Cell, Vol. 11, 223-232,
`
`May 1977, Copyright
`
`0 1977 by MIT
`
`of Purified Herpes Virus Thymidine
`Transfer
`Gene
`to Cultured Mouse Cells
`
`Kinase
`
`Lih-Syng
`and Richard
`
`Lee,
`Axel
`
`Saul Silverstein,
`Michael Wigler,
`Yung-chi
`Cheng*
`Angel Pellicer,
`Institute
`of Cancer
`Research
`and Pathology
`and Departments
`of Microbiology
`College
`of Physicians
`and Surgeons
`Columbia
`University
`New York, New York
`
`10032
`
`Summary
`
`in thy-
`L cells deficient
`of Ltk-, mouse
`Treatment
`endonu-
`midine
`kinase
`(tk), with Barn
`I restriction
`virus-l
`clease
`cleaved
`DNA
`from
`herpes
`simplex
`(HSV-1)
`produced
`tk+ clones
`with
`a frequency
`of
`1O-6/2 pg of HSV-1 DNA. Untreated
`cells or cells
`treated
`with Eco RI restriction
`endonuclease
`frag-
`ments
`produced
`no
`tk+
`clones
`under
`the same
`conditions.
`The
`thymidine
`kinase
`activities
`of four
`independently
`derived
`clones were
`characterized
`By
`by biochemical
`and
`serological
`techniques.
`to be
`these
`criteria,
`the
`tk activities
`were
`found
`identical
`to HSV-1
`tk and different
`from host wild-
`type
`tk. The
`tk+ phenotype
`was
`stable
`over
`sev-
`eral hundred
`cell generations,
`although
`the
`rate
`of reversion
`to
`the
`tk- phenotype,
`as
`judged
`by
`cloning
`efficiency
`in the presence
`of bromodeoxy-
`uridine,
`was high
`(l-5
`x 10m3). HSV-1 DNA Barn
`restriction
`fragments
`were
`separated
`by gel elec-
`trophoresis,
`and virtually
`all activity,
`as assayed
`by
`transfection,
`was
`found
`to
`reside
`in a 3.4 kb
`fragment.
`Transformation
`efficiency
`with
`the
`iso-
`lated
`fragment
`is 20 fold higher
`per gene
`equiva-
`lent
`than with
`the unfractionated
`total Barn digest.
`These
`results
`prove
`the usefulness
`of
`transfec-
`tion
`assays
`as a means
`for
`the
`bioassay
`and
`isolation
`of restriction
`fragments
`carrying
`specific
`genetic
`information.
`Cells
`expressing
`HSV-1
`tk
`may also provide
`a useful model
`system
`for
`the
`detailed
`analysis
`of eucaryotic
`and viral gene
`reg-
`ulation.
`
`Introduction
`
`of eucaryotic
`fragments
`of specific
`isolation
`The
`the
`structural
`DNA has permitted
`an analysis
`of
`organization
`of specific
`genes
`and may ultimately
`provide
`information
`on
`the mechanism
`regulating
`the expression
`of
`these
`genes.
`Cleavage
`of
`the
`genome
`with
`restriction
`endonucleases
`followed
`by molecular
`cloning
`of these
`fragments
`in bacte-
`rial plasmids
`has permitted
`the
`isolation
`and ampli-
`fication
`of
`specific
`genes
`(Cohen
`and Chang,
`1974).
`Identification
`of eucaryotic
`genes within
`re-
`* Department
`of Experimental
`Therapeutics
`and Grace
`Cancer
`Drug Center,
`Roswell
`Park Memorial
`Institute,
`New York
`State
`Department
`of Health,
`Buffalo,
`New York
`14263.
`
`mo-
`requires
`however,
`DNAs,
`plasmid
`combinant
`RNA or DNA
`purified
`with
`hybridization
`lecular
`with a sin-
`specifically
`reacting
`capable
`of
`probes
`of
`the biological
`activity
`of iso-
`gle gene. Analysis
`lated DNA
`fragments
`by
`transfection
`provides
`an
`alternate
`means
`of
`identifying
`specific
`eucaryotic
`genes. This approach
`further
`demonstrates
`that
`the
`DNA contained
`within
`a given
`fragment
`includes
`the
`information
`required
`to code
`for
`the entire
`structural
`gene,
`in a form
`recognizable
`by the
`tran-
`scriptional
`and
`translational
`machinery
`of the host
`ccl I.
`to iden-
`has been used
`design
`This experimental
`of the SV40 and adenovirus
`tify specific
`fragments
`the genes
`for malignant
`trans-
`genomes
`containing
`et al.,
`1975).
`Another
`gene
`formation
`(Graham
`by restriction
`endonuclease
`amenable
`to
`isolation
`with
`transfection
`assays
`is the
`cleavage
`in concert
`thymidine
`kinase
`(tk) gene of herpes
`simplex
`virus
`(HSV-1).
`Infection
`of cells with
`ultraviolet-irradi-
`and
`tated
`herpes
`virus
`results
`in
`the
`introduction
`stable
`expression
`of multiple
`viral gene
`functions
`(Macnab
`and Timbury,
`1976). HSV-1
`thymidine
`ki-
`nase activity
`has been
`transferred
`to
`tk-deficient
`mouse
`L cells
`(Ltk-)
`by
`infection
`with
`inactivated
`virions
`(Munyon
`et al., 1971).
`a specific
`to isolate
`We have
`therefore
`attempted
`kinase
`thymidine
`DNA
`fragment
`containing
`the
`gene
`from
`the HSV-I
`genome
`using
`transfection
`of
`this gene
`function
`as a bioassay.
`The choice
`of this
`system
`was
`dictated
`by several
`considerations.
