Cloning and expressing restriction endonucleases and modification methylases from caryophanon

Abstract: The present invention is directed to recombinant hosts which contain and express the Cla I Type-II restriction endonuclease and/or modification methylase genes. The present invention is also directed to vectors or DNA molecules which contain these gene, and to methods of producing the enzymes. One source of these enzyme is Caryophanon latum, although other microorganisms may be used to isolate the restriction endonuclease isoschizomers and modification methylase isoschizomers of the invention.

Description: Field of the Invention The present invention is in the field of genetic engineering and molecular biology. This invention is directed to an isolated DNA sequence coding for Cla I restriction endonuclease obtainable from Caryophanon . This invention is specifically directed to the recombinant hosts and vectors which contain the isolated DNA coding for the restriction endonuclease Cla I and its corresponding methylase. This invention is also directed to the cloned modification methylase. Background of the Invention Restriction endonucleases 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 affinity for different recognition sequences. About 100 kinds of different endonucleases have so far been isolated from many 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. The document "Nucleic Acid Research", 1981, 9 (19, pp. 4833-4845) discloses a crude preparation of activity Cla I, a site specific endonuclease activity from Caryophanon Latum which is free of any unspecific nuclease activity. However, said document fails to disclose the purified restriction enzyme suitable for cloning or the cloned gene encoding said enzyme. Furthermore, the document does not disclose methylation activity, a methylase or a gene encoding said methylase. The patent application WO-A-9 118 916 discloses a recombinant host which contains and expresses various Type II restriction endonuclease and/or modification methylase genes. The document does not mention the Cla I restriction-modification system and/or methods for cloning same. Restriction-modification systems can be cloned by several methods. A number of endonuclease and methylase genes have been cloned from endogenous plasmids: Eco RII (Kosykh et al ., Mol. Gen. Genet. 178 :717-718 (1980)), Eco RI (Newman et al., J. Biol. Chem. 256 :2131-2139 (1981)), Greene et al., J. Biol. Chem. 256 :2143-2153 (1981)), Eco RV (Bougueleret et al., Nucl. Acids Kes . 12 :3659-3676 (1984)), Pvu II (Blumenthal et al ., J. Bacteriol. 164 :501-509 (1985)), Kpn I (Hammond et al ., Gene 97 : 97-102 (1990)), and Pae R71 (Gingeras et al., Proc. Natl. Acad. Sci. USA 80 :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 . 213 :346-353 (1988)). Another procedure is based upon methylation protection and has been suggested by Mann et al., supra, and Szomolanyi et al., Gene 10 :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 et al ., J. Biol. Chem. 258 :1235-1241 (1983); Kiss et al ., Gene 21 :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 Dde I (Howard et al., Nucl. Acids Res. 14 :7939-7950 (1989)), Bam HI (Brooks et al ., Nucl. Acids Res. 17 :979-997 (1989)), Kpn I (Hammond et al ., supra ) and Cla I (disclosed herein). In each case, protection of the host with methylase expressed on a plasmid was necessary to stabilize a compatible vector containing the functional endonuclease gene. Wilson, supra, has proposed a model to explain why certain restriction-modification systems must be cloned utilizing a protected host. This model proposes that in order to establish a plasmid carrying a restriction-modification system, methylase protection must occur an 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. Although this model is a plausible explanation of plasmid establishment, it has yet to be determined whether continued independent expression of methylase from a separate plasmid is necessary to maintain the plasmid carrying the restriction-modification system during cell growth and replication. The object of the present invention was to provide means to obtain Clal enzyme in high quantities. Summary of the Invention The present invention is directed to an isolated DNA sequence coding for Cla I restriction endonuclease obtainable from Caryophanon and an isolated DNA sequence coding for Clal modification methylase obtainable from Caryophanon. The invention is further directed to vectors comprising an isolated DNA sequence coding for Cla I restriction endonuclease/ modification methylase obtainable from Caryophanon. The invention is further directed to recombinant host cells comprising an isolated DNA sequence/ vector coding for Cla I restriction endonudease/ modification methylase obtainable from Caryophanon. The invention is further directed to processes for obtaining Cla I restriction endonuclease/ modification methylase. According to the process of this invention, the restriction endonuclease and/or modification methylase is produced by culturing a recombinant host comprising an isolated DNA sequence coding for Cla I restriction endonuclease/ modification methylase and isolating enzymes from the recombinant host Brief Description of the Drawings Figure 1 shows schematic maps of the vectors pCP13, pBluescript KS, pUC-Kam, pCPClaIM1, and pCPClaIM6. The figure also shows the location of the plasmid genes which confer resistance to the antibiotics kanamycin (Km'), ampicillin (Ap'), and tetracycline (Tc'), as well as the location of the cohesive ends (COS) in pCP13. The size of each vector is indicated in kilobases (kb). Figure 2 shows a restriction map of C. latum DNA in the region of the Cla I restriction-modification system and the clones derived from this region. The figure also shows the location of probe "A" DNA sequences. Slashed lines (//) indicate that the DNA molecules are not shown in their entirety and extend beyond the scope of the diagram. Definitions 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. Substantially pure. As used herein means that the desired purified enzyme is essentially free from contaminating cellular components, said components being associated with the desired enzyme in nature, as evidenced by a single band following polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Contaminating cellular components may include, but are not limited to, phosphatases, exonucleases or other undesirable endonucleases. Restriction endonuclease isoschizomer. 