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` on May 10, 2016
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`http://science.sciencemag.org/
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`57. This work was supported by grants from the
`57. This work was supported by grants from the
`U.S. Public Health Service, National Institutes
`U.S. Public Health Service, National Institutes
`of Health, to R.A. (CA 16346 and CA 23767) and
`of Health, to R.A. (CA 16346 and CA 23767) and
`to S.S. (CA 17477).
`to S.S. (CA 17477).
`7 July 1980
`7 July 1980
`
`Expression of a Bacterial
`Expression of a Bacterial
`Gene in Mammalian Cells
`Gene in Mammalian Cells
`
`R. C. Mulligan* and P. Berg
`R. C. Mulligan* and P. Berg
`
`The most powerful tools now available
`The most powerful tools now available
`for studying the molecular anatomy of
`for studying the molecular anatomy of
`eukaryote genes and chromosomes, par-
`eukaryote genes and chromosomes, par-
`ticularly those of higher vertebrates, are
`ticularly those of higher vertebrates, are
`restriction enzymes,
`simple
`physical
`restriction enzymes, simple physical
`methods for separating and visualizing
`methods for separating and visualizing
`DNA molecules, molecular cloning, and
`DNA molecules, molecular cloning, and
`rapid DNA sequencing. In just a few
`rapid DNA sequencing. In just a few
`years, the application of these tech-
`years, the application of these tech-
`niques has produced a qualitative change
`niques has produced a qualitative change
`in our views of gene structure and organ-
`in our views of gene structure and organ-
`ization in mammalian and other et.
`ization in mammalian and other ey,-
`karyote organisms. Such newly coined
`karyote organisms. Such newly coined
`terms as gene libraries,
`split genes,
`terms as gene libraries, split genes,
`pseudogenes, and transposons and pic-
`pseudogenes, and transposons and pic-
`turesque references to "'shotgunning,"
`turesque references to "shotgunning,"
`or even ""walking and jumping along
`or even "walking and jumping along
`
`chromosomes," reflect this revolution
`chromosomes," reflect this revolution
`and enrich the lexicon of modern ge-
`and enrich the lexicon of modern ge-
`netics. Solving the nucleotide sequence
`netics. Solving the nucleotide sequence
`of the chromosomal locus encoding the
`of the chromosomal locus encoding the
`entire human 8-globin-like gene cluster,
`entire human 13-globin-like gene cluster,
`a region encompassing about 65 kilobase
`a region encompassing about 65 kilobase
`pairs (kbp) (1), would have been consid-
`pairs (kbp) (1), would have been consid-
`ered visionary 10 years ago, but now that
`ered visionary 10 years ago, but now that
`prospect looms on the horizon as a fea-
`prospect looms on the horizon as a fea-
`sible undertaking.
`sible undertaking.
`Nevertheless, in spite of this impres-
`Nevertheless, in spite of this impres-
`is worth considering
`sive progress it
`sive progress it is worth considering
`whether knowing the molecular anatomi-
`whether knowing the molecular anatomi-
`cal details of genes can, by itself, explain
`cal details of genes can, by itself, explain
`the subtleties of gene expression and reg-
`the subtleties of gene expression and reg-
`ulation during growth and development.
`ulation during growth and development.
`Put another way, can we deduce the
`Put another way, can we deduce the
`mechanisms of transcription initiation,
`mechanisms of transcription initiation,
`splicing, and polyadenylation from the
`splicing, and polyadenylation from the
`R. C. Mulligan is a predoctoral fellow and P. Berg
`R. C. Mulligan is a predoctoral fellow and P. Berg
`is Willson Professor of Biochemistry in the Depart-
`nucleotide sequences of isolated genes?
`is Willson Professor of Biochemistry in the Depart-
`nucleotide sequences of isolated genes?
`ment of Biochemistry, Stanford University Medical
`ment of Biochemistry, Stanford University Medical
`Can we expect to learn from the nucle-
`Can we expect to learn from the nude-
`Center, Stanford, California 94305.
`Center, Stanford, California 94305.
