Method for cloning and expression of BsaI restriction endonuclease and BsaI methylase in E. coli

The present invention relates to recombinant DNA encoding the BsaI restriction endonuclease (BsaI endonuclease or BsaI) as well as BsaI methyltransferases (BsaI methylases, M. BsaIA and M.BsaIB, or M. BsaIAB), as well as expression of BsaI endonuclease and methylase in E. coli cells containing the recombinant DNA.

BsaI endonuclease is found in the strain of Bacillus stearothermophilus 6-55 (New England Biolabs' strain collection #481; Beverly, Mass.) It binds to the double-stranded DNA sequence 5′GGTCTC3′ N1/N5 and cleaves downstream sequence at N1 (top strand) and N5 (bottom strand) to generate a 4-base 5′ overhang (/ indicates the cleavage of phosphodiester bond). BsaI methylases (M.BsaIA and M.BsaIB) are also found in the same strain. M.BsaIA is an adenine methylase, presumably modifying the adenine on the bottom strand (5′GAGACC3′). M.BsaIB is a C5 methylase and presumably modifies the top strand of BsaI site.

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

Restriction endonucleases recognize and bind particular sequences of nucleotides (the ‘recognition sequence’) on DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313 (1999)).

Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5′TTT/AAA3′, 5′PuG/GNCCPy3′ and 5′CACNNN/GTG3′ respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5′G/AATTC3′.

A second component of bacterial/viral restriction-modification (R-M) systems are the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign 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 particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.

With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by ‘shotgun’ procedures, when they occur at frequencies as low as 10 −3 to 10 −4 . Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.

A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. 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 expressions 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 genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.

Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. 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); Tsp45I: Wayne et al. Gene 202:83-88 (1997)).

A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421 (1985)). Since restriction-modification genes are often closely linked together, 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); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119 (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241,(1983)).

A more recent method, the “endo-blue method”, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403 (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that some positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.

There are three major groups of DNA methylases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632 (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer the DNA site resistant to restriction digestion. For example, Dcm methylase modification of 5′CCWGG3′ (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpG methylase can modify the CG dinucloetide and make the NotI site (5′GCGGCCGC3′) refractory to NotI digestion (New England Biolabs' Catalog, 2000-01, page 220; Beverly, Mass.). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.

Type II methylase genes have been found in many sequenced bacterial genomes (GenBank, http://www.ncbi.nlm.nih.gov; and Rebase, http://rebase.neb.com/rebase). Direct cloning and over-expression of ORFs adjacent to the methylase genes yielded restriction enzymes with novel specificities (Kong et al. Nucl. Acids Res. 28:3216-3223 (2000)). Thus microbial genome mining emerged as a new way of screening/cloning new type II restriction enzymes and methylases and their isoschizomers.

Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a great commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such over-expression strains should also simplify the task of enzyme purification.

The present invention relates to a method for cloning BsaI endonuclease gene (bsaIR) from Bacillus stearothermophilus 6-55 into E. coli by inverse PCR and direct PCR amplification from genomic DNA. The inverse PCR primer sequences are based on the BsaI methylase gene (bsaIMB) sequence that derived from methylase selection.

It proved difficult to clone the bsaIM genes by the standard methylase selection method. ApoI, Sau3AI, and NlaIII partial genomic DNA libraries were constructed using a modified cloning vector pRRS (Ap R ). No methylase positive clones were identified following BsaI challenge and methylase selection. To increase the selection efficiency, a second step of mung bean nuclease treatment was included following BsaI digestion, which destroyed the DNA ends and inactivated the β-lactamase gene. This additional step increased the methylase selection efficiency and generated 20 BsaI resistant clones.

Since restriction gene is usually located in close proximity to the cognate methylase gene in a particular R-M system, inverse PCR was employed to clone the adjacent DNA surrounding the bsaIMB gene. Open reading frames (ORF) were identified on both sides of the bsaIMB gene. A second methylase gene was found upstream and the restriction gene was found downstream.

BsmAI site (5′GTCTC) overlaps with BsaI site (5′GGTCTC) and M.BsmAI protects E. coli chromosome DNA against BsaI digestion. To express bsaIR gene in E. coli , a non-cognate methylase gene, bsmAIM gene (M1::M2 fusion) was first cloned in pBR322 to pre-modify T7 expression host ER2566. The bsaIR gene was amplified by PCR, ligated to pACYC-T7ter with compatible ends, and transformed into pre-modified host ER2566 [pBR322-BsmAIM]. Transformants were cultured and BsaI endonuclease activity was detected in IPTG-induced cell extracts. Three clones with high BsaI activity were sequenced and clone #5 was confirmed to contain the wild type sequence.

Two more expression strains were constructed using the cognate methylase genes, ER2566 [pBR-BsaIMA&B, pACYC-T7ter-BsaIR] or ER2683 [pACYC-BsaIMA&B, pUC19-BsaIR]. Neither strain generated more BsaI units per gram of wet cells. Therefore, the non-cognate methylase modified strain ER2556 [pBR322-BsmAIM1M2, pACYC-T7ter-BsaI] was used as the BsaI production strain.

FIG. 1 Gene organization of BsaI restriction-modification system. bsaIMA, BsaI methylase 1 gene; bsaIMB. BsaI methylase 2 gene; bsaIR, bsaI restriction endonuclease gene.

FIG. 2 DNA sequence of M.BsaIA gene (bsaIMA, 1710 bp) (SEQ ID NO:1) and its encoded amino acid sequence (SEQ ID NO:2).

FIG. 3 DNA sequence of M.BsaB gene (bsaIMB, 1188 bp) (SEQ ID NO:3) and its encoded amino acid sequence (SEQ ID NO:4).

FIG. 4 DNA sequence of BsaI endonuclease gene (bsaIR, 1635 bp) (SEQ ID NO:5) and its encoded amino acid sequence (SEQ ID NO:6).

FIG. 5 Recombinant BsaI restriction endonuclease activity assay using cell extract. Lane 1, positive control, T7 DNA digested with purified native BsaI, lanes 2 to 11, T7 DNA treated with diluted cell extract containing recombinant BsaI endonuclease; The dilution factors in lanes 2 to 11 were: 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200. Lane 12, 1 kb DNA size marker.

FIG. 6 Purified recombinant BsaI endonuclease protein on SDS-PAG gel. Lane 1, broad range protein size marker; lane 2, purified recombinant BsaI endonuclease. BsaI endonuclease apparent size=61 kDa; Predicted size=63.7 kDa.

It was difficult to isolate M.BsaI positive clones among the ApoI, Sau3AI, and NlaIII genomic DNA libraries. After BsaI challenge of ApoI, Sau3AI, and NlaIII plasmid DNA libraries, the digested DNA was transferred into ER2502 to screen M.BsaI positive clones. However, after screening 36 Ap R survivors, no M.BsaI positive clones were identified. In order to increase the BsaI challenge efficiency and reduce the background of transformation, the BsaI-digested DNA was further treated with mung bean nuclease. This nuclease removes the 4-base ends and causes open reading frame shift in the β-lactamase gene, thus inactivating the Ap R gene. This strategy proved successful in cloning the bsaIMB gene.

The method described herein by which the bsaIMA, bsaIMB, and bsaIR genes are preferably cloned and expressed in E. coli using the following steps:

1. Construction of Genomic DNA Libraries and Methylase Selection

Genomic DNA was prepared from Bacillus stearothermophilus 6-55 and digested with restriction enzymes ApoI, Sau3AI, and NlaIII. Genomic DNA libraries were constructed using a modified pRRS vector with two BsaI sites. The ligated DNA was transferred into restriction minus E. coli ER2502 competent cell by transformation. Approximately 10 4 transformants were pooled and amplified overnight in 0.5 L culture. Primary plasmid DNA libraries were prepared by Qiagen Maxi column method and challenged with BsaI and mung bean nuclease. Following digestion, the plasmids were transformed into ER2502. Plasmids were prepared from Ap R survivors and screened for resistance to BsaI digestion. The resistant clones were identified as true methylase positive clones by DNA sequencing. The entire insert was sequenced with pUC19 primers and custom-made primers. One ORF of 1188 bp was found and was named bsaIMB, encoding a 395-aa protein with predicted molecular mass of 44.4 kDa. M.BsaIB shows extensive homology with other C5 methylases. However, the conserved motifs IX and X are located at the N-terminus instead of at C-terminus. Inverse PCR was used to amplify the adjacent DNA sequence. After five rounds of inverse PCR two more genes were identified. The upstream gene, bsaIMA, is 1710 bp, encoding an amino-methyltransferase (569-aa protein) with predicted molecular mass of 65.7 kDa.

2. Cloning of bsaIR Gene by Inverse PCR

Inverse PCR primers were made based on the DNA sequence of bsaIMB gene. Genomic DNA was digested with restriction enzymes with 4-6 base recognition sequences and self-ligated. The circular DNA molecules were used as the templates for inverse PCR. Inverse PCR products downstream of bsaIMB were obtained, gel-purified and sequenced. An ORF of 1635 bp was found downstream of the bsaIMB gene. This ORF was named bsaIR gene. It encodes a 544-aa protein with predicted molecular mass of 63.7 kDa.

