CA1214737A - Genetic engineering in procaryotic organisms - Google Patents

Abstract

GENETIC ENGINEERING IN PROCARYOTIC ORGANISMS

ABSTRACT
A procaryotic microorganism and a method for its production is provided wherein the microorganism contains at least one stable foreign DNA portion in the chromosome. The disclosed microorganisms and their progeny are substantially free of genetic rearrangement involving the foreign DNA. In a preferred embodiment, cyanobacteria are employed. The microorganisms are produced by introducing into the cell an insertion vehicle that contains foreign DNA ligated between two portions of DNA homologous to adjacent portions of the recipient's chromosome.

Description

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GENETIC ENGINEERING IN PROCARYOTIC ORGANISMS

TECHNICAL FIELD
l he present invention relates to procaryotic organisms that have foreign DNA stably inser~ed in a chromosome and a method for their production~
3~ACKGROUND OF THE INVENTION
Practical methods for introducing oreign DNA in~o bacteria and other organismc are, of course, known in the art. These methods employ recombinant DNA technology to construc~ ohimeric plasmid DNA
molecules which contain pieces of foreign DNA. The chimeric plasmids are designed to replicate autonomously as an extrachromosomal DNA
species in a procaryotic host such as E. coli. In yeast, the chimeric plasmid may either replicate extrachromosomally or it rnay integrate into yeast chromosomes, ~though such integration tends to be unstable.
When ernploying such ~enetically engineered microorganisms to manufactllre produ~s, it is important to continously select ~or the presence of the foreign DNA in the recipient organism because the foreign DNA carried on a plasmid or in integrated form tends to be unstable and can be lost in dau~hter cell~.
Many applications of genetic engineering technology are centered around the production of proteins via genetically altered microorganisms (e.g., insulin or interferon). Production of a protein of~en requires only that a single gene encoding the specific protein be introduced into the recipient organism. The synthesis of more complex metobolic products, however, may require the introduction of several foreign DNA segments or genes to create new biochemical pathways. For example, the ~oduction of nonprotein products could be achieved if genes encoding ~Z3 '~737

-2-the production of enzymes which operate on nonprotein substrates to produce the desired product could be stably introducecl into a microorganism. It is difficult to maintain a multistep biochemical pathway using "traditional" technology because of the instability of the foreign DNA introduced via the chimeric plasmid DNA
molecules. Simply too many genes must be introduced and maintained collectively in the recipient organism. A means for introducing several foreign DNA segments or genes stably in a recipient organism would, therefore, be a valuable contribution to the art.
SUMMARY OF THE IN~ENTION
The present invention provides procaryotic micro-organisms which have at least one foreign DNA portion stably inserted in-to the chromosome.
The present invention also provides a unicellular photosynthetic microorganism which contains at least one stable ~oreign DNA portion in a chromosome.
The present invention includes a method for stably inserting a foreign gene in a chromosome of a unicellular microorganism.
The present invention also includes a method oE
producing chemicals, including chemicals other than proteins, employing microorganisms.
One aspect of the present invention is directed to a procaryotic microorganism containing at least one stable ~oreign DNA portion covalently bonded directly to chromosomal DNA of the microorganism, wherein said microorganism and its progeny are substantially free of genetic rearrangements involving said foreign DNA.
These microorganisms, which may be made by a process of the present invention, can contain multiple foreign DNA
portions or genes stably incorporated into the chromosomal genome. It is possible to stably encode complete biochemical pathways foreign to a microorganism into the chromosome so that useful chemicals, particularly chemicals other than proteins, can be made ln vivo by a practical method. In a preferred embodiment of the present invention the microorganism is a cyanobacterium. This allows useful products to be made photosynthetically. The present invention also encompasses i 7 3~

pure cultures of the above microorganisms which do not have to be subjected to constant selectiYe pressure in contrast to microorganisms containing foreign genes inserted into plasmids.
Another aspect oE the present inven~ion provides a method of producing a miCrQorganism having at least one stable foreign DNA portion in its chromosome, said method comprising: (a) providing a DNA insertion vehicle containing first and second DNA portions containing DNA
hornologous to adjacent portions o~ a chromosome in said microorganism, said homolo~ous DNA in said first and second DNA portions oriented in relation to each other in the same manner as said homologous chromosomal DNA portions in said microorgarlism; and a third DNA
portion containing DNA foreign to said microorganism, ~aid third DNA
portion located between and covalently bonded to said first and second DNA portions; and (b) in~roducing said DNA insertion vehicle inside the cell membrane of said microorganism to effect incorporation of the genetic material of said foreign DNA into the chromosomal genome of said microorg~nisms.
ln cne pre~erred aspect, the present invention contemplates a circular DNA insertion vehicle which facilltates the stable insertion of foreign DNA into the chromosome of a procaryo~ic microorganisrn, said circular DNA insertion vehicle comprisirlg: (a) a firs~ DNA segment comprising iirst and second DNA portions containing DNA homologous to adjacent portions of a chromosome in a microorganlsm, said first and second DNA portions oriented in relation to each other in the same manner as said homologous chromosomal DNA portions in said microorganism; and a third DNA portion containing DNA foreign to said microorganism that expresses a selectable phenotype located between and covalently bonded to said first and second DNA portions, and a sin~le restriction site in said first DNA segment for a particular restriction enzyme at a location nonessential to said expressable phenotype between said first and second DNA portions; and (b) a second DNA segrnent containing a DNA portion that is not homologous to the chromosomal DNA in said microorganism.
The various aspects of the present invention will be apparent to those skilled in the ar~ ~rom the following description.

