WO1998038205A1 - Compositions and methods for positive selection cloning - Google Patents

Abstract

Nucleic acid molecules are disclosed which comprise a toxic gene operably linked to a regulatory DNA sequence, wherein the regulatory DNA sequence contains one or more cloning sites allowing insertional inactivation of the toxic gene. Also disclosed are kits for cloning and selecting a gene which comprise the nucleic acid molecules of the invention. Also disclosed are methods of cloning and selecting nucleic acid molecules in a transformed host cell using the methods and compositions of the invention. In the practice of the invention, when a genetic insert is inserted into a cloning site within the nucleic acid molecule comprising the toxic gene, the toxic gene is rendered substantially non-toxic to the host cell, allowing for the isolation of viable transformants which comprise the gene to be cloned and selected. The invention also provides stable compositions for cloning and selecting a nucleic acid molecule, vectors and host cells comprising these nucleic acid molecules, kits for cloning and selecting a nucleic acid molecule, methods for producing recombinant polypeptides using these nucleic acid molecules and compositions, and recombinant polypeptides produced according to these methods.

Description

Compositions and Methods for Positive Selection Cloning

FIELD OF THE INVENTION

The present invention is in the field of molecular genetics. The invention relates generally to methods and compositions for cloning nucleic acid molecules and selecting transformants by positive selection. In particular, the invention is related to a nucleic acid molecule or genetic construct comprising a toxic gene linked to a promoter, to vectors and host cells comprising these nucleic acid molecules or genetic constructs, and to methods and kits for cloning and selecting a gene using these nucleic acid molecules or genetic constructs. The invention also relates to methods for producing a desired protein using the methods and compositions of the invention, and to proteins produced by these methods.

BACKGROUND OF THE INVENTION

Nucleic Acid Cloning

A variety of procedures are useful to clone genes. One such method entails analyzing a library of cDNA inserts (derived from a cell expressing the corresponding protein) for the presence of an insert which contains the desired gene. Such an analysis may be conducted by transfecting cells with the vector, inducing the expression of the protein, and then assaying for protein expression, for example, by immunoreaction with an antibody which is specific for the desired protein.

Alternatively, in order to detect the presence of the desired gene, one may employ an oligonucleotide (or set of oligonucleotides) which have a nucleotide sequence that is complementary to the oligonucleotide sequence or set of sequences that codes for the desired protein. Such oligonucleotides are used to detect and/or isolate the desired gene by selective hybridization. Techniques of nucleic acid hybridization are disclosed by Maniatis, T., et ai, In: Molecular Cloning, a Laboratory Manual, Cold Spring Harbor, NY (1982), and by Haymes, B.D., et al., In: Nucleic Acid Hybridization, a Practical Approach, TRL Press,

Washington, DC (1985), which references are herein incorporated by reference.

In addition to the above methods, most commonly used cloning vectors have an indicator gene which results in the expression of a specific phenotype in host cells containing the vector (e.g., blue colonies for host cells containing vectors that carry lacZa; see Maniatis, T., et al., Id.). Insertion of heterologous nucleic acid sequences into multiple cloning sites in such vectors interrupts or inactivates the indicator gene, resulting in non-expression of the phenotype (e.g., white colonies for the above-described host cells containing lacZa vectors). Such an approach provides a convenient means for differentiating recombinant clones

(i.e., those forming white colonies) from non-recombinant clones (i.e., those forming blue colonies). However, this approach does not prevent the growth of non-recombinant clones.

These and similar techniques have enabled the cloning of a variety of human genes, including those encoding aldehyde dehydrogenases (Hsu, L.C. et al., Proc. Natl. Acad. Sci. USA 52:3771-3775 (1985)), fibronectin (Suzuki, S. et al., Eur. Mol. Biol. Organ. J. 4:2519-2524 (1985)), the estrogen receptor (Walter, P. et al., Proc. Natl. Acad. Sci. USA 52:7889-7893 (1985)) and tissue- type plasminogen activator (Pennica, D. et al, Nature 307:214-221 (1983)) and human placental alkaline phosphatase (Kam, W. et al., Proc. Natl. Acad. Sci. USA

52:8715-8719 (1985)).

Restriction Endonucleases

Most cloning techniques employ restriction endonucleases, which are a class of enzymes that occur naturally in prokaryotic and eukaryotic organisms. When restriction endonucleases are purified away from other contaminating cellular components, the enzymes can be used in the laboratory to cleave DNA molecules in a specific and predictable manner. Thus, restriction endonucleases have proved to be indispensable tools in modern genetic research. Restriction endonucleases cleave DNA by recognizing and binding to particular sequences of nucleotides (the "recognition sequence") along the DNA molecule. The enzymes cleave both strands of the DNA molecule within, or to one side of, this recognition sequence. Different restriction endonucleases have affinities for different recognition sequences. Over 100 kinds of different endonucleases have so far been isolated from various microorganisms, each being identified by the specific base sequence it recognizes and by the cleavage pattern it exhibits. In addition, a number of restriction endonucleases, called restriction endonuclease isoschizomers, have been isolated from different microorganisms which in fact recognize the same recognition sequence as those restriction endonucleases that have previously been identified. These isoschizomers, however, may or may not cleave the same phosphodiester bond as the previously identified endonuclease.

Modification methylases are complementary to their corresponding restriction endonucleases in that they recognize and bind to the same recognition sequence. However, in contrast to restriction endonucleases, the modification methylases chemically modify certain nucleotides within the recognition sequence by the addition of a methyl group. Following this methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. Thus, in nature, methylases serve a protective function, i.e., to protect the DNA of an organism which produces its corresponding restriction enzyme.

Restriction enzymes and modification methylases can be purified from the host organism by growing large amounts of cells, lysing the cell walls, and purifying the specific enzyme away from the other host proteins by extensive column chromatography. However, the amount of restriction enzyme relative to that of the other host proteins is usually quite small. Thus, the purification of large quantities of restriction enzymes or methylases by this method is labor intensive, inefficient, and uneconomical. An alternative method for producing large quantities of restriction and modification enzymes is to clone the genes encoding the desired enzymes and overexpress the enzymes in a well studied organism, such as Escherichia coli (E. coli). In this way, the amount of restriction and modification enzymes, relative to that of the host proteins, may be increased substantially. The first cloning of a DNA endonuclease gene was described by Mann et al. Gene 3:97-112 (1978). Since then more than seventy DNA methylase and restriction endonucleases have been cloned. Thus far, the majority of the restriction endonuclease genes are closely linked to their corresponding methylase genes. Restriction-modification systems can be cloned by several methods. A number of endonuclease and methylase genes have been cloned from endogenous plasmids: EcoRII (Kosykh etα/., M>/. Gen. Genet. 775:717-718 (1980)), EcoRI (Newman et al, J. Biol Chem. 256:2131-2139 (1981)), Greene et al., J. Biol Chem. 256:2143-2153 (1981)), EcoRV (Bougueleret et al, Nucl Acids Res. 72:3659-3676 (1984)), Pvull (Blumenthal et al, J. Bacteriol 164:501-509

(1985)), Kpril (Hammond etal, Gene 97: 97-102 (1990)), andP eR71 (Gingeras etal, Proc. Natl. Acad. Sci. USA 50:402-406 (1983)). An alternative method of cloning is the phage restriction method in which bacterial cells carrying cloned restriction and modification genes survive phage infection (Mann et al, supra; Walder et al, Proc. Natl. Acad. Sci. U.S.A. 78: 1503-1507 (1981); Rodicio et al,

Mol. Gen. Genet. 273:346-353 (1988)). Another procedure is based upon methylation protection and has been suggested by Mann et al, supra, and Szomolanyietα/., Gene 70:219-225 (1980). This latter scheme involves digestion of a plasmid library with the restriction enzyme to be cloned. Only those plasmids with DNA sequences modified by the corresponding methylase will be resistant to digestion and will produce transformants in a suitable host. This selection method has been used to clone endonuclease and methylase genes together as well as to clone methylase genes alone (Szomolanyi et al. , supra; Janulaitis et al. , Gene 20:197-204 (1982); Walder etal, J. Biol. Chem. 255:1235-1241 (1983); Kiss et al, Gene 27:111-119 (1983); Wilson, Gene 74:281-289 (1988)). However, this technique sometimes yields only the methylase gene, even though the endonuclease and modifying genes are closely linked.