3. Cloning of bsmAIM Gene into pBR322 to Construct a Premodified Host

Since BsmAI recognition sequence (5′GTCTC′) overlaps with BsaI recognition sequence (5′GGTCTC3′), M.BsmAI cross-protects E. coli chromosome DNA and plasmid DNA against BsaI digestion. Efforts were made to over-express M.BsmAI first and to construct a pre-modified expression host (BsmAI restriction-modification filed Sep. 20, 2001 received U.S. Ser. No. 09/957,005).

The bsmAIM gene was amplified from the genomic DNA by PCR using two primers. The PCR DNA was digested with NheI and SphI and ligated to pBR322 with compatible ends. The pre-modified host ER2566 [pBR322-BsmAIM] was used for over-expression of bsaIR gene in E. coli.

4. Expression of bsaIR Gene in T7 Expression Vector pACYC-T7ter

A BamHI fragment containing bsaIR gene was cloned into pACYC-T7ter expression vector. The ligated DNA was transformed into pre-modified host ER2566 [pBR322-BsmAIM]. Ap R Cm R transformants were cultured and induced with IPTG. Recombinant BsaI activity was detected in the supernatant of IPTG-induced cell extracts. Plasmids were extracted from those clones with high BsaI activity. After sequencing the insert, the clone with wild type sequence was used for stability study and purification of BsaI endonuclease.

5. Expression of bsaIR Gene in Cognate Methylase-Modified Host.

A second expression strain was constructed using the cognate methylase genes, ER2566 [pBR-BsaIMA&B, pACYC-T7ter-BsaIR]. The recombinant BsaI yield from this expression clone was estimated at 0.7-1.4×10 6 units BsaI/g of wet cell. The first expression strain ER2566 [pBR322-BsmAIM1M2, pACYC-T7ter-BsaIR] produces 1.0-2.0×10 6 units of BsaI/g of wet cell. A third expression strain was also constructed in which the bsaIMA and bsaIMB genes were expressed from pACYC184 and the bsaIR gene was expressed from pUC19, ER2683 [pACYC-BsaIMA&B, pUC19-BsaIR]. This strain also generated less BsaI units per gram of wet cells. Therefore, the strain ER2556 [pBR322-BsmAIM1M2, pACYC-T7ter-BsaI] described in section 4 was used as the BsaI production strain.

6. Purification of BsaI Restriction Endonuclease

IPTG-induced cell extracts containing recombinant BsaI endonuclease was purified by heat treatment at 55° C. for one hour to denature E. coli proteins. The heat-denatured proteins were removed by centrifugation. The supernatant with BsaI activity was further purified by chromatography through Heparin-Sepharose and DEAE-Sepharose columns. The purified BsaI protein was analyzed on SDS-PAGE and the N-terminus of the recombinant BsaI protein was sequenced to obtain the first 20 aa residues. The actual aa sequence was in total agreement with the predicted aa sequence based on the DNA coding sequence.

The present invention is further illustrated by the following Example. This Example is provided to aid in the understanding of the invention and is not construed as a limitation thereof.

The references cited above and below are herein incorporated by reference.

Cloning of BsaI Restriction-Modification System in E. coli

1. Preparation of Genomic DNA and Restriction Digestion of Genomic DNA and Construction of Genomic DNA Libraries

Genomic DNA was prepared from Bacillus stearothermophilus 6-55 (New England Biolabs' collection #481; Beverly, Mass.) by the standard procedure consisting of the following steps:

(a) cell lysis by addition of lysozyme (2 mg/ml final), sucrose (1% final), and 50 mM Tris-HCl, pH 8.0;

(b) cell lysis by addition of 10% SDS (final concentration 0. 1%);

(c) further cell lysis by addition of 1% Triton X-100 and 62 mM EDTA, 50 mM Tris-HCl, pH 8.0;

(d) Phenol-CHCl 3 extraction of DNA 3 times (equal volume) and CHCl 3 extraction once;

(e) DNA dialysis in 4 liters of TE buffer, change 3 times; and

(f) RNA removal by RNase A treatment and genomic DNA precipitated with 95% ethanol, washed with 70% ethanol, vacuum dried and resuspended in TE buffer.

Restriction enzyme ApoI was diluted by 2-fold serial dilutions. Nine μg genomic DNA was digested partially with varying amounts of ApoI (2, 1, 0.5, 0.25, 0.125, 0.061 units) at 50° C. for 30 min. Good partial digestion was achieved at 0.5, 0.25, and 0.125 units of ApoI. The ApoI partially digested genomic DNA fragments in the range of 3 to 10 kb were gel-purified and ligated to CIP treated pRRS vector with compatible ends. The PRRS vector has been modified by insertion of a BsaI linker at the SspI site. The purpose of this modification was to increase the efficiency of methylase selection. Partial digestions were also carried out with Sau3AI and NlaIII on genomic DNA (2, 1, 0.5, 0.25, 0.125, 0.061 units of Sau3AI; 0.12, 0.06, 0.03, 0.015, 0.0075 units of NlaIII). Again, the DNA fragments in the range of 3 to 10 kb were gel-purified and ligated to BamHI and SphI digested pRRS, respectively. The ligated DNA was used to transform ER2502 electro-competent cells by electroporation. Ap R transformants were selected on Ap plates (100 μg/ml Ap).

2. Cloning of bsaIMB by Methylase Selection Method

More than 10,000 Ap R transformants were obtained from ApoI, NlaIII, and Sau3AI libraries. All of the colonies were pooled and amplified in 1 liter LB+Ap overnight culture. Plasmid DNA was prepared by the Qiagen Maxi-prep kit. 0.8 ng-0.2 μg of the library DNA was challenged with 100 units of BsaI at 50° C. for 2 hours. The challenged plasmid DNA was used for re-transformation into ER2502 and plated on Ap plates. Surviving Ap R transformants were selected. Thirty-six colonies were inoculated into 2 ml LB+Ap and cultured overnight. Plasmid DNA was prepared by Qiagen spin columns and screened for resistance to BsaI digestion. None of the 36 plasmids showed resistance to BsaI digestion.

In order to increase the methylase selection efficiency, after BsaI digestion of plasmid library, the digested plasmids were further treated with 100 units of mung bean nuclease at 37° C. for 1 hour. The mung bean nuclease removed the 4-base overhang and destroyed the AP R resistant gene (4-base deletion within the β-lactamase gene inactivates this gene). The same plasmid library was challenged with BsaI and split into two half. Half of the DNA was further treated with mung bean nuclease. Both DNA samples were used to transform ER2502 competent cells. It was found that the mung bean nuclease treated DNA gave rise to 5-times less transformants than non-treated DNA. Thirty-six individual transformants from the mung bean nuclease treated DNA were picked and inoculated into 2 ml LB+Ap and cultured overnight. Plasmid DNA was prepared and analyzed by BsaI digestion and agarose gel electrophoresis. Twenty out of 36 screened showed resistance. Isolates #7, #8, #10, #12, and #13 have a common 1.3 kb HindIII fragment by HindIII digestion. This common HindIII fragment was subcloned into pUC19 and sequenced, it was found to encode a C5 methylase. This gene is 1188-bp and was named bsaIMB gene, encoding M.BsaIB. Since BsaI recognition sequence is asymmetric and it was predicted that a second methylase should be present in this R-M system.

3. Inverse PCR Amplification and Sequencing of Adjacent DNA and Identification of bsaIR Gene

Since type II R-M genes are usually located in close proximity to each other, efforts were made to amplify the DNA sequence adjacent to bsaIMB gene. B. stearothermophilus 6-55 genomic DNA was digested with restriction enzymes with 4 to 6-bp recognition sequence to identify DNA fragments that encompass bsaIMB gene and the flanking DNA. B. stearothermophilus genomic DNA was digested with AatII, AseI, BamHI, BssHII, DraI, HaeII, MfeI, NcoI, NdeI, NspI, PstI, SacI, SpeI, SspI, XbaI, and XhoI, respectively. The genomic DNA fragments were self-ligated at a low concentration (2 μg/ml), and the ligated circular molecules were used as the template for inverse PCR. The inverse PCR primers have the following sequence:

Inverse PCR conditions were as follows: 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 3 min, running for 30 cycles with Vent® (exo − ) DNA polymerase. A 1.2 kb inverse PCR product was found in the NdeI template. The 1.2 kb fragment was gel-purified and sequenced directly with primers 258-98 and 99, generating approximately 700 bp new DNA sequence.

A second set of inverse PCR primers with the following sequences were synthesized:

AatII, AseI, BamHI, BssHII, DraI, HaeII, MfeI, NcoI, NdeI, NspI, PstI, SacI, SpeI, SspI, XbaI, and XhoI digested and self-ligated DNA molecules were used as the template for inverse PCR. Inverse PCR conditions were as follows: 94° C. for 30 sec, 55° C. for 30 sec. 72° C. for 3 min, running for 30 cycles with Deep Vent® (exo − ) DNA polymerase. A 600 bp inverse PCR product was found in the DraI template. The 600 bp fragment was gel-purified and sequenced directly with primers 259-137 and 138, generating approximately 200 bp new DNA sequence.

