which the sedimentary layers would iso-
` which the sedimentary layers would iso-
` late fluids from the surface for long peri-
` late fluids from the surface for long peri-
` ods.
` ods.
` In our previous examples of a strategy
` In our previous examples of a strategy
` for waste disposal in crystalline rocks,
` for waste disposal in crystalline rocks,
` the role of buoyancy-induced flow pro-
` the role of buoyancy-induced flow pro-
` duced by heat generated in the reposi-
` duced by heat generated in the reposi-
` tory has not been mentioned. This driv-
` tory has not been mentioned. This driv-
` ing mechanism could be significant for
` ing mechanism could be significant for
` inducing flows and transporting nuclear
` inducing flows and transporting nuclear
` wastes. Recent work (6), however, shows
` wastes. Recent work (6), however, shows
` that if the waste is allowed to cool for 40
` that if the waste is allowed to cool for 40
` to 60 or 70 years, depending on the waste
` to 60 or 70 years, depending on the waste
` type, the heat would be reduced to the
` type, the heat would be reduced to the
` point where buoyancy-induced flow
` point where buoyancy-induced flow
` would not be significant.
` would not be significant.
`
` Conclusions
` Conclusions
`
` The examples presented above illus-
` The examples presented above illus-
` trate relatively simple alternatives to the
` trate relatively simple alternatives to the
` current concept of a repository situated
` current concept of a repository situated
`
` in a single geologic medium. With very
` in a single geologic medium. With very
` little change in the present approach,
` little change in the present approach,
` additional natural barriers can be
` additional natural barriers can be
` brought into play. There are a number of
` brought into play. There are a number of
` barriers to migration of the wastes: (i)
` barriers to migration of the wastes: (i)
` the waste form and its capsule; (ii) engi-
` the waste form and its capsule; (ii) engi-
` neered barriers within the repository,
` neered barriers within the repository,
` such as a low-permeability, highly sorp-
` such as a low-permeability, highly sorp-
` tive backfill; and (iii) the migration path
` tive backfill; and (iii) the migration path
` back to the biosphere through the
` back to the biosphere through the
` ground-water flow system, which in our
` ground-water flow system, which in our
` examples includes flow through the crys-
` examples includes flow through the crys-
` talline rocks as well as overlying sedi-
` talline rocks as well as overlying sedi-
` mentary rocks in which sorption could
` mentary rocks in which sorption could
` provide yet an additional barrier.
` provide yet an additional barrier.
` By selecting an environment in which
` By selecting an environment in which
` a crystalline rock mass is beneath a
` a crystalline rock mass is beneath a
` sedimentary rock blanket with suitable
` sedimentary rock blanket with suitable
` hydrologic characteristics, one has the
` hydrologic characteristics, one has the
` advantage that (i) ground-water flow can
` advantage that (i) ground-water flow can
` be investigated with conventional, well-
` be investigated with conventional, well-
` understood technology; (ii) under favor-
` understood technology; (ii) under favor-
` able circumstances, the flow system can
` able circumstances, the flow system can
`
` work as an active barrier, so that a long
` work as an active barrier, so that a long
` migration and very slow path for the
` migration and very slow path for the
` wastes to the bIosphere can be assured;
` wastes to the bIosphere can be assured;
` and (iii) the wastes can be emplaced in a
` and (iii) the wastes can be emplaced in a
` setting in which the ground water is
` setting in which the ground water is
` nonpotable (salty) and not a potentially
` nonpotable (salty) and not a potentially
` attractive resource, thus minimizing the
` attractive resource, thus minimizing the
` possibility of future human intrusion at
` possibility of future human intrusion at
` the site. This is a disposal strategy wor-
` the site. This is a disposal strategy wor-
` thy of careful evaluation.
` thy of careful evaluation.
`
` References and Notes
` References and Notes
`
` 1. J. D. Bredehoeft, A. W. England, D. B. Stew-
` 1. J. D. Bredehoeft, A. W. England, D. B. Stew-
` art, N. J. Trask, I. J. Winograd, U.S. Geol.
` art, N. J. Trask, I. J. Winograd, U.S. Geol.
