartis Ex. 2009
`Page 4 of 10
`
`

`

`SV40 Enhances
`303
`
`Globin Gene Expression
`
`the Kpn
`into
`I fragment was cloned
`Kpn
`globin-gene
`(9 bp deleted)
`I site of the mutant SV40 DNAs 6-17
`and 1-l 1 (58 bp deleted)
`to yield
`the clones p6-17p+
`and ~1-11 p-
`(Figure
`1 D). HeLa
`cells were
`trans-
`fected
`and assayed
`by immunofluorescence
`for pro-
`duction
`of T antigen
`and
`rabbit
`/3 globin.
`Both p6-
`17p+
`(data not shown)
`and ~1-11
`/I-
`(Table
`1) effi-
`ciently expressed
`the rabbit P-globin
`gene.
`for
`To determine
`if T-antigen
`expression
`is required
`the enhancing
`effect, we
`tested
`the 1118
`bp SV40
`Hind
`Ill C
`fragment
`(map
`positions
`5171
`to 1046
`according
`to Appendix
`A of Tooze
`[1980];
`see Figures
`1 B and 4b), a segment
`containing
`the origin
`of repli-
`cation and adjacent
`“late’‘-region
`sequences
`but no
`sequences
`coding
`for T antigen.
`This
`fragment,
`con-
`taining a P-globin
`gene
`inserted
`at the Kpn
`I site, was
`obtained
`from a complete
`Hind
`Ill digest
`of pSVK+
`(Figure
`1 C) and was cloned
`into the Hind
`Ill site of the
`plasmid
`pJC-1
`in both orientations,
`resulting
`in the
`clones
`pSVHin+K+
`(Figure
`1 E) and pSVHin-K+.
`The same SV40 Hind
`Ill C fragment
`containing
`the p-
`globin gene
`in opposite
`orientation
`was obtained
`from
`the clone pSVK-
`(see
`legend
`to Figure 1 C), and was
`also cloned
`in both orientations
`into
`the plasmid
`pJC-
`1 to yield
`the clones pSVHin+K-
`and pSVHin-K-.
`The
`four different
`recombinants
`that
`resulted
`were
`transfected
`into HeLa cells. These plasmids
`showed
`a much
`lower expression
`of
`the
`,L?-globin gene
`than
`the parental
`recombinants
`pSVK+
`and pSVK-
`(data
`not shown). This suggested
`either
`that only part of the
`SV40 enhancing
`activity was present within
`the Hind
`Ill C
`fragment,
`or
`that
`the enhancing
`activity was
`disturbed
`by neighboring
`plasmid
`sequences.
`The
`latter was
`found
`to be the case, since digestion
`of the
`recombinants
`with Hind
`Ill prior
`to transfection
`led
`to
`very efficient
`globin
`production
`(Table
`2)
`thus
`indi-
`cating
`that T antigen
`is not required.
`Linear
`and cir-
`cular DNAs work equally well
`in transfection
`experi-
`ments
`(our unpublished
`data), presumably
`because
`linear DNA can be circularized
`within
`the nucleus
`of
`the
`transfected
`cell
`(Subramanian,
`1979). We also
`encountered
`another
`example
`of plasmid DNA
`inter-
`ference:
`the enhancing
`activity of SV40 was not de-
`tected when viral DNA was separated
`from
`the globin
`gene on both sides by one 3.7 kb copy of the plasmid
`pJC-1
`(our unpublished
`data). This kind of
`interfer-
`ence was not further
`investigated.
`Instead. most of the
`recombinants
`were made by inserting
`putative enhan-
`cer
`sequences
`between
`the
`two globin
`genes
`of
`p/32x.
`In these molecules,
`no negative
`effect
`from
`vector plasmid DNA was observed.
