Abstract:
In accordance with the present invention, there is provided a DNA (deoxyribonucleic acid) fragment which encodes the MmeI type II restriction endonuclease enzyme. This one polypeptide possesses two related enzymatic functions; namely an endonuclease activity which recognizes the DNA sequence 5′-TCC(Pu)AC-3′ and cleaves as indicated by the arrows:  
                                 5′-TCCRAC(N20)↓-3′                       3′-AGGYTG(N18)↑-5′                
 
     and a second enzymatic activity that recognizes the same DNA sequence, 5′-TCC(Pu)AC-3′, but modifies this sequence by the addition of a methyl group to prevent cleavage by the MmeI endonuclease activity.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    The present invention relates to a DNA (deoxyribonucleic acid) fragment, which fragment codes for one polypeptide possessing two related enzymatic functions, namely an enzyme which recognizes the DNA sequence 5′-TCC(Pu)AC-3′ and cleaves the phosphodiester bond between the 20th and 21st residues 3′ to this recognition sequence on this DNA strand, and between the 18th and 19th residues 5′ to the recognition sequence on the complement strand 5′-GT(Py)GGT-3′ to produce a 2 base 3′ extension (hereinafter referred to as the MmeI restriction endonuclease), and a second enzymatic activity that recognizes the same DNA sequence, 5′-TCC(Pu)AC-3′, but modifies this sequence by the addition of a methyl group to prevent cleavage by the MmeI endcnuclease. The present invention also relates to a vector containing the DNA fragment, a transformed host containing this DNA fragment, and an improved process for producing MmeI restriction endonuclease from such a transformed host. The present invention also relates to a process for identifying additional DNA fragments that encode enzymes having the same general properties as MmeI but potentially having unique DNA recognition sequences. This process depends on the use of the amino acid sequence of the MmeI enzyme presented in this application, or subsequently on the additional sequences identified through this process. The invention also relates to additional DNA fragments, identifiable through the process described, each of which encodes a polypeptide having significant amino acid sequence similarity to the MmeI polypeptide. The polypeptides encoded by these DNA fragments are predicted to perform similar functions to MmeI. Specifically, they are predicted to possess the dual enzymatic functions of cleaving DNA in a specific manner at a relatively far distance from the specific recognition sequence and also modifying their recognition sequences to protect the host DNA from cleavage by endonuclease activity. An example of such an enzyme identified by this process is CstMI (see U.S. application Ser. No. ______, filed concurrently herewith). CstMI was identified as a potential endonuclease because of its highly significant amino acid sequence similarity to MmeI. CstMI recognizes the sequence 5′-AAGGAG-3′ and cleaves the phosphodiester bond between the 20th and 21st residues 3′ to the recognition sequence on this DNA strand, and between the 18th and 19th residues 5′ to the recognition sequence on the complement strand 5′-CTCCTT-3′ to produce a 2 base 3′ extension.  
           [0002]    Restriction endonucleases are a class of enzymes that occur naturally in prokaryotes. There are several classes of restriction systems known, of which the type II endonucleases are the class useful in genetic engineering. When these type II endonucleases are purified away from other contaminating prokarial components, they can be used in the laboratory to break DNA molecules into precise fragments. This property enables DNA molecules to be uniquely identified and to be fractionated into their constituent genes. Restriction endonucleases have proved to be indispensable tools in modern genetic research. They are the biochemical ‘scissors’ by means of which genetic engineering and analysis is performed.  
           [0003]    Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the ‘recognition sequence’) along the DNA molecule. Once bound, the type II endonucleases cleave the molecule within, or to one side of, the sequence. Different restriction endonucleases have affinity for different recognition sequences. The majority of restriction endonucleases recognize sequences of 4 to 6 nucleotides in length, although recently a small number of restriction endonucleases which recognize 7 or 8 uniquely specified nucleotides have been isolated. Most recognition sequences contain a dyad axis of symmetry and in most cases all the nucleotides are uniquely specified. However, some restriction endonucleases have degenerate or relaxed specificities in that they recognize multiple bases at one or more positions in their recognition sequence, and some restriction endonucleases recognize asymmetric sequences. HaeIII, which recognizes the sequence 5′-GGCC-3′, is an example of a restriction endonuclease having a symmetrical, non-degenerate recognition sequence; HaeII, which recognizes 5′-(Pu)GCGC(Py)-3′ typifies restriction endonucleases having a degenerate or relaxed recognition sequence; while BspMI, which recognizes 5′-ACCTGC-3′ typifies restriction endonucleases having an asymmetric recognition sequence. Type II endonucleases with symmetrical recognition sequences generally cleave symmetrically within or adjacent to the recognition site, while those that recognize asymmetric sequences tend to cleave at a distance of from 1 to 20 nucleotides to one side of the recognition site. The enzyme of this application, MmeI, (along with CstMI) has the distinction of cleaving the DNA at the farthest distance from the recognition sequence of any known type II restriction endonuclease. More than two hundred unique restriction endonucleases have been identified among several thousands of bacterial species that have been examined to date.  
           [0004]    A second component of restriction 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 nucleotide recognition sequence as the corresponding restriction endonuclease, but instead of breaking 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 modified by virtue of the activity of its modification methylase and it is therefore 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. Modification methyltransferases are usually separate enzymes from their cognate endonuclease partners. In some cases, there is a single polypeptide that possesses both a modification methyltransferase function and an endonuclease function, for example, Eco57I. In such cases, there is a second methyltransferase present as part of the restriction-modification system. In contrast, the MmeI system of the present application has no second methyltransferase accompanying the endonuclease-methyltransferase polypeptide.  
           [0005]    Endonucleases are named according to the bacteria from which they are derived. Thus, the species Haemophilus aegyptius, for example synthesizes 3 different restriction endonucleases, named HaeI, HaeII and HaeIII. These enzymes recognize and cleave the sequences 5′-(W)GGCC(W)-3′,5′-(Pu)GCGC(Py)-3′ and 5′-GGCC-3′ respectively.  Escherichia coli  RY13, on the other hand, synthesizes only one enzyme, EcoRI, which recognizes the sequence 5′-GAATTC-3′.  
           [0006]    While not wishing to be bound by theory, 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 such as viruses and plasmids that would otherwise destroy or parasitize them. They impart resistance by binding to infecting DNA molecules and cleaving them in each place that the recognition sequence occurs. The disintegration that results inactivates many of the infecting genes and renders the DNA susceptible to further degradation by exonucleases.  
           [0007]    More than 3000 restriction endonucleases have been isolated from various bacterial strains. Of these, more than 240 recognize unique sequences, while the rest share common recognition specificities. Restriction endonucleases which recognize the same nucleotide sequence are termed “isoschizomers.” Although the recognition sequences of isoschizomers are the same, they may vary with respect to site of cleavage (e.g., XmaI v. SmaI, Endow, et al.,  J. Mol. Biol.  112:521 (1977); Waalwijk, et al.,  Nucleic Acids Res.  5:3231 (1978)) and in cleavage rate at various sites (XhoI v. PaeR7I, Gingeras, et al.,  Proc. Natl. Acad. Sci. U.S.A.  80:402 (1983)).  
           [0008]    Restriction endonucleases have traditionally been classified into three major classes; type I, type II and type III. The type I restriction systems assemble a multi-peptide complex consisting of restriction polypeptide, modification polypeptide, and specificity, or DNA recognition, polypeptide. Type I systems require a divalent cation, ATP and S-adenylosyl-methionine (SAM) as cofactors. Type I systems cleave DNA at random locations up to several thousand basepairs away from their specific recognition site. The type III systems generally recognize an asymmetric DNA sequence and cleave at a specific position 20 to 30 basepairs to one side of the recognition sequence. Such systems require the cofactor ATP in addition to SAM and a divalent cation. The type III systems assemble a complex of endonuclease polypeptide and modification polypeptide that either modifies the DNA at the recognition sequence or cleaves. Type III systems produce partial digestion of the DNA substrate due to this competition between their modification and cleavage activities, and so have not been useful for genetic manipulation.  
           [0009]    MmeI does not require ATP for DNA cleavage activity and it cleaves to completion; thus it can be classified as a type II endonuclease. Unlike other type II enzymes, however, MmeI consists of a single polypeptide that combines both endonuclease and modification activities and is sufficient by itself to form the entire restriction modification system. MmeI also cleaves the farthest distance from the specific DNA recognition sequence of any type II endonuclease (as does CstMI of this application). MmeI is quite large and appears to have three functional domains combined in one polypeptide. These consist of an amino-terminal domain which contains the endonuclease DNA cleavage motif and which may also be involved in DNA recognition, a DNA modification domain most similar to the gamma-class N6mA methyltransferases, and a carboxy-terminal domain presumed to be involved in dimer formation and possibly DNA recognition. The enzyme requires SAM for both cleavage and modification activity. The single MmeI polypeptide is sufficient to modify the plasmid vector carrying the gene in vivo to provide protection against MmeI cleavage in vitro, yet it is also able to cleave unmodified DNAs in vitro when using the endonuclease buffer containing Mg++ and SAM.  
           [0010]    There is a continuing need for novel type II restriction endonucleases. Although type II restriction endonucleases which recognize a number of specific nucleotide sequences are currently available, new restriction endonucleases which recognize novel sequences provide greater opportunities and ability for genetic manipulation. Each new unique endonuclease enables scientists to precisely cleave DNA at new positions within the DNA molecule, with all the opportunities this offers.  
         SUMMARY OF THE INVENTION  
         [0011]    In accordance with the present invention, there is provided a novel DNA fragment encoding a novel restriction endonuclease, obtainable from  Methylophilus methylotrophus  (NEB#1190). The endonuclease is hereinafter referred to as “MmeI”, which endonuclease:  
           [0012]    (1) recognizes the degenerate nucleotide sequence 5′-TCC(Pu)AC-3′ in a double-stranded DNA molecule as shown below:  
                                       5′-TCC(Pu)AC-3′                           3′-AGG(Py)TG-5′          
 
           [0013]     (wherein G represents guanine, C represents cytosine, A represents adenine, T represents thymine, (Pu) represents a purine, either A or G, and (Py) represents a pyrimidine, either C or T);  
           [0014]    (2) cleaves DNA in the phosphodiester bond following the 20th nucleotide 3′ to the recognition sequence 5′-TCC(Pu)AC-3 and preceding the 18th nucleotide 5′ to the complement strand of the recognition sequence 5′-GT(Py)GGA-3′ to produce a 2 base 3′ extension:  
                                       5′-TCC(Pu)AC(N20)/-3′                           3′-AGG(Py)GT(N18)/-5′;          
 
           [0015]    (3) methylates the recognition sequence specified in (1) in vivo to protect the host DNA from cleavage by the MmeI endonuclease activity;  
           [0016]    The invention further relates to additional DNA fragments, each of which is identified to encode polypeptides which share significant sequence similarity to the MmeI restriction-modification polypeptide. The DNA fragment encoding the MmeI polypeptide enables the identification of these additional potential endonucleases by using similarity searching of the MmeI sequence against sequences available in databases, such as GENBANK, using a program such as BLAST (Altschul, et al. Nucleic Acids Res. 25:3389-3402 (1997)). These DNA fragments, as well as any other fragments with such similarity to MmeI that may be deposited in the databases in the future, are candidates which may encode polypeptides that are similar to MmeI, in that the polypeptides encoded act as both restriction endonuclease and methyltransferase. These polypeptides may, like MmeI, cleave DNA at a similarly far distance from the recognition sequence, in the range of 18 to 20 nucleotides or more, which character is unique and useful in certain molecular biology technologies. Specifically these polypeptides contain amino acid motifs common to N6mA DNA methyltransferases in the middle of the polypeptide, have a motif common to restriction endonucleases and located in the aminoterminal section of the polypeptides, consisting of the amino acids D/E(X8-X12)D/EXK, and have a region of several hundred amino acids following the conserved methyltransferase motifs which are significantly similar to this region of MmeI and are believed to serve as a dimerization and possibly a DNA sequence recognition domain. An example of such a polypeptide, CstMI, is presented. CstMI has been shown to recognize the 6 base pair asymmetric sequence 5′-AAGGAG-3′ and to cleave the DNA in the same manner as MmeI; 5′-AAGGAGN20/Nl8-3′. The endonuclease encoded by these DNA fragments may be produced by the process used for MmeI, as described below.  
           [0017]    The present invention further relates to a process for the production of the restriction endonuclease MmeI. This process comprises culturing a transformed host, such as  E. coli , containing the DNA fragment encoding the MmeI restriction system polypeptide, collecting the cultured cells, obtaining a cell-free extract therefrom and separating and collecting the restriction endonuclease MmeI from the cell-free extract. The present invention further relates to a process for the production of the restriction endonucleases encoded by the DNA sequences identified as homologous to MmeI. This process comprises culturing a transformed host, such as  E. coli , containing the gene for these restriction systems, collecting the cultured cells, obtaining a cell-free extract therefrom and separating and collecting the restriction endonuclease from the cell-free extract. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0018]    [0018]FIG. 1—Agarose gel showing MmeI cleavage of lambda, T7, phiX174, pBR322 and pUC19 DNAs.  
         [0019]    [0019]FIG. 2—DNA sequence of the MmeI gene locus (SEQ ID NO:1).  
         [0020]    [0020]FIG. 3—Amino acid sequence of the MmeI gene locus (SEQ ID NO:2).  
         [0021]    [0021]FIG. 4—Agarose gel showing MmeI cleavage of pTBMmeI.1 DNA and unmodified DNA substrates.  
         [0022]    [0022]FIG. 5—Agarose gel showing MmeI cleavage of unmethylated, hemi-methylated and fully methylated DNA substrates.  
         [0023]    [0023]FIG. 6—Incorporation of labeled methyl groups into unmethylated, hemi-methylated and fully methylated DNA substrates.  
         [0024]    [0024]FIG. 7—Multiple sequence alignment of MmeI amino acid sequence (SEQ ID NO:3 through SEQ ID NO:14) and homologous polypeptides from public databases.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0025]    The recognition sequence and cleavage site of the endonuclease of the present invention were previously described (Boyd, Nucleic Acids Res. 14: 5255-5274 (1986)). However the MmeI enzyme proved difficult to produce from the native host,  Methylophilus methylotrophus , due to very low yield of the enzyme and the relative difficulty of growing the  M. methylotrophus  host in large quantity. To overcome these limitations to producing MmeI, the present application describes the identification of the DNA sequence encoding the MmeI gene and the expression of this MmeI gene in a suitable host, in the present instance  E. coli . This manipulation of the MmeI encoding DNA fragment results in both a significant increase in the amount of enzyme produced per gram of cells and a significant increase in ease of growth of large amounts of cells containing MmeI enzyme.  
         [0026]    Several standard approaches typically employed by persons skilled in the art of cloning were applied to the task of cloning of MmeI without success. Specifically, the methylase selection approach (Wilson, et al., U.S. Pat. No. 5,200,333) was attempted unsuccessfully. Several random libraries of  M. methylotrophus  DNA were constructed in  E. coli  and challenged by digesting with MmeI, but no MmeI methylase containing clones were obtained.  
         [0027]    A second approach was also attempted but failed. In this approach, antibodies specific for N6mA were used to screen a library of random clones constructed in a lambda phage replacement vector. The approach was successful in obtaining methylase positive clones, but all examined were found to express the methyltransferase of the second restriction system in  M. methylotrophus , the MmeII methylase (recognition sequence 5′-GATC-3′) rather than the desired MmeI methylase activity.  
         [0028]    The successful approach to obtain the desired DNA fragment encoding the MmeI restriction system involved several steps. First a novel purification procedure was developed to purify the MmeI endonuclease peptide to homogeneity from  M. methylotrophus . Once this ultra pure MmeI endonuclease polypeptide was successfully obtained in a significant amount, amino acid sequence from the amino terminus and from internal cyanogen-bromide degradation peptides was determined. Using the amino acid sequence obtained, degenerate DNA primers complementary to the DNA coding for the amino acid sequences were synthesized and used to PCR amplify a portion of the MmeI gene. The DNA sequence of this portion of the MmeI gene was determined. The entire MmeI endonuclease gene and surrounding DNA sequences were then obtained by applying the inverse PCR technique. A number of primers matching the DNA sequence obtained were designed, synthesized and used in combination with numerous different templates. The inverse PCR templates were produced by digesting  M. methylotrophus  genomic DNA with various restriction endonucleases and then ligating the cut  M. methylotrophus  DNA at low concentration to obtain circular molecules. The various primers were tried in combinations with the various templates to find primer-template combinations that produced a specific PCR amplification product. The products thus obtained were sequenced. Once the DNA sequence encoding the entire MmeI endonuclease gene was obtained, primers were designed to specifically amplify the gene from  M. methylotrophus  genomic DNA. The amplified gene was inserted into an expression vector and cloned into an  E. coli  host. The host was tested and found to both express MmeI endonuclease activity and to in vivo modify the recombinant expression vector such that it was protected against MmeI endonuclease activity in vitro.  
         [0029]    This finding that the single polypeptide encoding the MmeI endonuclease also provided in vivo protection against MmeI is in contrast to the previously published information on MmeI (Tucholski, Gene 223:293-302 (1998)). Specifically, this reference taught that the MmeI endonuclease polypeptide did not provide protection against MmeI endonuclease cleavage. This reference reported a separate methyltransferase of 48 kD as required to modify the MmeI site on both strands and thus block cleavage by the MmeI endonuclease. Specifically, the reference teaches that the MmeI endonuclease polypeptide modifies the adenine in the top strand of the recognition sequence only, 5′-TCCRAC-3′ and that such modified DNA is cut by the MmeI endonuclease. The DNA fragment of the present invention encodes the MmeI endonuclease gene, which when grown alone in an  E. coli  host renders the vector containing the MmeI endonuclease resistant to cleavage by the purified MmeI endonuclease. Further, the MmeI endonuclease produced from this fragment does not cleave a DNA fragment modified at the adenine of the top strand, 5′-TCC.RAC-3′ when no modification of the opposite, or bottom strand is present. This is in contrast to the teaching of the Tucholski reference. Also, the MmeI endonuclease of this application does cleave a DNA fragment in which the adenine residue in the bottom strand is modified 5′-GTYGGA-3′ in contrast to the teaching of the Tucholski reference. When both the top strand and the bottom strand are modified at the adenine residues, the MmeI endonuclease does not cleave the DNA. No second methyltransferase gene, such as reported in the Tucholski reference, was found adjacent to the MmeI endonuclease gene. There is an open reading frame immediately 3′ to the MmeI endonuclease gene which would encode a protein of approximately the reported size of such a second methyltransferase activity (48 kD). However, this potential polypeptide does not have the amino acid motifs found in methyltransferases, nor did it provide protection against MmeI endonuclease when cloned in  E. coli . While the Tulchoski reference taught the necessity of a second methyltransferase polypeptide to provide protection against MmeI endonuclease activity for the host cell, it is demonstrated in the present application that the DNA fragment encoding the MmeI endonuclease polypeptide is sufficient to provide such protection. Additionally, the eleven DNA fragments described herein which encode amino acid sequences similar to MmeI are not flanked by any recognizable DNA methyltransferase genes. This indicates that these polypeptides are also likely to provide both protection for the host DNA and endonuclease activity against unmodified DNA substrates on their own, without having a second methyltransferase as part of the restriction modification system. This contrasts with other type II restriction modification systems.  
         [0030]    The same group (Tucholski, Gene 223: 293-302 (1998), and Anna Podhajska, personal communication) had previously reported an amino acid sequence of eight residues for a single internal CnBr digestion fragment (sequence GRGRGVGV (SEQ ID NO: ______). PCR based on this sequence was attempted yet failed repeatedly. This sequence was found to be unrelated to MmeI once the actual MmeI amino acid sequence was determined in accordance with the present invention. Therefore correct internal amino acid sequences determination, which enabled the cloning of the MmeI gene, depended on the novel purification method described in this application for the production of sufficiently pure MmeI in large enough quantity to determine cyanogen bromide internal fragment amino acid sequences, as performed in this Application.  
         [0031]    In Example II we obtained MmeI by culturing a transformed host carrying the MmeI gene, such as  E. coli  ER2683 carrying pTBMmeI.1 and recovering the endonuclease from the cells. A sample of  E. coli  ER2683 carrying pTBMmeI.1 (NEB#1457) has been deposited under the terms and conditions of the Budapest Treaty with the American Type Culture Collection (ATCC) on Jul. 3, 2002 and bears the Patent Accession No. PTA-4521.  
         [0032]    For recovering the enzyme of the present invention  E. coli  carrying pTBMmeI.1 (NEB#1457) may be grown using any suitable technique. For example,  E. coli  carrying pTBMmeI.1 may be grown in Luria broth media containing 100 μg/ml ampicillin and incubated aerobically at 37° C. with aeration. Cells in the late logarithmic stage of growth are induced by adding 0.3 mM IPTG, grown for an additional 4 hours, collected by centrifugation and either disrupted immediately or stored frozen at −70° C.  
         [0033]    The MmeI enzyme can be isolated from  E. coli  carrying pTBMmeI.1 cells by conventional protein purification techniques. For example, cell paste is suspended in a buffer solution and treated by sonication, high pressure dispersion or enzymatic digestion to allow extraction of the endonuclease by the buffer solution. Intact cells and cellular debris are then removed by centrifugation to produce a cell-free extract containing MmeI. The MmeI endonuclease, along with its corresponding intrinsic methylase activity, is then purified from the cell-free extract by ion-exchange chromatography, affinity chromatography, molecular sieve chromatography, or a combination of these methods to produce the endonuclease of the present invention.  
         [0034]    The present invention also relates to methods for identifying additional DNA fragments, each of which encodes a polypeptide having significant amino acid sequence similarity to the MmeI polypeptide. The polypeptides encoded by these DNA fragments are predicted to perform similar functions to MmeI. Specifically, they are predicted to possess the dual enzymatic functions of cleaving DNA in a specific manner at a relatively far distance from the specific recognition sequence and also modifying their recognition sequences to protect the host DNA from cleavage by their endonuclease activity. Once the amino acid sequence of the MmeI endonuclease was determined as described in this application, sequences deposited in databases can be compared to this MmeI sequence to find those few sequences that are highly significantly similar to MmeI. This method is similar to that of U.S. Pat. No. 6,383,770 (Roberts, et al.), except that here we are searching for similarity to the MmeI endonuclease sequence, rather than searching for sequences that match a database of methyltransferase or endonuclease proteins and then examining any unidentified open reading frames next to potential methyltransferase open reading frames. Prior to identifying the MmeI amino acid sequence, the DNA sequences coding for proteins related to MmeI had not been included in the database of restriction and methyltransferase gene sequences utilized by Roberts, et al., supra since these sequences had not been linked to any known endonuclease function. The method disclosed herein of identifying potential MmeI-like endonucleases is thus more specific than the method of U.S. Pat. No. 6,383,770 (Roberts, et al.).  
         [0035]    Similarity searching of the MmeI sequence against sequences available in databases, such as GENBANK, is accomplished using a program such as BLAST (Altschul, et al. Nucleic Acids Res. 25:3389-3402 (1997)). A sequence with an expectation value (E) score of less than E=e-10 is considered a potential candidate endonuclease. Sequences that give expectation values that are much lower, such as less than E=e-30 is to be considered as highly likely to be endonucleases like MmeI. Such candidate MmeI-like peptides are further examined to see if they conform to the domain architecture that MmeI exhibits. A true candidate will contain an endonuclease fold motif, usually of the form (D/E)X8-X12(D/E)XK in the amino-terminal portion of the peptide, (Aravind et al. Nucleic Acid Res. 28:3417-3432 (2000)). A true candidate will contain methyltransferase motifs in the middle portion of the peptide similar to gamma class N6methyl adenine methyltransferases, and sequences similar to the carboxyl portion of MmeI in the carboxyl portion of the candidate peptide. Such a BLAST search performed on Jun. 12, 2003 returned the following sequences as highly significantly similar to MmeI:  
                                                                                     SEQ                       ID       GENBANK ACCESSION ID NO.   DESCRIPTION   SCORE   E VALUE   NO:                                1. gi|15794682|ref|NP_284504.1|   hypothetical protein   643   0.0   6           [Neisseri       2. gi|9945797|gb|AAG03371.1|   GcrY [ Corynebacterium     604    e−171   8           ii striatum       3. gi|16077744|ref|NP_388558.1|   similar to hypothetical   564    e−159   7           protei       4. gi|28373198|ref|NP_783835.1|   putative YeeA protein   531    e−l49   3           [Lactoba . . .       5. gi|23110638|gb|ZP_00096791.1|   hypothetical protein   426    e−118   10           [Novosph . . .       6. gi|27450519|gb|AA014619.1|AF465251_62   unknown [Lactobacillus . . .   217   9e−55   4       7. gi|15807258|ref|NP_295988.1|   DNA modification   213   1e−53   14           methyltransfe . . .       8. gi|15807788|ref|NP_285443.1|   conserved hypothetical   164   7e−39   13           protein . . .       9. gi|21231551|ref|NP_637468.1|   conserved hypothetical   142   2e−32   N/A           protein . . .       10. gi|20803963|emb|CAD31540.1|   PUTATIVE DNA METHYLASE   134   7e−30   11           PROTEIN . . .       11. gi|23451826|gb|AAN32874.1|AF461726_1   unknown [Pseudomonas f . . .   98   6e−19   9       12. gi|16125079|ref|NP_419643.1|   conserved hypothetical   92   3e−17   12           protein . . .       13. gi|10954534|ref|NP_044172.1|     M. jannaschii  predicted   76   2e−12   N/A           coding . . .                  
 
