POSITIVE SELECTION SYSTEM FOR STABILISATION OF EXPRESSION VECTORS

Abstract: The invention relates to a method for the construction of plasmids assuring the insertion of a nucleotide sequence into a chromosome and the simple separation of clones containing the transformed chromosome by using methods of genetic engineering known per se which comprises inserting a copy of a chromosomal segment corresponding to a chromosomal segment of the host cell between the transcription regulating sequences capable of expressing foreign gene into a plasmid containing transcription regulating sequences capable of expressing foreign gene and a resistance gene (R2) expressed independently from the said regulating sequences, then downstream inserting a resistance gene without own promoter (R1) different from (R2), and finally inserting a sequence containing at least one transcription terminator into the inserted copy of the chromosomal segment.

Description: POSITIVE SELECTION SYSTEM FOR STABILISATION OF EXPRESSION VECTORS This invention relates to a method for the construction of plasmids assuring insertion of any nucleotide sequence into a chromosome and simple separation of clones containing the transformed chromosome. The plasmids (expression vectors) prepared according to the method of the present invention extraordinarily facilitate the insertion of any DNA fragment into a selected gene on the chromosome of the host cell. With the insertion of the DNA fragment the gene on the chromosome is inactivated at once. The method is based on the homologuous recombination mechanism of the bacterial cell providing an advantage whereby a positive selection system can be created which makes possible to 7 R simultaneously test 10 -10 bacterial cells and to immediately select the recombiπaπt clone to be created. The well known method for inactivating the genes on the chromosome is also based on the homologuous recombination (Ruvkun, G.B. et al.: A general method for site-directed mutagenesis in procaryotes, Nature, 289, 85 (1981); Lee, CA. et al . : Use of cloned mtl genes of Escherichia coli to introduce tl deletion mutations into the chromosome, I.Bact. 153, 685 (1983); Jasin, M. et al.: Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments, 3 . Bact. , 139., 783 (1984); Virans, S.C. et al.: Site directed insertation and deletion mutagenesis with cloned fragments in Escherichia coli, 3. Bact., 161_, 1219 (1985)) it is, however, much more expensive and time consuming than the method according to the present invention. According to the known method, the gene to be inactivated was first cloned into a plasmid resistant to an antibiotic (further on R, resistance), then a DNA region containing another antibiotic resistance (further on R ? resistance) gene - genεraly a Tn5 transposon - was introduced into the copy of the gene coded on the plasmid. The cell was then transformed with another plasmid which is incompatible with the previous plasmid and contains a third resistance (further on R-,) gene. Cultivating the traπsfomed cells on a medium containing R and R-, antibiotics and testing the single colonies to R, resistance, there could be isolated R« and R., resistant R, sensitive clones involving the recombinants sought for. This method is very time consuming even in the case of testing some thousand clones. Moreover, if the gene to be inactivated is essential for the bacterium, this approach is applicable only in an enlarged form. Namely, an intact functional copy of the gene must be present in the cell. This can be introduced most simply into the cell by a further plasmid which is compatible with the second plasmid, but in this case the gene copy on the plasmid can also be the target of the double recombination. In this case the proper clone must be selected from a large number of false positive clones that makes the method even more expensive and time consuming. The method according to the present invention can also be used for introducing heterologuous genes into the chromosome. So far this was achieved by expression vectors based on lysogeπ phage (Molecular cloning of the T. genome - organization and expiration of the FRD-DNA ligase reaction, Mol. Gen. Genet. , r7_9. , (2), 227-239 (1980)). When using these vectors, however, the location of the insertion can not be chosen arbitrarily, there are strict limitations. In the known methods - if also the homologuous recombination was used for the integration - in the contrary to our method, the desired recombinant could be selected only by serious difficulties or the integration was even not possible to certain spots of the chromosome. Our aim was to develop a system that makes substantially easier the selection of the clones containing the properly transformed (recombinant) chromosome, furthermore, whereby changes can be performed on