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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention relates to the field of application of recombinant DNA techniques to the transformation of higher plants. More specifically, the invention relates to vectors useful in transforming higher plants and in conferring desired properties on them, and to methods of employing such vectors in transformations.
• the bacterial DNA transfor ing the infected plant cell is non-chromosomal DNA; and is transferred from a plasmid vector (Ti).
• Ti (tumor inducing) plasmid types which are designated according to the opines associated with them
• "nopaline" Ti plasmids such as pTiT37 (Depicker, A., et al, J Molec Appl Genet (1982) 1:561; Bevan, M., et al. Nucleic Acids Res (1983) .11:369) and pTiC58 (see, e.g., Shaw, C. H., et al.
• pTiB6S3 "octopine" plasmids such as pTiB6S3 (DeGreve, H., et al, J Molec Appl Genet (1982) 1 ⁇ :499). They are quite large (e.g., 132 Mdal or about 200 kilobases (kb) for pTiC58) and have a number of relevant features.
• a "virulence” (vir) region which appears to be associated with those functions permitting successful infection and integration of certain DNA portions into the host plant cell, i.e., this region is associated with encoding whatever products effect the attachment of the bacterium to a plant cell, transfer of the plasmid DNA into the plant cell, or other necessary functions required for infection.
• a second region of significance is the "transfer-DNA” or T-DNA region constituting that portion of the plasmid which, after infection, actually becomes integrated into the plant genome. In a wild type Ti plasmid, this T-region has a number of important characteristics.
• opine synthase region which, when expressed, provides the enzymes required for the synthesis of a characteristic opine—nopaline in the case of the nopaline-type plasmids, and octopine in the case of octopine-type plasmids.
• nopaline is a conjugate of arginine and alpha ketoglutaric acid
• octopine is a conjugate of arginine and pyruvic acid.
• synthesis of these opines serves a natural function in the normal course of infection—an additional region of the plasmid encodes enzymes which permit the degradation and metabolism of the particular opine encoded by the synthase in the T-DNA region.
• plant cells infected by a particular plasmid acquire the ability to synthesize a particular opine, i.e., nopaline or octopine, which product then is a nutrient for additional infecting cells. That is, the ability to metabolize a particular opine is encoded on the same plasmid as the synthase encoding region, but not integrated into the host cells* genome.
• the T-DNA region also encodes genes which appear to affect endogenous levels of the plant hormones cytokinin and auxin. This is doubly significant. On the positive side, this function is useful in selecting successful transformants in tissue culture, since generally only transformed cells have the capacity to synthesize these hormones and are capable of reproduction in their absence from the medium. On the other hand, the presence of these specific genes in the transferred DNA has natural consequences in the course of infection, and because of the increased and unbalanced amounts of plant hormones, transformed plant cells fail to regenerate into complete normal plants and infected intact plants develop crown galls. Indeed, it has been shown that if specific mutations are made in the T-DNA regions affecting the levels of these hormones, sufficient imbalance of the auxin-cytokinin ratio can be obtained to result in growth predominantly of roots or predominantly of shoots from the resulting tumor tissue.
• T-DNA is flanked by "border* sequences which contain approximately 25 nucleotide direct repeats. Because of the nature of these sequences, it is surmised, although not known, that these regions may be involved in the insertion of the intervening T-DNA into the chromosome of the infected cell.
• T-DNA region is integrated into the host cell chromosome, attempts have been made to utilize this region as a vehicle for transformation of plant host cells by foreign DNA. None of these attempts have been entirely successful, ⁇ i evaluated for the goal of obtaining the desired combination of an expressed foreign gene in the context of a normal plant.
• Shaw, C. H., et al. Gene (1983) 23:315 succeeded in transforming an axenic culture of tobacco seedlings by infecting them with A. tumefaciens presumably containing a recombined Ti plasmid. This plasmid was thought to result from an ⁇ n. vivo cross-over in the A. tumefaciens cells involving homologous regions of the T-DNA.
• the putative recombined vector was derived from a broad-host range intermediate vector containing the HindIII-23 right-end T region fragment of the nopaline plasmid pTiC58 into which the rabbit ⁇ -globin gene had been inserted, and the Ti plasmid (also pTiC58) indigenous to the Agrobacterium.
• the infected seedlings incorporated the ⁇ -globin DNA, but did not make any transcripts of it.
• 303:209 succeeded in obtaining expression of the coding sequence for a heterologous protein conferring chloramphenicol resistance in axenic cultures derived from Agrobacterium infection induced-tobacco seedling tumors.
• the coding sequence for chloramphenicol acetyl transferase (CAT) was linked to the promoter sequences of the nopaline synthase gene to obtai a NOS-CAT chimaeric gene and placed in a recombinant vector with a narrow host range (E. coli) origin of replication, a region of T-DNA homology, and the ampicillin resistance conferring (Amp R ) sequences. This plasmid was transformed into A.
• E. coli narrow host range
• Amp R ampicillin resistance conferring
• the present invention in one. aspect relates to vectors useful for expression of foreign gene sequences in plant tissues.
• a basic intermediate carrier vector behaves as a receiving unit which permits the introduction of any desired foreign gene sequence so as to be suitably disposed to take advantage of promoter and polyadenylation signal sequences which are operable in plant tissues.
• a wide variety of foreign genes can be introduced with facility.
• the resulting expression carrier vectors can be spliced using standard recombinant DNA restriction and ligation techniques to contain any desired number of expression cassettes for a variety of genes. Among these cassettes, it may be desirable to include a marker which will permit easy selection of successfully transformed cells.
• the present invention relates to expression carrier vectors effective in expressing the coding sequence of foreign genes in 5 recombinant plant cells and to the intermediate DNA sequences and vectors useful in constructing them.
• expression carrier vectors include:
• Recombinant vectors containing one or more expression cassettes each such expression cassette o comprising a promoter normally operable in plant cells and a polyadenylation signal operable in plant cells, both operably linked to a coding sequence for a desired foreign protein.
• the expression carrier vectors may contain more than one such expression cassette.
• These 5 vectors also contain at least one cassette-unique restriction site at the terminus of at least one expression cassette. This class of vectors is useful in obtaining the expression of the foreign coding sequences in plant cells after direct transformation of such cells • 0 with these vectors.
• Another class of expression carrier vectors comprises those of the preceding paragraph, but modified so as to contain at least one A. tumefaciens T-DNA border sequence proximate a terminus of the expression cassette. or if multiple expression cassettes are contained in the vector, at a terminus of these series.
• the single expression cassette, or the series of expression cassettes is framed by two such T-DNA border sequences.
• These vectors also contain, on their vector portions, a broad host range bacterial origin of replication. They are useful in transforming plant cells so as to obtain expression of foreign protein, either by the direct transformation methods employed in connection with the vectors of the previous paragraph, or in transformation methods which involve the mediation of the virulence regions of an Agrobacterium Ti plasmid.
• the invention relates to intermediate plasmids and DNA sequences employed in the construction of the foregoing vectors, to methods of transforming plants or plant cells using these vectors, to plants or plant cells transformed with such vectors, to methods of producing foreign proteins using plants so transformed, and to the proteins so produced.
• Figure 1 shows the construction of pCMC59 and pCMC60.
• Figure 2 shows the construction of ⁇ CMC121.
• Figure 3 shows the construction of pCMClOl.
• Figure 4 shows the construction of pCMC91.
• Figure 5 shows the construction of pCMC72.
• a promoter sequence "normally operable in plant cells” refers to a promoter sequence which is not only coincidentally compatible with plant cells in the sense of being minimally capable of initiating transcription of a coding sequence into mRNA, but which is found natively to effect gene expression in plant tissue.
• Such promoter may be of plant origin; however, more typicially in the embodiments of the invention it is, in fact, a bacterial sequence.
• the sequence may.be relatively silent in the bacterium and is designed to operate specifically in an infected plant cell.
• many of the vectors of the present invention utilize the promoter for nopaline synthase, which is carried on an Agrobacterium tumefaciens plasmid, but which has not been reported to express in the bacterium.
• the plasmid when the plasmid is introduced into infected plant cells, it operates in this context to express the nopaline synthase coding sequence.
