United States Patent (19)
`Cohen et al.
`
`11
`45
`
`Patent Number:
`Date of Patent:
`
`4,468,464
`"Aug. 28, 1984
`
`54) BIOLOGICALLY FUNCTIONAL
`MOLECULAR CHMERAS
`75) Inventors: Stanley N. Cohen, Menlo Park;
`Herbert W. Boyer, Mill Valley, both
`of Calif.
`73) Assignee: The Board of Trustees of The Leland
`Stanford Junior University, Stanford,
`Calif.
`The portion of the term of this patent
`subsequent to Dec. 22, 1998 has been
`disclaimed.
`21) Appl. No.: 959,288
`22 Filed:
`Nov. 9, 1978
`
`*) Notice:
`
`63
`
`Related U.S. Application Data
`Continuation of Ser. No. 687,430, May 17, 1976, aban
`doned, which is a continuation-in-part of Ser. No.
`520,691, Nov. 4, 1974, abandoned.
`51
`Int. Cl. ....................... C12N 1/00; C12N 15/00;
`C12N 1/20; C12P 21/00
`52 U.S.C. ...................................... 435/317; 435/68;
`435/253; 435/820; 435/172.3; 935/6; 935/29;
`935/56; 935/60, 935/67; 935/68; 935/73;
`935/84; 535/23
`58) Field of Search ................... 435/172, 68,317,91,
`435/253
`
`56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,237,224 12/1980 Cohen et al. ........................ 435/172
`OTHER PUBLICATIONS
`Lobban, Thesis for Degree of Doctor of Philosophy at
`Stanford University, May 1972.
`Hogness et al., published in "The Eukaryotic Genome',
`Australian National University, Press, Canberra Austra
`lia, 1975.
`Wensink et al.; Cell, vol. 3, pp. 315-325, Dec. 1974.
`Letter from Dr. Stanley N. Cohen to Dr. Donald Fre
`drickson, Sep. 6, 1977.
`Nature, vol. 284, p. 388, Apr. 3, 1980.
`Ziff, New Scientist, vol. 60, Oct. 25, 1973, pp. 274 and
`275.
`Lear, Recombinant DNA, The Untold Story, Crown
`Publishers Inc., New York, pp. 83-84, 1978.
`Cohen et al., Journal of Bacteriology, Revised Interpre
`
`tation of the Origin of the pSC101 Plasmid, Nov. 1977,
`pp. 734–737.
`Cohen, Scientific American, The Manipulation of
`Genes, Jul. 1975, pp. 113-121.
`Cohen et al., Proc. Nat. Acad. Sci., vol. 70, No. 11, pp.
`3240-3244, Nov. 1973.
`Acevedo et al.; Cancer, vol. 41, pp. 1217 to 1229, Apr.
`1978.
`Itakwa et al.; Science, vol. 198, pp. 1056-1063, 12-1977.
`Villa-Komanoff et al.; Proc. Natl. Acad. Sci., U.S.A.,
`vol. 75, No. 8, pp. 3727-3731, Aug. 1978.
`Novick, Bacteriological Reveiws, vol. 33, No. 2, pp.
`210, 211 & 248-252, Jun. 1969.
`Morgan et al.; Recombinant DNA and Genetic Experi
`mentation, Pergamon Press, pp. 21-45 (1979).
`Jackson et al.; Proc. Natl. Acad. Sci., U.S.A., vol. 69,
`No. 10, pp. 2904-2909, Oct. 1972.
`Mentz et al.; Proc. Natl. Acad. Sci., U.S.A., vol. 69, No.
`11, pp. 3370-3374, Nov. 1972.
`Lobban et al.; J. Mol. Biol., 78:453-471 (1973).
`Cohen et al.; Proc. Natl. Acad. Sci., U.S.A., vol. 69, No.
`8, pp. 2110-2114, Aug. 1972.
`Lederberg, J., Toward Century 21: Technology, Society,
`and Human Values, C. S. Wallia (editor), pp. 39-52,
`(1970).