         [0036]    Most of these proteins are labeled as hypothetical or putative in their database entries. A number of these appear to be full-length polypeptides, such as sequence #2 above: GcrY. Such candidates can be expressed as described in Roberts to identify the expected endonuclease activity. Some endonuclease genes may be inactive in the particular strain used for sequencing (Lin, et al. Proc. Natl. Acad. Sci. USA 98:2740-2745 (2001)). In such a circumstance it may prove possible to express functional endonucleases by repairing the mutations that have inactivated these genes. Several of the MmeI homologs, such as #7 (SEQ ID NO:14)(Deinococcus radiodurans DR2267) and #8 (SEQ ID NO:13)(Deinococcus radiodurans DR0119.1) have disruptions in the open reading frames. DR2267 has a stop codon, TAG, which prematurely terminates the open reading frame, in a position where MmeI has a glutamate amino acid coded for by the codon GAG. By changing this TAG stop codon to GAG it may be possible to reactivate this potential endonuclease gene. DR0119.1 is also disrupted, in that it has a frameshift that disrupts open reading frame. The MmeI sequence may be used as a guide to direct where to repair this frameshift by maximizing the similarity of the DR0119.1 sequence to the MmeI sequence. This may well restore DR0119.1 endonuclease activity.  
         [0037]    An alternative way to generate potential new endonucleases is to take advantage of their similar domain structure by performing domain swapping. One may be able to swap the amino terminal domain of an MmeI-like peptide, for the amino terminal domain in the MmeI protein, for example by swapping the sequence of the potential new gene up to the first methyltransferase motif (motif X, “Gly Ala His Tyr Thr Ser” into MmeI to replace this portion of MmeI up to the same sequence. This approach may be particularly useful when only a partial sequence is available or a potential gene has lost function due to multiple mutations. This approach will create a chimeric protein that potentially has endonuclease activity and cleaves at a distance away from the recognition sequence, like MmeI, but that recognizes a novel DNA sequence. One may also find sequences in the databases that are highly similar to MmeI but that are partial. For example, sequence #11 (SEQ ID NO:9) above (Pseudomonas fluorescens) is from a small fragment of DNA sequence in the database. To obtain a functional endonuclease like MmeI from this sequence one can use inverse PCR or other techniques to obtain DNA sequence adjacent to the fragment reported, then use that sequence to obtain an intact endonuclease gene.  
         [0038]    Once a sequence is identified the potential endonuclease can be expressed and characterized as described in Roberts, et al. supra. Here, however, there is no separate methyltransferase gene to express along with the endonuclease. Once such a potential endonuclease is cloned and expressed in a suitable host, such as in  E. coli , a cell free extract is prepared and analyzed to detect any endonuclease activity. Such an endonuclease assay must include the SAM cofactor required by these endonucleases. Once specific DNA cleavage activity is found the recognition sequence and cleavage site may be determined by standard methods. (Schildkraut, (1984) In Genet. Eng. (N Y) Vol 6. (Setlow J. K., Hollaender, A. Ed.). pp 117-140. Plenum Press, New York. “Screening for and characterizing restriction endonucleases.”)  
         [0039]    The enzymes so identified can be isolated from  E. coli  cells carrying the DNA fragment in a suitable vector by conventional protein purification techniques. For example, cell paste is suspended in a buffer solution and treated by sonication, high pressure dispersion or enzymatic digestion to allow extraction of the endonuclease by the buffer solution. Intact cells and cellular debris are then removed by centrifugation to produce a cell-free extract containing the enzyme. The endonuclease, along with its corresponding intrinsic methylase activity, is then purified from the cell-free extract by ion-exchange chromatography, affinity chromatography, molecular sieve chromatography, or a combination of these methods to produce the endonuclease of the present invention.  
         [0040]    These DNA fragments, as well as any other fragments with such similarity to MmeI that may be deposited in the databases in the future, are predicted to encode polypeptides that are similar to MmeI, in that the polypeptides encoded act as both restriction endonuclease and methyltransferase. These polypeptides may, like MmeI, cleave DNA at a similarly far distance from the recognition sequence, in the range of about 18 to 20 nucleotides or more, which character is unique and useful in certain molecular biology technologies.  
         [0041]    An example of such an enzyme identified by this process is CstMI (see U.S. application Ser. No. ______, filed concurrently herewith). CstMI was identified as a potential endonuclease because of its highly significant amino acid sequence similarity to MmeI. CstMI is encoded by sequence #2 above (SEQ ID NO:8), which gave highly significant Expectation value of e −171  when compared to MmeI by BLAST. CstMI recognizes the 6 base pair asymmetric sequence 5′-AAGGAG-3′ and cleaves the DNA in the same manner as MmeI: it cleaves the phosphodiester bond between the 20th and 21st residues 3′ to this recognition sequence on this DNA strand, and between the 18th and 19th residues 5′ to the recognition sequence on the complement strand 5′-CTCCTT3′ to produce a 2 base 3′ extension.  
         [0042]    The present invention is further illustrated by the following Examples. These Examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof.  
         [0043]    The references cited above and below are herein incorporated by reference.  
       EXAMPLE I Purification of MmeI Endonuclease  
       [0044]    A single colony of  Methylophilus methylotrophus  (NEB#1190) was grown for 24 hrs in 1 liter of medium M (0.08 μM CuSO 4 , 0.448 μM MnSO 4 , 0.348 μM ZnSO 4 , 6.0 μM FeCl 3 , 18 μM CaCO 3 , 1.6 mM MgSO 4 , 9.0 μM NaH 2 PO 4 , 10.9 mm K 2 HPO 4 , 13.6 mM (NH 4 ) 2 SO 4 ) for 24 hours. This culture was used to inoculate 100 liters of medium M. The cells were grown aerobically at 37° C., overnight, until stationary. Five 100-liter fermentations were required to harvest 752 grams of wet cell pellet. 750 gram of  M. methylotrophus  cell pellet was suspended in 2.25 liters of Buffer A (20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA, 5% Gycerol) and passed through a Gaulin homogenizer at ˜12,000 psig. The lysate was centrifuged at −13,000×G for 40 minutes and the supernatant collected.  
         [0045]    The supernatant solution was applied to a 500 ml Heparin Hyper-D column (BioSepra SA) which had been equilibrated in buffer A. A 1.0 L wash of buffer A was applied, then a 2 L gradient of NaCl from 0.05 M to 1 M in buffer A was applied and fractions were collected. Fractions were assayed for MmeI endonuclease activity by incubating with 1 μg Lambda DNA (NEB) in 50 μl NEBuffer 1, supplemented with 32 μM S-adenosyl-L-methionine (SAM) for 15 minutes at 370 C. MmeI activity eluted at 0.3 M to 0.4 M NaCl.  
         [0046]    The Heparin Hyper-D column fractions containing the Mme I activity were pooled, diluted to 50 mM NaCl with buffer A (without NaCl) and applied to a 105 ml Source 15 Q column (Amersham Biotech) which had been equilibrated with buffer A. A 210 ml wash with buffer A was applied followed by a 1.0 L gradient of NaCl from 0.05 M to 0.7 M in buffer A. Fractions were collected and assayed from Mme I endonuclease activity. The Mme I activity was found in the unbound fraction.  
         [0047]    The Source 15 Q pool was loaded onto a 22 ml AF-Heparin-TSK column (TosoHaas) which had been equilibrated with buffer A. A wash of 44 ml buffer A was applied, followed by a linear gradient of NaCl from 0.05 M to 1.0 M in buffer A. Fractions were collected and assayed from Mme I endonuclease activity. The Mme I activity eluted between 0.26 M and 0.29 M NaCl. The fractions containing activity were pooled and dialyzed against buffer B (20 mM NaPO 4  (pH 7.0), 50 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA, 5% Glycerol).  
         [0048]    The dialyzed AF-Heparin-TSK pool was loaded onto a 6 ml Resource 15 S column (Amersham Biotech) which had been equilibrated with buffer B. A wash of 12 ml buffer B was applied, followed by a linear gradient of NaCl from 0.05 M to 1.0 M in buffer B. Fractions were collected and assayed for Mme I endonuclease activity. Mme I activity eluted between 0.14 M and 0.17 M NaCl.  
         [0049]    This pool was applied to a 2 liter Superdex 75 sizing column (Amersham Biotech) which had been equilibrated with buffer C (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA, 5% Glycerol). Fractions were collected between 500 and 1500 ml elution with buffer C, then assayed by Mme endonuclease assay and polyacrylamide gel electrophoresis on 4-20% gradient gel, followed by protein staining with Coomassie Brilliant Blue dye. Fractions eluting between 775 and 825 ml corresponded to Mme I activity and a protein band of 105 kDa. These fractions were pooled and dialyzed against buffer D (20 mM NaPO 4  (pH 7.0), 50 mM NaCl, 1 mM DTT, 5% Glycerol).  
         [0050]    The dialyzed sizing pool was applied to a 16 ml Ceramic HTP column (BioRad) which had been equilibrated with buffer D. A 32 ml wash with buffer D was followed by a linear gradient from 0.02 M to 1.0 M NaPO 4  in buffer D. Fractions were collected and assayed by Mme endonuclease assay and polyacrylamide gel electrophoresis on a 4-20% gradient gel, followed by protein staining with Coomassie Brilliant Blue dye. Mme I eluted between 0.26 M and 0.3 M NaPO 4 . A portion of several fractions containing a single homogeneous protein band of 105 kDa were used for protein sequencing. The rest of the purified MmeI fractions were pooled (6 ml @0.36 mg/ml) and dialyzed against storage buffer (10 mM Tris (pH 7.9), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol). The purified MmeI enzyme was stored at −200C.  
         [0051]    Activity Determination:  
         [0052]    Samples from 1-4 μl were added to 50 μl substrate solution consisting of 1× NEBuffer 1, 32 μM S-adenosyl-L-methionine, and 1 μg DNA (lambda, PhiX174 or pUC19 DNAs). Reactions were incubated for 15 minutes at 370, received 20 μl stop solution and were analyzed by electrophoresis on a 1% agarose gel.  
         [0053]    Optimized Endonuclease Activity  
         [0054]    Following purification of MmeI from  M. methylotrophus , experiments were performed to determine the optimal reaction conditions for DNA cleavage. Endonuclease activity was found to be significantly enhanced by the presence of potassium in the reaction buffer. Reactions were performed at 4° C. to 37° C. and from 5 to 60 minutes with no appreciable change in the amount of DNA cleavage. Enzyme concentrations at or near stoichiometric equivalence to DNA sites were required for maximal cleavage. Large excess of enzyme blocked cleavage. These findings were used to reassess the activity of MmeI and to define a workable endonuclease unit.  
         [0055]    Unit Definition  
         [0056]    One unit of MmeI is defined as the amount of MmeI required to completely cleave 1 μg of PhiX174 DNA in 15 minutes at 37° C. in NEBuffer 4 (20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol (pH7.9 at 250C)) supplemented with 80 μM S-adenosyl-L-methionine (SAM).  
       EXAMPLE II  
     Cloning the MmeI Endonuclease  
       [0057]    1. DNA purification: Total genomic DNA of  Methylophilus methylotrophus  was prepared. 5 grams of cell paste was suspended in 20 ml of 25% sucrose, 0.05 M Tris-HCl pH 8.0, to which was added 10 ml of 0.25 M EDTA, pH 8.0. Then 6 ml of lysozyme solution (10 mg/ml lysozyme in 0.25 M Tris-HCl, pH 8.0) was added and the cell suspension was incubated at 40C for 16 hours. 25 ml of Lytic mix (1% Triton-X100, 0.05 M Tris, 62 mM EDTA, pH 8.0) and 5 ml of 10% SDS was then added and the solution incubated at 370C for 5 minutes. The solution was extracted with one volume of equilibrated phenol:chloroform:isoamyl alcohol (50:48:2, v/v/v) and the aqueous phase was recovered and extracted with one volume of chloroform:isoamyl alcohol (24:1, v/v) two times. The aqueous solution was then dialysed against four changes of 2 L of 10 mM Tris, 1 mM EDTA, pH 8.0. The dialysed DNA solution was digested with RNase (100 μg/ml) at 370C for 1 hour. The DNA was precipitated by the addition of {fraction (1/10)}th volume 5 M NaCl and 0.55 volumes of 2-propanol and spooled on a glass rod. The DNA was briefly rinsed in 70% ethanol, briefly air dried and dissolved in 20 ml TE (10 mM Tris, 1 mM EDTA, pH 8.0) to a concentration of approximately 500 μg/ml and stored at 4° C.  
         [0058]    2. The MmeI endonuclease was purified to homogeneity as described in Example I above.  
         [0059]    3. Amino acid sequences of the MmeI endonuclease were obtained for the amino terminus and for several internal cyanogen bromide digestion products of the MmeI polypeptide. The MmeI restriction endonuclease, prepared as described in Example I above, was subjected to electrophoresis and electroblotted according to the procedure of Matsudaira (Matsudaira. J. Biol. Chem. 262:10035-10038, 1987)), with modifications as previously described (Looney, et al. Gene 80:193-208 (1989)). The membrane was stained with Coomassie blue R250 and the protein band of approximately 105 kD was excised and subjected to sequential degradation on an ABI Procise 494 Protein/Peptide Sequencer with gas-phase delivery (Waite-Rees, et al. J. Bacteriol. 173:5207-5219 (1991)). The amino acid sequence of the first 14 amino terminal residues obtained was the following: ALSWNEIRRKAIEF (SEQ ID NO:15).  
         [0060]    An additional sample of the MmeI endonuclease, 20 μg in 20 μl, was treated with 2 μg of cyanogen bromide (Sigma) dissolved in 200 μl of 88% distilled formic acid for 24 hours in the dark at room temperature. This reaction mixture was evaporated to dryness and resuspended in 20 μl of loading buffer (1.5M Tris-HCl, pH 8.5, 12% glycerol, 4% SDS, 0.05% Serva Blue G, 0.05% Phenol Red) at 100° C. for 5 minutes. This sample was subjected to electrophoresis on a Tris-Tricine 10 to 20% polyacrylamide gradient gel (Invitrogen) for three hours and then transferred to a polyvinylidene difluoride (PVDF) membrane (Problott, Applied Biosystems Inc.) using 10 mM CAPS buffer (10 mM 3-[cyclohexylamino]-1propanesulfonic acid, 10% methanol, 0.05% SDS, 0.005% dithiotheritiol, adjusted to pH 11.0 with NaOH) for 18 hours at 200 volts in a tank electroblotter (TE52, Hoeffer). The membrane was stained with Coomassie blue R-250 and major bands of 25 kilodaltons (kD), 14 kD, 7.5 kD and 6 kD were observed, as well as smaller bands. These stained protein bands were excised from the membrane and each subjected to sequential degradation. The fragments other than the amino terminal fragment are derived from internal cleavage by cyanogen bromide at methionine residues from within the protein and thus should be preceded by a methionine. The first 29 residues of the 25 kD peptide corresponded to (M)KISDEFGNYFARIPLKSTXXIXEXNALQ (SEQ ID NO:16). Residues 20, 21, 23 and 25, labeled X, were not identified. The first 40 amino acid residues obtained from the 14 kD fragment were: (M)DAKKRRNLGAHYTSEANILKLI KPLLLDELWVVFXKVKN (SEQ ID NO:17). Residue 36 was not determined. The first 25 residues of the 7.5 kD peptide corresponded to (M)KSRGKDLDKAYDQALDYFSGIAER (SEQ ID NO:18). The 6 kD fragment was found to contain a mixture of three sequences.  
         [0061]    4. Amplification of a portion of the MmeI endonuclease: The peptide sequence data from the amino terminus, 25 kD, 14 kD and 7.5 kD peptides was used to construct a series of degenerate PCR primers corresponding to the codons for the amino acid residues. The order of the internal peptide fragments was unknown, so both forward (sense strand) and reverse (antisense strand) primers were made for these fragments. The primers were:  
         [0062]    25 kD fragment: residues DEFGNYFA (SEQ ID NO:19)  
         [0063]    Forward:  
                                                                         Forward:                1)   5′-GARTTYGGNAAYTAYTTYGC-3′   (SEQ ID NO:20)                        Reverse:                2)   5′-AARTARTTNCCRAAYTCRTC-3′   (SEQ ID NO:21)              
 
         [0064]    14 kD fragment: residues MDAKKR (SEQ ID NO:22)  
         [0065]    Forward A:  
                                                                                                     Forward A:                3)   5′-ATGGAYGCNAARAARCG-3′   (SEQ ID NO:23)                        Forward B:                4)   5′-ATGGAYGCNAARAARAG-3′   (SEQ ID NO:24)                        Reverse:                5)   5′-CGNCGYTTYTTNGCRTCCAT-3′   (SEQ ID NO:25)              
 