• These promoters are, although not of plant origin, "normally operable" therein.
• Polyadenylation signal operable in plant cells* refers to a sequence normally found 3* of a coding sequence which appears to be necessary for its transcription, transport of the polyA-mRNA from the nucleus and its stable accumulation in the cytoplasm, permitting subsequent translation in eukaryotic cells. It is currently believed that the function of these sequences is to signal polyadenylation of the messenger RNA transcribed from the coding sequence. However, the complete function of these signal sequences has not been unequivocally established. Therefore, as used herein, this term refers to those signals which follow the coding sequence in DNA and assure its expression, including translation, in the eukaryotic cellular environment. It is not at present clear whether this ability to assure expression in plants is confined to sequences which are found to be so utilized by plant cells in nature. Therefore, any sequence which in fact is successful in performing this function in plant cells is included in the definition.
• Polylinker refers to a DNA sequence which contains at least three closely associated restriction enzyme sites after ligation into a receptor DNA sequence.
• “Expression cassette” refers to a DNA sequence which is capable of effecting expression of a contained coding sequence in plants.
• a typical expression cassette contains a promoter sequence normally operable in plant cells, operably linked to the coding sequence and a polyadenylation signal operable in plant cells also operably linked to the coding sequence.
• the word “cassette” is also used in connection with other DNA sequences which are designed to be treated as a unit. For example, a series of "expression cassettes" would also be referred to as a "DNA sequence cassette".
• “Cassette-unique” restriction site refers to an endonuclease enzyme cleavage recognition site which is found only once in the referenced cassette or series of cassettes.
• Vector sequence or “vector segment” or “vector fragment” refers to DNA sequences on a plasmid that are not part of an expression cassette, DNA sequence cassette, or other "cassette” DNA sequence. Typical of “vector sequences” are origins of replication and T-DNA border sequences.
• “Narrow host range bacterial origin of replication” refers to an origin of replication which is capable of function only in a very limited number of bacterial species. Typically, in most recombinant work, such replicons are confined to functionality in such commonly used species as E. coli, and, in the context of the present invention, fail to replicate in foreign hosts such as Agrobacterium. Conversely, “broad host range bacterial origin of replication” refers to those replicons which are functional not only in their strain of origin but also in disparate bacterial species. In the context of the present invention, the most meaningful example would be those origins which are capable of replication both in E. coli and in Agrobacterium.
• T-DNA border sequences refers to repeating DNA sequences the same as, or analogous to, those bordering the T-DNA portion of the Ti plasmids of A. tumefaciens. In the wild type A. tumefaciens, these repeating sequences frame the T-DNA which is transferred into the chromosome of the infected cell.
• the definition of border sequences as used in this invention is not limited to these specific repeating segments, but includes any modifications thereof which still are functional in mediating the transfer of the intervening material, with the aid of the virulence region of a Ti plasmid, into a host cell genome.
• the abbreviations "LB" (left border) and "RB” (right border) are used to designate those sequences framing the T-DNA of the A. tumefaciens plasmid.
• Plants or “plant cells” refer to plant types which are capable of reproduction by formation of seeds, and these terms include the plant itself, the cells of the plant, protoplasts which derived from plant cells, other plant-derived cells such as tissue culture cells, tumor cells, calli and seeds, and progeny thereof. It is understood that progeny can undergo spontaneous or intentional modifications when cultured over several generations, and such modified progeny are intended to be included as long as they are derived from the plant cell or plant referred to.
• operably linked refers to sequences which are juxtaposed in such a way that their functionality is maintained.
• a promoter "operably linked" to a coding sequences refers to a disposition of a promoter which is capable of effecting the transcription and translation of this sequence.
• a polyadenylation signal “operably linked” to a coding sequence refers to a disposition of this signal which permits production of protein from the coding sequence.
• transformation techniques of the present invention utilize a system of vector constructs which permits facile manipulation of gene sequences in an interchanageable set of vectors of general utility.
• the seminal vector is an intermediate carrier vector which is designed to permit convenient insertion of any desired polypeptide encoding sequence.
• This vector comprises a "cassette" DNA sequence which includes, in order, proceeding 5'-3' in the sense strand: (a) a first cassette-unique restriction site;
• a second cassette-unique restriction site (e) a second cassette-unique restriction site.
• the presence of the polylinker permits easy manipulation in relation to coding sequences so as to permit insertion either of an ATG-initiated sequence encoding a "mature" protein or of a sequence into reading frame with a partial coding sequence operably included with the promoter. ⁇ pon insertion of such coding sequences this vector becomes an expression carrier vector capable of expressing the coding sequence when transformed into plant cells.
• Carrier vectors containing a wide variety of coding sequences for example, APH-I and APH-II (which encode proteins conferring resistance to the amirtoglycoside antibiotic G418 or kanamycin, which are toxic to yeast, plant and mammalian as well as to bacterial cells); beta interferon, which may, in fact, protect plants as well as mammals against viral infection (Orchansky, P. et al, Proc Natl Acad Sci (USA) (1982) 79_:2278; Bt-toxin which acts as an internal pesticide for plant tissues; and CAT which catalyzes an acetylation modification of chloramphenicol.
• APH-I and APH-II which encode proteins conferring resistance to the amirtoglycoside antibiotic G418 or kanamycin, which are toxic to yeast, plant and mammalian as well as to bacterial cells
• beta interferon which may, in fact, protect plants as well as mammals against viral infection (Orchansky, P.
• any coding sequence which can be provided with its own ATG start codon and framed by suitable restriction sites can be utilized.
• the intermediate vector which contains the promoter sequence also contains, operably linked to the promoter, codons which include an ATG start signal
• any sequence which can be placed in reading frame with the ATG can be expessed as a fusion protein.
• a portion of the 3-galactosidase coding sequence has been so ligated.
• a panoply of carrier expression vectors for many desired gene sequences can be constructed from an intermediate carrier vector.
• the initial single cassette carrier expression vectors which result from insertion of coding sequences into the intermediate carrier vector are further characterized in that the expression cassettes contained in them are interchangeable and combinations of these can be used to form carrier expression vectors which have two, three or any desired number of cassettes.
• the expression cassettes contained in them are interchangeable and combinations of these can be used to form carrier expression vectors which have two, three or any desired number of cassettes.
• the critical portions .of the expression carrier vector comprise one or more expression cassettes, each of which contains a desired coding- sequence operably linked both to a promoter normally operable in plant cells, and to a polyadenylation signal operable in plant cells.
• at least one of the desired coding sequences encodes a protein which results in a phenotypic characteristic enabling selection of transformed plant cells—i.e., a dominant selectable marker.
• a cassette-unique restriction site is located at one end of the expression cassette in a single expression cassette vector, and at the terminus of a series of expression cassettes or at the junction between two cassettes in a multiple cassette vector, and is, in this case, unique not only to the particular expression cassette of its location, but to the entire series or "DNA sequence cassette".
• the expression carrier vector is to be used as a donor vector for supplying an additional expression cassette to a recipient, it is further preferred that there be a second cassette-unique restriction site at the expression cassette's other terminus.
• two single expression cassette vectors are* used, one to provide simply an expression cassette (a donor) and the other to provide its own expression cassette as well as the vector sequences (the recipient).
• the recipient vector is cleaved with one restriction enzyme which cuts at a cassette-unique site at the terminus of its expression cassette;
• the donor expression cassette is excised by treating the donor plasmid with two restriction enzymes, one for each cassette-unique restriction site at the ends of the expression cassette.
• the excised expression cassette and the recipient plasmid are then ligated using standard conditions to yield the resulting multiple expression cassette vector.
• the inserted expression cassette can be oriented in either of two orientations, and the position of the regenerated- cassette-unique restriction site will depend on which orientation has resulted.
• the expression cassettes are bounded by Xhol and Sail restriction sites at the 5* and the 3 1 termini, respectively.
• the recipient vector is treated with Sail to form linear DNA with Sail sticky ends.
• the excised donor expression cassette bears Xhol and Sail sticky ends.
• an intermediate carrier vector is constructed and is part of the invention.
• a first restriction site It has, reading in the 5' to 3' direction of the sense strand, a first restriction site, a promoter normally operable in plants, a polylinker, a polyadenylation signal operable in plants, and a second restriction site.