A multi-step approach has been required to clone certain restriction- modification systems in E. coli, including Ddel (Howard et al, Nucl. Acids Res.

74:7939-7950 (1989)), BamΗI (Brooks et al, Nucl. Acids Res. 77:979-997 (1989)), Kpnl (Hammond etal, Gene 97: 97-102 (1990)) and Clal (U.S. Patent No. 5,312,746). A model has been proposed to explain why certain restriction- modification systems must be cloned utilizing a protected host (Wilson, Gene 74:281-289 (1988)). This model proposes that in order to establish a plasmid carrying a restriction-modification system, methylase protection must occur at a rate that is greater than the rate of endonuclease digestion. Otherwise, restriction enzymes would cleave unmethylated plasmid and/or genomic DNA and degrade the plasmid and/or kill the host. Patents describing the cloning of restriction endonuclease genes and modification methylases include without limitation U.S. Patent Nos. 4,960,707 (Dpnl and Dpnll); 5,000,333, 5,082,784 and 5,192,675 (Kpnl); 5,147,800 (NgoAlϊl and NgoAl); 5,179,015 (EspI andHαelll); 5,200,333 (Hαell and Taql); 5,248,605 (Hpαll); 5,312,746 (Clal); 5,231,021 and 5,304,480 (.YfcoIand.ATzoII); 5,334,526 (Alul); 5,470,740 (Nsiϊ); 5,534,428 (SstllSacl); 5,202,248 (Ncol);

5,139,942 (MM); and 5,098,839 (Pad). See also Wilson, G.G., Nucl Acids Res. 79:2539-2566 (1991); and Lunnen, K.D. et al, Gene 74:25-32 (1988).

Toxic Genes

As alluded to above, restriction endonucleases are toxic to the host cells that produce them. A variety of analogously toxic genes have been described in prokaryotic and eukaryotic cells. Toxic genes for eukaryotic cells include apoptosis-related genes such as ASK1 (Ichijo, Η., et al, Science 275:90-94

(1997)) and members of the bcl-2/ced-9 family (Davies, A.Η., Trends Neurosci. 75:355-358 (1995); WO 95/13292; WO 95/00160; U.S Patent Nos. 5,015,568, 5,523,393 and 5,539,094); retroviral genes including those of the human immunodeficiency virus (HIV); defensins (Chan, R.Y., et al, DNA Cell Biol. 73:311-319 (1994)); and inverted repeat or paired palindromic DNA sequences (Kieser, T., and Melton, R.E., Gene 65:83-91 (1988); Elhai, J., and Wolk, P.,

Gene 65: 119-138 (1988)). Toxic genes for prokaryotic cells include lytic genes ofbacteriophages (Valerie, K., et al, Proc. Natl. Acad. Sci. USA 52:4763-4767 (1985); Henrich, B., and Plapp, R., Gene 42:345-349 (1986); Henderson, E.E., etal.,Mutat. Res. 220: 151-160 (1989); Henrich, B., and Schmidtberger, B., Gene 754:51-54 (1995); Coleclough, C, Meth. Enzymol 277:152-170 (1993)); antibiotic and antimicrobial sensitivity genes (Hashimoto-Gotoh, T., etal, Gene 737:211-216 (1993); Kast, P., Gene 735:109-114 (1994)); plasmid killer genes (Bernard, P., et al, Gene 745:71-74 (1994)); and eukaryotic transcriptional factors which are toxic to bacteria (Trudel, P., et al, BioTechniques 20(4) :684- 693 (1996)).

PCR-based Cloning

The polymerase chain reaction (PCR) is a method for amplification and enrichment of specific nucleic acid sequences in vitro (see U.S. Patent Nos.

4,683,195; 4,683,202; and 4,800,159, all of which are fully incorporated herein by reference). It is often desirable to clone and characterize nucleic acid molecules amplified by PCR, and there are a number of methods available for performing such cloning and characterization.

In one such method, restriction enzyme sites can be incorporated into the

PCR primers; the PCR-generated nucleic acid molecules will thus contain these restriction sites. For cloning of these specific sequences, these amplified nucleic acid molecules can then be digested with restriction enzymes, the digested fragments ligated into an appropriate site within a plasmid vector, and the vector incorporated into a host cell. Alternatively, PCR products generated by Taq DNA polymerase, which typically contain an additional deoxyadenosine (dA) residue at their 3' termini, can be cloned into specific cloning vectors containing 3' deoxythymidine (dT) overhangs which provide a specific recognition sequence for the 3 ' A residue on the PCR product. This process, often referred to as "TA cloning," provides a means of directly cloning PCR-amplified nucleic acid molecules without the need for preparation of primers with specific restriction sites (see U.S. Patent No. 5,487,993, which is incorporated herein by reference in its entirety).

Blunt-end PCR fragments generated by cleavage with certain restriction enzymes (e.g., Smal, Pfu or Hindϊll) can be cloned into blunt-end insertion sites of cloning vectors (see, e.g., Ausubel, F.M., et al, eds., "Current Protocols in Molecular Biology," New York: John Wiley & Sons, Inc., pp. 3.16.1-3.16.11 (1995)), or PCR-amplified nucleic acid molecules can be cloned using uracil DNA glycosylase (UDG; see U.S. Patent No. 5,137,814, which is incorporated herein by reference in its entirety). Such blunt-end cloning may also be facilitated by treatment of 7 -7-amplified PCR products, which contain dA overhangs as described above, with T4 DNA polymerase to remove the dA overhangs (a procedure often termed "polishing") followed by insertion of the resulting blunt- end fragments into blunt-end vector insertion sites as generally described above.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods and compositions useful in cloning PCR-amplified DNA fragments into positive selection vectors. The present methods and compositions are useful in approaches such as blunt-end cloning, restriction site cloning and TA cloning. In addition, the invention provides methods for the simultaneous cloning of PCR fragments containing non-templated dA overhangs and blunt-end PCR fragments. Specifically, the present invention relates to a genetic construct comprising a toxic gene encoding a gene product that is toxic to a host cell operably linked to a regulatory sequence, wherein the regulatory sequence contains one or more cloning sites allowing insertional inactivation of the toxic gene. The invention also relates to a nucleic acid molecule which comprises a toxic gene encoding a gene product that is toxic to a host cell operably linked to a regulatory DNA sequence that regulates expression of the toxic gene, such as an inducible promoter, and a heterologous gene or DNA fragment inserted into a cloning site within the regulatory sequence, wherein the toxic gene is inactivated when introduced into a host cell.

The toxic gene according to the invention may be, without limitation, a restriction endonuclease, preferably Dpnl; an apoptosis-related gene, preferably ASK1 or a member of the bcl-2/ced-9 family; a bacteriophage lytic gene; a retroviral gene including those of the human immunodeficiency virus (HIV); an antibiotic sensitivity gene; an antimicrobial sensitivity gene; a plasmid killer gene; a eukaryotic transcriptional factor gene that produces a gene product toxic to bacteria; a defensin; or an inverted repeat or paired palindromic DNA sequence.