         [0066]    7.5 kD fragment: residues DKAYDQA (SEQ ID NO:26)  
         [0067]    Forward:  
                                                                         Forward:                6)   5′-GAYAARGCNTAYGAYCARGC-3′   (SEQ ID NO:27)                        Reverse:                7)   5′-GCYTGRTCRTANGCYTTRTC-3′   (SEQ ID NO:28)              
 
         [0068]    where  
         [0069]    Y=T,C  
         [0070]    R=A,G  
         [0071]    H=A,T,C  
         [0072]    S=G,C  
         [0073]    N=A,C,G,T  
         [0074]    Primers 1 and 2 are derived from the MmeI 25 kD CNBr peptide and were prepared to prime on the sense strand (1) or the antisense strand (2) of the gene. Primers 3 through 5 are derived from the 14 kD CNBr peptide and were prepared to prime on the sense strand (3 and 4) or the antisense strand (5) of the gene, with 3 and 4 differing in the codon usage for the arginine residue. Primers 6 and 7 are derived from the 7.5 kD CNBr peptide and were prepared to prime on the sense strand (6) or the antisense strand (7) of the gene.  
         [0075]    PCR amplification reactions were performed using the primer combinations of 1 with 5, 1 with 7, 3 with 2, 3 with 7, 4 with 2, 4 with 7, 6 with 2 and 6 with 7. A portion of the MmeI gene was amplified in a PCR reaction by combining:  
         [0076]    80 μl 10× Thermopol buffer (NEB)  
         [0077]    50 μl 4 mM DNTP solution (NEB)  
         [0078]    4 μl MmeI genomic DNA (500 μg/ml stock)  
         [0079]    16 μl 100 mM MgSO 4    
         [0080]    586 μl dH 2 O  
         [0081]    16 μl (32 units) Vent® exo-DNA polymerase (NEB).  
         [0082]    This master mix was divided into 8 aliquots of 90 μl, to which was added 5 μl forward primer (10 μM stock) and 5 μl reverse primer (10 μM stock). The cycling parameters were 95° C. for 3 minutes for one cycle, then 95° C. for 30 seconds, 46° C. for 30 seconds, 72° C. for 2 minutes, for 25 cycles.  
         [0083]    The amplification reactions were electrophoresed on a 1% agarose gel and analyzed. Major DNA amplification products of 450 base pairs (bp) (primers 2 with 4), 650 bp (primers 5 with 6) and 1100 bp (primers 2 with 6) were obtained. These fragment sizes are consistent with the 7.5 kD CnBr fragment being located nearest the amino end of the protein and approximately 650 bp away from the 14 kD CnBr fragment, with the 14 kD fragment between the 7.5 kD and the 25 kD fragment and adjacent to the 25 kD fragment. The amplified DNA fragments were gel purified and sequenced using the primers that were used for the amplification. A translation of the DNA sequence obtained matched the amino acid sequence derived from the purified MmeI endonuclease, indicating that a portion of the MmeI endonuclease gene DNA sequence had been successfully obtained.  
         [0084]    5. Determining the DNA sequence for the entire MmeI gene and adjacent DNA: The inverse PCR technique was used to extend the DNA sequence from both sides of the 1060 bp of the MmeI gene obtained above. To accomplish this a series of primers matching the MmeI gene DNA sequence and oriented for inverse PCR were designed and synthesized. MmeI genomic DNA was cut with a number of restriction endonucleases and ligated at low concentration to generate circular DNA templates.  
         [0085]    A. MmeI genomic DNA was digested with ten different restriction endonucleases and then circularly ligated to obtain DNA templates to amplify using the inverse PCR technique. The restriction enzymes used were:  
                                           BspHI   (T/CATGA)                           EcoRI   (G/AATTC)                       HindIII   (A/AGCTT)                       HinP1I   (G/CGC)                       MspI   (C/CGG)                       N1aIII   (CATG/)                       PstT   (CTGCA/G)                       SacI   (GAGCT/C)                       SphI   (GCATG/C)                       XbaI   (T/CTAGA)          
 
         [0086]    Restriction enzyme digests were performed by combining:  
         [0087]    5 μl 10× NEBuffer recommended for the enzyme (varied with enzyme)  
         [0088]    2 μM. methyloptrophus genomic DNA (1 μg)  
         [0089]    43 μl dH 2 O  
         [0090]    1 μl (10-20 units) restriction enzyme.  
         [0091]    The reactions were incubated for 1 hour at 37° C. The restriction endonuclease was inactivated by heating the reaction to 65° C. (80° C. for PstI) for 20 minutes. The digested DNA was then ligated into circular fragments by adding 50 μl 10× T4 DNA ligase buffer, 400 μl dH 2 O and 3 μl concentrated T4 DNA ligase (6000 units, New England Biolabs, Inc.) and incubating at 160C for 16 hours. The ligated DNA was then extracted with phenol and chloroform, precipitated with 2-propanol and resuspended in 100 μl TE buffer.  
         [0092]    B. Amplification of DNA adjacent to the 1060 bp fragment of the MmeI endonuclease gene: Two pairs of PCR primers were designed, one near each end of the 1060 bp sequence obtained from direct PCR with degenerate primers. The primer sequences were:  
                               primer IP 1:               5′-GTTGGATCCCGCACAGATTGCTCAGG-3′   (SEQ ID NO:29)               primer IP 2:       5′-GTTGGATCCTACGTTAATCTGAATAAGATG-3′   (SEQ ID NO:30)               primer IP 3:       5′-GTTGGATCCTGTTAATCTGAAACGCTGG-3′   (SEQ ID NO:31)               primer IP 4:       5′-GTTGGATCCTTATACCAAAATGTGAGGTC-3′   (SEQ ID NO:32)          
 
         [0093]    Inverse PCR reactions were performed on the 10 circularized templates produced above with the primer pairs of IP 1 with IP 2, IP 3 with IP 4, and IP 1 with IP 3. The amplification reactions were assembled by combining:  
         [0094]    80 μl 10× Thermopol buffer (NEB)  
         [0095]    50 μl 4 mM DNTP solution (NEB)  
         [0096]    40 μl IP primer (forward)  
         [0097]    40 μl IP primer (reverse)  
         [0098]    16 μl 1100 mM MgSO 4    
         [0099]    534 μl dH 2 O  
         [0100]    16 μl (32 units) Vent® exo-DNA polymerase (NEB).  
         [0101]    The master mix was aliquoted into ten tubes of 76 μl, to which was added 4 μl of the appropriate digested, circularly ligated template. The cycling parameters were 95° C. for 3 minutes for one cycle, then 95° C. for 30 seconds, 56° C. for 30 seconds, 72° C. for 3 minutes, for 25 cycles. Amplification products were analyzed by agarose gel electrophoresis.  
         [0102]    For primers IP 1 and IP 2 with the SphI template and the NlaIII template a product of approximately 825 bp was obtained. For primers IP 3 and IP 4 with the BspHI template a product of approximately 800 bp was obtained. For primers IP 1 and IP 3 with the EcoRI template a product of approximately 1500 bp was obtained. These amplified DNA fragments were gel purified, sequenced and assembled with that previously obtained. The assembled sequence did not contain the entire MmeI endonuclease open reading frame. The assembled sequence was used to direct synthesis of a second group of inverse PCR primer pairs. The sequences of these primers were:  
                                           primer IP 5:                   5′-TTCAGAAATACGAGCGATGC-3′   (SEQ ID NO:33)                       primer IP 6:           5′-GTCAAGCCATAAACACCATC-3′   (SEQ ID NO:34)                       primer IP 7:           5′-GAGGGTCAGAAAGGAAGCTG-3′   (SEQ ID NO:35)                       primer IP 8:           5′-GTCCAACTAACCCTTTATGG-3′   (SEQ ID NO:36)          
 
         [0103]    Inverse PCR amplification reactions were performed as above. Using primers IP 5 and IP 6, products were obtained from the NlaIII template (approximately 450 bp) and the MspI template (approximately 725 bp), but not from the other circular ligation templates. Using primers IP 7 and IP 8, products were obtained from the EcoRI template (approximately 500 bp), the SphI template (approximately 825 bp) and the BspHI template (approximately 750 bp). These DNA fragments were sequenced and the sequence was assembled with that previously obtained. The assembled sequence did not yet contain the entire MmeI endonuclease open reading frame, so another round of primer synthesis and inverse PCR was performed. Additional DNA templates were generated as above, but using the restriction enzymes ApoI (R/AATTY), AseI (AT/TAAT), BsaHI (GR/CGYC), MfeI (C/AATTG), SspI (AAT/ATT) and EcoRV (GAT/ATC) to digest  M. methylotrophus  genomic DNA. The sequences of this third round of primers were:  
         [0104]    primer IP 9:  
                                           primer IP 9:                   5′-TTCCTAGTGCTGAACCTTTG-3′   (SEQ ID NO:37)                       primer IP 10:           5′-GTTGCGTTACTTGAAATGAC-3′   (SEQ ID NO:38)                       primer IP 11:           5′-CCAAAATGGAACTTGTTTCG-3′   (SEQ ID NO:39)                       primer IP 12:           5′-GTGAGTGCGCCCTGAATTAG-3′   (SEQ ID NO:40)          
 
         [0105]    Inverse PCR amplification reactions were performed as above. Using primers IP 9 and IP 10, products were obtained from the NlaIII template (approximately 425 bp), the MfeI template (approximately 750 bp), the ApoI template (approximately 800 bp) and the MspI template (approximately 2100 bp). Using primers IP 11 and IP 12, products were obtained from the SphI template (approximately 875 bp), the BspHI template (approximately 925 bp) and the EcoRI template (approximately 950 bp). These DNA fragments were sequenced and the sequence was assembled with the sequences previously obtained. Further sequencing was performed on the IP 9, IP10 MspI 2100 bp product using three additional primers:  
                                           primer S1:                   5′-GCTTCATTTCATCCTCTGTGC-3′   (SEQ ID NO:41)                       primer S2:           5′-TAACCGCCAAAATTAATCGTG-3′   (SEQ ID NO:42)                       primer S3:           5′-CCACTATTCATTACAACACC-3′   (SEQ ID NO:43)          
 
         [0106]    The final assembled sequence (FIG. 2) contained the entire MmeI restriction gene, as well as 1640 bp of sequence preceding the gene and 1610 bp of sequence following the gene.  
         [0107]    6. Cloning the MmeI endonuclease gene in  E. coli : The putative MmeI endonuclease open reading frame was identified from the DNA sequence assembly obtained from sequencing the various inverse PCR amplified DNA fragments. The beginning of the open reading frame was identified on the basis of the match of the predicted amino acid sequence at the amino terminus of the open reading frame with the sequence determined from the MmeI endonuclease protein. The predicted end of the open reading frame would allow for the coding of an approximately 105 kD polypeptide, which matched the observed size of the native MmeI endonuclease. The amino acid sequence deduced from translation of this open reading frame contained conserved sequence motifs of N6mA DNA methyltransferases. However, no open reading frame containing sequence motifs conserved among DNA methyltransferases was observed adjacent to the MmeI endonuclease gene, as had been expected. It was decided to try to express the MmeI endonuclease in  E. coli  without having a second methyltransferase present to protect the  E. coli  host DNA from cleavage. Oligonucleotide primers were synthesized to specifically amplify the MmeI gene from  M. methylotrophus  genomic DNA for expression in the cloning vector pRRS (Skoglund, Gene 88:1-5 (1990)). The forward primer contained a PstI site for cloning, a stop codon in frame with the lacZ gene of the vector, a consensus  E. coli  ribosome binding site, the ATG start codon for translation (changed from the GTG used by  M. methylotrophus  to facilitate greater expression in  E. coli ) and 20 nucleotides that matched the  M. methylotrophus  DNA sequence:  
         [0108]    5′-GTTCTGCAGTTAAGGATAACATATGGCTTTAAGCTGGAACGAG-3′ (SEQ ID NO:44)  
         [0109]    The reverse primer contained a BamHI site for cloning and 22 nucleotides that matched the  M. methylotrophus  DNA sequence 3′ to the end of the MmeI open reading frame:  
         [0110]    5′-GTTGGATCCGTCGACATTAATTAATTTTTGCCCTTAG-3′ (SEQ ID NO:45)  
         [0111]    The MmeI gene was amplified in a PCR reaction by combining:  
         [0112]    50 μl 10× Thermopol buffer (NEB)  
         [0113]    30 μl 4 mM DNTP solution  
         [0114]    12.5 μl forward primer (10 μM stock)  
         [0115]    12.5 μl reverse primer (10 μM stock)  
         [0116]    5 μl MmeI genomic DNA (500 μg/ml stock)  
         [0117]    387 μl dH 2 O  
         [0118]    3 μl (6 units) Vent® DNA polymerase  
         [0119]    The reaction was mixed and aliquoted into 5 tubes of 80 μl each. MgSO 4  was added (100 mM stock) to bring the final concentration of Mg++ions to 2 mM, 3 mM, 4 mM, 5 mM and 6 mM respectively. The cycling parameters were 95° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 3 minutes, for 24 cycles. The reactions were analyzed by gel electrophoresis and the 3 mM through 6 mM Mg++reactions were found to contain a DNA band of the desired size of 2.8 kb. These reactions were pooled and the 2.8 kb band was gel purified. The 2.8 kb amplified MmeI gene fragment was digested with BamHI and PstI endonucleases (NEB) in the following reaction conditions:  
         [0120]    15 μl 10×BamHI reaction buffer (NEB)  
         [0121]    1.5 μl BSA (NEB)  
         [0122]    50 μl MmeI gene 2.8 kb amplified DNA fragment  
         [0123]    80 μl dH 2 O  
         [0124]    5 μl BamHI endonuclease (100 units)  
         [0125]    5 μl PstI endonuclease (100 units)  
         [0126]    The reaction was mixed and incubated for 1 hour at 37° C. The small fragments cleaved off the ends of the 2.8 kb DNA fragment were removed, along with the endonucleases, by purification on a Qiagen QiaPrep spin column according to the manufacturer&#39;s instructions.  
         [0127]    The cleaved MmeI gene DNA fragment was ligated to the pRRS vector as follows: 10 μl of the digested, purified 2.8 kb MmeI fragment was combined with 5 μl pRRS vector previously cleaved with BamHI and PstI and purified, 5 μl dH 2 O, 20 μl 2× QuickLigase Buffer (NEB), the reaction was mixed, and 2 μl of QuickLigase was added. The reaction was incubated at room temperature for 5 minutes. 5 μl of the ligation reaction was transformed into 50 μl chemical competent  E. coli  ER2683 cells and the cells were plated on L-broth plates containing 100 μg/ml ampicillin and incubated at 370C overnight. Approximately 200 transformants were obtained and 18 representatives were analyzed as follows: plasmid from each colony was isolated by miniprep procedures and digested with AlwNI and NdeI endonucleases to determine if they contained the correct size insert. 2 of the 18 transformants had the correct size insert of approximately 2800 bp. Both clones were tested to see if they produced MmeI endonuclease activity. The clones were grown overnight at 370C in 500 mL L-broth containing 100 μg/ml ampicillin. The cells were harvested by centrifugation, suspended in 10 mL sonication buffer (20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, pH7.5) and broken by sonication. The crude lysate was cleared by centrifugation and the supernatant was recovered. The lysate was assayed for endonuclease activity by serial dilution of the lysate in 1× reaction buffer NEBuffer 1 (New England Biolabs) containing 20 μg/ml lambda DNA substrate and supplemented with SAM at 100 μM final concentration. The reactions were incubated for 1 hour at 37° C. The reaction products were analyzed by agarose gel electrophoresis on a 1% agarose gel in 1× TBE buffer. One of the two clones had MmeI endonuclease activity. This active clone was designated strain NEB1457 and was used for subsequent production of MmeI. The plasmid construct expressing MmeI activity in this clone was designated pTBMmeI.1.  
       EXAMPLE III  
     The MmeI Endonuclease Provides In Vivo Protection Against MmeI Cleavage  
       [0128]    The plasmid pTBMmeI.1 was purified from NEB1457 using the Qiagen miniprep protocol. This plasmid has two MmeI sites in the vector backbone, and one site within the MmeI gene. The plasmid was digested with MmeI to test whether this DNA was resistant to MmeI endonuclease activity, which would indicate that the single MmeI gene was able to methylate DNA in vivo to protect the host DNA against its endonuclease activity. To test this the following were combined:  
         [0129]    10 μl pTBMmeI.1 miniprep DNA  
         [0130]    15 μl 10×NEBuffer 4  
         [0131]    15 μl SAM (1 mM stock solution)  
         [0132]    110 μl dH20  
         [0133]    1 μl MmeI endonuclease (15 units)  
         [0134]    The reaction was mixed and split in thirds. To one third was added 0.5 μl dH 2 O, to the second was added 0.5 μl pRRS vector and to the third was added 0.5 μl PhiX174 DNA as a positive control. The pTBMmeI.1 was not cleaved by the MmeI endonuclease activity, while the Phix174 and pRRS DNAs in the same reaction were cleaved, indicating that the three MmeI sites in the pTBMmeI.1 DNA are resistant to MmeI endonuclease activity (FIG. 4).  
       EXAMPLE IV  
     MmeI Endonuclease Sensitivity to Methylation  
       [0135]    The prior literature reports that MmeI endonuclease methylates just one strand of its recognition sequence, and that this hemi-methylation does not block subsequent cleavage of the DNA by the endonuclease (Tucholski, Gene 223 (1998) 293-302). To test this a set of four oligonucleotides were synthesized so that a DNA substrate could be formed that was either unmethylated (oligo 1+oligo 2), methylated in the top strand only (oligo 3+oligo 2), methylated in the bottom strand only (oligo 1+oligo 4), or methylated on both strands (oligo 3+oligo 4). The oligos synthesized were:  
                               Oligo 1:               5′-FAM-GTTTGAAGACTCCGACGCGATGGCCAGCGATCGGCGCCTCAGCTTT   (SEQ ID NO:46)       TG-3′               Oligo 2:       5′-FAM-CAAAAGCTGAGGCGCCGATCGCTGGCCATCGCGTCGGAGTCTTCA   (SEQ ID NO:47)       AAC-3′               Oligo 3:       5′-FAM-GTTTGAAGACTCCG (6mA) CGCGATGGCCAGCGATCGGCGCCTCAGCTT   (SEQ ID NO:48)       TTG-3′               Oligo 4:       5′-FAM-CAAAAGCTGAGGCGCCGATCGCTGGCCATCGCGTCGG (6mA) GTCTTCA   (SEQ ID NO:49)       AAC-3′          
 
         [0136]    (Other nucleotides outside the MmeI recognition sequence were also methylated for other studies, but since MmeI does not have any sequence specifity for these nucleotides this does affect MmeI activity and these other methylations are omitted here for clarity.) Duplex DNA was formed by mixing 100 μl top strand oligo (14 μM stock) with 100 μl bottom strand oligo (14 μM stock), heating to 85° C. and cooling slowly to 30° C. over a time of 20 minutes. MmeI was then used to cleave the oligo pairs in a 30 μl reaction of 1× NEBuffer4, 2.5 μM oligo, 100 μM SAM and 2.5 units MmeI. As a control, restriction endonuclease Hpy188I was also used to cleave the oligo DNA. The Hpy188I recognition sequence overlaps the first 5 nucleotides of the MmeI recognition sequence in this DNA, 5′-TCNGA-3′ and is blocked by methylation at the adenine in either strand of the DNA. MmeI was found to cleave unmethylated DNA as expected. In contrast to previous teaching (Tucholski, Gene 223:293302 (1998)) MmeI did not cleave the hemi-methylated DNA when the top strand only was methylated: 5′-TCCG(N6mA)C3′. When the bottom strand only was methylated MmeI did cleave the DNA. When both strands were methylated MmeI did not cleave the DNA. (FIG. 5) This finding is consistent with both the observed ability of the single MmeI enzyme to protect host DNA against cleavage in vivo and the observation that MmeI methylates only the top strand of its recognition sequence. We confirmed the report that MmeI enzyme methylates only the top strand of its recognition sequence by methylating the oligo pairs above with tritium labeled H 3 -SAM, washing away the unincorporated SAM and counting the radioactivity in the DNA. Both the unmethylated oligo DNA and the top unmethylated, bottom methylated DNAs had greater than 10-fold more counts than background, while the bottom unmethylated, top methylated DNA and the DNA with both strands methylated had counts near background (FIG. 6). These findings indicate that MmeI is a novel type of restriction modification system which does not require a separate methyltransferase enzyme to modify the host DNA to provide protection against the activity of the endonuclease, as is the case for the type IIG (also called type IV) enzymes such as Eco57I.  
       EXAMPLE V  
     DNA Sequencing and Analysis  
       [0137]    DNA Sequencing: DNA sequencing was performed on double-stranded templates on an ABI 373 or ABI 377 automated sequencer. Amplified DNA fragments and individual clones were sequenced with primers synthesized as above or from universal primers located in the vector.  
         [0138]    Computer analyses: Computer analyses of the DNA sequences obtained were performed with the Genetics Computer Group programs (Deverenx, et al., Nucleic Acids Res. 12:387-395 (1984)) and database similarity searches were performed via the internet at the National Center for Biotechnology Information site (http://www.ncbi.nlm.nih.gov/BLAST/) using the BLASTX and the BLASTP algorithms (Altschul, et al., J. Mol. Biol 215:403-410 (1990) and Gish, et al., Nature Genet. 3:266-722 (1993)).  
     