any site of the chromosome. The above problem could be solved by developing a new plasmid construction. It is therefore an object of the present invention to provide method for constructing plasmids assuring the insertion of any nucleotide sequence into the chromosome and the simple separation of the clones containing the transformed chromosome by using methods of genetic engineering known per se during the construction of the plasmids, which comprises introducing a copy of a chromosomal segment corresponding to a chromosomal segment of the host cell into a plasmid, between the transcription regulating sequences capable for expressing foreign gene, said plasmid containing transcription regulation sequences capable for expressing any foreign gene and a resistance gene (further on R ? ) expressed independently from the said regulating sequences, then downstream introducing a resistance gene without its own promoter (further on R,) differing from R„, finally, introducing a sequence containing at least one transcription terminator into the inserted copy of the chromosomal segment. The desired chromosome transformation can be performed easily by the plasmids according to the present invention and the clones containing the transformed chromosomes can be simply separated by cultivating the clones starting from the cells containing the plasmid according to the present invention on a medium containing an antibiotic corresponding to gene R 2 for 10-20 hours, then spreading the colonies grown out on a medium containing an antibiotic corresponding to gene R, , finally, separating and further cultivating the living colonies. The positive selection method according to the present invetion makes possible to screen 3-4 order of magnitudes more clones than those by the know methods based on negative selection. A further advantage provided by the present invention is that the intact copy of the gene transformed on the chromosome shouldn't be separately introduced into the cell even in the case of a gene of vital importance, since it is formed automatically on the plasmid at the moment of the double recombination event . The method according to the present invention can be widely used. The most important applications are shortly summarized as follows. 1. Targeted introduction of any DNA fragment into the chromosome of the host cell In contrary to the known methods, the possibilites concerning the location of introduction are little limited. When selecting the chromosomal segment to be inserted into the plasmid according the present invention, care should be only taken to the fact that it may not contain any transcription terminator. It has to be noted that according to the method of the present invention - similarly to the methods so far known - DNA fragments can be introduced into genes of vital importance complementable only in trans position. 2. Stabilisation of a plasmid by damaging a gene of vital importance The great advantage of the plasmid construction according to the present invention is making possible, on the plasmid, the formation of the copy of the gene damaged on " the chromosome after the double recombination event, thus the plasmid can not be lost from the cell, since the loss of the plasmid should result in ceasing the essential gene function. Thus, any plasmids, for example expression vectors can be stabilized against loss of plasmid by utilizing our method. This is a result of greatest economical importance coming from our method, since it makes unnecessary the expensive antibiotic selection during the fermentation. 3. Investigation of the events following the loss of gene function According to the method of the present invention artifical operons containing a regulatable promoter can also be constructed. In this case the gene function in question becomes regulatable. For example, if the cell is put into conditions where the promoter does not work, the events immediately following the loss of the gene function of vital importance can be investigated. Thus, the method according to the present invention can be used for both inactivating a chromosomal gene and making a gene function regulatable. The main steps of the method for preparing plasmids of new construction and the method for selecting the suitably transformed clones are outlined below. In the first step, an artificial bacterial operon consisting of two genes (A and R, in Fig. 1) is constructed on a plasmid using the method of in vitro DNA recombination. According to construction of Fig. 1 the promoter "Pr" controls the synthesis of a mRNA with two cistrons (A and R,) from which the R, antibiotic resistance gene can be expressed without difficulty, thus, the bacterial cell transformed by the plasmid becomes resistant to both antibiotics (R, and R«). In the second step, the gene "B" to be integrated