• the polylinker permits convenient insertion of a desired coding sequence to generate a single cassette expression carrier vector wherein the single cassette is bounded by the two restriction sites.
• Mutiple cassette expression carrier vectors can be constructed by excising the expression cassette of one such vector which has been cleaved at these two restriction sites and inserting it into another using just one of the restriction sites of the recipient vector, a procedure which can be repeated any desired number of times. In a particularly preferred embodiment, this process is made more efficient by utilizing combinations of restriction sites such that one of the sites is not regenerated after the ligation of two expression cassettes.
• the expression carrier vectors described above can be modified in their vector segments so as to adapt them for use in plant cell transformation mediated by bacteria. If A. tumefaciens is to be used as the infectious agent, the modified vector will have at least one A. tumefaciens T-DNA border sequence disposed proximate the terminus of the expression cassette or the series thereof. Preferably the expression cassette or series is framed by a pair of such border sequences.
• the vector segment contains a broad host range bacterial origin of replication. While these modifications do not impair the ability of the expression carrier vectors to transform plant cells directly, they are necessary to permit transformation using the offices of the intermediate bacterium.
• the preferred pivotal vector is comprised of an expression cassette containing a desired coding sequence (preferably that encoding a dominant selectable marker) operably linked both to a promoter normally operable in plant cells and a polyadenylation sequence operable in plant cells framed by the right border (RB) and left border (LB) sequences associated with A. tumefaciens.
• a cassette-unique restriction site is disposed between one of the border sequences and the terminus of the expression cassette.
• the vector segment contains a broad host range bacterial origin of replication.
• This basic expression carrier Agrobacterium- adapted vector is exemplified below by pCMC91.
• the coding sequence encodes the dominant selectable marker, a odifed APH-II, conferring resistance against G418 and kanamycin.
• Derivatives of the basic construction contain additional expression cassettes' so that these derivatives are useful in expressing the coding sequence of any desired foreign gene in the transformed plant cell, and, further, still retain the selectable marker feature of the original cassette.
• These expression cassettes can be conveniently derived from the vectors already described above in paragraph B.2. They are excised by use of the two cassette-unique restriction sites at either end of the cassette, and ligation of the cassette at the cassette- unique restriction site between the border sequence and the terminus of the APH-II cassette in the recipient vector.
• B.-4.a. Direct Transformation All of the expression carrier vectors of the invention can be used to transform plant cells directly. In this method, no bacterial infection is employed. Rather, plant cells are treated under suitable conditions directly with naked DNA, i.e., simply with a suspension of the appropriate vector. At present, one workable method to effect this transformation is to suspend plant cell protoplasts with the plasmid DNA in the presence of a suitable facilitator such as, for example, polyethylene glycol (PEG). This technique is described in greater detail in paragraph H. below. Another method, which has been used less frequently, employs microinjection into individual plant cells- This method is more tedious, but does not require the preparation of protoplasts. In- any event, the vectors of the invention are suitable * for any workable direct transformation technique and it should be understood that the invention is not limited to their use in the foregoing specific methods.
• a suitable facilitator such as, for example, polyethylene glycol (PEG).
• Transformations which are described in paragraph B.3 are suitable for use in bacterial mediated transformation of plant cell hosts. Two variations of such methods are commonly employed. Both share the same initial ste —transformation of a suitable bacterial host harboring a Ti plasmid bearing the virulence coding regions. The bacterial host, now containing both the expression carrier vector and the virulence-containing plasmid, is either co-cultivated with plant cells, preferably protoplasts, in tissue culture, or injected directly into wounded plant tissue. These alternatives are described in greater detail below.
• the infecting or co-cultivated Agrobacterium will contain both the expression carrier vector and a plasmid bearing the virulence region normally associated with a tumor- inducing plasmid. It is preferred, in order to prevent the resulting transformed cells from becoming tumorous, that the virulence region reside on a plasmid lacking the tumor-inducing functions normally carried in its T-DNA region.
• a disarmed virulence region containing plasmid may already be present in the host bacterial cell as in the example hereinbelow, or, perhaps less conveniently, a bacterial host devoid of tumor-inducing plasmids may be transformed both with the carrier expression vector and a disarmed virulence-encoding vector, either simultaneously or sequentially.
• an A. tumefaciens host already containing a disarmed Ti plasmid is used as the recipient for the expression carrier vector.
• the carrier expression vector is suspended under suitable conditions with plant cell protoplasts to permit direct transformation.
• the carrier expression vector is first transformed or conjugated into Agrobacterium containing a disarmed Ti vector and then the Agrobacterium co- cultivated with plant protoplasts in tissue culture.
• Plasmids containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.
• Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g. New England Biolabs, Product Catalog.
• suitable restriction enzyme or enzymes
• about 1 ⁇ g of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate. Partial digestions are occasionally specified. Incubation times of about one hour to two hours at about 37°C are workable, although variations can be tolerated.
• protein is removed by extraction with phenol/chloroform; this step may be followed by ether extraction and the nucleic acid recovered from aqueous fractions by precipitation with ethanol followed by running over a Sephadex G-50 spin column.
• size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology (1980) 6_5:499-560.
• Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four nucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20 to 25°C in 50 mM Tris pH 7.6,
• Synthetic oligonucleotides are prepared by the triester method of Matteucci, et al; (J Am Chem Soc (1981) 103:3185-3191).
• kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 1 n ole- substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl 2- 5 mM dithiothreitol, 1-2 mM ATP, 1.7 pmoles ⁇ 32 p ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.
• an excess e.g., approximately 10 units of polynucleotide kinase to 1 n ole- substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl 2- 5 mM dithiothreitol, 1-2 mM ATP, 1.7 pmoles ⁇ 32 p ATP (2.9 mCi/mmole), 0.1 mM spermidine
• Ligations are performed in 15-30 1 volumes under the following standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 ⁇ g/ml BSA, 10 mM-50 mM NaCl, and either 40 ⁇ M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end” ligation).
• Intermolecular "sticky end” ligations are usually performed at 33-100 ⁇ g/ml total DNA concentrations (5-100 nM total end concentration). Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at 1 ⁇ M total ends concentration.
• the vecto fragment is commonly treated with 5 bacterial alkaline phosphatase (BAP) in order to remove the 5* phosphate and prevent religation of the vector.
• BAP digestions are conducted at pH 8 in approximately 150 M Tris, in the presence of Na + and Mg + 2 using about 1 unit of BAP per ⁇ g of vector at 60° for about one hour.
• the preparation is extracted with phenol/chloroform and ethanol precipitated and desalted by application to a Sephadex G-50 spin column.
• religation can be prevented in vectors which have been double digested
• promoter sequences normally operable in plant cells can be used as well as other polyadenylation signals operable in plants.
• Suitable alternate promoters include the maize promoter for alcohol dehydrogenase-1 or alchohol dehydrogenase-2
• pCMCl served as the source of the NOS promoter, polyadenylation signal, and in the bacterial adapted plasmid derivatives, the "right" T-DNA border sequences.
• ⁇ CMC60 employs a number of intermediate plasmids as shown in Figure 1 and below. These are: pCMC30 which provides the NOS promoter modified- to contain a Hindlll site at its 3' terminus, pCMC39 which provides the NOS promoter modified to contain a Xhol site at its 5' terminus, and pCMC49 which provides the polylinker, the polyadenylation signal and a bacterial host replicon.
• D.l PCMC30 pCMC30 was constructed to contain the NOS promoter modified to contain a Hindlll site at the 3 * terminus.
• a primer repair reaction essentially as outlined by Messing , J . , Third Cleveland Symposium on
• the sequence of the Sau3AI insert was confirmed to comprise the NOS promoter and the sequences surrounding the 3' terminus of the promoter were confirmed by dideoxy sequencing to conform to the expected sequence as disclosed by Depicker, A., et al, J Mol Appl Genet (1982), Vol. 1, p. 566.
• the 3' terminus of the promoter was modified using a primer repair reaction, by annealing with the synthetic pentadecamer
• 5'-pTTGCAGATTATTTGG-OH 3' which complements this fragment so as to include the first A of the ATG codon and 14 upstream nucleotides.