The invention further relates to methods for producing a recombinant host comprising transforming a host cell with the genetic constructs or nucleic acid molecules of the invention, and to recombinant host cells made by these methods.

The invention also relates to kits for cloning and selecting a gene, comprising comprising a carrier means having in close confinement therein one or more container means, wherein a first container means contains the nucleic acid molecule of the invention. Further container means may contain a restriction endonuclease that is capable of linearizing the nucleic acid molecule at the cloning site, and/or a host cell, preferably a prokaryotic host cell (and most preferably an E. coli cell) or a eukaryotic host cell (more preferably an animal cell, still more preferably an insect cell, a nematode cell or a mammalian cell, and most preferably a human cell), capable of being transformed with the construct. The invention also relates to a method of cloning and selecting a nucleic acid molecule in a transformed host cell, comprising

(a) linearizing the nucleic acid molecule of the invention at a cloning site within the regulatory sequence; (b) ligating a nucleic acid molecule to be cloned and selected into the linearized nucleic acid molecule at the cloning site to give a ligated genetic construct;

(c) transforming a host cell with the ligated genetic construct; and

(d) isolating viable colonies of the transformed host cell. According to the invention, the cloning site is preferably a restriction site, and the host cell is preferably a prokaryotic host cell (and most preferably an E. coli cell) or a eukaryotic host cell (more preferably an animal cell, still more preferably an insect cell, a nematode cell or a mammalian cell, and most preferably a human cell) capable of being transformed with the ligated genetic construct. The invention also provides compositions and methods for use in cloning a nucleic acid molecule. Compositions according to this aspect of the invention comprise a 3'exo+ DNA polymerase, which is preferably a T4 DNA polymerase, and a DNA ligase, which is preferably a T4 DNA ligase. Preferably, the components of these compositions are present at working concentrations suitable for use with or without dilution and maintain activity upon storage for an extended time. In a particularly preferred embodiment of this aspect of the invention, the invention provides kits for use in cloning a nucleic acid molecule, comprising a carrier means having in close confinement therein one or more container means, such as vials, tubes, bottles and the like, wherein a first container means contains any one of the compositions described above.

Methods according to this aspect of the invention comprise contacting a nucleic acid molecule with the above-described compositions and with a linearized vector comprising 3' and 5' blunt ends, preferably a positive selection cloning vector or an expression vector, to produce a nucleic acid molecule with 3' and 5' blunt ends and to simultaneously insert the nucleic acid molecule into the vector.

Nucleic acid molecules cloned according to these methods may be amplified, preferably by the polymerase chain reaction (PCR) prior to being contacted with the compositions and vectors. The invention also provides vectors produced using these compositions and methods, methods for producing a recombinant host cell comprising inserting these nucleic acid molecules or vectors into a host cell, and recombinant host cells produced by these methods. Preferred host cells according to this aspect of the invention include bacterial cells (most preferably E. coli or Bacillus cells), yeast cells, plant cells and animal cells (more preferably insect cells, nematode cells or mammalian cells, and most preferably CHO cells, COS cells, VΕRO cells, BHK cells or human cells).

The invention also provides methods of producing a recombinant polypeptide comprising culturing any of the above-described host cells under conditions favoring the expression of a recombinant polypeptide by the host cells, and isolating the recombinant polypeptide. Also provided are recombinant polypeptides produced according to these methods.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A depicts the map of plasmid pAMPl containing the Dpnl gene under the control of the lac promotor.

Fig. IB is a table showing the results of experiments described in Example 1.

Fig. 2 depicts a number of restriction maps of construction containing engineered restriction sites and the results of experiments described in Example 3. Fig. 3 depicts the scheme used to test the possibility of using the EcoRV site for cloning of PCR amplified products as described in Example 4. Fig. 4 depicts the map of the multiple cloning region of the positive selection cloning vector pNGld.

DETAILED DESCRIPTION OF THE INVENTION

In the description that follows, a number of terms used in recombinant DNA technology are utilized extensively. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

Cloning vector. A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a DNA fragment may be spliced in order to bring about its replication and cloning. The cloning vector may further contain a marker suitable for use in the identification of cells transformed with the cloning vector. Markers, for example, provide tetracycline resistance or ampicillin resistance.

Expression vector. A vector similar to a cloning vector but which is capable of enhancing the expression of a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.

Promoter sequences may be either constitutive or inducible.

Restriction endonuclease isoschizonter. A restriction endonuclease isoschizomer is a term used to designate a group of restriction endonucleases that recognize and bind to the same recognition sequence but are isolated from different microbial sources. Restriction endonuclease isoschizomers may or may not cleave in the exact location as the restriction endonuclease with which it is being compared.

Modification methylase isoschizomer. A modification methylase isoschizomer is a term used to designate a group of modification methylases that recognize the same recognition sequence but are isolated from different microbial sources. Modification methylase isoschizomers may or may not chemically modify the same nucleotides within the recognition sequence as the restriction endonuclease with which it is being compared.

Recognition sequence. Recognition sequences are particular DNA sequences which a restriction endonuclease or a modification methylase recognizes and binds. Recognition sequences are typically four to six (and in some cases, eight) nucleotides in length with a two-fold axis of symmetry.

Recombinant Host According to the invention, a recombinant host may be any prokaryotic or eukaryotic cell which contains the desired nucleic acid construct. This term is also meant to include those cells that have been genetically engineered to contain the desired construct in the chromosome or genome of that cell.

Recombinant vector. Any cloning vector or expression vector which contains the desired cloned gene(s) or nucleic acid construct. Host Any prokaryotic or eukaryotic cell that is the recipient of a replicable expression vector or cloning vector. A "host," as the term is used herein, also includes prokaryotic or eukaryotic cells that can be genetically engineered by well known techniques to contain desired gene(s) in its chromosome or genome. For examples of such hosts, see Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold

Spring Harbor, New York (1982). Preferred prokaryotic hosts include, but are not limited to, bacteria of the genus Escherichia, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, Caryophanon, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest in the present invention include E. coli K12, DH10B (F, raD139 Δ (ara, leu) 7697, tJacXIΛ, galU, galK, mcrh, Δ(mrr hsdRMS tncrB), rpsL dor, φ 80 d lacZ ΔM15, end Al, rø/pG, recAl ), DH5 α and HB 101. Preferred eukaryotic hosts include, but are not limited to, yeast cells, plant cells and animal cells. Particularly preferred animal cells are insect cells such as Drosophila cells, Spodoptera Sf9 and Sf 1 cells and

Trichoplusa High-Five cells; nematode cells such as C. elegans cells; and mammalian cells such as COS cells, CHO cells, VΕRO cells, BHK cells and human cells.

Promoter. A DNA sequence generally described as the 5' region of a gene, located proximal to the start codon. The transcription of an adjacent gene(s) is initiated at the promoter region. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Examples of inducible promoters include the lac promoter (induced with LPTG), tac promoter, trc promoter, tet promoter, trp promoter, λpL promoter, T7 promoter, SP6 promoter and ara promoter. With respect to the gene coding for the restriction endonuclease, the promotor may be a homologous or heterologous promoter. The promoters used in the invention may be natural, mutant or synthetic promoter nucleic acid sequences, which may have altered (/^'. e. , enhanced or decreased) activities seen in the natural sequences.

Gene. A DNA sequence that contains information needed for producing a polypeptide or protein.

Structural gene. A DNA sequence that is transcribed into messenger RNA (mRNA) that is then translated into a sequence of amino acids characteristic of a specific polypeptide.

