RESTRICTION ENDONUCLEASE FseI

Abstract: A novel Type II restriction endonuclease isolated from Frankia species DDB13250120 is provided. The enzyme, designated FseI, recognizes the octanucleotide sequence (I), and cleaves the DNA at the sites indicated by the arrows.

Description: RESTRICTION ENDONUCLEASE Fsel INTRODUCTION Technical Field This invention relates to a new Type II restriction endonuclease designated Fsel. Background The ability to specifically cleave a duplex DNA molecule into discrete fragments is essential for the manipulation of DNA in vitro. Such specific cleavage is accomplished using restriction endonucleases which are enzymes capable of cutting a DNA molecule at or about a specific recognition site. Type II restriction endonucleases recognize and cleave DNA at site-specific sequences which often have an axis of rotational symmetry. The site of cleavage may be within the recognition sequence or may lie a fixed number of base pairs away from the sequence. Moreover, the cleavage may be cut straight across the duplex producing flush or blunt-ended fragments, or may be staggered to produce either 5'- or 3'-cohesive ends. To perform genetic manipulation in vitro and for mapping of a cell genome it is desirable to have a large number of restriction endonucleases able to cleave at preselected locations. Most restriction enzymes have recognition sites containing 4-6 bases. However, the use of restriction enzymes whose recognition sites contain a larger number of bases are of interest, since fewer and larger fragments of DNA are likely to be generated by treatment with such restriction enzymes. More contiguous genes can thus be visualized on a single fragment and the fragments generated are fewer and therefore more easily separated from each other. Relevant Literature Many restriction enzymes are reported in the literature. Two restriction enzymes have been described which recognize 8 bases, Sfil, Qiang, et al., Nucleic Acids Res. (1984) 12:4507 and No I, Barsetti, et al. , (unpublished) quoted in Roberts Nucleic Acids Res. (1984) 12:rl67-r204. For a compilation of restriction enzymes and their isoschizomers, see, for example, Roberts Nucleic Acids Res. (1988) 16:27113 (supplement) . SUMMARY OF THE INVENTION A novel restriction enzyme obtained from the bacterial strain Frankia sp. DDB13250120 is provided. The endonuclease designated Fsel, recognizes the sequence 3»retGGCCGG5' where the arrow indicates the cleavage site. Fsel digests Adenovirus 2 DNA into four fragments. The DNA of SV40, ΦX174, bacteriophages lambda and T7, and plasmids pBR322 and pUClδ are not cleaved by Fsel. Fsel can be isolated at a high degree of purity, being at least substantially free from contaminating exonuclease and endonuclease activities. DESCRIPTION OF SPECIFIC EMBODIMENTS Restriction endonuclease Fsel is a Type II restriction enzyme which can be isolated from bacterial strain Frankia sp. DDB13250120. The enzyme has been named in accordance with the nomenclature for Frankia of Lechevalier Canadian J. of Botany (1983) 61:29642967. Fsel can be characterized as recognizing and cleaving the octanucleotide sequence GGCCAGCC; optimal enzyme activity is obtained at about 26°-28°C in NaCl restriction buffer or KGB buffer. The enzyme is further characterized as capable of cleaving Adenovirus 2 DNA into four fragments, the cleavage site within the octanucleotide recognition sequence being as indicated b .y .t..he arrows 5 3 ', c GG c C tG C G G C G C - G C G C 5 3.'. Fsel may be obtained in a variety of ways. It is available from naturally occurring sources, such as Frankia sp. DDB13250120. Isolation of the enzyme may be performed using, for example, the purification procedure of Myertons, et al. , J. Industrial Microbiology (1987) 2:293-303. Cells are grown in a suitable culture medium, e.g., Bennett's broth, pelleted, for example by centrifugation, and suspended in an extraction buffer. The cell walls are lysed by brief incubation of the cells with lysozyme followed by disruption in a French pressure cell or sonication. The cell lysate is cleared of cell debris and unbroken cells by centrifugation, and the supernatant