A third set of inverse PCR primers with the following sequences were synthesized:

AatII, AseI, BamHI, BssHII, DraI, HaeII, MfeI, NcoI, NdeI, NspI, PstI, SacI, SpeI, SspI, XbaI, and XhoI digested and self-ligated DNA molecules were used as the template for inverse PCR. Inverse PCR conditions were as follows: 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 3 min, running for 30 cycles with Deep Vent® (exo − ) DNA polymerase. A 650 bp inverse PCR product was found in AseI template. The 650 bp fragment was gel-purified and sequenced directly with primers 259-139 and 140, generating approximately 400 bp new DNA sequence.

A fourth set of inverse PCR primers with the following sequences were synthesized:

B. stearothermophilus genomic DNA was digested with AciI, ApoI, BsaWI, BspHI, BstBI, HincII, HindIII, NlaIII, RsaI, SpeI, SspI, TaqI, TfiI, Tsp45I, and XmnI, respectively. The genomic DNA fragments were self-ligated at a low concentration (2 μg/ml), and the ligated circular molecules were used as the template for inverse PCR. Inverse PCR conditions were as follows: 94° C. for 30 sec. 55° C. for 30 sec, 72° C. for 3 min, running for 30 cycles with Vent® (exo − ) DNA polymerase and Taq DNA polymerase, respectively. A 1.2 kb inverse PCR product was found in SspI template. The 1.2 kb fragment was gel-purified and sequenced directly with primers 264-210 and 211, generating approximately 540 bp new DNA sequence.

A fifth set of inverse PCR primers with the following sequences were synthesized:

AciI, ApoI, BsaWI, BspHI, BstBI, HincII, HindIII, NlaIII, RsaI, SpeI, SspI, TaqI, TfiI, Tsp45I, and XmnI digested and self-ligated circular molecules were used as the template for inverse PCR. Inverse PCR conditions were as follows: 94° C. for 30 sec, 55° C. for 30 sec. 72° C. for 3 min, running for 30 cycles with Vent® (exo − ) DNA polymerase. 0.9, 1.6, and 1.2 kb inverse PCR products were found in ApoI, RsaI, and TaqI templates, respectively. The 1.2 kb fragment was gel-purified and sequenced directly with primers 270-223 and 224, generating approximately 600 bp new DNA sequence.

After five rounds of inverse PCR and sequencing of the original methylase positive clones, two additional ORFs were found upstream and downstream. The upstream ORF was named bsaIMA, encoding M.BsaIA, an adenine methyltransferase. Based on the amino acid sequence comparison with other amino-methylases, the M.BsaA methylase belongs to the γ type of N6A methylase.

Among C5 methylases, the conserved amino acid blocks IX and X are located at the C-terminus of the protein. Conserved blocks I to VIII and the variable region are located before blocks IX and X. However, in M.BsaIB, the C5 methylase blocks IX and X are located at the N-terminus, displaying circular permutation of the two blocks. Such circular permutation has been found in the BssHII methylase.

An ORF of 1635 bp was found downstream of the bsaIMA and bsaIMB genes. This ORF was named bsaIR gene. It encodes a 544-aa protein with predicted molecular mass of 63.7 kDa (FIG. 4 ). As demonstrated below, it is the bona fide R gene, coding for BsaI restriction endonuclease.

4. Cloning of bsmAIM1M2 Genes into pBR322 and Construction of a Pre-Modified Host

Since BsmAI recognition sequence (5′GTCTC′) overlaps with BsaI recognition sequence (5′GGTCTC3′), M.BsmAI cross-protects E. coli chromosome DNA against BsaI digestion. Efforts were made to over-express M.BsmAI first and to construct a pre-modified expression host. M.BsmAI is a fusion of M1 and M2. (BsmAI restriction-modification patent application U.S. Ser. No. 09/957,005 filed Sep. 20, 2001 The patent application for BsmAI restriction-modification is pending in United States.

Two primers were synthesized with the following sequence:

(SEQ ID NO:17)

5′ GGTGGT GCTAGC GGAGGTAAATAAATGAAAGAAAACACAGAAATTAA

TATAGAT 3′ (253-245, underlined nt, NheI site)

(SEQ ID NO:18)

5′ GGTGGT GCATGC CTAATATATTTCTTGGTACGTCATTTT 3′

(253-246, underlined nt, SphI site)

The bsmAIM gene was amplified from the genomic DNA in PCR using primers 253-245 and 253-246 under PCR condition of 95° C. 1 min, 55° C. 1 min, 72° C. 4 min for 25 cycles. The PCR DNA was purified through a Qiagen spin column and digested with NheI and SphI. The PCR fragment was purified again in low melting agarose gel and ligated to pBR322 with compatible ends. Ligated plasmid was transformed into ER2566 (T7 expression strain, NEB's collection). The Ap R transformants were pooled and plasmid DNA prepared. The plasmid mixture was challenged with BsmAI endonuclease and retransformed back into ER2566 cells. Four out of six clones were found to have the right size insert and resistant to BsmAI digestion. The pre-modified host ER2566 [pBR322-BsmAIM] was used for expression of the bsaIR gene in E. coli.

5. Expression of bsaIR Gene in T7 Expression Vector pACYC-T7ter

To construct a stable expression clone, the bsmAIM gene was expressed from a medium-copy-number vector pBR322 and the bsaIR gene was expressed from a low-copy-number vector pACYC-T7ter. The vector pACYC-T7ter contains a T7 promoter, Cm R gene, lacI gene, p15A replication origin, and four copies of transcription terminators upstream of T7 promoter to reduce run-off transcription from cryptic E. coli promoters.

NdeI and BamHI restriction sites were incorporated into the forward and reverse PCR primers respectively for amplification of bsaIR gene by PCR. The primers have the following sequence:

The bsaIR gene was amplified by PCR using Vent® DNA polymerase and primers 271-161 and 162 under conditions of 95° C. 1 min, 50° C. 1.5 min, 72° C. 1.5 min for 25 cycles. The PCR product was purified by Qiagen spin column and digested overnight with NdeI and BamHI. After purification from low-melting agarose gel and β-agarase treatment, the DNA was precipitated with ethanol and NaOAc. The PCR DNA was ligated to CIP-treated pACYC-T7ter with compatible ends. The ligated DNA was transformed into pre-modified host ER2566 [pBR322-BsmAIM] and selected for Ap R Cm R transformants. Individual transformants were then picked and cultured in 10 ml LB plus Ap (100 μg/ml) and Cm (33 μg/ml) to late log phase and induced with IPTG (0.5 mM final) for 3 h. Thirty-six cell extracts were assayed for BsaI activity. Seven clones (#2, #3, #5, #18, #21, #25, and #32) displayed high BsaI activity. Three plasmids (#2, #3, #5) from highly active clones were sequenced, and #5 were found to contain the wild type sequence and was used in subsequent large-scale purification of BsaI endonuclease protein.

6. Cloning of bsaIMA and bsaIMB Gene into pBR322 to Construct a Pre-Modified Host

Two primers were synthesized with the following sequences:

(SEQ ID NO:21)

5′ GGTGGT GGATCC GGAGGTAAATAAATGAGTAATGCTAAAAGTTTCTC

T3′

(270-148, underlined nt, BamHI site)

(SEQ ID NO:22)

5′ GGTGGT GCATGC TTATATTATCGCTAAACTGCTCAA 3′

(270-150, underlined nt, SphI site)

The bsaIMA and bsaIMB genes were amplified by Vent® DNA polymerase from genomic DNA in PCR using primers 270-148 and 270-150 under PCR condition of 95° C. 1 min, 55° C. 1.5 min, 72° C. 4 mmin for 25 cycles. The PCR DNA was gel purified through a Qiagen spin column and digested with BamHI and SphI. The PCR fragment was purified again in low melting agarose gel and ligated to pBR322 with compatible ends. Ligated plasmid was transformed into ER2566 (T7 expression strain, NEB's strain collection). The Ap R transformants were pooled and plasmid DNA prepared. The plasmid mixture was challenged with BsaI endonuclease and retransformed back into ER2566 cells. Plasmid DNA was prepared again and digested with BsaI endonuclease. One out of three clones were found to have the right size insert and resistant to BsaI digestion. The pre-modified host ER2566 [pBR322-BsaIMA&B] was used for expression of the bsaIR gene in E. coli.

The plasmid pACYC-T7ter-BsaIR isolated in section 4 was transferred into ER2566 [pBR322-BsaIMA&B] by transformation. Ap R and Cm R colonies were selected and transformants were grown in 10 ml LB plus Ap and Cm overnight. The 10 ml cells were inoculated into 500 ml LB plus Ap and Cm and cultured for five hours. Following addition of 0.5 mM IPTG (final concentration) cell growth was continued for 3 h. Cells were harvested and resuspended in a sonication buffer. Cell lysis was completed by sonication and cell debris was removed by centrifugation. Cell extract was diluted and assayed on T7 DNA. The recombinant BsaI yield from this expression clone was estimated at 0.7-1.4×10 6 units BsaI/g of wet cell. The recombinant BsaI activity from the previous expression strain ER2566 [pBR322-BsmAIM1M2, pACYC-T7ter-BsaIR] was 1.0-2.0×10 6 units of BsaI/g of wet cell. A third expression strain was also constructed in which the bsaIMA and bsaIMB genes were expressed from pACYC184 and the bsaIR gene was expressed from pUC19. This strain also generated less BsaI units per gram of wet cells. Therefore, the strain ER2556 [pBR322-BsmAIM1M2, pACYC-T7ter-BsaI] was used as the BsaI production strain.