• 50 ⁇ g of single-stranded mCMCIO DNA was digested with Haelll and the 597 bp fragment isolated.
• One ⁇ g of the isolated fragment was annealed with the pentadecamer (supra) and extended with T4 DNA polymerase I in the presence of dNTPs under standard conditions.- The remaining single- stranded region at the 3' end was simultaneously degraded to the 5' terminus of the annealed primer.
• the resulting primer-repaired double-stranded fragment thus terminates at the 3' end after the A of the ATG codon at position 1 of the NOS coding sequence.
• This double-stranded fragment was digested with EcoRI and the 320 bp EcoRI/ blunt DNA fragment purified and cloned into pUC13 (a freely available pBR322 derivative containing polylinkers; see Messing, J., et al. Gene (1982) 19:269) as follows: ⁇ UC13 (10 ⁇ g) was digested with Hindlll and then treated with DNA polymerase I (Klenow fragment) in the presence of dNTPs and then digested with EcoRI.
• the vector fragment was ligated with the 320 bp EcoRI/blunt promoter fragment above and the ligation mixture transformed into E. coli MM294.
• Successful transformants were selected by Amp R , and the required plasmid, pCMC30, confirmed by restriction analysis with Sstll/Hindlll to yield the expected 188 bp DNA fragment.
• pCMC30 provides the NOS promoter as a convenient EcoRI, Sstl, or Smal (Xmal)/ Hindlll cassette-
• D.2 pCMC39 pCMC39 is another intermediate plasmid and it contains the NOS promoter modified at the 5' terminus to contain a Xhol site.
• the Xhol site was excised from pBW14 (4375 bp), which is a pBR322 derivative into which a Xhol-containing octanucleotide (5'-CCTCGAGG-3') was ligated into a Klenow repaired, Hindlll digest of pBR322.
• pBW14 was digested with Xhol, treated with DNA polymerase I (Klenow fragment) in the presence of dNTPs and then treated with EcoRI.
• pCMC29 contains an unmodified NOS promoter whih was originally obtained from pCMCl by digestion of pCMCl with Sau3AI and inserted into the BamHI site of pUC13.
• the ligation mixture was used to transform E. coli MM294 and Amp R colonies were selected.
• Successful transformants were screened for the desired 3.1 kb plasmid, designated pCMC39.
• pCMC39 therefore, contains the NOS promoter modified at the 5' terminus to contain a Xhol site immediately upstream as shown in Figure 1. Its construction was confirmed by restriction analysis.
• D.3 pCMC49 pCMC49 was constructed to contain the NOS polyadenylation signal and a polylinker segment through the intermediate plasmid pCMC20, which, in turn, was constructed using a digest of pCMCl as the source of the relevant sequences, combined with rsal03 used as a source of the polylinker for the expression plasmid.
• pCMCl was digested with Sau3AI and the 250 bp fragment containing the polyadenylation signal from the NOS gene isolated.
• the isolated fragment was ligated with BamHI digested ⁇ UC8 (a freely available ⁇ BR322 derivative containing a polylinker for convenient constructions; see Messing, J., et al (supra)) and the ligation mixture was transformed into E. coli MM294.
• Successful (AmpR) transformants were screened for the expected 3.0 kb plasmid and construction was confirmed by restriction analysis.
• pCMC20 contains unique Sstl and Smal (Xmal) sites 5' to the polyadenylation signal and Sail, Pstl, Haell, Hindlll and BssHII sites 3' to the signal.
• pCMC49 pCMC20 was digested with Xmal, blunt-ended with Klenow fragment and the four dNTPs, and then digested with Sail.
• the 260 bp desired terminator signal fragment was purified and ligated to BamHI(Klenow repaired)/SalI digested rsal03 purified vector-plus- polylinker fragment and the ligation mixture transformed into E. coli MM294.
• Amp R transformants were selected and screened for the desired plasmid, pCMC49, which contains the signal fragment framed by a 5' polylinker containing Hindlll, Xbal, Bglll, Pstl, BamHI and XmaI/(SmaI) sites, and a 3' Sail site as well as the origin of replication from rsal03.
• rsal03 is a pBR322 derivative in which (i) a polylinker containing Hindlll Xbal, Bglll, Pstl, BamHI, and Sail sites is substituted for the 621 bp Hindlll/Sall vector fragment in the tetracycline resistance region, by insertion of the small Hindlll-BamHI fragment from VX (Seed, B., Nucleic Acid Res (1983) 11_:2427) into the large Hindlll-BamHI fragment of pBR322, and (ii) the 514 bp Rsal DNA fragment from bacteriophage M13RF containing the origin of replication is adapted with Hindlll linkers, cut with Hindlll and inserted at the Hindlll site. In rsal03, the M13RF origin of replication is oriented in the same direction as the bla gene on the pBR322 portion.)
• pCMC59 The 3' unmodified, XhoI-5' supplemented, NOS promoter from pCMC39 and the polylinker- ⁇ signal sequences along with the rsal03 vector fragment from pCMC49 were ligated to give pCMC59, the penultimate plasmid in the preparation of pCMC60, which is also a (modified) intermediate carrier vector.
• pCMC39 was digested with EcoRI and Hindlll, the digest ligated to EcoRI/Hindlll digested pCMC49 and the ligation mixture used to transform E. coli MM294. Amp R colonies were screened for the presence of a 4.48 kb plasmid containing unique EcoRI and Hindlll sites as well as the remaining expected restriction sites.
• the selected plasmid, pCMC59 contains the 3* unmodifed NOS promoter preceded by several 5' restriction sites which is upstream from the polylinker and the polyadenylation signals followed' at the 3' terminus by additional restriction sites as well as the vector fragment of rsal03, which includes the Amp R gene and replicon.
• pCMC59 is an alternate form of intermediate carrier vector which permits the insertion of a desired coding sequence so as to yield a fusion protein with the first approximately 15 amino acids of the NOS sequence provided the sequence is inserted in appropriate reading frame.
• the more convenient intermediate carrier vector, pCMC ⁇ O which permits the insertion of a coding sequence for a mature protein in front of the Hindlll site provided by the modified promoter was constructed employing pCMC59 and pCMC30.
• pCMC30 containing the 3'-modified NOS promoter was digested with" Sstll and Hindlll and the 188 bp promoter-containing fragment isolated.
• ⁇ CMC59 was also digested with Sstll and Hindlll and the vector fragment purified. A ligation mixture of these two purified fragments was transformed into E.
• pCMC60 contains a narrow host range bacterial host vector segment from rsal03 and a gene control sequence cassette which, in a 5' to 3' direction, contains a cassette-unique restriction site (a Xhol site, among others), the NOS promoter modified at its 3' end to contain a Hindlll site immediately preceding what would be the ATG initiation codon in the Ti plasmid nopaline synthase gene, a polylinker segment, and a polyadenylation signal derived from the NOS gene followed, finally, by a second cassette-unique restriction site.
• a cassette-unique restriction site a Xhol site, among others
• pCMC70 contains coding sequences, for a truncated and modified APH-I protein (herein designated mtAPH-I) encoding an aminoglycoside phosphotransf rase activity derived from the transposon Tn601 (also known as Tn903) (Sharp, P. A., et al, J Mol Biol (1973) 75:235; Oka, A., et al, J Mol Biol (1981) 147:217).
• the protein encoded by this sequence is capable of conferring resistance to the antibiotics G418 or kanamycin.
• ⁇ CMC71 contains the coding sequence for APH-II, an enzyme conferring"similar antibiotic resistance, which is encoded on the transposon Tn5 (Jorgensen, R. A., et al, Mol Gen Genet (1979) 177:65). Also constructed were: pCMC102 wherein the sequence insert encodes -interferon. pCMC103 wherein the insert encodes chloramphenicol acetyltransferase (CAT) which confers resistance to chloramphenicol. pCMC121 which encodes Bt-toxin, an endotoxin from Bacillus thuringiensis strain HD-1, which is lethal to the larvae of certain insects.
• CAT chloramphenicol acetyltransferase
• E.l PCMC70 pDG144 contains the modified coding sequence for a truncated, modified APH-I protein (mtAPH-I).
