# Patent

## Title
Mutated alpha virus

## Abstract

The present invention relates to recombinant DNA which encodes the SacI restriction endonuclease and modification methylase, and production of SacI restriction endonuclease from the recombinant DNA.

## Background
The present invention relates to recombinant DNA which encodes the SacI restriction endonuclease and modification methylase, and the production of SacI restriction endonuclease from the recombinant DNA.

Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other bacterial components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into precise fragments for molecular cloning and gene characterization.

Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the `recognition sequence`) along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over one hundred and eighty restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date.

Bacteria tend to possess at most, only a small number of restriction endonucleases per species. The endonucleases typically are named according to the bacteria from which they are derived. Thus, the species Deinococcus radiophilus for example, synthesizes three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences TTTAAA, PuGGNCCPy and CACNNNGTG respectively. Escherichia coli RY13, on the other hand, synthesizes only one enzyme, EcoRI, which recognizes the sequence GAATTC.

It is thought that in nature, restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteria to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They impart resistance by cleaving invading foreign DNA molecule each time that the recognition sequence occurs. The cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific nucleases.

A second component of bacterial protective systems are the modification methylases. These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer cleaved by the restriction endonuclease. The DNA of a bacterial cell is always fully modified by virtue of the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.

With the advent of genetic engineering technology, it is now possible to clone genes and to produce the proteins and enzymes that they encode in greater quantities than are obtainable by conventional purification techniques. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex `libraries`, i.e. populations of clones derived by `shotgun` procedures, when they occur at frequencies as low as 10.sup.-3 to 10.sup.-4. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.

Type II restriction-modification systems are being cloned with increasing frequency. The first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Molec. Gen. Genet, 178:717-719, (1980); HhaII: Mann et al., Gene, 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci., 78:1503-1507, (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phage. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.

Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acid. Res., 12:3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA, 80:402-406, (1983); Theriault and Roy, Gene, 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol., 164:501-509, (1985)).

A third approach, and one that is being used to clone a growing number of systems are now being cloned by selection for an active methylase gene (refer to EPO Publication No.: 0 193 413 published, Sep. 3, 1986 and BsuRI: Kiss et al., Nucl. Acid. Res., 13:6403-6421, (1985)). Since restriction and modification genes are often closely linked, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene, 10:219-225, (1980); Bcn I: Janulaitis et al, Gene, 20:197-204 (1982); Bsu RI: Kiss and Baldauf, Gene, 21:111-119, (1983); and Msp I: Walder et al., J. Biol. Chem., 258:1235-1241, (1983)).

A more recent method of cloning (the "endo-blue method") has been described for direct cloning of restriction endonuclease genes in E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., Nucl. Acids Res., 22:2399-2403 (1994)). This method utilizes the E. coli SOS response following DNA damage by the endonucleases or non-specific nucleases. A number of thermostable nuclease genes (Tth111I, BsoBI, Thermus nuclease) have been cloned by this method (patent application pending).

Another obstacle to cloning these systems in E. coli was discovered in the process of cloning diverse methylases. Many E. coli strains (including those normally used in cloning) have systems that resist the introduction of DNA containing cytosine methylation. (Raleigh and Wilson, Proc. Natl. Acad. Sci., USA, 83:9070-9074, (1986)). Therefore, it is also necessary to carefully consider which E. coli strain(s) to use for cloning.

Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for characterizing genes in the laboratory, there is a commercial incentive to obtain bacterial strains through recombinant DNA techniques that synthesize these enzymes in abundance. Such strains would be useful because they would simplify the task of purification as well as providing the means for production in commercially useful amounts.

When foreign restriction modification systems are cloned and introduced into E. coli, sometimes the methylase and endonuclease yield is very low compared to the native endonuclease-producing strain probably due to inefficient transcription or translation of the genes in E. coli. This is particularly true for cloning of Streptomyces genes into E. coli because of the different GC contents of the two microorganisms. It would be desirable to have a cloning system that Streptomyces genes can be sufficiently expressed in E. coli and selected for based on the efficient gene expression.

The present invention relates to a method for cloning the SacI restriction endonuclease gene from Streptomyces into E. coli by the methylase selection method. At first, the standard methylase gene selection method was tried to clone the SacI methylase gene using standard cloning vectors such as pBR322 and pUC19 during library construction. The SacI methylase gene was refractory to cloning by using the conventional cloning vectors, presumably due to the poor expression of the SacI methylase gene in E. coli. If the SacI methylase is not efficiently expressed in E. coli, the SacI sites on the plasmid will not be efficiently modified by the methylase. As a consequence, the plasmid will be cleaved and lost in the plasmid library after SacI endonuclease challenge. Since the standard methylase selection did not work, the "endo-blue method" was used to clone the SacI endonuclease gene. More then twenty blue clones were identified, but none of them yielded any detectable SacI endonuclease activity. A second approach using PCR DNA amplification was used to clone the beginning of the SacI endonuclease gene. Degenerate primers were made based on the SacI protein N-terminal sequence and used to amplify the 87 bp (29 codons) coding sequence. Short PCR products were obtained, cloned into pUC19, and DNA sequences determined. It was found that the PCR products are primer multimers.