       
       
         1 
         
           
             50  
           
           
             1  
             6010  
             DNA  
             Methylophilus methylotrophus  
             
               misc_feature  
               (800)..(800)  
               n is a, c, g, or t  
             
           
            1 

gaattccaga taggtagtcc tttggtactt ccatcccaac cagtgtcacg ttccgcgcca     60 

aaccaatcgg ttaaagtgta agaaagtctt gcactgaagt agctgtagga caaaccgaag    120 

ttaacctctg tggtatccca gcgaccacct ttaggtgttt gacggaagcc tgctgcgtca    180 

cctgccaagt tatatttctt ccatgaacca cctgggtaca ggtagctgat caaaccagca    240 

gtccaaccca agccttcaat agcaggaata gttccgttat acccaccata aatatcaatt    300 

tcggcagttg catcagggaa ggtatttggt gtcacgtttg aaccccatgc accgacataa    360 

aagccgctgt catgagtaat atcaataccg ccttgaacgg caggtttgtg ccagttttgt    420 

gaaataccac gagcatagta atctgaaaca aatccaacgt ttgcagtagc agcccaggct    480 

gatttttctt ctttagcctc ttcagctgcg tatgaaactt gggcaaaaga taatgtgctt    540 

aacactgctg tgagcaatat agattgacgc attatgagtc ctctctctgt gaaatctttg    600 

attaagttgt tgtaaacgag aatgaaacaa caaccacaaa gcaaagcacg tgccaaacta    660 

taaataacat tataatcaat tatttaaaat atatttataa tctaaaatat taaattaatt    720 

atttaataaa ctgtttttta ttgatttaac tctaaaacat atgggtgcaa ccaccctttt    780 

tactcactga taatgctaan atagccaaca aaggagcctt caccatgctg atttcaaatg    840 

aaaaaattca ggaattatct ttaaaaatca aacaactaat cgaatcaagc cccatttcag    900 

agctaaataa caacttgcat gcactaattc agggcgcact caccaaaatg gaacttgttt    960 

cgcgtgaaga attcgatatc caatctgcat tattagcgcg cacgcaagag caattaaaac   1020 

gtcttgaaga aaaaatcagc cagcttgaag aagggcaggc atccagaaag taaaaattaa   1080 

tttacaattg ttagcattcc attattgagg agtgcgctat gagtctggcg gtgttataca   1140 

gtcgcgcgtt aagcggcatg gaggcgccag aagtggtggt agaagtccac ttggcgaatg   1200 

gactacccag ctttaccatt gttgaaacat attgaaactt taagccttag cattttttca   1260 

aatatacaaa tgccccaagc tggtgcatta agaagaatgt aacaactccc tgcagactag   1320 

gaataacttc atgatttaac gaacatccct gagtttcaaa gtcgaatctt ctcgtgttgc   1380 

aaatttctac agcttccttt ctgaccctct tgcaccaaat tgcactatgg cgctaataaa   1440 

tcttctgcta tccaataatg tccaactaac cctttatgga ctcttaaaaa agatttaata   1500 

aatgattaag atgaattcaa ggaatttgat gcctggaaat atggcaaaag caaaaaggca   1560 

gcccagtgct gacttttttg ttttaacatt ggcccatata tccaatttca aataatttaa   1620 

aaattatcgg gagctaatct gtggctttaa gctggaacga gataagaaga aaagctattg   1680 

agttttctaa aagatgggaa gacgcctcag atgaaaacag tcaagccaaa ccctttttaa   1740 

tagatttttt cgaagttttt ggaataacta ataagagagt tgcaacattt gagcatgctg   1800 

tgaaaaagtt cgccaaggcc cataaggaac aatctcgagg attcgtagat ttgttttggc   1860 

ctggcattct tcttattgaa atgaaaagca gaggtaaaga cctcgacaaa gcgtatgacc   1920 

aggcacttga ttacttttct ggcattgcag aaagagactt acccagatac gttttagttt   1980 

gcgacttcca gcgtttcaga ttaacagacc taataacaaa agagtcagtt gaatttcttt   2040 

taaaggactt ataccaaaat gtgaggtctt ttggttttat agctggttat caaactcaag   2100 

taatcaagcc acaagaccct attaatatta aggcggctga acggatgggt aagcttcatg   2160 

acaccctgaa gttggttgga tatgagggac acgctttaga actttatcta gtgcgtttac   2220 

ttttttgctt attcgcagaa gacacaacta tttttgagaa aagtttattc caagaatata   2280 

tcgagacaaa gacgctagag gacggcagtg accttgcaca tcatatcaat acactttttt   2340 

atgttctcaa taccccagaa caaaaaagat taaagaatct agacgaacac cttgctgcat   2400 

ttccatatat caatggaaaa cttttcgagg agccacttcc gccagctcag tttgataaag   2460 

caatgagaga ggcattgctt gacttgtgct cattagattg gagcaggatt tcaccagcaa   2520 

tatttggaag tttattccaa agcattatgg atgctaaaaa gagaagaaat cttggggcac   2580 

actacaccag cgaagcaaat attctcaagt taatcaagcc attgtttctt gacgagctct   2640 

gggtagagtt cgagaaagtt aaaaataata aaaataaatt actagcgttc cacaaaaaac   2700 

taagaggact tacatttttc gaccctgcat gcggttgcgg aaattttctt gtaatcacat   2760 

accgagaact aagactttta gaaattgaag tgttaagagg attgcataga ggtggtcaac   2820 

aagttttgga tattgagcat cttattcaga ttaacgtaga ccagtttttt ggtatcgaaa   2880 

tagaggagtt tcccgcacag attgctcagg ttgctctctg gcttacagac caccaaatga   2940 

atatgaaaat ttcagatgag tttggaaact actttgcccg tatcccacta aaatctactc   3000 

ctcacatttt gaatgctaat gctttacaga ttgattggaa cgatgtttta gaggctaaaa   3060 

aatgttgctt catattagga aatcctccat ttgttggtaa aagtaaacaa acaccgggac   3120 

aaaaagcgga tttactatct gtttttggaa atcttaaatc cgcttcagac ttagacctag   3180 

ttgctgcttg gtatcccaaa gcagcacatt acattcaaac aaatgcaaac atacgctgtg   3240 

catttgtctc aacgaatagt attactcaag gtgagcaagt atcgttgctt tggccgcttc   3300 

tgctctcatt aggcataaaa ataaactttg ctcacagaac tttcagctgg acaaatgagg   3360 

cgtcaggagt agcggcggtt cactgcgtaa ttatcggatt tgggttgaag gattcagatg   3420 

aaaaaataat ctatgagtat gaaagtatta atggagaacc attagctatt aaggcaaaaa   3480 

atattaatcc atatttgaga gacggggtgg atgtgattgc ctgcaagcgt cagcagccaa   3540 

tctcaaaatt accaagcatg cgttatggca acaaaccaac agatgatgga aatttcctat   3600 

ttactgacga agaaaaaaac caatttatta caaatgagcc atcttccgaa aaatacttca   3660 

gacggtttgt gggcggggat gagttcataa acaatacaag tcgatggtgt ttatggcttg   3720 

acggtgctga catttcagaa atacgagcga tgcctttggt cttggctagg ataaaaaaag   3780 

tccaagaatt cagattaaaa agctcggcca aaccaactcg acaaagtgct tcgacaccaa   3840 

tgaagttctt ttatatatct cagccggata cggactatct gttgatacct gaaacatcat   3900 

ctgaaaacag acaatttatt ccaattggtt ttgttgatag aaatgtcatt tcaagtaacg   3960 

caacgtatca tattcctagt gctgaacctt tgatatttgg cctgctttca tcgaccatgc   4020 

acaactgctg gatgagaaat gtaggaggaa ggttagaaag tcgttataga tattctgcca   4080 

gcctggttta caacacgttt ccatggattc aacccaacga aaaacaatcg aaagcgatag   4140 

aagaagctgc atttgcgatt ttaaaagcta gaagcaatta tccaaacgaa agtttagctg   4200 

gtttatacga cccaaaaaca atgcctagtg agcttcttaa agcacatcaa aaacttgata   4260 

aggctgtgga ttctgtctat ggatttaaag gaccaaacac agaaattgct cgaatagctt   4320 

ttttgtttga aacataccaa aagatgactt cactcttacc accagaaaaa gaaattaaga   4380 

aatctaaggg caaaaattaa ttaatgtatt taacattaaa ccaccctgat ttatttcgaa   4440 

tagttcaaat gcttccatgt ggactaatcg ccttcaatca tattaaaaaa ccgacgctag   4500 

taataaaaac ttccaaagag gccatattaa ccgccaaaat taatcgtgaa tttaaaatat   4560 

atctttatca aaccacatcg gcttgtgttc tagtaagtgc attttttgac gattctgata   4620 

gtccactatt cattacaaca ccaattgttc gagatgacca acactcctta gacttgttaa   4680 

gatttttaat caacaatgat tttacgattt gcttctttga tgaactgaac cgagaatttc   4740 

tttccgttaa cgcaactggt aatttagtct ctatctttga gagcattcac ttgatgccac   4800 

tgccgagccc agaggaagcc cacaatgcat tgaatgaagc ggaattttgg ttcagtttac   4860 

gctcagctgc tgatgatgaa tcatctatcc aggtttcttt attggataat ctatttcctg   4920 

acgattttgt aatttatgac ctatcctcaa acaaaaacga tatgacatca ttggttagag   4980 

aaactaaacc aggatactat caggaagcag atattgcaaa gttactaaca agagctttta   5040 

gtttggaaag catttatcag aatccagtga aaacaagcga ttcaaaagag ttggcagacg   5100 

ttgtggtatt cggccaaaag gaaattttaa taattcaagc taaagatagt gaaaacaatc   5160 

agaaacaagt tttagaggtt tcgttagaca agaaatgcgc aaagtcttca aagaaacttt   5220 

ctgaagcttt ggcacaactc accgacacta tcttaacaat atccaataca ccaatagttg   5280 

atgttcgggt tggtaagaaa aaatgcactc tgaactttga gggaaagcag cttattggta   5340 

tcgtcgttgt taaagagctt tttaatgata tttacgataa atacagtcaa aaagtttttg   5400 

agcatgtaga gttgtctaaa gcacccattg tcttctttga ctatccagaa tttgcaagaa   5460 

tgacatttca ttgtaattct gaggaattat tactttatgc tttgcatagg atatttagtt   5520 

ctgcaataga aaatggaatg tataaacgat tgagatttac tcaacctatc ataactgatg   5580 

gtcatgacag ctacttcagg atacaaaaca ggccccattc tgatgaggcc tatttaattt   5640 

gcacagagga tgaaatgaag ctctcaaata agtttaaaga ctaaatttat attttcctca   5700 

gtatcttaaa aacaatattc attaaattgg aaagcccgca atgattgttg cagtatcaat   5760 

gcgggcatca gtatccagct cttgcaatac acggaagtat caagaagcga atcaggattc   5820 

taaccatacc tttttaattg caacaatcta atttccataa catgtgtagc tacatcgaaa   5880 

aaaagacctc gaagaggttg caagagcgtc cagctcgcgg catcaaaaga ccctagtctt   5940 

ttgacaaggg ggagccaaaa aactgaggtg gaggagcttg ccgacgaagc caggaagccc   6000 

cagcgtccgg                                                          6010 

 
           
             2  
             919  
             PRT  
             Methylophilus methylotrophus  
           
            2 

Met Ala Leu Ser Trp Asn Glu Ile Arg Arg Lys Ala Ile Glu Phe Ser 
1               5                   10                  15 

Lys Arg Trp Glu Asp Ala Ser Asp Glu Asn Ser Gln Ala Lys Pro Phe 
            20                  25                  30 

Leu Ile Asp Phe Phe Glu Val Phe Gly Ile Thr Asn Lys Arg Val Ala 
        35                  40                  45 

Thr Phe Glu His Ala Val Lys Lys Phe Ala Lys Ala His Lys Glu Gln 
    50                  55                  60 

Ser Arg Gly Phe Val Asp Leu Phe Trp Pro Gly Ile Leu Leu Ile Glu 
65                  70                  75                  80 

Met Lys Ser Arg Gly Lys Asp Leu Asp Lys Ala Tyr Asp Gln Ala Leu 
                85                  90                  95 

Asp Tyr Phe Ser Gly Ile Ala Glu Arg Asp Leu Pro Arg Tyr Val Leu 
            100                 105                 110 

Val Cys Asp Phe Gln Arg Phe Arg Leu Thr Asp Leu Ile Thr Lys Glu 
        115                 120                 125 

Ser Val Glu Phe Leu Leu Lys Asp Leu Tyr Gln Asn Val Arg Ser Phe 
    130                 135                 140 

Gly Phe Ile Ala Gly Tyr Gln Thr Gln Val Ile Lys Pro Gln Asp Pro 
145                 150                 155                 160 

Ile Asn Ile Lys Ala Ala Glu Arg Met Gly Lys Leu His Asp Thr Leu 
                165                 170                 175 

Lys Leu Val Gly Tyr Glu Gly His Ala Leu Glu Leu Tyr Leu Val Arg 
            180                 185                 190 

Leu Leu Phe Cys Leu Phe Ala Glu Asp Thr Thr Ile Phe Glu Lys Ser 
        195                 200                 205 

Leu Phe Gln Glu Tyr Ile Glu Thr Lys Thr Leu Glu Asp Gly Ser Asp 
    210                 215                 220 

Leu Ala His His Ile Asn Thr Leu Phe Tyr Val Leu Asn Thr Pro Glu 
225                 230                 235                 240 

Gln Lys Arg Leu Lys Asn Leu Asp Glu His Leu Ala Ala Phe Pro Tyr 
                245                 250                 255 

Ile Asn Gly Lys Leu Phe Glu Glu Pro Leu Pro Pro Ala Gln Phe Asp 
            260                 265                 270 

Lys Ala Met Arg Glu Ala Leu Leu Asp Leu Cys Ser Leu Asp Trp Ser 
        275                 280                 285 

Arg Ile Ser Pro Ala Ile Phe Gly Ser Leu Phe Gln Ser Ile Met Asp 
    290                 295                 300 

Ala Lys Lys Arg Arg Asn Leu Gly Ala His Tyr Thr Ser Glu Ala Asn 
305                 310                 315                 320 

Ile Leu Lys Leu Ile Lys Pro Leu Phe Leu Asp Glu Leu Trp Val Glu 
                325                 330                 335 

Phe Glu Lys Val Lys Asn Asn Lys Asn Lys Leu Leu Ala Phe His Lys 
            340                 345                 350 

Lys Leu Arg Gly Leu Thr Phe Phe Asp Pro Ala Cys Gly Cys Gly Asn 
        355                 360                 365 

Phe Leu Val Ile Thr Tyr Arg Glu Leu Arg Leu Leu Glu Ile Glu Val 
    370                 375                 380 

Leu Arg Gly Leu His Arg Gly Gly Gln Gln Val Leu Asp Ile Glu His 
385                 390                 395                 400 

Leu Ile Gln Ile Asn Val Asp Gln Phe Phe Gly Ile Glu Ile Glu Glu 
                405                 410                 415 

Phe Pro Ala Gln Ile Ala Gln Val Ala Leu Trp Leu Thr Asp His Gln 
            420                 425                 430 

Met Asn Met Lys Ile Ser Asp Glu Phe Gly Asn Tyr Phe Ala Arg Ile 
        435                 440                 445 

Pro Leu Lys Ser Thr Pro His Ile Leu Asn Ala Asn Ala Leu Gln Ile 
    450                 455                 460 

Asp Trp Asn Asp Val Leu Glu Ala Lys Lys Cys Cys Phe Ile Leu Gly 
465                 470                 475                 480 

Asn Pro Pro Phe Val Gly Lys Ser Lys Gln Thr Pro Gly Gln Lys Ala 
                485                 490                 495 

Asp Leu Leu Ser Val Phe Gly Asn Leu Lys Ser Ala Ser Asp Leu Asp 
            500                 505                 510 

Leu Val Ala Ala Trp Tyr Pro Lys Ala Ala His Tyr Ile Gln Thr Asn 
        515                 520                 525 

Ala Asn Ile Arg Cys Ala Phe Val Ser Thr Asn Ser Ile Thr Gln Gly 
    530                 535                 540 

Glu Gln Val Ser Leu Leu Trp Pro Leu Leu Leu Ser Leu Gly Ile Lys 
545                 550                 555                 560 

Ile Asn Phe Ala His Arg Thr Phe Ser Trp Thr Asn Glu Ala Ser Gly 
                565                 570                 575 

Val Ala Ala Val His Cys Val Ile Ile Gly Phe Gly Leu Lys Asp Ser 
            580                 585                 590 

Asp Glu Lys Ile Ile Tyr Glu Tyr Glu Ser Ile Asn Gly Glu Pro Leu 
        595                 600                 605 

Ala Ile Lys Ala Lys Asn Ile Asn Pro Tyr Leu Arg Asp Gly Val Asp 
    610                 615                 620 

Val Ile Ala Cys Lys Arg Gln Gln Pro Ile Ser Lys Leu Pro Ser Met 
625                 630                 635                 640 

Arg Tyr Gly Asn Lys Pro Thr Asp Asp Gly Asn Phe Leu Phe Thr Asp 
                645                 650                 655 

Glu Glu Lys Asn Gln Phe Ile Thr Asn Glu Pro Ser Ser Glu Lys Tyr 
            660                 665                 670 

Phe Arg Arg Phe Val Gly Gly Asp Glu Phe Ile Asn Asn Thr Ser Arg 
        675                 680                 685 

Trp Cys Leu Trp Leu Asp Gly Ala Asp Ile Ser Glu Ile Arg Ala Met 
    690                 695                 700 

Pro Leu Val Leu Ala Arg Ile Lys Lys Val Gln Glu Phe Arg Leu Lys 
705                 710                 715                 720 

Ser Ser Ala Lys Pro Thr Arg Gln Ser Ala Ser Thr Pro Met Lys Phe 
                725                 730                 735 

Phe Tyr Ile Ser Gln Pro Asp Thr Asp Tyr Leu Leu Ile Pro Glu Thr 
            740                 745                 750 

Ser Ser Glu Asn Arg Gln Phe Ile Pro Ile Gly Phe Val Asp Arg Asn 
        755                 760                 765 

Val Ile Ser Ser Asn Ala Thr Tyr His Ile Pro Ser Ala Glu Pro Leu 
    770                 775                 780 

Ile Phe Gly Leu Leu Ser Ser Thr Met His Asn Cys Trp Met Arg Asn 
785                 790                 795                 800 

Val Gly Gly Arg Leu Glu Ser Arg Tyr Arg Tyr Ser Ala Ser Leu Val 
                805                 810                 815 

Tyr Asn Thr Phe Pro Trp Ile Gln Pro Asn Glu Lys Gln Ser Lys Ala 
            820                 825                 830 

Ile Glu Glu Ala Ala Phe Ala Ile Leu Lys Ala Arg Ser Asn Tyr Pro 
        835                 840                 845 

Asn Glu Ser Leu Ala Gly Leu Tyr Asp Pro Lys Thr Met Pro Ser Glu 
    850                 855                 860 

Leu Leu Lys Ala His Gln Lys Leu Asp Lys Ala Val Asp Ser Val Tyr 
865                 870                 875                 880 

Gly Phe Lys Gly Pro Asn Thr Glu Ile Ala Arg Ile Ala Phe Leu Phe 
                885                 890                 895 

Glu Thr Tyr Gln Lys Met Thr Ser Leu Leu Pro Pro Glu Lys Glu Ile 
            900                 905                 910 

Lys Lys Ser Lys Gly Lys Asn 
        915 

 
           
             3  
             932  
             PRT  
             unknown  
             
               GenBank No. gi|28373198|ref|NP_783835.1  
             
           
            3 

Met Pro Thr Arg Gln Gln Ala Ala Arg Glu Phe Val Lys Thr Trp Ser 
1               5                   10                  15 

Ser Asp Lys Lys Gly Arg Glu Asp Ala Asp Arg Gln Thr Phe Trp Asn 
            20                  25                  30 

Asp Leu Leu Gln Arg Val Tyr Gly Ile Asp Asn Tyr Tyr Asp Tyr Ile 
        35                  40                  45 

Thr Tyr Glu Lys Asp Val Gln Val Lys Ala Asp Gly Lys Val Thr Thr 
    50                  55                  60 

Arg Arg Ile Asp Gly Tyr Ile Pro Ser Thr Lys Ile Met Val Glu Met 
65                  70                  75                  80 

Lys Gly Lys Asn Ile Lys Asp Leu Ser Lys Pro Ile Thr Gln Ser Gly 
                85                  90                  95 