• the sequence had been modified by destroying the Xma (Smal) site at the codons for amino acids 93/94 by converting the CCC codon encoding proline (93) to the also proline encoding CCT, and by destroying the Hindlll recognition site at amino acids 175/176 by converting the lysine encoding AAG codon to the also lysine encoding AAA.
• N-terminal sequence was truncated and placed immediately downstream from a Hindlll recognition site by altering the wild type sequence immediately before the ATG start codon from GGTGTT to AAGCTT, deleting the codons for amino acids 2-10 and fusing the ATG start codon to the codon for amino acid 11.
• pCMC71 pCMC71 encoding APH-II was constructed similarly.
• the APH-II gene was obtained by digestion of pAMl (de Fraramond, A., et al. Biotechnology (1983) 1 ⁇ :262- 269), a plasmid which confers kanamycin resistance, with Bglll and Smal to yield a 1.0 kb DNA fragment containing the desired APH-II coding sequence with an additional 36 bp preceding the translation initiation codon.
• the polylinker sequence in pCMC60 contains Bglll and Smal recognition sites in the correct orientation to accept and express the resulting APH-II fragment, and thus this fragment was ligated with a Bglll/Smal digest of pCMC60, the mixture digested with BamHI to inactivate unwanted fragments and used to transform E. coli MM294. Amp R and chloramphenicol sensitive colonies were screened for the presence of a 5.35 kb plasmid. .pCMC71 was analyzed by restriction analysis and shown to have lost the Smal site; the remainder of the structure was as expected.
• E.2.b Construction of pCMC72 pCMC72 was constructed from pCMC71 as shown in Figure 5. This construction removes an out of frame ATG codon in the 5'-noncoding region of the APH- II mRNA, and thus results in an elevated level of APH-II protein production relative to pCMC71.
• pCMC71 was partially digested with Pstl to open a Pstl site located within the APH-II coding region, 177 bp from the amino terminal ATG.
• the plasmid was then digested to completion with Bglll, and both the 207 bp Bglll/Pstl fragment containing the N-terminal coding sequences and a portion of the 5' untranslated region, and the almost full-length vector fragment (missing this 207 bp fragment) were isolated.
• the 207 bp fragment was then digested with Sau3AI, which removed an additional 24 nucleotides from the Bglll end but left a four nucleotide sticky end (GATC) which is compatible with the Bglll sticky end.
• the resulting Sau3AI/PstI fragment was then ligated with the isolated Bglll/Pstl vector fragment of pCMC71, and transformed into E.
• pCMC72 is identical to pCMC71 except for a 24 bp deletion at the 5' end of APH-II which removes an out of frame ATG codon and the bacterial ribosome binding site.
• E.3 PCMC1Q2 ⁇ CMC102 contains the ⁇ -interferon gene in operable linkage to the components of the carrier cassette.
• p£ltr ⁇ 3-4-l a plasmid containing the ⁇ -IFN coding sequence under the control of the trp promoter, was used as a source of the ⁇ -interferon gene. It was digested with Hindlll and XhoII and the 502 bp fragment containing the mature ⁇ -interferon coding sequence was isolated.
• pCMC60 was digested with Hindlll and Bglll and the fragments mixed.
• E.4 PCMC103 The gene encoding chloramphenicol acetyl transferase (CAT) which encodes a protein conferring chloramphenicol resistance was excised from pBR325 (Bolivar, F., et al. Gene (1978) 4_:121) by isolating a 775 bp fragment generated by digestion with Taql. The Taql fragment was subcloned into the Accl site of the polylinker in pUC13 to give an intermediate plasmid which contains the coding sequence for CAT between a Hindlll site 45 bp upstream of the ATG start codon and an Xmal site 100 bp 3' of the TAA termination codon.
• CAT chloramphenicol acetyl transferase
• the Hindlll/Xmal fragment was excised from the intermediate plasmid and purified.
• pCMC ⁇ O was digested with Xmal and Hindlll and the linear DNA ligated with the foregoing fragment, followed by Bglll digestion to inactivate undesired products.
• the ligation mixture was transformed into E. coli MM294 and successful colonies were selected for Amp R .
• the desired vector, pCMC103 was confirmed by restriction analysis.
• E.5 pCMC121 pCMC121 which is a one cassette carrier expression vector for Bt-toxin, was constructed using pCMC ⁇ O in a manner analogous to the foregoing constructions. Its construction is outlined in Figure 2.
• the recombinant plasmid, pESl Wang, et al, J Biol Chem (1983) 258:1960 and Schnepf and Whiteley, Proc Natl Acad Sci (USA) (1981) 2JL :2 893), is a derivative of pBR322 containing as an insert the coding sequences for the Bt-toxin. See also European Application
• pESl in E. coli K12/HB101 is deposited under the terms of the Budapest Treaty at the ATCC under No. 31995.
• pESl was mutagenized with the bacterial transposon, Tn5 according to the method of Guyer, M. S., et al. Methods Enzymol (1983) 101:362 to insert a Xhol recognition sequence 5' to the Bt-toxin.
• a 5.8 kb Xhol fragment was isolated from the pESl-Tn5 plasmid and inserted into the Sail site of pUC9. The ligation mixture was used to transform E.
• coli K12 JM83 available from BRL
• the successful transformants selected by Amp R and lac-.
• Successful transformants were screened for the presence of the desired 8.6 kb plasmid, and the construction of the desired pSYC823 confirmed by restriction analysis.
• pSYC823 was further modified to place the appropriate restriction sites at the termini of the coding sequences. To create a Hindlll site at the 5' terminus, pSYC823 was digested to completion with Ndel, and blunt ended with Klenow in the presence of dATP and dTTP. After complete digestion with Hindlll, the DNA was ligated into the phage vector M13mp8 which had been digested with Smal and Hindlll.. The desired phage
• the resulting modified vector was designated mp ⁇ BtRF.
• a 3' Hindlll site was placed adjacent the Bt-toxin coding region in pSYC823 as follows: Another sample of pSYC823 was partially digested with Ndel, completely digested with Pstl, and blunt ended using Klenow in the presence of all four dNTPs. The 7.0 kb fragment was purified, self-ligated, and then transformed into E. coli MM294.
• the desired intermediate plasmid is designated pCMC120 and represents pSYC823 with a 1.6 kb deletion of Bacillus sequences from the unique Pstl site of the vector to the Ndel site at the 3* end of the Bt-toxin coding region. This deletion placed the Hindlll site of the pUC9 vector directly adjacent the 3' end of the toxin coding region and destroyed the Pstl site.
• the Bt-toxin coding sequence was inserted into the pCMC60 intermediate vector in two portions, the 5' portion from the mp ⁇ BtRF 5' Hindlll modified segment, and the 3' portion from the pCMC120.3' Hindlll modified Bt-toxin coding sequence of the intermediate plasmid above.
• mp ⁇ BtRF was digested to completion with Hindlll and Sstl and the 1.4 kb fragment isolated.
• the second fragment was obtained by digesting pCMC120 to completion with Sstl, then partially with Hindlll and purifying the 2.3 kb DNA fragment from the Hindlll site of the pUC9 vector portion to the Sstl site located within the gene.
• the two foregoing fragments were ligated with Hindlll digested, -BAPed pCMC ⁇ O, and transformed into E. coli
• E.6 Construction of an Intermediate and Expression Carrier Plasmid Suitable for Expression of a Fusion Protein pCMClOl is analogous to pCMC60 except that, instead of the NOS promoter which terminates in a convenient Hindlll site suitable for the insertion of a mature coding sequence, pCMClOl has a chimaeric segment with the NOS promoter fused to coding sequences for 3-galactosidase. Subsequent insertion of desired coding sequence in reading frame with the ⁇ -galactosidase codons results in a fusion protein containing both ⁇ -galactosidase sequences and amino acid sequences corresponding to the inserted codons. It is also an expression carrier vector in that it is capable of effective expression of the NOS- ⁇ -galactosidase fusion.
• the construction is outlined in Figure 3.
• Plasmid pMC1403 (Casadaban, M. J., et al, J Bacteriol (1980) 143:971) contains the ⁇ -galactosidase coding sequence, with a polylinker at the 5' end. To provide a convenient 3' terminal restriction site, the lac-Z portion of pMC1403 was excised by digestion with EcoRI (which cleaves in the polylinker) and Clal which cleaves just 3' of the lac-Z gene (Buchel, D. E. , et al. Nature (1980) 2j$3.:541).