decanted. DNA and RNA_are removed, for example, by precipitation with streptomycin sulfate. The enzyme may be isolated from the supernatant by ion- exchange chromatography followed by Fast Protein Liquid Chromatography (FPLC) . Active fractions, as measured by ability to cleave Adenovirus 2 DNA, are pooled and dialyzed, and the concentrated enzyme may be stored at - 20°C or -70°C. The enzyme preparation obtained by this procedure is substantially free from contaminating exonuclease and endonuclease activity. Other techniques of enzyme purification may also find use; these techniques can include solvent extraction, gel permeation chromatography, reversed phase-HPLC, electrophoresis, or the like. For active fragments, various synthetic techniques may be employed, where the polypeptide will be synthesized on a solid support. A number of commercial synthesizers are available and may be used to advantage, for example, from SmithKline-Beckman, Applied Biosystems, etc. Alternatively, Fsel may be produced by first- isolating the gene which encodes Fsel and introducing the gene into an appropriate expression host. Methods for manipulating and identifying genetic material in vitro are now well known and need not be described in detail. Briefly, however, the Fsel gene may be isolated by first cleaving the chromosomal DNA, typically by endonuclease digestion or mechanical shearing, followed by cloning in an appropriate, usually bacterial, cloning vector. Clones carrying the Fsel gene may be selected and expanded. The Fsel gene may then be introduced into an expression host, either directly or by means of an expression vector. The Fsel gene product may then be isolated from the expression host by conventional means. The gene product will have an amino acid sequence which is substantially identical to the naturally-occurring product, usually differing by fewer than 5% of the amino acids. Fsel may be purified, whether from natural or synthetic sources, using affinity chromatography, either by the use of, for example, immobilized Adenovirus 2 DNA, or fragments thereof capable of binding to Fsel, or by the use of antibodies to Fsel, or fragments thereof, which can be coupled to a solid support in conventional ways. For the antibodies, either polyclonal or monoclonal antibodies can be used. The antibodies can be prepared in conventional ways, by using Fsel as an immunogen and injecting it into a mammalian host, e.g. mouse, cow, goat, sheep, rabbit, etc., particularly with an adjuvant, e.g. complete Freund's adjuvant, aluminum hydroxide gel, or the like. The host may then be bled and the blood employed for isolation of polyclonal antibodies. In the case of the mouse, the peripheral blood lymphocytes or splenic lymphocytes (B-cells) may be employed for fusion with an appropriate myeloma cell to immortalize the chromosomes for monoclonal expression of antibodies specific for the subject compounds. The antibodies can also be used to screen other cells for the presence of Fsel, or enzymes having at least substantial homology with Fsel, usually differing by fewer than 5% of the amino acids. In use, substrate DNA may be restricted by Fsel in a suitable buffer, e.g., Tris-HCl pH = 7.6, 10 mM MgCl 2 , 50 mM NaCl, 10 mM 3-mercaptoethanol, 100 g/ml bovine serum albumin or KGB buffer: 200 mM potassium glutamate, 50 mM tris-acetate pH = 7.6, 20 mM magnesium acetate, 1 mM /9-mercaptoethanol and 100 g/ml bovine serum albumin, at 37°C for a suitable time, e.g., 1 hour. McClelland, et al. , Nucleic Acids Res. (1988) 16:364. The recognition sequence for the restriction endonuclease can be determined by digesting susceptible DNA with the restriction enzyme alone and in conjunction with other restriction enzymes. The cut sites on the DNA in these double digests can be used to determine the recognition sequence; the primed synthesis reaction can be used to characterize the cleavage site. In this way it can be determined that the recognition sequence is an octanucleotide which comprises the bases guanine and cytosine. When using Adenovirus 2 DNA as substrate DNA, the probable sequence of the octanucleotide is 5'- GCCCGGCC-3'and the cleavage site is as indicated by the »™Mβ 5'GGCCGG-CC3' arrows 3 , CC GGCCGG5' . Fsel finds application in the genetic engineering of prokaryotes and eukaryotes for production of medical and industrial products. Fsel also finds use in determining restriction fragment polymorphisms in human diseases and in mapping of cellular genomes. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL Frankia sp. DDB132501250 was deposited in the Northern Regional Research Laboratory (NRRL) culture collection, Peoria, Illinois, and in the Rutgers University culture Collection, Waksman Institute, Piscataway, New Jersey. EXPERIMENTAL Isolation and Characterization of Fsel Growth of Cells and Preparation of Cell Extract Frankia cells were incubated statically at 27°-30°C for three to four weeks in a nutrient medium. The cells were harvested by centrifugation and resuspended in a solution containing 10 mM Tris-HCl pH- 8.0, 1 mM EDTA and 50% glycerol. Cells were frozen at - 70°C until processing for the enzyme. The frozen pellet was then thawed, and the cells fractured in a French pressure cell (IX at 1300 psi) . The crude extract was centrifuged to remove cell debris. A solution of 20% streptomycin sulfate in 100 mM Tris-HCl, pH 8.0 was then added to the supernatant to a final concentration of 2% to precipitate RNA and DNA. The supernatant was clarified by centrifugation and then purified as follows. DEAE-52 Chromatography The crude extract was loaded onto a 1.0 cm x 10 cm DEAE-52 column equilibrated with Buffer A (10 mM potassium phosphate, pH 7.4, 10 mM (3-mercaptoethanol, 0.1 M EDTA, 10% (v/v) glycerol). The restriction enzyme was eluted from the column with a linear 0-1.0 M KC1- buffer A gradient. One ml fractions were collected and assayed for activity by observing digestion of Adenovirus 2 DNA. Fsel activity eluted between fractions 13 and 16. FPLC The active fractions from the DEAE-52 column were pooled and dialyzed against Buffer B (20 mM tris- HC1 pH7.5, 1 mM EDTA pH8.0, 10% (v/v) glycerol, 10 mM / 9-mercaptoethanol) . The restriction enzyme was further fractionated using FPLC, using a MonoQ anion-exchange column (Pharmacia). Partially purified enzyme was eluted from the FPLC column using a 0-1 M KCl-buffer B linear gradient. One ml fractions were collected and assayed for activity by observing digestion of Adenovirus 2 DNA. The active fractions (elution occurs within 1 hour) were pooled, adjusted to a final concentration of 50% glycerol and stored at -20°C. Fsel Assay Conditions To assay for Fsel activity, suitable dilutions of the FPLC column-purified enzyme were incubated with Adenovirus 2 DNA in either NaCl or KGB restriction buffers at 26°-28°C for from one hour to overnight. The reactions were terminated by the addition of Ficoll stop dye (0.25% bro ophenol blue, 15% Ficoll type 400 and 50 mM EDTA) and by heating the reaction to 65°C for 5 minutes. The DNA fragments were separated by electrophoresis at 80 volts for several hours in a 0.8% agarose gel containing 1 /tg/ml ethidium bromide. Characterization of Fsel Complete Fsel digests of Adenovirus 2 DNA allowed sizes to be assigned to the four fragments obtained. Comparison of the approximate fragment sizes with the Adenovirus 2 genome nucleotide sequence allowed GGCCGGCC to be assigned as the probable recognition sequence for Fsel. Mapping of the cleavage sites to specific regions of the Adenovirus 2 genome was achieved by performing Fsel digests on Adenovirus 2 DNA alone and in conjunction with other restriction enzymes. These double digests localized the cut sites on the DNA and confirmed the recognition site as GGCCGGCC. The pri ed- synthesis reaction was used to characterize the Fsel cleavage site. An M13 clone containing a segment of the Adenovirus 2 genome with an Fsel site was extended using dideoxynucleoside triphosphates for the chain termination sequencing reaction. The primed-synthesis reaction was cleaved with Fsel, and the resulting single band, when compared to the control lanes of G, A, T, C, indicated that DNA cleavage of Adenovirus 2 DNA ., . ^ 4. . ^ 5'GGCCGGACC3' . ... occurred in the s i te 3»cctGGCCGG5' as ιndlcated --Y the