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 microorganism which contains the desired cloned genes on an expression vector or cloning vector. This term is also meant to include those microorganisms that have been genetically engineered to contain the desired gene(s) in the chromosome or genome of that organism. The term "recombinant host" is not meant to include the wild type Caryophanon strain which produces Cla I. Recombinant vector. Any cloning vector or expression vector which contains the desired cloned gene(s). Host. Any prokaryotic or eukaryotic microorganism 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 microorganisms that can be genetically engineered by well known techniques to contain desired gene(s) on 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). 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. Gene. A DNA sequence that contains information needed for expressing 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). Nomenclature for naming restriction endonucleases are in accord with the proposal of Smith et al., J. Mol. Biol. 81 :419-423 (1973). Briefly, the first letter "C" of Cla I designates the genus " Caryophanon " while the lower case letters "la" designate the species " latum ." Thus, the original strain found to produce Cla I was designated Caryophanon latum ("BRL 240"). Detailed Description of the Invention This invention is directed to isolated DNA sequences coding for the Type-II restriction endonuclease Cla I. Cla I recognizes the palindromic sequence 5' ATCGAT3', cleaving between the T and C residues from the 5' end, producing a two-base 5' extension. The double-stranded recognition site of Cla I is thus characterized as follows: (wherein A represents deoxyadenosine, T represents deoxythymidine, G represents deoxyguanosine, and C represents deoxycytidine) This present invention is further directed to isolated DNA sequences coding for me modification methylase genes which are complementary to the Cla I restriction endonuclease. These methylases chemically modify certain nucleotides with the recognition sequence by the addition of a methyl group, thus making the modified sequence resistant co cleavage by the complementary restriction endonuclease. Also provided by this invention are recombinant hosts which contain isolated DNA sequences coding for the restriction endonuclease and modification methylase of the present invention. Methods for producing the enzymes of the invention are also disclosed. I. Isolation of the Genes Coding for the Restriction Endonuclease and Modification Methylase or Isoschizomers thereof The restriction endonuclease ( Cla I) and its corresponding modification methylase may be obtained from any strain of C. latum . Genes coding for isoschizomers of these enzymes can be obtained from any genus including, but not limited to, Arthrobacter , Bacillus, Citrobacter, Enterobacter, Escherichia , Flavobacterium , Caryophanon , Klebsiella , Micrococcus , Xanthomonas , Nocardia , Pseudomonas , Salmonella, and Streptomyces. The preferred genus to isolate isoschizomers of the restriction endonuclease of the present invention is Caryophanon. Any strain of Caryophanon capable of producing restriction endonuclease isoschizomers of Cla I can be used for the purpose of this invention. For example, Caryophanon tenue may be used to obtain the genes expressing the restriction endonuclease isoschizomers of Cla I. Restriction endonuclease isoschizomers of Cla I may also be obtained from Bacillus aneurinolyticus ( Ban III), Bacillus subtilus ( Bst I), Achromobacter species ( Asp 707I), or Bacillus species ( Bsc I). The preferred species for obtaining the gene encoding the enzyme of the present invention is Caryophanon latum as described in the examples. II. Cloning and Expressing the Genes Coding for the Restriction Endonuclease and Modification Methylase or Isoschizomers thereof Cla I and Cla I methylase are preferably obtained by isolating the genes coding for the enzymes from Caryophanon latum and then cloning and expressing them. It is understood in this invention that genes coding for isoschizomers of the restriction endonucleases and modification methylases of the present invention may be obtained from any microorganism including the genus Caryophanon by using the recombinant techniques described herein. DNA molecules which code for Cla I and Cla I methylase, or isoschizomers thereof, can be recombined into a cloning vector and introduced into a host cell to enable the expression of the restriction endonuclease or modification methylase by that cell. DNA molecules may be recombined with vector DNA in accordance with conventional techniques, including restriction digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. a. Hosts for Cloning and Expressing The present invention encompasses the expression of the desired restriction endonuclease and modification methylase in prokaryotic and eukaryotic cells. Eukaryotic and prokaryotic hosts that may be used for