Expression. Expression is the process by which a polypeptide is produced from a structural gene. The process involves transcription of the gene into mRNA and the translation of such mRNA into polypeptide(s). Regulatory Region. Any nucleic acid sequence outside the coding sequence (the sequence from the start codon to the termination codon) that is involved in the expression of a gene, particularly a toxic gene, an antibiotic resistance gene (genes for tetracycline resistance, ampicillin resistance, kanamycin resistance, etc.) or a nutritionally required gene (e.g., an amino acid biosynthesis gene, a metabolically required gene, etc.). A regulatory region may comprise one or more sequences recognized as structurally distinct regions, including, for example, a promoter region, enhancer regions, repressor regions, regulatory regions of a repressor sequence, etc. More specifically, a regulatory region may comprise a promoter, a ribosomal-binding site, a transcription start site, and spacer nucleic acid sequences located between the promoter and the ribosomal-binding site and between the ribosomal-binding site and the transcription start site. A regulatory region may also be a repressor gene which controls the expression of an essential gene such as an antibiotic resistance marker or a nutritionally required gene. A regulatory region may also control a repressor gene which regulates the expression of another gene used for positive selection.

Gene Product An RN A molecule resulting from transcription of a gene, or a polypeptide or protein resulting from translation of an expressed gene.

Toxic Gene. A gene that encodes a gene product (a "toxic gene product") which, upon expression in a host cell, is lethal to the host cell.

Cloning Site. A particular location in a nucleic acid molecule where the nucleic acid molecule can be linearized. For example, a cloning site may be a sequence in the nucleic acid molecule recognized by a particular restriction endonuclease, such that upon treatment of the nucleic acid molecule with the restriction endonuclease the nucleic acid molecule is linearized at the recognition

(restriction) site. Alternatively, a cloning site may be a site of linearization achieved by PCR amplification of a nucleic acid molecule.

Insertional Inactivation. The insertion of a nucleic acid molecule comprising one or more nucleotides (or bases), preferably two or more nucleotides (or bases), most preferably about 10 bases to about 500 kilobases, into a regulatory sequence whereby the expression of the toxic gene (e.g. , transcription of the gene to produce mRNA, or translation of the mRNA to produce a polypeptide or protein) is inhibited or prevented in such a way as to allow a cell comprising the nucleic acid molecule to be viable while cells not comprising the insertion will not be viable.

Since the nucleotide sequences of many toxic genes and their regulatory regions are relatively well-understood, it would be advantageous to use these toxic genes in a method for positively selecting recombinant host cells by transforming the host cells with genetic constructs that down-regulate or inactivate these toxic genes such that the transformants are able to survive induction of the toxic gene product during selective culturing. The present invention is therefore directed to a nucleic acid molecule comprising a toxic structural gene operably linked to a regulatory DNA sequence, wherein the regulatory DNA sequence contains one or more cloning sites, which is preferably a restriction endonuclease site, allowing insertional inactivation of the toxic gene. Preferably, the nucleic acid molecule also comprises a genetic insert introduced into the cloning site. The genetic insert introduced into the cloning site may be of any size, but is preferably 10 basepairs - 500 kilobase pairs, 20 base pairs - 250 kilobase pairs, 100 base pairs - 100 kilobase pairs, 250 base pairs - 50 kilobase pairs, 500 base pairs - 25 kilobase pairs, or 1000 base pairs - 10 kilobase pairs. According to the invention, when the nucleic acid molecule is linearized (e.g., with a restriction endonuclease) and a genetic insert is ligated into the cloning site, the regulatory sequence of the toxic gene is disrupted, thereby rendering the construct substantially non-toxic to the host cell. The gene coding for the restriction endonuclease is considered substantially non-toxic if viable transformed host cell colonies may be identified when the host cells are cultured under conditions that typically permit expression of the toxic gene. In the absence of the insert, the toxic gene regulatory sequence is not disrupted and remains toxic to the host cell. Thus, the toxic gene regulatory sequence will be disrupted in recombinant host cells (i.e., those containing the inserted nucleic acid molecule), and such cells will be capable of growth in culture medium. Conversely, the toxic gene regulatory sequence will not be disrupted in host cells not containing the inserted nucleic acid molecule, and such cells will therefore be killed by expression of the toxic gene and thus not be capable of growth in culture medium.

The cloning site capable of accepting the insert is within the toxic gene regulatory sequence, preferably at the 5'-end. Thus, the restriction site may be located in a promotor region, in a transcription initiation region, in a translation initiation region, in a spacer region located between a promoter and a ribosome- binding region, or in a spacer region located between a ribosome-binding region and a translation initiation region, such that when the nucleic acid molecule is ligated into the cloning site of the nucleic acid molecule, the regulatory region is disrupted and the toxic gene is not expressed in the transformed host cell. In another embodiment of the present invention, aDpnl restriction enzyme gene is used as a toxic gene. The insertional inactivation of this gene can be accomplished by ligation of a DNA molecule into one or more restriction enzyme sites within the structural gene or within the regulatory sequence, rendering the toxic gene non-toxic to cells. The restriction enzyme site within the gene may be the natural sequence, or may be modified sequences resulting in new restriction enzyme sites within the gene without substantially changing the toxicity of the gene for host cell. The modifications can be in the form of single base mutations as well as more substantial insertions, deletions or substitutions.

In accordance with the invention, fusion genes made by combining the toxic structural gene (or portion thereof) to a second nucleic acid sequence may also be used, provided that said fusion gene encodes for a product that is toxic to the host cell. Insertional inactivation of the regulatory sequence of the fusion gene allows for positive selection in accordance with the invention. The nucleic acid molecule of the invention may be circular or linear so long as it is capable of transfecting a host cell. Thus, the invention concerns a nucleic acid molecule of the invention linearized at a cloning site located within the regulatory sequence. Methods for transfecting host cells with linear nucleic acid molecules are well known and are disclosed, for example, in U.S. Patent Nos.

5,521,291; 5,547,932; and 5,166,320, the disclosures of all of which are incorporated by reference herein in their entireties. Alternatively, the nucleic acid molecule may be prepared from, or in, a bacteriophage or cosmid vector, which is preferably an expression vector, capable of transfecting the desired host cell. A mutant toxic gene may be used in accordance with the invention so long as the gene product is toxic to the host cell. For example, the gene product may be a fusion protein which comprises one or more additional amino acids (e.g. , Tyr, Gly, Phe, Ala, Met, Ser, He, Leu, Thr, Val, Pro, Lys, His, Glu, Gin, Trp, Arg, Asp, or Cys) at the N-terminus, C-terminus or within an intervening region. Such fusion proteins can be of varying sizes, depending upon the size of the toxic gene and the product thereof as well as its tolerance (in terms of maintenance of toxic activity) for amino acid additions, deletions and substitutions within its sequence.

It will be recognized by one of ordinary skill in the art that some amino acid sequences of the toxic gene product can be varied without significant effect on the structure or function of the toxic gene product. If such differences in sequence are contemplated, it should be remembered that there will be critical areas in the sequence of the toxic gene product which determine activity. In general, it is possible to replace residues which form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the toxic gene product. Such variations include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral" amino acid substitutions will generally have little effect on the activity of the toxic gene product.

Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe and Tyr. Further guidance concerning which amino acid changes are likely to be phenotypically silent (i.e., are not likely to have a significant deleterious effect on the function of the toxic gene product) can be found in Bowie, J.U., et al,

"Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247.T306-1310 (1990).

Preferably, the cloning site in the nucleic acid molecules of the invention are cleavage recognition sites for restriction endonucleases ("restriction sites"). Examples of restriction sites that may be engineered into the constructs of the present invention include without limitation Pstl, EcoKV, EcoBl and Ndel.

