Patent to create E. coli 0157 using calcium chloride as an agent for genetic engineering -- the subject
invention provides a technique, whereby a replicon and gene can coexist in a plasmid, which is capable of
being introduced into a unicellular organism,
which could not exist in nature.  Conveniently, genes are
available, which provide for antibiotic or heavy metal resistance or polypeptide resistance, e.g.
colicin. Therefore, by growing the bacteria on a medium containing a bacteriostatic or bacteriocidal
substance, such as an antibiotic, only the transformants having the antibiotic resistance will survive.
Illustrative antibiotics include tetracycline, streptomycin, sulfa drugs, such as sulfonamide, kanamycin,
neomycin, penicillin, chloramphenicol, or the like.

This example is a real patent for a key procedure in genetic engineering. The patent claims a method to introduce
foreign genes into bacteria and enable their expression.  


United States Patent 4,237,224

Process for producing biologically functional molecular chimeras

APPL. NO.: 001021 PATENT TYPE: 1
ART REC. NO.: 172 APP FILL. DT: 19790104
ISSUE DATE: 19801202 NO.OF CLAIMS: 14  
EXEMPL.CLAIM: 1 PRI.EXAMINER: Tanenholtz; Alvin E.


Cohen; Stanley N. Portola Valley CA
Boyer; Herbert W. Mill Valley CA

NAME: Board of Trustees of the Leland Stanford Jr. University
CITY: Stanford

72 959288 19781109 0
82 687430 19760517 0
82 520691 19741104 0

ORG US CLS: 435/68 XREF CLASS: 435207
XREF CLASS: 435172 XREF CLASS: 2601125S
XREF CLASS: 435231  XREF CLASS:  260/27
XREF CLASS: 435183 XREF CLASS: 435212
XREF CLASS: 435317 INTL. CLASS: C12P/2100
XREF CLASS: 435820 FLD SRCH SUB: 1;28 N;28 R;112;78;79
FLD SRCH SUB: 68;172;231;183


3813316 19740500 Chakrabarty 195/28R


Morrow et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp. 3365-3369, Nov. 1972.
Morrow et al., Proc. Nat. Acad. Sci. USA, vol. 71, pp. 1743-1747, May 1974.

Hershfield et al., Proc. Nat. Acad. Sci. USA, vol. 71, pp. 3455 et seq. (1974).

Jackson et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp. 2904-2909, Oct. 1972.

Mertz et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp. 3370-3374, Nov. 1972.

Cohen, et al., Proc. Nat. Acad. Sci. USA, vol. 70, pp. 1293-1297, May 1973.

Cohen et al., Proc. Nat. Acad. Sci. USA, vol. 70, pp. 3240-3244, Nov. 1973.

Chang et al., Proc. Nat. Acad. Sci, USA, vol. 71, pp. 1030-1034, Apr. 1974.

Ullrich et al., Science vol. 196, pp. 1313-1319, Jun. 1977.

Singer et al., Science vol. 181, p. 1114 (1973).

Itakura et al., Science vol. 198, pp. 1056-1063 Dec. 1977.

Komaroff et al., Proc. Nat. Acad. Sci. USA, vol. 75, pp. 3727-3731, Aug. 1978.

Chemical and Engineering News, p. 4, May 30, 1977.

Chemical and Engineering News, p. 6, Sep. 11, 1978.


Attorney, Agent, or Firm - Bertram I. Rowland

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 to which is inserted a gene having
complementary termini, to provide a biologically functional replicon with a desired phenotypical property. The replicon is
inserted into a microorganism cell by transformation. Isolation of the transformants 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 and 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.

This application is a continuatin-in-part of applicatin Ser. No. 959,288, filed Nov. 9, 1978, which is a continuation of
application Ser. No. 687,430 filed May 17, 1976, now abandoned, which was a continuation-in-part of application Ser.
No. 520,691, filed Nov. 4, 1974, now abandoned.


1. Field of the Invention
Although transfer of plasmids among strains of E. coli and other Enterobacteriaceae has long been accomplished by
conjugation and/or transduction, it has not been previously possible to selectively introduce particular species of
plasmid DNA into these bacterial hosts or other microorganisms. Since microorganisms that have been transformed with
plasmid DNA contain autonomously replicating extrachromosomal DNA species having the genetic and molecular
characteristics of the parent plasmid, transformation has enabled the selective cloning and amplification of particular
plasmid genes.

The ability of genes derived from totally different 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 as nitrogen fixation, photosynthesis,
antibiotic production, hormone synthesis, protein synthesis, e.g. enzymes or antibodies, or the like--functions which are
indigenous to other classes of organisms--by linking the foreign genes to a particular plasmid or viral replicon.

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., 71, 1743 (1974); Novick,
Bacteriological Rev., 33, 210 (1969); and Hershfeld, et al., Proc. Nat. Acad. Sci., in press; Jackson, et al., ibid, 69, 2904

Methods and compositions are provided for genetically transforming microorganisms, particularly bacteria, to provide
diverse genotypical capability and producing recombinant plasmids. A plasmid or viral DNA is modified to form a linear
segment having ligatable termini which is joined to DNA having at least one intact gene and complementary ligatable
termini. The termini are then bound together to form a "hybrid" plasmid molecule which is used to transform susceptible
and compatible microorganisms. After transformation, 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 desired end product, or metabolities of enzymic conversion, or be lysed and the desired nucleic acids or
proteins recovered.

The process of this invention employs novel plasmids, which are formed by inserting DNAhaving one or more intact
genes into a plasmid in such 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
property. The plasmmid chimera is used to transform a susceptible and competent microorganism under conditions
where transformation occurs. The microorganism is then grown under conditions which allow for separation 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.

Preparation of Plasmid Chimera
In order to prepare the plasmid chimera, it is necessary to have a DNA vector, such as a plasmid or phage, 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. Of particular interest are those plasmids which have a
phenotypical property, which allow for ready separation of transformants from the parent microorganism. The plasmid
will be capable of replicating in a microorganism, particularly a bacterium which is susceptible to transformation. Various
unicellular microorganisms can be transformed, such as bacteria, fungii and algae. That is, those unicellular organisms
which are capable of being grown in cultures of 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; Pneumococcus; Streptococcus, and Haemophilus influenzae.

A wide variety of plasmids may be employed of greatly varying molecular weight. Normally, the plasmids employed will
have molecular weights in the range of about 1 X 106 to 50 X 106d, more usually from about 1 to 20 X 106 d, and
preferably, from about 1 to 10 X 106 d. 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 provides for a reasonable probability of recircularization
with the foreign gene(s) to form the recombinant plasmid chimera. Desirably, a restriction enzyme should be available,
which will cleave the plasmid without inactivating the replicator locus and system associated with the replicator locus.
Also, means must be provided for providing ligatable termini 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 transformed.
Therefore, the original plasmid will usually be derived from a member of the family to which the bacterium belongs.