Gly Asp Glu Leu Thr Pro Phe Glu Gln Ala Lys Arg Tyr Ala Asn Phe 
            100                 105                 110 

Leu Pro Asn Ser Glu Gln Pro Arg Trp Ile Leu Val Ser Asn Phe Asn 
        115                 120                 125 

Glu Ile Asp Ile His Asp Met Glu Arg Pro Leu Asp Glu Pro Lys Val 
    130                 135                 140 

Ile Lys Leu Glu Asp Leu Pro Lys Lys Val Lys Ser Leu Glu Phe Met 
145                 150                 155                 160 

Val Asp Ala Asn Gln Gln Gln Val Ile Asp Glu Lys Gln Leu Ser Val 
                165                 170                 175 

Asp Ala Gly Asn Leu Val Ala Lys Ile Tyr Asn Glu Leu Thr Asn Ala 
            180                 185                 190 

Tyr Ala Ala Gly Arg Gly Ile Asp Val Asn Glu Pro Arg Ile Gln Arg 
        195                 200                 205 

Ser Leu Asn Met Leu Ile Val Arg Leu Val Phe Leu Leu Tyr Ala Asp 
    210                 215                 220 

Asp Ser Asn Leu Phe Gly Lys Glu Asp Ile Phe Gln Ala Phe Ile Glu 
225                 230                 235                 240 

Arg Arg Glu Pro Arg Asp Ile Arg Arg Asp Leu Ser Glu Leu Phe Lys 
                245                 250                 255 

Val Leu Asp Gln Pro Glu Glu Gln Arg Asp Pro Tyr Leu Asp Asp Glu 
            260                 265                 270 

Phe Asn Gln Phe Ala Tyr Val Asn Gly Gly Met Phe Ser Asp Glu Asn 
        275                 280                 285 

Val Ile Ile Pro Gln Phe Thr Asp Glu Leu Lys Arg Leu Ile Val Glu 
    290                 295                 300 

Asp Ala Gly Arg Gly Phe Asp Trp Ser Gly Ile Ser Pro Thr Ile Phe 
305                 310                 315                 320 

Gly Ala Val Phe Glu Ser Thr Leu Asn Pro Glu Thr Arg Arg Ser Gly 
                325                 330                 335 

Gly Met His Tyr Thr Ser Ile Glu Asn Ile His Lys Val Ile Asp Pro 
            340                 345                 350 

Leu Phe Leu Asn Asp Leu His Asp Glu Phe Asp Lys Ile Gln Asn Met 
        355                 360                 365 

Gly Asn Arg Arg Gln Arg Val Thr Arg Ala Lys Ala Phe Arg Asp Lys 
    370                 375                 380 

Leu Gly Lys Leu Lys Phe Phe Asp Pro Ala Cys Gly Ser Gly Asn Phe 
385                 390                 395                 400 

Leu Thr Glu Thr Tyr Leu Ser Leu Arg Lys Met Glu Asn Glu Cys Leu 
                405                 410                 415 

Arg Ile Ile Val Gly Asn Gln Gly Ala Leu Ala Leu Thr Asp Glu Ser 
            420                 425                 430 

Glu Pro Lys Val Lys Ile Gln Asn Phe Tyr Gly Ile Glu Ile Asn Asp 
        435                 440                 445 

Phe Ala Val Ser Val Ala Arg Thr Ala Met Trp Ile Ala Glu Ser Gln 
    450                 455                 460 

Met Trp Glu Gln Thr Lys Asp Ile Thr Phe Ala Asn Lys Asp Phe Leu 
465                 470                 475                 480 

Pro Leu Asp Ser Asn Asp Ser Ile Tyr Glu Gly Asn Ala Leu Arg Met 
                485                 490                 495 

Asp Trp Asn Asp Ile Val Lys Pro Tyr Glu Leu Asp Tyr Ile Met Gly 
            500                 505                 510 

Asn Pro Pro Phe Val Gly Tyr Ser Leu Gln Thr Lys Glu Gln Lys Gln 
        515                 520                 525 

Asp Ile Lys Gln Glu Phe Phe Lys Tyr Thr Asp Lys Tyr Gly Lys Phe 
    530                 535                 540 

Asp Tyr Val Ser Gly Trp Tyr Ile Lys Gly Ala Lys Tyr Ile Gln Asn 
545                 550                 555                 560 

Ser Thr Ile Lys Val Gly Phe Val Ser Thr Asp Ser Ile Ile Gln Gly 
                565                 570                 575 

Glu Gln Ala Pro Glu Ile Trp Lys Val Leu Phe Asn Asp Phe His Ile 
            580                 585                 590 

Phe Ile Asn Tyr Gly Tyr Arg Ser Phe Glu Trp Asn Asn Glu Ala Ala 
        595                 600                 605 

Asn Lys Ala Lys Val Asp Val Val Ile Val Gly Phe Ser Thr Lys Glu 
    610                 615                 620 

Asp Lys Asn Pro Thr Ile Tyr Asp Glu Gln Lys Ile Ile Ser Ala Lys 
625                 630                 635                 640 

His Ile Asn Gln Tyr Met Tyr Asp Ser Asp Asn Ile Phe Ile Asp Thr 
                645                 650                 655 

Thr Arg Lys Tyr Ile Glu Ala Met Pro Lys Met Lys Thr Gly Asn Arg 
            660                 665                 670 

Pro Ala Asp Gly Gly Ala Leu Ile Leu Ser Pro Lys Glu Ala Lys Glu 
        675                 680                 685 

Leu Val Asn Glu Glu Pro Gln Ser Lys Gln Phe Ile Lys Lys Leu Thr 
    690                 695                 700 

Gly Ser Lys Glu Phe Ile Thr Gly Lys Tyr Arg Tyr Cys Leu Trp Leu 
705                 710                 715                 720 

Val Asn Val Thr Pro Lys Gln Leu Arg Ser Met Pro Leu Val Leu Lys 
                725                 730                 735 

Arg Val Glu Gln Cys Lys Glu Asn Arg Leu Ser Gly Ala Pro Asp Arg 
            740                 745                 750 

Gln Lys Leu Ala Ala Thr Pro His Leu Phe Arg Glu Gln Met Asn Pro 
        755                 760                 765 

Asp Asn Tyr Met Ile Val Pro Leu Val Thr Gly Cys Arg Arg Lys Tyr 
    770                 775                 780 

Val Pro Phe Gly Tyr Leu Gly Asn Asp Ile Ile Pro Thr Asn Leu Ala 
785                 790                 795                 800 

Thr Ile Ile Pro Glu Ala Asp His Tyr Ala Phe Gly Val Leu Glu Ser 
                805                 810                 815 

Ile Val His Met Ala Trp Met Arg Val Val Ala Gly Arg Lys Gly Thr 
            820                 825                 830 

Ser Tyr Arg Tyr Ser Lys Asn Leu Val Tyr Thr Asn Phe Pro Trp Pro 
        835                 840                 845 

Val Val Asp Ile Asn Gln Lys Glu Lys Ile Thr Ile Thr Ala Gln Asp 
    850                 855                 860 

Ile Leu Asn Ala Arg Asn Leu Tyr Pro Asp Ser Ser Leu Ala Asp Leu 
865                 870                 875                 880 

Tyr Asp Pro Leu Thr Met Pro Ile Glu Leu Arg Lys Ala His Glu Ala 
                885                 890                 895 

Asn Asp Lys Ala Val Leu Lys Ala Tyr Gly Leu Lys Pro Ser Ala Thr 
            900                 905                 910 

Glu Pro Glu Ile Val Gln His Leu Phe Lys Met Tyr Glu Lys Leu Thr 
        915                 920                 925 

Lys Lys Asp Trp 
    930 

 
           
             4  
             354  
             PRT  
             unknown  
             
               GenBank No. 
      gi|27450519|gb|AA014619.1|AF465251_62  
             
           
            4 

Val Leu Phe Asn Asp Phe His Ile Phe Ile Asn Tyr Gly Tyr Arg Ser 
1               5                   10                  15 

Phe Glu Trp Asn Asn Glu Ala Ala Asn Lys Ala Lys Val Asp Val Val 
            20                  25                  30 

Ile Val Gly Phe Ser Thr Lys Glu Asp Lys Asn Pro Thr Ile Tyr Asp 
        35                  40                  45 

Ser Ser Asn Ile Ser His Cys Lys Asn Ile Asn Gly Tyr Leu Phe Asp 
    50                  55                  60 

Gly Asn Asn Ile Phe Val Thr Asn Arg Pro Ala Pro Leu Ser Asn Val 
65                  70                  75                  80 

Pro Arg Met His Asn Gly Cys Lys Leu Leu Asp Gly Gly Phe Tyr Thr 
                85                  90                  95 

Leu Thr Ser Gln Glu Arg Lys Glu Ala Ile Ser Lys Asp Pro Tyr Ala 
            100                 105                 110 

Asp Lys Phe Ile Arg Pro Tyr Leu Gly Ala Lys Asn Phe Ile His Gly 
        115                 120                 125 

Thr Ala Arg Tyr Cys Ile Trp Leu Lys Asp Ala Asn Pro Lys Asp Ile 
    130                 135                 140 

His Gln Ser Pro Phe Ile Leu Asp Arg Ile Asn Lys Val Ala Glu Phe 
145                 150                 155                 160 

Arg Ser Gln Gln Lys Ser Lys Asp Thr Gln Lys Tyr Ala Lys Arg Pro 
                165                 170                 175 

Met Leu Pro Thr Arg Leu Ala Tyr Tyr Ser His Asp Glu His Thr Asp 
            180                 185                 190 

Met Leu Ile Val Pro Ala Thr Ser Ser Gln Arg Arg Glu Tyr Leu Pro 
        195                 200                 205 

Ile Gly Tyr Val Ser Glu Lys Asn Ile Val Ser Tyr Ser Leu Met Leu 
    210                 215                 220 

Ile Pro Asn Ala Ser Asn Phe Asn Phe Gly Ile Leu Glu Ser Lys Val 
225                 230                 235                 240 

His Tyr Ile Trp Leu Lys Asn Phe Cys Gly Arg Leu Lys Ser Asp Tyr 
                245                 250                 255 

Arg Tyr Ser Asn Thr Ile Ile Tyr Asn Asn Phe Pro Trp Pro Thr Val 
            260                 265                 270 

Gly Asp Lys Gln Glu Gln Asn Ile Ser Glu Thr Ala Gln Gly Ile Leu 
        275                 280                 285 

Asn Thr Arg Lys Leu Tyr Pro Asp Ser Ser Leu Ala Asp Leu Tyr Asp 
    290                 295                 300 

Pro Leu Thr Met Pro Val Glu Leu Arg Lys Ala His Glu Ala Asn Asp 
305                 310                 315                 320 

Lys Ala Val Leu Lys Ala Tyr Gly Leu Ser Pro Lys Ala Thr Glu Gln 
                325                 330                 335 

Glu Ile Val Glu His Leu Phe Lys Met Tyr Glu Lys Leu Thr Lys Gly 
            340                 345                 350 

Glu Arg 

 
           
             5  
             919  
             PRT  
             unknown  
             
               M. methylotrophus amino acid  
             
           
            5 

Met Ala Leu Ser Trp Asn Glu Ile Arg Arg Lys Ala Ile Glu Phe Ser 
1               5                   10                  15 

Lys Arg Trp Glu Asp Ala Ser Asp Glu Asn Ser Gln Ala Lys Pro Phe 
            20                  25                  30 

Leu Ile Asp Phe Phe Glu Val Phe Gly Ile Thr Asn Lys Arg Val Ala 
        35                  40                  45 

Thr Phe Glu His Ala Val Lys Lys Phe Ala Lys Ala His Lys Glu Gln 
    50                  55                  60 

Ser Arg Gly Phe Val Asp Leu Phe Trp Pro Gly Ile Leu Leu Ile Glu 
65                  70                  75                  80 

Met Lys Ser Arg Gly Lys Asp Leu Asp Lys Ala Tyr Asp Gln Ala Leu 
                85                  90                  95 

Asp Tyr Phe Ser Gly Ile Ala Glu Arg Asp Leu Pro Arg Tyr Val Leu 
            100                 105                 110 

Val Cys Asp Phe Gln Arg Phe Arg Leu Thr Asp Leu Ile Thr Lys Glu 
        115                 120                 125 

Ser Val Glu Phe Leu Leu Lys Asp Leu Tyr Gln Asn Val Arg Ser Phe 
    130                 135                 140 

Gly Phe Ile Ala Gly Tyr Gln Thr Gln Val Ile Lys Pro Gln Asp Pro 
145                 150                 155                 160 

Ile Asn Ile Lys Ala Ala Glu Arg Met Gly Lys Leu His Asp Thr Leu 
                165                 170                 175 

Lys Leu Val Gly Tyr Glu Gly His Ala Leu Glu Leu Tyr Leu Val Arg 
            180                 185                 190 

Leu Leu Phe Cys Leu Phe Ala Glu Asp Thr Thr Ile Phe Glu Lys Ser 
        195                 200                 205 

Leu Phe Gln Glu Tyr Ile Glu Thr Lys Thr Leu Glu Asp Gly Ser Asp 
    210                 215                 220 

Leu Ala His His Ile Asn Thr Leu Phe Tyr Val Leu Asn Thr Pro Glu 
225                 230                 235                 240 

Gln Lys Arg Leu Lys Asn Leu Asp Glu His Leu Ala Ala Phe Pro Tyr 
                245                 250                 255 

Ile Asn Gly Lys Leu Phe Glu Glu Pro Leu Pro Pro Ala Gln Phe Asp 
            260                 265                 270 

Lys Ala Met Arg Glu Ala Leu Leu Asp Leu Cys Ser Leu Asp Trp Ser 
        275                 280                 285 

Arg Ile Ser Pro Ala Ile Phe Gly Ser Leu Phe Gln Ser Ile Met Asp 
    290                 295                 300 

Ala Lys Lys Arg Arg Asn Leu Gly Ala His Tyr Thr Ser Glu Ala Asn 
305                 310                 315                 320 

Ile Leu Lys Leu Ile Lys Pro Leu Phe Leu Asp Glu Leu Trp Val Glu 
                325                 330                 335 

Phe Glu Lys Val Lys Asn Asn Lys Asn Lys Leu Leu Ala Phe His Lys 
            340                 345                 350 

Lys Leu Arg Gly Leu Thr Phe Phe Asp Pro Ala Cys Gly Cys Gly Asn 
        355                 360                 365 

Phe Leu Val Ile Thr Tyr Arg Glu Leu Arg Leu Leu Glu Ile Glu Val 
    370                 375                 380 

Leu Arg Gly Leu His Arg Gly Gly Gln Gln Val Leu Asp Ile Glu His 
385                 390                 395                 400 

Leu Ile Gln Ile Asn Val Asp Gln Phe Phe Gly Ile Glu Ile Glu Glu 
                405                 410                 415 

Phe Pro Ala Gln Ile Ala Gln Val Ala Leu Trp Leu Thr Asp His Gln 
            420                 425                 430 

Met Asn Met Lys Ile Ser Asp Glu Phe Gly Asn Tyr Phe Ala Arg Ile 
        435                 440                 445 

Pro Leu Lys Ser Thr Pro His Ile Leu Asn Ala Asn Ala Leu Gln Ile 
    450                 455                 460 

Asp Trp Asn Asp Val Leu Glu Ala Lys Lys Cys Cys Phe Ile Leu Gly 
465                 470                 475                 480 

Asn Pro Pro Phe Val Gly Lys Ser Lys Gln Thr Pro Gly Gln Lys Ala 
                485                 490                 495 

Asp Leu Leu Ser Val Phe Gly Asn Leu Lys Ser Ala Ser Asp Leu Asp 
            500                 505                 510 

Leu Val Ala Ala Trp Tyr Pro Lys Ala Ala His Tyr Ile Gln Thr Asn 
        515                 520                 525 

Ala Asn Ile Arg Cys Ala Phe Val Ser Thr Asn Ser Ile Thr Gln Gly 
    530                 535                 540 

Glu Gln Val Ser Leu Leu Trp Pro Leu Leu Leu Ser Leu Gly Ile Lys 
545                 550                 555                 560 

Ile Asn Phe Ala His Arg Thr Phe Ser Trp Thr Asn Glu Ala Ser Gly 
                565                 570                 575 

Val Ala Ala Val His Cys Val Ile Ile Gly Phe Gly Leu Lys Asp Ser 
            580                 585                 590 

Asp Glu Lys Ile Ile Tyr Glu Tyr Glu Ser Ile Asn Gly Glu Pro Leu 
        595                 600                 605 

Ala Ile Lys Ala Lys Asn Ile Asn Pro Tyr Leu Arg Asp Gly Val Asp 
    610                 615                 620 

Val Ile Ala Cys Lys Arg Gln Gln Pro Ile Ser Lys Leu Pro Ser Met 
625                 630                 635                 640 

Arg Tyr Gly Asn Lys Pro Thr Asp Asp Gly Asn Phe Leu Phe Thr Asp 
                645                 650                 655 

Glu Glu Lys Asn Gln Phe Ile Thr Asn Glu Pro Ser Ser Glu Lys Tyr 
            660                 665                 670 

Phe Arg Arg Phe Val Gly Gly Asp Glu Phe Ile Asn Asn Thr Ser Arg 
        675                 680                 685 

Trp Cys Leu Trp Leu Asp Gly Ala Asp Ile Ser Glu Ile Arg Ala Met 
    690                 695                 700 

Pro Leu Val Leu Ala Arg Ile Lys Lys Val Gln Glu Phe Arg Leu Lys 
705                 710                 715                 720 

Ser Ser Ala Lys Pro Thr Arg Gln Ser Ala Ser Thr Pro Met Lys Phe 
                725                 730                 735 

Phe Tyr Ile Ser Gln Pro Asp Thr Asp Tyr Leu Leu Ile Pro Glu Thr 
            740                 745                 750 

Ser Ser Glu Asn Arg Gln Phe Ile Pro Ile Gly Phe Val Asp Arg Asn 
        755                 760                 765 

Val Ile Ser Ser Asn Ala Thr Tyr His Ile Pro Ser Ala Glu Pro Leu 
    770                 775                 780 

Ile Phe Gly Leu Leu Ser Ser Thr Met His Asn Cys Trp Met Arg Asn 
785                 790                 795                 800 

Val Gly Gly Arg Leu Glu Ser Arg Tyr Arg Tyr Ser Ala Ser Leu Val 
                805                 810                 815 

Tyr Asn Thr Phe Pro Trp Ile Gln Pro Asn Glu Lys Gln Ser Lys Ala 
            820                 825                 830 

Ile Glu Glu Ala Ala Phe Ala Ile Leu Lys Ala Arg Ser Asn Tyr Pro 
        835                 840                 845 

Asn Glu Ser Leu Ala Gly Leu Tyr Asp Pro Lys Thr Met Pro Ser Glu 
    850                 855                 860 

Leu Leu Lys Ala His Gln Lys Leu Asp Lys Ala Val Asp Ser Val Tyr 
865                 870                 875                 880 

Gly Phe Lys Gly Pro Asn Thr Glu Ile Ala Arg Ile Ala Phe Leu Phe 
                885                 890                 895 

Glu Thr Tyr Gln Lys Met Thr Ser Leu Leu Pro Pro Glu Lys Glu Ile 
            900                 905                 910 

Lys Lys Ser Lys Gly Lys Asn 
        915 

 
           
             6  
             936  
             PRT  
             unknown  
             
               GenBank No. gi|15794682|ref|NP_284504.1  
             
           
            6 

Met Lys Thr Leu Leu Gln Leu Gln Thr Ala Ala Gln Asn Phe Ala Ala 
1               5                   10                  15 

Tyr Tyr Lys Asp Gln Thr Asp Glu Arg Arg Glu Lys Asp Thr Phe Asn 
            20                  25                  30 

Glu Phe Phe Ala Ile Phe Gly Ile Asp Arg Lys Asn Val Ala His Phe 
        35                  40                  45 

Glu Tyr Pro Val Lys Asp Pro Ala Asp Asn Thr Gln Phe Val Asp Ile 
    50                  55                  60 

Phe Trp Glu Gly Ile Phe Leu Ala Glu His Lys Ser Ala Asn Lys Asn 
65                  70                  75                  80 

Leu Thr Lys Ala Lys Glu Gln Ala Glu Arg Tyr Leu Gln Glu Ile Gly 
                85                  90                  95 

Arg Thr Lys Pro Ser Ala Leu Pro Glu Tyr Tyr Ala Val Ser Asp Phe 
            100                 105                 110 

Ala His Phe His Leu Tyr Arg Arg Val Pro Glu Glu Gly Ala Glu Asn 
        115                 120                 125 

Gln Trp Gln Phe Pro Leu Glu Glu Leu Pro Glu Tyr Ile Thr Arg Gly 
    130                 135                 140 

Val Phe Asp Phe Met Phe Gly Ile Glu Ala Lys Val Arg Gln Ile Gln 
145                 150                 155                 160 

Glu Glu Ala Asn Ile Gln Ala Ala Ala Thr Ile Gly Arg Leu His Asp 
                165                 170                 175 