7. Purification of BsaI Endonuclease

Cell extract was prepared by sonication of 4 grams of IPTG-induced cells resuspended in 20 ml sonication buffer (50 mM Tris-HCl, pH 7.8, 10 mM β-mercaptoethanol). Cell debris was removed by centrifugation at 15 k rpm for 30 min. The BsaI activity was measured. The cell extract was heated at 55° C. for one hour to denature E. coli thermolabile proteins. Denatured proteins were removed by centrifugation. The supernatant was loaded onto a 20 ml Heparin Sepharose column. Following extensive washing with low salt buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM β-mercaptoethanol, 0.1 mM EDTA), fractions were eluted with a NaCl gradient of 0.05 M-1 M. Fractions containing BsaI endonuclease as determined by an activity assay on λ DNA were pooled and dialyzed overnight in DEAE-Sepharose loading buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM β-mercaptoethanol, 0.1 mM EDTA). After dialysis, the protein mixture was loaded onto a DEAE Sepharose column equilibrated with the same buffer. Fractions were eluted with a 0.05 M-1 M NaCl gradient and those fractions containing purified BsaI were pooled. The recombinant BsaI was analyzed on SDS-PAGE. It was estimated to be >92% homogeneity (FIG. 6 ). A total of 10 6 units of functionally purified BsaI were obtained. The apparent molecular size of BsaI protein on the gel appeared to be 61 kDa, slightly smaller than the predicted size of 63.7 kDa. To determine the exact start amino acid of the recombinant BsaI endonuclease, the purified protein was subjected to N-terminal sequencing analysis. It was confirmed that the protein contained the correct N-terminal amino acid sequence:

(M) GKKAEYGQGHPIFLEYAEQ (SEQ ID NO:23).

The migration difference may arise from factors such as the aa residue composition and side chain charges.

The strain ER2566 [pBR322-BsmAIM, pACYC-T7ter-BsaIR] has been deposited under the terms and conditions of the Budapest Treaty with the American Type Culture Collection on Mar. 26, 2002 and received ATCC Accession No. PTA-4181.

<160> NUMBER OF SEQ ID NOS: 23

<210> SEQ ID NO 1

<211> LENGTH: 1710

<212> TYPE: DNA

<213> ORGANISM: Bacillus stearothermophilus 6-55

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1)..(1710)

<223> OTHER INFORMATION:

<400> SEQUENCE: 1

ttg agt aat gct aaa agt ttc tct ctt aac ga

#a aaa aca gaa gct aat       48

Leu Ser Asn Ala Lys Ser Phe Ser Leu Asn Gl

#u Lys Thr Glu Ala Asn

1               5

#                10

#                15

gct cta ata gat ttt att att gaa aaa tct aa

#t caa agt aaa gac ttg       96

Ala Leu Ile Asp Phe Ile Ile Glu Lys Ser As

#n Gln Ser Lys Asp Leu

20

#            25

#            30

ggt tat tgg tta caa aaa tca aaa gga cag tt

#t tat aca cat aat ttt      144

Gly Tyr Trp Leu Gln Lys Ser Lys Gly Gln Ph

#e Tyr Thr His Asn Phe

35

#        40

#        45

att ggt gaa aaa tta gtt aca gaa ata gta ga

#a aat ata aaa ttt aat      192

Ile Gly Glu Lys Leu Val Thr Glu Ile Val Gl

#u Asn Ile Lys Phe Asn

50

#    55

#    60

gat gat agt gaa gtt ata aaa ata att gac cc

#t ttt tgt gga gat gga      240

Asp Asp Ser Glu Val Ile Lys Ile Ile Asp Pr

#o Phe Cys Gly Asp Gly

65

#70

#75

#80

cgc tta ata tgc att ttg tta gat aaa ttt aa

#t gct ata aat aaa ttt      288

Arg Leu Ile Cys Ile Leu Leu Asp Lys Phe As

#n Ala Ile Asn Lys Phe

85

#                90

#                95

aga aat acc ttg tta gag att gaa ttt tgg ga

#t att gac cct gaa gct      336

Arg Asn Thr Leu Leu Glu Ile Glu Phe Trp As

#p Ile Asp Pro Glu Ala

100

#           105

#           110

gta gaa gtt gct tat acc aat ata aaa gag aa

#a gct aat gca tta gaa      384

Val Glu Val Ala Tyr Thr Asn Ile Lys Glu Ly

#s Ala Asn Ala Leu Glu

115

#       120

#       125

ttt aat gta caa cta aag ggg aga gta tgc ga

#t act ttt ctt ttt gct      432

Phe Asn Val Gln Leu Lys Gly Arg Val Cys As

#p Thr Phe Leu Phe Ala

130

#   135

#   140

caa gac tac ttt ggc tca tat gat atc tgt at

#t aca aac ccg cca tgg      480

Gln Asp Tyr Phe Gly Ser Tyr Asp Ile Cys Il

#e Thr Asn Pro Pro Trp

145                 1

#50                 1

#55                 1

#60

gta att ata aaa cca gat aaa aag gaa aag ga

#a agg tta agt aaa gaa      528

Val Ile Ile Lys Pro Asp Lys Lys Glu Lys Gl

#u Arg Leu Ser Lys Glu

165

#               170

#               175

gaa gag ata gag tat att gaa ata cta aag aa

#t ttt gat gat ttt ttg      576

Glu Glu Ile Glu Tyr Ile Glu Ile Leu Lys As

#n Phe Asp Asp Phe Leu

180

#           185

#           190

agt aga tac tat cct act tct tta cct aca aa

#a aaa tat gga gga tgg      624

Ser Arg Tyr Tyr Pro Thr Ser Leu Pro Thr Ly

#s Lys Tyr Gly Gly Trp

195

#       200

#       205

gga act aac tta gct aga tgt ggt aca gaa gt

#t gcg ctg aga tta att      672

Gly Thr Asn Leu Ala Arg Cys Gly Thr Glu Va

#l Ala Leu Arg Leu Ile

210

#   215

#   220

tca aaa gta ggg att tgt ggt ata gta tca cc

#a gca tcg ctt tta ggt      720

Ser Lys Val Gly Ile Cys Gly Ile Val Ser Pr

#o Ala Ser Leu Leu Gly

225                 2

#30                 2

#35                 2

#40

gac caa gta tcg gat aat ctt aga gtt tgg at

#g ttt aat aac tat gag      768

Asp Gln Val Ser Asp Asn Leu Arg Val Trp Me

#t Phe Asn Asn Tyr Glu

245

#               250

#               255

gtt tat agt att tct tac ttt gtt gca gaa gc

#t aaa tta ttt ggt aaa      816

Val Tyr Ser Ile Ser Tyr Phe Val Ala Glu Al

#a Lys Leu Phe Gly Lys

260

#           265

#           270

gta gac caa gca gtt att aca tta act tta tc

#a cct aaa tgt gat gat      864

Val Asp Gln Ala Val Ile Thr Leu Thr Leu Se

#r Pro Lys Cys Asp Asp

275

#       280

#       285

agt agt ggt gat att att ccg cat ctg ttt ta

#c tac gat aga gag cta      912

Ser Ser Gly Asp Ile Ile Pro His Leu Phe Ty

#r Tyr Asp Arg Glu Leu

290

#   295

#   300

ttt gaa aaa aga tat tat atg gat gaa tat ga

#t tgg cgt att ata aag      960

Phe Glu Lys Arg Tyr Tyr Met Asp Glu Tyr As

#p Trp Arg Ile Ile Lys

305                 3

#10                 3

#15                 3

#20

tca ctc aat tat gtt att cca att caa ttt gg

#t tta gag ata ata aaa     1008

Ser Leu Asn Tyr Val Ile Pro Ile Gln Phe Gl

#y Leu Glu Ile Ile Lys

325

#               330

#               335

atg aat aga tta ttt aag tct tta ccc act tt

#a ggg gac tta gag aac     1056

Met Asn Arg Leu Phe Lys Ser Leu Pro Thr Le

#u Gly Asp Leu Glu Asn

340

#           345

#           350

gaa aaa gaa ggg att tgg cta gga agg gaa ct

#a gat gaa acc gga ata     1104

Glu Lys Glu Gly Ile Trp Leu Gly Arg Glu Le

#u Asp Glu Thr Gly Ile

355

#       360

#       365

aag gaa aaa ttg gct aac aaa ggt caa tat cg

#t ttt atc aaa ggg aaa     1152

Lys Glu Lys Leu Ala Asn Lys Gly Gln Tyr Ar

#g Phe Ile Lys Gly Lys

370

#   375

#   380

atg gta gga aga tac aac cta att gaa gag tc

#t aat caa tat att gat     1200

Met Val Gly Arg Tyr Asn Leu Ile Glu Glu Se

#r Asn Gln Tyr Ile Asp

385                 3

#90                 3

#95                 4

#00

gtt aga aag ata gat aaa atc ccc aaa tct gt

#c gaa ttt tac aga ttg     1248

Val Arg Lys Ile Asp Lys Ile Pro Lys Ser Va

#l Glu Phe Tyr Arg Leu

405

#               410

#               415

gtc tgg aga gat gtc tca aga acg act caa aa

#a aga aga tta ata tca     1296

Val Trp Arg Asp Val Ser Arg Thr Thr Gln Ly

#s Arg Arg Leu Ile Ser

420

#           425

#           430

aca att att cca cct aaa tat att act ggc aa

#t tca ctt aat gta gct     1344

Thr Ile Ile Pro Pro Lys Tyr Ile Thr Gly As

#n Ser Leu Asn Val Ala

435

#       440

#       445

tat ttc aaa gat aat aat tta aag aaa tta aa

#a gct tta ctt gca atc     1392

Tyr Phe Lys Asp Asn Asn Leu Lys Lys Leu Ly

#s Ala Leu Leu Ala Ile

450

#   455

#   460

atg aat tca ttt gtt ttt gaa gct caa gta ag

#a gct aat tta tct aca     1440

Met Asn Ser Phe Val Phe Glu Ala Gln Val Ar

#g Ala Asn Leu Ser Thr

465                 4

#70                 4

#75                 4

#80

aat cat att tct ttg gga ata ata agg aga gc

#a cat ata ccg aag tta     1488

Asn His Ile Ser Leu Gly Ile Ile Arg Arg Al

#a His Ile Pro Lys Leu

485

#               490

#               495

gaa ggt aga gtt gtg gat gaa ctt tct caa ct

#a gtt gat aat tat gtt     1536

Glu Gly Arg Val Val Asp Glu Leu Ser Gln Le

#u Val Asp Asn Tyr Val

500

#           505

#           510

aat gaa gaa agc gaa tta cta tta gaa gta aa

#a gtt gct aaa gca tat     1584

Asn Glu Glu Ser Glu Leu Leu Leu Glu Val Ly

#s Val Ala Lys Ala Tyr

515

#       520

#       525

gga ctc tca ttt gaa gat ttt agt tca att ct

#t tct cta ttt gac aag     1632

Gly Leu Ser Phe Glu Asp Phe Ser Ser Ile Le

#u Ser Leu Phe Asp Lys

530

#   535

#   540

ata ggt aaa gac gaa aaa gaa aag ata cta ca

#a gta gca aaa aaa tat     1680

Ile Gly Lys Asp Glu Lys Glu Lys Ile Leu Gl

#n Val Ala Lys Lys Tyr

545                 5

#50                 5

#55                 5

#60

tta aag ggg gga ata aag aat gat tcc taa

#

#         1710

Leu Lys Gly Gly Ile Lys Asn Asp Ser

565

<210> SEQ ID NO 2

<211> LENGTH: 569

<212> TYPE: PRT

<213> ORGANISM: Bacillus stearothermophilus 6-55

<400> SEQUENCE: 2

Leu Ser Asn Ala Lys Ser Phe Ser Leu Asn Gl

#u Lys Thr Glu Ala Asn

1               5

#                10

#                15

Ala Leu Ile Asp Phe Ile Ile Glu Lys Ser As

#n Gln Ser Lys Asp Leu

20

#            25

#            30

Gly Tyr Trp Leu Gln Lys Ser Lys Gly Gln Ph

#e Tyr Thr His Asn Phe

35

#        40

#        45

Ile Gly Glu Lys Leu Val Thr Glu Ile Val Gl

#u Asn Ile Lys Phe Asn

50

#    55

#    60

Asp Asp Ser Glu Val Ile Lys Ile Ile Asp Pr

#o Phe Cys Gly Asp Gly

65

#70

#75

#80

Arg Leu Ile Cys Ile Leu Leu Asp Lys Phe As

#n Ala Ile Asn Lys Phe

85

#                90

#                95

Arg Asn Thr Leu Leu Glu Ile Glu Phe Trp As

#p Ile Asp Pro Glu Ala

100

#           105

#           110

Val Glu Val Ala Tyr Thr Asn Ile Lys Glu Ly

#s Ala Asn Ala Leu Glu

115

#       120

#       125

Phe Asn Val Gln Leu Lys Gly Arg Val Cys As

#p Thr Phe Leu Phe Ala

130

#   135

#   140

Gln Asp Tyr Phe Gly Ser Tyr Asp Ile Cys Il

#e Thr Asn Pro Pro Trp

145                 1

#50                 1

#55                 1

#60

Val Ile Ile Lys Pro Asp Lys Lys Glu Lys Gl

#u Arg Leu Ser Lys Glu

165

#               170

#               175

Glu Glu Ile Glu Tyr Ile Glu Ile Leu Lys As

#n Phe Asp Asp Phe Leu

180

#           185

#           190

Ser Arg Tyr Tyr Pro Thr Ser Leu Pro Thr Ly

#s Lys Tyr Gly Gly Trp

195

#       200

#       205

Gly Thr Asn Leu Ala Arg Cys Gly Thr Glu Va

#l Ala Leu Arg Leu Ile

210

#   215

#   220

Ser Lys Val Gly Ile Cys Gly Ile Val Ser Pr

#o Ala Ser Leu Leu Gly

225                 2

#30                 2

#35                 2

#40

Asp Gln Val Ser Asp Asn Leu Arg Val Trp Me

#t Phe Asn Asn Tyr Glu

245

#               250

#               255

Val Tyr Ser Ile Ser Tyr Phe Val Ala Glu Al

#a Lys Leu Phe Gly Lys

260

#           265

#           270

Val Asp Gln Ala Val Ile Thr Leu Thr Leu Se

#r Pro Lys Cys Asp Asp

275

#       280

#       285

Ser Ser Gly Asp Ile Ile Pro His Leu Phe Ty

#r Tyr Asp Arg Glu Leu

290

#   295

#   300

Phe Glu Lys Arg Tyr Tyr Met Asp Glu Tyr As

#p Trp Arg Ile Ile Lys

305                 3

#10                 3

#15                 3

#20

Ser Leu Asn Tyr Val Ile Pro Ile Gln Phe Gl

#y Leu Glu Ile Ile Lys

325

#               330

#               335

Met Asn Arg Leu Phe Lys Ser Leu Pro Thr Le

#u Gly Asp Leu Glu Asn

340

#           345

#           350

Glu Lys Glu Gly Ile Trp Leu Gly Arg Glu Le

#u Asp Glu Thr Gly Ile

355

#       360

#       365

Lys Glu Lys Leu Ala Asn Lys Gly Gln Tyr Ar

#g Phe Ile Lys Gly Lys

370

#   375

#   380

Met Val Gly Arg Tyr Asn Leu Ile Glu Glu Se

#r Asn Gln Tyr Ile Asp

385                 3

#90                 3

#95                 4

#00

Val Arg Lys Ile Asp Lys Ile Pro Lys Ser Va

#l Glu Phe Tyr Arg Leu

405

#               410

#               415

Val Trp Arg Asp Val Ser Arg Thr Thr Gln Ly

#s Arg Arg Leu Ile Ser

420

#           425

#           430

Thr Ile Ile Pro Pro Lys Tyr Ile Thr Gly As

#n Ser Leu Asn Val Ala

435

#       440

#       445

Tyr Phe Lys Asp Asn Asn Leu Lys Lys Leu Ly

#s Ala Leu Leu Ala Ile

450

#   455

#   460

Met Asn Ser Phe Val Phe Glu Ala Gln Val Ar

#g Ala Asn Leu Ser Thr

465                 4

#70                 4

#75                 4

#80

Asn His Ile Ser Leu Gly Ile Ile Arg Arg Al

#a His Ile Pro Lys Leu

485

#               490

#               495

Glu Gly Arg Val Val Asp Glu Leu Ser Gln Le

#u Val Asp Asn Tyr Val

500

#           505

#           510

Asn Glu Glu Ser Glu Leu Leu Leu Glu Val Ly

#s Val Ala Lys Ala Tyr

515

#       520

#       525

Gly Leu Ser Phe Glu Asp Phe Ser Ser Ile Le

#u Ser Leu Phe Asp Lys

530

#   535

#   540

Ile Gly Lys Asp Glu Lys Glu Lys Ile Leu Gl

#n Val Ala Lys Lys Tyr

545                 5

#50                 5

#55                 5

#60

Leu Lys Gly Gly Ile Lys Asn Asp Ser

565

<210> SEQ ID NO 3

<211> LENGTH: 1188

<212> TYPE: DNA

<213> ORGANISM: Bacillus stearothermophilus 6-55

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1)..(1188)

<223> OTHER INFORMATION:

<400> SEQUENCE: 3

atg att cct aat cat gtt tca tca aaa tta ag

#t gaa ctt gat atg tta       48

Met Ile Pro Asn His Val Ser Ser Lys Leu Se

#r Glu Leu Asp Met Leu

1               5

#                10

#                15

atc att aag cat gta cct cca gga ggt aat tg

#g aag gac att cca gaa       96

Ile Ile Lys His Val Pro Pro Gly Gly Asn Tr

#p Lys Asp Ile Pro Glu

20

#            25

#            30

tgg gtt cct tct aaa aga tta gaa caa ata cg

#a aaa agt tat gca gaa      144

Trp Val Pro Ser Lys Arg Leu Glu Gln Ile Ar

#g Lys Ser Tyr Ala Glu

35

#        40

#        45

gga aag gga agt cgt tct acg tat tat ggt ag

#a ctt ctt cca gat atg      192

Gly Lys Gly Ser Arg Ser Thr Tyr Tyr Gly Ar

#g Leu Leu Pro Asp Met

50

#    55

#    60

cct tct tat aca ata aac act tat ttt aat ag

#a cca gga aat ggt tgt      240

Pro Ser Tyr Thr Ile Asn Thr Tyr Phe Asn Ar

#g Pro Gly Asn Gly Cys

65

#70

#75

#80

cat ata cat tat gag caa gat agg aca tta tc

#t caa cgt gag gca gct      288

His Ile His Tyr Glu Gln Asp Arg Thr Leu Se

#r Gln Arg Glu Ala Ala

85

#                90

#                95

aga ctt cag tca ttt cct gat gat ttt att tt

#t tat gga agt aaa aca      336

Arg Leu Gln Ser Phe Pro Asp Asp Phe Ile Ph

#e Tyr Gly Ser Lys Thr

100

#           105

#           110

gcc ata aat aat cag att gga aat gca gta cc

#g ccg tta tta gcg tat      384

Ala Ile Asn Asn Gln Ile Gly Asn Ala Val Pr

#o Pro Leu Leu Ala Tyr

115

#       120

#       125

caa ata gct aaa gca ttt ccc ttt aaa gga ca

#a ttt gtc gac ttg ttt      432

Gln Ile Ala Lys Ala Phe Pro Phe Lys Gly Gl

#n Phe Val Asp Leu Phe

130

#   135

#   140

agt ggt gca gga ggt ctt tct cta gga ttt tt

#a tgg gca ggt tgg aaa      480

Ser Gly Ala Gly Gly Leu Ser Leu Gly Phe Le

#u Trp Ala Gly Trp Lys

145                 1

#50                 1

#55                 1

#60

cct ata att gca aat gat att gat aaa tgg gc

#a cta aca act tac atg      528

Pro Ile Ile Ala Asn Asp Ile Asp Lys Trp Al

#a Leu Thr Thr Tyr Met

165

#               170

#               175

aat aac ata cat aat gaa gtt gtt tta ggg ga

#t ata aga gat gaa aaa      576

Asn Asn Ile His Asn Glu Val Val Leu Gly As

#p Ile Arg Asp Glu Lys

180

#           185

#           190

gta tca gaa aca atc att caa aaa tgc cta at

#a gca aag aaa agc aat      624

Val Ser Glu Thr Ile Ile Gln Lys Cys Leu Il

#e Ala Lys Lys Ser Asn

195

#       200

#       205

cca gat aga cca ttg ttt gtt tta ggt gga cc

#a cct tgt caa ggt ttt      672

Pro Asp Arg Pro Leu Phe Val Leu Gly Gly Pr

#o Pro Cys Gln Gly Phe

210

#   215

#   220

tct act gct gga aaa aag cgt agc ata gta ga

#t gaa aga aac tgg ctt      720

Ser Thr Ala Gly Lys Lys Arg Ser Ile Val As

#p Glu Arg Asn Trp Leu

225                 2

#30                 2

#35                 2

#40

ttt gaa tct tac gta tca ata tta aaa gaa gt

#t aaa cca gat gga ttt      768

Phe Glu Ser Tyr Val Ser Ile Leu Lys Glu Va

#l Lys Pro Asp Gly Phe

245

#               250

#               255

att ttt gaa aat gta aca ggt tta tta agt at

#g gaa aaa ggt gcg ttt      816

Ile Phe Glu Asn Val Thr Gly Leu Leu Ser Me

#t Glu Lys Gly Ala Phe

260

#           265

#           270

ttt gaa atg gtt aag tca gaa tta agt aaa ac

#a gtt tct aac cta ttt      864

Phe Glu Met Val Lys Ser Glu Leu Ser Lys Th

#r Val Ser Asn Leu Phe

275

#       280

#       285

gta tat aaa cta aat agt gta gat tat ggg gt

#t cct cag aga aga aat      912

Val Tyr Lys Leu Asn Ser Val Asp Tyr Gly Va

#l Pro Gln Arg Arg Asn

290

#   295

#   300

agg gta gta atc ata ggt gac tca acg ggt ac

#t aaa aac agt gaa cca      960

Arg Val Val Ile Ile Gly Asp Ser Thr Gly Th

#r Lys Asn Ser Glu Pro

305                 3

#10                 3

#15                 3

#20

cca att cct att aca tct cta aaa ggt gag aa

#a aca tta ttt gat gcc     1008

Pro Ile Pro Ile Thr Ser Leu Lys Gly Glu Ly

#s Thr Leu Phe Asp Ala

325

#               330

#               335

ctt tca tca gcc ata tca gta aaa gaa gct tt

#a tct gat tta cca ttg     1056

Leu Ser Ser Ala Ile Ser Val Lys Glu Ala Le

#u Ser Asp Leu Pro Leu

340

#           345

#           350

ctt tct cct aat gaa gat ggc tct tgg aaa aa

#t tat gtt tgt gag cca     1104

Leu Ser Pro Asn Glu Asp Gly Ser Trp Lys As

#n Tyr Val Cys Glu Pro

355

#       360

#       365

cag aat atc tat caa agt ttt atg agg aaa aa

#g att act gct caa caa     1152

Gln Asn Ile Tyr Gln Ser Phe Met Arg Lys Ly

#s Ile Thr Ala Gln Gln

370

#   375

#   380

tat ata gaa atg ttg agc agt tta gcg ata at

#a taa

#     1188

Tyr Ile Glu Met Leu Ser Ser Leu Ala Ile Il

#e

385                 3

#90                 3

#95

<210> SEQ ID NO 4

<211> LENGTH: 395

<212> TYPE: PRT

<213> ORGANISM: Bacillus stearothermophilus 6-55

<400> SEQUENCE: 4

Met Ile Pro Asn His Val Ser Ser Lys Leu Se

#r Glu Leu Asp Met Leu

1               5

#                10

#                15

Ile Ile Lys His Val Pro Pro Gly Gly Asn Tr

#p Lys Asp Ile Pro Glu

20

#            25

#            30

Trp Val Pro Ser Lys Arg Leu Glu Gln Ile Ar

#g Lys Ser Tyr Ala Glu

35

#        40

#        45

Gly Lys Gly Ser Arg Ser Thr Tyr Tyr Gly Ar

#g Leu Leu Pro Asp Met

50

#    55

#    60

Pro Ser Tyr Thr Ile Asn Thr Tyr Phe Asn Ar

#g Pro Gly Asn Gly Cys

65

#70

#75

#80

His Ile His Tyr Glu Gln Asp Arg Thr Leu Se

#r Gln Arg Glu Ala Ala

85

#                90

#                95

Arg Leu Gln Ser Phe Pro Asp Asp Phe Ile Ph

#e Tyr Gly Ser Lys Thr

100

#           105

#           110

Ala Ile Asn Asn Gln Ile Gly Asn Ala Val Pr

#o Pro Leu Leu Ala Tyr

115

#       120

#       125

Gln Ile Ala Lys Ala Phe Pro Phe Lys Gly Gl

#n Phe Val Asp Leu Phe

130

#   135

#   140

Ser Gly Ala Gly Gly Leu Ser Leu Gly Phe Le

#u Trp Ala Gly Trp Lys

145                 1

#50                 1

#55                 1

#60

Pro Ile Ile Ala Asn Asp Ile Asp Lys Trp Al

#a Leu Thr Thr Tyr Met

165

#               170

#               175

Asn Asn Ile His Asn Glu Val Val Leu Gly As

#p Ile Arg Asp Glu Lys

180

#           185

#           190

Val Ser Glu Thr Ile Ile Gln Lys Cys Leu Il

#e Ala Lys Lys Ser Asn

195

#       200

#       205

Pro Asp Arg Pro Leu Phe Val Leu Gly Gly Pr

#o Pro Cys Gln Gly Phe

210

#   215

#   220

Ser Thr Ala Gly Lys Lys Arg Ser Ile Val As

#p Glu Arg Asn Trp Leu

225                 2

#30                 2

#35                 2

#40

Phe Glu Ser Tyr Val Ser Ile Leu Lys Glu Va

#l Lys Pro Asp Gly Phe

245

#               250

#               255

Ile Phe Glu Asn Val Thr Gly Leu Leu Ser Me

#t Glu Lys Gly Ala Phe

260

#           265

#           270

Phe Glu Met Val Lys Ser Glu Leu Ser Lys Th

#r Val Ser Asn Leu Phe

275

#       280

#       285

Val Tyr Lys Leu Asn Ser Val Asp Tyr Gly Va

#l Pro Gln Arg Arg Asn

290

#   295

#   300

Arg Val Val Ile Ile Gly Asp Ser Thr Gly Th

#r Lys Asn Ser Glu Pro

305                 3

#10                 3

#15                 3

#20

Pro Ile Pro Ile Thr Ser Leu Lys Gly Glu Ly

#s Thr Leu Phe Asp Ala

325

#               330

#               335

Leu Ser Ser Ala Ile Ser Val Lys Glu Ala Le

#u Ser Asp Leu Pro Leu

340

#           345

#           350

Leu Ser Pro Asn Glu Asp Gly Ser Trp Lys As

#n Tyr Val Cys Glu Pro

355

#       360

#       365

Gln Asn Ile Tyr Gln Ser Phe Met Arg Lys Ly

#s Ile Thr Ala Gln Gln

370

#   375

#   380

Tyr Ile Glu Met Leu Ser Ser Leu Ala Ile Il

#e

385                 3

#90                 3

#95

<210> SEQ ID NO 5

<211> LENGTH: 1635

<212> TYPE: DNA

<213> ORGANISM: Bacillus stearothermophilus 6-55

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1)..(1635)