`0036-8075180/0919-1422$01.50/0
`Copyright X) 1980 AAAS
`0036-8075/80/0919-1422501.50/0 Copyright 0 1980 AAAS
`
`1422
`1422
`
`otide sequence of the human y- and /-
`otide sequence of the human y- and $-
`globin genes why the former is expressed
`globin genes why the former is expressed
`only during fetal life and the latter only in
`only during fetal life and the latter only in
`adulthood? Most likely not; at least not -
`adulthood? Most likely not; at least not -
`without an assay for the biological activi-
`without an assay for the biological activi-
`ty of the genes in question.
`ty of the genes in question.
`About 10 years ago we began to con-
`About 10 years ago we began to con-
`sider how the biological activity of isolat-
`sider how the biological activity of isolat-
`ed genes could be assayed. That interest
`ed genes could be assayed. That interest
`coincided with another preoccupation
`coincided with another preoccupation
`concerned with devising a virus-mediat-
`concerned with devising a virus-mediat-
`ed transducing system for cultured mam-
`ed transducing system for cultured mam-
`malian cells. The overlap of these two in-
`malian cells. The overlap of these two in-
`terests culminated in a general approach
`terests culminated in a general approach
`for introducing isolated genes and their
`for introducing isolated genes and their
`modified derivatives into the genomes of
`modified derivatives into the genomes of
`cultured mammalian cells (2). Our imme-
`cultured mammalian cells (2). Our imme-
`diate goal was, and still is, to character-
`diate goal was, and still is, to character-
`ize the physical state, expression, and
`ize the physical state, expression, and
`regulation of the new genes in their
`regulation of the new genes in their
`transduced hosts.
`transduced hosts.
`Because bacteriophages had proved to
`Because bacteriophages had proved to
`be so versatile for transducing genes in-
`be so versatile for transducing genes in-
`to bacterial cells (3), simian virus 40
`to bacterial cells (3), simian virus 40
`(SV40), a mammalian virus, was adopted
`(SV40), a mammalian virus, was adopted
`as the vector to mediate the gene trans-
`as the vector to mediate the gene trans-
`fer. SV40 was chosen because its mini-
`fer. SV40 was chosen because its mini-
`chromosome propagates vegetatively or
`chromosome propagates vegetatively or
`becomes stably integrated into selected
`becomes stably integrated into selected
`host cell genomes. SV40 was also at-
`host cell genomes. SV40 was also at-
`tractive because its genes and their cor-
`tractive because its genes and their cor-
`responding functions had been identified
`responding functions had been identified
`and experiments were under way to map
`and experiments were under way to map
`the genes to specific regions of the vi-
`the genes to specific regions of the vi-
`rus's DNA. Subsequently, the entire
`rus's DNA. Subsequently, the entire
`5243-base pair (bp) sequence of the cir-
`5243-base pair (bp) sequence of the cir-
`cular viral DNA was solved (4, 5), and
`cular viral DNA was solved (4, 5), and
`SCIENCE, VOL. 209, 19 SEPTEMBER 1980
`SCIENCE, VOL. 209, 19 SEPTEMBER 1980
`Merck Ex. 1002, pg 69
`
`Mylan v. Genentech
`IPR2016-00710
`Merck Ex. 1120, Pg. 1
`
`

`

`Downloaded from http://science.sciencemag.org/ on May 10, 2016
`Downloaded from
` on May 10, 2016
`
`http://science.sciencemag.org/
`
`extensive information about the replica-
`extensive information about the replica-
`tion, expression, and regulation of the
`tion, expression, and regulation of the
`viral genome in different cells has be-
`viral genome in different cells has be-
`come available (6).
`come available (6).
`To avoid a dependence on biologically
`To avoid a dependence on biologically
`generated transducing viruses and to in-
`generated transducing viruses and to in-
`crease the probability of obtaining spe-
`crease the probability of obtaining spe-
`cific trafisducing genomes, we elected to
`cific transtnicing genomes, we elected to
`construct, in vitro, recombinants be-
`construct, in vitro, recombinants be-
`tween SV40 DNA and the gene of inter-
`tween SV40 DNA and the gene of inter-
`est. This was accomplished by ligating
`est. This was accomplished by ligating
`appropriate DNA fragments to whole (2)
`appropriate DNA fragments to whole (2)
`or subgenomic segments (7, 8) of viral
`or subgenomic segments (7, 8) of viral
`DNA via enzymatically synthesized ho-
`DNA via enzymatically synthesized ho-
`mopolymeric cohesive termini (2,
`9).