Methods for inserting restriction endonuclease recognition sites into sequences by oligonucleotide-mediated mutagenesis are well known. See Molecular Cloning, A Laboratory Manual, 2nd edition, Sambrook et al. (eds.), Cold Spring Harbor Laboratory Press, Chapter 15 (1989); Directed Mutagenesis,

A Practical Approach, M.J. McPherson (ed.), Oxford University Press, New York, NY (1991). A preferred method for inserting the restriction site is by PCR. Methods of performing PCR are well known to those of ordinary skill in the art. See U.S. Patent Nos.4,683, 195; 4,683,202; 4,889,818; 5,079,352; 5,352,600; and 5,512,462, all of which are incorporated fully by reference herein. In this embodiment, one or more of the oligonucleotide primers complementary to the gene encompass the restriction site. When PCR is carried out, a modified gene sequence is obtained which comprises the restriction site. See "PCR Technology, Current Innovations," Griffin and Griffin (eds.), CRC Press, Boca Raton, FL, Chapter 10 (1994). Preferably, the restriction site is within the regulatory sequence of the toxic gene, although the restriction site may be placed in the structural gene.

In addition to a toxic gene, other genes may be used for positive selection according to the invention. These genes include, but are not limited to, antibiotic resistance genes (for example, those genes conferring tetracycline resistance, ampicillin resistance, kanamycin resistance, etc., on cells comprising the antibiotic resistance genes), nutritionally required genes (for example, amino acid biosynthesis genes, metabolically required genes, etc.), repressor genes which control the expression of an essential gene such as an antibiotic resistance gene or a nutritionally required gene, and the like. Other art-known genes suitable for use in positive selection according to the invention will be apparent to one of ordinary skill.

Atoxic gene encoding atoxic gene product for use in the present invention may be any gene that encodes an RNA transcript, a polypeptide or protein that is toxic to, preferably lethal to or otherwise deleterious to the health of, the host cell. Preferred toxic genes include, but are not limited to, apoptosis-related genes, preferably ASK1 or members of the bcl-2/ced-9 family; retroviral genes including those of the human immunodeficiency virus (HIV); defensins such as NP- 1 ; inverted repeat or paired palindromic DNA sequences; restriction endonuclease genes, most preferably those encoding Dpnl; bacteriophage lytic genes such as those from φXl 74 or bacteriophage T4; antibiotic sensitivity genes such as rpsL; antimicrobial sensitivity genes such aspheS; plasmid killer genes such as the ccdB gene of plasmid F; and eukaryotic transcriptional factor genes that produce a gene product toxic to bacteria, such as GAT A- 1. Other toxic genes and gene products which may advantageously be used in the methods of the present invention are known to one of ordinary skill in the art.

Many genes coding for restriction endonucleases operably linked to inducible promoters are known, and may be used in the present invention. See, e.g. J.S. Patent Nos. 4,960,707 (Dpnl and Dpnll); 5,000,333, 5,082,784 and 5, 192,675 (Kpnl); 5, 147,800 (NgoAlll andNgoAl); 5, 179,015 (Fspl andHαeϊll); 5,200,333 (H elland Tα^I); 5,248,605 (H/?αII); 5,312,746 (Cfol); 5,231,021 and 5,304,480 (Xhol and Xholl); 5,334,526 (AM); 5,470,740 (Nsiϊ); 5,534,428 (SstVSαcl); 5,202,248 (Tvcol); 5, 139,942 (MM); and 5,098,839 (Pad). See also

Wilson, G.G., Nucl. Acids Res. 79:2539-2566 (1991); and Lunnen, K.D. et al, Gene 74:25-32 (1988).

In the practice of the invention, the nucleic acid molecule comprising the toxic gene, is linearized in the regulatory sequence at a cloning site, preferably a restriction site. A nucleic acid molecule to be cloned and selected is then ligated into the linearized nucleic acid molecule comprising the toxic gene, to form a genetic construct. Host cells are then transformed with the genetic construct, the transformed host cells are cultured under conditions favoring the expression of the genetic construct (i.e., favoring the expression of the toxic gene), and viable colonies of transformed host cells are identified. If desired, the colonies may be tested for the presence of the nucleic acid molecule by northern blotting. These viable cells are then cultured as necessary, the cell walls disrupted, and the genetic construct is isolated. The desired nucleic acid molecule may then be isolated from the genetic construct by cutting again with the restriction endonuclease and isolating the nucleic acid molecule, which may then be ligated into a suitable expression plasmid or other vector.

The practice of the method of the invention more specifically comprises: (a) linearizing the nucleic acid molecule of the invention at a cloning site within a regulatory sequence with a restriction endonuclease; (b) ligating a nucleic acid molecule to be cloned and selected into the linearized nucleic acid molecule to give a ligated genetic construct;

(c) transforming a host cell with the ligated genetic construct; and

(d) isolating viable colonies of the transformed host cell. While the use of a restriction enzyme is a common method for rendering a vector linear, there are other methods well known in the art that may also be used for preparation of a linearized vector. For example, the Polymerase Chain Reaction (PCR) has been used to produce a linear DNA fragment from a circular plasmid DNA (Rashtchian et al. PCR Methods and Applications 2: 124-130

(1992)).

In addition, the linearized vector can be modified to contain one or more 3' overhangs, which are preferably overhangs of dT or a derivative thereof (e.g., dideoxythymidine (ddT)), suitable for cloning of PCR-amplified DNA which often contain 3' dA overhangs. Methods suitable for the preparation ofdT vectors are well-known in the art and may be applied to the preparation of dT vectors with positive selection capability in accordance with the invention (see U.S. Patent No. 5,487,993, which is directed to methods of cloning nucleic acid molecules using dT vectors). The invention also is directed to the production of a recombinant polypeptide using the present methods, compositions and nucleic acid molecules. A number of recombinant DNA strategies exist for production of the polypeptide encoded by the cloned or isolated nucleic acid molecule in eukaryotic or prokaryotic hosts. These strategies, which will be appreciated by one of ordinary skill in the art, utilize high copy number cloning vectors, expression vectors, inducible high copy number vectors, etc. See Molecular Cloning, A Laboratory Manual, 2nd edition, Sambrook et al. (eds.), Cold Spring Harbor Laboratory Press, (1989).

Suitable host cells for use in the present invention include, but are not limited to, bacterial cells, preferably Escherichia, Salmonella, Bacillus,

Agrobacterium and Streptomyces, and most preferably Escherichia coli, Bacillus subtilis and Bacillus megaterium; yeast cells; plant cells; and animal cells, preferably insect cells such as Spodoptera Sf9 or Sf21 cells or Trichoplusa High- Five cells, nematode cells such as those from C. elegans, and mammalian cells, more preferably CHO cells, COS cells, VERO cells and BHK cells and most preferably human cells.

To isolate the expressed protein, the host cells may be collected, dispersed in a suitable buffer, and broken down by ultrasonic treatment to allow extraction of the protein by the buffer solution. After removal of the residue by ultracentrifugation, the desired protein can be purified by extraction, ion-exchange chromatography, molecular-sieve chromatography, affinity chromatography, and the like.