cloning and expressing the enzymes of the invention are well known in the art. Vectors which replicate in such host cells are also well known (Maniatis et al ., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982)). Preferred prokaryotic hosts include, but are not limited to, bacteria of the genus Eschenchia , 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, and DH10B (F - , ara D139 Δ ( ara, leu ) 7697, Δ lac X74 , gal U, gal K, mcr A, Δ(mrr hsd RMS mcr B), rps L dor ,  80 d lac Z ΔM15, end A1, nup G, rec A1). It has been found that E. coli has several mechanisms (restriction systems) for identifying foreign DNA and destroying it. This can be a significant problem in cloning experiments, resulting in reduced recovery of the desired sequences. In particular, it has been found that E . coli contains restriction systems that degrade DNA when it is methylated, either on cytosine residues or adenine residues. Specifically, the well known methylcytosine-specific systems include mcr A ( rgl A), and mcr B ( rgl B) (Revel et al ., Virology 31 :688-701 (1967); Raleigh et al., Proc. Natl. Acad. Sci. USA 83 :9070-9074 (1986)). The methyladenine-specific restriction system has been designated mrr (Heitman et al., J. Bacterial. 169 :3243-3250 (1987)). Thus, the preferred host for cloning and expressing the genes encoding for the enzymes of the present invention are hosts in which these types of restriction systems have been inactivated through mutation or loss. b. Methods for Cloning and Expressing Cla I and Cla I methylase, or isoschizomers thereof, are preferably obtained by isolating the genes coding for the enzymes and then cloning and expressing them. Four different techniques for isolating and cloning restriction endonucleases and modification methylases have been described in a review by Wilson, Gene 74 :281-289 (1988). The four methods reviewed include: (1) subcloning of natural plasmids; (2) selection based on phage restriction; (3) selection based on vector modification involving methylation protection; and (4) multi-step isolation. The methylation protection method for cloning restriction endonuclease genes relies on the proximity of the methylase and restriction enzyme genes to each other and on the expression of both genes in the host cell such as E. coli. First, a library is constructed by ligating fragmented genomic DNA from the source organism into a vector. For this library, one chooses a vector having one or, preferably, more recognition sites of the restriction enzyme one wishes to clone. Preferably, vector pCP13 is used to construct the plasmid library (Darzins, A. et al ., J. Bacterial. 159 :9-18 (1984)). Generally, library inserts are prepared by only partially digesting the genomic DNA in order to obtain a portion of DNA fragments which contain the intact gene of interest. Second, this library is introduced into and grown in a suitable host such as E. coli . Vector DNA that is subsequently isolated from these transformed and grown cells is called the plasmid library. The plasmid library is a mixture of different DNA molecules, having virtually all possible inserts and thus, is representative of most, if not all, DNA sequences contained in the source organism. The vector/insert combinations having a methylase gene will have methylated the recognition sequences within the vector/insert DNA and the host chromosomal DNA if the methylase is expressed in the host used, preferably, E. coli. The isolated plasmid library DNA is then digested with the restriction enzyme. Unmethylated vector/insert combinations are degraded and methylated combinations survive the endonuclease treatment. The endonuclease-treated DNA is then introduced into a fresh host cell. Degraded vector/insert combinations do not become established. Methyl-protected vector/insert combinations, which survived the endonuclease treatment, can establish and maintain themselves in the new E. coli host cells, thereby forming clones. Cell extracts of these clones are then assayed for restriction endonuclease activity in order to identify clones which express the desired restriction enzyme. Thus, genes for a methylase-restriction system can be cloned on a single recombinant DNA molecule, provided that the restriction endonuclease gene is closely linked to the methylase gene. There are a number of reasons why the above method might not work with a particular endonuclease-methylase system. (1) The two genes (methylase and endonuclease) may not be closely linked. In that case, both genes cannot be on the same DNA fragment insert. (2) The cloned fragment may, by chance, contain only the methylase gene. For example, a closely linked endonuclease gene might be inactivated by being cut by the restriction enzyme that generated the DNA library. Similarly, the methylase and endonuclease genes may have been separated from each other by a cleavage at an intervening restriction site. (3) The level of expression of the endonuclease may be high relative to the expression level of the methylase. In this situation, before the expressed methylase can protect the host DNA, the expressed endonuclease destroys the vector/insert combination as well as degrades the chromosome(s) and may kill the host cell. Alternatively, a deletion(s) resulting in loss of part or all of the endonuclease gene from the vector/insert combination may allow the host to survive. (4) The methylase gene may not be expressed in the new host, leading to lack of protection of DNA from the endonuclease. (5) The endonuclease gene may not be expressed in the new host. In situations (1) and (3), if the endonuclease is expressed in the host, there will be no methylase enzyme activity to protect DNA in the host cell and the attempt to clone the