• the EcoRI/Clal 3.4 kb fragment was cloned into EcoRI/Clal digested pOG2326, a bifunctional replicon capable of replication in both E. coli and B__ subtilis and derived from pBR322, to obtain pDH5425.
• pDH5425 was digested with BamHI to obtain a 3.8 kb fragment spanning the BamHI site of the polylinker sequence at the lac-Z 11th codon to the BamHI of the Tet R gene of the vector plasmid.
• the BamHI fragment was subcloned into the BamHI site of ⁇ UC13 to give pCMC9163 which thus has a 5' terminal polylinker on the lac-Z gene as follows:
• the pCMC29 intermediate plasmid (paragraph D.2), which contains the unmodified NOS promoter, was used.
• This plasmid has a polylinker region following the codon 16 of the original NOS coding sequence as follows:
• the Xbal sites of pCMC29 and pCMC9163 are in identical reading frames. Further, there is a unique Hindlll site at the 3 * end of the polylinker in pCMC29 and at the 3 1 end of the ⁇ -galactosidase coding sequences in pCMC9163. Therefore, both plasmids were digested to completion with Hindlll and Xbal, the fragments ligated, and the ligation mixture was transformed into E. coli MM294. Successful colonies were selected for Amp R .
• the resulting plasmid, pCMC17 thus comprises the restriction sites EcoRI, Sstl, Smal(Xmal) of the pUC13 polylinker, the NOS promoter and the amino terminal coding region for NOS fused in correct reading frame to the lac-Z gene, the coding sequences for ⁇ -galactosidase downstream from codon 18, followed by a Hindlll site.
• pCMC ⁇ O was digested with EcoRI and Hindlll and the vector fragment ligated with the 3.4 kb fragment obtained from pCMC17 by digestion with EcoRI and Hindlll containing the NOS promoter and amino terminal codons and fused lac-Z gene.
• the resulting vector, pCMClOl was obtained from successful E. coli MM294 transformants selected for Am ⁇ R by screening for the desired 7.9 kb plasmid and confirmed by restriction analysis.
• pCMC102 was digested to completion with Xhol and Sail, and the desired 1.1 kb DNA fragment purified. The fragment was ligated with Sail digested, BAPed ⁇ CMC71, and the ligation mixture was transformed into E. coli MM294. Amp R colonies were screened for the presence of the desired 6.5 kb plasmid, pCMC77. The correct construction was confirmed by restriction analysis.
• the resulting vector has Xhol and Sail sites which remain "cassette-unique" at the 5' and 3* termini of the series, if the donor expression cassette is unidirectional with the recipient vector DNA sequences; the Sail site is between the expression cassettes if the opposite orientation results (see paragraph B.2 above).
• Similar Xhol/Sall digestions were used to prepare the CAT cassette from pCMC103, the mtAPH-I cassette from pCMC70, and the Bt-toxin cassette from pCMC121. These fragments were ligated into Sail digested pCMC71 to form the double-cassette expression carrier vectors below:
• pCMC78 containing the APH-II and CAT cassettes
• pCMC79 containing the APH-II and mtAPH-I cassettes
• pCMC75 containing the APH-II nd Bt-toxin cassettes
• the NOS-lac-Z fusion cassette was obtained from pCMClOl, because there was no Xhol site 5' to the gene, by digestion instead with EcoRI (a 5' cassette-unique site), and Sail.
• EcoRI a 5' cassette-unique site
• Sail a 5 r EcoRI/3'SalI galactosidase chimaera was ligated into pCMC71 which had.been digested with EcoRI and Xhol to give a double cassette recombinant plasmid (pCMC76), selected following minipreps.
• pCMC90, pCMC91 and pCMC92 are single cassette expression vectors for the mtAPH-I or APH-II genes framed by border sequences and having a broad host range bacterial origin of replication. These constructions are outlined in Figure 4. As shown in Figure 4, pCMC91 is constructed from pCMC90 by replacing the mtAPH-I coding region with the APH-II coding sequence from pCMC71. pCMC92 is formed from pCMC90 using pCMC72 instead of pCMC71.
• pCMC90 pCMC90 was constructed from the intermediate vectors described below by inserting a 2.8 kb EcoRI/Sall cassette from pCMC80, which contained the mtAPH-I coding sequences with suitable control segments along with right border (RB) T-DNA into a broad spectrum replicating host plasmid bearing the left border (LB) regions, pCMC25.
• G.l.a Construction of pCMC80 pCMC70, prepared as described in paragraph E.l, is a carrier expression vector for the modified, truncated (mt) dominant selectable marker, mtAPH-I. It contains the NOS promoter as an EcoRI/Hindlll cassette immediately preceding the coding sequence. In pCMCSO, this cassette is replaced by a larger EcoRI/Hindlll cassette, which cassette contains the RB sequence from pTiT37 along with the NOS promoter modified to have a Hindlll site at the 3' end of the promoter as in pCMC70 preceding the site for translation initiation as in pCMC70.
• pCMCl was digested with Hindlll, treated with Poll Klenow fragment in the presence of dNTPs, and then digested with Sstll.
• the 1.1 kb Hindlll(blunt)/ Sstll fragment was isolated and ligated with DNA fragments obtained by digesting pCMC70 with Xhol, treating with Klenow and the dNTPs, and then digesting with Sstll. Approximately 200 ng of the DNA from the ligation mixture was used to transform E. coli MM294 to Amp R .
• pCMC ⁇ O exemplifies a carrier expression vector for a foreign gene with a narrow host range baterial origin of replication, containing a right border sequence properly placed with respect to the transcription cassette.
• G.l.b. Construction of pCMC25 pCMC25 was constructed as follows: The LB sequence was obtained from pTiT37 by digesting with EcoRI and isolating the 1.6 kb fragment 29 using standard methods. This fragment was cloned into the EcoRI site of pAMl, a derivative of pBR325 in which a 1.5 kb Hindlll/ Sail DNA fragment from transposon Tn5 containing the APH-II antibiotic resistance gene had been substituted for the Hindlll/Sall region of the Tet R gene.
• the ligation mixture containing the thus modified pAMl vector was cloned into E. coli MM294 and selected for Amp R .
• Successful colonies were screened for the desired 8.1 kb plasmid, pCMC5, which contained the LB region of T-DNA contiguous with the APH-II gene.
• pCMC5 was further modified to provide a broad spectrum bacterial origin of replication by utilizing the vector portions of RSF1010 (Bagdasarian, M., et al. Gene (1981) 16_:237), a broad host range, multicopy plasmid containing a sulfonamide resistance (Su R ) gene and capable of replicating both in E. coli and in A. tumefaciens.
• pCMC5 was partially digested with Bglll and then further digested with Sail to produce the desired 3.4 kb Bglll/Sall fragment containing the T-DNA LB sequence and adjacent APH-II gene.
• the fragment was repaired with Klenow and dNTPs to provide a blunt-ended fragment having the sequence 5'- GATCT-LB-APH-II-GTCGA-3'.
• This blunt-ended fragment was ligated with Sstl digested, Klenow repaired RSFIOIO which thus provided a vector sequence with 5 1 C and 3' G terminal nucleotides.
• the ligation mixture was transformed into E.
• pf pCMC15 was confirmed by restriction analysis: the desired construction contains a single Sail site at the point of ligation of the APH-II DNA but has lost the Sstl and Bglll sites.
• pCMC15 was further modified to insert a polylinker region adjacent the T-DNA-LB segment. The polylinker was derived from pUC13 by digestion with EcoRI, repaired with Klenow and all four dNTPs, followed by further digestion with Sail.
• the ligation mixture was trans ⁇ formed into E. coli MM294 to Su R and subsequently transformants were screened for kanamycin sensitivity. The transformants were screened for the presence of the desired 10.2 kb plasmid, pCMC25, which contains a polylinker region proximal to the LB sequences.