<223> OTHER INFORMATION:

<400> SEQUENCE: 5

ttg gga aaa aaa gct gaa tat gga cag gga ca

#t cca ata ttt tta gaa       48

Leu Gly Lys Lys Ala Glu Tyr Gly Gln Gly Hi

#s Pro Ile Phe Leu Glu

1               5

#                10

#                15

tat gct gaa cag ata atc caa cat aaa gaa ta

#c caa ggt atg cca gac       96

Tyr Ala Glu Gln Ile Ile Gln His Lys Glu Ty

#r Gln Gly Met Pro Asp

20

#            25

#            30

tta aga tac cct gat gga aga att cag tgg ga

#a gca cct tca aat aga      144

Leu Arg Tyr Pro Asp Gly Arg Ile Gln Trp Gl

#u Ala Pro Ser Asn Arg

35

#        40

#        45

aaa agc ggt ata ttt aaa gac act aac att aa

#g cga aga aaa tgg tgg      192

Lys Ser Gly Ile Phe Lys Asp Thr Asn Ile Ly

#s Arg Arg Lys Trp Trp

50

#    55

#    60

gaa caa aaa gct att tct atc ggg ata gac cc

#t tct agt aat caa tgg      240

Glu Gln Lys Ala Ile Ser Ile Gly Ile Asp Pr

#o Ser Ser Asn Gln Trp

65

#70

#75

#80

att agt aag aca gcc aaa tta att cat cca ac

#a atg aga aaa cct tgc      288

Ile Ser Lys Thr Ala Lys Leu Ile His Pro Th

#r Met Arg Lys Pro Cys

85

#                90

#                95

aaa aag tgc gga agg ata atg gat tta cga ta

#t agc tat cct acc aaa      336

Lys Lys Cys Gly Arg Ile Met Asp Leu Arg Ty

#r Ser Tyr Pro Thr Lys

100

#           105

#           110

aat ctt att aaa cgc att aga aaa tta cct ta

#t gtt gat gaa tcg ttt      384

Asn Leu Ile Lys Arg Ile Arg Lys Leu Pro Ty

#r Val Asp Glu Ser Phe

115

#       120

#       125

gaa att gat tca cta gaa cat att tta aaa ct

#t att aaa cgt tta gtt      432

Glu Ile Asp Ser Leu Glu His Ile Leu Lys Le

#u Ile Lys Arg Leu Val

130

#   135

#   140

cta caa tat ggt gat aaa gta tac gat gat tt

#g cct aaa cta cta act      480

Leu Gln Tyr Gly Asp Lys Val Tyr Asp Asp Le

#u Pro Lys Leu Leu Thr

145                 1

#50                 1

#55                 1

#60

tgc aaa gca gtt aaa aat att cca aga ttg gg

#a aac gat tta gat act      528

Cys Lys Ala Val Lys Asn Ile Pro Arg Leu Gl

#y Asn Asp Leu Asp Thr

165

#               170

#               175

tgg tta aat tgg att gat tct gtt tat att cc

#g agt gaa cct agt atg      576

Trp Leu Asn Trp Ile Asp Ser Val Tyr Ile Pr

#o Ser Glu Pro Ser Met

180

#           185

#           190

ctt agt cca ggc gct atg gca aat ccg cca ga

#t aga tta gat ggg ttc      624

Leu Ser Pro Gly Ala Met Ala Asn Pro Pro As

#p Arg Leu Asp Gly Phe

195

#       200

#       205

cac tca tta aat gaa tgc tgt aga agt cat gc

#t gat aga ggt aga tgg      672

His Ser Leu Asn Glu Cys Cys Arg Ser His Al

#a Asp Arg Gly Arg Trp

210

#   215

#   220

gaa aaa aac ctg cgg tca tat aca aca gat ag

#a aga gca ttt gaa tac      720

Glu Lys Asn Leu Arg Ser Tyr Thr Thr Asp Ar

#g Arg Ala Phe Glu Tyr

225                 2

#30                 2

#35                 2

#40

tgg gtt gat gga gat tgg gtt gca gct gac aa

#a ctt atg gga cta ata      768

Trp Val Asp Gly Asp Trp Val Ala Ala Asp Ly

#s Leu Met Gly Leu Ile

245

#               250

#               255

aga aca aat gaa cag ata aag aag gaa act tg

#t tta aat gat aac cac      816

Arg Thr Asn Glu Gln Ile Lys Lys Glu Thr Cy

#s Leu Asn Asp Asn His

260

#           265

#           270

cca ggc cct tgt tct gca gac cat att ggt cc

#t att tcc ctt ggt ttt      864

Pro Gly Pro Cys Ser Ala Asp His Ile Gly Pr

#o Ile Ser Leu Gly Phe

275

#       280

#       285

gtt cac agg cca gag ttt caa ttg cta tgt aa

#c tca tgt aac agt gcc      912

Val His Arg Pro Glu Phe Gln Leu Leu Cys As

#n Ser Cys Asn Ser Ala

290

#   295

#   300

aaa aat aat cga atg act ttt tca gat gtc ca

#a cac tta att aat gcg      960

Lys Asn Asn Arg Met Thr Phe Ser Asp Val Gl

#n His Leu Ile Asn Ala

305                 3

#10                 3

#15                 3

#20

gaa aat aat gga gaa gag gtt gca tct tgg ta

#c tgt aaa cat ata tgg     1008

Glu Asn Asn Gly Glu Glu Val Ala Ser Trp Ty

#r Cys Lys His Ile Trp

325

#               330

#               335

gat tta aga aaa cat gat gtt aaa aac aac ga

#a aac gca ttg aga tta     1056

Asp Leu Arg Lys His Asp Val Lys Asn Asn Gl

#u Asn Ala Leu Arg Leu

340

#           345

#           350

agt aaa att tta aga gat aat cgc cat aca gc

#t atg ttt ata tta agt     1104

Ser Lys Ile Leu Arg Asp Asn Arg His Thr Al

#a Met Phe Ile Leu Ser

355

#       360

#       365

gag tta tta aaa gat aat cac tat ctt ttc ct

#t tca act ttt tta gga     1152

Glu Leu Leu Lys Asp Asn His Tyr Leu Phe Le

#u Ser Thr Phe Leu Gly

370

#   375

#   380

tta cag tat gca gaa agg tct gta agt ttt tc

#a aat ata aaa att gaa     1200

Leu Gln Tyr Ala Glu Arg Ser Val Ser Phe Se

#r Asn Ile Lys Ile Glu

385                 3

#90                 3

#95                 4

#00

aat cat att atc aca gga caa att tct gaa ca

#a cca aga gat aca aaa     1248

Asn His Ile Ile Thr Gly Gln Ile Ser Glu Gl

#n Pro Arg Asp Thr Lys

405

#               410

#               415

tac act gaa gag caa aaa gcc aga aga atg ag

#a att gga ttc gaa gca     1296

Tyr Thr Glu Glu Gln Lys Ala Arg Arg Met Ar

#g Ile Gly Phe Glu Ala

420

#           425

#           430

ctt aaa agc tat att gaa aaa gaa aac aga aa

#t gcc tta tta gta ata     1344

Leu Lys Ser Tyr Ile Glu Lys Glu Asn Arg As

#n Ala Leu Leu Val Ile

435

#       440

#       445

aat gat aaa att att gat aaa att aac gaa at

#a aaa aat ata tta caa     1392

Asn Asp Lys Ile Ile Asp Lys Ile Asn Glu Il

#e Lys Asn Ile Leu Gln

450

#   455

#   460

gat att cca gac gaa tat aaa ctt tta aat ga

#a aaa atc tct gag caa     1440

Asp Ile Pro Asp Glu Tyr Lys Leu Leu Asn Gl

#u Lys Ile Ser Glu Gln

465                 4

#70                 4

#75                 4

#80

ttc aac agt gaa gaa gtt tct gat gaa cta ct

#c aga gat tta gtt act     1488

Phe Asn Ser Glu Glu Val Ser Asp Glu Leu Le

#u Arg Asp Leu Val Thr

485

#               490

#               495

cat ctc cca act aaa gaa agt gaa cca gct aa

#c ttt aaa ttg gct aga     1536

His Leu Pro Thr Lys Glu Ser Glu Pro Ala As

#n Phe Lys Leu Ala Arg

500

#           505

#           510

aaa tac ttg caa gaa ata atg gaa ata gta gg

#a gat gaa ctt tca aaa     1584

Lys Tyr Leu Gln Glu Ile Met Glu Ile Val Gl

#y Asp Glu Leu Ser Lys

515

#       520

#       525

atg tgg gaa gat gaa cga tac gta aga caa ac

#a ttt gca gat tta gat     1632

Met Trp Glu Asp Glu Arg Tyr Val Arg Gln Th

#r Phe Ala Asp Leu Asp

530

#   535

#   540

taa

#

#

#           1635

<210> SEQ ID NO 6