`mopolymeric cohesive termini (2, 9).
`Today, ligation of two DNA molecules is
`Today, ligation of two DNA molecules is
`usually accomplished via ends that are
`usually accomplished via ends that are
`generated by restriction enzyme cleav-
`generated by restriction enzyme cleav-
`age of natural or engineered restriction
`age of natural or engineered restriction
`sites (10).
`sites (10).
`Initially,
`the recombinant genomes
`Initially, the recombinant genomes
`were propagated as virions and trans-
`were propagated as virions and trans-
`duction occurred concomitantly with vi-
`duction occurred concomitantly with vi-
`rus infection (7, 8, 10). Since this experi-
`rus infection (7, 8, 10). Since this experi-
`mental design requires that the recombi-
`mental design requires that the recombi-
`nant genomes replicate, they must con-
`nant genomes replicate, they must con-
`tain the origin of SV40 DNA replication
`tain the origin of SV40 DNA replication
`(ori). Furthermore, to encapsidate the
`(ori). Furthermore, to encapsidate the
`recombinant genome into a virion, the
`recombinant genome into a virion, the
`DNA molecule must be smaller than 5.3
`DNA molecule must be smaller than 5.3
`kbp, that is, about one mature viral DNA
`kbp, that is, about one mature viral DNA
`length. Since the vector lacks genetic
`length. Since the vector lacks genetic
`functions coded by the excised DNA
`functions coded by the excised DNA
`segment, the recombinant genomes are
`segment, the recombinant genomes are
`defective and, therefore, must be propa-
`defective and, therefore, must be propa-
`gated with a helper virus that supplies
`gated with a helper virus that supplies
`the missing gene product or products. In
`the missing gene product or products. In
`our protocol (7, 8, 10) the recombinant
`our protocol (7, 8, 10) the recombinant
`genome retains at least one functioning
`genome retains at least one functioning
`virus gene, and consequently, it can
`virus gene, and consequently, it can
`complement a defective gene in the help-
`complement a defective gene in the help-
`er virus. For example, recombinants in
`er virus. For example, recombinants in
`which the DNA insert replaces all or part
`which the DNA insert replaces all or part
`of SV40's late region can be propagated
`of SV40's late region can be propagated
`with SV40 mutants that have a defective
`with SV40 mutants that have a defective
`early region (for example, tsA mutants at
`early region (for example, tsA mutants at
`high temperature); similarly, recombi-
`high temperature); similarly, recombi-
`nants in which early region segments are
`nants in which early region segments are
`replaced by the DNA implant can be
`replaced by the DNA implant can be
`propagated with a helper that is defective
`propagated with a helper that is defective
`in its late region (for example, tsB mu-
`in its late region (for example, tsB mu-
`tants at high temperature).
`tants at high temperature).
`Our early attempts to obtain expres-
`Our early attempts to obtain expres-
`sion of cloned segments as distinct mes-
`sion of cloned segments as distinct mes-
`senger RNA's (mRNA) and proteins
`senger RNA's (mRNA) and proteins
`were negative (7, 8). Subsequently, re-
`were negative (7, 8). Subsequently, re-
`combinant genomes containing either a
`combinant genomes containing either a
`1B-globin complementary DNA
`rabbit
`rabbit 13-globin complementary DNA
`(cDNA) (11), a Drosophila melanogaster
`(cDNA) (11), a Drosophila melanogaster
`gene for histone H2b (12) or a cDNA
`gene for histone H2b (12) or a cDNA
`coding for mouse dihydrofolate reduc-
`coding for mouse dihydrofolate reduc-
`tase (DHFR) (13), in place of portions of
`tase (DHFR) (13), in place of portions of
`SV40's late region, were constructed.
`SV40's late region, were constructed.
