JPS6181791A - Combined vector - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to genetically engineered cells that have desired properties, such as the ability to produce desired compounds.

In producing organisms with properties imparted by genetic engineering, it is desirable to integrate all genetically engineered genes into the chromosomes of cells.

Modified copies of genes present on extrachromosomal elements (plasmids) tend to mutate or disappear from the cell during cell proliferation, reducing the yield of the product. In comparison, chromosomal DNA tends to be retained reliably during replication, so that genetically engineered markings of genes present on chromosomes are stably retained in the progeny of genetically engineered cells. Ru.

Genetically engineered DNA is generally cloned when the vehicle is capable of self-replication in the cell.
Since it is not integrated into the chromosome of the host cell, the ultimate host for the engineered gene must be one in which the vehicle cannot replicate. Therefore, methods for altering genes present in chromosomes are generally performed using both hosts. A cloning vehicle with genetically engineered genes? The first type of host cell in which it can replicate is obtained. Then for incorporation into cell staining algae,
Transfer this to the second tights host.

Usually, the DNA introduced into the second host is unable to replicate because it lacks a suitable replication origin or because the second host is unable to initiate plasmid replication. One such situation is Escherichia coli (E, Co11), a known host with favorable optical properties for turasmid manipulation.
) and different types of microorganisms (e.g., yeast or Bacillus subtilis) which cannot cause replication of E. coli plasmids.
) plasmids that are intended to be integrated into the chromosome. For example, Haldenpunk (Haldenwan)
g) et al. C1980) J, Bact-eriology
l 42 (1): 90-98 Sierra (5ch
(1979) Proc, Hat'
l, Acad.

Sci, USA 76(to):4951-4955k
> Teru.

If the entire genetically engineered gene is to be integrated into the chromosome of a single embryo of the same type as that used as the first host, other measures are required, such as mutating the second host. , the filling should make it impossible to duplicate plasmid DNA (eg, Gutterson and K.
oshland) “Replacement and amplification of bacterial genes by in vitro modified sequences”, P.
roc, Nat (1, Acad, 5ci1Vo1
．． 80, pp. 4894-4898. August 1983.

Generally, the present invention relates to vectors used as cloning vectors for genetically engineered genes intended to integrate into bacterial chromosomes. The vectors of the invention contain replication-specific sequences, ie, sequences that are capable of autonomous replication and are stably maintained in bacterial host cells.

The vector also contains restriction/donuclease cleavage sites that flank the replication-specific sequences, so that the replication-specific sequences can be conveniently excised. Additionally, the vectors of the invention also have genetically engineered DNA sequences that integrate into the bacterial host chromosome.

By separating replication-specific sequences, genetically engineered DNA sequences can integrate into the chromosomes of bacterial host cells of the same type as those used for self-replication, and Stable maintenance is also possible, thus allowing the genetically engineered sequences to replicate as part of the bacterial chromosome. This vector further provides a means for detecting all bacterial cells that have genetically engineered sequences on their chromosomes.
Contains A sequence.

A second aspect of the present invention is a precursor of the vector.

The precursor lacks all genetically engineered sequences and has cleavage sites recognized by restriction endonucleases that do not recognize any intervening sites of replication-specific sequences.

In the third aspect, the present cry is a genetically produced DNA.
The NQ'1 method is aimed at replicating the A sequence extrachromosomally in one type of bacterial host cell and then integrating into the chromosome of the same type of bacterial cell. The steps of this method consist of providing the vector in a bacterial host cell, excising all replication-specific sequences from this genetically engineered construct, and recircularizing the remaining DNA. The remaining DNA allows bacterial J
The cells are transformed into host cells, and the genetically engineered constructs integrate into the cell chromosome by total recombination, replacing the chromosomal DNA sequence.

In a preferred embodiment, the replication-specific sequence is a plasmid-derived origin of replication; the host Δdj bubble is, for example, E. coli and the replication-specific sequence is the origin of replication of pBR322. The DNA sequences that provide a means to detect all bacterial cells carrying the genetically engineered sequences are sequences that confer resistance to antibiotics. It is also preferred that at least some of the steps involved in genetically engineering the DNA sequence are performed on the vector while it is maintained in the host bacterial cell. Genetically engineered DNA has flanking sequences at both ends, and these flanking sequences are identical in position to each positioning sequence at both ends of the chromosomal DNA to be located.

