United States Patent
`Comai
`
`[19]
`
`[11] Patent Number:
`
`4,762,785
`
`[45] Date of Patent:
`
`Aug. 9, 1988
`
`[54] NOVEL METHOD AND COMPOSITIONS
`FOR INTRODUCTING ALIEN DNA IN VIVO
`
`[75]
`
`Inventor:
`
`Luca Comai, Davis, Calif.
`
`[73] Assignee: Calgene, Inc., Davis, Calif.
`[2]] Appl. No.: 796,484
`[22] Filed:
`Nov. 6, 1985
`
`[51]
`
`[63]
`
`Related US. Application Data
`Continuation of Ser. No. 407,525, Aug. 12, 1982, aban-
`doned.
`V
`Int. cu ....................... Cl2N 15/on; Cl2_N 1/20;
`Cl2P 21/00; C12P 19/34
`[52] U.S. Cl. ................................... 435/172.3; 435/68;
`435/91; 435/172.1; 435/253; 435/320; 935/23;
`935/27; 935/29; 935/55; 935/56; 935/64;
`935/67; 935/72; 935/73
`[58] Field of Search .................. 435/68, 70, 91, 172.3,
`435/253, 317, 320, 172.1; 935/23, 27, 29,55, 56,
`64, 67, 72, 73
`
`[56]
`
`References Cited
`PUBLICATIONS
`
`Hooykaas, “Plasmid Genes Essential for the Interac-
`tions of Argobacteria and Rhizobia with Plant cells”, in
`Molecular Genetics of the Bacteria—PIam‘ Interaction,
`Puhler
`(ed.), Springer—Verlag, Berlin,
`1983, pp.
`229-239.
`-
`Thomashow et al., “Integration and Organization of Ti
`
`Plasmid Sequences in Crown Gall Tumors", Cell, 19:
`729, (1980).
`Petit et al.: Nature, 271, 570, (1978).
`Low et al.: Ann. Rev. Genet., 12, 249, (1978).
`Eisenstark: Ann. Rev. Genet., 11,369, (1977).
`Matzke et al.: Chem. Abstr. 95:ll1256u, (1981), of J.
`Mol. Appl. Genet, 1, 39, (1981).
`Ditta et al., Prac. Natl. Acad. Sci.
`77:7347—735l,
`v
`Ruvkun and Ausubel, Nature, (1981), 289:85—88.
`Primary Examiner—James Martinell
`Attorney. Agent, or F1'rm—Bertram I. Rowland
`
`(1930),
`
`USA,
`
`ABSTRACT
`[57]
`Method and compositions are provided for introducing
`alien DNA into a nucleic acid in vivo by providing a
`plasmid capable of replication in a first host, which is
`capable of conjugation with a second host into which
`the alien DNA is to be introducted. The plasmid is -
`characterized by being capable of maintenance in said
`first host, being incapable of maintenance in said second
`host, having a region of homology with DNA present
`in said second host, not self-transmissable but capable of
`mobilization by a helper plasmid and having a marker,
`which may be the alien DNA, for selection in said sec-
`ond host. The invention finds particular application for
`introducing alien DNA into the Ti plasmid of Agrobac-
`terium tumefaciens for introduction into plant cells.
`
`7 Claims, No Drawings
`
`Sanofi/Regeneron Ex. 1035, pg 966
`
`Mylan Ex. 1035, pg 966
`
`

`
`1
`
`4,762,785
`
`2
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`NOVEL METHOD AND COMPOSITIONS FOR
`INTRODUCTING ALIEN DNA IN VIVO
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation of U.S. application
`Ser. No. 407,525, filed Aug. 12, 1982, now abandoned,
`which disclosure is incorporated herein by reference.
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`There are many situations where it is desirable to
`introduce alien DNA into a particular segment of a
`chromosome or
`extrachromosomal
`element. Fre-
`quently, this can be very cumbersome because of the
`numerous steps involved in cloning the alien DNA,
`isolating the alien DNA, finding appropriate restriction
`sites and introducing the alien DNA into the host of
`interest. There are many aspects to be considered in a
`useful method. Small plasmids are desirable, since they
`are easy to work with, readily manipulated, and gener-
`ally have few restriction sites for a particular restriction
`enzyme, frequently providing a number of unique re-
`striction sites. Thus, they are readily manipulatable.