` Surv. Circ. 779 (]978).
` Surv. Circ. 779 (]978).
` 2. J. H. Healy, W. H. Jackson, J. R. Van Schaack,
` 2. J. H. Healy, W. H. Jackson, J. R. Van Schaack,
` U.S. Geol. Surv. Open-File Rep. 832 (1966).
` U.S. Geol. Surv. Open-File Rep. 832 (1966).
` 3. W. F. Brace, Int. J. Rock Mech. Min. Sci. 17,
` 3. W. F. Brace, Int. J. Rock Mech. Min. Sci. 17,
` 24] (]980).
` 24] (]980).
` 4. R. A. Freeze and P. A. Witherspoon, Water
` 4. R. A. Freeze and P. A. Witherspoon, Water
` Resous. Res. 2, 64] (1966).
` Resous. Res. 2, 64] (1966).
` 5. , ibid. 3, 628 (]967).
` 5. , ibid. 3, 628 (]967).
` 6. United Kingdom Atomic Energy Authority,
` 6. United Kingdom Atomic Energy Authority,
` Rep. ND-R-514R (]980).
` Rep. ND-R-514R (]980).
` 7. H. E. Vokes and J. Edwards, Jr., Md. Geol.
` 7. H. E. Vokes and J. Edwards, Jr., Md. Geol.
` Surv. Bull. 19 (]957).
` Surv. Bull. 19 (]957).
` 8. P. B. King, The Tectonics of Middle North
` 8. P. B. King, The Tectonics of Middle North
` America (Princeton Univ. Press, Princeton,
` America (Princeton Univ. Press, Princeton,
` N.J., ]95]).
` N.J., ]95]).
`
`
`
` Dissections and Reconstructions of Dissections and Reconstructions of
`
`
`
` Genes and Chromosomes Genes and Chromosomes
`
`
`
` Paul Berg Paul Berg
`
` The Nobel lecture aSords a welcome
` The Nobel lecture aSords a welcome
` opportunity to express my gratitude and
` opportunity to express my gratitude and
` admiration to the numerous students and
` admiration to the numerous students and
` colleagues with whom I have worked
` colleagues with whom I have worked
` and shared, alternately, the elation and
` and shared, alternately, the elation and
` disappointment of venturing into the un-
` disappointment of venturing into the un-
` known. Without their genius, persever-
` known. Without their genius, persever-
` ance, and stimulation much of our work
` ance, and stimulation much of our work
` would not have flourished. Those who
` would not have flourished. Those who
` have worked with students and experi-
` have worked with students and experi-
` enced the discomfort of their curiosity,
` enced the discomfort of their curiosity,
` the frustrations of their obstinacy, and
` the frustrations of their obstinacy, and
`
` Copyright t ]98] by the Nobel Foundation.
` Copyright t ]98] by the Nobel Foundation.
` The author is the Willson Professor of Biochemis-
` The author is the Willson Professor of Biochemis-
` try in the Department of Biochemistry, Stanford
` try in the Department of Biochemistry, Stanford
` University School of Medicine, Stanford, California
` University School of Medicine, Stanford, California
` 94305. This article is the lecture he delivered in
` 94305. This article is the lecture he delivered in
` Stockholm on 8 December ]980 when he received
` Stockholm on 8 December ]980 when he received
` the Nobel Prize in Chemistry, which he shared with
` the Nobel Prize in Chemistry, which he shared with
` Frederick Sanger and Walter Gilbert. The article is
` Frederick Sanger and Walter Gilbert. The article is
` published here with permission from the Nobel
` published here with permission from the Nobel
` Foundation and will also be included in the complete
` Foundation and will also be included in the complete
` volume of Les Prix Nobel en 1980 as well as in the
` volume of Les Prix Nobel en 1980 as well as in the
` series Nobel Lectures (in English) published by
` series Nobel Lectures (in English) published by
` Elsevier Publishing Company, Amsterdam and New
` Elsevier Publishing Company, Amsterdam and New
` York. The lectures by Dr. Sanger and Dr. Gilbert
` York. The lectures by Dr. Sanger and Dr. Gilbert
` will be published in a subsequent issue.