In order to increase SacI methylase gene expression in E. coli, a high-copy-number plasmid containing a lacUV5 promoter called pRRS (Skoglund et al. Gene, 88:1-5, 1990) was used to clone the SacI methylase gene and used for methylase selection. The SacI methylase gene was successfully cloned in pRRS in four steps: (1) ligation of Sau3AI partially digested genomic DNA and BamHI-cleaved and CIP treated pRRS and transformation of the ligated DNA into E. coli RR1 competent cells; (2) preparation of mixed plasmid library; (3) SacI digestion of plasmid DNA library and retransformation of the challenged DNA into RR1 cells; (4) Screening SacI resistant plasmid(s) among the survivors. After the SacI methylase gene was cloned, efforts were made to clone DNA fragments next to the methylase gene. Usually methylase gene and endonuclease genes in a particular restriction-modification system are located next to each other. Colony hybridization was used to screen plasmid library for large insert including the SacI methylase gene and the adjacent DNA. The effort failed to identify any large insert. It was realized that in order to clone the SacI endonuclease gene, one has to increase the SacI methylase gene expression in a compatible plasmid. A good ribosome binding with 6 bp spacing was engineered in front of the ATG start codon (GGAGGTAAATAA (SEQ ID NO:1)). The SacI methylase gene with a good ribosome binding site was cloned into the SalI site of the Tet.sup.R gene of pLG339 (a medium-copy-number vector) so that the saclM gene is driven by the Tet promoter. E. coli chromosomal DNA is fully modified by the SacI methylase. After the pre-protected host was made, the SacI endonuclease gene was cloned by two steps of inverse PCR. The first inverse PCR covered the 70% of the SacI endonuclease gene. The second inverse PCR contained the remaining of the gene. The entire SacI endonuclease gene was cloned by amplifying the gene with polymerase chain reaction from genomic DNA and inserted into pRRS vector.

FIG. 1 is a scheme for cloning and producing the SacI restriction endonuclease.

FIG. 2 is the DNA sequence (SEQ ID NO:2 and corresponding amino acid sequence SEQ ID NO:3) of saclM gene and its encoded protein sequence.

FIG. 3 is the DNA sequence (SEQ ID NO:4 and corresponding amino acid sequence SEQ ID NO:5) of saclR gene and its encoded protein sequence.

The method described herein by which the SacI methylase gene and endonuclease gene are cloned and expressed is illustrated in FIG. 1 and includes the following steps:

1. The genomic DNA of Streptomyces achromogenes is purified.

2. The DNA is digested partially with a restriction endonuclease such as Sau3AI, or any of its isoschizomers, that generates a DNA fragment(s) containing the entire SacI methylase gene. Alternatively, one could make a library which contains the entire restriction-modification system such as ApaLI, SacII, or SalI genomic library. The fragment(s) should also be of clonable size, that is, between 2-20 kb.

3. The Sau3AI-digested genomic DNA is preferably ligated into BamHI-cleaved/CIP treated pUC19 cloning vector. The resulting mixtures are used to transform an appropriate host, i.e. a hsdR.sup.-, mcrBC.sup.-, mrr.sup.- strain, such as E. coli strain RR1. The DNA/cell mixtures are plated on ampicillin selective media for transformed cells. After incubation, the transformed cell colonies are harvested together to form the primary cell library.

4. The recombinant plasmids are purified in toto from the primary cell library to make primary plasmid library. The purified plasmid library is then digested to completion in vitro with SacI endonuclease, or any SacI isoschizomer. SacI endonuclease digestion causes the selective destruction of unmodified, non-methylase-containing clones, resulting in an increase in the relative frequency of SacI methylase-carrying clones.

5. Identification of SacI methylase clone: The digested plasmid library DNA is transformed back into a host such as E. coli strain RR1 and transformed colonies are again obtained by plating on ampicillin plates. The colonies are picked and their plasmid DNAs are prepared and analyzed for the presence of the SacI methylase gene by incubating purified plasmid DNA in vitro with SacI endonuclease to determine whether it is resistant to SacI digestion.

6. Once it has been established that the methylase gene has been cloned, the clone is analyzed by restriction mapping and deletion mapping. The entire insert is sequenced to determine the DNA sequence of the SacI methylase gene.

7. Once the methylase gene sequence is determined, two sets of primers are designed which anneal to the beginning and to the end of the methylase gene. Inverse PCR is used to amplify DNA sequence that is adjacent to the methylase gene

8. Streptomyces achromogenes genomic DNA is preferably digested with ApaLI, BsrFI, EaeI, EagI, HaeII, NgoMI, PstI, SalI, SacII or TaqI restriction enzymes or any other restriction enzymes that will give rise to reasonable size template DNA (less than 3 kb) for inverse PCR reaction. The digested DNA are self-ligated at a low DNA concentration (less than 2 microgram per ml). The ligated circular DNA is used as templates for inverse PCR reaction using a set of primers that annealed to the end of the SacI methylase gene.