`Other considerations are efficiency of transformation
`or conjugation, stability or maintenance of the alien
`DNA in the host, the genetic environment and control
`of the alien DNA, and the like. Further considerations
`include the ease with which the presence of the alien
`DNA can be determined, purification of the alien DNA,
`by itself and in conjunction with DNA segments to
`which it is attached, and the ease of introduction and
`excision of the alien DNA into and out of a vector.
`Exemplary of the problems described above is the
`modification of large naturally occurring plasmids, such
`as the Ti plasmid of A. tumefaciens. The large size of
`the plasmid makes manipulation difficult, impedes isola-
`tion and purification, as well as identification of the
`introduction of alien DNA and the opportunity for
`excision.
`2. Description of the Prior Art
`Ditta et al. PNAS USA (l980) 77:7347—735l describe
`a broad host range DNA cloning system for Gram-neg-
`ative bacteria. Ruvkun and Ausubel, Nature (1981)
`289:85—88 describe a general method for site directed
`mutagenesis in prokaryotes. Thomashow et al. Cell
`(1980) l9:729—739 describe the integration and organi-
`zation of Ti plasmid sequences in crown gall tumors, as
`well as the construction of the plasmid pNW33C-19-1.
`SUMMARY OF THE INVENTION
`
`A unicellular microorganism is transformed with an
`extrachromosomal element capable of maintenance in
`such first host, where the extrachromosomal element is
`characterized by being self-transmissable or capable of
`mobilization with a second helper extrachromosomal
`element; being capable of conjugal transfer to a host of
`interest (second host); having a narrow host range repli-
`cation system; containing predetermined DNA seg-
`ments comprising alien DNA and DNA homologous
`with a DNA sequence in said second host, either chro-
`mosomal or extrachromosomal and a marker allowing
`for selection.
`
`5
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`35
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`65
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`In accordance with the subject invention, methods
`and compositions are provided for introduction of alien
`DNA in vivo by conjugation between a first host and a
`second host. The introduction is achieved with an extra-
`chromosomal element characterized by having a repli-
`con having a narrow host range, being self-transmissa-
`ble or mpable of mobilization with a helper plasmid,
`being incapable of maintenance in the exconjugant or
`target host, having a region of homology with target
`DNA in the second host, having alien DNA capable of
`replication,
`transcription and usually translation in a
`host cell, and having one or more markers selectable in
`the target host.
`The two hosts are unicellular microorganisms, partic-
`ularly prokaryotes. Of particular interest is the use of E.
`coli as the first host and A. tumefaciens as the exconju-
`gant or transconjugant second host.
`The extrachromosonal elements which are the com-
`positions of this invention have a vector having a repli-
`con or replication system having a narrow host range.
`That is, they are capable of maintenance and replication
`in a first unicellular host which is capable of conjuga-
`tion with a second host of interest having the target
`DNA, but the vector is incapable of maintenance in the
`target or second host, so that the target host is rapidly
`cured. In this way, in the absence of integration of at
`least a portion of the extrachromosomal composition,
`the extra chromosomal DNA will be lost in the second
`host. Thus, the DNA of the extrachromosomal element
`is rescued by integration with a stable genetic element
`of the target host.
`The replicon may come from a plasmid, virus, or
`other source which allows for stable maintenance in the
`first host. The first host will normally be a prokaryote,
`particularly E. coli. By employing a vector capable of
`replication in E. coli and other enteric hosts (narrow
`range), the vector will not be replicated and maintained
`in non-enteric Gram negative bacteria. The extrachro-
`mosomal element may therefore be a plasmid or virus
`(phage) or other DNA, which provides the requisite
`properties.
`The extrachromosomal element will also have a mo-
`bilization locus, and will be self-transmissable, or capa-
`ble of transfer to another host with a helper plasmid
`having the necessary genes for transfer, e.g. tra.
`'
`Another characteristic is the presence of a DNA
`sequence homologous with DNA present in the target
`host to provide for efficient integration. The homolo-
`gous DNA in the target or second host which serves as
`the recombination locus may be chromosomal or extra
`chromosomal. Of particular interest for target DNA is
`the Ti plasmid in A.