` will be published in a subsequent issue.
`
` .
` .
`
` the exhilaration of their growth know
` the exhilaration of their growth know
` firsthand the magnitude of their contri-
` firsthand the magnitude of their contri-
` butions. Each in our common effort left a
` butions. Each in our common effort left a
` mark on the other and, I trust, each
` mark on the other and, I trust, each
` richer from the experience. I have also
` richer from the experience. I have also
` been fortunate to have two devoted re-
` been fortunate to have two devoted re-
` search assistants, Marianne Dieckmann
` search assistants, Marianne Dieckmann
` and June Hoshi, who have labored dili-
` and June Hoshi, who have labored dili-
` gently and effectively, always with un-
` gently and effectively, always with un-
` derstanding and sympathy for my idio-
` derstanding and sympathy for my idio-
` syncracles.
` syncracles.
` I have also been blessed with an amaz-
` I have also been blessed with an amaz-
` ing group of colleagues at Stanford Uni-
` ing group of colleagues at Stanford Uni-
` versity who have created as stimulating
` versity who have created as stimulating
` and liberating an environment as one
` and liberating an environment as one
` could long for. Their many achievements
` could long for. Their many achievements
` have been inspirational, and without
` have been inspirational, and without
` their helpintellectually and material-
` their helpintellectually and material-
` ly-my efforts would have been severely
` ly-my efforts would have been severely
` handicapped. I am particularly grateful
` handicapped. I am particularly grateful
` to Arthur Kornberg and Charles Yan-
` to Arthur Kornberg and Charles Yan-
` ofsky, both longtime close personal
` ofsky, both longtime close personal
` friends, for their unstinting interest, en-
` friends, for their unstinting interest, en-
`
` couragement, support, and criticism of
` couragement, support, and criticism of
` my work, all of which enabled me to
` my work, all of which enabled me to
` grow and thrive. And finally, there is my
` grow and thrive. And finally, there is my
` wife, Millie, without whom the rare tri-
` wife, Millie, without whom the rare tri-
` umphs would have lost their luster. ller
` umphs would have lost their luster. ller
` strength, assent, and encouragement
` strength, assent, and encouragement
` freed me to immerse myself in research.
` freed me to immerse myself in research.
` Certainly my work could not have
` Certainly my work could not have
` taken place without the generous and
` taken place without the generous and
` enlightened support of the U.S. National
` enlightened support of the U.S. National
` Institutes of Health, the National Sci-
` Institutes of Health, the National Sci-
` ence Foundation, the American Cancer
` ence Foundation, the American Cancer
` Society, and numerous foundations and
` Society, and numerous foundations and
` individuals who invested their wealth in
` individuals who invested their wealth in
` our research.
` our research.
`
`
` Although we are sure not to know every- Although we are sure not to know every-
`
` thing and rather likely not to know very thing and rather likely not to know very
`
` much, we can know anything that is known to much, we can know anything that is known to
`
` man, and may, with luck and sweat, even find man, and may, with luck and sweat, even find
`
` out some things that have not before been out some things that have not before been
`
` known to man. J. ROBERT OPPENHEIMER known to man. J. ROBERT OPPENHEIMER
`
`
` Although the concept that genes trans- Although the concept that genes trans-
`
` mit and control hereditary characteris- mit and control hereditary characteris-
`
` tics took hold early in this century, igno- tics took hold early in this century, igno-
`
` rance about the chemical nature of genes rance about the chemical nature of genes
`
` forestalled most inquiries into how they forestalled most inquiries into how they
`
` function. All of this changed as a result function. All of this changed as a result