endonuclease would fail. Surprisingly, the usual methods for cloning restriction system genes were unsuccessful for the Cla I restriction/modification system. Hybridization analyses indicated that the structure of the Cla I methylase gene isolated from Caryophanon latum genomic libraries differed from the structure of the Cla I methylase gene in the C. latum chromosome. One hypothesis that could explain this unexpected result was that the Cla I endonuclease gene was closely linked to the Cla I methylase gene in the C. latum chromosome but that endonuclease gene expression was toxic to the host. In this case, the only clones which would survive would be those in which the link between Cla I and Cla I methylase was disrupted by a rearrangement of the DNA. Thus, an intact clone of the Cla I endonuclease and methylase genes might be obtained if Cla I methylase was present in the recombinant host before introducing the genomic library. In the present invention, genomic DNA was isolated from a Cla I producing strain of Caryophanon latum . Using standard techniques well-known to those in the art, a recombinant DNA library was constructed, the library was introduced into a bacterial host, and DNA insert/vector molecules were isolated from host cells. A portion of the first DNA library was digested with Cla I and the resulting DNA digest was introduced into fresh host cells. Clones were selected and DNA samples from these clones were screened for resistance to digestion with Cla I. Clones containing DNA resistant to Cla I harbored insert/vector combinations that carried the Cla I methylase. The Cla I methylase gene was then subcloned into a different vector which contained at least one Cla I site and introduced into a bacterial host. The resulting transformants express Cla I methylase but not Cla I endonuclease. A second genomic library was constructed by introducing C. latum genomic DNA into the recombinant host which harbored the Cla I methylase vector. In order to distinguish transformants containing Cla I endonuclease gene sequences from transformants which merely carried the protecting Cla I methylase vector, it was necessary to devise a DNA probe which codes for DNA sequences that are contiguous with, but distinct from, the Cla I methylase gene sequences. For example, oligonucleotide synthesis or polymerase chain reaction technology may be used to produce probes with sequences corresponding to the sequence in the Bam HI/ Sau 3AI- Hind III fragment of pBSClaIM6 that is not present in pBSClaIM4 (Probe A; Figure 2). An appropriate DNA probe was used to identify transformants which harbor a plasmid coding for the Cla I endonuclease gene. Cells harboring these clones were then assayed for Cla I endonuclease activity. Although the steps outlined above are the preferred mode for practicing the present invention, it will be apparent to those skilled in the art that the above-described approach can vary in accordance with techniques known in the art For example, once the Cla I methylase and/or restriction genes are cloned based on the information disclosed herein, these gene sequences or synthetic oligonucleotides of these sequences may be used in hybridization experiments with genetic material from different organisms to obtain clones which contain these genes. See Maniatis et al ., supra. Furthermore, one of ordinary skill in the art, using standard hybridization techniques, can utilize these sequences to isolate genes which code for isoschizomers of the Cla I restriction and modification enzymes by altering the hybridization stringencies. c. Methods for Enhancing Expression Once the desired restriction endonuclease and modification genes have been isolated, a number of recombinant DNA strategies exist for enhanced production of the desired protein in eukaryotic or prokaryotic hosts. These strategies, which will be appreciated by those skilled in the art, utilize high copy number cloning vectors, expression vectors, inducible high copy number vectors, etc. Enhanced production of the restriction endonuclease and modification methylase can be accomplished, for example, by operably linking the desired gene to a strong prokaryotic promoter, although the natural restriction or modification methylase gene promoter may be used. Such well known promoters may be either constitutive or inducible. Examples of constitutive promoters include the int promoter of bacteriophage λ, and the bla promoter of the β-lactamase gene of pBR322, etc. Examples of inducible prokaryotic promoters include the major left and right promoters of bacteriophage λ (P L and P R ), the trp , rec A, lac Z, gal, trc, and tac promoters of E. coli , the α-amylase (Ulmanen, I., et al ., J. Bacteriol. 162 :176-182 (1985)), the δ 28 -specific promoters of B. subtilis (Gilman, M.Z., et al., Gene 32 :11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, J.M., et al., Mol. Gen. Genet. 203 :468-478 (1986)). Prokaryotic promoters are reviewed by Glick, B.R., ( J. Ind. Microbiol. 1 :277-282(1987)); Cenatiempo, Y. ( Biochimie 68 :505-516 (1986)); and Gottesman, S. ( Ann . Rev. Genet. 