`sequences
`coding
`Another
`DNA
`fragment
`lacking
`for T antigen was also
`tested. The small Hind
`Ill-Kpn
`I fragment
`of SV40 DNA
`(366 bp, positions
`5171
`to
`294; see Figures
`16 and 4c) was
`inserted
`between
`the
`two globin
`genes of pp2 x
`(Figure
`1 F), in either
`orientation,
`by means of Hind
`Ill linkers. These DNAs
`
`Table 2. Production
`Cells
`
`of T Antigen
`
`and p Globin
`
`in Transfected
`
`HeLa
`
`Total Cells
`(6 x 6 mm)
`
`Cells Positive
`for T Antigen
`
`Cells
`Positive
`Globin
`
`for p
`
`Transfecting
`
`DNA
`
`(Hind
`pSVHin+K+
`Ill-digested)
`
`pSVHin+K-
`Ill-digested)
`
`(Hind
`
`pSVHin+K-
`(digested),
`pBSV3x
`(Control)
`
`pBSV3x
`
`(Control)
`
`pSVK+
`
`(Control)
`
`psxfl+
`
`PSXP-
`
`72 bp Repeats
`Present
`
`2 (Wild-type)
`
`It-72
`
`bp)
`
`‘h (-105
`
`bp)
`
`32,000
`
`36,000
`
`32,000
`
`36,000
`
`42.000
`
`37,000
`
`30,000
`
`40,000
`
`42,000
`
`36,000
`
`0
`
`0
`
`266
`
`(0.9%)
`
`415(1.1%)
`
`4200
`
`(13%)
`
`6600
`
`(17%)
`
`310
`
`(1%)
`
`0
`
`6200
`
`(15%)
`
`436
`
`(1%)
`
`0
`
`516
`
`(1.4%)
`
`333
`
`(1 .l%)
`
`7900
`
`(20%)
`
`6300
`
`(15%)
`
`504
`
`(1.3%)
`
`331
`
`(0.6%)
`
`9 (0.03%)
`
`0
`
`’ Every
`
`fl-globin-positive
`
`cell was also positive
`
`for SV40 T antigen.
`
`or
`into HeLa cells either undigested
`transfected
`were
`I or Xba
`I to release @globin
`dimers
`digested with Kpn
`or monomers,
`respectively,
`containing
`the 366 bp of
`SV40 DNA
`(Figure
`1G). All of
`these DNAs gave en-
`hanced
`expression
`of
`the
`,lI-globin
`gene
`(data
`not
`shown). No globin-positive
`cells were
`found with
`the
`parental
`plasmid pp2 x , whether
`used undigested
`(Ta-
`ble 1) or digested with Kpn
`I or Xba
`I (data not shown).
`The phenomenon
`of enhanced
`globin gene expres-
`sion was not restricted
`to linkage
`of SV40 and
`the /?-
`globin
`gene at their Kpn
`I sites. A 2.1 kb
`long Bgl
`II
`fragment
`containing
`the
`rabbit
`/I-globin
`gene was
`cloned
`in both orientations
`into
`the SV40 early
`region
`by means
`of Xho
`I linkers
`(Figure
`1H). Both
`these
`clones
`(pSXP+
`and pSXp-)
`gave high
`levels of globin
`gene
`expression
`upon
`transfection
`into HeLa
`cells
`(Table 2). Viral
`“enhancer”
`sequences
`were also ac-
`tive when
`inserted
`at the Xba
`I or Eco RI sites (Figure
`1A) downstream
`from
`the globin
`gene
`(data
`not
`shown).
`
`Sequences
`“Enhancer”
`of the SV40
`Localization
`fi-
`that enhances
`The 366 bp segment
`of SV40 DNA
`globin gene expression
`contains,
`apart
`from
`the func-
`tion as origin
`of replication,
`which was
`found
`to be
`dispensable,
`some peculiar
`structural
`features:
`a 17
`bp segment
`containing
`only
`the bases adenine
`and
`thymine,
`two repeated-sequence
`motifs of 21 bp and
`the 72 bp repeat
`(Figure 4a). Promoter
`information
`for
`
`Novartis Ex. 2009
`Page 5 of 10
`
`

`

`Cell
`304
`
`a
`
`h
`
`C
`
`d
`
`e
`
`g
`
`h
`
`origin
`
`Intact
`OrIgInof
`replmtion
`+
`
`5200
`
`50
`
`200
`
`250
`
`300
`
`-9
`
`-56
`
`H/ndU
`,
`
`Hind fE
`
`. . . . . . .