`
` of several dramatic developments during of several dramatic developments during
`
` the 1940's to 1960's. First, Beadle and the 1940's to 1960's. First, Beadle and
`
` Tatum's researches (1-3) lent strong sup- Tatum's researches (1-3) lent strong sup-
`
` port for earlier (4) and widespread specu- port for earlier (4) and widespread specu-
`
` lations that genes control the formation lations that genes control the formation
`
` of proteins (enzymes); indeed, the dic- of proteins (enzymes); indeed, the dic-
`
` tum, "one geneone protein," intensi- tum, "one geneone protein," intensi-
`
` fied the search for the chemical defini- fied the search for the chemical defini-
`
` tion of a gene. The discovery by Avery tion of a gene. The discovery by Avery
`
` and his colleagues (S) and subsequently and his colleagues (S) and subsequently
`
` by Hershey and Chase (6) that genetic by Hershey and Chase (6) that genetic
`
` information is encoded in the chemical information is encoded in the chemical
`
` 296 296
`
`
`
` 0036-8075/8]/07]7-0296$02.00/0 Copyright (¢ ]98] AAAS 0036-8075/8]/07]7-0296$02.00/0 Copyright (¢ ]98] AAAS
`
`
`
` SCIENCE, VOL. 2 ] 3, ] 7 JULY ] 98 ] SCIENCE, VOL. 2 ] 3, ] 7 JULY ] 98 ]
`
`This content downloaded from 128.97.27.20 on Wed, 25 May 2016 17:20:18 UTC
`All use subject to http://about.jstor.org/terms
`
`Merck Ex. 1004, pg 80
`
`

`

` structure of deoxyribonucleic acid
` (DNA) provided the first clue. Watson
` and Crick's solution (7) of the molecular
` structure of DNA-the three dimension-
` al arrangement of the polymerized nucle-
` otide subunits-not only revealed the
` basic design of gene structure but also
` the outlines of how genes are replicated
` and function. Suddenly, genes shed their
` purely conceptual and statistical charac-
` terizations and acquired defined chemi-
` cal identities. Genetic chemistry, or mo-
` lecular biology as it has frequently been
` called, was born.
` Until a few years ago, much of what
` was known about the molecular details
` of gene structure, organization, and
` function had been learned in studies with
` prokaryote microorganisms and the vi-
` ruses that inhabit them, particularly the
` bacterium Escherichia coli and the T and
` lambdoid bacteriophages. These orga-
` nisms were the favorites of molecular
` biologists because they can be propagat-
` ed readily and rapidly under controllable
` laboratory conditions. More significant-
` ly, utilizing several means of natural
` genetic exchanges characteristic of these
` organisms and phages, the mapping and
` manipulation of their relatively small ge-
` nomes became routine. As a conse-
` quence, discrete DNA molecules, con-
` taining one or a few genes, were isolated
` in sufficient quantity and purity to permit
` extensive characterizations of their nu-
` cleotide sequences and chromosomal or-
` ganization. Moreover, such isolated ge-
` netic elements provided the models, sub-
` strates, and reagents needed to investi-
` gate a wide range of basic questions: the
` chemical basis of the genetic code; muta-
` genesis; the mechanisms of DNA and
` chromosome replication, repair, and re-
` combination; the details of gene expres-
` sion and regulation.
` The astounding successes in defining
` the genetic chemistry of prokaryotes
` during the l950's and 1960's were both
` exhilarating and challenging. Not sur-
` prisingly, I and others wondered wheth-
` er the more complex genetic structures
` of eukaryote organisms, particularly
` those of mammalian and human cells,
` were organized and functioned in analo-
` gous ways. Specifically, did the require-
` ments of cellular differentiation and in-
` tercellular communication, distinctive
` characteristics of multicellular orga-
` nisms, require new modes of genome
` structure, organization, function, and
` regulation? Were there just variations of
` the prokaryote theme or wholly new
` principles waiting to be discovered in
` explorations of the genetic chemistry o
` higher organisms? It seemed important
` to try to find out.