18 :415-442 (1984)). In order to enhance the production of the desired restriction endonuclease in a prokaryotic cell, it is important to maintain expression of the corresponding modification methylase gene sufficient to protect the DNA of the recombinant host against cleavage with the cloned restriction endonuclease. Therefore, it may be necessary to enhance the level of methylase expression in conjunction with increased endonuclease activity. Furthermore, those skilled in the art will recognize that various combinations of maintaining both the modification and restriction genes within the same recombinant host can be constructed. The only requirement, when cloning restriction endonuclease genes, is that the recombinant host contain and express the methylase gene corresponding to the endonuclease gene being cloned. III. Isolation and Purification of the Restriction Endonuclease and Modification Methylase from Recombinant Hosts The enzymes of this invention, Cla I and Cla I methylase, or isoschizomers thereof, are preferably produced by fermentation of the recombinant host (prokaryotic or eukaryotic) containing and expressing the cloned restriction endonuclease and/or modification methylase genes. The recombinant host, such as E. coli , producing the cloned proteins, can be grown and harvested according to techniques well known in the art. After culturing, the recombinant host cells of this invention can be separated from the culture liquid, for example, by centrifugation. The modification methylase and/or restriction enzymes produced by this host can be extracted and purified by using known protein purification techniques commonly employed for these types of enzymes. In general, the collected microbial cells are dispersed in a suitable buffer, and then broken down by ultrasonic treatment to allow extraction of the enzyme by the buffer solution. After removal of the residue by ultracentrifugation, desired enzyme can be purified by extraction, ion-exchange chromatography, molecular-sieve chromatography, affinity chromatography, and the like, giving the restriction endonuclease of this invention. According to the present invention, assays to detect the presence of the restriction endonucleases and modification methylases can be used during the conventional biochemical purification methods to determine the presence of these enzymes. The restriction endonuclease can be identified on the basis of the cleavage of its recognition sequence. For example, lambda (λ) DNA can be used as a substrate. After digestion with endonuclease, the DNA fragments are separated electrophoretically in agarose gels in the buffer systems conventional for fragment separation and in the presence of ethidium bromide (EtdBr). Demonstration of modification methylase activity can be, but is not limited to, a two-step identification process. First, substrate DNA (λ DNA) that contains the recognition sequence is incubated with column fractions to be tested for methylase activity. Second, this DNA is then challenged with the corresponding restriction activity to identify those fractions which contain methylase activity. For example, while assaying for Cla I methylase, the DNA samples will be challenged with Cla I. Thus, DNA samples which do not exhibit cleavage with Cla I contain Cla I methylase activity. The recombinant host ( E. coli DH10B) containing the genes encoding for Cla I (pCPClaI402) and Cla I methylase (pBSClaIM4) was put on deposit with the Patent Culture Collection, Northern Regional Research Center, USDA, 1815 N. University Street, Peoria, IL 61604 USA (NRRL) as deposit No. NRRL B-21039 (deposit date: December 23, 1992). 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 Bacterial Strains and Growth Conditions Caryophanon latum BRL 240 producing the Cla I restriction endonuclease was grown in a 20 liter (I) fermentor at 30°C to mid-log phase in nutrient media (pH 7.4) consisting of 10 gm/l malt extract ND 201 (Premier Malt Extract), 10 gm/l Hycase S.F. (Sheffield), 5 gm/l sodium acetate, 5 gm/l sodium butyrate, 1 gm/l dibasic potassium phosphate, and 7 gm/l sodium hydroxide. These cells were centrifuged and stored at -70°C as a cell pellet prior to total genomic DNA isolation. E. coli strains were grown at 37°C in YET broth (5 gm/l yeast extract, 10 gm/l tryptone, and 5 gm/l NaCl) with antibiotic supplements of 100 mg/l ampicillin; 20 mg/l tetracycline; or 50 mg/l kanamycin, as appropriate. E. coli strains DH10B and DH10B/pBSClaIM4 were used for cloning the Cla I genes. DH10B is a rec Al - , end A - ,  80 d lacZ ΔM15 derivative of MC1061 (Casadaban et al., J. Mol. Biol. 138 :179-207 (1980)). Competent and electrocompetent E. coli strains were either obtained from Life Technologies, Inc. (LTI; 8717 Grovemont Circle, Gaithersberg, MD 20884-9980) or made by a protocol described by Hanahan, J. Mol . Biol . 166 :557-580 (1983). Electroporation was performed at 2.5 kV in a 0.15 cm gap with a pulse length of about 4 milliseconds. EXAMPLE 2 Construction of pCP13, pBluescript KS, and pUC-Kam Since a methylase protection scheme was to be used to clone Cla I restriction and modification genes, it was necessary to use vectors containing Cla I site(s). Plasmid pBluescript KS, containing a single Cla I site, was obtained from Stratagene (Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037). The cosmid pCP13 has two Cla I sites, one of which is located in the kanamycin-resistance gene (Darzins et al., J. Bacteriol. 59 :9-18 (1984)). The plasmid pUC-Kam is a pUC19 derivative in which the kanamycin resistance gene on a Pst I fragment from pUC4K (Vieira et al ., Gene 19 :259-268 (1982)) was inserted into the Sca I site of pUC19 (James Hartley, Life Technologies, Inc.). EXAMPLE 3 DNA Isolation Small scale plasmid DNA isolations were performed by an alkaline lysis method (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)). For large scale preparations, alkaline lysis was followed by a standard CsCl (cesium chloride)-ethidium bromide (EtdBr) gradient centrifugation. C. latum total genomic DNA was isolated by resuspending 2 grams of frozen C. latum cells in 8 ml of TNE buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 10 mM EDTA). A 10 mg/ml lysozyme solution in TNE buffer was added to the cell suspension to a final concentration of 1 mg/ml. After a 1 hr incubation at 37°C, 10% sodium dodecyl sulfate (SDS) was added to a 2% final