The original plasmid should desirably have a phenotypical property which allows for the separation of transformant
bacteria from parent bacteria. Particularly useful is a gene, which provides for survival selection. Survival selection can
be achieved by providing resistance to a growth inhibiting substance or providing a growth factor capability to a
bacterium deficient in such capability.

Conveniently, genes are available, which provide for antibiotic or heavy metal resistance or polypeptide resistance, e.g.
colicin. Therefore, by growing the bacteria on a medium containing a bacteriostatic or bacteriocidal substance, such as
an antibiotic, only the transformants having the antibiotic resistance will survive. Illustrative antibiotics include
tetracycline, streptomycin, sulfa drugs, such as sulfonamide, kanamycin, neomycin, penicillin, chloramphenicol, or the

Growth factors include the synthesis of amino acids, the isomerization of substrates to forms which can be metabolized
or the like. By growing the bacteria on a medium which lacks the appropriate growth factor, only the bacteria which have
been transformed and have the growth factor capability will clone.

One plasmid of interest derived from E. coli is referred to as pSC101 and is described in Cohen, et al., Proc. Nat. Acad.
Sci., USA, 70, 1293 (1972), (referred to in that article as Tc6-5). Further description of this particular plasmid and its use
is found in the other articles previously referred to.

The plasmid pSC101 has a molecular weight of about 5.8 X 106 d and provides tetracycline resistance.

Another plasmid of interest is colicinogenic factor EI (ColE1), which has a molecular weight of 4.2 X 106 d, and is also
derived from E. coli. The plasmid has a single EcoRI substrate site and provides immunity to colicin E1.

In preparing the plasmid for joining with the exogenous gene, a wide variety of techniques can be provided, including the
formation of or introduction of cohesive termini. Flush ends can be joined. Alternatively, the plasmid and gene may be
cleaved in such a manner that the two chains are cleaved at different sites to leave extensions at each end which serve
as cohesive termini. Cohesive termini may also be introduced by removing nucleic acids from the opposite ends of the
two chains or alternatively, introducing nucleic acids at opposite ends of the two chains.

To illustrate, a plasmid can be cleaved with a restriction endonuclease or other DNA cleaving enzyme. The restriction
enzyme can provide square ends, which are then modified to provide cohesive termini or can cleave in a staggered
manner at different, but adjacent, sites on the two strands, so as to provide cohesive termini directly.

Where square ends are formed such as, for example, by HIN (Haemophilus influenzae RII) or pancreatic DNAse, one can
ligate the square ends or alternatively one can modify the square ends by chewing back, adding particular nucleic acids,
or a combination of the two. For example, one can employ appropriate transferases to add a nucleic acid to the 5' and 3'
ends of the DNA. Alternatively, one can chew back with an enzyme, such as a λ-exonuclease, and it is found that there
is a high probability that cohesive termini will be achieved in this manner.

An alternative way to achieve a linear segment of the plasmid with cohesive termini is to employ an endonuclease such
as EcoRI. The endonuclease cleaves the two strands at different adjacent sites providing cohesive termini directly.

With flush ended molecules, a T 4 ligase may be employed for linking the termini. See, for example, Scaramella and
Khorana, J. Mol. Biol. 72: 427-444 (1972) and Scaramella, DNAS 69: 3389 (1972), whose disclosure is incorporated
herein by reference.

Another way to provide ligatable termini is to leave employing DNAse and Mn++ as reported by Lai and Nathans, J. Mol.
Biol, 89: 179 (1975). The plasmid, which has the replicator locus, and serves as the vehicle for introduction of a foreign
gene into the bacterial cell, will hereafter be referred to as "the plasmid vehicle."

It is not necessary to use plasmid, but any molecule capable of replication in bacteria can be employed. Therefore,
instead of plasmid, viruses may be employed, which will be treated in substantially the same manner as the plasmid, to
provide the ligatable termini for joining to the foreign gene.

If production of cohesive termini is by restriction endonuclease cleavage, the DNA containing the foreign gene(s) to be
bound to the plasmid vehicle will be cleaved in the same manner as the plasmid vehicle. If the cohesive termini are
produced by a different technique, an analogous technique will normally be employed with the foreign gene. (By foreign
gene is intended a gene derived from a source other than the transformant strain.) In this way, the foreign gene(s) will
have ligatable termini, so as to be able to covalently bonded to the termini of the plasmid vehicle. One can carry out the
cleavage or digest of the plasmids together in the same medium or separately, combine the plasmids and recircularize
the plasmids to form the plasmid chimera in the absence of active restriction enzyme capable of cleaving the plasmids.

Descriptions of methods of cleavage with restriction enzymes may be found in the following articles: Greene, et al.,
Methods in Molecular Biology, Vol. 9, ed. Wickner, R. B., (Marcel Dekker, Inc., New York), "DNA Replication and
Biosynthesis"; Mertz and Davis, 69, Proc. Nat. Acad. Sci., USA, 69, 3370 (1972);

The cleavage and non-covalent joining of the plasmid vehicle and the foreign DNA can be readily carried out with a
restriction endonuclease, with the plasmid vehicle and foreign DNA in the same or different vessels. Depending on the
number of fragments, which are obtained from the DNA endonuclease digestion, as well as the genetic properties of the
various fragments, digestion of the foreign DNA may be carried out separately and the fragments separated by
centrifugation in an appropriate gradient. Where the desired DNA fragment has a phenotypical property, which allows
for the ready isolation of its transformant, a separation step can usually be avoided.

Endonuclease digestion will normally be carried out at moderate temperatures, normally in the range of 10° to 40° C. in
an appropriately buffered aqueous medium, usually at a pH of about 6.5 to 8.5. Weight percent of total DNA in the
reaction mixture will generally be about 1 to 20 weight percent. Time for the reaction will vary, generaly being from 0.1 to
2 hours. The amount of endonuclease employed is normally in excess of that required, normally being from about 1 to 5
units per 10 μg of DNA.

Where cleavage into a plurality of DNA fragments results, the course of the reaction can be readily followed by
electrophoresis. Once the digestion has gone to the desired degree, the endonuclease is inactivated by heating above
about 60° C. for five minutes. The digestion mixture may be worked up by dialysis, gradient separation, or the like, or
used directly.