The invention also relates to kits for cloning and selecting a gene, comprising a carrier means such as a box, carton, tube or the like having in close confinement therein one or more container means such as vials, tubes, jars, ampules and the like. A first container means may contain the nucleic acid molecule of the invention in linear or circular form. Such a linear nucleic acid molecule is preferably linearized at a cloning site within the regulatory sequence. A second container means may contain a restriction endonuclease that is capable of cleaving the nucleic acid molecule of the invention at a cloning site. A third container means may contain a cell capable of being transformed with the nucleic acid molecule of the invention, which is preferably one of the host cells described above. The contents of the container means may be supplied as solutions in buffers which may be frozen. See U.S. Patent No. 4,981,797, that discloses methods for the preparation of frozen competent host cells. Alternatively, the contents may be in dry form. In the case of dry reagents and cells, the reagents may be in lyophilized or air-dried form together with an agent which protects the reagents or cells from degradation. Such agents include sugars such as trehalose, sucrose and mannitol, and non-glass forming polymers such as acacia gum and polyvinylpyrrolidone.

The invention also relates to compositions and methods for use in cloning a nucleic acid molecule. Compositions according to this aspect of the invention comprise an enzyme having 3' exonuclease activity (a "3'exo+ enzyme") and a DNA ligase. Preferred 3'exo+ enzymes for use in these compositions are any enzyme with 3' proofreading (3'exo+) capability, more preferably DNA polymerases having 3' exonuclease activity (a "3'exo+ DNA polymerase"), including but not limited to Klenow DNA polymerase, E. coli poll DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, and thermostable DNA polymerases including, but not limited to, Pfu DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, and DNA polymerases from Thermotoga maritima (Tma) and Thermotoga neapolitana (Tne). It will be apparent to one of ordinary skill in the art that other enzymes having 3' exonuclease activity may also be used in the compositions and methods of the invention.

Similarly, any DNA ligase may be used in the present compositions, including but not limited to T4 DNA ligase and E. coli DNA ligase, as well as thermostable DNA ligases such as those isolated from Thermus aquaticus (Taq) and Thermus thermophilus (Tth). Other DNA polymerases and DNA ligases suitable for use in these compositions will be apparent to one of ordinary skill in the art.

The components of these compositions are preferably stable and are provided at working concentrations. The terms "stable" and "stability" as used herein generally mean the retention by a composition, such as an enzyme composition, of at least 70%, preferably at least 80%, and most preferably at least 90%, of the original enzymatic activity (in units) after the enzyme or composition containing the enzyme has been stored for about four weeks at a temperature of about 20-25 ° C, about six months at a temperature of about 4 ° C or about one year at a temperature of -20 °C. As used herein, the term "working concentration" means the concentration of a reagent that is at or near the optimal concentration used in a solution to perform a particular function (such as amplification, sequencing or digestion of nucleic acids). In a particularly preferred embodiment of this aspect of the invention, the stable compositions described above are provided in the form of kits suitable for use in cloning a nucleic acid molecule. Kits according to this embodiment comprise carrier means having in close confinement therein one or more container means, such as vials, tubes, bottles and the like, wherein a first container means contains a stable composition comprising a DNA polymerase having 3' exonuclease activity (a "3'exo+ DNA polymerase") and a DNA ligase. Preferred DNA polymerases and ligases for use in these kits include, but are not limited to, those described above. The compositions and kits of this aspect of the invention may be used in methods of cloning a nucleic acid molecule. Such methods comprise mixing a double-stranded nucleic acid molecule having one or more 3' overhangs, which are preferably overhangs of deoxythymidine or a derivative thereof (e.g., dideoxythymidine) at one or both of its termini with any of the above-described compositions and with the linearized vector or nucleic acid molecule of the invention to simultaneously produce 3' and 5' blunt ends on the nucleic acid molecule and insert the nucleic acid molecule into the linearized vector or nucleic acid molecule of the invention. The method is also applicable to cloning blunt- ended nucleic acid molecules into a linearized vector or nucleic acid molecule of the invention. The fact that the 3 'exo+ DNA polymerase and the DNA ligase are admixed in the present compositions facilitates this simultaneous blunt-ending, and insertion into the vector, of the nucleic acid molecule; these steps are traditionally done separately thereby increasing cost and time required for successful cloning. Nucleic acid molecules to be cloned according to these methods may be amplified, preferably by the polymerase chain reaction (PCR), prior to being contacted with the compositions and vectors. Following cloning and selection, the nucleic acid molecules may be inserted into a host cell to produce a recombinant host cell, and a recombinant polypeptide produced from these host cells as described above. Preferred host cells according to this aspect of the invention include bacterial cells (most preferably E. coli or Bacillus cells), yeast cells, plant cells and animal cells (more preferably insect cells, nematode cells or mammalian cells, and most preferably CHO cells, COS cells, VΕRO cells, BHK cells or human cells).

Having now generally described the invention, the same will be more readily understood through reference to the following Examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

Example 1: Dpnl Toxicity to E. coli Cells

The gene coding for Dpnl restriction enzyme was cloned and expressed under the control of lac promoter. Figure 1 shows the map of plasmid pAMPl containing the Dpnl gene under the lac promoter. The expression of this gene is regulated by lac repressor in E. coli and is inducible with LPTG. In the absence of IPTG, lac repressor binds to the lac operator and prevents expression of Dpnl gene. If the gene is induced by addition of IPTG, Dpnl protein is produced in the cell and digests the cellular DNA resulting in cell death. This was demonstrated by growing the cells harboring plasmid pDpnl in the absence of IPTG on bacterial agar plates. The cells were then transferred to agar plates with and without IPTG.

After overnight incubation at 37 °C, the plates were examined for growth. It was observed that while E. coli DH5 α cells containing Dpnl could grow in the absence of IPTG, no growth was observed on plates with IPTG. This demonstrates the inducible nature of this gene in this plasmid and its toxicity to E. coli cells when induced with IPTG.

Example 2: Use of the Dpnl Gene as a Toxic Gene for Positive Selection Cloning

Based on the plasmid pDpnl, a vector was constructed for positive selection cloning. The Dpnl gene in the plasmid pDpnl was modified by insertion of a restriction enzyme site in the coding sequences of Dpnl gene. It was demonstrated that insertion of two amino acids immediately following the ATG start codon did not alter the function of Dpnl as demonstrated by the E. coli toxicity assay. It was demonstrated that when the gene was induced by IPTG, production of Dpnl gene product resulted in cell death. This plasmid was then used as a vector for positive selection cloning of DNA fragments into the Pstl site as the cloning site. In order to test the suitability of this site for cloning, the vector DNA was digested with Pstl, extracted with phenol and chloroform and precipitated with ethanol. Lambda DNA digested with Pstl was used as insert for cloning experiments. 60 ng of Pstl-digested vector DNA was mixed with approximately 10 ng of Pstl digested lambda DNA and were ligated overnight at 16°C. The ligations were performed in a final volume of 10 μl according to the instructions of the manufacturer and contained 5 units of ligase in 50 mM TRIS- HC1, pH 7.6, 10 mM MgCl₂, lmM ATP, 1 mM DTT and 5% (w/v) PΕG-8000. A portion of the ligation mix (3 μl) was then used for transformation of DH5α competent E. coli cells. Transformants were selected on L agar plates with ampicillin (100 μg/ml) and IPTG (1 mM). Transformants were then analyzed for the presence of insert fragment cloned in the Pstl site of plasmid pDpnl. A control reaction with 60 ng of vector DNA but containing no insert was also tested as negative control.

Table 1 Cloning of DNA Fragments into the Pstl Site ofpDpnPI

Example 3: Insertionallnactivation by Disruption of Translation for Positive Selection Cloning

Example 2 demonstrated that insertional inactivation of Dpnl gene within the coding sequences of this gene is a useful tool for positive selection. We also examined the possibility of disrupting expression of this gene by disruption of translational elements required for expression of this gene. A series of vectors were developed by modifying the DNA sequences residing between the ribosome- binding site and the translation initiation site (i.e., the ATG start codon). These modifications involved replacing these sequences for those which result in restriction enzyme sites. A number of vectors were developed with a number of restriction enzyme sites (Figure 2). These plasmids were demonstrated to harbor functional and inducible Dpnl gene and the cells harboring these plasmids were susceptible to induction of Dpnl gene and did not grow in the presence of IPTG.