`Primary Examiner-Alvin E. Tanenholtz
`Attorney, Agent, or Firm-Bertram I. Rowland
`57
`ABSTRACT
`Method and compositions are provided for replication
`and expression of exogenous genes in microorganisms.
`Plasmids or virus DNA are cleaved to provide linear
`DNA having ligatable termini, which are bound to a
`gene having complementary termini, to provide a bio
`logically functional replicon with a desired phenotypi
`cal property. The replicon is inserted into a microor
`ganism cell by transformation. Isolation of the transfor
`mants provides cells for replication and expression of
`the DNA molecules present in the modified plasmid.
`The method provides a convenient and efficient way to
`introduce genetic capability into microorganisms for
`the production of nucleic acids are proteins, such as
`medically or commercially useful enzymes, which may
`have direct usefulness, or may find expression in the
`production of drugs, such as hormones, antibiotics, or
`the like, fixation of nitrogen, fermentation, utilization of
`specific feedstocks, or the like.
`The invention was supported by generous grants of
`NIH, NSF and the American Cancer Society.
`
`11 Claims, No Drawings
`
`Page 1
`
`Page 1
`
`KASHIV EXHIBIT 1014
`IPR2019-00791
`
`

`

`1.
`
`BIOLOGICALLY FUNCTIONAL MOLECULAR
`CHMERAS
`
`5
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`This is a continuation of application Ser. No. 687,430
`filed May 15, 1976, now abandoned which is a CIP of
`Ser. No. 520,691 filed Nov. 4, 1974 now abandoned.
`10
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`Although transfer of plasmids among strains of E.
`coli and other Enterobacteriaceae has long been accom
`plished by conjugation and/or transduction, it has not
`15
`been previously possible to selectively introduce partic
`ular species of plasmid DNA into these bacterial hosts
`: or other microorganisms. Since microorganisms that
`have been transformed with plasmid DNA contain au
`tonomously replicating extrachromosomal DNA spe-20
`cies having the genetic and molecular characteristics of
`the parent plasmid, transformation has enabled these
`lective cloning and amplification of particular plasmid
`genes.
`The ability of genes derived from totally different 25
`biological classes to replicate and be expressed in a
`particular microorganism permits the attainment of
`interspecies genetic recombination. Thus, it becomes
`practical to introduce into a particular microorganism,
`genes specifying such metabolic or synthetic functions
`30
`as nitrogen fixation, photosynthesis, antibiotic produc
`tion, hormone synthesis, protein synthesis, e.g. enzymes
`or antibodies, or the like-functions which are indige
`nous to other classes of organisms-by linking the for
`eign genes to a particular plasmid or viral replicon.
`35
`2. Brief Description of the Prior Art
`References which relate to the subject invention are
`Cohen, et al., Proc. Nat. Acad, Sci., USA, 69, 2110
`(1972); ibid, 70, 1293 (1973); ibid, 70, 3240 (1973); ibid,
`71, 1030 (1974); Morrow, et al., Proc. Nat. Acad. Sci., 40
`71, 1743 (1974); Novick, Bacteriological Rev., 33,210
`(1969); and Hershfeld, it al., Proc. Nat. Acad. Sci., in
`press; Jackson, et al., ibid, 69,2904 (1972);
`SUMMARY OF THE INVENTION
`45
`Methods and compositions are provided for geneti
`cally transforming microorganisms, particularly bac
`teria, to provide diverse genotypical capability and
`producing recombinant plasmids. A plasmid or viral
`DNA is modified to form a linear segment having liga
`table terminai which is joined to DNA having at least
`one intact gene and complementary ligatable termini.