After preparation of the two double stranded DNA sequences, the foreign gene and vector are combined for annealing
and/or ligation to provide for a functional recombinant DNA structure. With plasmids, the annealing involves the
hydrogen bonding together of the cohesive ends of the vector and the foreign gene to form a circular plasmid which has
cleavage sites. The cleavage sites are then normally ligated to form the complete closed and circularized plasmid.

The annealing, and as appropriate, recircularization can be performed in whole or in part in vitro or in vivo. Preferably,
the annealing is performed in vitro. The annealing requires an appropriate buffered medium containing the DNA
fragments. The temperature employed initially for annealing will be about 40° to 70° C., followed by a period at lower
temperature, generaly from about 10° to 30° C. The molar ratio of the two segments will generally be in the range of
about 1-5:5-1. The particular temperature for annealing will depend upon the binding strength of the cohesive termi.
While 0.5 hr to 2 or more days may be employed for annealing, it is believed that a period of 0.5 to 6 hrs may be
sufficient. The time employed for the annealing will vary with the temperature employed, the nature of the salt solution,
as well as the nature of the cohesive termini.

The ligation, when in vitro, can be achieved in conventional ways employing DNA ligase. Ligation is conveniently carried
out in an aqueous solution (pH 6-8) at temperatures in the range of about 5° to 40° C. The concentration of the DNA will
generally be from about 10 to 100 g/ml. A sufficient amount of the DNA ligase or other ligating agent e.g. T4 ligase, is
employed to provide a convenient rate of reaction, generally ranging from about 5 to 50 U/ml. A small amount of a
protein e.g. albumin, may be added at concentrations of about 10 to 200 g/ml. The ligation with DNA ligase is carried out
in the presence of magnesium at about 1-10 mM.

At the completion of the annealing or ligation, the solution may be chilled and is ready for use in transformation.

It is not necessary to ligate the recircularized plasmid prior to transformation, since it is found that this function can be
performed by the bacterial host. However, in some situations ligation prior to transformation may be desirable.

The foreign DNA can be derived from a wide variety of sources. The DNA may be derived from eukaryotic or prokaryotic
cells, viruses, and bacteriophage. The fragments employed will generally have molecular weights in the range of about
0.5 to 20 X 106 d, usually in the range of 1 to 10 X 106 d. The DNA fragment may include one or more genes or one or
more operons.

Desirably, if the plasmid vehicle does not have a phenotypical property which allows for isolation of the transformants,
the foreign DNA fragment should have such property. Also, an intact promoter and base sequences coding for initiation
and termination sites should be present for gene expression.

In accordance with the subject invention, plasmids may be prepared which have replicons and genes which could be
present in bacteria as a result of normal mating of bacteria. However, the subject invention provides a technique,
whereby a replicon and gene can coexist in a plasmid, which is capable of being introduced into a unicellular organism,
which could not exist in nature. The first type of plasmid which cannot exist in nature is a plasmid which derives its
replicon from one organism and the exogenous gene from another organism, where the two organisms do not exchange
genetic information. In this situation, the two organisms will either be eukaryotic or prokaryotic. Those organisms which
are able to exchange genetic information by mating are well known. Thus, prior to this invention, plasmids having a
replicon and one or more genes from two sources which do not exchange genetic information would not have existed in
nature. This is true, even in the event of mutations, and induced combinations of genes from different strains of the
same species. For the natural formation of plasmids formed from a replicon and genes from different microorganisms it
is necessary that the microorganisms be capable of mating and exchanging genetic information.

In the situation, where the replicon comes from a eukaryotic or prokaryotic cell, and at least one gene comes from the
other type of cell, this plasmid heretofore could not have existed in nature. Thus, the subject invention provides new
plasmids which cannot naturally occur and can be used for transformation of unicellular organisms to introduce genes
from other unicellular organisms, where the replicon and gene could not previously naturally coexist in a plasmid.

Besides naturally occurring genes, it is feasible to provide synthetic genes, where fragments of DNA may be joined by
various techniques known in the art. Thus, the exogenous gene may be obtained from natural sources or from synthetic

The plasmid chimera contains a replicon which is compatible with a bacterium susceptible of transformation and at least
one foreign gene which is directly or indirectly bonded through deoxynucleotides to the replicon to form the circularized
plasmid structure. As indicated previously, the foreign gene normally provides a phenotypical property, which is absent
in the parent bacterium. The foreign gene may come from another bacterial strain, species or family, or from a plant or
animal cell. The original plasmid chimera will have been formed by in vitro covalent bonding between the replicon and
foreign gene. Once the originally formed plasmid chimera has been used to prepare transformants, the plasmid chimera
will be replicated by the bacterial cell and cloned in vivo by growing the bacteria in an appropriate growth medium. The
bacterial cells may be lysed and the DNA isolated by conventional means or the bacteria continually reproduced and
allowed to express the genotypical property of the foreign DNA.

Once a bacterium has been transformed, it is no longer necessary to repeat the in vitro preparation of the plasmid
chimera or isolate the plasmid chimera from the transformant progeny. Bacterial cells can be repeatedly multiplied which
will express the genotypical property of the foreign gene.

One method of distinguishing between a plasmid which originates in vivo from a plasmid chimera which originates in vitro
is the formation of homoduplexes between an in vitro prepared plasmid chimera and the plasmid formed in vivo. It will be
an extremely rare event where a plasmid which originates in vivo will be the same as a plasmid chimera and will form
homoduplexes with plasmid chimeras. For a discussion of homoduplexes, see Sharp, Cohen and Davidson, J. Mol. Biol.,
75, 235 (1973), and Sharp, et al, ibid, 71, 471 (1972).

The plasmid derived from molecular cloning need not homoduplex with the in vitro plasmid originally employed for
transformation of the bacterium. The bacterium may carry out modification processes, which will not affect the portion of
the replicon introduced which is necessary for replication nor the portion of the exogenous DNA which contains the gene
providing the genotypical trait. Thus, nucleotides may be introduced or excised and, in accordance with naturally
occurring mating and transduction, additional genes may be introduced. In addition, for one or more reasons, the
plasmids may be modified in vitro by techniques which are known in the art. However, the plasmids obtained by
molecular cloning will homoduplex as to those parts which relate to the original replicon and the exogenous gene.

II. Transformation
After the recombinant plasmid or plasmid chimera has been prepared, it may then be used for the transformation of
bacteria. It should be noted that the annealing and ligation process not only results in the formation of the recombinant
plasmid, but also in the recircularization of the plasmid vehicle. Therefore, a mixture is obtained of the original plasmid,
the recombinant plasmid, and the foreign DNA. Only the original plasmid and the DNA chimera consisting of the plasmid
vehicle and linked foreign DNA will normally be capable of replication. When the mixture is employed for transformation
of the bacteria, replication of both the plasmid vehicle genotype and the foreign genotype will occur with both genotypes
being replicated in those cells having the recombinant plasmid.