Furthermore, in a preferred embodiment, the vectors of the invention contain a selectable marker between the first and second restriction enzyme cleavage sites, which can indicate the presence of replication-specific sequences and thus have the ability to replicate on-integrators. allows for differentiation from cells with extrachromosomal plasmids. Alone or in combination with the selection markers described above, the replication-specific sequences and other DNA to be separated are positioned to divide the gene encoding one selection marker into two non-functional fragments, and these non-functional A functional fragment becomes functional upon cleavage of said fragment. In this way, when the replication-incompetent DNA is integrated into the host chromosome, the second selection marker is expressed.

Therefore, by selecting based on the presence of the second selection marker, it is possible to remotely separate cells that have undergone the desired integration. Most preferably, the DNA to be integrated has a marker that is selectable and the same as said second selection marker, such as tetracycline resistance, so that once colonies exhibiting the property have been established, the property can be reversed. Microorganisms from which marker genes have been separated can be isolated by selection. This simplifies the method of identifying the final target cells, since the identification will be performed with a population containing a high percentage of the desired cells.

The method of the invention is particularly used to integrate a genetically engineered gene encoding a desired protein into a chromosome and cause a second host to produce the desired protein; or to remove a gene present in the chromosome from a bacterial cell. , two separate DNA sequences in the correct position, one naturally located 5' to the gene to be removed and the other 3' to the gene to be removed.
It can also be used to construct hybrid DNA consisting of (naturally occurring in The DNA molecule can be inserted into a vector, from which the origin of replication can be conveniently excised. After cutting out the origin of replication from the genetically engineered construct, the remaining DNA'z is circularized again and newly obtained Genetically engineered copies can be integrated at or near the targeted gene locus.

Modification of genes present in the chromosome ensures that the modification is stably maintained in the progeny of the genetically engineered cells. The use of vectors that are prepared and stably maintained in the same type of host cell as the one that will ultimately carry the alterations present in the chromosomes poses problems inherent in systems using two different types of hosts, e.g. , restriction modification system disorder (re) between heterologous cell types.
restriction modification s
system barrier) f to overcome. The vectors of the present invention and their methods of use simplify the modification of genes present in chromosomes and expand the scope of such methods.

In general, the vectors of the invention are vectors of the present invention that contain an origin of replication flanked on both sides by restriction endonuclease sites (e.g., pKB701 in Figure 2 or pKB701 shown in Figure 44).
843). These two restriction endonuclease sites may be sites recognized by the same or different enzymes. Outside the replication-specific sequence there is a cleavage site for insertion of the DNA sequence to be genetically engineered. In a preferred embodiment, this restriction endonuclease cleavage site is recognized by an enzyme that does not recognize either of the first two sites. The vector also contains a DNA sequence or sequence segments which together encode the property of resistance to antibiotics and thereby provide a means for detecting bacterial cells having the genetically engineered sequences in their chromosomes. is given.

The structures of two specific examples of such precursors C'1) KB701 and pKBF343) are explained below with a description of their preparation.

Precursor Production The following description of the construction of pKB701 and pKB843 describes the method of vector production; to the extent not specifically described herein, for example, Maniatis, MolecuL
Standard recombinant DNA techniques were used as described in CLONING C. Cold Spring Harbor Laboratory (IJ-S. 1982).