is cloned into the target sequence "A" followed by a terminator sequence T (Fig. 2). (In special cases cloning merely the terminator sequence "T" is enough.) The cell transformed with the Plasmid/2 according to Fig.2, becomes sensitive to R, antibiotic, since the transcription starting from promoter Pr will stop at the terminator T. The plasmid can be maintained with antibiotic R 2 - If the double recombination shown in Figures 3a and 3b occurs DNA sequence "B" together with the terminator integrates into sequence "A" on the chromosome, the state according to Fig. 1 reforms on the plasmid and resistance R, is expressed again. Accordingly, the occurrence of the double recombination event can easily be traced. " The rec + (able to reco binate) clone containing the intermediate plasmid (plasmid/2) according to Fig2, suitably an E.coli strain, is grown on a medium containing antibiotic R„, then 150-200 ,ul of overnight culture is spread on a medium containing antibiotic R, . On the plate containing antibiotic R, only the R-, resistant clones resulting from the desired double recombination grow. The frequency of the double recombination depends on the length of the segment of sequence A preceeding and following, respectively, the insert "B+T". If both regions have a length of more than 300-400 basepairs, the frequency of recombination is _ 10 ~ /cell, according to literature data. 200,ul of overnight culture contains about ID cells, thus, according to a rough estimate, about 100 positive clones grow on one plate. Further details of our invention are shown in the following examples, without limiting the scope of the invention to the facts described in the examples. Example 1 Construction of the plasmid A restriction fragment TaqI with 750 nucleotides originating from plasmid pBR329 (MNG 0029 " 9), containing the CAT gene without its own promoter was cloned to the unique Clal restriction site of plasmid pER VI/23 PLH4 (NCAIM B (P)001016) . The clones containing the plasmid were resistant to chloramphenicol (Intermediate I). The resulting plasmid was digested with Xbal enzyme, then about 20-20 nucleotides were eliminated in both directions from the Xbal site by treating the linearized plasmid with Bal31 exonuclease. In this way Hindlll and Bglll restriction sites were eliminated from the plasmid, thus the Hindlll site between the < χ _-peptide and CAT gene became unique (Intermediate II). The EcoRI, HphI fragment with 1300 nucleotides containing the 3'-end of the L10 ribosomal protein gene and the intact LI ribosomal protein gene was cut out from plasmid pNo2016 /Proc. Natl. Acad. Sci. USA, 1696-1701 (1979)/. The fragment was treated with T. polymerase and Klenow-polymerase enzymes for transforming the sticky ends to blunt ends, then a Hindlll linker was ligated to both ends of the fragment and following a Hindlll digestion the fragment was cloned to the unique Hindlll site of Intermediate II plasmid. In case of correct orientation of insertion the artificial operon contained three genes: ø -L10 fusion gene, LI gene, CAT gene. The cells transformed with the plasmid were chlorampheπicol resistant further on (Intermediate III). A DNA fragment with 320 nucleotides containing BamHI linker as well as terminators T-, and T- of the rrnB operon was cloned to the unique Bglll site (inside the LI gene) of the Intermediate III plasmid. The resulting plasmid was named pER 23 "C" LI T-, T 2 « When isolating chloramphenicol sensitive clones, it turned out that they contained 2-3 terminator fragments tandemly repeated. If one fragment was inserted, the clones still grew on chloramphenicol. This fact does, however, not affect the applicability of the plasmids. The structure of the plasmids was determined by restriction enzyme mapping and DNA sequencing. In an experiment (growing on YTB+ampicilliπ , then spreading to chloramphenicol) chloramphenicol resistant recombiπaπt clones could be isolated. The recombinaπt clones were controlled by the "genomic Southern" method and it was proved that the LI gene on the chromosome contained terminator T,T ? insert, indeed. The recombiπant clones maintained their plasmid in 100% after a cultivation without antibiotics for 24 hours, contrary to the 45% of the control clones, Chemicals and other preparations The generally used laboratory chemicals were of pro anal quality, commercially available from Reanal, Sigma and Merck. The specific activity of tπe /jT~ 32p ATP and ^- 2 P-7dATP preparations (Hungarian Isotope Institute) were 100-150 TBq/mM. Restriction endonucleases BamHI , EcoRI, Hindlll, Bglll, Xbal were purified by the coworkers of the Nucleic Acids group in the Hungarian Academy of Sciences, Biochemical Institute, according to published methods /Methods in Enzymology (Ed.: L. Grossmann, V. Moldave/ Vol. £5 pages 89-180/. Enzymes