Various techniques exist for transformation of a bacterial cell with plasmid DNA. A technique, which is particularly useful
with Escherichia coli, is described in Cohen, et al., ibid, 69, 2110 (1972). The bacterialcells are grown in an appropriate
medium to a predetermined opticaldensity. For example, with E. coli strain C600, the optical density was 0.85 at 590 nm.
The cells are concentrated by chilling, sedimentation and washing with a dilute salt solution. After centrifugation, the
cells are resuspended in a calcium chloride solution at reduced temperatures (approx. 5°-15° C.), sedimented,
resuspended in a smaller volume of a calcium chloride solution and the cells combined with the DNA in an appropriately
buffered calcium chloride solution and incubated at reduced temperatures. The concentration of Ca++ will generally be
about 0.01 to 0.1 M. After a sufficient incubation period, generally from about 0.5-3.0 hours, the bacteria are subjected
to a heat pulse generally in the range of 35° to 45° C. for a short period of time; namely from about 0.5 to 5 minutes.
The transformed cells are then chilled and may be transferred to a growth medium, whereby the transformed cells
having the foreign genotype may be isolated.

An alternative transformation technique may be found in Lederberg and Cohen, I. Bacteriol., 119, 1072 (1974), whose
disclosure is incorporated herein by reference.

III. Replication and Transcription of the Plasmid
The bacterial cells, which are employed, will be of such species as to allow replication of the plasmid vehicle. A number
of different bacteria which can be employed, have been indicated previously. Strains which lack indigenous modification
and restriction enzymes are particularly desirable for the cloning of DNA derived from foreign sources.

The transformation of the bacterial cells will result in a mixture of bacterial cells, the dominant proportion of which will not
be transformed. Of the fraction of cells which are transformed, some significant proportion, but normally a minor
proportion, will have been transformed by recombinant plasmid. Therefore, only a very small fraction of the total number
of cells which are present will have the desired phenotypical characteristics.

In order to enhance the ability to separate the desired bacterial clones, the bacterial cells, which have beeen subjected
to transformation, will first be grown in a solution medium, so as to amplify the absolute number of the desired cells. The
bacterial cells may then be harvested and streaked on an appropriate agar medium. Where the recombinant plasmid
has a phenotype, which allows for ready separation of the transformed cells from the parent cells, this will aid in the
ready separation of the two types of cells. As previously indicated, where the genotype provides resistance to a growth
inhibiting material, such as an antibiotic or heavy metal, the cells can be grown on an agar medium containing the
growth inhibiting substance. Only available cells having the resistant genotype will survive. If the foreign gene does not
provide a phenotypical property, which allows for distinction between the cells transformed by the plasmid vehicle and
the cells transformed by the plasmid chimera, a further step is necessary to isolate the replicated plasmid chimera from
the replicated plasmid vehicle. The steps include lysing of the cells and isolation and separation of the DNA by
conventional means or random selection of transformed bacteria and characterization of DNA from such transformants
to determine which cells contain molecular chimeras. This is accomplished by physically characterizing the DNA by
electrophoresis, gradient centrifugation or electron microscopy.

Cells from various clones may be harvested and the plasmid DNA isolated from these transformants. The plasmid DNA
may then be analyzed in a variety of ways. One way is to treat the plasmid with an appropriate restriction enzyme and
analyze the resulting fragments for the presence of the foreign gene. Other techniques have been indicated above.

Once the recombinant plasmid has been replicated in a cell and isolated, the cells may be grown and multiplied and the
recombinant plasmid employed for transformation of the same or different bacterial strain.

The subject process provides a technique for introducing into a bacterial strain a foreign capability which is genetically
mediated. A wide variety of genes may be employed as the foreign genes from a wide variety of sources. Any intact
gene may be employed which can be bonded to the plasmid vehicle. The source of the gene can be other bacterial
cells, mammalian cells, plant cells, etc. The process is generally applicable to bacterial cells capable of transformation. A
plasmid must be available, which can be cleaved to provide a linear segment having ligatable termini, and an interact
replicator locus and system, preferably a system including a gene which provides a phenotypical property which allows
for easy separation of the transformants. The linear segment may then be annealed with a linear segment of DNA
having one or more genes and the resulting recombinant plasmid employed for transformation of the bacteria.

By introducing one or more exogeneous genes into a unicellular organism, the organism will be able to produce
polypeptides and proteins ("poly(amino acids)") which the organism could not previously produce. In some instances the
poly(amino acids) will have utility in themselves, while in other situations, particularly with enzymes, the enzymatic product
(s) will either be useful in itself or useful to produce a desirable product.

One group of poly(amino acids) which are directly useful are hormones. Illustrative hormones include parathyroid
hormone, growth hormone, gonadotropins (FSH, luteinizing hormone, chorionogonadatropin, and glycoproteins), insulin,
ACTH, somatostatin, prolactin, placental lactogen, melanocyte stimulating hormone, thyrotropin, parathyroid hormone,
calcitonin, enkephalin, and angiotensin.

Other poly(amino acids) of interest include serum proteins, fibrinogin, prothrombin, thromboplastin, globulin e.g. gamma-
globulins or antibodies, heparin, antihemophilia protein, oxytocin, albumins, actin, myosin, hemoglobin, ferritin,
cytochrome, myoglobin, lactoglobulin, histones, avidin, thyroglobulin, interferin, kinins and transcortin. Where the genes
or genes produce one or more enzymes, the enzymes may be used for fulfilling a wide variety of functions. Included in
these functions are nitrogen fixation, production of amino acids, e.g. polyiodothyronine, particularly thyroxine, vitamins,
both water and fat soluble vitamins, antimicrobial drugs, chemotheropeutic agents e.g. antitumor drugs, polypeptides
and proteins e.g. enzymes from apoenzymes and hormones from prohormones, diagnostic reagents, energy producing
combinations e.g. photosynthesis and hydrogen production, prostaglandins, steroids, cardiac glycosides, coenzymes,
and the like.

The enzymes may be individually useful as agents separate from the cell for commercial applications, e.g. in detergents,
synthetic transformations, diagnostic agents and the like. Enzymes are classified by the I.U.B. under the classifications
as I. Oxidoreductases; II. Transferases; III. Hydrolases; IV. Lyases; V. Isomerases; and VI. Ligases.

In order to demonstrate the subject invention, the following experiments were carried out with a variety of foreign genes.

(All temperatures not otherwise indicated are Centrigrade. All percents not otherwise indicated are percents by weight.)