`less
`First,
`the viral genome
`is orders
`of magnitude
`complex
`than
`the eucaryotic
`genome.
`This greatly
`enhances
`the prospects
`for successful
`transfection
`and allows
`the possibility
`of purification
`of active
`restriction
`fragments
`by size alone. Second,
`the tk+
`phenotype
`can
`be efficiently
`selected
`over
`a
`tk-
`phenotypic
`background
`by utilizing
`growth
`condi-
`tions
`in which
`the salvage
`pathway
`enzyme
`thymi-
`dine
`kinase
`is necessary
`for survival.
`There
`exist
`cell
`lines deficient
`in tk with
`low
`rates of sponta-
`neous
`reversion
`to tk+ which
`can be used as recipi-
`ents. Third,
`the
`tk gene
`is an
`ideal
`subject
`for
`mutational
`analysis
`because
`the
`tk+ or the
`tk- phe-
`notype
`can be selected
`under
`various
`conditions.
`Fourth,
`the gene
`product,
`thymidine
`kinase,
`is a
`well characterized
`viral protein
`of known
`function.
`In this
`report, we demonstrate
`the stable
`transfer
`of HSV-1
`tk activity
`to mouse
`L cells
`(Ltk-)
`by trans-
`fection with HSV-1 DNA cleaved
`by restriction
`en-
`donuclease
`Barn
`I. The
`tk gene
`can be transfected
`using
`an electrophoretically
`pure
`fragment
`3.4 kb
`in
`length.
`The
`transformed
`mouse
`cells with
`re-
`stored
`tk activity
`synthesize
`an enzyme with
`anti-
`genie
`and electrophoretic
`properties
`identical
`to
`that
`coded
`for by
`the herpes
`virus
`genome.
`Fur-
`thermore,
`this gene
`function
`is stably
`expressed
`
`Merck Ex. 1029, pg 1058
`
`

`
`Cell
`224
`
`selective
`under
`erations.
`
`pressure
`
`for several
`
`hundred
`
`gen-
`
`ABCDEFG
`
`of tk Activity with Fragments
`
`of HSV
`
`Endonu-
`
`1. Digestion
`
`of HSV-I
`
`DNA with DNA Restriction
`
`Figure
`cleases
`restriction
`with 3 U of various
`incubated
`1 .O pg of HSV-1 DNA was
`resultant
`DNA
`fragments
`were
`enzymes
`for
`3 hr at 37°C. The
`analyzed
`by electrophoresis
`on a 17 cm 0.5%
`agarose
`slab gel.
`Gels were
`stained
`with ethidium
`bromide
`and photographed
`un-
`der short-wave
`ultraviolet
`illumination.
`(A) Hpa
`I; (6) Bgl
`II; (C) Sal
`I; (D) Hind
`Ill; (E) Earn
`I; (F) Barn
`I + Eco RI; (G) Eco RI.
`
`phos-
`calcium
`with
`co-precipitated
`DNA
`HSV-1
`Salmon
`1973).
`(Graham
`and
`van der Eb,
`phate
`sperm DNA was used as carrier
`to yield a total DNA
`concentration
`of 20 pg/ml.
`After a 30 min exposure
`to DNA, cells were
`fed growth medium.
`24 hr
`later,
`cultures
`were
`refed
`growth
`medium
`containing
`HAT and subsequently
`fed HAT medium
`every 2-3
`days.
`After
`2 weeks,
`surviving
`colonies
`were
`counted.
`Table
`1 summarizes
`the
`results
`from
`four
`experiments.
`In all four experiments,
`untreated
`cul-
`tures, or cultures
`treated with either Salmon
`sperm
`DNA alone
`or with Eco Rl-digested
`HSV-1,
`ex-
`hibited
`no surviving
`colonies
`in HAT. We estimate
`from
`these
`and other
`experiments
`that
`the
`rever-
`sion
`rate of
`these
`cells
`to
`the
`tk+ phenotype
`is
`<lO-8.
`By contrast,
`cultures
`treated
`with Barn
`l-
`digested
`HSV-1 DNA consistently
`displayed
`surviv-
`ing colonies
`at a frequency
`of approximately
`1 col-
`ony per
`IO6 cells per 2 pg HSV-1 DNA.
`
`in-
`
`ki-
`for
`to
`
`in-
`et
`
`Results
`Transfection
`DNA
`the HSV-1
`of
`fragment
`of a specific
`isolation
`The
`gene
`re-
`kinase
`genome
`containing
`the
`thymidine
`endonuclease
`quires
`that we
`identify
`a restriction
`capable
`of digesting
`HSV DNA, which makes no
`ternal
`cleavages
`within
`the
`tk gene.
`Identification
`of such a DNA
`fragment
`in addition
`requires
`a cell
`line
`that will
`stably
`express
`the
`tk
`function
`upon
`competent
`transfection.
`Ltk-
`clone
`d, a clone
`of
`mouse
`cells
`resistant
`to
`bromo-deoxyuridine
`(BdUrd)
`and deficient
`in cytoplasmic
`thymidine
`nase
`(Kit et al., 1963) was
`therefore
`chosen
`transfection
`experiments.
`Ltk-
`cells are unable
`grow
`in medium
`containing
`HAT
`(hypoxanthine,
`de-
`aminopterin
`and
`thymidine),
`in which
`survival
`pends
`upon
`the presence
`of both salvage
`pathway
`enzymes
`thymidine
`kinase
`and
`hypoxanthine-
`guanosine
`phosphoribosyl
`transferase
`(Littlefield,
`1963). The cells have a very
`low
`rate of spontane-
`by
`ous
`reversion
`to
`the
`tk+ phenotype,
`as judged
`ability
`to form colonies
`in HAT-containing
`medium,
`and were
`used as host
`recipients
`to demonstrate
`that ultraviolet-inactivated
`HSV-1
`virions
`could
`fect and
`stably
`confer
`HSV
`tk activity
`(Munyon
`al., 1971).