`The termini are then covalently bound to form a "hy
`brid' plasmid molecule which is used to transform sus
`ceptible and compatible microorganisms. After trans
`55
`formation, the cells are grown and the transformants
`harvested. The newly functionalized microorganisms
`may then be employed to carry out their new function;
`for example, by producing proteins which are the de
`sired end product, or metabolites of enzymic conver
`60
`sion, or be lysed and the desired nucleic acids or prote
`ins recovered.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`The process of this invention employs novel plas
`mids, which are formed by covalently inserting DNA
`having one or more intact genes into a plasmid in such
`
`4,468,464
`2
`a location as to permit retention of an intact replicator
`locus and system (replicon) to provide a recombinant
`plasmid molecule. The recombinant plasmid molecule
`will be referred to as a "hybrid” plasmid or plasmid
`"chimera.” The plasmid chimera contains genes that are
`capable of expressing at least one phenotypical prop
`erty. The plasmid chimera is used to transform a suscep
`tible and competent microorganism under conditions
`where transformation occurs. The microorganism is
`then grown under conditions which allow for separa
`tion and harvesting of transformants that contain the
`plasmid chimera.
`The process of this invention will be divided into the
`following stages:
`I. preparation of the recombinant plasmid or plasmid
`chimera;
`II. transformation or preparation of transformants;
`and
`III. replication and transcription of the recombinant
`plasmid in transformed bacteria.
`I. Preparation of Plasmid Chimera
`In order to prepare the plasmid chimera, it is neces
`sary to have a plasmid, which can be cleaved to provide
`an intact replicator locus and system (replicon), where
`the linear segment has ligatable termini or is capable of
`being modified to introduce ligatable termini. A small
`number of such plasmids presently exist. Of particular
`interest are those plasmids which have a phenotypical
`property, which allow for ready separation of transfor
`mants from the parent microorganism. The plasmid will
`be capable of replicating in a microorganism, particu
`larly a bacterium, which is susceptible to transforma
`tion. Various unicellular microorganisms can be trans
`formed, such as bacteria, fungi and algae. That is, those
`unicellular organisms which are capable of being grown
`in cultures or fermentation. Since bacteria are for the
`most part the most convenient organisms to work with,
`bacteria will be hereinafter referred to as exemplary of
`the other unicellular organisms. Bacteria, which are
`susceptible to transformation, include members of the
`Enterobacteriaceae, such as strains of Escherichia coli;
`Salmonella; Bacillaceae, such as Bacillus subtilis; Pneu
`mococcus; Streptococcus, and Haemophilus influenzae.
`A wide variety of plasmids may be employed of
`greatly varying molecular weight. Normally, the plas
`mids employed will have molecular weights in the
`range of about 1X106 to 50x10d, more usually from
`about 1 to 20x10d, and preferably, from about 1 to
`10x10d. The desirable plasmid size is determined by a
`number of factors. First, the plasmid must be able to
`accommodate a replicator locus and one or more genes
`that are capable of allowing replication of the plasmid.
`Secondly, the plasmid should be of a size which pro
`vides for a reasonable probability of recircularization
`with the foreign gene(s) to form the recombinant plas
`mid chimera. Desirably, a restriction enzyme should be
`available, which will cleave the plasmid without inacti
`vating the replicator locus and system associated with
`the replicator locus. Also, means must be provided for
`providing ligatable terminal for the plasmid, which are
`complementary to the termini of the foreign gene(s) to
`allow fusion of the two DNA segments.
`Another consideration for the recombinant plasmid is
`that it be compatible with the bacterium to be trans
`formed. Therefore, the original plasmid will usually be
`derived from a member of the family to which the bac
`terium belongs.
`
`50
`
`65
`
`Page 2
`
`Page 2
`
`

`

`5
`
`15
`
`4,468,464
`4.
`3
`two strands at different adjacent sites providing cohe
`The original plasmid should desirably have a pheno
`sive termini directly.
`typical property which allows for the separation of
`With flush ended molecules, a T4 ligase may be em
`transformant bacteria from parent bacteria. Particularly
`ployed for linking the termini. See, for example, Sgara
`useful is a gene, which provides for survival selection.
`mella and Khorana, J. Mol. Biol. 72: 427–444(1972) and
`Survival selection can be achieved by providing resis
`Sgaramella, DNAS 69; 3389(1972), whose disclosure is
`tance to a growth inhibiting substance or providing a
`incorporated herein by reference.