concentration and the suspension was shaken gently until lysis was complete. After cell lysis, the lysate was extracted once with phenol and twice with phenol:chloroform:isoamyl alcohol (25:24:1). DNA was spooled with a glass rod under two volumes of cold ethanol. The spooled DNA was dissolved in TE (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) and purified by CsCl-EtdBr gradient centrifugation. EXAMPLE 4 DNA Sequencing Plasmids were sequenced by the cycle sequencing method using The Double Stranded DNA Cycle Sequencing System (LTI, cat # 8196SA) which is based on the method described in Krishnan, et al., Nucl . Acids. Res. 19 :1153 (1991). Plasmid pBSClaIM6 was sequenced using oligonucleotide #1648(SEQ ID NO: 1; 5' GATTTTGATGACGAGCGTAATGGCTGG 3'), which corresponds to the sequence of the kanamycin resistance gene in pCP13 immediately upstream from the Hind III site (Oka et al ., J. Mol. Biol. 147:217 -226 (1981)). The location of the oligonucleotide #1648 DNA sequence in pBSClaIM6 is indicated as segment #1 in Figure 2. Plasmid pBSClaM4 was sequenced using the M13 forward and reverse primers described in Krishnen et al ., supra. EXAMPLE 5 Colony and Southern Hybridization Southern hybridizations were performed using biotinylated probe and photo-gene detection using the Photo Gene Nucleic Acid Detection System (LTI). Fragments generated by endonuclease digestion of the appropriate plasmid were separated by agarose gel electrophoresis and isolated from agarose gel slices by the Gene-Clean procedure (Bio101 Inc, P.O. Box 2284, La Jolla, CA 92038-2284). Fragments were then labeled with biotin by octamer-primed synthesis in the presence of biotinylated nucleotides using the BioPrime DNA Labeling System (LTI). Colony Hybridizations were performed using a radioactive probe essentially as described by Maniatis et al ., supra, except that a phosphorimager (Molecular Dynamics, Inc., 880 E. Arques Ave., Sunnyvale, CA 94086) was used to detect bound radioactivity. The radioactive probe was labeled with [α- 32 P]dCTP in a polymerase chain reaction (PCR) described herein. The 50 µl PCR mixture contained 50 mM KCl, 25 mM Tris (pH 8.3), 5 mM MgCl 2 , 200 µM dATP, 200 µM dGTP, 50 µM dCTP, 200 µM dTTP, 100 ng pBSClaIM6, 1 µM oligonucleotide #1718 (SEQ ID NO: 2; 5' TCACAAATTTTGACTTTTACAAAC 3'), 1 µM oligonucleotide #1719 (SEQ ID NO: 3; 5' TCCTGAATTTTGGTACTCTACTG 3'), 5 µCi [α- 32 P]dCTP, and 1 unit of Taq DNA polymerase (Perkin Elmer Cetus, 761 Main Ave., Norwalk, CT 06859) in water. The 0.6 ml PCR tube was placed in a Techne thermal cycler (LTI) and subjected to the following regimen: 5 min at 94°; 30 cycles of 15 sec at 94°; 15 sec at 55°; 15 sec at 72°; and 2 min at 72°. The locations of the DNA sequences corresponding to oligonucleotides #1718 and #1719 in pBSClaIM6 are indicated as segments #2 and #3, respectively, in Figure 2. EXAMPLE 6 Construction of Genomic Libraries A cosmid library of C. latum genomic DNA was constructed in pCP13. Plasmid pCP13 DNA was digested with an enzyme indicated in Table 1 and dephosphorylated using calf intestine alkaline phosphatase (Boehringer Mannheim). Genomic DNA of C. latum was partially digested with the enzyme indicated in the Table 1. One µg of cosmid vector DNA was ligated with 6 µg of the appropriate chromosomal DNA using 2 units of T4 DNA ligase in 1 x ligase buffer (LTI) overnight at room temperature. One fourth of the ligated DNA was packaged using LTI's Lambda Packaging System and transfected into DH10B. Approximately 10 4 tetracycline resistant (Tc R ) colonies were pooled and an aliquot was inoculated into 500 ml of Circlegrow media (Bio101) containing tetracycline. After a 5 hour growth at 30°, the cells were harvested and plasmid DNA was purified according to Example 3. Libraries of C. latum DNA in cosmid pCP13 and pBluescript Library Insertion site in vector Partial digestion of C. latum genomic DNA ClaI methylase clones obtained Pst I pCP13 Pst I Pst I none Sal I pCP13 Sal I Sal I none Hind III pCP13 Hind III Hind III pCPClaIM1 Sau 3AI pCP13 BamHI Sau 3AI pCPClaIM8 Sph I pUS-Kam Sph I Sph I none For example, the Sph I library was constructed by completely digesting C. lotion genomic DNA with Sph I, isolating 8-16 kb DNA fragments using agarose gel electrophoresis, and inserting the DNA fragments into the dephosphorylated Sph I site of pUC-Kam. About 10 4 colonies were pooled, and inoculated into 500 ml of Circlegrow medium containing kanamycin. After 8 hours of growth at 30°C, the cells were harvested and plasmid DNA was prepared as described. EXAMPLE 7 Selection of Clones Expressing ClaI Methylase Procedure for selection of ClaI Methylase Clones Since the selection for Cla I methylase is dependent upon methylation of the Cla I restriction sites, plasmid libraries of C. latum DNA in pCP13 (Figure 1) were subjected to Clal digestion to eliminate the background of clones lacking the gene for Cla I methylase (" Cla I selection procedure"). In the Cla I selection procedure, 3-5 µg of genomic DNA were digested overnight with 120 units of Cla I. The digested DNA was dephosphorylated, extracted with phenol:chloroform followed by sec-butanol, and precipitated with ethanol. One-tenth of the digested DNA was used to transform E. coli DH10B electrocompetent cells. Plasmid DNA samples isolated from clones selected on plates containing tetracycline or ampicillin were tested for resistance to Cla I. Protection of the resident plasmid and the host chromosomal DNA from digestion with Cla I indicated the presence of methylase activity. A potential problem in the use of the Cla I selection procedure is that loss of Cla I sites in the vector due to deletions increases the background of clones not containing the Cla I methylase. The