`
` ]7 JULY ]98]
`
` SV40's Minichromosome
`
` Sometime during 1965 to 1966 I be-
` came acquainted with Renato Dulbec-
` co's work on the then newly discovered
` polyoma virus. The growing sophistica-
` tion of animal cell culture methods had
` made it possible for Dulbecco's labora-
` tory to monitor and quantify the virus'
` growth cycle in vitro (8). Particularly
` significant was the discovery that the
` entire virus genome resided in a single,
` relatively small, circular DNA molecule,
` one that could accommodate about five
` to eight genes (9). l was intrigued by the
` resemblance between polyoma' s life-
` styles and those of certain bacterio-
` phages. On the one hand, polyoma re-
` sembled lytic bacteriophages in that the
` virus could multiply vegetatively, kill its
` host, and produce large numbers of virus
` progeny (8). There was also a tantalizing
` similarity to lysogenic bacteriophages,
` since some infections yielded tumorigen-
` ic cells (10, 11). The acquisition of new
` morphologic and growth characteristics,
` as well as certain virus-specific proper-
` ties, suggested that tumorigenesis and
` cell transformation resulted from cova-
` lent integration of viral DNA into the
` cell's chromosomal DNA and the conse-
` quent perturbation of cell growth control
` by the expression of virus genes (12, 13).
` These discoveries and provocative
` speculations, together with an eagerness
` to find an experimental - model with
` which to study the mechanisms of mam-
` malian gene expression and regulation,
` prompted me to spend a year's sabbati-
` cal leave (1967 to 1968) in Dulbecco's
` laboratory at the Salk Institute. The
` work and valuable discussions we car-
` ried on during that time (14) reinforced
` my conviction that the tumor virus sys-
` tem would reveal interesting features
` about mammalian genetic chemistry.
` For somewhat technical reasons when
` I returned to Stanford, I adopted SV40, a
` related virus, to begin our own research
` program. SV40 virions are nearly spheri-
` cal particles whose capsomers are orga-
` nized in icosahedral symmetry (Fig. la).
` The virions contain three viral coded
` polypeptides and a single double-strand-
` ed circular DNA molecule (Fig. lb), that
` is normally associated with four his-
` tones, H2a, H2b, H3, and H4 in the form
` of condensed (Fig. lc) or beaded (Fig.
` ld) chromatin-like structures. The SV40
` DNA contains 5243 nucleotide pairs
` [5.24 kilobase pairs (kbp)], the entire
` sequence of which is known from studies
` in the laboratories of S. Weissman (15)
` and W. Fiers (16). Coding information for
` five (and possibly six) proteins is con-
` tained in the DNA nucleotide sequence.
`
` Three of the proteins occur in mature
` virions, possibly as structural compo-
` nents of the capsid shell, although one
` might be associated with the DNA and
` have a regulatory function (17). Of the
` two nonvirion proteins encoded in the
` DNA sequence, one is localized in the
` cell nucleus (large T antigen) and func-
` tions in viral DNA replication and cell
` transformation; the other, found in the
` cytoplasm (small t antigen), enhances
` the efficiency of cell transformation (18).
` Other proteins related in structure to
` large T antigen have been speculated
` about but their structures and functions
` are unclear.
` Restriction endonucleases have
` played a crucial role in defining the phys-
` ical and genetic organization of the SV40
` genome (19, 20). The restriction or cleav-
` age sites served as coordinates for a
` physical map of the viral DNA; the avail-
` ability of such map coordinates made it
` possible to locate, accurately, particular
` physical features and genetic loci. In this
` system of map coordinates, the single
` Eco RI endonuclease cleavage site
` serves as the reference marker and is
` assigned map position 0/1.0; other posi-
` tions in the DNA are assigned coordi-
` nates in DNA fractional length units
` measured clockwise from 0/1.0 (see Fig.
` 2). At the present time, knowledge of the
` entire nucleotide sequence has made
` possible a more precise set of map coor-
` dinates: nucleotide pair number. Thus,
` nucleotide 0/5243 is placed within ori,
` the site where DNA replication is initiat-
` ed, and the other nucleotide pairs are
` numbered consecutively in the clock-
` wise direction (see Fig. 2).
` The SV40 minichromosome is ex-
` pressed in a regulated temporal sequence
` after it reaches the nucleus of infected
` primate cells. Initially, transcription in
` the counterclockwise direction of one
` strand (the E strand) of about one-half of
` the DNA (the early region) yields the
` early messenger RNA's (mRNA's) (Fig.