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BRIEF_DESCRIPTION OF THE DRAWINGS
FiF,ure I is a schema~ic representation of the preferred circular chimeric DNA insertion vehicle of the present invention in its simplest form.
Figure 2 is a schema~ic representation of the circular chimeric l)NA molecule from which the vehicle of Figure I is made.
DETAILED l)ESCRIPTION OF THE INVENTION
The present inventlon i5 direc~ed to procaryotic microorganisms which have at least one foreign DNA portion s~ably inser~ed in the chromosomes. Genetically engineered microorganisms of the prior art generally contain foreign genes either in plasmids or unstably integrated, as in yeas~, into the chromosomes. Plasmids can be lost upon reproduction in some daughter cells and unstable foreign genes integrated into chromosomes are subject to deletion. In contrast, the foreign DNA
in the microorganisms of the present invention is ligated ti.e., covalently bonded) directly to the chromosomal DNA of the microorganism, not to plasmid DNA or other DNA such as viral DNA or transposable elements.
Typically, the foreign DNA in the present invention is contained within nonessçn~ial DNA in the chromosome.
A DNA segment or portion is a linear length o~ DNA. A DNA
segment as used herein is a longer length of DNA than a DNA portion.
~ oreign DNA, as used herein, includes synthetic DNA that does not sxist in nature, chromosomal or plasmid DNA that is derived from a genus other than the recipient organism's genus, or a Yiral DNA length that does not have the ability to naturally infect or transfect the recipient organism. Excluded from foreign DNA, however, is a DNA
lengSh that is a transposable element (~ ~ ~ transposon or insertion sequence) in the recipient organism. A transposable element is a length of DNA that interacts with a second DNA molecule (~ ~ chromosome of the recipient) by site-specific recombination to produce new linkage relationships in the second DNA molecule, including inversion or delçtion of DNA in the second DNA molecule, or addition of the transposable element to the second DNA molecule. See C. M. Radding, (1978~ Am.
Rev. Biochem. 47: 847-880; DNA Insertion Elements, Plasmids ~ Episomes (A. Bukhari, J. Shapiro ~ S. Adhya eds., Cold Spring Harbor Labratory 1977). DNA "derived from a ~enus other than the recipient organism's genus" is DNA found in nature only in organisms of other genera. Even if the particular copy of DNA employed in the present invention was t not immediately derived from another genus, the DNA is still "derived from" another genus if it is found in nature only in a genus or ~enera other thar the genus of the recipient organism.
Viral DNA that can infect or transEect a recipient is excluded from the term foreign DNA because such viruses can reversibly integrate into the chromosome of the recipient and/or kill the recipient.
Transposable elements are excluded from the ~erm forei~n DNA because they can cause genetic rearrangement such as inserting replicas at other sites in the recipient, excising themselves from the chromosome causing inversion or deletion mutations. Transposable elements and viral DNA
tha~ can infect or transfect the recipients are unstable and, therefore, unsuited ~o the cs)nstruction of a microorganism that contains at least one forei~n DNA portion in the chromosome. This is particularly true when multiple foreign DNA portions are inserted.
The stably inserted foreign DNA of the present invention is actually incorporated into the chromosomal genome of the microorganism. Stably inserted foreign DNA refers to foreign DNA that is not involved in genetic rearrangement in the recipient microorganism or its progeny.
The microor~anisms of the present invention and ~heir progeny are substantially free of genetic rearrangement involving the foreign DNA.
By genetic rearran8ement involving the foreign DNA is mean~
recombination between homologous portions of the recipient's chromosome leading to loss of foreign DNA, insertion o~ replicas c>f the foreign DNA
in the ~enome of the recipient, or excision o$ the forei~n DNA.
Foreign DNA that has been stably inserted in micro~rganisms of the present invention undergoes a functional change only as a result of mutagenic processes provoked by the repair or replication of l)NA. The rate of such mutagenic processes can be defined by the rate at which auxotrophic mutations accumulate in a population of recipient organisrns that has been transformed by the foreign DNA. Fcr a discussion of auxotrophic mutations and methods for determining mutation rates, see J. Miller, E~periments in Molecular Genetics 121-B2 (Cold Spring Harbor Laboratory 1972). A convenient test for the frequency of an auxotrophicmutation in a recipierlt is ~he frequency of chlorate-resistant mutations which can be measured as described in C. MacGregor et al. (lg71) 3.
Bacteriol. i0B: S64-570. The function of foreign DNA stably contained in the chromosome of a microorganism of the present invention is lost from the microorganism at a ~requency that is not subs~antially ~reater than the highest frequency of a auxotrophic mutation in the organism.
The frequency o~ mutagenesis is substantially lower than the frequency of recombination between homologous chromosomal DNA. After growin~
a culture for forty generations without seiective pressure, for example, nearly 1,000 microorganisms of the present invention were tes~ed for loss of function of the foreign DNA. All o~ the microsrganisms tested exhibited the foreign DNA function. In contrast, when DNA was inserted into the chromosome in a manner that allowed recombination between homologous regions o~ the chromosome, at least 22% of the microorganisms tested had lost the inserted DNA function after forty generations in the absence of selective pressure. (See Example 2, infra.) Viral DNA or transposable elements that have the ability to integrate or provoke rearran~ement in the recipient l)NA are per se unstabie and have7 therefore, are excluded from the defirlition of foreign DNA as discussed above.
The microorganisms of the present inven~ion are procaryotes (i.e.3 organisms within the kingdom Monera). Procaryotic oganisms lack a nuclear envelope around their chromosome~ such as is found in eucaryotic organisms. Procaryotic organisms are generally divided into the phyla schi~ophyta (all bacteria) and cyanophy~a (blue-green algae or cyanobac~eria). Typical examples o~ bacterial genera to which the present invention is applicable include, inter ~ Bacillus, Pseudomonas, Escherichia, Azotobacter, Rhizobium, Rhodo~seudomon~ Streptococcus, HaemoPhiius and Klebsiella. Examples of cyanobacteria to which the present invention is directed include, inter ~ the genera APhanocapsa, Anabaena, Nostoc, Oscillatoria, Synechococcus, G oeocapsa, A~,menellum, Scvtonema7 Mastigocladus, Arthrosprira and Haplosiphon.
The preferred microorganisms o~ the present invention are cyanobacteriaJ which are gram-negative procaryotes. Cyanobacteria are L~7 3~

~hotosynthetic unicellular or~anisms which are either free-living or in symbiotic association with bacteria, fungi, plants, or animals. They are found in lakes, rivers, oceans, mineral hot springs, soll (tropical to arctic) and on rocks and buildings. Some are filamentous organisms and several species can fix nitrogen.
Cyanobacteria have the ability to synthesize chemicals from air, water and inorganic salts utilizing the energy of sunlight. In a par~icularly preferred embodiment of the present invention, therefore, photosynthetic cyanobacteria are contemplated whose chromosomal structure has been altered. Cyanobacteria can produce me~abolites, such as carbohydrates, proteins, lipids and nucleic acids, from C02 (from the air), water, inorganic salts and light. With a nitrogen fixing cyanobacteria, nitrogen containing salts need not be added because the cyanobac~eria can fix N2 from the air. By introducing into a recipient cyanobacteria foreign DNA portions ~hat encode new biochemical pathways, important and usef ul products can oe produced photosyn~hetically. If the precursor of the foreign biochemical pathway ls a metabolite of cyanobacteria, the product of the pathway can be produced solely from air, water, inorganic salts and sunlight. Additional precursor may be added, howesrer, to increase the product yield. If the precursor is not a metabolite of the cyanobacteria, then precursor can be added to cultures oI the cyanobacteria. In this case, i~ is preferred that the precursor be a readily available and inexpensive material such as a waste byproduct of other processes (e.~. Iignosulfonates or casein).
Since the microorganisms of the present invention are readily produced by introducing a DNA insertion vehicle wi~hin the cell, preferably a circular vehicle comprised of a chimeric plasmid, a consideration in selecting a recipient organism is the ease with which it takes up exogenous DNA. Particularly preferred organisms in the pr~sent invention are those cyanobacteria which have been shown to have a naturally occurring system for the uptake exogenous DNA such as Gloecapsa alpicola, A~menellum quadruplicatum and Anacystis nidulans.
If a bacterium is employed, bacteria which spontaneously take up exo~enous DNA, such as Bacillus subtilis, are preferred. The insertion vehicle employed in present invention, however, can also be introduced ~Z~3~