9. After obtaining the inverse PCR products, the DNA is treated with T4 DNA kinase and DNA polymerase and cloned into HincII-cleaved/CIP treated pUC19 vector. The entire inserts are sequenced and the DNA sequences are translated into amino acid sequences in all six reading frames and then compared with the amino acid sequences to the SacI N-terminus protein sequence. One open reading frame of 804 bp is identified that has 27 amino acids identity with the SacI protein sequence. No stop codon was found at the end of the 804 bp sequence, indicating the SacI endonuclease gene has not ended yet.

10. Streptomyces achromogenes genomic DNA is digested with AatII, ApaI, BsaWI, BssHII, EaeI, HaeII, MseI, NcoI, NlaIII, PvuI or TaqI restriction enzymes or any other restriction enzymes that will give rise to reasonable size template DNA (less than 3 kb) for inverse PCR reaction. The digested DNA are self-ligated at a low DNA concentration (less than 2 mg/ml). The ligated circular DNA is used as templates for inverse PCR reaction using a set of primers that annealed to the end of the 804 bp open reading frame. After getting the inverse PCR products, the DNA is treated with T4 DNA kinase and DNA polymerase and cloned into HincII-cleaved/CIP treated pUC19 vector. The insert is sequenced until a stop codon is found. The entire SacI endonuclease gene was found to be 1077 bp. It was cloned by two steps of inverse PCR.

11. The SacI methylase gene is then cloned into a pSC101 derivative to premodify E. coli host. The entire SacI endonuclease gene is amplified by PCR with two primers. The forward primer contains the ribosome binding site and 7 bp spacing before the ATG start codon. The SacI endonuclease gene is then cloned into expression vector pRRS and transformed into SacI methylase premodified cells.

The following example is given to illustrate embodiments of the present invention as it is presently preferred to practice. It will be understood that this example is illustrative, and that the invention is not to be considered as restricted thereto except as indicated in the appended claims

1. Using the "endo-blue method" to clone SacI endonuclease gene.

It was reasoned that if the SacI endonuclease gene is poorly expressed in E. coli cell, one may use the "endo-blue method" to clone the endonuclease gene directly into E. coli without the SacI methylase protection. Sau3AI partially digested SacI genomic DNA was ligated to BamHI-digested/CIP treated pUC19 and transformed into E. coli indicator strain carrying din1::lacZ fusion and plated on X-gal indicator plates. Twenty-six blue colonies were found among 10,000 Ap.sup.R transformants. Plasmid DNA were prepared from all these blue isolates and used to hybridize a degenerate probe that was designed from the SacI N-terminus protein sequence. It was found that one plasmid hybridized to the probe. The DNA sequence was determined for the insert in this plasmid. No significant identity was found between the translated amino acid sequences and the SacI protein sequence except a stretch of four amino acid identity. The degenerate probe was designed from amino acid sequence that includes these four amino acid residues. No SacI activity was found in the cell extracts of all the blue isolates.

2. Using PCR to amplify the beginning of the SacI endonuclease gene.

The N-terminus sequence of SacI protein was determined as (M)GI TIKKSE(or T)AEQVLRKAYEAAASDDVFLEF(or D) (SEQ ID NO:6). Degenerate primers were designed from amino acid residues ITIKK (SEQ ID NO:7) and DDVFLEF (SEQ ID NO:8) and used to amplify 84 bp beginning of the SacI endonuclease gene in a PCR reaction (95.degree. C. 1 min, 30.degree. C. 1 min, 40 cycles). The PCR products were treated with T4 DNA kinase and DNA polymerase and cloned into HincII-cleaved/CIP treated pUC19. The insert DNA was sequenced. It was found that all the inserts are concatemers of primers (primer-dimers).

3. SacI methylase selection using pUC19 as a cloning vector.

AatII, BamHI, NarI, Sau3AI and SphI genomic DNA libraries were constructed using pUC19 as a cloning vector as following: AatII, BamHI, NarI, and SphI digested S. achromogenes genomic DNA were ligated to AatII, BamHI, NarI, and SphI-cleaved/CIP treated pUC19 vector; Sau3AI partially digested S. achromogenes genomic DNA were ligated to BamHI cleaved/CIP treated pUC19 DNA. Ligated DNA were transformed into RR1 competent cells and plated on Ampicillin plates. A total of 1-5.times.10.sup.4 cells were found in each transformation. Plasmid DNAs were prepared from each primary cell library and cleaved with SacI restriction endonuclease. The SacI-digested DNAs were retransformed into RR1 competent cells. Plasmid DNAs were isolated again from the surviving transformants and digested with SacI restriction enzyme to see if the plasmid DNA is resistant to SacI digestion. 108 plasmids were checked for resistance to SacI digestion from each library. Fourteen resistant clones were found that have lost the SacI site in the vector. No true resistant clones were found in all five libraries.