Sph I library in pUC-Kam did not yield clones which expressed Cla I methylase, but many clones contained vector DNA in which the Cla I site had been deleted. Similarly, the Sal I and Pst I libraries in pCP13 did not yield Cla I methylase clones, while many of the surviving colonies contained plasmids which were about the size of the cloning vector pCP13 but lacked Clal sites. Identification of Methylase Clones The pCP13 Hin dIII library was subjected to the Cla I selection procedure and plasmid DNA was isolated from Tc R clones. All 24 clones tested were identical, and expressed methylase activity as determined by resistance to Cla I restriction digestion of the host DNA. One clone (pCPClaIM1) was chosen for further study. Of 24 clones which survived the Cla I selection from the pCP13 Sau 3AI library, two expressed methylase activity and one was chosen for further study (pCPClaIM8). Subcloning of the ClaI Methylase Gene A deletion derivative of the cosmid pCPClaIM1 was generated by digesting pCPClaIM1 with Bam HI in order to remove a 6.5 kb Bam HI fragment. The Bam HI fragment was remote from the part of pCPClaIM1 shown in Figure 2. After circularizing the modified plasmid (designated "pCPClaMB1") with DNA ligase, plasmid DNA was introduced into DH10B cells. Partial digestion of pPCPlaMB1 with Sau3AI produced 2-4 kb fragments which were subcloned into the BamHI site of pBluescript KS. The library thus formed was selected for resistance to Clal, and several Cla I methylase subclones were obtained, including pBSClaIM4 and pBSClaIM6. Restriction mapping indicated that the C. latum DNA inserts in pBSClaIM4 and pBSClaIM6 correspond to the C. latum DNA insert near one of the two Hind III junctions with pCP13 (Figure 2). The 2 kb overlap between the two cloned regions closely defined the location of the Cla I methylase gene, which was estimated to be between 0.8 and 1.5 kb in length. Further inspection revealed that pBSClaIM6, but not pBSClaIM4, contained the Hin dIII junction between C. latum DNA and pCP13. Thus, a DNA probe was constructed from this segment of pBSClaIM6 which may be used to detect pCP13 clones containing DNA sequences contiguous with the methylase gene and the probe would not cross-hybridize with pBSClaIM4. As discussed below, this discovery made it possible to design a novel recombinant host which harbored the pBSClaIM4 plasmid and produced the protecting enzyme activity of Cla I methylase. The DNA probe was constructed and designated probe "A" (Figure 2). Chromosomal Localization the Clal Methylase Gene The C. latum genome contiguous to the Cla I methylase gene was mapped to identify which regions were present or missing in the cosmid clones pCPClaIM1, pCPClaIM2 and pCPClaIM3. A restriction map of the C. latum chromosome in the region of the Cla I methylase gene was determined using Southern blot analysis of C. latum chromosomal DNA digested with various restriction enzymes (Figure 2). Not one of the three Cla I methylase clones contained a DNA insert representing an intact segment of that region of the chromosome. Failure to clone this region of DNA intact suggested the presence of a functional gene for Cla I endonuclease and/or other lethal gene(s). If the endonuclease gene was present, it was reasonable to assume that previous attempts to clone both genes on a common fragment resulted in the isolation of deletions or other rearrangements which removed the endonuclease gene. To establish a plasmid expressing both restriction and modification genes in an E. coli host, methylation of the host DNA must precede endonuclease digestion. Evidently, the Cla I restriction-modification system cloned in E. coli did not exhibit this coordinated expression. Thus, it was hypothesized that utilization of a host already protected with the Cla I methylase would be required to circumvent this problem. EXAMPLE 8 Selection of Clones Expressing ClaI Endonuclease Selection of Cla I endonuclease clones required the use of a protected host expressing Cla I methylase. A cosmid library of C. latum genomic DNA was constructed in pCP13 using C. latum DNA partially digested with Sau 3AI. The pCP13 cosmid library packaging mix was introduced into DH10B/pBSClaIM4 (i.e., DH10B cells containing the pBSClaIM4 plasmid). The 2000 Ap R Tc R transformants obtained were subjected to colony hybridization using probe A derived from the C. latum DNA which was unique to pBSClaIM6 (Figure 2). As discussed above, this probe has no homology to pBSClaIM4, but was localized to the region of interest. Therefore, it was used to identify cosmid clones with large chromosomal inserts that might contain the endonuclease gene. Plasmid DNA samples from colonies which hybridized to probe A were tested for the ability to produce a PCR signal from oligonucleotides #1 and #2. Two out of eleven cultures did so, and these were tested for Cla I endonuclease activity. Both clones exhibited Cla I restriction endonuclease activity. The cosmid isolated from the strain containing the Cla I restriction and methylase genes was designated, pCPClaI402B. EXAMPLE 9 Assay for Restriction Enzyme Overnight cultures (20 ml) were harvested and resuspended in 1 ml buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM β-mercaptoethanol and 1 mM EDTA. Cells were sonicated on ice 3 times with 10 second bursts with a micro-tip probe. Lambda DNA substrate (1.0 µg) was digested in 1 X REact™ 1 buffer (LTI) with serial dilutions of cell extract for 1 hour at 37°C. DNA was fractionated by electrophoresis and visualized by EtdBr staining. Activity was determined by the presence of the appropriate size bands associated with a Cla I digestion of lambda DNA. Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention, which is defined by the following Claims. All publications and patent applications mentioned in this specification are indicative of the level of skill of those in the art to which the invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in their entirety. SEQUENCE LISTING (LT6303SL.SEQ) (1) GENERAL INFORMATION: (i) APPLICANT: LIFE TECHNOLOGIES, Inc. (ii) TITLE OF INVENTION: Cloning And Expressing Restriction Endonucleases And Modification Methylases From caryophanon (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: LIFE TECHNOLOGIES, Inc.,SG2-Corp. R&D (B) STREET: 8717 Grovemont Circle, P.O. Box 6009 (C) CITY: Gaithersburg (D) STATE: Maryland (E) COUNTRY: united States of America (F) ZIP: 20877 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: herewith (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Kuhnen, Wacker & Partners (B) REGISTRATION NUMBER: Association No 5 (C) REFERENCE/DOCKET NUMBER: (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 08161/930-0 (B) TELEFAX: 08161/930-100 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Claims: Isolated DNA sequence coding for Cla I restriction endonuclease obtainable from Caryophanon. A DNA sequence according to claim 1 obtainable by screening genetic material of bacteria belonging to the genus Caryophanon by using (i) the DNA insert of pCPClal402 carried by the microorganism identified by accession number NRRL B-21039 or (ii) synthetic oligonucleotides thereof as hybridization probe. A recombinant vector comprising a DNA sequence according to claim 1 or 2. The vector of claim 3, wherein the endonuclease gene is under control of a Cla I endonuclease gene promoter; and/or wherein said endonuclease gene is under control of an inducible promoter; and/or wherein said promoter is a lambda P L promoter; and/or wherein said promoter is a tac promoter. A recombinant host cell comprising a DNA sequence according to claim 1 or 2. The host cell of claim 5, wherein the endonuclease gene is obtained from Caryophanon. latum , in particular from Caryophanon latum BRL 240; and/or wherein said host cell is E. coli . A method of producing Cla I restriction endonuclease comprising: (a) culturing a recombinant host cell comprising a DNA sequence according to claim 1 or 2; and (b) isolating Cla I restriction endonuclease from said host cell. The method of claim 7, wherein said DNA sequence is obtained from Caryophanon latum , in particular from Caryophanon latum BRL 240; and/or wherein said host cell is E. coli. The method of claim 7 or 8, wherein said DNA sequence is contained in a vector. The method of any one of claims 7 to 9, wherein the endonuclease gene is under control of a Cla I endonuclease gene promoter; and/or wherein said endonuclease gene is under control of an inducible promoter; and/or wherein said promoter is a lambda P L promoter; and/or wherein said promoter is a tac promoter. A method of using a vector according to claim 3 or 4, to prepare Cla I restriction endonuclease, said method comprising: (a) introducing said vector into a host cell to produce a recombinant host cell; (b) culturing said recombinant host cell; and (c) isolating Cla I restriction endonuclease from said recombinant host cell. Isolated DNA sequence coding for Cla I modification methylase obtainable from Caryophanon. A DNA sequence according to claim 12 obtainable by screening genetic material of bacteria belonging to the genus Caryophanon by using (i) the DNA insert of pBSClalM4 carried by the microorganism identified by accession number NRRL B-21039 or (ii) synthetic oligonucleotides thereof as hybridization probe. A recombinant vector comprising a DNA sequence according to claim 12 or 13. The vector of claim 14, wherein the modification methylase gene is under control of a Cla I modification methylase gene promoter; and/or wherein said modification methylase gene is under control of an inducible promoter; and/or wherein said promoter is a lambda P L promoter; and/or wherein said promoter is a tac promoter. A recombinant host cell comprising a DNA sequence according to claim 12 or 13. The host cell of claim 16, wherein the modification methylase gene is obtained from Caryophenon latum , in particular from Caryophanon latum BRL 240; and/or wherein said host cell is E. coli. A method of producing Cla I modification methylase comprising: (a) culturing a recombinant host cell comprising a DNA sequence according to claim 12 or 13; and (b) isolating Cla I modification methylase from said host cell. The method of claim 18, wherein said gene is obtained from Caryophanon latum , in particular from Caryophanon latum BRL 240; and/or wherein said host cell is E. coli . The method of any one of claims 18 or 19, wherein said DNA sequence is contained in a vector. The method of any one of claims 18 to 20, wherein the modification methylase gene is under control of Cla I modification methylase gene promoter; and/or wherein said modification methylase gene is under control of an inducible promoter; and/or wherein said promoter is a lambda P L promoter; and/or wherein said promoter is a tac promoter. A substantially pure enzyme obtainable by the process according to any one of claims 18 to 21. A method of using a vector according to claim 14 or 15, to prepare Cla I modification methylase, said method comprising: (a) introducing said vector into a host cell to produce a recombinant host cell; (b) culturing said recombinant host cell; and (c) isolating Cla I modification methylase from said recombinant host cell.

Inventors: CHATTERJEE DEB K, LONGO MARY C, SMITH MICHAEL D, CHATTERJEE, DEB K., LONGO, MARY C., SMITH, MICHAEL D.
Applicants: INVITROGEN CORP, INVITROGEN CORPORATION
Application year: 1993
Main classification: C12N15
IPC codes: C12N9, C12N15, C12N1