`from
`was extracted
`transfection
`for
`Viral DNA
`free of
`and
`purified
`in Vero
`cells
`virions
`grown
`sedi-
`by velocity
`host
`sequences
`contaminating
`density
`centrifuga-
`or CsCl equilibrium
`mentation
`tion. Purity was monitored
`by isopycnic
`centrifuga-
`a
`tion
`in CsCI. This DNA was
`then
`cleaved
`with
`series of restriction
`endonucleases.
`The DNA prod-
`ucts of these digestions
`were separated
`by electro-
`phoresis
`on 0.5% agarose
`slab gels
`(Figure
`1). Al-
`though
`all
`the enzymes
`used
`for cleavage
`require
`the recognition
`of a unique
`hexanucleotide
`pair
`for
`activity,
`significant
`differences
`in
`the number
`of
`cleavage
`sites
`for
`the different
`enzymes
`is appar-
`ent. The gel profiles
`shown
`in Figure
`1 reflect
`com-
`plete
`digestion
`by
`the endonuclease
`since
`first,
`incubation
`for an additional
`3 hr
`results
`in no
`change
`in the band pattern;
`second,
`the addition
`of
`a second
`dose
`of enzyme
`at 3 hr
`followed
`by a
`second
`incubation
`did not alter
`the digestion
`pro-
`file; and
`third,
`adenovirus
`2 DNA was completely
`digested
`under
`our
`reaction
`conditions.
`In initial experiments,
`Barn
`I- and Eco RI-digested
`in
`HSV DNA
`(Figure
`1, slots E and G) were
`used
`transfection
`assays. Cells were plated
`at a density
`of 6 x 105/100 mm petri
`dish
`in growth
`medium.
`Culture medium
`was
`removed
`24 hr
`later, and cell
`monolavers
`were
`overlaid
`with
`restriction-cleaved
`
`Merck Ex. 1029, pg 1059
`
`

`
`Thymidine
`
`Kinase Gene
`
`in Mouse Cells
`
`Herpes
`225
`
`Table
`
`1. Summary
`
`of Transfection
`
`Experiments
`
`with Unfractionated
`
`Restriction
`
`Endonuclease
`
`Digests
`
`of HSV-1 DNA
`
`Experiment
`
`1
`
`Experiment
`
`2
`
`Experiment
`
`3
`
`Experiment
`
`4
`
`pg HSV-1
`DNA per
`Dish”
`
`4 .oo
`
`2 .oo
`1 .oo
`
`0.50
`0.25
`
`4.00
`2 .oo
`
`1 .oo
`
`0.50
`0.25
`
`2.00
`
`Z/Xb
`
`o/2
`o/2
`
`o/2
`o/2
`
`o/2
`
`o/5
`O/5
`
`Eco RI-Digested
`HSV-I DNA
`
`l-Digested
`Barn
`HSV-1 DNA
`
`I-, Eco RI-
`Barn
`Digested
`HSV-1 DNA
`Salmon
`Sperm DNA
`Untreateda
`
`Specific
`Activityc
`
`0.00
`
`Specific
`Activity
`
`0.00
`
`0.50
`
`0.25
`
`0.00
`0.00
`
`2.00
`
`xx
`
`o/2
`012
`
`o/2
`o/2
`
`o/2
`
`412
`l/2
`
`o/2
`o/2
`
`l/2
`
`o/5
`O/5
`
`WI
`
`Specific
`Activity
`
`X/P
`
`Specific
`Activity
`
`o/5
`
`0.00
`
`0.90
`1.50
`
`1 .oo
`0.00
`
`3.00
`
`712
`
`612
`212
`
`o/2
`314
`
`o/5
`
`315
`
`0.60
`
`015
`o/5
`
`0.00
`
`a All dishes
`cultures,
`b P/P =
`e Specific
`
`10 pg DNA
`received
`which
`did not
`receive
`total
`number
`of colonies
`activity
`= HAT-resistant
`
`in 0.5 ml using
`even
`salmon
`per number
`colonies
`
`sperm DNA as carrier,
`salmon
`DNA.
`sperm
`dishes.
`of replicate
`per
`lo6 ceils exposed
`
`as described
`
`in Experimental
`
`Procedures,
`
`except
`
`“untreated”
`
`per pg of HSV DNA.
`
`of HSV-1
`I cleavage
`that Barn
`These data suggest
`contain-
`DNA generates
`at least one DNA
`fragment
`gene.
`ing
`information
`for
`the entire
`tk structural
`Eco RI fragments
`display
`no activity
`in transfection
`assay, presumably
`because
`cleavage
`occurs within
`the gene. To verify
`this,
`the
`infectivity
`of HSV DNA,
`which was digested
`with both Barn
`I and Eco RI,
`was assayed
`by
`transfection.
`This preparation
`of
`doubly
`cleaved
`fragments
`was not capable
`of gen-
`erating
`HAT-resistant
`colonies.
`
`tk Activity
`HSV
`Cells Express
`Transformed
`df tk in Transformed
`Cells
`Mobility
`Electrophoretic
`Proof
`that
`transformation
`of the mouse
`Ltk- pheno-
`type
`results
`from
`the
`introduction
`and expression
`of viral DNA
`fragments
`requires
`us to demonstrate
`the viral
`origin
`of
`the
`tk expressed
`in
`the
`trans-
`fected
`clones.
`To
`this end,
`colonies
`were
`picked
`from different
`culture
`dishes,
`grown
`into mass cul-
`tures and
`further
`analyzed.