`growth factor capability to a bacterium deficient in such
`Another way to provide ligatable termini is to cleave
`capability.
`employing DNAse and Mn+ as reported by Lai and
`conveniently, genes are available, which provide for
`Nathans, J. Mol. Biol, 89: 179(1975).
`antibiotic or heavy metal resistance or polypeptide re
`10
`The plasmid, which has the replicator locus, and
`sistance, e.g. colicin. Therefore, by growing the bac
`serves as the vehicle for introduction of a foreign gene
`teria on a medium containing a bacteriostatic or bacteri
`into the bacterial cell, will hereafter be referred to as
`ocidal substance, such as an antibiotic, only the transfor
`"the plasmid vehicle.'
`mants having the antibiotic resistance will survive. Il
`It is not necessary to use plasmid, but any molecule
`lustrative antibiotics include tetracycline, streptomycin,
`capable of replication in bacteria can be employed.
`sulfa drugs, such as sulfonamide, kanamycin, neomycin,
`Therefore, instead of plasmid, viruses may be em
`penicillin, chloramphenicol, or the like.
`ployed, which will be treated in substantially the same
`Growth factors include the synthesis of amino acids,
`manner as the plasmid, to provide the ligatable termini
`the isomerization of substrates to forms which can be
`for joining to the foreign gene.
`20
`metabolized or the like. By growing the bacteria on a
`If production of cohesive termini is by restriction
`medium which lacks the appropriate growth factor,
`endonuclease cleavage, the DNA containing the for
`only the bacteria which have been transformed and
`eign gene(s) to be bound to the plasmid vehicle will be
`have the growth factor capability will clone.
`cleaved in the same manner as the plasmid vehicle. If
`One plasmid of interest derived from E. coli is re
`the cohesive termini are produced by a different tech
`25
`ferred to as pSC101 and is described in Cohen, et al.,
`nique, an analogous technique will normally be em
`Proc. Nat. Acad. Sci., USA, 70, 1293 (1972), (referred
`ployed with the foreign gene. (By foreign gene is in
`to in that article as Tc6-5). Further description of this
`tended a gene derived from a source other than the
`particular plasmid and its use is found in the other arti
`transformant strain.) In this way, the foreign gene(s)
`cles previously referred to.
`will have ligatable termini, so as to be able to be cova
`The plasmid pSC101 has a molecular weight of about
`lently bonded to the termini of the plasmid vehicle. One
`5.8x 106d and provides tetracycline resistance.
`can carry out the cleavage or digest of the plasmids
`Another plasmid of interest is colicinogenic factor EI
`together in the same medium or separately, combine the
`(ColE1), which has a molecular weight of 4.2X 106d,
`plasmids and recircularize the plasmids to form the
`and is also derived from E. coli. The plasmid has a
`plasmid chimera in the absence of active restriction
`single EcoRI substrate site and provides immunity to
`enzyme capable of cleaving the plasmids.
`colicin E1.
`Descriptions of methods of cleavage with restriction
`In preparing the plasmid for ligation with the exoge
`enzymes may be found in the following articles:
`nous gene, a wide variety of techniques can be pro
`Greene, et al., Methods in Molecular Biology, Vol. 9, ed.
`vided, including the formation of or introduction of
`Wickner, R.B., (Marcel Dekker, Inc., New York),
`40
`cohesive termini. Flush ends can be joined. Alterna
`“DNA Replication and Biosynthesis”; Mertz and Da
`tively, the plasmid and gene may be cleaved in such a
`vis, 69, Proc. Nat. Acad. Sci., USA, 69, 3370 (1972);
`manner that the two chains are cleaved at different sites
`The cleavage and non-covalent joining of the plasmid
`to leave extensions at each end which serve as cohesive
`vehicle and the foreign DNA can be readily carried out
`termini. Cohesive termini may also be introduced by
`with a restriction endonuclease, with the plasmid vehi
`45
`removing nucleic acids from the opposite ends of the
`cle and foreign DNA in the same or different vessels.