A. Preparation of pSC101 Plasmid

Covalently closed R6-5 DNA was sheared with a Virtis stainless steel microshaft in a one milliliter cup. The R6-5 DNA
was sheared at 2,000 r.p.m. for 30 minutes in TEN buffer solution (0.02 M Tris-HCl (pH 8.0)-1 mM EDTA (pH 8.0)-0.02 M
NaCl), while chilled at 0°-4°.

The sheared DNA sample was subjected to sucrose gradient sedimentation at 39,500 r.p.m. in a Spinco SW 50.1 rotor
at 20°. A 0.12 mil fraction was collected on a 2.3 cm diameter circle of Whatman No. 3 filter paper, dried for 20 minutes
and precipitated by immersion of the disc in cold 5% trichloroacetic acid, containing 100 μg/ml thymidine. The precipitate
was filtered and then washed once with 5% trichloroacetic acid, twice with 99% ethanol and dried. pSC101 was the 27S
species having a calculated molecular weight of 5.8.times.106 d.

B. Generalized Transformation Procedure
E. coli strain C600 was grown at 37° in H1 medium to an optical density of 0.85 at 590 nm. At this point the cells were
chilled quickly, sedimented and washed once in 0.5 volume 10 nM NaCl. After centrifugation, the bacteria was
resuspended in half the original volume of chilled 0.03 M calcium chloride, kept at 0° for 20 minutes, sedimented, and
then resuspended in 0.1 of the original volume of 0.03 M of calcium chloride solution. Chilled DNA samples in TEN buffer
were supplemented with 0.1 M calcium chloride to a final concentration of 0.03 M. 0.2 ml of competent cells treated with
calcium chloride was added to 0.1 ml of DNA solution with chilled pipets and an additional incubation was done for 60
minutes at 0°. The bacteria were then subjected to a heat pulse at 42° for two minutes, chilled, and then either placed
directly onto nutrient agar containing appropriate antibiotics or, where indicated, diluted 10 times in L-broth and
incubated at 37° before plating. The cell survival is greater than 50% after calcium chloride treatment and heat pulse.
Drug resistance was assayed on nutrient agar plates with the antibiotics indicated in specific experiments.

EXAMPLE I: Construction of Biologically Functional
Bacterial Plasmids in vitro
A. Covalently closed R6-5 plasmid DNA was cleaved by incubation at 37° for 15 minutes in a 0.2 ml reaction mixture
containing DNA (40 μg/ml, 100 mM Tris.HCl (pH 7.4)), 5 mM MgCl2, 50 mM NaCl, and excess (2 U) EcoRI endonuclease
in 1 μl volume. An additional incubation at 60° for 5 minutes was employed to inactivate the endonuclease.

The resulting mixture of plasmid fragments was employed for transformation of E. coli strain C600 in accordance with the
procedure previously described. A single clone was examined further which was selected for resistance to kanamycin
and was also found to carry resistance to neomycin and sulfonamide, but not to tetracycline, chloramphenicol, or
streptomycin after transformation of E. coli by EcoRI generated DNA fragments of R6-5. Closed circular DNA obtakined
from this isolate (plasmid designation pSC102) by CsCl-ethidium bromide gradient centrifugation had an S value of 39.5
in neutral surcrose gradients.

Treatment of pSC102 plasmid DNA with EcoRI resistriction endonuclease in accordance with the above-described
procedure resulted in the formation of 3 fragments that were separable by electrophoresis in agarose gels. Intact
pSC102 plasmid DNA and pSC101 plasmid DNA, which had been separately purified by dye-buoyant density
centrifugation, were treated with EcoRI endonuclease followed by annealing at 0°-2° for about six hours. The mixture
was then subjected to ligation with pSC101 and pSC102 in a ratio of 1:1 respectively, by ligating for 6 hours at 14° in 0.2
ml reaction mixtures containing 5 mM MgCl2, 0.1 mM NAD, 100 μg/ml of bovine-serum albumin (BSA), 10 mM ammonium
sulphate (pH 7.0), and 18 U/ml of DNA ligase. (J. Mertz and Davis, Proc. Nat. Acad. Sci., USA, 69, 3370 (1972); and
Modrich, et al., J. Biol. Chem., 248, 7495 (1973). Ligated mixtures were incubated at 37° for 5 minutes and then chilled
in ice water. Aliquots containing 3.3-6.5 μg/ml of total DNA were used directly for transformation.

Transformation of E. coli strain C600 was carried out as previouslydescribed. For comparison purposes, transformation
was also carried outwith a mixture of pSC101 and pSC102 plasmid DNA, which had been subjectedto EcoRI
endonuclease, but not DNA ligase. The antibiotics used for selection were tetracycline (10 μg/ml) and kanamycin (25
μg/ml). The results are reported as transformants per microgram of DNA. The following table indicates the results.

Transformation of E. coli C600 by a mixture
of pSC101 and pSC102 DNA
Transformation frequency for
antibiotic resistence markers
of DNA Tetracycline Kanamycin Tetracycline +
None 2 X 105 1 X 105 2 X 102
EcoRI 1 X 104  1.1 X 103 7 X 101
DNA ligase 1.2 X 104 1.3 X 103 5.7 X 102  

Kanamycin resistance in the R65 plasmid is a result of the presence of the enzyme kanamycin
monophosphotransferase. The enzyme can be isolated from the bacteria by known procedures and employed in an
assay for kanamycin in accordance with the procedure described in Smith, et al., New England J. Medicine, 286, 583

In the preparation for the enzyme extracts, the E. coli are grown in ML-broth and harvested in a late logarithm phase of
growth. The cells are osmotically shocked (see Nossal, et al., J. Biol. Chem. 241, 3055 (1966), washed twice at room
temperature with 10 ml 0.01 M Tris and 0.03 M NaCl, pH 7.3, and the pellet suspended in 10 ml 20% sucrose, 3 X 103
MEDTA and 0.033 M Tris (pH 7.5), stirred for 10 minues at room temperatureand centrifuged at 16,000 g for 5 minutes.
The pellet is then suspended in 2 ml of cold 5 X 10-4 M MCl2, stirred for 10 minutes at 2° and centrifuged at 26,000 g
for 10 minutes to yield a supernatant fluid referred to as the osmotic shockate. The solution should be stored at -20° or
lower. (See Benveneste, et al., FEBS Leters, 14 293 (1971).

The osmotic shockate may then be used in accordance with the procedure of Smith, et al., supra.