` 2). These mRNA's, which encode the
` large T and small t antigen polypeptides
` (the stippled portion of the mRNA's indi-
` cate the protein coding regions), have 5'
` ends originating from nucleotide se-
` quences near the site marked ori, and 3'-
` polyadenylated [poly(A)] ends from near
` map position 0.16. Synthesis of large T
` antigen triggers the initiation of viral
` DNA replication at ori, a specific site in
` the DNA (Fig. 2 identifies ori at map
` position 0.67 or nucleotide position 0/
` 5243); replication then proceeds bidirec-
` tionally, terminating about 180° away
` near map position 0.17, yielding cova-
` lently closed circular progeny ONA.
` New viral mRNA's appear in the polyri-
`
` 297
`
`This content downloaded from 128.97.27.20 on Wed, 25 May 2016 17:20:18 UTC
`All use subject to http://about.jstor.org/terms
`
`Merck Ex. 1004, pg 81
`
`

`

`
`
` .t . # . : -b
`
` bosomes concomitantly with DNA repli-
` cation; these are synthesized in the
` clockwise direction from the L strand
` of the other half of the virus DNA (the
` late region) and are referred to as late
` mRNA's. Transcription of the late
` mRNA's, which code for the virion pro-
` teins VP1, VP2, and VP3 (the stippled
` regions designate the protein coding re-
` gions of these proteins), begins from
` multiple positions between map posi-
` tions 0.68 to 0.72 and terminates at about
` map position 0.16. Finally, the accumu-
` lation of progeny DNA molecules and
` virion proteins culminates in death of the
` cell and release of mature virion parti-
` cles.
` SV40 possesses an alternative life cy-
` cle when the virus infects rodent and
` other nonprimate cells. The same early
` events take place the E strand
` mRNA's and large T and small t antigens
` are synthesized but DNA replication
` does not occur, and late strand mRNA's
` and virion proteins are not made. Fre-
` quently, replication of cell DNA and
` mitosis are induced after infection, and
` most infected cells survive with little
` evidence of prior infection. Generally, a
` small proportion of the cells (less than 10
` percent) acquire the ability to multiply
` under culture conditions that restrict the
` growth of normal cells; moreover, these
`
` transformed cells can produce tumors
` after inoculation into appropriate ani-
` mals. Invariably, the transformed cells
` contain all or part of the viral DNA
` covalently integrated into the cell's chro-
` mosomal DNA and produce the
` mRNA's and proteins coded by the early
` genes.
` During the 1970's several different ap-
` proaches, carried on in many labora-
` tories including my own, clarified the
` arrangement of SV40's genes on the
` DNA and revealed how they function
` during the virus' life cycle (21-23). Ini-
` tially, viral genes were mapped on the
` DNA relative to restriction sites by lo-
` calizing the regions from which early and
` late mRNA's were transcribed. Subse-
` guently, more precise mapping was
` achieved by correlating the positions of
` discrete deletions and other alterations
` in the viral DNA with specific physiolog-
` ic defects. But with the nucleotide se-
` guence map, the boundaries of each
` SV40 gene and the nucleotide segments
` coding for each polypeptide can be spec-
` ified with considerable precision (Fig. 2).
` As expected, the availability of a precise
` genetic and physical map of SV40's mini-
` chromosome has shifted the research
` emphasis to explorations of the molecu-
` lar mechanisms governing each gene's
` expression and function, the replication
`
` and maturation of the viral minichromo-
` some, recombination between the viral
` and host DNA, and how virus and host
` gene products interact to cause transfor-
` mation of normal into tumorigenic cells.
` Excellent and more detailed summaries
` and analyses of the molecular biology of
` SV40 and polyoma, containing acknowl-
` edgments to the inzportant contriblltions
` made by many individuals, can be found
` in several recent monographs (21-23).
`
` SV40 as a Transducing Virus
`
` The analysis of the organization,
` expression, and regulation of bacterial
` genomes was greatly aided by the use of
` bacteriophage-mediated transfer of
` genes between cells. Indeed, specialized
` transducing phages of A, +80, P22, and
` others permitted the cloning and amplifi-
` cation of specific segments of bacterial
` DNA, thereby making it possible to con-
` struct cells with unusual and informative
` genotypes and to obtain valuable sub-
` strates and probes for exploring mecha-
` nisms of transcription, translation, and
` regulation.