Clal, TaqI, HphI were from New England Biolabs. Bal31 nuclease and Kleπow enzyme (E.coli DNA-polymerasel Large Fragment) were from New England Biolabs Bacterial alkaline phosphatase (BAP) was from Worthington, pancrease RN-ase and lysozyme were from Reanal. T * induced polynucleotide ligase was prepared according to the method of Murray et al. /Murray, N.E., Bruce Ξ.A. and Murray, K.: Molecular cloning of the DNA ligase gene from bacteriophage T^. I. Mol. Bio., l 493-505 (1979)/. Strains E.coli K12 3M107 /Yanisch-Perron, C. et al.: Improved M13 phage cloning vectors and host strains nucleotide sequences of the M13 mp 18 and pUC19 vectors. Gene, 21- 103-119 (1985)/. Escherichia coli strains were grown in complete culture medium containing 10 g of Bacto-tryptone , 5 g of Bacto Yeast extract and 5 g of NaCl per litre. A solid culture medium was prepared by adding 15 g of Bacto-agar per litre to the above medium. Cells containing a plasmid comprising /b-lactamase gene were grown in a medium containing 100 ug/ml ampicillin. Isolation of plasmid DNA was carried out by growing a bacterial strain containing the proper plasmid in a medium containing 100,ug/ml ampicillin and adding 170,ug/ml chloramphenicol to the culture to amplify the plasmid at 0D 6nn =0.7-0.8. Preparative scale isolation of plasmid DNA was carried out by preparing a clear lysate by the method of Clewell and Helinski /Clewell, D.B. and Helinski, D.R., (1969) Supercoiled circular DNA-proteiπ complex in Escherichia coli: purification and induced conversion to an open circular DNA form, Proc. Natl. Acad. Sci. USA, £2, 1159-1166/ then purifying plasmid DNA on Sephacryl S1000 (Pharmacia) column or by ultracentrifugation on caesium chloride/ethidium bromide density gradient. Analytical scale isolation of plasmid DNA from 1.0-1.5 ml of bacterium culture was carried out according to the potassium-acetate method elaborated by Doly and Birnboim and modified by Ish-Horowitz /Maniatis, T. , Fritsch, E.F., Sambrook, I. (1982) Molecular cloning, Cold Spring Harbor Lab., New York/. Cleavage of DNA samples with restriction endonucleases was carried out under the conditions suggested by New England Biolabs. Single stranded DNA ends were made blunt ended by alcohol-precipitating the DNA after digestion with restriction enzyme, then dissolving in a buffer containing 50 mM Tris-HCl pH=7.4; 7 mM MgCl 2 ; 1 mM dithiothreitol; 0.1 mM dATP; 0.1 mM dCTP; 0.1 mM dGTP; 0.1 mM dTTP (final volume 10-20,ul, DNA concentration 200-250,ug/ml) and incubating with 1-2 units of DNA polymerase I Klenow fragment at 37°C for 15 minutes. When joining DNA fragments with sticky ends, the reaction mixture (30-40,ul) contained 0.5-1.0/ug of DNA; 66 mM Tris-HCl (pH=7 ' .6); 5 mM MgCl 2 ; 5 mM dithiothreitol; 1 mM ATP and 1 unit of induced T 4 polynucleotide ligase. The ligation was conducted at 14°C for 2-3 hours /Maniatis T., Fritsch, E.F., Sambrook, I. (1982) Molecular Cloning, Cold Spring Harbor Lab., New York/. Joining fragments with blunt ends was carried out in a buffer containing 30-40 / ug/ml DNA, 25 mM Tris-HCl (pH=7.4), 5 mM MgCl ? , 5 mM dithiothreitol, 0.25 M spermidiπ, 1 M ATP, 10,ug/ml BSA (Sigma, Type V). After adding 4-6 units of induced T^ polynucleotide ligase to the reaction mixture, it was incubated at 14°C for 8-12 hours. Gel electrophoresis of the DNA samples was performed in 0.8-0.2% agarose gels (Sigma, Type I) in horizontal electrophoresis apparatuses according to the method described by Helling et al. (1974). The polyacrylamide gel electrophoresis - using 4 and 8% 1 mm thick vertical gels - was performed according to the method of Maniatis, T., Jeffrey, A. and van de Sande, H. (1985) /Chain length determination of small double and single stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry, l±, 3787-3794/. Isolation of DNA fragments from agarose and polyacrylamide gels was performed according to the method of Winberg G., Hammarskjbld, M. , using DEAE paper /Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adeπovirus type 16 DNA. NAR, 8., 253 (1980)/. The DNA fragments were digested with BAL31 nuclease in a reaction mixture with a final concentration of 100,ug/ml containing 600 mM NaCl, i2 mM CaCl 2 , 20 mM Tris-HCl (pH=8.0), 1.0 M EDTA at 30°C. Depending on the desired degree of the shortening 0.4-1.2 units of enzyme were added to 1.0,ug of DNA. Test reaction was made in each case with the given DNA fragment and the available BAL31 enzyme to determine the degree of shortening of the fragment by gel-electrophoretic analysis of the samples taken after different incubation periods. The enzymatic reaction was stopped by extracting the reaction mixture with phenol and after ethanol- -precipitation of the DNA the ends were made blumt-ended by Klenow polymerase under reaction conditions suitable for labelling 3'-ends /Maniatis, T., Fritsch., E.F., Sambrook, K. (1982) Molecular cloning, Cold Spring Harbor Lab., New York/. Cells competent for the transformation of the bacterium strain 3M107 were prepared according to the CaCl 2 method described by Mandel and Higa /Maniatis, T., Fritsch, E.F., Sambrook, I. (1982) Molecular Cloning, Cold Spring Harbor Lab., New York/. The dephosphorylation of 5'-ends of the DNA fragments, end-labelling with polynucleotide kinase and 7} ~ - P ATP and the determination of the nucleotide sequence were performed according to the protocol described by Maxam, A. and Gilbert, W. (1980) /Sequencing end-labeled DNA with basespecific chemical cleavages. Meth. Enzymol. 