`two chains or alternatively, introducing nucleic acids at
`Depending on the number of fragments, which are
`opposite ends of the two chains.
`obtained from the DNA endonuclease digestion, as well
`To illustrate, a plasmid can be cleaved with a restric
`as the genetic properties of the various fragments, diges
`tion endonuclease or other DNA cleaving enzyme. The
`tion of the foreign DNA may be carried out separately
`50
`restriction enzyme can provide square ends, which are
`and the fragments separated by centrifugation in an
`then modified to provide cohesive terminior can cleave
`appropriate gradient. Where the desired DNA fragment
`at different, but adjacent, sites on the two strands, so as
`has a phenotypical property, which allows for the ready
`to provide cohesive termini directly.
`isolation of its transformant, a separation step can usu
`Where square ends are formed such as, for example,
`ally be avoided.
`by HIN (Haemophilus influenzae RII) or pancreatic
`Endonuclease digestion will normally be carried out
`DNAse, one can ligate the square ends or alternatively
`at moderate temperatures, normally in the range of 10
`one can modify the square ends by chewing back, add
`to 40 C. in an appropriately buffered aqueous medium,
`usually at a pH of about 6.5 to 8.5. Weight percent of
`ing particular nucleic acids, or a combination of the
`two. For example, one can employ appropriate transfer
`total DNA in the reaction mixture will generally be
`about 1 to 20 weight percent. Time for the reaction will
`ases to add a nucleic acid to the 5' and 3' ends of the
`vary, generally being from 0.1 to 2 hours. The amount
`DNA. Alternatively, one can chew back with an en
`of endonuclease employed is normally in excess of that
`zyme, such as a N-exonuclease, and it is found that there
`required, normally being from about 1 to 5 units per
`is a high probability that cohesive termini will be
`10pug of DNA.
`achieved in this manner.
`65
`Where cleavage into a plurality of DNA fragments
`An alternative way to achieve a linear segment of the
`results, the course of the reaction can be readily fol
`plasmid with cohesive termini is to employ an endonu
`lowed by electrophoresis. Once the digestion has gone
`clease such as EcoRI. The endonuclease cleaves the
`
`30
`
`35
`
`55
`
`60
`
`Page 3
`
`Page 3
`
`

`

`4,468,464
`5
`6
`to the desired degree, the endonuclease is inactivated by
`have existed in nature. This is true, even in the event of
`heating above about 60° C. for five minutes. The diges
`mutations, and induced combinations of genes from
`tion mixture may then be worked up by dialysis, gradi
`different strains of the same species. For the natural
`ent separation, or the like, or used directly.
`formation of plasmids formed from a replicon and genes
`The plasmid vehicle and foreign DNA fragments are
`from different microorganisms it is necessary that the
`microorganisms be capable of mating and exchanging
`then allowed to combine to form hydrogen bonds and
`recircularize. This process is referred to as annealing
`genetic information.
`and DNA ligation. An appropriate buffered medium is
`In the situation, where the replicon comes from a
`employed containing the DNA fragments, DNA ligase,
`eukaryotic or prokaryotic cell, and at least one gene
`and appropriate cofactors. The temperature employed
`comes from the other type of cell, this plasmid hereto
`10
`initially for annealing will be about -5 to 15° C. When
`fore could not have existed in nature. Thus, the subject
`DNA segments hydrogen bond, the DNA ligase will be
`invention provides new plasmids which cannot natu
`able to introduce a covalent bond between the two
`rally occur and can be used for transformation of unicel
`segments. Where the two ends of each of the segments
`lular organisms to introduce genes from other unicellu
`are hydrogen bonded to one another, they may be li
`lar organisms, where the replicon and gene could not
`15
`previously naturally coexist in a plasmid.
`gated to form a circularized recombinant plasmid. The
`mole ratio of the two segments will generally be in the
`Besides naturally ocurring genes, it is feasible to pro
`range of 1-5:5-1. The particular temperature for anneal
`vide synthetic genes, where fragments of DNA may be
`ing will depend upon the binding strength of the cohe
`joined by various techniques known in the art. Thus, the
`sive termini. While 0.5 to 2 or more days have been
`exogenous gene may be obtained from natural sources
`20
`employed for annealing, it is believed that only a short
`or from synthetic sources.