`Four clones were cho-
`sen, and
`their
`tk activity was characterized
`by elec-
`trophoretic
`mobility,
`immunologic
`neutralization
`and substrate
`specificity.
`fractions
`the cytosol
`of
`Electrophoretic
`profiles
`(a mouse
`leukemic
`line),
`Ltk-,
`derived
`from
`L1210
`12 hr post-infection
`with
`Vero and Ltk-HSV+
`(Ltk-
`HSV-1 at IO pfu/cell)
`are presented
`in Figure
`2. As
`expected,
`Ltk-
`showed
`a small
`peak of activity
`of
`
`In contrast,
`of 0.9.
`tk at an Rf value
`mitochondrial
`infected
`cell
`homogenate,
`has an
`the
`Ltk-HSV+,
`peak migrating
`with an Rf of 0.45. This
`additional
`value
`is in agreement
`with previous
`studies
`of the
`HSV-l-induced
`tk
`(Cheng
`and Ostrander,
`1976).
`cell
`The
`L1210
`homogenate
`showed
`the normal
`pattern
`of mouse
`cytoplasmic
`and mitochondrial
`tk
`with activity migrating
`at an Rf of 0.2 and 0.9, and
`with no detectable
`activity with an Rr of 0.45. Vero
`cells,
`in which
`the HSV-1 was grown,
`similarly
`re-
`vealed
`no
`tk activity
`at an R, of 0.45. The second
`peak of activity
`at Rf = 0.55
`in the electrophoretic
`pattern
`of Vero
`could
`be due
`to
`the mitochondrial
`tk and has an electrophoretic
`mobility
`similar
`to the
`human mitochondrial
`tk (Lee and Cheng,
`1976).
`Studies
`were
`performed
`on
`the electrophoretic
`mobility
`of
`four
`herpes-transformed
`cell
`lines:
`LHlA2-1,
`LH2-1,
`LH5-1
`derived
`from
`cultures
`ex-
`posed
`to 4,2 and 0.25 pg DNA (Table 1, experiment
`to
`2), and LH5C2-2
`derivgd
`from
`cultures
`exposed
`0.5 pg DNA (experiment
`3). These
`lines were main-
`tained
`in continuous
`culture
`in medium
`containing
`HAT
`for approximately
`30 cell doublings.
`The main
`tk activity was consistently
`found
`a.t the position
`Rf
`= 0.45,
`in agreement
`with
`that of Ltk-HSV+
`(Figure
`3). Although
`mouse mitochondrial
`tk
`is present
`at
`Rf = 0.9, no mouse
`cytoplasmic
`tk is found
`in these
`lines.
`
`Merck Ex. 1029, pg 1060
`
`

`
`Cell
`226
`
`;
`
`?
`
`z
`
`5
`
`5
`u
`
`%
`a
`5
`
`2
`s
`
`0.25
`
`0.2
`
`0.15
`
`0.1
`
`0.05
`
`0.05
`0.03
`0.01
`
`0.4
`0.3
`0.2
`0.1
`
`C
`
`I
`0.2
`
`0.4
`
`0.6
`
`0.8
`
`I
`
`0.2
`
`0.4
`
`0.6
`
`0.8
`
`I
`
`Kinase
`
`R f
`of Thymidine
`Pattern
`2. Electrophoretic
`Figure
`of Various
`Cell Lines
`from Cytoplasmic
`Fractions
`to 5%
`applied
`of homogenates
`were
`30,000
`x g supernatants
`polyacrylamide
`disc
`gels. Gels were
`sliced,
`and each
`slice was
`assayed
`for
`thymidine
`kinase
`activity
`as described
`previously
`(Lee
`and Cheng,
`1976). Specific
`activities
`of samples
`are as
`indicated
`in Table
`2. Electrophoretic
`mobilities
`(R, values)
`were
`calculated
`with
`reference
`to the electrophoretic
`mobility
`of bromphenol
`blue.
`(A) Vero;
`(B) L1210;
`(C) Ltk-;
`(D) Ltk-HSV+.
`
`Activities
`
`Cells
`tk in Transformed
`of
`Identity
`Antigenic
`tk effectively
`raised
`against
`purified
`HSV
`Antisera
`the enzymatic
`activity
`of viral,
`but not
`neutralizes
`(Klemperer
`et al.,
`1967). We would
`tk
`cellular,
`predict
`that
`the
`tk activity
`of our
`trans-
`therefore
`fected
`clones
`should
`be completely
`neutralized
`by
`these antisera.
`The experiments
`shown
`in Table
`2
`indicate
`that
`the
`tk activity
`four
`transformed
`of
`lines,
`LH2-1,
`LH5-1,
`LH5C2-2
`and LHlA2-I,
`can be
`neutralized
`by antisera
`to purified
`HSV-1
`tk. The
`tk
`activities
`of Vero,
`Ltk- and A9, a mouse
`L cell with
`wild-type
`tk+, are not neutralized
`by the same anti-
`sera. These
`data demonstrate
`that
`the
`tk activity
`present
`in the
`transformed
`cell
`lines
`is antigenically
`related
`to purified
`HSV-1
`tk. The
`residual
`activity
`remaining
`after
`neutralization
`of
`the
`transformed
`cell extracts may represent
`mouse mitochondrial
`activity.