EXAMPLE II: Genome Construction between
Bacterial Species in vitro: Replication and Expression
of Staphylococcus Plasmid Genes in E. coli
S. aureus strain 8325 contains the plasmid pI258, which expresses resistance to penicillin, erythromycin, cadmium and
mercury. (Lindberg, et al., J. Bacteriol., 115, 139 (1973)). Covalently closed circular pSC101 and pI258 plasmid DNA
were separately cleaved by incubation at 37° for 15 minutes in 0.2 ml reaction mixtures by EcoRI endonuclease in
accordance with the procedure described previously. Aliquots of the two cleaved species were mixed in a ratio of 3 μg of
pI258:1 μg of pSC101 and annealed at 2°-4° for 48 hours. Subsequent ligation was carried out for six hours at 14% as
described previously and aliquots containing 3.3-6.5 μg/ml of total DNA were used directly in the transformation as
described previously.

Other transformations were carried out employing the two plasmids independently and a mixture of the two plasmids.
Selection of transformants was carried out at antibiotic concentrations for tetracycline (Tc, 25 μg/ml) or pencillin (Pc, 25
OU/ml). The transformation was carried out with E. coli strain C600 rK-mK-. The following table indicates the results.

Transformation of C600 rK- mK- by pSC101
and pI258 Plasmid DNA

Transformants/μg DNA
PSC101 closed circular 1 X 106 <3
pI258 closed circular <3.6 <3.6
pSC101 + pI258 untreated 9.1 X 105 <5
pSC101 + pI258 EcoRI-treated 4.7 X 103 10

The above table demonstrates that bacteria can be formed which have both tetracycline resistance and penicillin
resistance. Thus, one can provide the phenotypical property penicillin resistance in bacteria from DNA, which is
indigenous to another biological organism. One can thus use E. coli for the production of the enzyme, which imparts
penicillin resistance to bacteria, and assay for penicillin in a manner similar to that employed for kanamycin. Penicillinase
is used for destroying penicillin in blood serum of patients treated with penicillin in order to determine whether
pathogenic organisms whose growth is inhibited by penicillin may be present.

EXAMPLE III: Replication and Transcription of
Eukaryotic DNA in E. coli
The amplified ribosomal DNA (rDNA) codeing for 18S and 28S ribsomal RNA of the South African toad, Xenopus laevis
was used as a source of eukaryotic DNA for these experiments. Dawid, et al., J. Mol. Biol., 51, 341 (1970). E. coli-X.
laevis recombinant plasmids were constructed in vitro as follows:

The reaction mixture (60 μl) contained 100 mM Tris.HCl (pH 7.5) 50 mM NaCl, 5 mM MgCl2, 1.0 μg of pSC101 plasmid
DNA and 2.5 μg of X. laevis rDNA, and excess EcoRI restriction endonuclease (1 μl, 2 U). After a 15 minute incubation at
37°, the reaction mixture was placed at 63° for 5 minutes to inactivate EcoRI endonuclease. The product was then
refrigerated at 0.5° for 24 hours, to allow association of the short cohesive termini.

The reaction mixture for ligation of phosphodiester bonds was adjusted to a total volume of 100 μl and contained in
addition to the components of the endonuclease reaction, 30 mM Tris.HCl (pH 8.1), 1 mM sodium EDTA, 5 mM MgCl2,
3.2 nM NAD, 10 mM ammonium sulphate, 5 μg BSA, and 9 U of E. coli DNA ligase. All components were chilled to 5°
before their addition to the reaction mixture. The ligase reaction mixture was incubated at 14° for 45 minutes, and then
at 0.5° for 48 hours. Additional NAD and ligase were added and the mixture incubated at 15° for 30 minutes and then for
15 minutes at 37°. The ligated DNA was used directly in the plasmid transformation procedure previously described. The
DNA was used to transform E. coli strain C600 rK- mK- and tetracycline resistant transformants (3.3 X 103 /μg of
pSC101 DNA) were selected and numbered consecutively CD1, CD2, etc. Plasmid DNA was isolated from a number of
the transformants.

32 P-labeled 18 S and 28 S X. laevis rRNA were hybridized with DNA obtained from the plasmids CD4, CD18, CD30, and
CD42. CD4 DNA annealed almost equally with both the 18 S and 28 S rRNA species. CD18 plasmid DNA hybridized
principally with 28 S X. laevis rRNA, while the DNA of plasmids CD30 and CD42 annealed primarily with 18 S rRNA.
These data indicate that portions of the X. laevis rDNA were, in fact, incorporated into a plasmid recombinant with
pSC101, which was capable of transforming E. coli, so as to be capable of replicating X. laevis rDNA.

Transcription of X. laevis DNA was also carried out in E. coli minicells. The minicell producing E. coli strain P678-54 was
transformed with plasmid DNA isolated from E. coli strain C600 rK- mK- containing CD4, CD18, or CD42. Many cells
containing the plasmids were isolated and incubated with [3 H] uridine; RNA purified from such minicells was hybridized
with X. laevis rDNA immobilized on nitrocellulose membranes in order to determine whether the X. laevis rDNA linked to
the pSC101 replicon is transcribed in E. coli. The results in the following table show that RNA species capable of
annealing with purified X. laevis rDNA are synthesized in E. coli minicells carrying the recombinant plasmids, CD4, CD18,
and CD42, but not by minicells carrying the pSC101 plasmid alone.

Minicells containing plasmids were isolated as described by Cohen, et al., Nature New Biol., 231, 249 (1971). They were
incubated with [3 H] uridine (50 μCi/ml, 30 Ci/mol) as described by Roozen, et al., J. Bacteriol., 107, 21 (1971) for 10
minutes at 37°. Minicells collected by centrifugation were resuspended in Tris.HCl (20 mM, pH 7.5)-5 mM MgCl2-1 mM
EDTA pH 8.0 and rapidly frozen and thawed 3 times. RNA was extracted as described in Cohen, et al., J. Mol. Biol., 37,
387 (1968). Hybridization assays were carried out in nitrocellulose membranes as described in Cohen, et al., ibid, at
saturating levels of pSC101 DNA. Hybridizations involving X. laevis DNA were not performed at DNA excess. Counts
bound to blank filters (5-10 c.p.m.) were substracted from experimentally determined values. 3 H count eluted from filters
containing X. laevis DNA were rendered acid soluble by ribonuclease A 20 μg/ml, 0.30 M NaCl-0.030 M sodium citrate, 1
hour, 37°. The following table indicates the results.