`period of 0.5 to 6 hours may be sufficient, since anneal
`The plasmid chimera contains a replicon which is
`ing and ligation can occur under ligating conditions.
`compatible with a bacterium susceptible of transforma
`The time employed for the annealing will vary with the
`tion and at least one foreign gene which is directly or
`temperature employed, the nature of the salt solution, as
`indirectly bonded through deoxynucleotides to the re
`25
`well as the nature of the sticky ends or cohesive termini.
`plicon to form the circularized plasmid structure. As
`The foreign DNA can be derived from a wide variety
`indicated previously, the foreign gene normally pro
`of sources. The DNA may be derived from eukaryotic
`vides a phenotypical property, which is absent in the
`or prokaryotic cells, viruses, and bacteriophage. The
`parent bacterium. The foreign gene may come from
`fragments employed will generally have molecular
`another bacterial strain, species or family, or from a
`30
`weights in the range of about 0.5 to 20x10°d, usually in
`plant or animal cell. The original plasmid chimera will
`the range of 1 to 10x 106d. The DNA fragment may
`have been formed by in vitro covalent bonding between
`the replicon and foreign gene. Once the originally
`include one or more genes or one or more operons.
`Desirably, if the plasmid vehicle does not have a
`formed plasmid chimera has been used to prepare trans
`phenotypical property which allows for isolation of the
`formants, the plasmid chimera will be replicated by the
`35
`transformants, the foreign DNA fragment should have
`bacterial cell and cloned in vivo by growing the bac
`such property.
`teria in an appropriate growth medium. The bacterial
`The covalent joining can be achieved in conventional
`cells may be lysed and the DNA isolated by conven
`ways employing a DNA ligase. Ligation is conveniently
`tional means or the bacteria continually reproduced and
`carried out in an aqueous solution (pH, 6-8) attempera
`allowed to express the genotypical property of the for
`40
`eign DNA.
`tures in the range of 5 to 40°C. The concentration of the
`DNA will generally be from about 10 to 100pg/ml. A
`Once a bacterium has been transformed, it is no
`longer necessary to repeat the in vitro preparation of
`sufficient amount of the DNA ligase or other ligating
`agent, e.g. T4 ligase, is employed to provide a conve
`the plasmid chimera or isolate the plasmid chimera from
`nient rate of reaction, generally ranging from 5 to 50
`45
`the transformant progeny. Bacterial cells can be repeat
`edly multiplied which will express the genotypical
`U/ml. Small amounts of a protein e.g. albumin, may be
`property of the foreign gene.
`added at concentrations of 10 to 200 pg/ml. The liga
`tion with DNA ligase is carried out in the presence of
`One method of distinguishing between a plasmid
`Mg++ at abut 1-10 mM.
`which originates in vivo from a plasmid chimera which
`At the completion of the ligation, the solution may be
`originates in vitro is the formation of homoduplexes
`5
`between an in vitro prepared plasmid chimera and the
`chilled and is ready for use in transformation.
`In accordance with the subject invention, plasmids
`plasmid formed in vivo. It will be an extremely rare
`may be prepared which have replicons and genes which
`event where a plasmid which originates in vivo will be
`could be present in bacteria as a result of normal mating
`the same as a plasmid chimera and will form homodu
`of bacteria. However, the subject invention provides a
`plexes with plasmid chimeras. For a discussion of
`55
`technique, whereby a replicon and gene can coexist in a
`homoduplexes, see Sharp, Cohen and Davidson, J. Mol.
`plasmid, which is capable of being introduced into a
`Biol., 75, 235 (1973), and Sharp, et al, ibid, 71, 471
`unicellular organism, which could not exist in nature.
`(1972).