• the desired pCMC90 was constructed as follows: pCMC25 was digested with with EcoRI and Sail to open the vector next to the polylinker region. ⁇ CMC80 was digested with EcoRI and Sail to release the mtAPH-I expression cassette which was purified and ligated with the opened pCMC25 vector. The ligation mixture was used to transform E. coli MM294 to Su R , and Amp s transformants were screened for the presence of the desired 12.9 kb expression carrier plasmid, pCMC90.
• pCMC90 is a multicopy, Su R Amp s derivative of the wide host range plasmid, RSFIOIO, which contains an expression cassette for the modified mtAPH-I sequence bordered by the T-DNA right and left border sequences.
• the inserted cassette contains a unique Sail site at its 3' end between the polyadenylation signal and the LB sequence.
• pCMC90 was digested with Hindlll and Sail, and the approximately 11.5 kb vetor fragment was purified.
• pCMC71 was digested with Hindlll and Sail, and the 1.3 kb fragment containing the APH-II coding region and NOS termination region was also purified.
• the 11.5 kb vector and 1.3 kb APH-II fragments were then ligated and transformed into E. coli MM294 to Su R .
• the correct construction of pCMC91 was confirmed by restriction analysis and pCMC91 was deposited with ATCC on or about 10 April 1985 and has accession no.
• pCMC92 was constructed in a manner exactly analogous to that described for pCMC91 except that pCMC72 was used instead of pCMC71 as the source of the APH-II gene.
• pCMC92 has a modified 5' untranslated region for the APH-II gene lacking the out of frame ATG and the bacterial ribosome binding site.
• pCMC92 was deposited with ATCC on or about 10 April 1985 and has accession no.
• Carrier Vectors pCMC112 contains a T-DNA bordered double expression cassette for APH-II and ⁇ -IFN on a vector with a broad host range replicon. It was prepared by digesting pCMC91 with Sail, treating with BAP, and ligating the linearized DNA with the 1.1 kb fragment isolated after Xhol/Sall digestion of pCMC102. The ligation mixture was transformed into E. coli MM294, and Su R colonies screened for the desired 13.8 kb ⁇ CMCH2. It contains a unique Sail site between the IFN cassette and the LB sequence.
• pCMC114 containing the APH-II and mtAPH-I cassettes; pCMC113 containing the APH-II and CAT cassettes;
• PCMC123 containing the APH-II and Bt-toxin cassettes.
• pCMC123 was deposited with ATCC on or about 10 April 1985 and has accession no. .
• the cassette series is framed by right border and left border T-DNA sequences, resides on a plasmid containing a broad host range origin of replication, and in most cases contains a cassette-unique (Sail) site between the polyadenylation signal of the cassette series and the LB sequence.
• Sail cassette-unique
• an EcoRI(blunt)/SalI cassette can be isolated from pCMClOl (paragraph E.6) and ligated into Sall/Smal digested pCMC91.
• pCMClOl can be digested with Sail, and this plasmid can be ligated with Sail digested pCMC91, generating a cointegrate plasmid (pCMC91/101) in which the entire galactosidase expression vector is inserted into the vector, pCMC91.
• the following examples illustrate the use of the expression carrier vectors prepared above and the results therefrom.
• paragraph H is illustrated the direct transformation of plant cells using pCMC71, pCMC91, and pCMC77. Of course, any of the other vectors could as well have been used in the same fashion.
• Paragraph I illustrates the use of pCMC91, pCMClll, and pCMC123 in Agrobacterium-mediated transformation. All of the vectors whose construction was illustrated in paragraph G above could as well have been used.
• expression carrier vector refers to each of these three plasmids.
• N. tabacum var. H425 protoplasts were prepared from sterile shoots as described by Krens, et al. Nature (1982) 296:72, and suspended in a K3 medium containing 0.4 M sucrose, naphthalene acetic acid (NAA) at 0.1 mg/1 and kinetin at 0.2 mg/1.
• NAA naphthalene acetic acid
• the protoplasts (5 x 10 5 cells in 1 ml) were combined with 0.5 ml of 40% (w/v) polyethylene glycol 6000 dissolved in an uptake buffer (UB) consisting of 140 mM NaCl, 5 mM NaCl, 5 mM KC1, 0.75 M Na2P ⁇ 4, 5 mM glucose and 125 mM CaCl2 * 2H2° at P H 7.0.
• UB uptake buffer
• 50 ⁇ l solution containing 1 to 10 ⁇ g expression carrier vector and 0 to 50 ⁇ g of purified N. tabacum var. H425 DNA, added to protect against degradation. (Higher rates of transformation were observed in the presence of the purified plant DNA.)
• the protoplasts were incubated at 25°C for 30 min with very gentle shaking. At 5 rain intervals thereafter, 2 ml aliquots of the UB medium were added dropwise to the solution, until a total of 10 ml new UB medium had been added. The protoplasts were pelleted by low speed centrifugation and the supernatant was removed. The pellet was resuspended in 10 ml K3 medium and plated on sterile Whatman #2 (qualitative) filter paper discs, and separated from a feeder layer of wild type N. tabacum var. H425 suspension cells by an additional layer of filter paper. The cells were grown on a standard M3 nutrient medium (Marton, et al. Nature (1979) 277:129) containing phytohormones.
• Cells were plated at a density of 10 2 to 10 4 per 10 cm Petri plate. The cells were incubated in the dark for 24 hr and then grown for 2 weeks at 2,000 lux in 12 hr photoperiods at 25°C. Cell survival was approximately 50% after this time period. The upper filter layer on which small cell clumps were grown was then transferred to M3 medium supplemented with phytohormones (0.1 mg/1 NAA and 0.2 mg/1 kinetin) and G418 (25 mg/1) or kanamycin (100 mg/1) and incubation was continued at 25°C with 12 hr photoperiods. Alternatively, transformed cells were plated at a density of l ⁇ 5/ml in T-75 culture flasks.
• Rooted plantlets were placed in soil under high humidity for several days, followed by routine greenhouse growth conditions.
• DNA from transformed and non-transformed plant cells and from cassette vector pCMC71 are digested to completion with Hindlll and Sail. Standard pCMC71 digests are carried out in the presence of 5 ⁇ g calf thymus DNA per well as carrier. The digested DNAs are loaded into 1x8x8 mm wells in a horizontal.0.6% agarose gel prepared in Tris-acetate buffer (Chilton, M. D., et al. Cell (1977) 11:263). Agarose-gel electrophoresis and transfer of gel- fractionated DNA to nitrocellulose are carried out as described in Thomashaw, et al, Proc Natl Acad Sci (USA) (1980) 72:6448. Plant DNA samples are loaded at 5 ⁇ g per well, and the pCMC71 DNA at various concentrations corresponding to 1 to 100 genome equivalents.
• Hybridization probes are generated by nick-translation of purified Hindlll/Sall 1.3 kb DNA fragment from plasmid pCMC71, according to the general method of Maniatis, T., et al, Proc Natl Acad Sci (USA) (1975) 22. : -_._- 84 _ The probe is used at a specific activity of approximately 10 ⁇ cpm/ ⁇ g DNA for Southern blot analysis of nitrocellulose-bound DNA fractions (Chirgwin, T., et al. Biochemistry (1979) 18 ⁇ :5294).
• Results of the Southern analyses show the presence of a Hindlll/Sall 1.3 kb DNA gene fragment in DNA from regenerated, transformed plants, corresponding in size to the DNA fragment from pCMC71 which encodes the APH-II protein. No similarly sized DNA fragment is observed in the DNA of normal (untransformed) tobacco plants.
• H.l.b. Transcription of pCMC71 Total RNA fractions from young transformed and normal plants are isolated according to the method of Chirgwin (supra), as modified by Bevan, et al, J Mol Appl Genet (1982) 1 ⁇ :539. Leaves and stems of young plants (5 to 50 grams per extraction) are frozen in liquid nitrogen, powdered, and lyophilized until dry.
• the tissue is homogenized in extraction solution containing 4 M guanidine thiocyanate, 25 mM Tris-Cl, pH 7.0, 0.5% sarkosyl and 0.03% Antifoam A Sigma.
• Sarkosyl is a trademark for sodium lauryl sarcosinate and was obtained from Sigma.
• the horaogenate is prepared to a concentration of 1 gram initial tissue weight/ml extraction solution.