`It is also apparent
`from
`these data
`that
`
`tk
`the
`
`“IB
`
`012
`
`d.4
`
`1
`0.6
`
`I
`0.8
`
`ICI5
`
`*
`
`1
`
`0.6
`
`1
`
`0.0
`
`I
`
`z
`
`c
`
`0.2
`
`0.4
`
`0.8
`
`I
`
`0.6
`R f
`Kinase
`of Thymidine
`Pattern
`of Four HSV-1 DNA-Transformed
`
`3. Electrophoretic
`Figure
`from Cytoplasmic
`Fractions
`Lines
`lines of mouse
`transfected
`four
`of
`x g supernatants
`The 30,000
`following
`gel
`kinase
`activity
`thymidine
`Ltk-
`ceils were assayed
`for
`activities
`of the
`2. Specific
`in Figure
`electrophoresis
`as described
`samples
`are
`indicated
`in Table 2. (A) LH5C2-2;
`(B) LH5-1;
`(C) LH2-
`1; (D) LHIAP-1.
`
`Activities
`Cell
`
`tk
`
`the
`
`lines have at least 20 times
`transformed
`four
`activity
`of the parental
`Ltk- cell.
`Cells
`Substrate
`Specificity
`tk in Transformed
`of
`As a final
`criterion
`of
`identification,
`the substrate
`specificity
`of the
`tk activity
`found
`in the
`four
`trans-
`formed
`cell
`lines was analyzed.
`Two known
`inhibi-
`tors of herpes-specific
`tk were used, 5-ethyl
`deox-
`yuridine @-ethyl
`dUrd)
`and 5-ally1
`dUrd
`(Cheng
`et
`al., 1976b).
`They
`inhibited
`phosphorylation
`of thy-
`midine
`by 80 and 60%,
`respectively,
`in all
`four cell
`lines. These
`same drugs
`inhibited
`the
`tk activity
`in
`Ltk-HSV+
`cells, but had no effect on extracts
`from
`Vero or A9 cells
`(Table
`3).
`
`tk+ Phenotype
`of the
`Stability
`2
`from experiments
`colonies
`were picked
`Fourteen
`and 3 (Table
`1) and grown
`in HAT medium.
`Of these
`fourteen
`colonies,
`two displayed
`multiple
`abortive
`
`Merck Ex. 1029, pg 1061
`
`

`
`Thymidine
`
`Kinase Gene
`
`in Mouse
`
`Cells
`
`Herpes
`227
`
`Table 2. Specific
`
`Neutralization
`
`of HSV-1 Thymidine
`
`Kinase
`
`with
`Activity
`Preimmune
`
`Serum
`
`with
`
`Activity
`Antiserum
`
`Source
`
`of tk
`
`Units
`per ml
`
`Units
`per mg
`
`Units
`per mg
`
`% Residual
`Activity
`
`Vero
`
`A9
`Ltk-
`
`Ltk-HSV+
`LHI A2-1
`LH2-1
`
`1 .o
`1.67
`
`0.06
`20.0
`1 .I
`1 .I5
`
`0.9
`
`0.25
`1.50
`
`0.007
`2.60
`0.19
`0.15
`
`0.14
`
`0.37
`1.50
`
`0.007
`0.13
`0.017
`0.019
`
`0.015
`
`150
`100
`
`100
`5
`9
`12
`
`IO
`
`Table
`Activity
`
`3. Effect
`Derived
`
`of Pyrimidine
`from Various
`
`Analogs
`Sources
`
`on Thymidine
`
`Kinase
`
`% Activity
`
`in the Presence
`
`of Analogs
`
`Source
`
`of
`
`tk
`
`5-Ethyl
`
`dUrd
`
`5-Ally1 dUrd
`
`Vero
`
`A9
`Ltk-
`
`Ltk-HSV+
`LHlA2-1
`
`105
`100
`
`86
`20
`
`20
`28
`
`85
`100
`
`36
`39
`
`42
`48
`
`LH5-1
`
`0.015
`
`1 .o
`
`0.15
`
`LH5C2-2
`(S-30)
`of homogenates
`x g supernatants
`30,000
`lines were mixed with preimmune
`sera or antisera
`1 tk, and
`tk activity
`was
`assayed
`as described
`Procedures.
`Activity
`is expressed
`both as units
`units
`oer ma orotein
`within
`the S-30
`fraction.
`
`10
`
`cell
`various
`from
`HSV-
`to purified
`in Experimental
`per ml of S-30 and
`
`is
`
`which
`colonies
`small
`formed
`is,
`(that
`colonies
`replated,
`than
`fifty ceils) when
`grew
`larger
`never
`be grown
`for at least 25 cell dou-
`but
`twelve
`could
`blings
`under
`continuous
`selective
`pressure.
`Of
`these
`twelve,
`four were
`chosen
`for
`further
`study
`and have now been carried
`in HAT medium
`for over
`6 months.
`Similarly,
`eleven
`colonies
`were
`picked
`from
`later experiments
`(see below,
`Table
`6). One
`colony
`formed
`abortive
`colonies
`on
`replating,
`and
`ten
`lines
`have
`now
`been maintained
`for several
`months
`of continuous
`passage
`in HAT medium. We
`conclude
`that
`the acquisition
`of the HAT resistance
`phenotype
`and,
`presumably,
`the
`tk+ phenotype
`stable
`under
`selective
`conditions.
`under
`tk phenotype
`To assess
`the stability
`of the
`cells,
`after approxi-
`other
`conditions,
`transformed
`mately
`50 doublings
`in continuous
`culture
`in HAT
`medium,
`were
`plated
`at
`low density
`into one
`of
`three media,
`and
`their
`cloning
`efficiency
`in
`these
`media was examined.
`The media were
`unsupple-
`mented
`growth
`medium,
`growth
`medium
`supple-
`mented with HAT, which
`selects
`for
`the
`tk+ pheno-
`type, or growth medium
`supplemented
`with BdUrd,
`which
`selects
`for
`the
`tk- phenotype.