4. SacI methylase selection using pRRS as a cloning vector.

The methylase selection method requires that the SacI methylase gene to be expressed to a reasonable level in vivo so that the methylase can modify the SacI site on the vector that carries the gene. It is known that Streptomyces genes are poorly expressed in E. coli due to the different GC contents of the two microorganisms. In order to express the SacI methylase gene to a high level, another high-copy-number vector pRRS was used as a cloning vehicle. Plasmid pRRS carries the lacUV5 promoter which is stronger than the regular lac promoter on pUC19. Genes cloned into the multiple cloning sites are driven by the lacUV5 promoter. Sau3AI partially digested S. achromogenes genomic DNA was ligated to the BamHI digested/CIP treated pRRS vector and the ligated DNA transformed into RR1 competent cells. A total of 10000 of Ap.sup.R transformants were obtained as the primary cell library. Plasmid DNA was prepared from the primary cell library, cleaved with SacI restriction enzyme, and transformed back into RR1 competent cells. The surviving transformants were picked and cultured. Plasmid DNA was isolated from these cells. To examine if any one of these plasmids contain SacI methylase gene, the individual plasmid was digested with SacI endonuclease. One plasmid was found to be truly resistant to SacI digestion. This plasmid carries approximately 1500 bp insert. Deletion of a PstI fragment (about 320 bp) or a HincII fragment (about 1200 bp) of the insert inactivate the SacI methylase gene and render the deletion clones sensitive to SacI endonuclease cleavage.

The entire SacI methylase gene was sequenced using deletion clones (PstI fragment deletion, HincII fragment deletion, EagI/BamHI fragment deletion, EagI/EcoRI deletion, XbaI/AflII fragment deletion, and Exonuclease III/Mung bean nuclease deletions). The DNA sequence and the predicted amino acid sequence are shown in FIG. 2.

5. Cloning of SacI endonuclease gene

Construction PvuII genomic DNA library: A Southern blot of genomic DNA analysis indicated that a 10 kb PvuII restriction fragment should cover both the SacI methylase gene and the endonuclease gene. PvuII fragments within 8 to 12 kb were gel-purified and ligated into HincII cleaved/CIP treated pUC19 and the ligated DNA transformed into RR1 competent cells. Colony hybridization was performed using SacI methylase gene as a probe. However, no true positive clones were identified.

Cloning of SacI endonuclease gene by inverse PCR: Inverse PCR is an efficient way to clone adjacent DNA to the known DNA sequence. S. achromogenes genomic DNA was digested with AatII, AvrII, BamHI, ClaI, EcoRI, EcoRV, HindIII, KpnI, MluI, NcoI, NarI, PstI, PvuII, SmaI, SphI, SalI, XbaI, or XhoI. Each digested DNA was self-ligated to circularize at a low DNA concentration (2 .mu.g/ml in 500 .mu.l total volume). The ligated DNA was extracted once with Phenol-CHCl.sub.3, and once with CHCl.sub.3 and precipitated with 95% ethanol. The DNA was used as a template for inverse PCR reaction (95.degree. C. 1 min, 60.degree. C. 1 min, 72.degree. C. 5 min, 30 cycles). One set of primers that annealed to the end of the methylase gene was designed as following: forward primer, 5'-GAGTGAAGCGCGAGGTGCAGCGGCAGA-3'(SEQ ID NO:9), reverse primer, 5'-AAACGAACTCTCAGGGAAAGTCATG A-3'(SEQ ID NO:10). A 900 bp and 4000 bp inverse PCR products were found in the self-ligated PstI genomic DNA and Sphl genomic DNA respectively.