In order to test the suitability of the restriction sites in the ribosome binding site/ ATG start codon region, we used plasmids pECP as well as pENP. Each of the plasmids were digested with appropriate restriction enzyme and was mixed with Lambda DNA digested with the same restriction enzyme. The procedure for ligation and transformation were as described in Example 2. Table 2 shows the results of cloning Lambda DNA fragments using positive selection methodology. It can be seen that insertion of DNA fragments into the restriction enzyme sites within the translational elements of these plasmids disrupted the expression of the Dpnl gene and allowed survival of the cells harboring recombinant plasmids. Examination of these recombinant plasmids for presence of insert DNA showed that virtually all transformants isolated after induction with IPTG had a cloned fragment. In addition various restriction enzyme sites such as EcoRV, EcoRI and Ndel could be used as cloning sites. Control reactions without insert DNA indicated very low or negligible vector background.

Table 2 Positive Section Cloning of DNA Inserts by Disruption of Translation

Example 4: Positive selection cloning of PCR amplified DNA

Example 3 showed that the restriction enzyme sites within the translational elements of plasmid were effective sites for positive selection cloning. These experiments also demonstrated that the EcoRV site of plasmid pΕCP could be used effectively for cloning of blunt ended DNA fragments. We examined the possibility of using EcoRV site for cloning of PCR amplified DNA fragments. We accomplished this in two different ways: 1) cloning of blunt-ended PCR fragments such as those produced by Pfu DNA polymerase; and 2) cloning of PCR fragments from Taq DNA polymerase by modification of the 3' ends of EcoRV digested plasmid to allow cloning by TA methodology (Figure 3).

Cloning of blunt-ended PCR fragments was achieved by amplification of a 1.3 kb DNA fragment of the human beta globin gene by Pfu polymerase using the following primers: primer I: 5' TTA GGC CTT AGC GGG CTT AGA C 3' (SΕQ ID NO: 1) primer II: 5' CCA GGA TTT TTG ATG GGA CAC G 3' (SΕQ LD NO:2)

EcoRV-digested pΕCP was used as the cloning vector. The TA cloning of PCR amplified DNA generated with Taq polymerase was accomplished by converting the EcoRV- digested plasmid DNA into the TA cloning vector. EcoRV-digested pΕCP (10 μg) was incubated with 10 units of Taq DNA polymerase for 2 hours at 70°C (2 mM dTTP, 20 mM Tris-HCl, pH 8.4, 50 mM KC1, and 1.5 mM

MgCl₂). The vector was then extracted with phenol and chloroform, precipitated with ethanol, and redissolved in TΕ. The protocol for ligation of insert fragments into the cloning vector was as described in Example 2.

The results of cloning PCR-amplified DNA fragments is shown in Table 3. As it can be seen, this vector DNA resulted in highly efficient cloning of PCR amplified DNA generated with Taq polymerase. However, as expected, this vector was not as effective for cloning of PCR fragments generated with Pfu DNA polymerase. Table 3 Cloning of PCR Amplified DNA in pECP Positive Section Vector

Example 5: Simultaneous Cloning of Blunt PCR Products and PCR Products with 3 ' Overhangs

Two methods were used for cloning of blunt PCR products and PCR products with 3' overhangs. One method involved preparing a mixture of vector DNA consisting of EcoRV-digested pΕCP (blunt vector) and the same vector with 3' T overhangs. This premixed vector allowed cloning of PCR fragments regardless of the DNA polymerase used for amplification. The conditions for ligation of DNA fragments were as described in Example 2.

Another method used to clone blunt PCR products and PCR products with 3' overhangs simultaneously was to develop a method for conversion of 3' overhang PCR products into blunt fragments (end polishing) prior to ligation into blunt vector. A mixture of T4 DNA polymerase and T4 DNA ligase was used for cloning of a variety of DNA fragments into the EcoRV digested (blunt) pΕCP. 25 ng of blunt vector DNA was mixed with a number of different PCR fragments generated with Taq polymerase (3 ' overhang) or Pfu DNA polymerase (blunt) and were treated with T4 DNA ligase and T4 DNA polymerase mixture. The buffer used for simultaneous polishing and ligation was 50 mM potassium glutamate pH 7.6, 12.5 mM Tris-acetate, pH 7.6, 5 mM of Mg acetate, 25 μg/ml BSA, 0.25 mM beta mercaptoethanol, 1 mM ATP, and 0.1 mM each of dTTP, dATP, dCTP and dGTP. Ligations were performed at room temperature or at 16°C for one hour to overnight. As shown in Table 4, both blunt and 3' overhang PCR products were successfully cloned using this methodology.

Table 4 Cloning of PCR Amplified DNA into Eco RV Digested pECP (Blunt)

'A+ insert DNAs were prepared using Taq polymerase; blunt inserts were prepared using Pfu polymerase

²"NT" = not tested. Example 6: Insertional Inactivation by Disruption of Transcription for Positive Selection Cloning

In Example 2, we demonstrated that insertional inactivation of Dpnl gene within the coding sequences of this gene is a useful tool for positive selection. Example 3 demonstrated that insertional inactivation by disruption of translation for positive selection cloning. We also examined the possibility of disruption expression of this gene by disruption of transcription. Vectors were developed by modifying the DNA sequences in RNA polymerase binding site. In order to test the suitability of this site for cloning, plasmid pNGld (Figure 4) was used. The recombinant host strain comprising plasmid pNGl d, E. coli DH5α(pNGl d), was deposited on February 26, 1997, with the Collection, Agricultural Research Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA, as Deposit No. NRRL B-21659. Plasmid pNGld DNA was digested with Pstl, extracted with phenol and chloroform and precipitated with ethanol. Lambda DNA digested with Pstl was used as the insert for cloning experiments. 50 ng of

Pstl digested vector DNA was mixed with 50 ng of Pstl-digested lambda DNA were ligated overnight at 16°C. The ligations were performed in a final volume of 10 μl according to the instructions of the manufacturer and contained 1 unit of ligase in 50 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 1 mM ATP, 1 mM DTT and 5% (w/v) PEG-8000. A portion of the ligation mix (2 μl) was then used for transformation of DH5 alpha competent E. coli cells. Transformants were selected on L agar plates with ampicillin (100 μg/ml) and IPTG (1 mM). Transformants were then analyzed for the presence of insert fragment cloned in the Pstl site of plasmid pNGl d. A control reaction with 50 ng of vector DNA but containing no insert was also tested as negative control. O

Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Life Technologies, Inc.

(B) STREET: 9800 Medical Center Drive

(C) CITY: Rockville

(D) STATE: Maryland

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP) : 20850

(ii) TITLE OF INVENTION: Compositions and Methods for Positive Selection Cloning

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS -DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(v) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (To be assigned)

(B) FILING DATE: 26-FEB-1998

(C) CLASSIFICATION:

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/039,044 (B) FILING DATE: 28-FEB-1997

(C) CLASSIFICATION:

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/041,213

(B) FILING DATE: 26-FEB-1997 (C) CLASSIFICATION:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TTAGGCCTTA GCGGGCTTAG A 21

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

CCAGGATTTT TGATGGGACA C 21

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13£>w)

A. The indications made below relate to the microorganism referred to in the description on page 33, line JO.

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet D

Name of depositary institution

Agricultural Research Culture Collection

Address of depositary institution (including postal code and country)

1815 North University Street Peoria, Illinois 61604 United States of America

Date of deposit Accession Number

February 26, 1997 NRRL B-21659

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet D

PLASMID pNGld, E. coli DH5α(pNGld)

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications art not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS fl, UωΛ a »« applicable)

The indications listed below will be submitted to the international Bureau later (specify the general nature of the indications, e.g., "Accession Number of Deposit")

For International Bureau use only

□ This sheet was received by the International Bureau on:

Authorized officer

Form PCT/RO/134 (July 1992)

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid molecule comprising a toxic gene operably linked to a regulatory sequence, wherein said regulatory sequence contains one or more cloning sites allowing insertional inactivation of said toxic gene.