Ala Leu Lys Glu Glu Gly Ile Tyr Glu Glu His Glu Leu Arg Leu Phe 
            180                 185                 190 

Ile Thr Arg Leu Leu Phe Leu Phe Phe Ala Asp Asp Ser Ala Val Phe 
        195                 200                 205 

Arg Arg Asn Tyr Leu Phe Gln Asp Phe Leu Glu Asn Cys Lys Glu Ala 
    210                 215                 220 

Asp Thr Leu Gly Asp Lys Leu Asn Gln Leu Phe Glu Phe Leu Asn Thr 
225                 230                 235                 240 

Pro Asp Gln Lys Arg Ser Lys Thr Gln Ser Glu Lys Phe Lys Gly Phe 
                245                 250                 255 

Glu Tyr Val Asn Gly Gly Leu Phe Lys Glu Arg Leu Arg Thr Phe Asp 
            260                 265                 270 

Phe Thr Ala Lys Gln His Arg Ala Leu Ile Asp Cys Gly Asn Phe Asp 
        275                 280                 285 

Trp Arg Asn Ile Ser Pro Glu Ile Phe Gly Thr Leu Phe Gln Ser Val 
    290                 295                 300 

Met Asp Ala Gln Glu Arg Arg Glu Ala Gly Ala His Tyr Thr Glu Ala 
305                 310                 315                 320 

Ala Asn Ile Asp Lys Val Ile Asn Gly Leu Phe Leu Glu Asn Leu Arg 
                325                 330                 335 

Ala Glu Phe Glu Ala Val Lys Ala Leu Lys Arg Asp Lys Ala Lys Lys 
            340                 345                 350 

Leu Ala Ala Phe Tyr Gln Lys Ile Gln Asn Leu Gln Phe Leu Asp Pro 
        355                 360                 365 

Ala Cys Gly Cys Gly Asn Phe Leu Ile Val Ala Tyr Asp Arg Ile Arg 
    370                 375                 380 

Ala Leu Glu Asp Asp Ile Ile Ala Glu Ala Leu Lys Asp Lys Ala Asp 
385                 390                 395                 400 

Gly Leu Phe Asp Ser Pro Ser Val Gln Cys Arg Leu Lys Gln Phe His 
                405                 410                 415 

Gly Ile Glu Ile Asp Glu Phe Ala Val Leu Ile Ala Arg Thr Ala Met 
            420                 425                 430 

Trp Leu Lys Asn His Gln Cys Asn Ile Arg Thr Gln Ile Arg Phe Asp 
        435                 440                 445 

Gly Glu Val Ala Cys His Thr Leu Pro Leu Glu Asp Ala Ala Glu Ile 
    450                 455                 460 

Ile His Ala Asn Ser Leu Arg Thr Pro Trp Gln Ala Ala Asp Tyr Ile 
465                 470                 475                 480 

Phe Gly Asn Pro Pro Phe Ile Gly Ser Thr Tyr Gln Thr Lys Glu Gln 
                485                 490                 495 

Lys Asn Asp Leu Glu Ser Ile Cys Gly His Ile Lys Gly Tyr Gly Leu 
            500                 505                 510 

Leu Asp Tyr Val Cys Asn Trp Tyr Val Lys Ala Ala Gly Ile Met Ala 
        515                 520                 525 

Gln His Pro Gln Val Gln Thr Ala Phe Val Ser Thr Asn Ser Ile Cys 
    530                 535                 540 

Gln Gly Gln Gln Val Glu Ile Leu Trp Gly Ser Leu Leu Asn Gln Gly 
545                 550                 555                 560 

Ile Glu Ile His Phe Ala His Arg Thr Phe Gln Trp Thr Ser Gln Ala 
                565                 570                 575 

Ala Gly Lys Ala Ala Val His Cys Ile Ile Val Gly Phe Arg Gln Lys 
            580                 585                 590 

Pro Pro Met Pro Ser Glu Lys Thr Leu Tyr Asp Tyr Pro Asp Ile Lys 
        595                 600                 605 

Gly Glu Pro Glu Lys His Ala Val Ala Asn Ile Asn Pro Tyr Leu Ile 
    610                 615                 620 

Asp Ala Pro Asp Leu Ile Ile Ala Lys Arg Ser Arg Pro Ile His Cys 
625                 630                 635                 640 

Glu Pro Asp Met Val Asn Gly Ser Lys Pro Thr Glu Gly Gly Asn Leu 
                645                 650                 655 

Ile Leu Ser Thr Ala Glu Lys Asp Ala Leu Ile Ala Ala Glu Pro Leu 
            660                 665                 670 

Ala Glu Gln Tyr Ile Arg Pro Phe Ile Gly Ala Asp Glu Phe Leu Asn 
        675                 680                 685 

Gly Lys Thr Arg Trp Cys Leu Trp Phe His Gly Val Ser Asp Val Lys 
    690                 695                 700 

Arg Asn His Asp Leu Lys Gln Met Pro Gln Val Gln Ala Arg Ile Gln 
705                 710                 715                 720 

Ala Val Lys Thr Met Arg Glu Ala Ser Ser Asp Lys Gln Thr Gln Lys 
                725                 730                 735 

Asp Ala Ala Thr Pro Trp Leu Phe Gln Lys Ile Arg Gln Pro Ser Asp 
            740                 745                 750 

Gly Asn Tyr Leu Ile Ile Pro Ser Val Ser Ser Glu Ser Arg Arg Phe 
        755                 760                 765 

Ile Pro Ile Gly Tyr Leu Ser Phe Glu Thr Val Val Ser Asn Leu Ala 
    770                 775                 780 

Phe Ile Leu Pro Asn Ala Thr Leu Tyr His Phe Gly Ile Leu Ser Ser 
785                 790                 795                 800 

Thr Met His Asn Ala Phe Met Arg Thr Val Ala Gly Arg Leu Lys Ser 
                805                 810                 815 

Asp Tyr Arg Tyr Ser Asn Thr Val Val Tyr Asn Asn Phe Pro Phe Pro 
            820                 825                 830 

Glu Ser Cys Arg Leu Pro Ser Glu Asn Asp Arg Pro Asp Pro Leu Arg 
        835                 840                 845 

Ala Ala Val Glu Ala Ala Ala Gln Thr Val Leu Asp Ala Arg Gly Gln 
    850                 855                 860 

Tyr Arg Arg Glu Ala Gln Glu Ala Gly Leu Pro Glu Pro Thr Leu Ala 
865                 870                 875                 880 

Glu Leu Tyr Ala Pro Asp Ala Gly Tyr Thr Ala Leu Asp Lys Ala His 
                885                 890                 895 

Ala Thr Leu Asp Lys Ala Val Asp Lys Ala Tyr Gly Tyr Lys Thr Gly 
            900                 905                 910 

Lys Asn Thr Asp Asp Glu Ala Glu Arg Val Ala Phe Leu Phe Glu Leu 
        915                 920                 925 

Tyr Arg Lys Ala Ala Ala Ile Ala 
    930                 935 

 
           
             7  
             879  
             PRT  
             unknown  
             
               GenBank No. gi|16077744|ref|NP_388558.1  
             
           
            7 

Met Ala Leu Ile Asp Leu Glu Asp Lys Ile Ala Glu Ile Val Asn Arg 
1               5                   10                  15 

Glu Asp His Ser Asp Phe Leu Tyr Glu Leu Leu Gly Val Tyr Asp Val 
            20                  25                  30 

Pro Arg Ala Thr Ile Thr Arg Leu Lys Lys Gly Asn Gln Asn Leu Thr 
        35                  40                  45 

Lys Arg Val Gly Glu Val His Leu Lys Asn Lys Val Trp Phe Lys Glu 
    50                  55                  60 

Ala Lys Lys Gly Lys Leu Phe Asp Ala Leu Ile Asp Ile Glu Gln Gln 
65                  70                  75                  80 

Val Glu Tyr Leu Ser Ala Lys Pro Arg Tyr Leu Leu Val Thr Asp Tyr 
                85                  90                  95 

Asp Gly Val Leu Ala Lys Asp Thr Lys Thr Leu Glu Ala Leu Asp Val 
            100                 105                 110 

Lys Phe Glu Glu Leu Pro Gln Tyr Phe Asp Phe Phe Leu Ala Trp Lys 
        115                 120                 125 

Gly Ile Glu Lys Val Glu Phe Glu Lys Glu Asn Pro Ala Asp Ile Lys 
    130                 135                 140 

Ala Ala Glu Arg Phe Ala Arg Ile Tyr Asp Val Leu Arg Lys Glu Asn 
145                 150                 155                 160 

Asn Ile Ile Glu Thr Asn Arg Gly Leu Asp Leu Phe Leu Ile Arg Leu 
                165                 170                 175 

Leu Phe Cys Phe Phe Ala Glu Asp Thr Asp Ile Phe Lys Arg Asn Ser 
            180                 185                 190 

Phe Thr Asn Leu Ile Lys Thr Leu Thr Glu Glu Asp Gly Ser Asn Leu 
        195                 200                 205 

Asn Lys Leu Phe Ala Asp Leu Phe Ile Val Leu Asp Lys Asn Glu Arg 
    210                 215                 220 

Asp Asp Val Pro Ser Tyr Leu Lys Glu Phe Pro Tyr Val Asn Gly Gln 
225                 230                 235                 240 

Leu Phe Thr Glu Pro His Thr Glu Leu Glu Phe Ser Ala Lys Ser Arg 
                245                 250                 255 

Lys Leu Ile Ile Glu Cys Gly Glu Leu Leu Asn Trp Ala Lys Ile Asn 
            260                 265                 270 

Pro Asp Ile Phe Gly Ser Met Ile Gln Ala Val Ala Ser Glu Glu Ser 
        275                 280                 285 

Arg Ser Tyr Leu Gly Met His Tyr Thr Ser Val Pro Asn Ile Met Lys 
    290                 295                 300 

Val Ile Lys Pro Leu Phe Leu Asp Lys Leu Asn Gln Ser Phe Leu Asp 
305                 310                 315                 320 

Ala Tyr Asp Asp Tyr Thr Lys Leu Glu Asn Leu Leu Thr Arg Ile Gly 
                325                 330                 335 

Lys Ile Lys Phe Phe Asp Pro Ala Cys Gly Ser Gly Asn Phe Leu Ile 
            340                 345                 350 

Ile Thr Tyr Lys Glu Leu Arg Arg Met Glu Ile Asn Ile Ile Lys Arg 
        355                 360                 365 

Leu Gln Glu Leu Leu Gly Glu Tyr Leu Tyr Val Pro Ser Val Thr Leu 
    370                 375                 380 

Ser Gln Phe Tyr Gly Ile Glu Ile Glu Asp Phe Ala His Asp Val Ala 
385                 390                 395                 400 

Lys Leu Ser Leu Trp Ile Ala Glu His Gln Met Asn Glu Glu Leu Lys 
                405                 410                 415 

Asn Glu Val His Asn Ala Val Arg Pro Thr Leu Pro Leu His Thr Ala 
            420                 425                 430 

Gly Asp Ile Arg Cys Ala Asn Ala Ile Arg Val Glu Trp Thr Glu Val 
        435                 440                 445 

Cys Pro Ala Gln Gly Ser Glu Glu Val Tyr Val Phe Gly Asn Pro Pro 
    450                 455                 460 

Tyr Leu Gly Ser Lys Lys Gln Asn Lys Glu His Lys Ser Asp Met Leu 
465                 470                 475                 480 

Ser Ile Phe Gly Lys Val Lys Asn Gly Lys Met Leu Asp Tyr Ile Ser 
                485                 490                 495 

Ala Trp Phe Tyr Phe Gly Ala Lys Tyr Ala Ser Thr Thr Asn Ala Lys 
            500                 505                 510 

Val Ala Phe Val Ser Thr Asn Ser Val Thr Gln Gly Glu Gln Val Ser 
        515                 520                 525 

Ile Leu Trp Asn Glu Leu Phe Lys Phe Gly Ile Gln Ile Asn Phe Ala 
    530                 535                 540 

Tyr Lys Ser Phe Lys Trp Ala Asn Asn Ala Lys Asn Asn Ala Ala Val 
545                 550                 555                 560 

Ile Val Val Ile Val Gly Phe Gly Pro Leu Asp Thr Lys Val Asn Lys 
                565                 570                 575 

Tyr Leu Phe Val Asp Glu Thr Lys Lys Leu Val Ser Asn Ile Ser Pro 
            580                 585                 590 

Tyr Leu Thr Asp Gly Glu Asn Ile Leu Val Ser Ser Arg Thr Lys Pro 
        595                 600                 605 

Ile Ser Asp Leu Pro Lys Leu His Phe Gly Asn Met Pro Asn Asp Gly 
    610                 615                 620 

Gly Gly Leu Leu Phe Thr Ile Thr Glu Tyr Thr Asp Ala Ile Asn Lys 
625                 630                 635                 640 

Tyr Pro Glu Leu Val Pro Tyr Phe Lys Lys Phe Ile Gly Ser Val Glu 
                645                 650                 655 

Phe Ile Asn Gly Gly Leu Arg Tyr Cys Leu Trp Leu Asn Glu Ala Lys 
            660                 665                 670 

Tyr Glu Lys Ile Lys Ser Asn Pro Leu Ile Gln Glu Arg Ile Ser Ile 
        675                 680                 685 

Ser Lys Asn His Arg Glu Lys Ser Thr Asp Lys Gly Thr Asn Lys Leu 
    690                 695                 700 

Ala Leu Thr Pro Trp Lys Phe Arg Asp Thr His Glu Thr Thr Asn Tyr 
705                 710                 715                 720 

Ser Ile Val Val Pro Ser Val Ser Ser Glu Asn Arg Phe Tyr Ile Pro 
                725                 730                 735 

Met Gly Leu Ala Gly Ala Asp Thr Ile Leu Ser Asn Leu Ile Tyr Val 
            740                 745                 750 

Ile Tyr Asp Ala Glu Ile Tyr Leu Leu Gly Ile Leu Met Ser Arg Met 
        755                 760                 765 

His Met Thr Trp Val Lys Ala Val Ala Gly Arg Leu Lys Thr Asp Tyr 
    770                 775                 780 

Arg Tyr Ser Ala Gly Leu Cys Tyr Asn Thr Phe Pro Ile Pro Glu Leu 
785                 790                 795                 800 

Ser Thr Arg Arg Lys Asn Glu Ile Glu Glu Ala Ile Leu Glu Ile Leu 
                805                 810                 815 

Asp Leu Arg Glu Glu Gln Gly Gly Thr Leu Ala Glu Leu Tyr Asn Pro 
            820                 825                 830 

Ser Thr Met Pro Ile Glu Leu Lys Val Ala His Glu Lys Leu Asp Gly 
        835                 840                 845 

Ile Val Glu Arg Ala Tyr Arg Gln Lys Gln Phe Glu Ser Asp Glu Glu 
    850                 855                 860 

Arg Leu Glu Val Leu Leu Lys Leu Tyr Gln Glu Met Thr Glu Arg 
865                 870                 875 

 
           
             8  
             952  
             PRT  
             unknown  
             
               GenBank No. gi|9945797|gb|AAG03371.1  
             
           
            8 

Met Val Met Ala Pro Thr Thr Val Phe Asp Arg Ala Thr Ile Arg His 
1               5                   10                  15 

Asn Leu Thr Glu Phe Lys Leu Arg Trp Leu Asp Arg Ile Lys Gln Trp 
            20                  25                  30 

Glu Ala Glu Asn Arg Pro Ala Thr Glu Ser Ser His Asp Gln Gln Phe 
        35                  40                  45 

Trp Gly Asp Leu Leu Asp Cys Phe Gly Val Asn Ala Arg Asp Leu Tyr 
    50                  55                  60 

Leu Tyr Gln Arg Ser Ala Lys Arg Ala Ser Thr Gly Arg Thr Gly Lys 
65                  70                  75                  80 

Ile Asp Met Phe Met Pro Gly Lys Val Ile Gly Glu Ala Lys Ser Leu 
                85                  90                  95 

Gly Val Pro Leu Asp Asp Ala Tyr Ala Gln Ala Leu Asp Tyr Leu Leu 
            100                 105                 110 

Gly Gly Thr Ile Ala Asn Ser His Met Pro Ala Tyr Val Val Cys Ser 
        115                 120                 125 

Asn Phe Glu Thr Leu Arg Val Thr Arg Leu Asn Arg Thr Tyr Val Gly 
    130                 135                 140 

Asp Ser Ala Asp Trp Asp Ile Thr Phe Pro Leu Ala Glu Ile Asp Glu 
145                 150                 155                 160 

His Ile Glu Gln Leu Ala Phe Leu Ala Asp Tyr Glu Thr Ser Ala Tyr 
                165                 170                 175 

Arg Glu Glu Glu Lys Ala Ser Leu Glu Ala Ser Arg Leu Met Val Glu 
            180                 185                 190 

Leu Phe Arg Ala Met Asn Gly Asp Asp Val Asp Glu Ala Val Gly Asp 
        195                 200                 205 

Asp Ala Pro Thr Thr Pro Glu Glu Glu Asp Glu Arg Val Met Arg Thr 
    210                 215                 220 

Ser Ile Tyr Leu Thr Arg Ile Leu Phe Leu Leu Phe Gly Asp Asp Ala 
225                 230                 235                 240 

Gly Leu Trp Asp Thr Pro His Leu Phe Ala Asp Phe Val Arg Asn Glu 
                245                 250                 255 

Thr Thr Pro Glu Ser Leu Gly Pro Gln Leu Asn Glu Leu Phe Ser Val 
            260                 265                 270 

Leu Asn Thr Ala Pro Glu Lys Arg Pro Lys Arg Leu Pro Ser Thr Leu 
        275                 280                 285 

Ala Lys Phe Pro Tyr Val Asn Gly Ala Leu Phe Ala Glu Pro Leu Ala 
    290                 295                 300 

Ser Glu Tyr Phe Asp Tyr Gln Met Arg Glu Ala Leu Leu Ala Ala Cys 
305                 310                 315                 320 

Asp Phe Asp Trp Ser Thr Ile Asp Val Ser Val Phe Gly Ser Leu Phe 
                325                 330                 335 

Gln Leu Val Lys Ser Lys Glu Ala Arg Arg Ser Asp Gly Glu His Tyr 
            340                 345                 350 

Thr Ser Lys Ala Asn Ile Met Lys Thr Ile Gly Pro Leu Phe Leu Asp 
        355                 360                 365 

Glu Leu Arg Ala Glu Ala Asp Lys Leu Val Ser Ser Pro Ser Thr Ser 
    370                 375                 380 

Val Ala Ala Leu Glu Arg Phe Arg Asp Ser Leu Ser Glu Leu Val Phe 
385                 390                 395                 400 

Ala Asp Met Ala Cys Gly Ser Gly Asn Phe Leu Leu Leu Ala Tyr Arg 
                405                 410                 415 

Glu Leu Arg Arg Ile Glu Thr Asp Ile Ile Val Ala Ile Arg Gln Arg 
            420                 425                 430 

Arg Gly Glu Thr Gly Met Ser Leu Asn Ile Glu Trp Glu Gln Lys Leu 
        435                 440                 445 

Ser Ile Gly Gln Phe Tyr Gly Ile Glu Leu Asn Trp Trp Pro Ala Lys 
    450                 455                 460 

Ile Ala Glu Thr Ala Met Phe Leu Val Asp His Gln Ala Asn Lys Glu 
465                 470                 475                 480 

Leu Ala Asn Ala Val Gly Arg Pro Pro Glu Arg Leu Pro Ile Lys Ile 
                485                 490                 495 

Thr Ala His Ile Val His Gly Asn Ala Leu Gln Leu Asp Trp Ala Asp 
            500                 505                 510 

Ile Leu Ser Ala Ser Ala Ala Lys Thr Tyr Ile Phe Gly Asn Pro Pro 
        515                 520                 525 

Phe Leu Gly His Ala Thr Arg Thr Ala Glu Gln Ala Gln Glu Leu Arg 
    530                 535                 540 

Asp Leu Trp Gly Thr Lys Asp Ile Ser Arg Leu Asp Tyr Val Thr Gly 
545                 550                 555                 560 

Trp His Ala Lys Cys Leu Asp Phe Phe Lys Ser Arg Glu Gly Arg Phe 
                565                 570                 575 

Ala Phe Val Thr Thr Asn Ser Ile Thr Gln Gly Asp Gln Val Pro Arg 
            580                 585                 590 

Leu Phe Gly Pro Ile Phe Lys Ala Gly Trp Arg Ile Arg Phe Ala His 
        595                 600                 605 

Arg Thr Phe Ala Trp Asp Ser Glu Ala Pro Gly Lys Ala Ala Val His 
    610                 615                 620 

Cys Val Ile Val Gly Phe Asp Lys Glu Ser Gln Pro Arg Pro Arg Leu 
625                 630                 635                 640 

Trp Asp Tyr Pro Asp Val Lys Gly Glu Pro Val Ser Val Glu Val Gly 
                645                 650                 655 