into procaryotes, such as E. coli, that ~ake up exogenous DNA only through artificial manipuJation. Methods for introducing exogenous DNA
lnto other procaryotes are known in the art.
Placement of the foreign DNA into the insertional DNA of the unloaded insertion vehicle rnay be accomplished using recombinant DNA
technology. While ~he insertion Yehicle can be either linear or circular, it is technically difficult to load a linear insertion vehicle with foreign l)NA. Loading linear vehicles in vivo is not feasible because linear DNA cannot ~e maintained due to exonuclease activity in cells. Loading linear insertion vehicles in vitro can be accomplished but is inefficient because cleavage of the unloaded linear vehicle prior to insertion of the ~oreign DNA can result in shu~fling ~he orientation of the insertional DNA fragments upon rejoining. From the standpoint of efficiency, these problems are obviated If circular vehicles, such as depicted in Figures I and 2J are employed. For efficient loading, the transformation vehicle desirably contains one or more unique restriction cleavage si~es, or "loading sites," located in or near the central portion of the insertional DNA (i.e., sites in the insertional DNA for an endonuclease that cleaves at no other sites~.
P~ "loaded" drcular DNA insert;on vehicle employed in the production of microorganisms of the present invention in its simplest form is comprised of three components as is shown in Figure 1. The loaded insertion /ehicle is made from an unloaded insertion vehicle or plasmid as depic~ed in Figure 2. The structure of the unloaded vehicle is substantially the same as that of the loaded vehicle described in Figure 1, excepting that there is no foreign DNA portion E~ and there is a restriction site 1 at the junction of insertional DNA portions A and A'.
Portions A and A' are termed insertional DNA. The insertional ONA is homolo~ous ~o a stable region of DNA in a chromosome of the recipient organism. Portions A and A' are homologous to adjacent chromosomal DNA regions in the recipient and are ideally derived from a single ~ragment of chromosomal DNA ~rom the recipient organism that has been cleaved at a centrally located restriction site (the loading site). "Adjacent" regions in the chromosomal DNA c~ the recipient ~z~

means ~hat either ~1) chromosomal DNA regions that are irnmediately adjacent to each other, or (2) chromosomal DNA regions that are separated only by nonessential DNA. Portions A and A' are, relative to each other, In the same orientation in the insertion vehicle as are the hornologues of A and A' in the chromosome of the recipient organism.
In other words, the ends of the homologous regions in ~he chromosome closest to each other correspond to the end portions of A and A' in contact with l)NA portion 13 in Figure 1 (or restriction site I in Figure 2).
The insertional DNA should have a5 long a length as practical on either side of the loading si$e. For example, it was found that when foreign DNA portions were loaded randomly into lnsertional DNA segments having an average length between 80,000 and 120~000 base pairs in a linear insertion vehicle so that the great majority of ~oreign DNA was located thousands of base pairs from the ends of the insertion vehicles.
There was a 400-fold increase in transformation efficiency (based on transform~ions per foreign DNA se~ment) with these linear insertion vehicles over an insertional DNA se~ment 4~100 base pairs long with a single loading site S00 base pairs from one end in a circular insertion vehicle. Insertional DNA should generally have a~ least 100 base pairs on either side of $he loading site to obtain reasonable transformation efficiency. The length of the insertional l)NA has not been found to affect the stability of the inserted foreign DNA.
Foreign DNA portion B, also referred to herein as interrupting DNA, is ligated between DNA portions A and A'. Useful foreign DiNA
which can be inserted into the chrs~mosome of the recipient organism include genes which encode the production of proteins (e.g., an enzyme in a metabolic pathway) or regions that regulate gene expression (e.g., promoters and operators). Transformation efficiency has been ~ound to drop as the length ~ the interrupting (foreign) DNA increases. For example, when the interrupting ~NA is 1,300 base pairs long, there is a 7-fold increase in transformation efficiency over ~oreign DNA 4,000 base pairs long, all other conditions being equal. Although it is possible to ir-sert quite long forelgn DNA portions (e.g., up to at least 20,000 base pairs), it is desirable to use the shortest DNA length that will 73~