To further increase the efficiency of inverse PCR reaction, S. achromogenes genomic DNA was digested with more frequent cutting restriction enzymes such as BsrFI, EaeI, HaeII, TaqI and NgoMI restriction enzyme whose recognition sequence is all GC base pairs. It is desirable to use restriction enzymes that will give rise to reasonable size template DNA (less than 3 kb) for efficient inverse PCR reaction. Amplified products were found in BsrFI, EaeI, HaeII, PstI, TaqI and NgoMI digested and self-ligated DNA in the inverse PCR reaction. The NgoMI and HaeII inverse PCR products were treated with T4 DNA kinase, DNA polymerase and cloned into pUC19. The inserts were sequenced and the new sequence was translated in all six frames. The translated amine acid sequences were compared with SacI N-terminus protein sequence. It was found that the SacI endonuclease gene is 22 bp downstream of the SacI methylase gene. One open reading frame of 804 bp is identified that has 27 amine acids identity with the SacI protein sequence. No stop codon was found at the end of the 804 bp sequence, indicating the SacI endonuclease gene has not ended yet. To clone the rest of the SacI endonuclease gene, another set of primers were designed to annealed to the end of the known DNA sequence as follows: forward primer, 5'-GAAGTGCGTGGAAAGAGCGTG TCG-3' (SEQ ID NO:11); reverse primer, 5'-GCAATGGATCGCCG TCCAAAATCA-3' (SEQ ID NO:12). S. achromogenes genomic DNA was digested with AatII, ApaI, BsaWI, BssHII, EaeI, HaeII, MseI, NcoI, NlaIII, PvuI or TaqI restriction enzymes. The digested DNAs were self-ligated at a low DNA concentration (less than 2 .mu.g/per ml). The ligated circular DNA was used as templates for inverse PCR reaction (95.degree. C. 1 min, 60.degree. C. 1 min, 72.degree. C. 1 min, 30 cycles). Inverse PCR products were found in the ligated DNA of AatII, BsaW1, BssHII, NlaIII, PvuI, and HaeII digested/self-ligated DNA. The inverse PCR products from HaeII and PvuI were treated with T4 DNA kinase and DNA polymerase and cloned into HincII-cleaved/CIP treated pUC19 vector. The insert was sequenced until a stop codon was found. The entire SacI endonuclease gene was found to be 1077 bp, encoding a protein of 41 kDa.