arrows. To determine the type of cut created, the primed-synthesis reaction cleaved with Fsel was incubated with DNA polymerase I-Klenow fragment. A comparison of the single fragment obtained and the control lanes of G, A, T, C, indicated that Fsel cleaves to produce cohesive DNA ends with a four-base 3'- extension. The pH and temperature optima, and the ideal assay conditions were tested. The pH range examined was pH 6.0-8.5. The temperature range investigated was 25°- 37°C. Ten different assay buffers were checked. The pH for optimum enzyme activity was pH * 7.6 and the ideal temperature was in the range 26°-28°C. The optimum restriction buffers were NaCl and KGB as described above. Example II Cloning Fsel Gene Using Expression of Fsel The genes encoding the Fsel restriction— modification system are cloned using standard procedures (Wilson, G.G., Gene (1988) 74:281-289). First, clones encoding the methylase gene are isolated using the direct methylase selection method. A vector that contains two Fsel sites is constructed by modification of the vector pUC19. This is then used as a host for a shotgun of Frankia species DNA. The mixed population of recombinant plasmids is grown and restricted with Fsel. Surviving colonies are tested for resistance to Fsel which indicates that they contain the methylase gene. Methylase positive colonies are grown up, extracts prepared and tested for the presence of the Fsel enzyme using digestion of Adenovirus 2 DNA (see Example I). Other strategies, that have been successful previously for cloning, will be used as outlined (Wilson, G.G., Gene (1988) 74:281-289) as needed. Selection for clones containing the methylase gene using the temperature-sensitive rocrB selection system (Piekarowicz, et al.. Gene (1988) 74:233-235) can also be used, as can the direct methylase selection method employing Streptomyces lividans as a host rather than E_ ; _ coli. Example III Cloning Fsel Gene Using Synthetic Oligonucleotides Corresponding to Conserved Coding Region Motifs Several conserved protein sequence motifs are found in all known 5-methyl-cytosine methylases. Two mixed sets of oligonucleotides that correspond to conserved motifs four and eight (Posfai, et al., Nucleic Acids Res. (1989) 17:2421-2435) are synthesized. One set of oligonucleotides corresponds to the coding region of conserved motif four. The other set of oligonucleotides corresponds to the noncoding strand of conserved motif eight. These two motifs typically lie 250 to 350 nucleotides apart in cytosine methylases. The mixed sets of oligonucleotides are used in a PCR reaction to produce an amplified product. This product is sequenced and tested for homology with the sequences of other known cytosine methylases. In the event that is appears homologous, a new pair of specific oligonucleotides is prepared that correspond respectively to the coding and non-coding strands from within the sequenced region. Total Frankia DNA is restricted ijn vitro with a variety of different restriction enzymes, and the products circularized at low concentration with DNA ligase. For each different restriction enzyme, inverse PCR (Triglia, et al. , Nucleic Acids Res. (1988) 16:8186; Ochman, et al.. Genetics (1988) 120:621-623) is performed so as to amplify a segment of the genome of Frankia species DNA that contains the methylase gene. Sequence analysis of the amplified DNA is used to identify adjacent open reading frames that are likely to encode the Fsel restriction enzyme. The adjacent open reading frames in both directions are then subcloned either from the initial inverse PCR products or by a fresh amplification using inverse PCR with oligonucleotides derived from either of the two open reading frames. The open reading frame is positioned behind a strong E. coli promoter such as ptac and is cloned into an E. coli strain expressing the Haelll methylase gene for protection. Transformants expressing the Fsel restriction endonuclease gene are detected by plating colonies on a lawn of a recombinant bacteriophage lambda, containing Fsel sites, and looking for nibbled colonies as an indicator of restriction (Walsder, et al., J. Biol. Chem. (1983) 