`The data pre-
`sented
`in Table
`4 are summarized
`as follows;
`the
`cloning
`efficiency
`of the
`tk+-transformed
`cells was
`the same
`in either
`(HAT) or nonselective
`media,
`and
`resembled
`that of
`the parental
`Ltk-
`line
`in nonse-
`lective media
`(30%). The cloning
`efficiency
`of these
`lines
`in the presence
`of BdUrd was
`reduced
`by two
`orders
`of magnitude
`to between
`0.1 and 0.45%.
`It is
`of
`interest
`that
`this
`level of cloning
`efficiency
`is
`some 100
`fold higher
`than
`that of A9, a mouse
`L cell
`derivative,
`or other
`tk+ mouse
`cells.
`In this
`respect,
`these
`cells are similar
`to
`those
`tk+ cells produced
`by
`infection
`of
`tk- cells with ultraviolet-inactivated
`virus
`(Davidson,
`Adelstein
`and Oxman,
`1973).
`
`LH2-1
`LH5-1
`LH5C2-2
`
`26
`24
`
`39
`44
`
`of
`Assay
`described
`with
`respect
`
`out
`carried
`(100 PM) was
`of analogs
`effect
`the
`is calculated
`in Experimental
`Procedures.
`% activity
`to the activity
`in the absence
`of an analog.
`
`as
`
`Efficiency
`4. Cloning
`Table
`Selective
`and Nonselective
`
`of VariousCell
`Media
`
`Lines
`
`in a Variety
`
`of
`
`Cell Line
`
`MEM”
`
`A9
`
`Ltk-
`LH2-1
`LH5-1
`LH5C2-2
`
`3.6 x
`
`IO-’
`
`IO-’
`3.0 x
`2.9 x 10-l
`2.0 x
`IO-’
`2.9 x
`IO-’
`
`HATb
`
`ND
`<IO-’
`
`lo-’
`2.2 x
`2.3 x 10-l
`3.8 x 10-l
`
`BdUrd’
`
`<I
`
`.o x 10-S
`2.7 x
`lo-’
`
`1.0 x 10-z
`2.0 x 10-s
`4.5 x 10-J
`
`a Nonselective
`b tk+
`selective
`hypoxanthine,
`glycine).
`c tk-
`selective
`BdUrd).
`in
`plated
`Cells were
`counted
`after 2 weeks
`
`medium
`
`fetal calf serum).
`5%
`(MEM,
`medium
`5%
`fetal
`calf
`serum,
`(MEM,
`medium
`1 @g/ml aminopterin,
`5 yglml
`thymidine,
`
`15 pg/ml
`15 *g/ml
`
`(MEM,
`
`5%
`
`fetal
`
`calf
`
`serum,
`
`30 @g/ml
`
`and
`replicate,
`as described
`
`were
`colonies
`in Experimental
`
`and
`stained
`Procedures.
`
`and
`
`Isolation
`
`of tk Active
`
`Barn
`
`I
`
`Identification
`Fragment
`of Barn
`the DNA products
`that
`The observation
`tk activity
`cleavage
`of HSV DNA can stably
`transfect
`the spe-
`suggests
`the use of
`this assay
`to
`identify
`kinase
`cific DNAfragment
`containing
`the
`thymidine
`chosen
`gene.
`The experimental
`design
`we have
`involves
`the electrophoretic
`separation
`of specific
`groups
`of DNA and
`ultimately
`of
`individual
`DNA
`fragments.
`The
`fragment
`in which
`the
`tk gene
`re-
`sides
`is then
`readily
`identified
`by transfection
`with
`these
`fractionated
`populations
`of DNA. To this end,
`a Barn
`I digest
`of HSV DNA
`(Figure
`1) was
`fraction-
`ated by electrophoresis
`on a 45 cm, 1% agarose
`slab gel. These DNA
`fragments
`were divided
`into
`five size classes
`and extracted
`from
`the agarose
`slab. DNA was purified
`free of agarose
`by hydroxyl-
`apatite
`and Sephadex
`G-50
`chromatography,
`and
`was again analyzed
`by agarose
`gel electrophoresis
`(Figure
`4).
`The
`isolated
`
`size classes
`
`seen
`
`in Figure
`
`4 were
`
`Merck Ex. 1029, pg 1062
`
`

`
`Cell
`228
`
`13.6-
`
`2.7 -
`2.3
`-
`
`A
`
`V
`
`IV
`
`IJJ
`
`I I
`
`I
`
`5. Transfection
`
`with Size Classes
`
`of Barn
`
`I-Cleaved
`
`HSV-1
`
`Table
`DNA
`
`pg Equivalents
`of HSV-I DNA
`per Dish
`
`Size Class
`
`I
`II
`
`III
`
`IV
`V
`
`4.0
`4.0
`
`4.0
`
`4.0
`4.0
`
`zm
`
`O/5
`313
`
`93/3
`
`O/5
`o/5
`
`Specific
`Activityb
`
`0.0
`0.25
`
`7.75
`
`0.0
`0.0
`
`1.
`a See Table
`calculated
`b Specific
`activity
`lents of HSV-1 DNA.
`
`as before,
`
`but based
`
`on pg equiva-
`
`A
`
`I
`
`2
`
`3
`
`4
`
`5
`
`III
`
`HSV-I DNA
`l-Cleaved
`of Barn
`4. Fractionation
`Figure
`result-
`the
`with Barn
`I endonuclease,
`and
`HSV-1 DNA was digested
`slab gel.
`ant
`fragments
`were
`separated
`on a 45 cm 1% agarose
`This
`preparative
`gel was
`sliced,
`and
`the DNA
`corresponding
`to
`five discrete
`size classes
`was extracted
`from
`the gel and electro-
`phoresed
`on a 0.5% agarose
`slab. The
`five size classes
`of DNA are
`shown
`in slots
`I-V. Slot A contains
`a preparation
`of Eco RI-
`digested
`Ad2 DNA as a size marker
`(Pettersson
`et al., 1973).