6. Expression of SacI endonuclease in E. coli.

The SacI methylase gene was subcloned into a pSC101 derivative pLG339 to premodify E. coli host. The entire SacI endonuclease gene was amplified by PCR with two primers. The forward primer contains the ribosome binding site and 7 bp spacing before the ATG start codon (forward primer, 5'-CATGGGAAGCTTGGAGGTTTAAAAATGGGAATAA CAATTAAAAAGAGCACG-3' (SEQ ID NO:13); reverse primer, 5'-TCTGG ATCCCGGCG ATACATTGCCTCAGGAAAG-3' (SEQ ID NO:14)). The SacI endonuclease gene flanked by HindIII and BamHI sites was cloned into expression vector pRRS and transformed into SacI methylase premodified cells. 500 ml of cells (NEB#963) carrying pRRS-SacIR.sup.+ and pLG-SacIM.sup.+ was grown to 120 klett units at 30.degree. C. in LB plus Ap (100 .mu.g/ml) and Km (50 .mu.g/ml) and the SacI production was induced for 4 hours by addition of IPTG to 0.5 mM. Cells were harvested and resuspended in 30 ml of sonication buffer. Cell lysis was completed by addition of lysozyme to 100 .mu.g/ml and sonication. Cell debris was removed by centrifugation. The cell extract was diluted 10, 100, 1000, and 10000-fold in TE buffer. 5 .mu.l of the diluted extract was used to digest 1 .mu.g HindIII-cleaved .lambda. DNA for 1 hour at 37.degree. C. The digested DNA was resolved in an 0.8% agarose gel. It was found that the clone makes 1.5.times.10.sup.6 units of SacI endonuclease/gram of wet E. coli cells. A sample of E. coli RR1 containing the SacI restriction modification system (NEB#963) has been deposited under the Budapest Treaty with the American Type Culture Collection on Mar. 22, 1995 and received ATCC Accession No. 69767 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 14 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGAGGTAAATAA12 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1168 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1167 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GTGAACCATGAGCTTCCTGTGATCAGTCTGTTCTCCGGGGCCGGTGGC48 MetAsnHisGluLeuProValIleSerLeuPheSerGlyAlaGlyGly 1 51015 CTCGACTGCGCCATCGAGAGCTGCGCTGAGCCACCGCTGGTCCAGGAC96 LeuAspCysAlaIleGluSerCysAlaGluProProLeuValGlnAsp 202530 GGCTCCGGGTCTCCGCTACGTGTCGCGGTTGCCACTGACTATGAGCAG144 GlySerGlySerProLeuArgValAlaValAlaThrAspTyrGluGln 35 4045 ACCGCTCTGGATACACTGTCGGCCAACTTCCCGCACACCAAGACGCTG192 ThrAlaLeuAspThrLeuSerAlaAsnPheProHisThrLysThrLeu 5055 60 TGTGGGGATATCCAAACGATCCCGACCGCAGAGTTGCTGGAAGCCGGC240 CysGlyAspIleGlnThrIleProThrAlaGluLeuLeuGluAlaGly 6570 7580 GGGCTGAAGCCCGGCGATCCCACCCTGGTCATCGGTGGTCCTCCTTGT288 GlyLeuLysProGlyAspProThrLeuValIleGlyGlyProProCys 8590 95 ACGCCGTTCAGTAAGTCCGGATTCTGGATCGAGGAGAAGCGCAACAGC336 ThrProPheSerLysSerGlyPheTrpIleGluGluLysArgAsnSer 100105 110 GCCGATCCCAATGCCTCCCTCCTTGACGAGTACGTCCGTGTGGTTCGG384 AlaAspProAsnAlaSerLeuLeuAspGluTyrValArgValValArg 115120 125 GAGAGCAAGCCCGAAGCCTTCATCCTGGAAAATGTGCAGGGCCTGACG432 GluSerLysProGluAlaPheIleLeuGluAsnValGlnGlyLeuThr 130135140 TAC AAGACCCATCAGGCTCAGTTCGACCGACTCATTGCGGGCCTTAAG480 TyrLysThrHisGlnAlaGlnPheAspArgLeuIleAlaGlyLeuLys 145150155160 GATGCGGGCTACAACCCGACGTTCCGTGTGCTCCTTGCGGCCGAGTAT528 AspAlaGlyTyrAsnProThrPheArgValLeuLeuAlaAlaGluTyr 165170175 G GGGTTCCGCAGCTCCGCAGGCGAGTATTCGTTGTGGGGCGACGTGAC576 GlyValProGlnLeuArgArgArgValPheValValGlyArgArgAsp 180185190 GGTAA GGCTTTCCACTTCCCCGAAACCACGCACTCTGGCGAGTCGGAG624 GlyLysAlaPheHisPheProGluThrThrHisSerGlyGluSerGlu 195200205 CGGGATCGGGTG ATTGATCACACCAAGATTCCGTTCACGAGTCTTCGC672 ArgAspArgValIleAspHisThrLysIleProPheThrSerLeuArg 210215220 GAGGCACTTGCCGGACTGCCG GACGTTCCTGAGGCTGGAGAAGTTGTT720 GluAlaLeuAlaGlyLeuProAspValProGluAlaGlyGluValVal 225230235240 GAGGGAACCTACGCCGAAC TCGCTGCGGAAGTTCCTCCTGGGCAAAAC768 GluGlyThrTyrAlaGluLeuAlaAlaGluValProProGlyGlnAsn 245250255 TACCTATGGCACACCGACCG ATATGGCGGTCGCAACGAGTTCAAATGG816 TyrLeuTrpHisThrAspArgTyrGlyGlyArgAsnGluPheLysTrp 260265270 CGTAGCCGCTATTGGACCTTCCTG CTGAAGGCGGACCCAGACCGCCCT864 ArgSerArgTyrTrpThrPheLeuLeuLysAlaAspProAspArgPro 275280285 TCTACGACGCTGCAGGCTCAGCCAGGTCCA TGGGTCGGACCATTTCAC912 