<211> LENGTH: 544

<212> TYPE: PRT

<213> ORGANISM: Bacillus stearothermophilus 6-55

<400> SEQUENCE: 6

Leu Gly Lys Lys Ala Glu Tyr Gly Gln Gly Hi

#s Pro Ile Phe Leu Glu

1               5

#                10

#                15

Tyr Ala Glu Gln Ile Ile Gln His Lys Glu Ty

#r Gln Gly Met Pro Asp

20

#            25

#            30

Leu Arg Tyr Pro Asp Gly Arg Ile Gln Trp Gl

#u Ala Pro Ser Asn Arg

35

#        40

#        45

Lys Ser Gly Ile Phe Lys Asp Thr Asn Ile Ly

#s Arg Arg Lys Trp Trp

50

#    55

#    60

Glu Gln Lys Ala Ile Ser Ile Gly Ile Asp Pr

#o Ser Ser Asn Gln Trp

65

#70

#75

#80

Ile Ser Lys Thr Ala Lys Leu Ile His Pro Th

#r Met Arg Lys Pro Cys

85

#                90

#                95

Lys Lys Cys Gly Arg Ile Met Asp Leu Arg Ty

#r Ser Tyr Pro Thr Lys

100

#           105

#           110

Asn Leu Ile Lys Arg Ile Arg Lys Leu Pro Ty

#r Val Asp Glu Ser Phe

115

#       120

#       125

Glu Ile Asp Ser Leu Glu His Ile Leu Lys Le

#u Ile Lys Arg Leu Val

130

#   135

#   140

Leu Gln Tyr Gly Asp Lys Val Tyr Asp Asp Le

#u Pro Lys Leu Leu Thr

145                 1

#50                 1

#55                 1

#60

Cys Lys Ala Val Lys Asn Ile Pro Arg Leu Gl

#y Asn Asp Leu Asp Thr

165

#               170

#               175

Trp Leu Asn Trp Ile Asp Ser Val Tyr Ile Pr

#o Ser Glu Pro Ser Met

180

#           185

#           190

Leu Ser Pro Gly Ala Met Ala Asn Pro Pro As

#p Arg Leu Asp Gly Phe

195

#       200

#       205

His Ser Leu Asn Glu Cys Cys Arg Ser His Al

#a Asp Arg Gly Arg Trp

210

#   215

#   220

Glu Lys Asn Leu Arg Ser Tyr Thr Thr Asp Ar

#g Arg Ala Phe Glu Tyr

225                 2

#30                 2

#35                 2

#40

Trp Val Asp Gly Asp Trp Val Ala Ala Asp Ly

#s Leu Met Gly Leu Ile

245

#               250

#               255

Arg Thr Asn Glu Gln Ile Lys Lys Glu Thr Cy

#s Leu Asn Asp Asn His

260

#           265

#           270

Pro Gly Pro Cys Ser Ala Asp His Ile Gly Pr

#o Ile Ser Leu Gly Phe

275

#       280

#       285

Val His Arg Pro Glu Phe Gln Leu Leu Cys As

#n Ser Cys Asn Ser Ala

290

#   295

#   300

Lys Asn Asn Arg Met Thr Phe Ser Asp Val Gl

#n His Leu Ile Asn Ala

305                 3

#10                 3

#15                 3

#20

Glu Asn Asn Gly Glu Glu Val Ala Ser Trp Ty

#r Cys Lys His Ile Trp

325

#               330

#               335

Asp Leu Arg Lys His Asp Val Lys Asn Asn Gl

#u Asn Ala Leu Arg Leu

340

#           345

#           350

Ser Lys Ile Leu Arg Asp Asn Arg His Thr Al

#a Met Phe Ile Leu Ser

355

#       360

#       365

Glu Leu Leu Lys Asp Asn His Tyr Leu Phe Le

#u Ser Thr Phe Leu Gly

370

#   375

#   380

Leu Gln Tyr Ala Glu Arg Ser Val Ser Phe Se

#r Asn Ile Lys Ile Glu

385                 3

#90                 3

#95                 4

#00

Asn His Ile Ile Thr Gly Gln Ile Ser Glu Gl

#n Pro Arg Asp Thr Lys

405

#               410

#               415

Tyr Thr Glu Glu Gln Lys Ala Arg Arg Met Ar

#g Ile Gly Phe Glu Ala

420

#           425

#           430

Leu Lys Ser Tyr Ile Glu Lys Glu Asn Arg As

#n Ala Leu Leu Val Ile

435

#       440

#       445

Asn Asp Lys Ile Ile Asp Lys Ile Asn Glu Il

#e Lys Asn Ile Leu Gln

450

#   455

#   460

Asp Ile Pro Asp Glu Tyr Lys Leu Leu Asn Gl

#u Lys Ile Ser Glu Gln

465                 4

#70                 4

#75                 4

#80

Phe Asn Ser Glu Glu Val Ser Asp Glu Leu Le

#u Arg Asp Leu Val Thr

485

#               490

#               495

His Leu Pro Thr Lys Glu Ser Glu Pro Ala As

#n Phe Lys Leu Ala Arg

500

#           505

#           510

Lys Tyr Leu Gln Glu Ile Met Glu Ile Val Gl

#y Asp Glu Leu Ser Lys

515

#       520

#       525

Met Trp Glu Asp Glu Arg Tyr Val Arg Gln Th

#r Phe Ala Asp Leu Asp

530

#   535

#   540

<210> SEQ ID NO 7

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 7

agataaatta gctcttactt gagcttc

#

#             27

<210> SEQ ID NO 8

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 8

ggagagcaca tataccgaag ttag

#

#                24

<210> SEQ ID NO 9

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 9

gctcttcagt gtattttgta tctc

#

#                24

<210> SEQ ID NO 10

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 10

tgagaattgg attcgaagca ctta

#

#                24

<210> SEQ ID NO 11

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 11

gagatagagt atattgaaat acta

#

#                24

<210> SEQ ID NO 12

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 12

ttacccatgg cgggtttgta atac

#

#                24

<210> SEQ ID NO 13

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 13

tctgcagaac aagggcctgg gtgg

#

#                24

<210> SEQ ID NO 14

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 14

catattggtc ctattttccc ttggt

#

#               25

<210> SEQ ID NO 15

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 15

caatttaacc aagtatctaa atcg

#

#                24

<210> SEQ ID NO 16

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: inverse PCR primers

<400> SEQUENCE: 16

ttctgtttat attccgagtg aacc

#

#                24

<210> SEQ ID NO 17

<211> LENGTH: 54

<212> TYPE: DNA

<213> ORGANISM: PCR primers

<400> SEQUENCE: 17

ggtggtgcta gcggaggtaa ataaatgaaa gaaaacacag aaattaatat ag

#at           54

<210> SEQ ID NO 18

<211> LENGTH: 39

<212> TYPE: DNA

<213> ORGANISM: PCR primers

<400> SEQUENCE: 18

ggtggtgcat gcctaatata tttcttggta cgtcatttt

#

#    39

<210> SEQ ID NO 19

<211> LENGTH: 33

<212> TYPE: DNA

<213> ORGANISM: PCR primers

<400> SEQUENCE: 19

ggtggtcata tgggaaaaaa agctgaatat gga

#

#         33

<210> SEQ ID NO 20

<211> LENGTH: 36

<212> TYPE: DNA

<213> ORGANISM: PCR primers

<400> SEQUENCE: 20

ggtggtggat cctcattaat ctaaatctgc aaatgt

#

#       36

<210> SEQ ID NO 21

<211> LENGTH: 48

<212> TYPE: DNA

<213> ORGANISM: PCR primers

<400> SEQUENCE: 21

ggtggtggat ccggaggtaa ataaatgagt aatgctaaaa gtttctct

#                48

<210> SEQ ID NO 22

<211> LENGTH: 36

<212> TYPE: DNA

<213> ORGANISM: PCR primers

<400> SEQUENCE: 22

ggtggtgcat gcttatatta tcgctaaact gctcaa

#

#       36

<210> SEQ ID NO 23

<211> LENGTH: 20

<212> TYPE: PRT

<213> ORGANISM: Bacillus stearothermophilus 6-55

<400> SEQUENCE: 23

Met Gly Lys Lys Ala Glu Tyr Gly Gln Gly Hi

#s Pro Ile Phe Leu Glu

1               5

#                10

#                15

Tyr Ala Glu Gln

20