encode the desired information.
DNA segment C, or flanking DNA, is ligated to the opposite ends of DNA portions A and A'. Flanking DNA is so-called because it flanks ~nd does not interrupt the insertional DNA. Flanking DNA is derived ideally frsm a plasmid compatable with and having the ability to replicate in a microorganism other than the recipient organism. The flanking DNA segment is required only in circular insertion vehicles. Since a linear vehicle only need be csmprised of homolo~ous DNA portions A
and A' and interrupting DNA portion B.
When the flanking DNA i5 derived from a plasmid which is compatable with and replicates in a second microorganism, the flanking DNA is preferably derived from an entire plasmid which has been cleaved at a single restriction site. A functioning replicon in the flanking portion allows the transformation vehicle to be cloned in a microorganism other than the recipient organism in large quantities. No replicon in the insertion vehicle (includlng any replicon in the flanking DNA), however, should be f unctional in the recipient organism since it is the ~unction of ~he insertion vehicle to transform the chromosome and not to replicate automonously in the recipient organism. It is also desirable to use a plasmid which çontains a gene for a selectable phenotype (e.g., antibiotic resistance) to ai~ in the isolation of plcasrnids.
To stably insert the foreign gene or DNA portion contained in the interrupting DNA into the chromosomal 8encme of a recipient or~anism, the loaded transformation vehicle is introduced inside the ce~l membrane of the recipient. 1~ the recipient organism naturally takes up exo~enous DNA, the loaded transformation vehicle need only be introduced into a culture of the recipient or~anism. If the recipient organism does not naturally take up exogenous DNA, the loaded plasmid can be introduced into the cell by conventional methods known in the art (e.g., conjugation or treatment with (::aC12). See M. Suzuki ~ A.
Szalay, (1979~ Meth. Enzymol. ~8: 331-341.
If the insertion vehicle is circular, the stably inserted transformant will usually have to be distinguished from two other types of unstable transformants. In one type of unstable transformant, the ~lanking DNA
and the insertional DNA are added (iOe., an addltive recombinant event) to the chromosome. If the flanking DNA expresses a xelectable phenotype, this type of transf ormant is readily distinguished. In the second type of unstable transformant, the entire vehicle (interrupting, insertional and flan3sing DNA) i5 added to the chromosome. If both the interrupting (foreign) IDNA and flanking Dr'lA express selectable phenotypes, this type of transformant is also readily Isolated. The instability of these types is believed to ~ise because there are two homologous regions in the chromosome; insertional DNA has been added to the chromosome rather than undergoing recombinant exchange. This would lead to loss of the interrupting DNA through recombination between the two homologous regions in the chromosome.
Pure cultures of the recipient organism with stably inserted foreign DNA can be obtained by selecting those organisms which have been stably transformed. Selection methods, well known in the art, include testing colonies grown from individual cells for product or other expressable phenotype, and colony hybridization. See M. Grunstein et al., (1975) Proc. Natl. Acad. Sci USA 72: 3962-3965. By pure culture is meant a culture of a species of microorganisms containing foreign DNA stably inserted into the chromosome that ls substantially free of the same microorganism without foreign DNA in the chromosome.
Applican~s have demonstrated that when a transformation vehicle according to the present invention is employed, foreign DNA can be stably lnserted into the chromosomes. Once inside the cell membrane, the insertional DNA portions undergo recombina~i~n with the chromosome of the recipient organism at the site of homology between ~he insertional DNA and the chromosomal DNA. Since the foreign DNA is ligated between the adjacent portions of insertional DNA, the foreign DNA is carried into the chromosomal DNA of the recipient organism. ~oreign DNA so inserted has been ~ound to be a stable component of the chromosomal genome.
Applicants believe, but do not wish to be bound by their theory, that the actual insertion of foreign DNA into a chromosome takes advantage of a natural recombination event which occurs in a wide variety of both eucaryotic and procaryotic organisms. In fact, the method of the present invention should be generally applicable to ~ 2:LL~ 7~
-- 12 _ eucaryotic organisrns by introducing viable loaded insertion vehicles of the present invention inside the nuclear membrane of eucaryotes. The rerombination event involves recombination between two molecules of DNA, identical in a portion of the molecules except for ~he presence in one of the molecules of a heterologous insertion. Recombination results in the transfer of the latter ~rom the first DNA molecule to the second.
By stably inserting several foreign genes Into the chromosomes of a microorganisrn, multistep biosynthetic pathways can be introduced which synthesize ronproteins. After identifying and isolating 311 the genes necessary in a desired biochemical pa~hway, the method of ~he present invention can be straightforwardly applied to produce a microorganism that will produce the desired product. Biological systems exist which produce chemicals of industrial importance, such as butanol, acetone, polysaccharides, carotenoids, hydrocarbons and molecular hydrogen. Genes for other biological systems can be isolated or synthesized. An ~n vivo method of producing such chemicals, particularly photosyn~hetically, is obviously of great value.
There are a multitude of applications for the present inventionO
For example, the genes for butanol-acetone fermentation (which produces butanol and ace~one from glucose) are found in Clostridium acetobutylicum. See G. Got~schalk, Bacterial Metabolism ~82 SSpringer Verlag 1979). Stable introduction of those genes into a cyanobac$erium provides a blue-green algae with a capability of producing butanol and acetone. All microorganisms produce ~lycerol. See, ~2 A. Newman, Glycerol (CRC Press 1968); 1. Stryer, Biochemistry 292 (Freeman 1975).
Glycerol production can be enhanced in a selected microorganism by introducing foreign DNA that affects the expression of the genes responsible for glycerol production. Produstion of hydrogen from water by a cyanobacteria is possible by mutating ferredoxin-NADP
oxidoreductase, so that electrons produced by photosynthesis accumulate on ferrodxin, and by inserting a foreign gene encoding a hydrogenase into the mutated cyanobacteria. See, ~ J.R. Benemann et al., (1982) Proc. Natl. Acad. Scl. lJSA 70: 2317-2320.
-When several foreign genes are introduced into a recipient to, for - 13 _ example, lntroduce a new biochemical pathway, several transforrnation steps may be employed. In such cases where the foreign DNA inserted in one step encodes no selectable phenotype in the recipient organism9 it is desirable to link the foreign DNA to a selectable gene to aid in ~he iden~ification of s~able ~ransformants. A selectable gene can be spliced into the insertional DNA such that a unique loading site is located either within a nonessential region c~f the selectable marker, or at the molecular juncture between the selectable marker and one of the insertional DNA portions. In the latter case, the unloaded vehicle has the same configuration as in Figure 2, except that an additional foreign gene that expresses a selectable phenotype is located between restriction site I and either DNA portion A or Al. Pre~errably, there is only one restriction site for the endonuclease of site I in the unloaded insertion vehicle~
In one preferred embodiment of the invention, insertion vehicles can be constructed by the following, relatively slmple procedure.
First, insertional DNA is isolated ~rcm the chromosome of the recipient organism by deavage with a restriction enzyme. DNA
fragmen.s of the ~esired length, preferably 5000 to about 10,000 base pairs lon~, are isolated by electrophoresis. The desired fragments are cloned into a restric$ion site of a plasmid (which becomes the flar king DNA) from another microorganism (host organism). The plasmid from the host organism desirably expresses a selectable phenotype in the host and the recipient. The recombinant plasrnids are used to transform the host organism and transformants are iso!lated accordin~ to phenotypes encoded on the plasmid. Milli~ram quantities of the mixture of recombinant plasmids are isolated from the host organism.
In the next step, it is necessary ~o isolate acceptable unloaded insertion vehicles from the mixed recombinant plasmids collected above.
Acceptable insertion vehicles are those having centrally-loca1 ed loading sites that are not wiehin insertional DNA $hat is homologous to an essential gene in the recipient organism. One method of isola~ing acceptable insertion vehicles is to construct restriceion cleavage maps of a number of the recombinant plasmids. This approach is unnecessarily laborious.