To produce the vector pKB701c (see Figure 2),
A pBR322 derivative was prepared in which -Kpn (linker) was inserted at the site adjacent to the replication origin and sandwiching this. This was carried out by partial digestion with 3iy and u3A on the side of the replication origin adjacent to the beta-lactamase gene. First, H was inserted between the beta-lactamase gene and the starting point.
ind [[IJ / car inserted. Next, this Hi
nd III site using a J(pnl') linker.
It was converted to pn I VA position. Similarly, pBR32
The Tt part of 2 was converted into a Kpni site using KpnIIJ:/car. This manufacturing recipe allows the Ncoi site (Kpnl linker and Tthllll
1 site) and an Nde 1 site (between two adjacent KpnII) anchors that appear in the structure). Two linker inserts were incorporated onto one plasmid, resulting in a construct containing Ndel and EcoRI. As a result, a small amount of components between the Ndel and Tthll I sites of pBR322 are present in the final vector ~
lost from. Hind m! Neither the original insert nor the deletion of these minor components, including the loss of minor components, affects the usefulness of the vector.

A second precursor vector, pKB843, was produced from the elements shown in Figure 4 and contains certain improvements that make its use more effective. Details of the elements of pKB843 are revealed from the structural description below. Simply put, p
KB843 contains two selection markers that aid in the selection of cells that have undergone the desired integration. When using pKB701, the origin of replication must be completely deleted; otherwise, it is not possible to use a selective marker to distinguish between colonies where integration has occurred and colonies where the marker is present on an extrachromosomal element. It would be impossible.

Plasmid pKB843 overcomes this problem, as described below.

Plasmid pKB843 carries the gene encoding the selected OT disease phenotype on a segment that is deleted along with the collar start. As shown in Figure 42, pKB843 is the origin of replication from piece BVCpBR322, and αr
Contains rtpR. Therefore, the presence of ampicillin resistance suggests that extrachromosomal plasmid pKB843 is present along with its origin of replication.

Plasmid pKB843 was selected for the second selection OfE phenotype (
It also contains genes encoding tetracycline resistance). This gene has been split into two inactive fragments from piece BK. Once the origin of replication is deleted and the resulting vector is integrated, the two fragments are fused to produce a functional tet gene, which is used to distinguish integrants from cells harboring plasmids carrying the origin of replication. be able to. As described below, one can also be used to reverse sort against tel to isolate cells that have undergone detachment.

Plasmid pKB843 also contains suitable restriction endonuclease cleavage sites for the insertion of sequences to be integrated into the host chromosome; for example, EcoR (FIG. 4).

To produce pKB843, the three separate segments shown in Figure 4 were prepared and the recombinant D
Connect together using NA manipulation techniques.

Piece A is a segment containing the 5' portion of the tet cryogene, e.g. pBE322, with EcoRl and BamH.
This is a 375 bp fragment obtained by digestion with I.

The piece Ev'1 is a 2.1.4 kb segment of pER322, which includes the region from Tthll to EcoRr, and has a DNA' segment with a similar function to the replication origin and the Anpinli7 injection tolerance gene. The segments are EcoRl and Tthll
The ends of the segment are linked by linkers, first to the Kpnl end and then to the f3 end.
amHI end.

Piece C is Ba adjacent to the 3' part of the tel gene.
mH (any suitable area that covers the part, piece A
tetR gene to generate a functional tetR gene. p
A suitable fragment or precursor of BR322, e.g.
A fragment of BamH [from pME9 CATCC37019] in which the H7)α site was replaced with EcoRIVCK by a linker can be used.

Plasmids pKB701 and pKB84, lj: deposited in E. coli YMC9 at the American Tissue-Type Culture Collection/No. 39772, respectively.
and 53181. The term of these deposits is such that they may be distributed to persons authorized by the depository under 37 CFRl. Ru. Applicant and his successors shall transfer the deposit to comply with the requirements of the Budapest Treaty.

Effects of the Invention In general, the main body vector is used as follows;
In FIGS. 1α and 1b, a DNA sequence A is inserted into the entity vector before carrying an origin of replication RO and a marker M to confer antibiotic resistance and identify cells carrying the vector. Insertion of sequence A into the precursor is made possible by the presence of a cleavage site on the precursor that is recognized by an enzyme that does not recognize any sites that sandwich the origin of replication.