`tumefaciens, which may be wild
`strain, a mutant, or containing alien DNA as a result of
`rDNA or other technique for introduction of alien
`DNA, e.g. transposon. More particularly of interest, is
`the T-region of the Ti plasmid of A. tumefaciens.
`The region of homology will generally be at least
`about 600 bp, usually 1000 bp or more, usually not ex-
`ceeding about 5000 bp, and more usually not exceeding
`about 3000 bp. The region of homology may include
`regions encodingproteins, regulatory regions, or non-
`encoding regions and need not have perfect homology.
`One or more segments of alien DNA will be included
`in the plasmid, normally encoding one or more proteins
`of interest. The DNA will be alien to the prokaryote
`
`Sanofi/Regeneron Ex. 1035, pg 967
`
`Mylan Ex. 1035, pg 967
`
`

`
`4,762,785
`
`3
`involved in the mating. The protein of interest may be
`any protein derived from any convenient source, either
`prokaryotic or eukaryotic, including lower or higher
`orders of eukaryotes, e.g. fungi, algae, protozoa, mam-
`mals, plants, etc. The protein expressed may provide a
`new function for the host organism or a product which
`is desired separate from the host organism, e.g. a mam-
`malian protein such as a mammalian hormone.
`There can be two types of integration: a single cross-
`over or a double crossover. In the single crossover, the
`entire extrachromosomal element becomes integrated
`into the DNA of the target host. With a double cross-
`over, a portion of the extrachromosomal element
`is
`substituted for a portion of the target host DNA.
`By having regions of homology with the target DNA
`on both sides of the alien DNA and in close proximity
`to the termini of the alien DNA, the alien DNA may be
`inserted into the target host DNA without including
`much of the remaining DNA of the extrachromosomal
`element.
`With the Ti plasmid, there is the further interest of
`introducing the alien DNA into a plant susceptible to
`tumor induction by the Ti plasmid. Thus, the subject
`method provides the opportunity to introduce alien
`DNA into a plant, where the alien DNA may be ex-
`pressed under the control of the regulatory signals pres-
`ent in the Ti plasmid. The subject method therefore
`provides a simple technique for modifying the genome
`of the plant cell through the use of a modified Ti plas-
`mid as the modifying agent.
`The alien DNA may be solely a gene encoding a
`protein of interest or may be present with other genes,
`regulatory signals, or the like. For example, a promoter
`may be provided which is native to the Ti plasmid or
`the ultimate plant cell, where the alien gene would not
`be capable of expression in the prokaryote but only in
`the eukaryotic plant cell. The promoter may provide
`for regulation in the plant cell or constitutive produc-
`tion. Various genes may be combined with the gene of
`interest to provide for amplification of the gene of inter-
`est, where the other genes respond to stress by resulting
`in tandem reiteration of such other gene in conjunction
`with flanking DNA regions, e.g., genes expressing
`DHFR, metallothioneins, etc.
`While not always essential, particularly where the
`alien DNA provides a means for selection, it will usu-
`ally be desirable to provide a marker as part of the
`extrachromosomal composition of this invention. The
`marker will allow for selection of exconjugants. A wide
`and diverse variety of markers may be employed.
`Conveniently, antibiotic resistance may be employed,
`which allows for selection of exconjugants by culturing
`the cells on a medium containing the particular antibi-
`otic. Antibiotic resistance can be provided to ampicillin,
`penicillin, tetracycline, kanamycin, etc. Resistance can
`also be provided to heavy metals by employing genes
`that express metallothioneins. These genes may also be
`employed as amplifying genes. Genes may be provided
`for selecting against toxins, such as colicin. Other genes
`provide immunity to viruses.
`Alternatively, prototrophy can be provided to an
`auxotrophic host. By culturing the exconjugants in a
`culture medium lacking the essential metabolite,
`the
`exconjugants will be selected for by being capable of
`multiplication. In many instances, it will be desirable to
`have a plurality of markers, particularly if one of the
`markers has a restriction site for insertion of the alien
`DNA. The loss of the property provided by the marker
`
`l0
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`4
`allows for detection of the extrachromosomal DNA
`into which the alien DNA has been inserted by double
`selection.