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A simpler approach is to allow ~he recipient organism itself to isolate useful insertion vehicles. First, the mixture of recombinant plasmids is cleaved with a restric~ion enzyme that has sites only within t~ the insertional DNA. The enzyme should not have restriction sites within the ~lanking DNA or at the junctions between the insertional DNA and $he flanking DNA. Second, interrupting (~oreign) DNA is ligated into the cleaved plasmids. Ths interrupting DNA desirably contains a gene which expresses a selectable pheno~ype distingui`shable from the selection marker in the flanking DNA.
The plasmids containing interrupting DNA are then used to transform a cul~ure o~ recipient microorganisms. Isolated transformants which exhibit the selectable phenotype of only the interrupting DNA
have obviously been transformed by a chimeric DNA molecule ~hat is active as an inser~ion vehicle. The insertion vehicle itself, of course, is destroyed in the process of integration of the interrupting DNA into the recipient chromosome.
TQ reconstruct insertion vehicles, chromosomal DNA is isolated from several of the transformants. Cleaving the recovered chromosomal DNA from the trans~ormants with the restriction enzyme originally used to isolate chromosomal DNA from the recipient organism (to be çmployed as insertional DNA) and then liga~ing the cleaved chromosomal DNA to cleaved plasmid 13NA (flanking DNA) that contains a selec~ion marker will yield a mixture that contains acceptable loaded transformation vehicles admixed with ligation products. By transf orming the host microorganism and selec~ing for the phenotypes expressed by both the interrupting DNA and the ~lanking DNA, acceptable insertion vehicles which have a loading site in a nonessential region and which have been demonstrated to be ef~ective can be isolated.
If it is desired to insert other foreign DNA into the reconstructed insertion vehicle, the loaded insertion vehicles can be unloaded by cleavage of the junctions between the insertional DNA and the intermpting DNA with the restriction enzyme described above which has restriction sites only within the insertional DNA. Alternatively, it may be desired ~o retain the foreign gene with its selec~ion marker and load the insertion vehicle wi~h nonselectable foreign DNA. In this case, it t~3~7 is preferred to eliminate from the insertion vehicle one of the restriction sites at the junctions between interrupting and insertional DIYA portions.
To do this, the insertion vehicle loaded with the selection marker (foreign DNA) is partially di~ested w;th the endonuclease active at the restriction sites on either side of the selection marker. Only enough enzyme is used to cleave about 30% of the vehicles at one site. The DNA is then purified and linear molecules the length of a circular vehicle tha$
has been cleaved at only one point are isolated by electrophoresis. DNA
polymerase is then employed to blunt the trailing ends ("sticky" ends) of ~he linear DNA molecules. This procedure destroys the restriction site. DNA molecules with the blunted ends are then ligated in a solution which has a very low concentration of DNA and a very high concentration of T4 DNA ligase to join linear molecules into a circular form. This mixture is then used to transf orm a host mioorganism and several transformants are chosen for further examination. Transformants containing acceptable insertion vehicies (i.e., an insertion vehicle with a selection marker between a insertional DNA portion and a single loading site) are identified by isolating plasmid DNA, digesting it with restriction enzymes, and subjecting the linear digestion products to electrophoresis to identify vehicles that have been cleaved at a single site.
The following examples are included for illustrative purposes only and are not intended to limit the scope of thls invention.

EXAMPLE I
The following example describes the cons~ruction of a loaded transformation vehicle suitable for the transformation of A. nidulans.
An A. nidulans strain (A. nidulans R-2 isolated by S. Y. Shestakov et al., (1970~ Molec. Gen. Genet. 107: 372-375) containing a ~ene within the chromosome capable of complimentin~ the ~hi-l mu~ation in E. coli was Iysed and DNA was isolated by dye-bouyant density centrifugation.
Plasmids pBR322 and pACYC184 were isolated by dye-bouyant density centrifugation of Iysozyme-sarkosyl Iysates of appropriate E. coli strains.
Plasmid p~R322 is a self-replicating E. coli plasmid which contains a selection marker which encodes resistance to ampicillin and is employed as the flanking DNA. The replicons in p~R322 and pACYC184 are not functional in A. nidulans. Plasmid pACYC1841 which encodes resistance to chloramphenicol, is employed as the Eoreign or interrupting DNA.
A mixture of broken plasmids and chromosomal DNA from A.
nidulans, at a concentration of 50 micrograms of DNA per rnilliliter was dl~ested with Sau3A resrtriction endonuclease wlder conditions specified by Bethesda Research Laboratories, Inc. (aRI ). The enzyme concentration, 6 units p~r milliliter, was chosen ~o provide Incomplete digestion a$ter a 30 minute incubation at 37C. To 5top the reaction, .080 ml of 0.25 M EDTA was added to the 2 ml reaction mlxture. The partially digested DNA was fractionated by electrophoresis through a 0.796 agarose gel and C)NA fragments 5000 base pairs and larger were recovered from the gel by electroelu~ion as described by Yang et al9 ~1979) Meth. EnzymoL 68: 17~182.
The purified A. nidulans DNA ragments were ligated to pBR322 DNA cJeaved beforehand with BamHI and treated with bacterial alkaline phosphatase. The 0.220 ml ligation mixture contained pBR322 DNA at a concentration of 15 ug/ml and A. nidulans DNA at 8.6 ug/ml. The ligatisn mix~ure was incubated at 14C for 12 hours under ligation conditions specified by Boehringer-Mannheim. The reaction was terminated by addition of Q011 ml of .25 M EDTA. The ligated DNA
was used to transform E. coli HB101 according to the method of Bolivar and Backman, (1979) Meth. EnzylmoL 68 245. A total of 5 x 10 Jndependent transformants resistan~ to ampicillin were obtained. C;reater than 99.9% of the transf ormants ~ontained chimeric plasmids with insertions of A. nidulans DNA. ~ mixed culture of the transformants -is designated as a "gene library" of A. nidulans DNA in E. coli.
E. coli HBIOI which contains the gene library plasmids, has a thi-I mutation that bloclcs the biosynthesis of thiamine. By plating the gene library culture on medium lacking thiamine, a chimeric plasmid designa~ed pKW1006 thi~ was identified.
Plasmid pKW1006 thi+ contained only one cleavage site for BamHI
located at one of the junctions between flanking DNA pBR322 and the cloned insertional DNA fragment from A. nidulans. To eliminate this cleavage si~e, the plasmid was digested exhaustively with BamHI and - ~2~