6f_, 499-560/. The mutation of the chromosomal gene was proved by the "genomic Southern" method /Southern, E.: Detection of specific sequences among DNA fragments separated by gel electrophoresis, 3 . Mol. Biol., 98 , 503 ( 1975 ) / . Other methods and processes for preparing the expression vectors were carried out according to the prescriptions of Maniatis T., Fritsch, E.F. , Sambrook, K. (1982) /Molecular Cloning, Cold Spring Harbor Lab., New York/. Legends to the figures Figure 1 The plasmid depicted assures resistance against both antibiotics R, and R 2 , promoter Pr controls the synthesys of mRNA with two cistroπs (a+R-,). A - cloned chromosomal gene or other chromosomal segment R,- resistance gene without own promoter R ? - resistance gene with own promoter Figure 2 The plasmid depicted assures resistance only against antibiotic R . B - gene or other sequence to be integrated into the chromosome T - transcription terminator sequence, where the transcription starting from promoter Pr stops Figures 3a and 3b The figures represent the double recombination event and its consequences. The symbols are the same as above. Data of deposition The symbol of the Number of Date of deposition strain deposited deposition IM 107/pER 23 "C" LI 13.07.1988 8 50 AM IM 207/pER 23 "C" LI T T 13.07.1988 8 50 AM

Claims: CLAIMS 1. Method for constructing plasmids assuring insertion of a nucleotide sequence into a chromosome and simple separation of the clones containing the transformed chromosome which comprises inserting a copy of a chromosomal segment corresponding to a chromosomal segment of the host cell between the transcription regulating sequences capable of expressing foreign gene into a plasmid containing transcription regulating . sequences capable of expressing foreign gene and a resistance gene (R 2 ) expressed independently from the said regulating sequences, then downstream inserting a resistance gene without own promoter (R,) different from R 2 and, finally inserting ' a sequence containing at least one transcription terminator into the inserted copy of the chromosomal segment. 2. A method according to Claim 1, characterized in that the chromosomal segment of the host cell is a gene of vital importance, .or a part thereof, from the viewpoint of the host cell. 3. A method according to any of Claims 1 and 2, characterized in that Escherichia coli is used as a host cell. 4. Method for introducing any nucleotide sequence into a chromosome and for simply separating the clones containing the transformed chromosomes which comprises growing the clones started from the cells containing the plasmid vector prepared according to the methods of any of Claims 1 to 3 in a medium containing an antibiotic corresponding to gene R. for 10 to 20 hours, scattering the colonies grown out on a medium containing an antibiotic corresponding to gene R,, finally, separating and further cultivating the living clones. 5. Plasmid assuring introduction of a nucleotide sequence into a chromosome and simple separation of the clones containing the transformed chromosome which comprises containing a copy of a chromosomal segment corresponding to a chromosomal segment of the host cell in two parts after a promoter capable of expressing foreign genes, containing a segment comprising at least one transcription terminator- between the two parts, containing a resistance gene (R-,) without own promoter downstream from the said sequences located within the same operon and containing a further resistance gene (R 2 expressed independently from the said operon. 6. A plasmid according to Claim 4, characterized in that the chromosomal segment of the host cell is a gene or part thereof of vital importance from the viewpoint of the host cell. 7. A plasmid according to any of Claims 5 and 6, characterized in that the host cell is an Escherichia coli bacterium.

Inventors: BALIKO GABRIELLA, GAAL TAMAS, LUKACSOVICH TAMAS, VENETIANER PAL, BALIKO, GABRIELLA, GAAL, TAMAS, LUKACSOVICH, TAMAS, VENETIANER, PAL
Applicants: RICHTER GEDEON VEGYESZET, RICHTER GEDEON VEGYESZETI GYAR R.T.
Application year: 1989
Main classification: C12N15
IPC codes: C12N15