`After infection of monkey cells, each of
`After infection of monkey cells, each of
`the im-
`the recombinants expresses
`im-
`the recombinants expresses the
`planted gene sequence as novel hybrid
`planted gene sequence as novel hybrid
`19 SEPTEMBER 1980
`19 SEPTEMBER 1980
`
`mRNA's; moreover, the proteins 13-glo-
`mRNA's; moreover, the proteins 13-glo-
`bin (10), histone H2b (14), and mouse
`bin (10), histone H2b (14), and mouse
`DHFR (15) are synthesized at levels
`DHFR (15) are synthesized at levels
`comparable to those of SV40 late pro-
`comparable to those of SV40 late pro-
`teins. Similar successes in obtaining ex-
`teins. Similar successes in obtaining ex-
`pression of cloned genes have been
`pression of cloned genes have been
`achieved by Hamer and Leder with re-
`achieved by Hamer and Leder with re-
`combinants carrying the mouse genomic
`combinants carrying the mouse genomic
`,3-globin (16) or a-globin (17) genes, a re-
`13-globin (16) or a-globin (/7) genes, a re-
`sult that established proper splicing of
`sult that established proper splicing of
`the globin intervening sequences and
`the globin intervening sequences and
`translation of the resulting mRNA's in a
`translation of the resulting mRNA's in a
`heterologous host.
`heterologous host.
`
`E. coli XGPRT differs from the anal-
`E. coli XGPRT differs from the anal-
`ogous mammalian enzyme, hypoxan-
`ogous mammalian enzyme, hypoxan-
`thine-guanine phosphoribosyltransferase
`thine-guanine phosphoribosyltransferase
`(HGPRT), in that xanthine is consid-
`(HGPRT), in that xanthine is consid-
`erably more active as a substrate than
`erably more active as a substrate than
`hypoxanthine in nucleotide
`synthesis
`hypoxanthine in nucleotide synthesis
`(19). By contrast, mammalian HGPRT
`(19). By contrast, mammalian HGPRT
`does not utilize xanthine efficiently as a
`does not utilize xanthine efficiently as a
`substrate; indeed, mammalian cells do
`substrate; indeed, mammalian cells do
`not convert xanthine toxainthylic acid or
`not convert xanthine to.xanthylic acid or
`to guanylic acid at a significant rate.
`to guanylic acid at a significant rate.
`In this article we describe the isolation
`In this article we describe the isolation
`ofEcogpt and its recombination with ap-
`of Ecogpt and its recombination with ap-
`
`Summary. Transfection of cultured monkey kidney cells with recombinant DNA
`Summary. Transfection of cultured monkey kidney cells with recombinant DNA
`constructed with a cloned Escherichia coli gene that codes for xanthine-guanine
`constructed with a cloned Escherichia colt gene that codes for xanthine-guanine
`phosphoribosyltransferase and several different SV40 DNA-based vectors, results in
`phosphoribosyltransferase and several different SV40 DNA-based vectors, results in
`the synthesis of readily measurable quantities of the bacterial enzyme. Moreover, the
`the synthesis of readily measurable quantities of the bacterial enzyme. Moreover, the
`physiological defect in purine nucleotide synthesis characteristic of human Lesch-
`physiological defect in purine nucleotide synthesis characteristic of human Lesch-
`Nyhan cells can be overcome by the introduction of the bacterial gene into these cells.
`Nyhan cells can be overcome by the introduction of the bacterial gene into these cells.