SerThrThrLeuGlnAlaGlnProGlyProTrpValGlyProPheHis 290295300 TGGGAGAACGTGAAGAACGCGAACGGGGAAGAGCGAGCGC GTAGGTTT960 TrpGluAsnValLysAsnAlaAsnGlyGluGluArgAlaArgArgPhe 305310315320 CGCGTTGCCGAAATGAAGCGGATCATGACTTTCCCTGA TGAGTTCGTT1008 ArgValAlaGluMetLysArgIleMetThrPheProAspGluPheVal 325330335 TTCACCGGAGTGAAGCGCGAGGTGCAGCGGCAGATCGG TAATCCCGTC1056 PheThrGlyValLysArgGluValGlnArgGlnIleGlyAsnProVal 340345350 CCGGTGGAGTTGGGGAAGGTCGTCGTCCGGGCCCTGATGGA ACAACTC1104 ProValGluLeuGlyLysValValValArgAlaLeuMetGluGlnLeu 355360365 GGCTATCTTGATTCTCGCGGTACTACTATTCCTAGCCAGGCTGGACA C1152 GlyTyrLeuAspSerArgGlyThrThrIleProSerGlnAlaGlyHis 370375380 GAACAGCTTGAATTGA1168 Glu GlnLeuGluLeu 385 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 389 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: MetAsnHisGluLeuProValIleSerLeuPheSerGlyAlaGl yGly 151015 LeuAspCysAlaIleGluSerCysAlaGluProProLeuValGlnAsp 202530 GlySe rGlySerProLeuArgValAlaValAlaThrAspTyrGluGln 354045 ThrAlaLeuAspThrLeuSerAlaAsnPheProHisThrLysThrLeu 50 5560 CysGlyAspIleGlnThrIleProThrAlaGluLeuLeuGluAlaGly 65707580 GlyLeuLysProGlyAspProThrLeuVa lIleGlyGlyProProCys 859095 ThrProPheSerLysSerGlyPheTrpIleGluGluLysArgAsnSer 100105 110 AlaAspProAsnAlaSerLeuLeuAspGluTyrValArgValValArg 115120125 GluSerLysProGluAlaPheIleLeuGluAsnValGlnGlyLeuThr 130135140 TyrLysThrHisGlnAlaGlnPheAspArgLeuIleAlaGlyLeuLys 145150155160 AspAlaGlyTy rAsnProThrPheArgValLeuLeuAlaAlaGluTyr 165170175 GlyValProGlnLeuArgArgArgValPheValValGlyArgArgAsp 180 185190 GlyLysAlaPheHisPheProGluThrThrHisSerGlyGluSerGlu 195200205 ArgAspArgValIleAspHisThrLysIle ProPheThrSerLeuArg 210215220 GluAlaLeuAlaGlyLeuProAspValProGluAlaGlyGluValVal 225230235 240 GluGlyThrTyrAlaGluLeuAlaAlaGluValProProGlyGlnAsn 245250255 TyrLeuTrpHisThrAspArgTyrGlyGlyArgAsnGluPheLys Trp 260265270 ArgSerArgTyrTrpThrPheLeuLeuLysAlaAspProAspArgPro 275280285 SerThrThrLe uGlnAlaGlnProGlyProTrpValGlyProPheHis 290295300 TrpGluAsnValLysAsnAlaAsnGlyGluGluArgAlaArgArgPhe 305310 315320 ArgValAlaGluMetLysArgIleMetThrPheProAspGluPheVal 325330335 PheThrGlyValLysArgGluValGln ArgGlnIleGlyAsnProVal 340345350 ProValGluLeuGlyLysValValValArgAlaLeuMetGluGlnLeu 355360 365 GlyTyrLeuAspSerArgGlyThrThrIleProSerGlnAlaGlyHis 370375380 GluGlnLeuGluLeu 385 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1077 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1074 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATGGGAATAACAATTAAAAAGAGCACGGCGGAGCAGGTTTTGAGG AAG48 MetGlyIleThrIleLysLysSerThrAlaGluGlnValLeuArgLys 151015 GCGTACGAGGCGGCCGCTTCGGATGATGTATTCCTCGAAGATTGGAT A96 AlaTyrGluAlaAlaAlaSerAspAspValPheLeuGluAspTrpIle 202530 TTTCTGGCTACTTCGCTACGCGAGGTAGATGCTCCAAGGACTTACACG144 PheLeuAlaThrSerLeuArgGluValAspAlaProArgThrTyrThr 354045 GCTGCGCTTGTGACAGCACTGCTCGCGCGTGCATGTGACGACAGGGTT192 AlaAlaL euValThrAlaLeuLeuAlaArgAlaCysAspAspArgVal 505560 GACCCACGCTCTATCAAAGAGAAGTACGACGATCGCGCCTTCTCTCTG240 AspProArgSerIleLys GluLysTyrAspAspArgAlaPheSerLeu 65707580 CGTACCCTTTGCCACGGTGTAGTGGTTCCGATGTCCGTAGAACTTGGA288 ArgThrLeuCysHisG lyValValValProMetSerValGluLeuGly 859095 TTTGATCTGGGGGCTACGGGCCGCGAGCCGATCAACAATCAGCCATTC336 PheAspLeuGlyAlaThr GlyArgGluProIleAsnAsnGlnProPhe 100105110 TTCCGTTACGACCAATACAGCGAGATCGTCAGGGTCCAGACGAAGGCG384 PheArgTyrAspGlnTyrSer GluIleValArgValGlnThrLysAla 115120125 CGCCCCTATCTGGACCGAGTAAGCAGTGCCCTTGCTAGGGTCGATGAA432 ArgProTyrLeuAspArgValSerSerA laLeuAlaArgValAspGlu 130135140 GAAGACTATTCCACTGAAGAGTCGTTCCGAGCGCTTGTCGCTGTCTTG480 GluAspTyrSerThrGluGluSerPheArgAlaLeuVa lAlaValLeu 145150155160 GCCGTGTGTATCTCTGTGGCGAACAAAAAGCAGCGCGTTGCCGTCGGG528 AlaValCysIleSerValAlaAsnLysLysGlnArg ValAlaValGly 165170175 AGTGCCATCGTCGAGGCGAGCCTAATCGCTGAGACTCAGAGCTTCGTT576 SerAlaIleValGluAlaSerLeuIleAlaGluThr GlnSerPheVal 180185190 GTGAGTGGCCACGACGTTCCACGAAAGCTGCAGGCTTGTGTTGCGGCT624 ValSerGlyHisAspValProArgLysLeuGlnAlaCysV alAlaAla 195200205 GGTCTTGACATGGTCTACAGCGAGGTCGTCTCGCGGAGGATCAATGAC672 GlyLeuAspMetValTyrSerGluValValSerArgArgIleAsnAs p 210215220 CCTTCTCGCGACTTTCCTGGTGACGTCCAAGTGATTTTGGACGGCGAT720 