[3 H] RNA synthesized by E. coli minicells
[3 H] RNA counts hybridized to
Plasmid  X. laevis rDNA  
carried by
minicells Input
cpm 0.2μg 0.4μg pSC101 DNA
CD42 4810 905(19%) 1436 (30%) 961 (20%)
CD18 3780 389 (10%) -- 1277 (34%)
CD4 5220 789 (15%) -- 1015 (19%)
pSC101 4170 0 (0%) -- 1500 (36%)

EXAMPLE IV: Plasmid ColEl as a Molecular Vehicle
for Cloning and Amplification of Trp Operon
In a volume of 200 μl (100 mM Tris.HCl (pH 7.5)-5 mM MgCl2 -50 mM NaCl), 5.7 μg of ColEl (E. coli JC411Thy- /ColEl)
(Clewell, et al., Proc. Nat. Acad. Sci., USA, 62, 1159 (1969) and 6.0 μg DNA from bacteriophage .phi.80pt190 (Deeb, et
al., Virology, 31, 289 (1967) were digested to completion with homogeneously purified EcoRI endonuclease, monitoring
the digestion by electrophoresis of the fragments in an agarose gel. The endonuclease was inactivated by heating at
65° for 5 minutes, the digest dialyzed overnight against 5 mM Tris.HCl, pH 7.5, and the sample concentrated to 50 μl.
The fragments were ligated as described in Dugaiczyk, et al., Biochemistry, 13, 503 (1974) at a concentration of 75
pmoles/ml of fragments.

Transformation was carried out as previously described except that the cells were grown to A590=0.600 and following
exposure to DNA were incubated in L-broth for 90 minutes. The cells were collected and resuspended in 10 mM NaCl
before plating. Cells employed as recipients for the transformations were E. coli strains C600 trpR', δtrpE5(MVl), C600
trpR- trpE 10220 recA(MV2), C600 δtrpE5(MV10) and C600 δtrpE5 recA(MV12). (trpR- is the structural gene for the trp
repressor and δtrpE5 is a trp operon deletion entirely within trpE and removing most of the gene.) Approximately 2 μg of
the DNA was used to transform the cells.

Cultures were plated on Vogel-Bonner agar supplemented with 50 μg/ml of the non-selective amino acids, 0.2% glucose
and 5 μg/ml of required vitamins. Transformants to colicin immunity were initially selected on a lawn of a culture of a
mutant strain carrying ColE1. Clones were then selected for their ability to grow in the absence of tryptophan. Cells
capable of producing tryptophan were isolated, which could be used for the production of exogenous tryptophan. The
subject example demonstrates the introduction of a complete operon from foreign DNA to provide a transformant
capable of replicating the operon and transcribing and translating to produce enzymes capable of producing an
aromatic amino acid.

EX. V: Cloning of Synthetic Somatostatin Gene
The deoxyribonucleotide sequence for the somatostatin gene was prepared in accordance with conventional
procedures. (Itakura et al, Science, 198 1056 (1977)). To prepare the recombinant plasmid, plasmid pBR 322 was
digested with Eco RI. The reaction was terminated by extraction with a mixture of phenol and chloroform, the DNA
precipitated with ethanol and resuspended in 50 μl of T4 DNA polymerase buffer. The reaction was started by the
addition of 2 units of T4 DNA polymerase. After 30 min at 37°, the mixture was extracted with phenol and chloroform and
the DNA precipitated with ethanol. The λplac5 DNA (3 μg) was digested with the endonuclease Hae III and the digested
pBR 322 DNA blunt end ligated with the Hae III-digested λplac5 DNA in a final volume of 30 μl with T4 DNA ligase
(hydroxylopatite fraction) in 20 mM tris-HCl pH 7.6), 10 mM MgCl2, 10 mM dithiothreitol and 0.5 mM ATP for 12 hrs at
12°. The ligated DNA mixture was dialyzed against 10 mM tris-HCl (pH 7.6) and used to transform E. coli strain RR1.
Transformants were selected for tetracycline resistance and ampicillin resistance on antibiotic (20 μg/ml) X-gal (40
μg/ml) medium. Colonies constitutive for the synthesis of .beta.-galactosiodase were identified by their blue color and of
45 colonies so identified, 3 of them were found to contain plasmids with 2 Eco R1 sites separated by ~200 base pairs.

The plasmid so obtained pBH10 was modified to eliminate the Eco R1 site distal to the lac operator and plasmid pBH20
was obtained.

Plasmid pBH20 (10 μg) was digested with endonucleases Eco R1 and Bam HI and treated with bacterial alkaline
phosphatase (0.1 unit of BAPF, Worthington) and incubation was continued for 10 min at 65°. After extract with a phenol-
chloroform mixture, the DNA was precipiated with ethanol. Somatostatin DNA (50 μl containing 4 μg/ml) was ligated with
the Bam HI-Eco R1 alkaline phosphatase=treated pBH20 DNA in a total volume of 50 μl with 4 units of T4 DNA ligase for
2 hrs at 22° and the recombinant plasmid used to transform E. coli RR1. Of the Tcr transformants isolated (10), four
plasmids has Eco R1 and Bam HI sites. Base sequence analysis indicated that the plasmid pSOM1 had the desired
somatostatin DNA fragment inserted. Because of the failure to detect somatostatin activity from cultures carrying plasmid
pSOM1, a plasmid was constructed in which the somatostatin gene could be located at the COOH-terminus of the .beta.-
galactosidase gene, keeping the translation in phase. For the construction of such a plasmid, pSOMI (50 μg) was
digested with restriction enzymes Eco RI and Pst I. A preparative 5 percent polyacrylamide gel was used to separate the
large Pst I-Eco RI fragment that carries the somatostatin gene from the small fragment carrying the lac control elements
(12). In a similar way plasmid pBR322 DNA (50 μg) was digested with Pst I and Eco RI restriction endonucleases, and
the two resulting DNA fragments were purified by preparative electrophoresis on a 5 percent polyacrylamide gel. The
small Pst I-Eco RI fragment from pBR322 (1 μg) was ligated with the large PstI-Eco RI DNA fragment (5 μg) from pSOM1.
The ligated mixture was used to transform E. coli RR1, and transformants were selected for Apr on X-gal medium.
Almost all the Apr transformants (95 percent) gave white colonies (no lac operator) on X-gal indicator plates. The
resulting plasmid, pSOM11, was used in the construction of plasmid pSOM11-3. A mixture of 5 μg of pSOM11 DNA and
5 μg of λplac5 DNA was digested with Eco RI. The DNA was extracted with a mixture of phenol and chloroform; the
extract was precipitated by ethanol, and the precipitate was resuspended in T4 DNA ligase buffer (50 μl) in the presence
of T4 DNA ligase (1 unit). The ligated mixture was used to transform E. coli strain RR1. Transformants were selected for
Apr on X-gal plates containing ampicillin aidn screened for constitutive.beta.-galactosidase production. Approximately 2
percent of the colonies were blue (such as pSOM11-1 and 11-2). Restriction enzyme analysis of plasmid DNA obtained
from these clones revealed that all the plasmids carried a new Eco RI fragment of approximately 4.4 megadaltons, which
carries the lac operon control sites and most of the .beta.-galactosidase gene (13, 14). Two orientations of the Eco RI
fragment are possible, and the asymmetric location of a Hind III restriction in this fragment can indicate which plasmids
had transcription proceeding into the somatostatin gene. The clones carrying plasmids SOM11-3, pSOM11-5, pSOM11-
6, and pSOM11-7 contained the Eco RI fragment in this orientation.