`
`Until recently, our principal focus has
`Until recently, our principal focus has
`been to exploit the ability of the recombi-
`been to exploit the ability of the recombi-
`nant genomes to replicate in the virus's
`nant genomes to replicate in the virus's
`permissive host. During vegetative mul-
`permissive host. During vegetative mul-
`tiplication, the transduced genomes are
`tiplication, the transduced genomes are
`amplified about 104- to 105-fold, thereby
`amplified about 104- to 105-fold, thereby
`ensuring high yields of the gene prod-
`ensuring high yields of the gene prod-
`ucts. Such studies have been informative
`ucts. Such studies have been informative
`about the necessity and mechanistic sub-
`about the necessity and mechanistic sub-
`tleties of RNA splicing, the rules govern-
`tleties of RNA splicing, the rules govern-
`ing expression of coding sequences in-
`ing expression of coding sequences in-
`serted at different loci in SV40 DNA, and
`serted at different loci in SV40 DNA, and
`about many facets of SV40 gene expres-
`about many facets of SV40 gene expres-
`sion itself. But this experimental design
`sion itself. But this experimental design
`also has a distinct shortcoming: The
`also has a distinct shortcoming: The
`cell is killed during the course of the
`cell is killed during the course of the
`infection, thereby precluding the oppor-
`infection, thereby precluding the oppor-
`tunity to monitor the transduced gene's
`tunity to monitor the transduced gene's
`expression in continuously multiplying
`expression in continuously multiplying
`cells. Moreover, our current collection
`cells. Moreover, our current collection
`of recombinants can only be studied in
`of recombinants can only be studied in
`permissive cells, that is, those that can
`permissive cells, that is, those that can
`amplify the recombinant genomes. This
`amplify the recombinant genomes. This
`constraint excludes many specialized
`constraint excludes many specialized
`and differentiated animal cells as hosts
`and differentiated animal cells as hosts
`for the transduced genes.
`for the transduced genes.
`To circumvent these disadvantages,
`To circumvent these disadvantages,
`we sought to develop transducing vec-
`we sought to develop transducing vec-
`tors that could be introduced and main-
`tors that could be introduced and main-
`tained in a variety of cells. Our initial at-
`tained in a variety of cells. Our initial at-
`tempts in this direction indicated that our
`tempts in this direction indicated that our
`approach would be facilitated by the
`approach would be facilitated by the
`availability of a gene whose function
`availability of a gene whose function
`could be selected for. Since our experi-
`could be selected for. Since our experi-
`ence indicated that the proper position-
`ence indicated that the proper position-
`ing of protein coding sequences in SV40
`ing of protein coding sequences in SV40
`vectors would ensure their expression in
`vectors would ensure their expression in
`transduced cells (18), we explored the
`transduced cells (18), we explored the
`possibility of using a bacterial gene for
`possibility of using a bacterial gene for
`that purpose. The gene chosen was the
`that purpose. The gene chosen was the
`Escherichia coli gene (Ecogpt) coding
`Escherichia coli gene (Ecogpt) coding
`for the enzyme xanthine-guanine phos-
`for the enzyme xanthine-guanine phos-
`phoribosyltransferase
`(XGPRT).
`The
`phoribosyltransferase
`(XGPRT). The
`
`propriate SV40 DNA based transducing
`propriate SV40 DNA based transducing
`vectors. Transfection of a variety of cul-
`vectors. Transfection of a variety of cul-
`tured mammalian cells with such re-
`tured mammalian cells with such re-
`combinant DNA's results in the forma-
`combinant DNA's results in the forma-
`tion of readily measurable quantities of
`tion of readily measurable quantities of
`coli XGPRT. Moreover, human
`E.
`E. coli XGPRT. Moreover, human
`Lesch-Nyhan cells that have been trans-
`Lesch-Nyhan cells that have been trans-
`fected with vectors containing Ecogpt
`fected with vectors containing Ecogpt
`DNA grow under conditions in which the
`DNA grow under conditions in which the
`parental cells do not survive. This result
`parental cells do not survive. This result
`indicates that E. coli XGPRT can over-
`indicates that E. coli XGPRT can over-
`come the Lesch-Nyhan cell's physiologi-
`come the Lesch-Nyhan cell's physiologi-
`cal defect in purine nucleotide synthesis,
`cal defect in purine nucleotide synthesis,
`and suggests that Ecogpt may be a gener-
`and suggests that Ecogpt may be a gener-
`ally useful selectable marker for mam-
`ally useful selectable marker for mam-
`malian cells.
`malian cells.