was used to transform E. coli HBl01 to ampicillin resistance. From one of ~he transformants7 a new plasmid, p~Wl034 thi~, was recovered which was identical to pKWl006 thi~ except for a deletion of about 700 base pairs bracke~ing She ~3arnHl cleava~e siteO The forei8n interrupting DNA, pACYCl84, had been cleaved with ~amHl and, therefore, could only be inserted at a BamHl cleava~e site in the insertional DNA. Since pKWl034 t + contained no BamHl cleavage sites in the insertional DNA, BamHl sites were created at various positions using BamHl linkers.
Plasmid pKWl034 thi~ was cleaved with Haelll (BRL) in a 5 ml reaction mixture containing 50 micrograms of the plasmid DNA, 0.38 units of Haelll and other ingredients as specified by BRL. Incubation was at 37bC for 60 minutes. The reaction was terminated by the addition of 0O300 ml of 0.25 M EDTA. The reaction mixture was extracted once with phenol, the volume was reduced to abGut 0.5 ml by extraction with n-butanol and the DNA was dialyzed against TE (10 mM Tris-HCl pH
7.5, 0.1 mM EDTA). Only 8% of the plasmids were cleaved by HaelII
as de$ermined by agarose gel electrophoresis. The partial digestt therefore, presumably consisted of a population of full length linear molecules having termini at varlous Haelll cleavage sites. BamHl linkers (CGGATCCG; BioLogicals) were added to the Haelll partial digest. The 0.36 ml reaction mixture contained 9 nl~lS pKW1034 thi ' DNA, 220 nM
BamHl linker, lû0 units of T~ DNA li~sase per millili$er (Boehringer-Mannheim1 and other ingredients as specified by Boehringer-Mannheim for ligation. lncubation was carried out at I~C ~or 15 hours. The reaction was terminated by the addition of .040 ml of .25 M EDTA, extracted once with phenol and the DNA was dialyzed a~ainst rE
~described above). Ei~ht micrograms of the dialyzed DNA was digested with BamHl (250 units per milliliter) in a voluFne of .200 ml under conditions specified by Boehringer-Mannheim. The reaction mixture was incubated at 37C for 4.5 l~urs, followed by l0 minutes at 65C. Then .020 ml of .25 M EDTA was added and the DNA was dialyzed against TE as described above.
To the dialyzed DNA that had been digested with BamHl (an unloaded insertion vehicle cleaved in the insertional DNA portion3, pACYCI84 was ligated. The pACYCl84 DNA (foreign or interruptin~

73~

DNA) had been cleaved beforehand with BamHI and treated with bacterial alkaline phosphatase. A 0.100 ml reaction mixture containing 2.7 micrograms of the pKW1034-derived insertion vehicle, .35 micrograms of pACYC184 DNA and 2 ~i~s of T4 DNA ligase was incubated for 17 hours at 15C. The DNA in the incubated mixture was used directly to transform E. coli HB101. A plasmid, designated pKW1039 thi- was recovered from the transformants resistan~ to both chloramphenicol and ampicillin. The pKW1039 thi- plasmid contained pACYC184 in ~he interrupting position and p~R322 in the flanking position. The plasmid was unable to compliment the E. coli thi-l mutation, presumably because the complimenting function was destroyed by the insertion of pACYC184 into the insertional DNA derived from A. nidulans DNA.
While plasmid pKW1039 thi is suitable for inserting pACYC:184 plasmid fragment into A. nidulan~ it may be desirable at times to insert only a portion of a foreign plasmid fragment. To demonstrate this, pACYC184 was digested exhaustively with Haell and the clevage products were fractioned by electrophoresis through a 1.4% agarose gel. The largest fragment, which was 1,270 base pairs long and contained the chloramphenicol resistance gene~ was purified according to the method of M. Albring et al., (1982) Anal. Biochem (in press~ which is described below.
A Haell digest of pACYC184 DNA (150 ug of DNA) was fractiona~ed by electrophoresis at 2 volts per cm for 15 hours through a 64 percent agarose gel (Low Melting Point agarose; BRL). The gel was stained with ethidiurn bromide, the gel portion (5 ml) containin~, the slowest migrating band of DNA (1.3 kb) was excised, and ~he agarose containing the DNA was dissolved by stirring for 15 min in 20 ml of 5~% urea (w/w) plus 5 g of urea crystals. All operations were performed at room temperature in siliconized glassware and centrifugations were at 2000 x g for 5 min. After the agarose was dissolved, the DNA was extracted from the agaro~e by adding 16 ml of DHA solution (40 ml. of n-butanol;
0.8 ml of ~lacial ~cetic acid; 4.6 ml o~ 2,2'-diethyldihexylamine from Eastman Kodak). The mixture was stirred vigorously for 5 min. The emulsion was centrifuged, the butanol phase (top) containing the DNA
was recovered and saved, and the aqueous phase was extracted again with 11 ml of DHA solution. The second butanol phase was pooled with the first to give a volume OI ca 30 ml. To extract the DNA from the butanol, the pool butanol phases were extracted with 6 ml of 1.25 M ammonium acetate for 5 min, the emulsion was centrifuged, the bottom aqueous phase was saved, and the upper phase was extracted again with 6 ml of 1.25 M ammonium acetate. The aqueous phases were pooled and were concentrated to about 0.4 ml by repeated extraction with n~butanol. The 0.4 ml sample was dialyzed against TE (described above).
To prepare flush termini, 5 micrograms o the purified fragment was incubated in a reaction mixture (.454 ml) con$aining ~0 mM Tris-HCI, 10 mM MgC12, 1 mM 2-mercaptoethanol, 10 micromolar each of dATP, dGTP, dCTP, dTTP, and 18 units of E. coli DNA polymerase I
large fragment (New England Bio. Labs) at a pH of 7.5 and a temperature of 37C for 4 hours, followed by I hour at lgC. The reactlon volume was increased to ~478 ml by the addition of .013 ml of 20 rnM ATP, .0045 ml 1 M dithiothreithol, .004 ml of 14 micromolar BamHI linkers, and 45 units of T4 DNA ligase. This mixture was incu~ated at 15~C
for 4 hours and terminated by the additlon of .027 ml of .25 M EDTA
and heating to 65C and holding that temperature for 10 minutes. The reaction mixture containing DNA t.505 ml) was mixed with .037 ml of water, .0056 ml of I M MgC12/ .012 ml of 1 M Tris-HCl (pH ~.0), .012 ml of 5 M NaCI, .0021 ml of 14.3 M 2-mercaptoethanol, .006 ml of bovine serum albumin (20 mg per ml held at 7~DC for 30 minutes) and 120 units of BamHI. This mixture was incubated at 37C for 14 hours and terminated by the addition of .040 ml of .25 M EDTA followed by holding the mixture at 65C for 10 minutes and dialyzing it against TE
(described above). Among the reaction products are DNA fragments encoding resistance to chloramphenicol and having single-stranded termini complementary to cleavage sites recognized by BamHl.
Plasmid pKW1039 thi at a concentration of 30 u~ml was digested exhaustively with BamHI, diluted ten-fold and ligated with T4 DNA ligase to remove pACYC184 from the interrupting position. This ligated DNA
was used to transform E. coli HBIOI and one transformant resistant to ampicillin and sensitive to chloramphenicol was recovered. Plasmid DNA