2. The nucleic acid molecule of claim 1, further comprising a genetic insert introduced into said cloning site.

3. The nucleic acid molecule of claim 2, wherein said genetic insert has a size selected from the group of sizes consisting of 10 basepairs - 500 kilobase pairs, 20 base pairs - 250 kilobase pairs, 100 base pairs - 100 kilobase pairs, 250 base pairs - 50 kilobase pairs, 500 base pairs - 25 kilobase pairs, and

1000 base pairs - 10 kilobase pairs.

4. The nucleic acid molecule of claim 1, wherein said cloning site is located at the 5'-end or the 3'-end of said toxic gene.

5. The nucleic acid molecule of claim 1, wherein said regulatory sequence is a promoter sequence.

6. The nucleic acid molecule of claim 5, wherein said promoter sequence is an inducible promoter sequence.

7. The nucleic acid molecule of claim 5, wherein said promoter sequence is a lac promoter DNA sequence.

8. The nucleic acid molecule of claim 1, wherein said cloning site is a restriction site.

9. The nucleic acid molecule of claim 8, wherein said restriction site is a Pstl site.

10. The nucleic acid molecule of claim 1, wherein said cloning site is located in said regulatory sequence in a location selected from the group consisting of a promoter, a ribosome-binding region, a transcription initiation region, a translation initiation region, a region between a promoter and a ribosome binding region, and a region between a ribosome-binding region and a translation initiation region.

11. The nucleic acid molecule of claim 1, wherein said toxic gene is selected from the group consisting of a restriction endonuclease gene, an apoptosis-related gene, a bacteriophage lytic gene, a retroviral gene, an antibiotic sensitivity gene, an antimicrobial sensitivity gene, a plasmid killing gene, a eukaryotic transcription gene producing a gene product that is toxic to bacterial cells, a defensin gene and an inverted repeat DNA sequence.

12. The nucleic acid molecule of claim 11, wherein said toxic gene is a restriction endonuclease gene.

13. The nucleic acid molecule of claim 12, wherein said restriction endonuclease gene encodes Dpnl.

14. The nucleic acid molecule of claim 11, wherein said apoptosis- related gene is a member of the bcl-2/ced-9 family.

15. A vector comprising the nucleic acid molecule of claim 1.

16. The vector of claim 15, wherein said vector is linearized at the cloning site within said regulatory sequence.

17. The linearized vector of claim 16, wherein said linearized vector comprises one or more 3' overhangs of deoxythymidine or a derivative thereof.

18. The linearized vector of claim 16, wherein said linearized vector has blunt ends.

19. A recombinant host cell comprising the nucleic acid molecule of claim 1.

20. A recombinant host cell comprising the vector of claim 15.

21. A kit for cloning and selecting a gene, comprising one or more containers, wherein a first container contains the nucleic acid molecule of claim 1.

22. The kit of claim 21, wherein a second container contains a restriction endonuclease that is capable of linearizing said nucleic acid molecule at said cloning site.

23. The kit of claim 22, wherein a third container contains a cell capable of being transformed with said nucleic acid molecule.

24. The kit of claim 23, wherein said cell is an E. coli cell.

25. A kit for cloning and selecting a nucleic acid molecule comprising one or more containers, wherein a first container contains the vector of claim 15.

26. The kit of claim 25 , wherein said vector is linearized at said cloning site within said regulatory sequence.

27. The kit of claim 26, wherein said linearized vector comprises one or more 3 Overhangs of deoxythymidine or a derivative thereof.

28. The kit of claim 26, wherein said linearized vector has blunt ends.

29. A method of cloning and selecting a nucleic acid molecule in a transformed host cell, comprising

(a) linearizing the nucleic acid molecule of claim 1 at a cloning site within said regulatory sequence;

(b) ligating a nucleic acid molecule to be cloned and selected into said linearized nucleic acid molecule at said cloning site to give a ligated genetic construct;

(c) transforming a host cell with said ligated genetic construct; and (d) isolating viable colonies of said transformed host cell.

30. The method of claim 29, wherein said genetic construct is inserted in a vector.

31. The method of claim 29, wherein said cloning site is at the 5'-end or the 3 '-end of said toxic gene.

32. The method of claim 29, wherein said regulatory sequence is a promoter sequence.

33. The method of claim 32, wherein said promoter sequence is a lac promoter sequence.

34. The method of claim 29, wherein said cloning site is a restriction site.

35. The method of claim 29, wherein said linearized nucleic acid molecule contains 3' overhangs of deoxythymidine or a derivative thereof.

36. The method of claim 29, wherein said linearized nucleic acid molecule has blunt ends.

37. The method of claim 34, wherein said restriction site is a Rstl site.

38. The method of claim 34, wherein said restriction site is located in said regulatory sequence in a location selected from the group consisting of a promoter, a ribosome-binding region, a transcription initiation region, atranslation initiation region, a region between a promoter and a ribosome binding region, and a region between a ribosome-binding region and a transcription initiation region.

39. The method of claim 29, wherein said toxic gene is selected from the group consisting of a restriction endonuclease gene, an apoptosis-related gene, a bacteriophage lytic gene, a retro viral gene, an antibiotic sensitivity gene, an antimicrobial sensitivity gene, a plasmid killing gene, a eukaryotic transcription gene producing a gene product that is toxic to bacterial cells, a defensin gene and an inverted repeat DNA sequence.

40. The method of claim 39, wherein said toxic gene is a restriction endonuclease gene.

41. The method of claim 40, wherein said restriction endonuclease gene encodes Dpnl.

42. The method of claim 39, wherein said apoptosis-related gene is a member of the bcl-2/ced-9 family.

43. A composition for use in cloning a nucleic acid molecule, said composition comprising a 3'exo+ enzyme and a DNA ligase.

44. The composition of claim 43, wherein said 3'exo+ enzyme is a

3'exo+ DNA polymerase.

45. The composition of claim 43, wherein the components of said composition are present in working concentrations suitable for use with or without dilution prior to use.

46. The composition of claim 44, wherein said 3'exo+ DNA polymerase is T4 DNA polymerase.

47. The composition of claim 43 , wherein said D A ligase is T4 DNA ligase.

48. A method for producing a recombinant vector, comprising contacting a nucleic acid molecule to be cloned and selected with the composition of claim 43 and with the linearized vector of claim 16 to simultaneously produce 3' and 5' blunt ends on said nucleic acid molecule and insert said nucleic acid molecule into said linearized vector.

49. The method of claim 48, wherein said nucleic acid molecule is amplified prior to being contacted with said composition and said linearized vector.

50. The method of claim 49, wherein said amplification is accomplished by PCR amplification.

51. The method of claim 50, wherein said PCR amplification is accomplished with Taq DNA polymerase.

52. A nucleic acid molecule comprising a Dpnl gene operably linked to a regulatory sequence, wherein said Dpnl gene contains one or more cloning sites allowing insertional inactivation of said gene.