Sequence A may be any sequence that can be genetically engineered into sequence A' for integration into the chromosome, e.g., an enzyme that catalyzes a reaction in a pathway to a necessary protein or desired product. It may be a combination of a structural gene encoding the gene and appropriate regulatory sequences for the expression of that gene. Sequence A has flanking regions at each end thereof, which are homologous and in the same orientation as the corresponding regions of the host chromosome sequence Ac that are intended to be replaced by sequence A'. These flanking regions need to be sufficiently long to allow integration of sequence A' into the chromosome by recombination events. Specifically, the efficiency of introducing structure A' into the chromosome increases as the size of the homologous lateral tilt increases.

In Figure 1c, the DNA sequence is modified by genetic engineering to produce a desired change in sequence A.
Genetically engineered to change to ’. In Fig. Lc, the vector containing the modified sequence A' can autonomously replicate in host cells and is stably maintained because of the presence of the replication origin RO on the vector. At all stages, vectors and genes that are genetically engineered are
It can ultimately be maintained in cells of the same type as those aimed at changing its chromosomes.

To achieve integration of the modified sequence into the chromosome of the host cell, a plasmid is isolated. The origin of replication RO is then excised by restriction endonuclease digestion (Figure 1d).
, separated from the rest of the DNA, and the remainder is treated with DNA ligase to recircularize.

The vector is taken up into the host cell and the vector/
'i+Ia' integrates at or near the sequence Ac (for which the vector has a genetically engineered copy A') of the chromosome (FIG. 1e). Multiple #
Only structures that have lost their starting point are integrated into the cell chromosome. The ratio of changes produced as a result of integration (A chromosome to wild type (Ac) chromosomes) is greatest when the sizes of the flanking regions are generally equal to each other; This is because it is convenient to equalize the recombination rate.

In Figure 1f, when a cross-recombination event occurs, the modified sequence A' is present in the cell chromosome and the native sequence Ac is present in the vector (Figure 1g).

The construction and use of a specific vector (pKB719) illustrates the use of the above method for the deletion of genes present in the chromosome. In FIG. 3, the E. coli phe A gene was cloned and used to obtain E. coli in which the entire chromosomal sequence including the pheA structural gene was deleted. phe
A small piece of DNA containing components from both sides of the A gene was constructed. Hinc from EcoRi as one side
II and Ba as the other side.
The area from nl to Xmn ■ was used. These regions were combined to form pl (B2O2 EcoRI and Pvu
pKB719 was obtained by cloning between the 11 sites.

pKB719fKpnl was digested, and the resulting fragments were separated by polyacrylamide gel electrophoresis. The large fragment carrying the genetically engineered phe A deletion was circularized using DNA ligase and transformed into E. coli ston YMC9CATCC33927). Ampicillin-resistant transformants were selected. This is p
Transformation of YMC9 with BR322 occurred at a 100- to 1000-fold lower rate. Transformants were analyzed for the presence of plasmid DNA. If the prepared DNA fragment is mixed with the original fragment or incompletely digested pKB719, the resulting transformant will have a plasmid identical to pKB719. The frequency of recovery of pKE719-form pJIr conversion was determined by converting circularized DNA into restriction endonuclease Ndel.
(which linearizes DNA molecules with TFI replication origin fragments) causes a sharp decrease. Several ampicillin-resistant isolates were identified in which the presence of the plasmid was not detected, and cultures of these isolates were enriched for phenylalanine autotrophs (mutants) by cycloserine enhancement. Such phenylalanine autotrophs simultaneously lost their ampinrin resistance, suggesting that these were generated by the mechanism described by the aLf diagram.

The above problems regarding the presence of transformants with the same plasmid as pKB719 or other plasmids with the αmpR selection marker on the replication OT-competent extrachromosomal element can be overcome by using the pKB843-derived plasmid. The issue was resolved due to improvements in the selection mechanism for microorganisms affected by the phenomenon. After inserting the desired chromosome-inserted fragment into pKE843, and after the start point has been deleted as described above, the resulting plasmid is inserted into a host microorganism and tetracyclyclytic/resistant transformants are selected. do. It has a Schiffglu copy of tet. A suitable amount of tetracycline for the cell is used, most preferably about 3 μmμ.