• the homogenate is filtered through cheesecloth, followed by centrifugation at 5000 x g for 10 min to remove particulate matter, and the supernatant liquid adjusted to pH 5.0 with 1 M acetic acid; one volume absolute ethanol is added to precipitate nucleic acids.
• the precipitate is pelleted by centrifugation at 5000 x g for 30 min and redissolved in 25 ml of 7.5 M guanidine hydrochloride, 25 mM Tris-Cl, pH 7.0. The pH is adjusted to 5.0 with 1 M acetic acid, and 18 ml absolute ethanol added. The precipitate which forms after about 12 hr at -20°C is collected by centrifugation, washed twice with 70% ethanol, and dissolved in 25 ml of 25 mM EDTA, 0.1% diethyl pyrocarbonate, pH 8.0. This solution is extracted with water saturated phenol:chloroform mixture (6:5) and precipitated with 2 volumes ethanol.
• RNA pellet resulting from centrifugation is fractionated by oligo-dT cellulose chromatography (Bantle, et al. Anal Biochem
• Polyadenylated RNA (polyA RNA) is denatured with 1 M glyoxal at 50°C for 1 hr (McMaster, et al, Proc Natl Acad Sci (USA) (1977) ⁇ 4_:4835). The RNA is electrophoresed and subjected to Northern blot analysis, as described by Bevan, et al, J Mol Appl Genet (1982)
• Tobacco ribosomal RNAs are used as molecular weight standards on gels.
• the pCMC71 probe shows no hybridization to polyA RNA in normal (untransformed) tobacco, but a hybridization band is observed in mRNA isolated from plants regenerated from pCMC71-transformed tissue cultures.
• the estimated size of the APH-II mRNA is in excess of 1000 nucleotides.
• Effective selection for G418 (or kanamycin) resistance to the transformed plant cells implies that the introduced gene is expressed. This is confirmed, by showing the presence of APH-II protein in transformed plant cells by immunoassay.
• Tissue samples from the regenerated plants are grown under conditions of brief radioactive amino acid labeling with 3H glycine.
• Plant supernatant proteins are extracted by the method of Barton, K., et al, J Biol Chem (1982) 257:6089.
• Supernatant proteins 500 ⁇ g/ml
• Supernatant proteins 500 ⁇ g/ml
• Immunopecipitation is carried out at 37°C for 4 hr.
• the immunoprecipitates are pelleted by centrifugation, dissolved in sodium dodecyl sulfate (SDS) and analyzed by SDS-PAGE.
• the radioactive banding pattern was determined by fluorography (Laskey, R. A., et al, Eur J Biochem (1975) 56:335) .
• All of the expression carrier vectors of the invention which contain the T-DNA border sequences, which mediate Agrobacterium assisted transformation, can be used in the procedures of this paragraph I. Construction of such vectors is illustrated in paragraph G, above. The description of this paragraph is set forth in terms of pCMC91, however, any of the described vectors or other vectors of the invention which contain T-DNA border sequences and broad host bacterial origins of replication can be used.
• the procedure illustrates the use of an Agrobacterium mutant which contains a disarmed Ti plasmid to supply the virulence regions. However, alternate means of supplying such sequences, absent the tumor- producing sequences in the T-DNA region of the Ti plasmid, such as cotransformation of an "empty" -56-
• Agrobacterium strain with both a disarmed Ti plasmid and an expression carrier vector of the invention could be used.
• the disarmed Ti vector employed was that harbored by A. tumefaciens strain LBA4404, available from Dr. Paul Hooykaas at the University of Lyden (Ooms, G., Hooykaas, P. J. J., et al. Gene (1981) 14 ⁇ :33).
• This strain is non-oncogenic as it harbors only pAL4404, an avirulent deletion mutant derivative of the octopine Ti plasmid, pTiAch ⁇ , which has a Tn904 insert in Smal fragment 3A, and has suffered a deletion extending from this point rightward to Smal fragment 6.
• This plasmid thus lacks the entire octopine T-DNA and octopine metabolism functions, but retains an intact virulence region on the left side of the T-DNA (See de Framond, A. J., et al. Biotechnology (May 1983) 262-269; Hoekema, A., et al. Nature (1983) 303:179).
• A. tumefaciens harboring both pCMC91 and the disarmed Ti plasmid pAL4404 was obtained by transforming A. tumefaciens strain LBA4404 with pCMC91 by the freeze-thaw method of Holsters, et al, Mol Gen Genet (1978) 163:181, and selecting for Su R .
• PCMC91 and the Disarmed Ti Plasmid Stems of Nicotiana tabacum, variation Havana 425 (H425) were surface sterilized with 7% commercial Chlorox and 80% ethanol, then rinsed with sterile distilled water and cut into 1 cm long segments.
• the segments were placed basal end up in Petri dishes containing complete MS medium (Binns, A., et al, Planta (1979) 145:365) with hormonal supplements of 2 mg/1 naphthalene acetic acid (NAA) and 0.3 mg/1 kinetin.
• the basal end of each stem segment was punctured repeatedly with syringe needles and inoculated with A. tumefaciens strain LBA4404 transformed with pCMC91.
• MS medium MS basal medium supplemented with 2.0 mg/1 NAA and 0.3 mg/1 kinetin.
• the medium also contained 200 mg/1 carbenicillin and 500 mg/1 vancomycin to kill the inoculating bacteria, and antibiotics to select for transformed plant cells.
• the selection of antibiotics routinely included kanamycin (100 mg/1) or G418 (25 mg/1) although neomycin or lividomycin are also acceptable alternatives.
• the calli After three transfers (at approximately 4-week intervals) of the small clumps of - viable cells which were resistant to kanamycin or G418, the calli are sufficiently enriched in transformed cells relative to non-transformed cells to enable assay for the presence of heterologous DNA in the cells, transcription of the heterologous gene, and heterologous-DNA gene expression.
• the calli grown under the above conditions reach approximately 1 mm in diameter, they are picked by scalpel point and placed on M3 medium solidified with 0.4% agar containing phytohormones and kanamycin (100 mg/1). Once calli reaches 2 mm or more in diameter, small pieces are transferred to M3 medium containing 0.1 mg/1 NAA and 0.5 mg/1 to 1 mg/1 kinetin to induce shoot formation.
• Rooted plantlets are placed in soil under high humidity for several days, followed by routine greenhouse growth conditions.
• N. tabacum variation H425 seedlings were grown under sterile conditions and young leaves were treated to generate viable protoplasts, as described in paragraph H.l.
• the protoplasts were cultured in a K3 medium supplemented with NAA at 0.1 mg/1 and kinetin at 0.2 mg/1 for 24 hr in the dark, followed by 48 hr incubation at 2,000 lux.
• the cells, with regenerating cell walls, were mixed with A. tumefaciens LBA4404 transformed with pCMC91 at l ⁇ 5 plant cells and 10? bacterial cells per ml.
• the cells were incubated in K3 medium supplemented with phytohormones as above for approximately 24 hours at 20°C at 2,000 lux, after which time bacteria were removed by repeated washing, with the final wash in K3 medium supplemented with phytohormones as above, and 200 mg/1 carbenicillin and 250 mg/1 vancomycin.
• Cells were then placed on Whatman #2 filters over feeder layers (as described above for single cell cloning) in M3 medium containing phytohormones, carbenicillin at 200 mg/1 and vancomycin at 250 mg/1. Plant cell densities were varied between 1Q and 10 cells per 10 cm Petri plates, the higher dilutions giving rise to colonies resulting from a single cell.
• the deposited plasmids have been assigned the indicated ATCC deposit numbers.
• the plasmids have also been deposited with the Master Culture Collection (CMCC) of Cetus Corporation, Emeryville, California, U.S.A., the assignee of the present application, and assigned the indicated CMCC deposit numbers: Plasmid and Host CMCC Deposit No. ATCC Deposit N p ⁇ ltrp3-4-l in E. coli K-12/MM294 1730 39646 pCMCl in E. coli K-12WM294 1985 39641 pCMC15 in E. coli K-12/MM294 2002 39654 PDG144 in E. coli K-12/MM294 1960 39579 pDH5425 in E.
• CMCC Master Culture Collection

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