`
`..a...,
`
`. . . . . . .
`
`sm.....
`
`Hind IIf
`t
`
`Kpnl
`
`..***..
`
`. . , , . . .
`
`.a..,..
`
`. . . , . . .
`
`I
`
`+
`
`-
`
`-
`
`+
`
`+
`
`-72
`
`-105
`
`I
`
`I
`
`_I
`
`1
`
`T antagen
`expression
`
`enhancer of
`a-globm gene
`
`+
`
`I
`
`+
`
`+
`
`+
`
`+
`
`+
`
`-
`
`-
`
`+
`
`+
`
`+
`
`(-)
`
`Region near
`“Enhancer”
`of the Transcriptional
`Figure 4. Mapping
`its sequence
`features
`of
`(a) Wild-type
`DNA with
`the characteristic
`(b) The 1116 bp Hind
`Ill C fragment
`(map positions
`5171
`to 1046).
`(c) The 366 bp Hind
`Ill-Kpn
`I fragment
`(5171
`to 294).
`(Gluzman
`(d-h) Various
`deletion mutants
`constructed
`by Y. Gluzman
`to 160; Rio et al., 1980).
`(i) The 311 bp Eco RII G fragment
`(5092
`
`of Replication
`the SV40 Origin
`indicated
`(nucleotide
`numbering
`
`according
`
`to Appendix
`
`A of Tooze
`
`[1980])
`
`et al., 1980;
`
`see
`
`text
`
`for
`
`further
`
`details).
`
`also
`of the viral early and probably
`transcription
`the
`is present within
`the 366 bp segment.
`late region
`the
`to localize
`the enhancer
`activity
`further
`We were able
`by linking
`the P-globin
`gene
`to the cloned Eco RII G
`fragment
`of SV40
`(Rio et al., 1980;
`a gift
`from R.
`Tjian),
`and
`to
`two SV40 mutants
`that had deletions
`located
`in
`the 72 bp
`repeat
`region
`(a gift
`from Y.
`Gluzman).
`The deletion mutants
`had been cloned
`via
`the SV40 Eco RI site
`into
`the vector plasmid.
`These
`recombinants
`were opened
`at the Kpn
`I site within
`the
`SV40 DNA, and
`the /3-globin-gene
`Kpn
`I fragment was
`inserted
`there.
`The ability
`of
`these SV40
`deletion
`mutants
`to act as
`long-distance
`“enhancers”
`of the
`globin gene was directly
`related
`to the presence
`of at
`least one
`intact 72 bp element.
`The mutant
`in which
`one of the 72 bp repeated motifs had been precisely
`eliminated
`by deletion
`of the small Sph
`I-Sph
`I restric-
`tion
`fragment
`(positions
`131
`to 202; Y. Gluzman,
`personal
`communication)
`did not show a significant
`decrease
`in enhancing
`activity
`(Figure
`49; Table 2).
`However,
`the mutant with an additional
`deletion
`of
`about 33 bp, removing
`sequences
`around
`the remain-
`ing Sph
`I site as shown
`in Figure 4h, was unable
`to
`enhance @globin
`gene expression
`(Table 2). In addi-
`tion,
`the cloned Eco RI1 G fragment
`of SV40
`(positions
`5092
`to 160)
`containing
`the origin of replication
`and
`most of one 72 bp element
`(Figure
`4i), was
`inserted
`in both orientations
`into
`the Eco RI site 650 bp down-
`stream
`from
`the globin
`gene
`polyadenylation
`site.
`These
`clones
`did not show enhanced
`globin
`gene
`activity. Thus we assume
`that at least one copy of the
`72 bp
`repeated-sequence
`element
`is essential
`for
`
`enhancement
`
`of globin
`
`gene expres-
`
`transcriptional
`sion.
`
`Discussion
`
`of a rabbit
`expression
`transient
`the
`We have studied
`it into HeLa cells. We
`@globin
`gene by transfecting
`of SV40 DNA,
`including
`found
`that a small segment
`motif near
`the origin of
`the 72 bp repeated-sequence
`transcription
`of the
`linked
`replication,
`enhanced
`the
`P-globin
`gene by
`two orders
`of magnitude,
`and
`that
`the
`transcripts
`were
`translated
`into hemoglobin
`/I-
`chain
`protein.