The use of the cLrrLp marker allows selected transformants to be characterized by an integrated tel gene;
This is to ensure that the original fragment is not characterized by the tet gene on the replication-competent plasmid (the original fragment joins the two replication-inactive fragments, resulting in a replication-competent plasmid carrying the let gene). may occur in rare cases).

Finally, from the tetracytalline resistant clones, subclones can be obtained which have undergone excision of the tetracycline gene and thus also caused the desired excision of the homologous portion of the original chromosome (Ac in Figure 5), or, for example, pKB.
It is possible to select those that have undergone undesired cleavage of the homologous segment A' derived from 843. tet
For example, Bochner
) et al. (1980) J, Bacteriologi'l
143:926-933.

By selecting a population highly enriched in cells that have undergone the desired cleavage, the identification of such cells can be greatly facilitated, for example by screening or sorting for phenotypic properties that emerge as a result of the cleavage; Or whole cell D
A simple screen showing the correct structure can also be used, such as restriction/donuclease digestion of NA followed by Southern blot analysis.

Figure 5 shows a separable reproduction-determining DNA segment (R
O) into two parts (ML and MR respectively in Fig.
) generally describes the use of a vector having a first marker gene that is split into two. The replication-determined segment also has a second marker gene, M2. In other respects, the labels are similar to those described above with respect to FIG. 1 of FIG. The process is the same, except that p
For example, the selection operation described above for KE843 is performed in step 5.
f to determine the absence of M2 and the presence of MLR, and in step 5 a selection against MLH was performed.

Further embodiments are set out in the claims. For example, the method of the invention can be used to insert genetically engineered genes encoding the production of any protein.

[Brief explanation of drawings]

Figures 1α to 1h are flowcharts of the steps of a method for introducing genetically engineered genes into the chromosomal DNA of bacterial cells.
Figure 2 is a schematic diagram of vector pKE701, showing its restriction enzyme/donuclease site;
FIG. 4 is a schematic diagram showing the structure of pKB843; FIG. 5α to FIG. 1 is a flowchart of the process of the method. Patent attorney Kyozo Yuasa, -:f (5 others) Jokichi of the drawings (no changes to the content) A1 IG 4 Procedures Amendment Written by the Patent Office on the 2nd day of April 1939 Government Uga Michi Number of copies 1 Indication of the case 1927 Patent Application No. 10267 2 Title of the invention 8 Tachikeni 56be 2/- 6 Person making the amendment Relationship with the case Patent applicant address 2 Ki l :'+λtef?-p・in71-rshi7cho、shi・i・corposhi-fu7F゛4、agent

Claims (1)