ProSerArgAspPheProGlyAspValGlnValIleLeuAspGlyAsp 225 230235240 CCATTGCTTACCGTTGAAGTGCGTGGAAAGAGCGTGTCGTGGGAGGGG768 ProLeuLeuThrValGluValArgGlyLysSerValSerTrpGluGly 245250255 CTTGAGCAATTCGTCAGCAGTGCAACGTATGCCGGCTTCCGACGAGTT816 LeuGluGlnPheValSerSerAlaThrTyrAlaGlyPheArgArgVal 260265270 GCGCTCATGGTCGATGCGGCGTCGCACGTGTCGCTTATGTCGGCCGAT864 AlaLeuMetValAspAlaAlaSerHisValSerLeuMetSerAlaAsp 275 280285 GATCTGACCAGCGCGCTTGAGCGGAAGTACGAGTGCATCGTCAAGGTG912 AspLeuThrSerAlaLeuGluArgLysTyrGluCysIleValLysVal 290 295300 AACGAGAGCGTGTCGAGCTTCCTGCGTGACGTATTCGTCTGGTCGCCT960 AsnGluSerValSerSerPheLeuArgAspValPheValTrpSerPro 305310 315320 CGCGACGTCCACTCGATTCTTTCGGCCTTTCCTGAGGCAATGTATCGC1008 ArgAspValHisSerIleLeuSerAlaPheProGluAlaMetTyrArg 325 330335 CGCATGATAGAGATCGAGGTGCGGGAGCCGGAGTTGGATCGGTGGGCT1056 ArgMetIleGluIleGluValArgGluProGluLeuAspArgTrpAla 340 345350 GAGATCTTCCCTGAAACGTGA1077 GluIlePheProGluThr 355 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 358 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: MetGlyIleThrIleLysLysSerThrAlaGluGlnValLeuArgLys 151015 AlaTyrGluAla AlaAlaSerAspAspValPheLeuGluAspTrpIle 202530 PheLeuAlaThrSerLeuArgGluValAspAlaProArgThrTyrThr 35 4045 AlaAlaLeuValThrAlaLeuLeuAlaArgAlaCysAspAspArgVal 505560 AspProArgSerIleLysGluLysTyrAspAspArgAlaPhe SerLeu 65707580 ArgThrLeuCysHisGlyValValValProMetSerValGluLeuGly 85909 5 PheAspLeuGlyAlaThrGlyArgGluProIleAsnAsnGlnProPhe 100105110 PheArgTyrAspGlnTyrSerGluIleValArgValGlnThrLysAla 115120125 ArgProTyrLeuAspArgValSerSerAlaLeuAlaArgValAspGlu 130135140 GluAspTyrSerThrGluGluSer PheArgAlaLeuValAlaValLeu 145150155160 AlaValCysIleSerValAlaAsnLysLysGlnArgValAlaValGly 165 170175 SerAlaIleValGluAlaSerLeuIleAlaGluThrGlnSerPheVal 180185190 ValSerGlyHisAspValProArgLysLeuGlnA laCysValAlaAla 195200205 GlyLeuAspMetValTyrSerGluValValSerArgArgIleAsnAsp 210215220 ProSer ArgAspPheProGlyAspValGlnValIleLeuAspGlyAsp 225230235240 ProLeuLeuThrValGluValArgGlyLysSerValSerTrpGluGly 245250255 LeuGluGlnPheValSerSerAlaThrTyrAlaGlyPheArgArgVal 260265270 AlaLeuMetValAsp AlaAlaSerHisValSerLeuMetSerAlaAsp 275280285 AspLeuThrSerAlaLeuGluArgLysTyrGluCysIleValLysVal 290295 300 AsnGluSerValSerSerPheLeuArgAspValPheValTrpSerPro 305310315320 ArgAspValHisSerIleLeuSerAlaPheProGluA laMetTyrArg 325330335 ArgMetIleGluIleGluValArgGluProGluLeuAspArgTrpAla 34034535 0 GluIlePheProGluThr 355 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: MetGlyIleThrIle LysLysSerGluAlaGluGlnValLeuArgLys 151015 AlaTyrGluAlaAlaAlaSerAspAspValPheLeuGluPhe 2025 30 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: IleThrIleLysLys 15 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AspAspValPheLeuGluPhe 15 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GAGTGAAGCGCGAGGTGCAGCGGCAGA27 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: AAACGAACTCTCAGGGAAAGTCATGA26 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GAAGTGCGTGGAAAGAGCGTGTCG24 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GCAATGGATCGCCGTCCAAAATCA 24 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GATGGGAAGCTTGGAGGTTTAAAAATGGGAATAACAATTAAAAAGAGCACG 51 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TCTGGATCCCGGCGATACATTGCCTCAGGAAAG 33 __________________________________________________________________________

## Claims

1. Isolated DNA coding for the SacI restriction endonuclease, wherein the isolated DNA is obtainable from the host Streptomyces achromogenes.

2. A recombinant DNA vector comprising a vector into which a DNA segment coding for the SacI restriction endonuclease has been inserted.

3. Isolated DNA coding for the SacI restriction endonuclease and methylase, wherein the isolated DNA is obtainable from ATCC No. 69767.

4. A cloning vector which comprises the isolated DNA of claim 3.

5. A host cell transformed by the cloning vector of claim 2 or 4.

6. A method of producing an SacI restriction endonuclease comprising culturing a host cell transformed with the vector of claim 2 or 4 under conditions suitable for expression of said endonuclease.