`The first type of plasmid which cannot exist in nature is
`The plasmid derived from molecular cloning need
`a plasmid which derives its replicon from one organism
`not homoduplex with the in vitro plasmid originally
`60
`and the exogenous gene from another organism, where
`employed for transformation of the bacterium. The
`the two organisms do not exchange genetic informa
`bacterium may carry out modification processes, which
`will not affect the portion of the replicon introduced
`tion. In this situation, the two organisms will either be
`eukaryotic or prokaryotic. Those organisms which are
`which is necessary for replication nor the portion of the
`able to exchange genetic information by mating are well
`exogenous DNA which contains the gene providing the
`65
`known. Thus, prior to this invention, plasmids having a
`genotypical trait. Thus, nucleotides may be introduced
`or excised and, in accordance with naturally occurring
`replicon and one or more genes from two sources
`which do not exchange genetic information would not
`mating and transduction, additional genes may be intro
`
`Page 4
`
`Page 4
`
`

`

`O
`
`15
`
`4,468,464
`8
`7
`subjected to transformation, will first be grown in a
`duced. In addition, for one or more reasons, the plas
`solution medium, so as to amplify the absolute number
`mids may be modified in vitro by techniques which are
`of the desired cells. The bacterial cells may then be
`known in the art. However, the plamids obtained by
`harvested and streaked on an appropriate agar medium.
`molecular cloning will homoduplex as to those parts
`Where the recombinant plasmid has a phenotype,
`which relate to the original replicon and the exogenous
`which allows for ready separation of the transformed
`gene.
`cells from the parent cells, this will aid in the ready
`II. Transformation
`separation of the two types of cells. As previously indi
`After the recombinant plasmid or plasmid chimera
`cated, where the genotype provides resistance to a
`has been prepared, it may then be used for the transfor
`growth inhibiting material, such as an antibiotic or
`mation of bacteria. It should be noted that the annealing
`heavy metal, the cells can be grown on an agar medium
`and ligation process not only results in the formation of
`containing the growth inhibiting substance. Only avail
`the recombinant plasmid, but also in the recirculariza
`able cells having the resistant genotype will survive. If
`tion of the plasmid vehicle. Therefore, a mixture is
`the foreign gene does not provide a phenotypical prop
`obtained of the original plasmid, the recombinant plas
`erty, which allows for distinction between the cells
`mid, and the foreign DNA. Only the original plasmid
`transformed by the plasmid vehicle and the cells trans
`and the DNA chimera consisting of the plasmid vehicle
`formed by the plasmid chimera, a further step is neces
`and linked foreign DNA will normally be capable of
`sary to isolate the replicated plasmid chimera from the
`replication. When the mixture is employed for transfor
`replicated plasmid vehicle. The steps include lysing of
`mation of the bacteria, replication of both the plasmid
`the cells and isolation and separation of the DNA by
`vehicle genotype and the foreign genotype will occur
`20
`with both genotypes being replicated in those cells
`conventional means or random selection of transformed
`bacteria and characterization of DNA from such trans
`having the recombinant plasmid.
`formants to determine which cells contain molecular
`Various techniques exist for transformation of a bac
`chimeras. This is accomplished by physically character
`terial cell with plasmid DNA. A technique, which is
`izing the DNA by electrophoresis, gradient centrifuga
`particularly useful with Escherichia coli, is described in
`25
`tion or electron microscopy.
`Cohen, et al., ibid, 69, 2110 (1972). The bacterial cells
`Cells from various clones may be harvested and the
`are grown in an appropriate medium to a predetermined
`plasmid DNA isolated from these transformants. The
`optical density. For example, with E. coli strain C600,
`plasmid DNA may then be analyzed in a variety of
`the optical density was 0.85 at 590 nm. The cells are
`ways. One way is to treat the plasmid with an appropri
`concentrated by chilling, sedimentation and washing
`ate restriction enzyme and analyze the resulting frag
`with a dilute salt solution. After centrifugation, the cells
`ments for the presence of the foreign gene. Other tech
`are resuspended in a calcium chloride solution at re
`niques have been indicated above.