` This background led me to consider,
` soon after beginning work with the tu-
` mor viruses, whether SV40 could be
` used to transduce new genes into mam-
`
` t w t {
`
` * w * :
`
` rvh s 4 % w
`
` *wo w i
`
` * / e. \-s
` *+s ,+w _. f 8
`
` eS
`
` a
`
` XX' eX g623
`
` Fig. I (left). Electron micrographs of: (a) SV40 virions; (b) SV40 DNA; (c) "condensed" SV40 minichromosomes; (d) ''relaxed-beaded" SV40
` minichromosome. [Photo by J. Griffith] Fig. 2 (right). A physical and genetic map of SV40 DNA. The inner circle symbolizes the closed
` circular DNA molecule; indicated within the circle are the nucleotide-pair map coordinates starting and ending at 0/5243. Also shown by small
` arrows within the circle are the sites at which five restriction endonucleases cleave SV40 DNA once. Arrayed around the outside of the circle are
` the map coordinates, expressed in fractional lengths, beginning at the reference point 0/1.0 (the Eco RI endonuclease cleavage site) and
` proceeding clockwise around the circle. The coding regions for the early and late proteins are shown as stippled arrows extending from the
` nucleotide pair of the first codon to the nucleotide pair that specifies termination of the protein coding sequence. Each of the coding regions is em-
` bedded in an mRNA, the span of which is indicated by dotted or dashed S' ends and wavy poly(A) 3' ends. The jagged or saw-toothed portions of
` each mRNA indicate the portions of the transcript that are spliced in forming the mature mRNA's.
`
` 298
`
` SCIENCE, VOL. 213
`
`This content downloaded from 128.97.27.20 on Wed, 25 May 2016 17:20:18 UTC
`All use subject to http://about.jstor.org/terms
`
`Merck Ex. 1004, pg 82
`
`

`

`
`
` P p P p D
`
` malian cells;. Initially, I had serious res-
` ervations about the success of such a
` venture because of the predictably low
` probability of generating specific recom-
` binants between virus and cell DNA and
` the limited capability for selecting or
` screening animal cells that had acquired
` specific genetic properties. But it
` seemed that one possible way out of this
` difficulty, at least one worth trying, was
` to produce the desired SV40 transducing
` genomes synthetically. Consequently, in
` about 1970, I began to plan the construc-
` tion in vitre of recombinant DNA mole-
` cules with SV40 and selected nonviral
` DNA segments. The goal was to propa-
` gate such recombinant genomes in suit-
` able animal cells, either as autonomously
` replicating or integrated DNA mole-
` cules. At the time there were few if any
` animal genes available for recombination
` with SV40 DNA, but I anticipated that a
` variety of suitable genes would eventual-
` ly be isolated. Therefore, the first task
` was to devise a general way to join
` together in vitro any two diSerent DNA
` molecules.
` Hershey and his colleagues had al-
` ready shown that A phage DNA could be
` circularized or joined end to end in vitro
` (24). This occurred because A phage
` DNA has cohesive ends, that is, single-
` stranded, overlapping, complementary
` DNA ends (25). So, it seemed that if
` cohesive ends could be synthesized onto
` the ends of DNA molecules, they could
` be covalently joined in vitro with DNA
` ligase.