`The extrachromosomal DNA into which the alien
`DNA is introduced will desirably have at least one and
`preferably a plurality of unique restriction sites. A wide
`variety and increasing number of restriction enzymes
`are commercially available, so that one has the opportu-
`nity to sequence the extrachromosomal DNA and/or
`prepare a restriction map to develop a synthetic scheme
`for the final composition. A wide variety of restriction
`enzymes are known, such as EcoRI, Pstl, Hindl, II, and
`III, Haell, Kpnl, Hpal, Xhol, and Smal, as illustrative
`but not exhaustive of restriction enzymes.
`As already indicated, the subject composition must be
`self-transmissible or capable of conjugal
`transfer by
`trans-complementation with a helper plasmid. Conve-
`niently, the extrachromosomal DNA and the helper
`plasmid will be initially employed in different cells,
`employing tripartite rather than dual mating, rather
`than requiring the in vitro introduction of both the
`extrachromosomal DNA and the helper plasmid into
`the first host cell prior to conjugal transfer. The helper
`plasmid will have the necessary function for conjugal
`mobilizability (transfer), as well as a functioning replica-
`tion system.
`The subject extrachromosomal DNA will normally
`be prepared from a vector derived from a plasmid or
`virus, more usually a plasmid, by modification of the
`plasmid to provide the desired properties, and inserting
`the appropriate DNA sequences in the appropriate
`order in available restriction sites. However, various
`techniques can be used, such as primer repair or in vitro
`mutagenesis for introduction of DNA into the vector.
`The manner of preparing the extrachromosomal com-
`position is conventional in that the technique will nor-
`mally involve restriction at specific sites to provide for
`blunt or cohesive ends, direct introduction of the DNA
`of interest, or through the use of linkers, tailing, or the
`like, ligation, cloning, followed by repeating the process
`for introduction of additional DNA sequences. Thus,
`after each addition of new DNA into the vector, the
`vector will be recircularized and cloned, followed by
`selecting for the vector having the newly introduced
`DNA sequence.
`Once the extrachromosomal DNA has been com-
`pleted, it will be introduced into the first host. Transfer
`can then be achieved by combining the first host, the
`second target or recipient host, and the host having the
`helper or mobilizing plasmid under conditions to pro-
`vide for mating of the three strains. One then selects for
`the ex- or transconjugant by the markers introduced
`into the vector. In the case of A.
`tumefaciens, the Ti
`plasmid may be restricted with appropriate restriction
`enzymes and compared with the wild strain or target Ti
`plasmid to demonstrate the introduction of the alien,
`DNA.
`The following examples are offered by way of illus-
`tration and not by way of limitation.
`EXPERIMENTAL
`Methods and Materials
`
`A 4.6 Kb fragment from the Ti plasmid pTiA6 gener-
`ated by digestion with endonuclease BamHl is referred
`to as “Baml9”. Plasmid pNW33C-19-l is constructed
`by the insertion of Baml9 in pBR325 (Thomashow et
`al., 1980, supra; Bolivar, Gene (1978) 4:121-126). Plas-
`
`Sanofi/Regeneron Ex. 1035, pg 968
`
`Mylan Ex. 1035, pg 968
`
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`
`4,762,785
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`l0
`
`6
`5
`in
`mid pCGN402 is a derivative of pNW33C-19-l
`mixture containing 2 pl of DNA, 2 ul of enzyme, 2 ul of
`which a kanamycin resistance determinant is inserted in
`buffer, and 14p] of water to provide a final volume of 20
`the unique Smal site of Baml9. pNW33C-19-1 is di-
`u] and the digest carried out at 37° for 100 min. The
`gested with Smal and BglII linkers inserted to give
`digestion was stopped by the addition of l0 ul of track-
`pCGN40l, which has a Bglll site replacing the Smal
`ing dye and the entire sample loaded on 0.7% agarose
`site. The kanamycin resistance gene obtained from
`and the electrophoresis carried out at 100 v for 180 min.