`duced temperatures (approx. 5-15 C.), sedimented,
`Once the recombinant plasmid has been replicated in
`resuspended in a smaller volume of a calcium chloride
`a cell and isolated, the cells may be grown and multi
`solution and the cells combined with the DNA in an
`35
`plied and the recombinant plasmid employed for trans
`appropriately buffered calcium chloride solution and
`formation of the same or different bacterial strain.
`incubated at reduced temperatures. The concentration
`The subject process provides a technique for intro
`of Cat + will generally be about 0.01 to 0.1M. After a
`ducing into a bacterial strain a foreign capability which
`sufficient incubation period, generally from about
`is genetically mediated. A wide variety of genes may be
`0.5-3.0 hours, the bacteria are subjected to a heat pulse
`employed as the foreign genes from a wide variety of
`generally in the range of 35° to 45° C. for a short period
`sources. Any intact gene may be employed which can
`of time; namely from about 0.5 to 5 minutes. The trans
`be bonded to the plasmid vehicle. The source of the
`formed cells are then chilled and may be transferred to
`gene can be other bacterial cells, mammalian cells, plant
`a growth medium, whereby the transformed cells hav
`cells, etc. The process is generally applicable to bacte
`ing the foreign genotype may be isolated.
`45
`rial cells capable of transformation. A plasmid must be
`An alternative transformation technique may be
`available, which can be cleaved to provide a linear
`found in Lederberg and Cohen, I. Bacteriol., 119, 1072
`segment having ligatable termini, and an intact replica
`(1974), whose disclosure is incorporated herein by ref.
`tor locus and system, preferably a system including a
`erence,
`gene which provides a phenotypical property which
`III. Replication and Transcription of the Plasmid
`50
`allows for easy separation of the transformants. The
`The bacterial cells, which are employed, will be of
`linear segment may then be annealed with a linear seg
`such species as to allow replication of the plasmid vehi
`ment of DNA having one or more genes and the result
`cle. A number of different bacteria which can be en
`ing recombinant plasmid employed for transformation
`ployed, have been indicated previously. Strains which
`of the bacteria.
`lack indigenous modification and restriction enzymes
`55
`By introducing one or more exogenous genes into a
`are particularly desirable for the cloning of DNA de
`unicellular organism, the organism will be able to pro
`rived from foreign sources.
`duce polypeptides and proteins ("poly(amino acids)')
`The transformation of the bacterial cells will result in
`which the organism could not previously produce. In
`a mixture of bacterial cells, the dominant proportion of
`some instances the poly(amino acids) will have utility in
`60
`which will not be transformed. Of the fraction of cells
`themselves, while in other situations, particularly with
`which are transformed, some significant proportion, but
`enzymes, the enzymatic product(s) will either by useful
`normally a minor proportion, will have been trans
`in itself or useful to produce a desirable product.
`formed by recombinant plasmid. Therefore, only a very
`One group of poly(amino acids) which are directly
`small fraction of the total number of cells which are
`useful are hormones. Illustrative hormones include pa
`present will have the desired phenotypical characteris
`rathyroid hormone, growth hormone, gonadotropins
`tics.
`(FSH, luteinizing hormone, chorionogonadatropin, and
`In order to enhance the ability to separate the desired
`glycoproteins), insulin, ACTH, somatostatin, prolactin,
`bacterial clones, the bacterial cells, which have been
`
`30
`
`65
`
`Page 5
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`Page 5
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`

`15
`
`25
`
`30
`
`EXPERIMENTAL
`In order to demonstrate the subject invention, the
`following experiments were carried out with a variety
`of foreign genes.
`35
`(All temperatures not otherwise indicated are Centi
`grade. All percents not otherwise indicated are precents
`by weight.)
`
`4,468,464
`10
`placental lactogen, melanocyte stimulating hormone,
`0.2 ml of competent cells treated with calcium chlo
`hyrotropin, parathyroid hormone, calcitonin, enkepha
`ride was added to 0.1 ml of DNA solution with chilled
`lin, and angiotensin.
`pipets and an additional incubation was done for 60
`Other p