`
`the
`following
`experiments
`in transfection
`then used
`total
`same protocol
`as was used
`for unfractionated
`digest.
`The
`results
`of
`this experiment
`are summa-
`rized
`in Table 5. Transfection
`activity
`is restricted
`to
`size class
`III. The small
`amount
`of activity
`seen
`in
`size class
`II probably
`results
`from
`the contamina-
`tion of that class with size class
`III, as can be seen
`in Figure
`4. Of particular
`interest
`is the observation
`that
`the specific
`activity
`of size class
`Ill is about
`colonies
`per
`lo6 ceils per pg genome
`equivalent,
`which
`is about
`20
`times
`the specific
`activity
`of the
`unfractionated
`genome.
`These
`data
`indicate
`that
`the
`tk gene
`is located
`on one of five well
`resolved
`fragments
`ranging
`in molecular
`weight
`from
`2.5
`3.7 kb. This size class was
`further
`fractionated
`into
`its five discrete
`fragments
`(Figure
`5). From
`the mo-
`lar yield of these
`fragments
`after Barn
`I digestion
`of
`
`IO
`
`of the Barn
`
`I Fragment
`
`of HSV-I DNA Contain-
`
`Ill
`
`Isolation
`5.
`Figure
`tk Gene
`ing
`the
`I digest
`4) of a Barn
`Ill (Figure
`in size class
`present
`The DNA bands
`fragments
`on 45 cm
`into
`five
`fractionated
`of HSV-1 DNA were
`were analyzed
`by elec-
`fragments
`agarose
`slab gels. The
`isolated
`slab gel. Slot A contains
`ECO RI-
`trophoresis
`on a 1% agarose
`weight markers.
`Slots 1-5 contain
`cleaved
`Ad2 DNA as molecular
`class
`Ill (see
`Figure
`4). Slot
`the
`isolated
`fragments
`of size
`contains
`the unfractionated
`DNA of size class
`Ill.
`that each of
`total HSV DNA
`(Figure
`l),
`it is probable
`these
`fragments
`represents
`a homogeneous
`spe-
`cies of DNA. These
`individual
`fragments
`obtained
`from
`two separate
`preparations
`of HSV DNA were
`then
`assayed
`for
`their
`ability
`to
`transfect
`the
`tk
`gene
`(Table
`6). Experiments
`1 and 2 both
`indicate
`
`Merck Ex. 1029, pg 1063
`
`

`
`Thymidine
`
`Kinase Gene
`
`in Mouse
`
`Cells
`
`Herpes
`229
`
`in
`only
`resides
`activity
`transfection
`that significant
`frag-
`purified
`fragment
`2 of size class
`III. The other
`little
`ments of class
`III as well as class
`II DNA have
`or no activity. The structural
`gene
`for
`tk is therefore
`in
`contained
`within
`a single DNA
`fragment
`3.4 kb
`length.
`Again
`it should
`be noted
`that
`the specific
`activity
`of band
`2 is about
`20
`fold
`higher
`per ge-
`nome
`equivalent
`than
`unfractionated
`total digest,
`with colonies
`appearing
`in cultures
`treated with as
`little as 0.05 pg HSV-1 DNA equivalents
`or about
`1
`ng of DNA.
`
`Discussion
`
`of
`consists
`virus
`simplex
`of herpes
`The genome
`to
`about
`lo* daltons
`of DNA, sufficient
`information
`direct
`the synthesis
`of 50-100
`proteins
`(Kieff, Bach-
`enheimer
`and Roizman,
`1971). The enzyme
`thymi-
`dine kinase
`consistently
`appears
`shortly
`after
`infec-
`tion, and considerable
`evidence
`indicates
`that
`it is
`coded
`for by the viral genome
`(Honess
`and Watson,
`1974;
`Summers,
`Wagner
`and
`Summers,
`1975).
`Considering
`the
`relatively
`small
`size of
`the
`viral
`genome,
`we would
`expect
`that a specific
`fragment
`tk
`could
`be obtained
`which
`could
`transfer-viral
`the
`activity
`to
`tk- cells.
`In this
`report, we describe
`isolation
`of a 3.4 kb fragment
`of HSV-1 DNA which
`contains
`the structural
`gene
`for
`tk and which
`can
`stably
`transfect
`cells
`lacking
`this activity.
`The ease
`with which
`this gene
`can be
`identified
`in transfec-
`tion experiments
`suggests
`that
`this approach
`may
`be applied
`to the
`localization
`of any gene
`for which
`selection
`criteria
`are available.
`cells
`of Ltk-
`Our studies
`indicate
`that
`treatment
`with Barn
`l-digested
`HSV-1 DNA
`results
`in the sta-
`ble
`transformation
`of a population
`of cells
`now
`expressing
`a tk+ phenotype
`and capable
`of forming
`colonies
`in HAT selection
`media.
`Eco RI-treated
`HSV-1 DNA does not show
`this activity,
`presumably
`because
`this enzyme
`cleaves within
`the
`tk gene.
`is possible,
`however,
`that Eco RI does not
`fragment
`the
`tk gene
`but
`liberates
`a series
`of
`fragments
`capable
`of transfecting
`lytic
`functions
`of HSV. This
`would
`result
`in cell death
`and obviously
`obscure
`the
`transfer
`of tk activity.
`If this were
`the case, we
`would
`expect
`that digestion
`of HSV DNA with both
`Barn
`I and Eco RI should
`leave
`the
`tk structural
`gene
`intact and
`result
`in fragmentation
`of the DNA
`coding
`for
`lytic
`functions.