`The results of the various digests are as follows. The
`pUC5 by BamHI digestion is then inserted into the
`BamHl pattern from pTiA6-El showed two new bands
`Bglll site. The particular KAN’ gene is chosen because
`of convenient Sall restriction sites at its termini, allow-
`of about 6 kb each, which were pBR325 and a 4.6 kb
`Baml9 fragment containing a 1.4 kb Kan’ insert. The
`ing for subsequent excision. Four different transfor-
`mants of E. coli are prepared by introduction in accor-
`EcoRI pattern gave a new 9.5 kb fragment which was
`dance with conventional techniques of four different
`part Eco7 with a kanr insert. With Hindlll, a new 8.6 kb
`plasmids: (1) pBR325; (2) pNW33C-19-l; (3) pCGN402;
`band was present while a 12.6 kb band was missing. The
`and (4) pRK20l3.
`missing one was Hindl, while the new one was part of
`Hindl plus a portion of Kan’.
`The strains employed have the following characteris-
`tics: E. coli K12 HBIOI (F-, pro, leu, thi, loc Y str’,
`Other new fragments appeared to be present but
`hsdR, hsdM, endoI, recA, ara14, galK2, xyl5, mtll,
`could not be readily characterized since they comi-
`supE44). (Boyer and Roulland, J. Mol. Bio.
`(1969)
`grated with other fragments generated from pTiA6-El.
`However, the observed differences clearly differentiate
`4l:459—472). E. coli K12 mm294 (F—, pro, thi, endoI,
`hsdR). (Gilbert et 9.]. Gene (1980) 12:235-240. A. tumo-
`pTiA6-El from the wild type plasmid pTiA6 and dem-
`faciens A348 (pTiA6) rif', cm’, nal'. (Watson et al., J.
`onstrates that pCGN402 has integrated into pTiA6.
`Bacteriol. (1975) l23:255-264; Chilton, et al, Genetics
`In accordance with the subject invention, a simple,
`(1976) 83:609—6l8)
`efficient method is provided for introducing DNA into
`a host, particularly into an extrachromosomal element
`Matings were performed as follows. Each of the
`strains was cultured in l0 ml of YT (trystone medium)
`of a host. By employing a readily available safe host as
`the donor strain, for which a large number of well char-
`overnight, except for the A.
`tumefaciens which was
`cultured in LB/AE. The matings were performed by
`acterized plasmid or viral vectors are available, foreign
`DNA can be readily transferred to a wide variety of
`combining 4ml of A. tumgfaciens A348, 2ml of E. coli
`HB 101 (pRK20l3) and 21111 of the donor strain. The cell
`prokaryotic hosts and alien DNA introduced into the
`suspensions were mixed in a Falcon tube and then fil-
`DNA of such prokaryotic hosts in a stable manner.
`tered onto a 0.45p. millipore filter. The filters were
`Furthermore, by employing a recipient strain, such as
`incubated overnight on LB/AE at 30' C., followed by
`A. tumefizciens, which is capable of transferring DNA to
`removal of the filter from the medium and washing in
`a higher cell order, namely plant cells, one can transfer
`0.5 ml water.
`.
`alien DNA from a simple prokaryote, such as E. coli, to
`The cells were then diluted to various dilutions and
`the higher order plant cells through the intermediacy of
`plated on selective media.
`the Ti plasmid. High efficiencies of integration can be
`The matings were carried out as described above and
`achieved through the stable integration of large DNA
`segments of alien DNA.
`the following table indicates the strains employed, the
`selective media, and the observations at various cell
`Although the foregoing invention has been described
`dilutions.
`40 in some detail by way of illustration and example for
`
`TABLE I
`
`20
`
`25
`
`30
`
`35
`
`C19
`
`ds
`
`ds
`
`X
`
`X
`
`X
`
`X
`
`-2000
`colonies
`
`Observatior|3v‘l
`Selection:
`Matifl Strains‘
`Re-
`Rif
`Kan
`Carb
`Dilution Cells
`
`Donor Mab.