Lt~ 7 isolated from this transformant was designated pKW1048 thi-. The plasmid pKW1048 thi- contains a sin~le BamHI cleavage slte at the in$errupting position defined in the parental plasmid pKW1039 thi-.
Plasmid pKW1048 thl- was deaved with BamHI; cxtracted with phenol and dialyzed against TF (desibed above). The cleaved pKW1048 thi plasmid (3.6 ug/ml) was ligated to the 1,270 base pair fragment of pACYC184 (1.6 uglml) in a volume of .200 ml. The ligated DNA was used to transf~rm E. coli HB101 and from among the transformants, a new plasmid designated pK~1065 ~hi was recovered that specified resistance to both ampicillin and chlorarnphenicol. Plasmid pKW1065 thi contained paR322 in the flanking position and a 1,270 base pair ~ragment encoding chloramphenicol resistance in the interrupting position. The new plasmid was unable to complement the E. coli thi-l mutation.
A more simplified method of constructing the above loaded insertion vehicle for A. nidulans is as follows.
A. nidulans chromosomal DNA can be deaved with ~ II and fragments between 5~000 and 10,000 nucleotides lon~ isolated by electrophoresis. These fragments can then be cloned into the ~amHl site o~ the plasmid paR322. A partial homology between the cleava~e sites of B~lll and BamHl makes it possible to liga$e together DNA
fragments produced by these endonucleases. The hybrid junctlons are not cleaved by either of the endonucleases. The recombinant plasmid can then be used to transform a strain of E._coli to ampicillin resistance.
Milligram quantities of the plasmid DNA can then be isolated from a mixed culture of the transformed cells. Next, the mixed plasmids are screened for those having loading sites located in the insertional DNA
using the recipient organism (A nidulans) to screen. First, the mixture of recombinant plasmids would be cleaved with BamHI. This enzyme will only cleave within the insertional DNA. Plasmid pBR322 in the recombinant plasmid has no BamHI restriction sites; the junctions between the insertional and flanking DNA are not cleavable by BamHI. Next, ~he mixture of cleaved DNA can be ligated to the purified 1,270 nucleotide base pair fragment that encodes chloramphenicol resistance.
This fragment can be conveniently isolated by electrophoresis of a BamHI
di~est of the plasmid pKW1065 employed in Example 1. ~he mixture of ligated DNA will contain loaded insertion vehiclesO l he entire mixture can then be used to trans~orm a strain of E. coli. Transformants resistant to both ampicillin and chloramphenicol will contain various recombinant pla~mids in which the 1,270 base pair fragment 5s linked to insertional DNA. A preparative quantity of the mix$ure of recombinant plasmlds can then be isolated from the resistant E. coli cultures.
This entire mixture of plasmids can then be used So transform a culture OI A. nidulans. Type I trar sformants, those resistant to chloramphenicol but sensiSive to ampicillin, isolated. Chromosomal DNA
can be recovered from several different transformants and cleaved with ~11. The cleaved DNA can then be ligated to paR322 cleaved with BamHI. This ligated DNA can then be used to transform a culture of E. coli. Transformants resistant to both chloramphenicol and ampicillin will con$ain useful insertion vehicles.

This example describes the transformation OI A. nidulans with plasmid pKW1065 thi produced by the method of Example 1.
In the example below, cell concentrations were estimated spectroscopically. An absorbance of 0.25 at 730 nM corresponds to about I x lo8 cells per milliliter as determined by microscopic examination.
An actively growing culture of A. nidulans at a density of 3 x 108 cells per milliliter was diluted to 2.5 x 107 cells per milliliter in 400 ml of fresh BG-ll medium prepared according to Rippka et al. (1979) J.
Gen. Microbiol~ 61. The diluted culture, perfused with air at a rate o~ abGut 3 ml per minute, was incubated overnight at 37~C under 1,800 lux of "warm white" iluoressent light plus 100 lux from a 60 watt tungsten bulb. To sterilize the air, it was passed through a solution of 1% CuS04, a filter of activated charcoal (Gelman3 and two membrane filters with a .20 um pore size (Gelman). When the culture reached a density of 1.4 x 10~ cells per milliliter, J40 ml of cells were harvested by centrifugation at 5,000 x g for 15 minutes at room temperature.
The cell pellet was suspended in 20 ml of fresh BG-II medium aS a concentra~ion of I x 109 cells per milliliter.

3~ 73~

To 1.2 ml of competent cells $rom the fresh suspension in a 10 x 13 mm clear ~lass ~est tube was added .12 ug of pKW1065 thi-(from Example 1) in .006 ml of TE (as described in Example 1). The transformation mixture was incubated at 3PC for 10 hours under the li~hting conditions described above. The test tube was agitated intermitently to preven$ se$~1ir~ of the cells. Aliquots of ~he mixture were spread onto the surface of membrane filters (Nuclepore Membra-Fil filters) .45 um pore size, cut to 8 cm diameter~ resting on solid BG-ll medium in plastic petri plates. The solid medium was prepared by mixing equal volume of autoclaved BG-ll liguid ~2x concentrated) and autoclaved Difco Bacto Agar (3% in water). Membrane filters were first stçrilized by autoclaving in water. The cells on the fil~ers were incubated under the growth conditions described above for 20 hours and then the filters were transferred to solid medium containing either chloramphenicol (5 ug/ml) or ampicillln (0.2 ug/ml), or both. The plates were then incubated for 10 days and the numbers of each type of transformant were tabula$ed. It was found that the cells had a pla~ing efficiency of about 40%.
Three types of transformants were found. Type I transformants were the most common type and were resistant to chloramphenicol only.
Of the total cells in the transformation mixture, one cell in one thousand was a type I transformant. Out of each 266 transformants, however, 250 transformants were of type 1, 15 were of type II and 1 was of type III. As determined by Southern hybridization analysis, E. Southern, (1975) J. Mol. Biol. 98: 503-517, type I transformants contain a stably inserted sin~,le copy of the interruptin~ (foreign) DNA ~egment which ls inte~rated in the recipient chromosome at a site homologous to the position of interrupting DNA in pKW1065 thi-. After ~rowth cf type I transformants for 40 generations in the absence of chlorampherlicol, 948 cells were tested for chloramphenicol resistance. All of the cells tested exhibited chloramphenicol resistance, indica~ing that the foreign gene had been retained. The Type I transformants exhibited a mutant colony morpholo~y; they formed colonies of very small size. The rnutant phenotype is related ~o the location of interruptin~ DNA in the recipient chromosome (i.e., destruction of the thi-l complimenting function).