`Almost
`all of
`the
`transcripts
`had
`the
`same 5’ end as rabbit
`/I-globin mRNA. Neither SV40
`T antigen
`nor a functional
`origin
`of DNA
`replication
`was required
`for the enhancing
`effect. Therefore
`both
`readthrough
`transcripts
`from SV40 promoters
`and a
`copy-number
`effect due to replication
`of a linked SV40
`replicon
`are unlikely
`to be
`responsible
`for
`the en-
`hanced globin gene expression.
`It is also unlikely
`that
`SV40 sequences
`act solely by increasing
`uptake and/
`or by stabilization
`of
`linked DNA, because
`similar
`amounts
`of recombinant
`DNA were
`found within
`the
`HeLa
`cell nuclei
`60 hr after
`transfection
`whether
`or
`not SV40 sequences
`were
`linked
`to the P-globin
`gene
`(data not shown). Nor is it likely
`that there
`is a general
`stimulation
`of macromolecular
`synthesis
`or a stabili-
`zation of mRNA by some diffusible
`SV40
`factor, since
`SV40 DNA acts only
`in cis.
`activate
`could
`sequences
`The SV40
`“enhancer”
`possible
`gene by several
`the promoter
`of a remote
`mechanisms,
`including
`reorganizing
`the chromatin
`
`Novartis Ex. 2009
`Page 6 of 10
`
`

`

`SV40 Enhances
`305
`
`Globin Gene Expression
`
`the
`of
`density
`the superhelical
`changing
`structure;
`to the nuclear
`DNA (reviewed
`by Smith, 1981); binding
`matrix
`(Cook
`and Brazell,
`1975)
`where
`viral DNA
`(Buckler-White
`et al., 1980)
`and
`transcriptional
`com-
`plexes
`(Jackson
`et al., 1981)
`seem
`to be located;
`and
`providing
`an entry
`site
`for RNA polymerase
`II (dis-
`cussed by Grosschedl
`and Birnstiel,
`1981).
`Another
`possible mechanism
`of SV40 DNA action
`has been
`suggested
`by Capecchi
`(1980)
`who
`re-
`ported
`an enhancing
`effect
`of SV40 DNA on cell
`transformation
`mediated
`by the herpesvirus
`thymidine
`kinase
`gene.
`In a transient
`expression
`assay, how-
`ever,
`the
`thymidine
`kinase
`gene without SV40 DNA
`appeared
`highly active. He concluded
`that
`the SV40
`DNA
`increased
`the number
`of stable
`transformants
`by
`facilitating
`integration
`of
`the
`thymidine
`kinase
`gene
`into
`recipient
`cell DNA.
`In our
`transient
`expression
`assay
`the /?-globin gene
`is quite
`inactive unless
`linked
`to the SV40
`“enhancer.”
`This could be taken
`to mean
`that globin genes have
`to be integrated
`into HeLa cell
`DNA
`in order
`to be efficiently
`expressed.
`If so,
`then
`the cells expressing
`the P-globin
`gene should do
`this
`in a more stable
`fashion
`than
`the cells expressing
`T
`antigen,
`since
`the SV40
`early
`region
`can be well
`expressed
`from extrachromosomal
`DNA (reviewed
`by
`Acheson,
`1980). However,
`in our
`time-course
`exper-
`iment
`(Table 1) the expression
`of both p globin
`and T
`antigen was
`transient,
`and
`the proportion
`of globin-
`positive
`cells
`declined
`even more
`rapidly
`than
`the
`proportion
`of T-antigen-positive
`cells.
`It should
`be
`out
`pointed
`that our
`findings
`do not argue against
`a
`“chromosomal
`integrator”
`activity of SV40 DNA, but
`against
`requisite
`integration
`for efficient
`globin
`gene
`expression.
`of globin gene activation
`the mechanism
`Whatever
`observed
`in our assay,
`it is a peculiarity
`neither
`of the
`rabbit P-globin
`gene,
`nor of SV40 DNA. A histone
`gene cluster of the s