`cipient
`Sug/ml
`so ug/ml
`so pg/ml
`10-3
`10-4
`10-5
`402
`2013
`348a
`X
`X
`bgd + -200
`bgd + ~49
`bgd + 2
`larger white
`larger white
`larger white
`colonies
`colonies
`colonies
`bgd of ~1ooo
`bgd + 5
`bgd
`colonies + 50
`larger white
`larger white
`colonies
`colonies
`confluent lawn
`
`325
`
`ds
`
`ds
`
`bgd of very small
`white colonies +
`-300 larger white
`colonies
`
`lDonor 4l)2—E. L‘o1imm294 (pCGN402) Cl9—E. coli HBl0l (pNW33C-19-l) 325—E. coli l-[B101 (pBR3Z5) Mab. 20l3 E. coli HBl0l
`gpRK20l3) Recipient 348a~A. Tumefaciem A3482:
`-Rxf—rifampicin Kan—-kanamyein Carb—carbenillin
`3hgd—-hmrkground
`‘Selection for ampicillin resistance in A. tumq/'ucienr strain 348:: leads to the isolation ofsponlaneous resistance mutants. For this reason. the
`experiments using C19 and 325 as donors are inconclusive.
`
`The selection of exconjugants was complicated by
`the appearance of spontaneous resistant colonies of
`Agrobacterium. These could, however, be distin-
`guished from true exconjugants as they appeared, on
`average, a day later.
`A restriction digest of Ti plasmid resulting from the
`mating with the donor 402 was compared with a restric-
`tion digest of the wild type Ti plasmid, employing
`EcoRI, BamH1, XhoI and HindIII using a restriction
`
`60
`
`65
`
`purposes of clarity of understanding, it will be obvious
`that certain changes and modifications may be practiced
`within the scope of the appended claims.
`The strain E. coli K12 mm294 (pCGN4-02) was depos-
`ited for patent purposes at the American Type Culture
`Collection, Rockville, MD, USA on Aug. 5, l982 and
`given accession no. 39171.
`What is claimed is:
`
`Sanofi/Regeneron Ex. 1035, pg 969
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`Mylan Ex. 1035, pg 969
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`4,762,785
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`7
`1. A method for stably inserting alien DNA into the
`DNA of the Ti plasmid of A. tumefaciens,
`said method comprising:
`mating with said A.
`tumefaciens a first E. coli host
`containing, a first extrachromosomal element char-
`acterized as having: a narrow range replication
`system incapable of maintenance in said A. turm.:fa-
`ciens; a DNA sequence homologous with a DNA
`sequence of the T region of the Ti plasmid present
`in said A. tumefacienx mobilization capability being
`self-transmissible or capable of transfer with a
`helper plasmid; a segment comprising alien DNA;
`and a marker capable of selection in said A. tumefa-
`ciens with the proviso that if the first extrachromo-
`somal element is not self-transmissible, the first E.
`coli host also contains a second extrachromosomal
`
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`4. A method for stably inserting alien DNA into the
`T region of the Ti plasmid, said method comprising:
`mating an A. tumefaciens host containing a Ti plasmid
`with an E. Cali host containing first and second
`extrachromosomal elements,
`said first element:
`having a replication system recognized by said E. coli,
`but not by A. tumefacieris;
`having a region of homology of at least 800 bp with
`said T region;
`having alien DNA;
`containing a mobilization locus, either being self-
`transmissible or using a second element as a helper
`plasmid; and
`having a marker capable of selection in said A. tume-
`faciens;
`said second element:
`having a replication system recognized by E. colt’, but
`not A. tumefaciens; and
`having genes capable of imparting transmissibility to
`said first element, and
`selecting for an A. tumefaciens exconjugant contain-
`ing said alien DNA by means of said marker.
`5. A method according to claim 4, wherein said alien
`DNA is inserted into said region of homology.
`6. A method according to claim 4, wherein said sec-
`ond extrachromosomal element is introduced into said
`E. coli by mating.
`7. A method according to any one of claims 4, 5, or 6,
`3- Am€th0d3CC01'dl718t0°1aim 1: lT1C1“din8 the addi' 30 including the additional step of infecting a plant cell
`tional step of infecting a plant cell with said exconju-
`with said exconjugant.I
`*
`*
`*
`I
`gant.
`
`element as said helper plasmid having a narrow
`range replication system and incapable of replica-
`tion in said A.
`tumefacians: and genes capable of
`imparting transmissibility to said first and second
`extrachromosomal elements; and
`selecting by means of said marker an exconjugant.
`2. A method according to claim 1, wherein said sec-
`ond extrachromosomal element is introduced into said
`E. coli host by mating with a host capable of conjuga-
`tion with said E. coli host.
`
`20
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Sanofi/Regeneron Ex. 1035, pg 970
`
`Mylan Ex. 1035, pg 970