Gln Ser Ile Asn Ala Tyr Leu Val Asp Gly Pro Asn Val Leu Val Asp 
            660                 665                 670 

Lys Ser Arg His Pro Ile Ser Ser Glu Ile Ser Pro Ala Thr Phe Gly 
        675                 680                 685 

Asn Met Ala Arg Asp Gly Gly Asn Leu Leu Val Glu Val Asp Glu Tyr 
    690                 695                 700 

Asp Glu Val Met Ser Asp Pro Val Ala Ala Lys Tyr Val Arg Pro Phe 
705                 710                 715                 720 

Arg Gly Ser Arg Glu Leu Met Asn Gly Leu Asp Arg Trp Cys Leu Trp 
                725                 730                 735 

Leu Val Asp Val Ala Pro Ser Asp Ile Ala Gln Ser Pro Val Leu Lys 
            740                 745                 750 

Lys Arg Leu Glu Ala Val Lys Ser Phe Arg Ala Asp Ser Lys Ala Ala 
        755                 760                 765 

Ser Thr Arg Lys Met Ala Glu Thr Pro His Leu Phe Gly Gln Arg Ser 
    770                 775                 780 

Gln Pro Asp Thr Asp Tyr Leu Cys Leu Pro Lys Val Val Ser Glu Arg 
785                 790                 795                 800 

Arg Ser Tyr Phe Thr Val Gln Arg Tyr Pro Ser Asn Val Ile Ala Ser 
                805                 810                 815 

Asp Leu Val Phe His Ala Gln Asp Pro Asp Gly Leu Met Phe Ala Leu 
            820                 825                 830 

Ala Ser Ser Ser Met Phe Ile Thr Trp Gln Lys Ser Ile Gly Gly Arg 
        835                 840                 845 

Leu Lys Ser Asp Leu Arg Phe Ala Asn Thr Leu Thr Trp Asn Thr Phe 
    850                 855                 860 

Pro Val Pro Glu Leu Asp Glu Lys Thr Arg Gln Arg Ile Ile Lys Ala 
865                 870                 875                 880 

Gly Lys Lys Val Leu Asp Ala Arg Ala Leu His Pro Glu Arg Ser Leu 
                885                 890                 895 

Ala Glu His Tyr Asn Pro Leu Ala Met Ala Pro Glu Leu Ile Lys Ala 
            900                 905                 910 

His Asp Ala Leu Asp Arg Glu Val Asp Lys Ala Phe Gly Ala Pro Arg 
        915                 920                 925 

Lys Leu Thr Thr Val Arg Gln Arg Gln Glu Leu Leu Phe Ala Asn Tyr 
    930                 935                 940 

Glu Lys Leu Ile Ser His Gln Pro 
945                 950 

 
           
             9  
             168  
             PRT  
             unknown  
             
               GenBank No. gi|23451826|gb|AAN32874.1  
             
           
            9 

Pro Ala Asp Glu Arg Ser Gln Met Asp Ala Gly Gly Lys Pro Val Glu 
1               5                   10                  15 

Gly Gly Asn Leu Leu Phe Ala Glu Glu Glu Lys Gln Arg Leu Val Glu 
            20                  25                  30 

Gly Asn Val Asp Val Val Lys Phe Leu Lys Arg Val Tyr Gly Ala Ser 
        35                  40                  45 

Glu Tyr Ile Arg Gly Glu Val Arg Phe Cys Leu Trp Ile Ser Asp Ser 
    50                  55                  60 

Gln Glu Gln Glu Ala Lys Ser Asn Ser Asp Ile Asn Cys Lys Leu Asn 
65                  70                  75                  80 

Ala Val Ala Ala Phe Arg Leu Lys Ser Pro Lys Ala Ala Thr Lys Lys 
                85                  90                  95 

Gly Ala Ala Trp Pro His Lys Phe Glu Glu Val Lys Gln Ile Gly Asn 
            100                 105                 110 

Glu Val Val Thr Ile Val Pro Lys Val Ser Ser Glu Ser Arg Glu Tyr 
        115                 120                 125 

Leu Pro Val Gly Leu Leu Pro Arg Gly Ser Ile Val Thr Asp Leu Ala 
    130                 135                 140 

Phe Ala Leu Tyr Asp Ala Pro Leu Trp Asn Met Ala Leu Ile Ala Ser 
145                 150                 155                 160 

Arg Leu His Leu Val Trp Ile Gly 
                165 

 
           
             10  
             909  
             PRT  
             unknown  
             
               GenBank No. gb|23110638|gb|ZP00096791.1  
             
           
            10 

Met Asn Pro Val Glu Ile Glu Glu Ala Val Ser Asp Leu Ala Arg Ala 
1               5                   10                  15 

Pro Tyr Asp Ala Ser Glu Phe Pro Phe Gln Phe Leu Ala Ala Phe Gly 
            20                  25                  30 

Asn Lys Gln Thr Thr Leu Gln Arg Leu Arg Ala Gly Asn Ser Asn Gln 
        35                  40                  45 

Ser Asp Leu Pro Gly Ala Val Leu Gln Arg Asn His Ile His Ile Ala 
    50                  55                  60 

Thr Cys Asp Ala Gly Asn Val Asp Arg Thr Leu Ala Ala Leu Arg Lys 
65                  70                  75                  80 

Ser Pro Lys Thr Ala Ser Gln Lys Ala Arg Phe Ile Leu Ala Thr Asp 
                85                  90                  95 

Gly Val Ala Phe Gln Ala Glu Asp Met Ala Ser Gly Glu Thr Val Ala 
            100                 105                 110 

Cys Asn Tyr Ala Ala Phe Pro Asp Lys Phe Ala Phe Phe Leu Pro Leu 
        115                 120                 125 

Ala Gly Ile Thr Thr Val Gln Gln Ile Arg Glu Ser Ser Phe Asp Ile 
    130                 135                 140 

Lys Ala Thr Gly Arg Leu Asn Lys Leu Tyr Val Glu Leu Leu Lys Asp 
145                 150                 155                 160 

Asn Pro Asp Trp Ala Ser Arg Ser Glu Asp Met Asn His Phe Met Ala 
                165                 170                 175 

Arg Leu Ile Phe Cys Phe Phe Ala Glu Asp Thr Asp Ile Phe Val Gly 
            180                 185                 190 

Glu Gly Leu Phe Ser Arg Thr Val Glu Thr Met Ser Ala Arg Asp Ala 
        195                 200                 205 

Ser Asp Thr His Met Val Ile Ala Glu Ile Phe Arg Ala Met Asp Thr 
    210                 215                 220 

Arg Leu Ala Asp Arg Ala Ala Ala Gly Ile Lys Ser Trp Ala Asp Val 
225                 230                 235                 240 

Phe Pro Tyr Val Asn Gly Gln Leu Phe Ser Gly Ser Thr Glu Cys Pro 
                245                 250                 255 

Arg Phe Ser Lys Ile Ala Arg Ser Tyr Leu Leu His Ile Gly Ser Leu 
            260                 265                 270 

Asp Trp Gln Lys Ile Asn Pro Asp Ile Phe Gly Ser Met Ile Gln Ala 
        275                 280                 285 

Val Ala Asp Asp Glu Glu Arg Gly Ala Leu Gly Met His Tyr Thr Ser 
    290                 295                 300 

Val Pro Asn Ile Leu Lys Val Leu Asn Pro Leu Phe Leu Asp Asp Leu 
305                 310                 315                 320 

Arg Ala Lys Leu Glu Glu Ala Gly Asp Asn Ser Arg Lys Leu Leu Asn 
                325                 330                 335 

Leu Arg Asn Arg Met Ala Lys Ile Arg Val Phe Asp Pro Ala Cys Gly 
            340                 345                 350 

Ser Gly Asn Phe Leu Val Ile Ala Tyr Lys Gln Met Arg Glu Leu Glu 
        355                 360                 365 

Ala Glu Ile Asn Arg Arg Arg Gly Glu Ala Asp Arg Arg Ser Asp Ile 
    370                 375                 380 

Pro Leu Thr Asn Phe Arg Gly Ile Glu Leu Arg Asn Phe Pro Ala Glu 
385                 390                 395                 400 

Ile Ala Arg Leu Ala Leu Ile Ile Ala Glu Tyr Gln Cys Asp Val Leu 
                405                 410                 415 

Tyr Arg Gly Gln Lys Glu Ala Leu Ala Glu Phe Leu Pro Leu Asp Ser 
            420                 425                 430 

Gln Asn Trp Ile Thr Cys Gly Asn Ala Leu Arg Leu Asp Trp Leu Ser 
        435                 440                 445 

Ile Cys Pro Pro Thr Gly Thr Ala Val Lys Leu Gln Ala Asn Asp Leu 
    450                 455                 460 

Phe Glu Met Pro Leu Asp Gln Ala Glu Ile Asp Phe Glu Asn Glu Gly 
465                 470                 475                 480 

Gly Glu Thr Tyr Ile Cys Gly Asn Pro Pro Tyr Leu Gly Ala Lys Lys 
                485                 490                 495 

Lys Ser Ser Asp Gln Ile Glu Asp Met Lys Arg Val Gly Leu Asp Lys 
            500                 505                 510 

Ala Gln Leu Leu Asp Tyr Val Ser Ala Phe Ile Val Arg Gly Leu Pro 
        515                 520                 525 

Leu Val Ala Gln Gln Arg Cys Asp Met Ala Leu Val Ser Thr Ser Ser 
    530                 535                 540 

Ile Cys Gln Gly Glu Gln Val Ser Leu Ile Trp Pro Arg Ile Leu Lys 
545                 550                 555                 560 

Ser Ala Asn Val Lys Phe Ala Tyr Arg Pro Phe Arg Trp Ser Asn Ser 
                565                 570                 575 

Ala Ala Asn Asn Ala Gly Val Tyr Cys Thr Ile Ile Gly Leu Thr Gly 
            580                 585                 590 

Ser Glu Val Ser Asn Lys Lys Leu Phe Gly Glu Gly Ser Val Val Glu 
        595                 600                 605 

Cys Ser Ser Ile Ala Pro Tyr Leu Val Pro Gly Pro Glu Ile Ile Cys 
    610                 615                 620 

Ala Pro Arg Gln Ser Ser Ile Ser Gly Phe Ala Arg Met Val Met Gly 
625                 630                 635                 640 

Ser Asn Pro Val Asp Gly Lys Arg Leu Ile Phe Glu Gln Asp Glu Lys 
                645                 650                 655 

Glu Ser Val Val Ala Ala Asp Pro Arg Ser Glu Arg Phe Phe Lys Arg 
            660                 665                 670 

Tyr Gly Gly Thr Gln Glu Leu Val Asn Gly Val Asp Arg Trp Cys Leu 
        675                 680                 685 

Trp Ile Asn Asp Asp Gln Val Asp Asp Ala Lys Ala Ile Ala Glu Ile 
    690                 695                 700 

Ala Lys Val Leu Glu Ser Cys Arg Ser Tyr Arg Gln Gly Ala Gly Arg 
705                 710                 715                 720 

Asp Ala Gln Lys Ala Ala Asn Arg Pro His Ser Phe Cys Tyr Arg Thr 
                725                 730                 735 

Phe Gln Glu Asn Ile Gly Ile His Val Gly Leu Thr Ile Gly Asn Gly 
            740                 745                 750 

Leu Ser His Val Pro Ala Asp Leu Lys Ser Ser Gly Phe Val Ser Ser 
        755                 760                 765 

His Thr Ala Tyr Met Ile Tyr Gly Trp His Pro Val Glu Phe Ala Leu 
    770                 775                 780 

Leu Asn Ser Arg Leu Met Leu Val Trp Thr Glu Thr Val Gly Gly Arg 
785                 790                 795                 800 

Leu Gly Asn Gly Met Arg Phe Ser Asn Thr Ile Val Tyr Asn Thr Phe 
                805                 810                 815 

Pro Val Pro Ser Leu Thr Asp Gln Asn Lys Ala Asp Leu Thr Arg Cys 
            820                 825                 830 

Ala Glu Asp Ile Leu Leu Ala Arg Glu Ser His Phe Pro Ala Thr Ile 
        835                 840                 845 

Ala Asp Leu Tyr Asp Pro Glu Thr Met Pro Glu Ser Leu Arg Ala Ala 
    850                 855                 860 

His Asp Arg Asn Asp Glu Val Leu Glu Arg Ile Tyr Ile Gly Arg Arg 
865                 870                 875                 880 

Phe Arg Asn Asp Thr Glu Arg Leu Glu Lys Leu Phe Glu Leu Tyr Thr 
                885                 890                 895 

Lys Met Thr Gly Gly Arg Ser Ser Glu Gly Gly Ala Ala 
            900                 905 

 
           
             11  
             1048  
             PRT  
             unknown  
             
               GenBank No. gi|20803963|emb|CAD31540.1  
             
           
            11 

Met Ser Leu Gly Ala Ala Gly Leu Thr Pro Ile Thr Pro Ala Ala Phe 
1               5                   10                  15 

Ile Lys Lys Trp Arg Lys Ser Glu Leu Gly Glu Arg Gln Ala Ala Gln 
            20                  25                  30 

Glu His Phe Leu Asp Ile Cys Ser Leu Val Gly His Pro Ser Pro Ser 
        35                  40                  45 

Asp Glu Asp Pro Thr Gly Ala Phe Phe Ala Phe Glu Lys Gly Ala Asn 
    50                  55                  60 

Lys Leu Gly Gly Gly Lys Gly Phe Ala Asp Val Trp Lys Lys Gly His 
65                  70                  75                  80 

Phe Ala Trp Glu Tyr Lys Arg Lys Lys Gly Asn Leu Asp Glu Ala Leu 
                85                  90                  95 

Leu Gln Leu Met Arg Tyr Ala Pro Ala Leu Leu Ser Pro Pro Leu His 
            100                 105                 110 

Ile Val Cys Asp Ile Glu Arg Leu Arg Ile His Thr Ala Trp Thr Asn 
        115                 120                 125 

Thr Val Pro Ser Thr Tyr Val Ile Thr Leu Asp Asp Leu Ala Glu Pro 
    130                 135                 140 

Ser Ala Arg Glu Met Leu His Asn Val Phe Phe Ser Pro Glu Lys Leu 
145                 150                 155                 160 

Arg Pro Thr Arg Thr Arg Ala Ala Val Thr Lys Glu Ala Ala Asp Lys 
                165                 170                 175 

Phe Ser Ala Ile Ala Leu Arg Val Gln Gly Arg Gly Thr Pro Asp Glu 
            180                 185                 190 

Ile Ala His Phe Val Asn Gln Leu Val Phe Cys Phe Phe Ala Gln Ser 
        195                 200                 205 

Val Ser Leu Leu Pro Asp Gly Leu Phe Thr Lys Leu Leu Lys Arg Ser 
    210                 215                 220 

Ala Arg Ala Pro Glu Arg Ala Met Ser Tyr Leu Asp Lys Leu Phe Glu 
225                 230                 235                 240 

Ala Met Glu Arg Gly Gly Glu Phe Asp Leu Thr Asp Ile Thr Trp Phe 
                245                 250                 255 

Asn Gly Gly Leu Phe Asp Gly Arg Arg Ala Leu Arg Leu Asp Asp Gly 
            260                 265                 270 

Asp Ile Gly Leu Leu Val Ala Ala Asp Ser Leu Asp Trp Gly Leu Ile 
        275                 280                 285 

Asp Pro Thr Ile Phe Gly Thr Leu Phe Glu Arg Phe Leu Asp Pro Glu 
    290                 295                 300 

Lys Arg Ala Gln Ile Gly Ala His Tyr Thr Asp Pro Glu Lys Ile Met 
305                 310                 315                 320 

Arg Leu Val Asp Pro Val Ile Leu Arg Pro Leu Arg Gln Glu Trp Glu 
                325                 330                 335 

Gln Ala Arg Arg Glu Ile Val Glu Leu Leu Asn Gly Asn Arg Lys Pro 
            340                 345                 350 

Pro Met Arg Arg Gln Gln Ser Arg Arg Met Lys Arg Glu Glu Ala Ala 
        355                 360                 365 

Ala Glu Val Arg Ser Arg Phe Thr Glu Arg Leu Arg Lys Leu Arg Ile 
    370                 375                 380 

Leu Asp Pro Ala Cys Gly Ser Gly Asn Phe Leu Tyr Leu Ala Leu Gln 
385                 390                 395                 400 

Gly Val Lys Asp Ile Glu His Arg Ala Asn Leu Asp Cys Glu Met Leu 
                405                 410                 415 

Gly Met Pro Ala Gln Leu Pro Leu Val Gly Pro Glu Ile Leu Arg Gly 
            420                 425                 430 

Ile Glu Ile Asn Met Met Ala Ala Glu Leu Ala Arg Thr Thr Ile Trp 
        435                 440                 445 

Ile Gly Asp Ile Gln Trp Gln Ile Lys Asn Gly Ile Arg Ser Lys Ser 
    450                 455                 460 

Ile Pro Ile Leu Arg Lys Leu Asp Ala Ile Glu Arg Arg Asp Ala Leu 
465                 470                 475                 480 

Val Arg Gln Ala Gln Asp Val Asp Thr Ala Arg Asp Ala Gln Gly Asp 
                485                 490                 495 

Leu Leu Ala Ala Leu Gln Pro Val Ser Glu Asp Ala Glu Ala Glu Trp 
            500                 505                 510 

Pro Glu Ala Glu Phe Ile Val Gly Asn Pro Pro Phe Val Gly Val Arg 
        515                 520                 525 

Leu Met Arg Gln Ala Leu Gly Asp Pro Thr Val Asp Arg Leu Phe Asp 
    530                 535                 540 

Val Tyr Asp Gly Arg Val Ser Arg Glu Ala Asp Leu Val Cys Tyr Trp 
545                 550                 555                 560 

Val Glu Lys Ser Arg Ala Ala Val Ala Ala Asp Arg Thr Arg Arg Val 
                565                 570                 575 

Gly Leu Val Thr Thr Asn Ser Ile Arg Gly Gly Ala Asn Arg Arg Val 
            580                 585                 590 

Leu Asp Arg Ile Ile Ala Glu Ser Arg Leu Phe Glu Ala Trp Ser Asp 
        595                 600                 605 

Glu Pro Trp Val Val Asp Gly Ala Ala Val Arg Val Ser Leu Ile Cys 
    610                 615                 620 

Phe Gly His Gly Glu Asp Pro Leu Cys Leu Asp Gly Arg Thr Val Ala 
625                 630                 635                 640 

Gln Ile Asn Ala Asp Leu Thr Ala Gly Val Thr Asp Leu Thr Lys Ala 
                645                 650                 655 

Arg Arg Leu Ser Glu Asn Gln Asn Val Ala Phe Met Gly Asp Thr Lys 
            660                 665                 670 

Gly Gly Ala Phe Asp Val Pro Gly Ser Leu Ala Arg Ala Trp Leu Ser 
        675                 680                 685 

Met Pro Met Asn Pro Asn Gly Arg Pro Asn Ser Asp Val Leu Arg Pro 
    690                 695                 700 

Trp Arg Asn Gly Met Asp Val Ala Arg Arg Gly Arg Asp Met Trp Ile 
705                 710                 715                 720 

Val Asp Phe Gly Trp Glu Met Ser Glu Gln Glu Ala Ala Leu Tyr Glu 
                725                 730                 735 

Ala Pro Phe Gln His Ile Arg Glu His Val Phe Pro Glu Arg Ser Lys 
            740                 745                 750 

Asn Arg Arg Asp Ala Tyr Arg Glu Arg Trp Trp Arg His Val Glu Pro 
        755                 760                 765 

Arg Pro Ala Phe His Ala Ser Leu Gln Gly His Ser Arg Tyr Met Ala 
    770                 775                 780 

Thr Pro Arg Val Ala Lys His Arg Thr Phe Val Trp Leu Asp Gln Ala 
785                 790                 795                 800 

Ile Val Pro Asp Ser Arg Ile Phe Ala Phe Ser Arg Ser Asp Asp Val 
                805                 810                 815 

Phe Phe Gly Ile Leu His Ser Arg Phe His Glu Ala Trp Ser Phe Gly 
            820                 825                 830 

Thr Cys Ser Trp His Gly Val Gly Asn Asp Pro Thr Tyr Asn Ser Ala 
        835                 840                 845 

Gly Val Phe Glu Thr Phe Pro Phe Pro Glu Gly Leu Thr Pro Asp Ile 
    850                 855                 860 

Pro Ala Val Arg Tyr Glu Lys Asp Ser Arg Ala Ile Ala Ile Ser Lys 
865                 870                 875                 880 

Ala Ala Lys Arg Leu Asp Asp Ile Arg Asn Ala Trp Leu Asn Pro Ser 
                885                 890                 895 

Asp Leu Val Gln Ile Lys Pro Glu Val Val Pro Gly Tyr Pro Asp Arg 
            900                 905                 910 

Ile Leu Pro Lys Asp Ile Ala Ser Asp Ala Ile Leu Arg Asp Arg Thr 
        915                 920                 925 