` During 1971-1972, using then available
` enzymes and relatively straightforward
` enzymologic procedures, David A. Jack-
` son? Robert H. Symons, and I (26) and,
` independently and concurrently, Peter
` E. Lobban and A. D. Kaiser (27), de-
` vised a way to synthesize synthetic co-
` hesive termini on the ends of any DNA
` molecules, thereby paving the way for
` constructing recombinant DNA's in vi-
` tro. We developed a procedure (Fig. 3)
` using as the model "foreign" DNA, a
` bacterial plasmid that contained some
` bacteriophage A DNA and three E. coli
` genes that specify enzymes required for
` galactose utilization (28). Circular SV40
` DNA (5.24 kbp) and Adv gal plasmid
` DNA (about 10 kbp) were each cleaved
` with a specific endonuclease to convert
` them to linear molecules. Then, after a
` brief digestion with A exonuclease to
` remove about 50 nucleotides from the 5'
` termini? it was possible for deoxynucleo-
` tidyl terminal transferase to add short
` "tails" of either deoxyadenylate or
` deoxythymidylate residues to the 3' ter-
` mini. After mixing and annealing under
` appropriate conditions, the two DNA's
`
` 17 JULY 1981
`
` were joined and cyclized via their com-
` plementary "tails" (Fig. 3). The gaps
` that occur where the two DNA mole-
` cules are held together, were filled in
` with DNA polymerase I and deoxynu-
` cleoside triphosphate substrates, and the
` resulting mjolecules were covalently
` sealed with DNA ligase; exonuclease III
` was present to permit repair of nicks or
` gaps created during the manipulations.
` The resulting hybrid DNA was ap-
` proximately three times the size of SV40
` DNA and, therefore, could not be propa-
` gated as a chromosome in a virus capsid.
` But we intended to test whether the E.
` coli galactose genes would be expressed
` after introduction into the chromosomes
` of cultured animal cells. Moreover, since
` the Adv gal plasmid could replicate au-
` tonomously in E. coli (28), we also
` planned to determine whether SV40
` DNA would be propagated in E. coli
` cells and whether any SV40 genes would
` be expressed in the bacterial host. A1-
` though the SV40-Adv gal recombinant
` DNA shown in Fig. 3 could not have
` replicated in E. coli-a gene needed for
` replication of the plasmid DNA in E. coli
` had been inactivated by the insertion of
` the SV40 DNA a relatively simple
` modification of the procedure, namely,
` the use of Adv gal dimeric DNA as
` acceptor for the SV40 DNA insert, could
` have circumvented this difficulty. Nev-
` ertheless, because many coileagues ex-
` pressed concern about the potential risks
` of disseminating E. coli containing SV40
` oncogenes, the experiments with this
` recombinant DNA were discontinued.
` Since that time there has been an
`
` explosive growth in the application of
` recombinant DNA methods for a number
` of novel purposes and challenging prob-
` lems. This impressive progress owes
` much of its impetus to the growing so-
` phistication about the properties and use
` of restriction endonucleases, the devel-
` opment of easier ways of recombining
` different DNA molecules, and, most im-
` portantly, the availability of plasmids
` and phages that made it possible to prop-
` agate and amplify recombinant DNA's in
` a variety of microbial hosts [see (29, 30)
` for a collection of notable examples].
` By 1975, extensive cloning experi-
` m-ents had produced elaborate libraries
` of eukaryote DNA segments containing
` single genes or clusters of genes from
` many species of organisms. As expect-
` ed, studies of their molecular anatomy
` and chromosomal arrangement have pro-
` vided new insights about possible mech-
` anisms of gene regulation in normal and
` developmentaliy interesting animal sys-
` tems. But, it seemed likely from the
` beginning that ways would be needed to
` assay isolated genes for their biological
` activity in vivo. Consequently, I re-
` turned to the original goal of using SV40
` to introduce cloned genes into cultured
` mammalian cells. But this time we ex-
` plored a somewhat different approach.
` During 1972 to 1974 Janet Mertz and I
` (31) learned how to propagate SV40 dele-
` tion mutants by complementation, using
` appropriate SV40 temperature-sensitive
` (ts) mutants as helpers. This advance
` made it feasible to consider propagating
` genomes containing exogenous DNA in
` place of specific regions of SV40 DNA.
`
` 3
` p I
` 'I
`
` 3'
`
` 3' I
`
` Endonucleose W
`
` 3' P + 3'
` P 3' P
`
` A exonucleose |
`
` p 3 3, P E p
`
` d4TP Terminol tronsferose dTTP
`
` + (dA)n + (dT) n
`
` (dA)n \ (dT}n /
`
` Fig. 3. The construction of
` SV40-Advgal recombinant
` DNA. See text for com-
` ment on individual steps.
` 4dTP, 4 deoxy triphos-
` phates; DPN, nicotinamide
` ad