`Preliminary
`results
`from
`our
`laboratory
`ind:,ate
`that
`this doubly
`digested
`DNA
`is not competent
`in transfection
`assays,
`sug-
`gesting
`that Eco RI
`indecj
`clee
`ies within
`the
`tk
`gene. More
`definitive
`data will
`be obtained
`by
`transfection
`with
`the Eco RI cleavage
`productions
`of the 3.4 kb Barn
`I fragment.
`It is of obvious
`importance
`activity
`transfected
`by Barn
`viral origin.
`The
`spontaneous
`
`tk
`that
`to demonstrate
`l-cleaved
`HSV DNA
`is of
`rate of
`reversion
`of
`
`It
`
`6. Transfection
`Table
`Cleaved
`HSV-1 DNA
`
`with Fractionated
`
`Size Class
`
`III of Barn
`
`I-
`
`Kl
`Equivalents
`per Dish
`
`Experiment
`
`1
`
`Experiment
`
`2
`
`Specific
`Activityb
`
`S/I
`
`Specific
`Activity
`
`XP
`
`2.0
`
`0.7
`
`2.0
`0.7
`1.5
`
`0.5
`
`0.15
`0.05
`1 .o
`2.0
`0.7
`1 .5
`
`2.0
`0.7
`1.5
`
`2.0
`0.7
`
`Size Class
`
`II
`
`Ill
`
`Size Class
`Band 1
`
`Band 2
`
`Band 3
`
`Band 4
`
`Band 5
`
`a See Table
`b See Table
`
`1.
`5.
`
`o/2
`
`o/2
`
`l/2
`
`o/2
`
`19/2
`6/2
`
`513
`
`712
`312
`
`o/2
`o/2
`
`012
`o/2
`
`0.0
`
`0.0
`
`0.25
`
`0.0
`
`19.0
`18.0
`
`30.0
`
`1.75
`2.0
`
`0.0
`0.0
`
`0.0
`0.0
`
`4814
`
`8.0
`
`o/4
`
`0.0
`
`o/4
`
`0.0
`
`o/4
`
`0.0
`
`in
`
`on
`
`it
`is <1O-8. Nevertheless,
`cells
`Ltk-
`recipient
`the
`to characterize
`carefully
`the tk activ-
`was necessary
`by the
`transformed
`cell clones.
`The
`tk
`ity expressed
`activity
`of these HAT-resistant
`clones
`is at least 20
`times greater
`than
`the activity
`detected
`in the Ltk-
`parent.
`The biochemical
`and antigenic
`properties
`of
`this enzyme
`were
`characterized
`by examining
`the neutralization
`of activity
`by specific
`antisera
`raised
`against HSV-1
`tk;
`the electrophoretic
`mobil-
`ity of the enzymatic
`activity;
`and
`the selective
`inhi-
`bition
`of
`tk activity
`by agents
`specific
`for
`the viral
`enzyme.
`For all
`these parameters,
`the enzyme was
`indistinguishable
`from HSV-1
`tk and differed
`from
`either mouse
`or monkey
`cell
`tk. The
`conclusion
`that
`the
`tk activity
`appearing
`in
`transfected
`Ltk-
`cells
`results
`from
`the
`introduction
`and expression
`firm.
`of viral DNA
`therefore
`appears
`transfection
`Barn
`l-treated
`DNA,
`competent
`25 bands
`assays,
`can be
`resolved
`into about
`agarose
`gels. Thrcugh
`a series of electrophoretic
`fractionations
`in concert
`with
`transfection
`assays,
`we have
`identified
`and
`isolated
`a 3.4 kb fragment
`of
`viral DNA containing
`the
`tk gene. This
`fragment
`is
`capable
`of efficiently
`transfecting
`tk activity
`to Ltk-
`cells
`in the absence
`of any additional
`HSV
`informa-
`tion.
`frequency
`The
`2 and 3
`in Table
`
`of
`1
`
`transfection
`is clearly
`
`from experiments
`too
`low
`to allow
`
`a
`
`Merck Ex. 1029, pg 1064
`
`

`
`Cdl
`230
`
`is
`
`tk
`
`I-
`
`dependence.
`of dose
`analysis
`statistical
`reliable
`the purified
`is performed
`with
`transfection
`When
`transfection
`fragment,
`the
`frequency
`of successful
`emerges.
`increases
`and a clear
`dose dependence
`clearly
`The precise
`nature
`of the dose dependence
`broad
`requires
`additional
`experiments
`spanning
`ranges
`of gene
`concentrations.
`These experiments
`may provide
`useful
`information
`for understanding
`the mechanism
`of
`transfection,
`which
`is currently
`not understood.
`Nevertheless,
`several
`interesting
`questions
`emerge
`as a result
`of our data. The effi-
`ciency
`of transfection
`we observe with
`the purified
`fragment
`of Barn
`I digests
`of HSV-1 DNA
`is about
`10
`colonies
`per
`fig DNA per
`lo6 cells. Transfer
`of
`tk
`activity
`occurs with about
`2% the efficiency
`lytic
`of
`transfection
`as assayed
`by plaque
`formation
`using
`purified
`intact HSV-1 DNA
`(personal
`observations).
`It is possible
`that
`this difference
`results
`in part
`from
`the
`fact
`that
`lytic
`transfection
`requires
`only
`the
`transient
`expression
`of viral
`functions
`in recipient
`cells. Detection
`of the
`transfer
`of tk activity
`requires
`the stable
`expression
`of this enzyme
`through
`sev-
`eral cell doublings.
`the
`that
`the observation
`Of
`further
`interest
`is
`tk gene
`containing
`the
`isolated
`Barn
`I fragment
`as-
`effective
`in
`transfecti