It is evident from the above results, that both DNA from a eukaryotic source and RNA transcribed from the eukaryotic
DNA can be formed in a bacterial cell and isolated. Thus, the subject process provides a simple technique for producing
large amounts of eukaryotic DNA and/or RNA without requiring the reproduction and maintenance of the eukaryotic
organism or cells. The employment of DNA for production of ribosomal RNA is merely illustrative of using a genome from
a eukaryotic cell for formation of a recombinant plasmid for replication in a bacteria. Genomes from a eukaryotic cell for
formation of genotypical properties, such as the production of enzymes, could have equivalently been used. As
evidenced by the transformation with DNA from a bacteriophage, and entire operon can be introduced into a bacterial
cell and the cell becomes capable of its transcription, translation, and production of a functional gene product. Thus, a
wide variety of auxotrophic properties can be introduced into a bacterial cell.

In accordance with the subject invention, DNA vehicles are provided, which are covalently closed circular
extrachromosomal replicons or genetic elements, including plasmids and viral DNA. The vehicles generally will have
molecular weights in the range of about 1 to 20X106 and are characterized by having an intact replicon, which includes
a replicator locus and gene. The vehicle is capable of clevage by a restriction enzyme to provide a linear segment
having an intact replicon and cohesive termini, which may be directly obtained by the cleavage or by subsequent
modification of the termini of the linear segment. The vehicle will be capable of transforming a bacterial cell and to that
extent is compatible with the cell which will provide replication and translation. Preferably, the vehicle will have a
phenotypical property which will allow for segregation of the transformant cells. Phenotypical properties include
resistance to growth inhibiting materials, such as antibiotics, peptides and heavy metals, morphological properties, color,
or the like, and production of growth factors, e.g. amino acids.

The vehicle is combined with DNA indigenous to a biological organism other than the cell which provides replication and
provides a genotypical or phenotypical property which is alien to the cell. The source of the DNA can be prokaryotic or
eukaryotic, thus including bacteria, fungi, vertebrates, e.g. mammals, and the like.

The plasmid vehicle and the alien DNA having complementary cohesive termini can be annealed together and
covalently linked to provide a recombinant plasmid, which is capable of transforming a bacterial cell, so as to be capable
of replication, transcription, and translation. As a result, a wide variety of unique capabilities can be readily introduced
into bacteria, so as to provide convenient ways to obtain nucleic acids and to study nucleic acids from a foreign host.
Thus, the method provides the ability to obtain large amounts of a foreign nucleic acid from bacteria in order to be able
to study the function and nature of the nucleic acid. In addition, the subject method provides means for preparing
enzymes and enzymic products from bacteria where the natural host is not as convenient or efficient a source of such
product. Particularly, bacteria may allow for more ready isolation of particular enzymes, uncontaminated by undersirable
contaminants, which are present in the original host. In addition, the products of the enzymic reactions may be more
readily isolated and more efficiently produced by a transformant than by the original host. Besides enzymes, other
proteins can be produced such as antibodies, antigens, albumins, globulins, glycoproteins, and the like.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of
clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of
the appended claims.

We claim:

1. A method for replicating a biologically functional DNA, which comprises:

transforming under transforming conditions compatible unicellular organisms with biologically functional DNA to form
transformants; said biologically functional DNA prepared in vitro by the method of:

(a) cleaving a viral or circular plasmid DNA compatible with said unicellular organism to provide a first linear segment
having an intact replicon and termini of a predetermined character;
(b) combining said first linear segment with a second linear DNA segment, having at least one intact gene and foreign to
said unicellular organism and having termini ligatable to said termini of said first linear segment,wherein at least one of
said first and second linear DNA segments has a gene for a phenotypical trait, under joining conditions where the
termini of said first and second segments join to provide a functional DNA capable of replication and transcription in said
unicellular organism;
growing said unicellular organisms under appropriate nutrient conditions; and
isolating said transformants from parent unicellular organisms by means of said phenotypical trait imparted by said
biologically functional DNA.

2. A method according to claim 1, wherein said unicellular organisms are bacteria.

3. A method according to claim 2, wherein said transformation is carried out in the presence of calcium chloride.

4. A method according to claim 3, wherein said phenotypical trait is resistance to growth inhibiting substance, and said
growth is carried out in the presence of a sufficient amount of said growth inhibiting substance to inhibit the growth of
parent unicellular organisms, but insufficient to inhibit the growth of transformants.

5. A method according to claim 1, wherein said unicellular organism is E. coli.

6. A method according to claim 1, wherein said predetermined termini are staggered and cohesive.

7. A method according to claim 6, wherein said joining conditions includes enzymatic ligation.

8. A method according to claim 6, wherein said cohesive ends are formed by staggered cleavage of said viral or circular
plasmid DNA and a source of said second segment with a restriction enzyme.

9. A method acording to claim 6 wherein said cohesive termini are formed by addition of nucleotides.

10. A method according to claim 1, wherein said predetermined termini are blunt end and said joining conditions include
enzymatic ligation.

11. A method for replicating a biologically functional DNA comprising a replicon compatible with a host unicellular
organism joined to a gene derived from a source which does not exchange genetic information with said host organism,
said method comprising:

isolating said biologically functional DNA from transformants prepared in accordance with claim 1;
transforming unicellular microorganisms with which said replicon is compatible with said isolated DNA to provide second
transformants; and

growing said second transformants under appropriate nutrient conditions to replicate said biologically functional DNA.

12. A method for producing a protein foreign to a unicellular organism by means of expression of a gene by said
unicellular organism, wherein said gene is derived from a source which does not exchange genetic information with said
organism, said method comprising:

growing transformants prepared in accordance with any of claims 1 and 11 under appropriate nutrient conditions,
whereby said organism expresses said foreign gene and produces said protein.
13. A method according to claim 12, wherein said protein is an enzyme.

14. A method according to claim 11, wherein said method is repeated substituting said biologically functional DNA from
transformants prepared in accordance with claim 1 with second or subsequent transformants to produce additional


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