`
`Isolation of Ecogpt for Introduction into
`Isolation of Ecogpt for Introduction into
`SV40 DNA Vectors
`SV40 DNA Vectors
`Ecogpt was obtained by a series of ma-
`Ecogpt was obtained by a series of ma-
`nipulations and subcloning operations
`nipulations and subcloning operations
`(Fig. 1). The existence and availability of
`(Fig. 1). The existence and availability of
`the transducing phage Xgpt (see legend to
`the transducing phage Xgpt (see legend to
`Fig. 1) greatly facilitated the isolation in
`Fig. 1) greatly facilitated the isolation in
`that it provided a more enriched source
`that it provided a more enriched source
`of the gpt gene than E. coli DNA itself.
`of the gpt gene than E. coli DNA itself.
`Equally crucial to the success of the gene
`Equally crucial to the success of the gene
`isolation was the availability of suitable
`isolation was the availability of suitable
`Gpt- mutants ofE. coli (20) whose trans-
`Gpt- mutants of E. coli (20) whose trans-
`formation to Gpt+ could be readily mon-
`formation to Gpt+ could be readily mon-
`itored. With one notable exception, the
`itored. With one notable exception, the
`subcloning procedure outlined in Fig. 1
`subcloning procedure outlined in Fig. 1
`employs the same strategy used in ob-
`employs the same strategy used in ob-
`taining Ecotdk, the gene coding for thy-
`taining Ecotdk, the gene coding for thy-
`midine kinase (8). In this instance, how-
`midine kinase (8). In this instance, how-
`ever, the 5' and 3' ends of the gpt-con-
`ever, the 5' and 3' ends of the gpt-con-
`taining DNA fragment has been modified
`taining DNA fragment has been modified
`to contain either a Hind III or a Bam HI
`to contain either a Hind III or a Bam HI
`cohesive end. As a result, ligation of the
`cohesive end. As a result, ligation of the
`gpt fragment to the corresponding cohe-
`gpt fragment to the corresponding cohe-
`sive ends of the pBR322 plasmid and
`sive ends of the pBR322 plasmid and
`1423
`1423
`Merck Ex. 1002, pg 70
`
`Merck Ex. 1120, Pg. 2
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`
`hours after infection with SVGT5-gpt(l)
`hours after infection with SVGTS-gpt(1)
`contain at least 40 times more XPRT ac-
`contain at least 40 times more XPRT ac-
`tivity than uninfected CVl extracts (in-
`tivity than uninfected CV1 extracts (in-
`set in Fig. 3A). Insignificant XPRT activ-
`set in Fig. 3A). Insignificant XPRT activ-
`ity was detected in comparable extracts
`ity was detected in comparable extracts
`from cells infected with SV40 or SVGT5.-
`from cells infected with SV40 or SVGT5-
`gpt(e). Also, Fig. 3A shows that in cells
`gpt(e). Also, Fig. 3A shows that in cells
`infected with SVGT5-gpt(l) nearly half of
`infected with SVGTS-gpt(1) nearly half of
`the enzymatic activity that converts 14C-
`the enzymatic activity that converts '4C-
`labeled guanine to 14C-labeled guanosine
`labeled guanine to '4C-labeled guanosine
`monophosphate (GMP) resembles E. coli
`monophosphate (GMP) resembles E. coli
`XGPRT in its insensitivity to inhibition
`XGPRT in its insensitivity to inhibition
`by unlabeled hypoxanthine. By contrast,
`by unlabeled hypoxanthine. By contrast,
`the same reaction with uninfected cell
`the same reaction with uninfected cell
`extracts or extracts obtained after infec-
`extracts or extracts obtained after infec-
`tions with either SV40 or SVGT5-gpt(e)
`tions with either SV40 or SVGT5-gpt(e)
`is completely inhibited by unlabeled
`is completely inhibited by unlabeled
`hypoxanthine, a property characteristic
`hypoxanthine, a property characteristic
`of the cellular HGPRT.
`of the cellular HGPRT.
`Electrophoretic evidence also sup-
`Electrophoretic evidence also sup-
`ports the contention that E. coli XGPRT
`ports the contention that E. coli XGPRT
`is formed after infection with SVGT5-
`is formed after infection with SVGTS-
`gpt(l) (Fig. 3B). After polyacrylamide gel
`gpt(1) (Fig. 3B). After polyacrylamide gel
`electrophoresis of extracts from cells in-
`electrophoresis of extracts from cells in-
`