[Scope of Claims] (1) A replication-specific sequence; a first cleavage site adjacent to one end of the replication-specific sequence and recognized by a restriction endonuclease; the other end of the replication-specific sequence a second cleavage site adjacent to and recognized by a restriction endonuclease; said first and second sites are the only sites on said vector that are recognized by a restriction endonuclease that recognizes said first or second cleavage site; a genetically engineered DNA sequence to be integrated into the chromosome of the bacterial cell; and means for detecting bacterial cells having the genetically engineered sequence; in the chromosome of a bacterial cell of the same type as the cell in which the vector is capable of stable maintenance and self-replication of the treated sequence and, by cleavage of the replication-specific sequence, the vector can be stably maintained. A vector characterized in that a genetically engineered sequence is integrated, and the genetically engineered sequence is then capable of replicating as part of the chromosome. (2) The vector according to claim 1, wherein the restriction endonuclease that recognizes the first cleavage site is the same restriction endonuclease that recognizes the second cleavage site. (3) The vector according to claim 1, wherein the detection means is a gene that confers resistance to antibiotics. (4) The vector according to claim 1, wherein the bacterial cell in which the vector can replicate is Escherichia coli. (5) The vector according to claim 4, wherein the replication-specific sequence is the replication initiation site of pBR322. (6) The genetically engineered sequence has a first side DNA sequence at one end and a second side DNA sequence at the other end.
NA sequence, the bacterial chromosome contains a DNA sequence to be replaced, and the DNA to be replaced
Claim 1, wherein the sequence has a sequence homologous to the first side DNA sequence at one end thereof and a DNA sequence homologous to the second side DNA sequence at the other end. Vectors listed. (7) The chromosomal sequence to be replaced includes a gene encoding protein expression, and the genetically engineered sequence includes a genetically engineered gene from the chromosomal gene. vector. (8) The chromosomal sequence to be replaced includes a gene flanked by a homologous chromosomal sequence on one side and a second homologous chromosomal sequence on the other side, and the genetically engineered gene is substantially 7. The vector according to claim 6, comprising first and second flanking sequences. (9) The vector according to claim 1, wherein the genetically engineered sequence contains a structural gene encoding the protein to be produced. (10) The vector has a marker encoding a selectable expression characteristic for marking the presence of a replication-specific sequence between the first restriction cleavage site and the second restriction cleavage site. Vector described in Section (11) The vector contains a marker gene encoding a selectable expression characteristic,
The gene is divided into two non-functional fragments by a portion of the vector between the first and second restriction cleavage sites, and the gene is expressed when said portion is separated. The vector described in Section 1. (12) The vector according to claim 10 or 11, wherein the expression characteristic is selected for its presence or absence. (13) The vector according to claim 12, wherein the expression characteristic is tetracycline resistance. (14) The vector according to claim 10 or 11, wherein the expression characteristic is ampicillin resistance. (15) Vector is pKB701, ATCC deposit number N
o. 39772, or pKB843, ATCC Deposit No. 53181. (16) [1] D genetically engineered onto the vector according to claim 1 in a first bacterial cell.
[2] exposing the vector to a restriction endonuclease;
and removing the DNA segment containing the replication-specific sequence, [3] recircularizing the remaining DNA, and [4] transforming a second bacterial cell of the same type as the first cell with the recircularized remaining DNA. and introducing the genetically engineered DNA into the chromosome of the cell by recombination to replace the target segment of the chromosomal DNA,
and [5] identifying progeny of the transformed cell in which the genetically engineered DNA has been integrated into the chromosome; A method of replacing segments of chromosomal DNA. (17) The method according to claim 16, wherein at least a part of the artificial genetic engineering treatment of the genetically engineered DNA is performed in the first bacterial cell. (18) The method of claim 16, wherein the transformed bacterial cell progeny are identified by antibiotic resistance conferred by the presence of the vector. (19) The method according to claim 16, wherein the bacterial cells are of the species E. coli. (20) [1] In the natural state, one part of the DNA sequence exists immediately on the 5' side of the gene to be deleted, and the other part normally exists on the 3' side of the gene. Two separate DNA sequences are linked in the correct order. [2] inserting the hybrid DNA molecule into a vector from which the DNA sequences necessary for replication can be readily excised; [3] The sequences necessary for replication are separated from the vector and the remaining DNA is
[4] transforming a bacterial cell with said circularized residual DNA, introducing said hybrid DNA molecule by integration into the chromosome of said cell, thereby replacing the gene present in said chromosome, and [5] A patent for specifically deleting a gene present on a chromosome from a bacterial cell by identifying progeny of said cell that has integrated said hybrid DNA molecule into the cell chromosome in place of the replaced gene. The method according to claim 16. (21) The genetically engineered sequence on the vector has a structural sequence encoding a protein, and the desired protein is produced by culturing a second bacterial cell or its progeny to obtain the protein. A method according to claim 16. (22) a replication-specific sequence; a first cleavage site adjacent to one end of said replication-specific sequence and recognized by a restriction endonuclease; a first cleavage site adjacent to the other end of said replication-specific sequence and recognized by a restriction endonuclease; a second cleavage site recognized by an endonuclease; and at least one cleavage site recognized by a third restriction endonuclease that is external to said replication-specific sequence and does not recognize said first or second cleavage site; The first and second cleavage sites are the only sites on the vector that are recognized by a restriction endonuclease that recognizes the first or second cleavage site; Precursor vector for the vector. (23) pKB701, ATCC deposit no. 397
72, pKB843, ATCC Deposit No. 5318
23. The vector according to claim 22, which is 1 or a derivative thereof.