258: 1235-1241). Extracts are then prepared from individual colonies and used to digest Adenovirus 2 DNA in vitro. According to the subject invention, a novel restriction endonuclease is provided which is capable of cleaving the sequence 5'GGCCGGCC-3' . The enzyme may be isolated from Frankia species DDB13250120 substantially free from contaminating enzymes. Most restriction enzymes recognize sequences of four to six nucleotides, hence when they cleave large genomes many small, indiscernible fragments of DNA are produced. Cleavage of large genomes by Fsel will yield fewer and larger DNA fragments which can be better resolved by gel electrophoresis. In addition, several contiguous genes can be isolated on a single fragment. The use of Fsel is thus advantageous for mapping large genomes, such as the E. coli genome and the human genome. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A restriction endonuclease capable of recognizing and cleaving DNA comprising an octanucleotide having the sequence 5'-GGCCGGCC-3' . 2. The restriction endonuclease according to Claim 1, wherein said DNA is Adenovirus 2 DNA and said octanucleotide is cleaved at a position indicated by the arrows: 5'GGCCGG1CC3' 3'CCtGGCCGG5' 3. The restriction endonuclease according to Claim 2, obtained by isolation from a culture of Frankia sp. DDB13250120. 4. A restriction endonuclease capable of isolation from Frankia sp. DDB13250120. 5. The restriction endonuclease according to Claim 4, wherein said endonuclease has a recognition sequence which comprises an octanucleotide. 6. The endonuclease according to Claim 4, wherein said octanucleotide comprises G and C. 7. The restriction endonuclease according to Claim 6, wherein said octanucleotide sequence is 5'- GGCCGGCC-3' . 8. The restriction endonuclease according to Claim 7, wherein said endonuclease cleaves DNA comprising said recognition sequence at a position indicated by the arrows: 5'GGCCGG4CC3' 3'CCTGGCCGG5'. 9. The restriction endonuclease according to Claim 5, wherein said DNA is Adenovirus 2 DNA. 10. A restriction endonuclease having at least substantially the same amino acid sequence as Fsel obtainable from Frankia sp. DDB13250120. 11. The restriction endonuclease according to Claim 7, wherein said endonuclease recognizes and cleaves a DNA sequence comprising 5'-GGCCGGCC-3' . 12. ' A restriction endonuclease obtained by isolation from a culture of Frankia sp. DDB13250120. 13. A method for obtaining a restriction endonuclease, said method comprising: lysing cells of Frankia sp. DDB13250120; freeing the resulting lysate of cell debris; and isolating said endonuclease from the supernatant . 14. The restriction endonuclease according to Claim 10, wherein said restriction enzyme is Fsel. 15. Fsel prepared in accordance with the method of Claim 10. 16. A method for preparing a restriction endonuclease, said method comprising: growing a host cell transformed with an expression cassette comprising a heterologous DNA sequence encoding a restriction endonuclease capable of recognizing and cleaving DNA comprising an octanucleotide having the sequence 5'-GGCCGGCC-3', under conditions whereby said heterologous DNA sequence is expressed; lysing said host cell and freeing the resulting lysate of cell debris; and isolating Fsel from the supernatant. 17. The method according to Claim 16, wherein the genome of Frankia sp. DDB13250120 comprises said heterologous DNA sequence. 18. Fsel prepared in accordance with the method of Claim 16. 19. An expression cassette comprising a heterologous DNA sequence encoding a restriction endonuclease capable of recognizing and cleaving DNA comprising an octanucleotide having the sequence 5'— GGCCGGCC-3' . 20. The expression cassette according to Claim 19, wherein the genome of Frankia sp. DDB13250120 comprises said heterologous DNA sequence.

Inventors: LECHEVALIER MARY P, MEYERTONS JANISE L, ROBERTS RICHARD J, LECHEVALIER, MARY, P., MEYERTONS, JANISE, L., ROBERTS, RICHARD, J.
Applicants: COLD SPRING HARBOR LAB, UNIV RUTGERS, COLD SPRING HARBOR LABORATORY, RUTGERS UNIVERSITY
Application year: 1990
Main classification: C12N9
IPC codes: C12N9, C12N15