~z~ 37 - 23 _ Type 11 and type 111 transformants were also observed. Type 11 transformar,ts are those in which the flanking DNA has been inserted in the chromosome o~ the recipient organism. Type 11 transformants demons~rate a resistance to ampicillin, but are sensitive to chlorampheticol. Type 111 transformants are resistane to both chloramphenicol and ampicillin. A ~ype III transformant arises by the addi~ion to ~he chromosome of at least one copy of the loaded plasmid, including the foreign DNA, the flankir~ DNA and ~he insertlonal DNA.
After growth of type 11 transformants for 40 generations in the absence of ampicillin, 22% of the cells had lost the ampicillin resistant phenotype ~pecified by the foreign DNA. A possible explanation for this loss could be that in type 11 transformants, both the insertional DNA and ~he flanking DNA are added to the chromosome. This would lead to two homologous areas in the chromosome (the insertional DNA
and ~he chromosomal DNA to which it i5 homologous) which could lead to excision of the foreign DNA.
After growth of a type 111 ~ransformant for 40 generations in ehe absence of antibiotics, it was estimated that approximately 35% lost resistance to one or both antibiotics. As in the case of type 11 transformants, the addition of the loaded plasmid results in multiple homologous regior~ in the chromosome which would lead to excision of the foreign DNA.

The following example demonstrates the transformation of A.
nidulans with a linear insertion vehicle.
Plasmid pKW1065 th~ was cleaved with the restriction enzyme Hindlll. The plasmid contained one Hindlll site located within one portion of the insertional DNA and another site approximately 300 base pairs into the flanking DNA from the end of the other insestional DNA
portion. The deaved DNA was used to transform a culture of A.
nidulans in substantially the same manner as in Example ~. A 3-fold decrease in the number of stable type I transformants was observed vis-a-vis transformations with uncleaved pKW1065 thi- as in Example 1. No type 11 or 111 transformants were observed.

_ ~4 -Since modifications will be apparent to those skilled in the art, it is intended that the lnvention be limited only by the 5COpe of the appended claims.

Claims (27)

1. A procaryotic microorganism containing at least one stable foreign DNA portion covalently bonded directly to chromosomal DNA of said microorganism wherein said microorganism and its progeny are substantially free of genetic rearrangement involving said foreign DNA.

2. The microorganism of claim 1 that is a photo-synthetic microorganism.

3. The microorganism of claim 1 that is a cyanobacterium.

4. The microorganism of claim 3 that is a cyanobacterium that takes up exogenous DNA.

5. The microorganism of claim 4 that is selected from the group consisting of Gloeocapsa alpicola, Agmenellum quadruplicatum and Anacystis nidulans.

6. The microorganism of claim 4 that is Anacystis nidulans.

7. A pure culture of the microorganism of claim 1, 2 or 3.

8. A method for producing a procaryotic microorganism having at least one stable foreign DNA chromosomal portion that comprises:
(a) providing a DNA insertion vehicle containing first and second DNA portions containing DNA homologous to portions of a chromosome in said microorganism, said homologous DNA in said first and second DNA portions oriented in relation to each other in the same manner as said homologous chromosomal DNA portions in said micro-organism; and a third DNA portion containing DNA foreign to said microorganism, said third DNA portion located between and covalently bonded to said first and second DNA portions and;
(b) inserting said DNA insertion vehicle inside the cell membrane of said microorganism to effect incorporation of the genetic material of said foreign DNA into the chromosomal genome of said microorganisms.

9. The method of claim 8 wherein said insertion vehicle is circular and said insertion vehicle cannot autonomously replicate in said microorganism.

10. The method of claim 9 wherein said first, second and third DNA portions in said insertion vehicle are located in a first DNA segment and a second DNA segment containing DNA
that is not homologous to said microorganism is covalently bonded to the ends of said first DNA segment to form a circle.

11. The method of claim 10 wherein said second DNA segment in said circular vehicle contains a replicon functional in an organism other than said microorganism.

12. The method of claim 8 or 9 wherein said microorganism is a cyanobacterium.

13. The method of claim 10 or 11 wherein said microorganism is a cyanobacterium.

14. The method of claim 8 or 9 wherein said microorganism is a cyanobacterium selected from the group consisting of Anacystis nidulans, Gloeocapsa alpicola and Agmenellum quadruplicatum.

15. The method of claim 10 or 11 wherein said microorganism is selected from the group consisting of Anacystis nidulans, Gloeocapsa alpicola and Agmenellum quadruplicatum.

16. The method of claim 8 or 9 wherein said microorganism is Anacystis nidulans.

17. The method of claim 10 or 11 wherein said microorganism is Anacystis nidulans.

18. The method of claim 11 wherein said microorganism is a cyanobacterium and said replicon is functional in a bacterium.

19. The method of claim 18 wherein said cyanobacterium is Anacystis nidulans and said bacterium is E. coli.

20. A circular DNA insertion vehicle comprising:
(a) a first DNA segment comprising first and second DNA portions containing DNA homologous to adjacent portions of a chromosome in a procaryotic microorganism, said first and second DNA portions oriented in relation to each other in the same manner as said homologous chromosomal DNA
portions in said microorganism; and a third DNA
portion containing DNA foreign to said microorganism that expresses a selectable phenotype located between and covalently bonded to said first and second DNA portions, and a single restriction site in said first DNA segment for a particular restriction enzyme at a location nonessential to said expressable phenotype between said first and second DNA portions; and (b) a second DNA segment containing a DNA portion that is not homologous to the chromosomal DNA in said microorganism.

21. The insertion vehicle of claim 20 wherein said second DNA segment contains replicons which are functional only in an organism other than said microorganism.

22. The insertion vehicle of claim 21 wherein said first and second DNA portions in said first DNA segment are derived from a single piece of chromosomal DNA.

23. The insertion vehicle of claim 21 or 22 wherein said microorganism is a cyanobacterium.

24. The insertion vehicle of claim 21 or 22 wherein said microorganism is a cyanobacterium selected from the group of Anacystis nidulans, Gloeocapsa alpicola and Agmenellum quadruplicatum.

25. The insertion vehicle of claim 21 or 22 wherein said microorganism is Anacystis nidulans.

26. The insertion vehicle of claim 21 or 22 wherein said microorganism is a cyanobacterium and said replicon is functional in a bacterium.

27. The insertion vehicle of claim 21 or 22 wherein said microorganism is Anacystis nidulans and said replicon is functional in a bacterium E. coli.