Leu Thr Asn Leu Tyr Asn Arg Arg Pro Gln Trp Leu Val Asp Ala His 
    930                 935                 940 

Ser Asp Leu Asp Ala Ala Val Ala Gly Ala Tyr Gly Trp Pro Ala Asp 
945                 950                 955                 960 

Ile Ser Glu Asp Glu Ala Leu Ala Asn Leu Leu Glu Leu Asn Leu Ala 
                965                 970                 975 

Arg Glu Ala Phe Asn Glu His Ala Lys Ser Gly Leu Lys Thr Arg Lys 
            980                 985                 990 

Pro Arg Arg Arg Pro Thr Pro Glu  Glu Val Arg Arg Ala  Pro Gln Met 
        995                 1000                 1005 

Lys Leu  Pro Ile Ala Gly Gly  Arg Lys Ser Val Val  Gly Pro Gln 
    1010                 1015                 1020 

Gln Leu  Thr Thr Lys Asp Arg  Glu Asn Gln Pro Thr  Ser Ala Glu 
    1025                 1030                 1035 

Arg Pro  Arg Asn Thr Lys Arg  Arg Thr Ser 
    1040                 1045 

 
           
             12  
             959  
             PRT  
             unknown  
             
               GenBank No. gi|16125079|ref|NP_419643.1  
             
           
            12 

Asp Leu Cys Arg Met Leu Glu Val Pro Thr Pro Ala Glu Asp Asp Pro 
1               5                   10                  15 

Leu Gly Glu Arg Tyr Cys Phe Glu Arg Gly Ala Ala Lys Thr Gly Gly 
            20                  25                  30 

Gly Asp Gly Trp Ala Asp Val Trp Arg Lys Gly Cys Phe Gly Trp Glu 
        35                  40                  45 

Tyr Lys Gly Lys His Lys Asn Leu Asp Ala Ala Leu Arg Gln Leu Gln 
    50                  55                  60 

Ala Tyr Ala Leu Asp Leu Gln Asn Pro Pro Tyr Leu Val Val Ser Asp 
65                  70                  75                  80 

Met Glu Arg Ile Ile Val His Thr Asn Trp Thr Asn Thr Ile Ser Arg 
                85                  90                  95 

Lys Ile Glu Phe Thr Leu Asp Asp Leu His Glu Pro Glu Lys Leu Ala 
            100                 105                 110 

Met Leu Arg Gln Val Phe Asp Gly Ser Asp Ser Leu Lys Pro Lys Ile 
        115                 120                 125 

Ser Pro Gln Glu Leu Thr Ala Lys Val Ala Gln Arg Phe Gly Asp Leu 
    130                 135                 140 

Gly Arg Arg Leu Gln Glu Arg Gly His His Pro Arg Asp Val Ala His 
145                 150                 155                 160 

Phe Leu Asn Arg Val Val Phe Cys Met Phe Ala Glu Asp Ala Lys Leu 
                165                 170                 175 

Leu Pro Glu Gly Leu Phe Thr Arg Leu Thr Arg Ser Met Gln Met Arg 
            180                 185                 190 

Pro Pro Ala Glu Ala Ala Pro Gln Phe Asp Ala Leu Phe Ala Met Met 
        195                 200                 205 

Arg Ala Gly Gly Met Phe Gly Ala Asp Ile Val His Trp Phe Asn Gly 
    210                 215                 220 

Gly Leu Phe Asp Glu Lys Pro Ala Leu Pro Leu Glu Arg Ala Asp Ile 
225                 230                 235                 240 

Lys Leu Ile His Asp Thr Ala Ala Glu His Asp Trp Ser Asp Leu Asp 
                245                 250                 255 

Pro Ser Val Phe Gly Asn Met Phe Glu Glu Ala Leu Lys Ala Thr Arg 
            260                 265                 270 

Glu Arg Ala Ala Leu Gly Ala His Tyr Thr Asp Arg Glu Lys Ile Leu 
        275                 280                 285 

Lys Ile Ile Asp Pro Val Ile Thr Trp Pro Leu Met Ala Gln Trp Glu 
    290                 295                 300 

Thr Ala Leu Ala Glu Ile Arg Ala Ala Leu Asp Ala Arg Ala Ala Ala 
305                 310                 315                 320 

Glu Ala Glu Arg Lys Ala Val Leu Glu Ala Ala Ala Glu Ala Met Arg 
                325                 330                 335 

Ala Asp Pro Val Lys Ala Lys Ala Gly Glu Ala Ala Arg Arg Lys Thr 
            340                 345                 350 

Leu Thr Ala Ile Ala Lys Arg Ser Asp Ala Ala Leu Gly Gln Ala Lys 
        355                 360                 365 

Asp Arg Leu Glu Ala Phe Leu Ser Arg Leu Ala Ala Phe Arg Val Leu 
    370                 375                 380 

Asp Pro Ala Cys Gly Ser Gly Asn Phe Leu Tyr Val Ala Leu His Ala 
385                 390                 395                 400 

Leu Lys Asp Ile Glu Arg Arg Ala Leu Val Asp Ala Glu Arg Leu Gly 
                405                 410                 415 

Leu Glu Val Pro Thr Pro Arg Val Gly Leu Ala Cys Val Arg Gly Ile 
            420                 425                 430 

Glu Ile Glu Glu Tyr Ala Ala Glu Leu Ala Arg Val Thr Leu Trp Ile 
        435                 440                 445 

Gly Asp Leu Gln Trp His Ala Lys Asn Asn Tyr Arg Gly Phe Ala Glu 
    450                 455                 460 

Pro Ile Leu Ser Ser Leu Asp Gln Ile Glu Cys Arg Asp Ala Leu Leu 
465                 470                 475                 480 

Asn Ala Asp Gly Thr Glu Ala Gln Trp Pro Ala Val Asp Val Ile Val 
                485                 490                 495 

Gly Asn Pro Pro Phe Leu Gly Ser Lys Arg Leu Arg Asp Gly Leu Gly 
            500                 505                 510 

Asn Asp Tyr Val Glu Arg Leu Phe Ser Thr Tyr Arg Gly Lys Val Pro 
        515                 520                 525 

Ala Glu Ala Asp Phe Val Ala Tyr Trp Ile Ala Lys Ala Trp Glu Leu 
    530                 535                 540 

Val Gln Ala Gln Gln Gly Arg Arg Ala Gly Leu Val Thr Thr Asn Ser 
545                 550                 555                 560 

Val Arg Gly Gly Ala Ser Arg Lys Val Leu Asp Pro Ile Ala Asp Ala 
                565                 570                 575 

Gly Ala Leu Met Glu Ala Trp Ala Asp Glu Pro Trp Ala Leu Glu Gly 
            580                 585                 590 

Ala Ala Val Arg Val Ser Met Phe Gly Phe Gly Asp Gly Phe Ala Glu 
        595                 600                 605 

Arg Arg Leu Glu Gly Arg Lys Ala Glu His Leu His Ser Asp Phe Arg 
    610                 615                 620 

Gly Ala Ser Thr Asp Val Thr Lys Ala Leu Arg Leu Lys Glu Asn Ala 
625                 630                 635                 640 

Ser Ile Ala Phe Met Gly Asp Thr Lys Gly Gly Ala Phe Asp Val Ser 
                645                 650                 655 

Gly Glu Ile Ala Arg Glu Trp Leu Arg Leu Pro Leu Asn Pro Asn Gly 
            660                 665                 670 

Arg Pro Asn Ser Asp Val Leu Lys Pro Trp Arg Asn Ala Met Asp Met 
        675                 680                 685 

Thr Arg Arg Ser Ser Asp Lys Trp Ile Ile Asp Phe Gly Trp Thr Met 
    690                 695                 700 

Ser Glu Ala Asp Ala Ala Leu Phe Glu Thr Pro Phe Arg His Val Leu 
705                 710                 715                 720 

Leu His Val Lys Pro Glu Arg Asp Arg Asn Asn Arg Glu Met Tyr Arg 
                725                 730                 735 

Leu Asn Trp Trp Lys His Val Glu Pro Arg Gln Gly Leu Met Lys Arg 
            740                 745                 750 

Val Pro Ala Leu Ser Arg Leu Leu Val Thr Pro Glu Val Ser Lys His 
        755                 760                 765 

Arg Leu Phe Ile Trp Leu Asp Ala Arg Val Leu Pro Asp His Lys Leu 
    770                 775                 780 

Gln Val Val Thr Leu Asp Asp Asp Cys Ser Phe Gly Val Leu His Ser 
785                 790                 795                 800 

Arg Phe His Glu Val Trp Ala Leu Ala Ala Gly Ser Trp His Gly Ser 
                805                 810                 815 

Gly Asn Asp Pro Arg Tyr Thr Ile Ser Thr Thr Phe Glu Thr Phe Pro 
            820                 825                 830 

Phe Pro Glu Gly Leu Thr Pro Asn Ile Ala Ala Val Asp Tyr Glu Gly 
        835                 840                 845 

Asp Pro Arg Ala Gln Ala Ile Ala Ala Ala Ala Ala Glu Leu Asn Arg 
    850                 855                 860 

Leu Arg Glu Ala Trp Leu Asn Pro Pro Asp Leu Val Arg Ile Glu Pro 
865                 870                 875                 880 

Glu Val Val Pro Gly Tyr Pro Asp Arg Val Leu Pro Val Ser Pro Glu 
                885                 890                 895 

Ala Gly Ala Glu Leu Lys Lys Arg Thr Leu Thr Asn Leu Tyr Asn Gln 
            900                 905                 910 

Arg Pro Ala Trp Leu Asp Met Ala His Gln Arg Leu Asp Ala Ala Val 
        915                 920                 925 

Ala Ala Ala Tyr Gly Trp Pro Asp Gly Leu Thr Asp Asp Glu Ile Leu 
    930                 935                 940 

Glu Arg Leu Phe Ala Leu Asn Gln Glu Arg Ala Ala Ala Gly Arg 
945                 950                 955 

 
           
             13  
             909  
             PRT  
             unknown  
             
               GenBank No. gi|15807788|ref|NP_285443.1  
             
           
            13 

Met His Pro Gln Glu Phe Ala Asp Thr Trp Ser Arg Arg Ala Leu Lys 
1               5                   10                  15 

Ala Thr Glu Arg Asp Ser Tyr Val Gln His Trp Leu Asp Leu Cys Gln 
            20                  25                  30 

Leu Leu His His Glu Ala Pro Gly Ala Asp Pro Asp Tyr Lys Phe Glu 
        35                  40                  45 

Arg Arg Val Thr Lys Val Gly Thr Lys Asp Lys Gly Phe Ala Asp Val 
    50                  55                  60 

Phe Lys Lys Ala His Phe Ile Thr Glu Tyr Lys Arg Pro Gly Ser Asp 
65                  70                  75                  80 

Leu Gly Ala Ala Leu Gln Gln Ala Thr Leu Tyr Ser Arg Asp Leu Gly 
                85                  90                  95 

Asn Pro Pro Leu Leu Leu Thr Ser Asp Phe Gln Arg Ile Glu Ile Asn 
            100                 105                 110 

Thr Ala Phe Thr Gly Thr Ser Pro Lys Ser Tyr Leu Ile Thr Leu Asp 
        115                 120                 125 

Asp Ile Ala Glu Asn Arg Val Val Gly Gly Asn Asp Val Pro Ala Leu 
    130                 135                 140 

Gln Ile Leu His Ser Ala Leu His Gln Pro Tyr Asp Leu Asp Pro Arg 
145                 150                 155                 160 

Leu Phe Arg Glu Arg Ile Thr Thr Asp Ala Thr Arg Gln Val Gly Leu 
                165                 170                 175 

Val Ala Arg Arg Leu Gly Glu Arg Glu Gly Arg Thr Arg Ala Ala His 
            180                 185                 190 

Met Met Met Arg Val Val Phe Ala Leu Phe Ala Glu Asp Thr Gly Met 
        195                 200                 205 

Leu Glu Arg Gly Ile Val Thr Arg Leu Leu Glu Arg Ala Arg Ala Pro 
    210                 215                 220 

Pro Gly Glu Asp Gln Leu Tyr Phe Gln Asp Leu Phe Gly Ala Met Lys 
225                 230                 235                 240 

Gly Gly Gly Glu Phe Trp Gly Thr Asp Ile Arg His Phe Asn Gly Gly 
                245                 250                 255 

Leu Phe Asp Ser Glu Asp Ala Leu Ala Leu Thr Ser Glu Asp Ala Ala 
            260                 265                 270 

Ala Leu Ile Ile Ala Ala Lys Leu Asp Trp Ser Glu Val Glu Pro Ser 
        275                 280                 285 

Ile Phe Gly Thr Leu Phe Glu Asn Ser Leu Asp Val Asp Thr Arg Ser 
    290                 295                 300 

Arg Arg Gly Ala His Tyr Thr Ser Val Asn Asp Ile Glu Arg Ile Val 
305                 310                 315                 320 

Asp Arg Val Val Met Glu Pro Leu Trp Ala Glu Trp Asp Ala Leu Arg 
                325                 330                 335 

Leu Ser Leu Pro Glu Leu Lys Lys Asn Val Arg Leu Glu Arg Leu Phe 
            340                 345                 350 

Ala Phe Gln Asp Arg Leu Thr Ala Val Arg Ile Leu Asp Pro Ala Cys 
        355                 360                 365 

Gly Ser Gly Asn Phe Leu Phe Val Ala Leu Lys Lys Leu Leu Asp Leu 
    370                 375                 380 

Glu Tyr Gln Val Arg Met Ala Ala Val Met Asn Asp Ile Gly Glu Phe 
385                 390                 395                 400 

Glu Met Pro Pro Leu Val His Pro Gln Gln Met Leu Gly Ile Glu Ile 
                405                 410                 415 

Glu Thr Phe Ala His Glu Leu Ala Ser Ile Thr Leu Trp Met Gly Tyr 
            420                 425                 430 

Phe Gln Trp Lys Arg Ala His Gly Gly His Trp Glu Thr Pro Ile Leu 
        435                 440                 445 

Gln Arg Leu Asp Asn Ile Gln Asn Arg Asp Ala Leu Leu Asn Pro Asp 
    450                 455                 460 

Gly Thr Glu Ala Thr Trp Pro Arg Ala Asp Phe Ile Val Gly Asn Pro 
465                 470                 475                 480 

Pro Phe Leu Gly Asp Lys Met Met Arg Ser Gln Leu Gly Glu Ala Tyr 
                485                 490                 495 

Thr Thr Gln Leu Arg Glu Thr Phe Lys Asp Arg Leu Pro Gly Gln Ser 
            500                 505                 510 

Asp Leu Val Cys Tyr Trp Pro Glu Lys Ala Arg Ala Leu Ile Glu Ala 
        515                 520                 525 

Gly Val Thr Thr Arg Ala Gly Phe Val Thr Thr Asn Ser Ile Arg Gly 
    530                 535                 540 

Gly Lys Asn Arg Val Val Leu Glu Arg Ile Lys Ala Thr Gly Asp Leu 
545                 550                 555                 560 

Phe Met Ala Trp Pro Asp Glu Pro Trp Gln Gln Asn Gly Ala Ala Val 
                565                 570                 575 

Arg Val Ser Leu Phe Gly Phe Asp Asn Gly Thr Glu Thr Leu Arg Thr 
            580                 585                 590 

Leu Asn Asp Gly His Val Gly Val Ile Asn Ala Asp Leu Asn Ala Gly 
        595                 600                 605 

Thr Asp Val Lys Gln Ala Gln Lys Leu Pro Glu Asn Ala Gly Val Ser 
    610                 615                 620 

Phe Ile Gly Thr Gln Lys Gly Gly Ala Phe Asp Ile Pro Gly Asp Leu 
625                 630                 635                 640 

Ala Arg Ser Trp Leu Ser Val Pro Asn Pro Asp Arg Val Ser Asn Ala 
                645                 650                 655 

Asp Val Leu Lys Pro Trp Val Asn Gly Met Asp Leu Thr Arg Arg Pro 
            660                 665                 670 

Ser Gly Arg Trp Ile Ile Asp Phe Ala Gln Met Asp Glu Gly Glu Ala 
        675                 680                 685 

Arg Gln Tyr Leu Gln Pro Met Ala Tyr Val Glu Gln Lys Ile Arg Pro 
    690                 695                 700 

Glu Arg Ala Thr Asn Ser Asp Arg Pro Ser Arg Glu Arg Trp Trp Leu 
705                 710                 715                 720 

His Gln Arg Ser Arg Pro Glu Leu Arg Glu Ala Thr Ile Glu Leu Asp 
                725                 730                 735 

Arg Phe Ile Gly Ile Pro Arg Val Ala Lys His Leu Leu Pro Val Trp 
            740                 745                 750 

Leu Pro Glu Gly Thr Leu Pro Asp Ser Gln Val Val Val Ile Ala Arg 
        755                 760                 765 

Asp Asp Asp Phe Ile Phe Gly Val Leu Ala Ser Thr Ile His Arg Ser 
    770                 775                 780 

Trp Ala Arg Met Gln Gly Thr Tyr Met Gly Val Gly Asn Asp Leu Arg 
785                 790                 795                 800 

Tyr Thr Pro Ser Thr Cys Phe Glu Thr Phe Pro Val Pro Ala Pro Thr 
                805                 810                 815 

Asp Glu Gln Arg Ala Glu Ile Glu Lys Trp Ala Lys Tyr Ile Val Gln 
            820                 825                 830 

Leu Arg Glu His Leu Leu Asn Gln Asp Ala Lys Gly Thr Leu Thr Gly 
        835                 840                 845 

Ile Tyr Asn Gln Leu Glu Lys Leu Arg Asn Ser Pro Asp Ala Ala His 
    850                 855                 860 

Pro Val Ser Ala Leu Ala Thr Ala His Asp Lys Leu Asp Gln Ala Val 
865                 870                 875                 880 

Ala Thr Ala Tyr Gly Trp Glu Trp Pro Leu Asn Glu Asp Gln Val Leu 
                885                 890                 895 

Glu Arg Leu Leu Ala Leu Asn Leu Glu Arg Cys Pro Ala 
            900                 905 

 
           
             14  
             955  
             PRT  
             unknown  
             
               GenBank No. gi|15807258|ref|NP_295988.1  
             
           
            14 

Met Pro Gln Thr Glu Thr Ala Gln Arg Met Glu Asp Phe Val Ala Tyr 
1               5                   10                  15 

Trp Arg Thr Leu Lys Gly Asp Glu Lys Gly Glu Ser Gln Val Phe Leu 
            20                  25                  30 

Asp Arg Leu Phe Gln Ala Phe Gly His Ala Gly Tyr Lys Glu Ala Gly 
        35                  40                  45 

Ala Glu Leu Glu Tyr Arg Val Ala Lys Gln Gly Gly Gly Lys Lys Phe 
    50                  55                  60 

Ala Asp Leu Leu Trp Arg Pro Arg Val Leu Ile Glu Met Lys Lys Arg 
65                  70                  75                  80 

Gly Glu Lys Leu Ala Asn His Tyr Gln Gln Ala Phe Asp Tyr Trp Leu 
                85                  90                  95 

Lys Leu Val Pro Asp Arg Pro Arg Tyr Ala Val Leu Cys Asn Phe Asp 
            100                 105                 110 

Glu Leu Trp Val Tyr Asp Phe Asn Gln Gln Leu Asp Glu Pro Met Asp 
        115                 120                 125 

Arg Leu Arg Ile Glu Glu Leu Pro Glu Arg Tyr Thr Val Leu Asn Phe 
    130                 135                 140 

Met Phe Glu Gln Glu Arg Ala Pro Leu Phe Gly Asn Asn Arg Val Asp 
145                 150                 155                 160 

Val Thr Arg Glu Ala Ala Asp Ser Val Ala Lys Val Leu Asn Ser Val 
                165                 170                 175 

Ile Ala Arg Gly Glu Asp Arg Ala Arg Ala Gln Arg Phe Leu Leu Gln 
            180                 185                 190 

Cys Val Met Ala Met Phe Ala Glu Asp Phe Glu Leu Ile Pro Arg Gly 
        195                 200                 205 

Phe Phe Thr Glu Leu Ala Asp Asp Ala Arg Ala Gly Arg Gly Ser Ser 
    210                 215                 220 

Phe Asp Leu Phe Gly Gly Leu Phe Arg Gln Met Asn Thr Ser Glu Arg 
225                 230                 235                 240 

Ala Arg Gly Gly Arg Phe Ala Pro Ile Pro Tyr Phe Asn Gly Gly Leu 
                245                 250                 255 

Phe Arg Ala Val Asp Pro Ile Glu Leu Asn Arg Asp Glu Leu Tyr Leu 
            260                 265                 270 

Leu His Lys Ala Ala Leu Glu Asn Asn Trp Ala Arg Ile Gln Pro Gln 
        275                 280                 285 

Ile Phe Gly Val Leu Phe Gln Ser Ser Met Asp Lys Lys Glu Gln His 
    290                 295                 300 

Ala Lys Gly Ala His Tyr Thr Ser Glu Ala Asp Ile Met Arg Val Val 
305                 310                 315                 320 

Leu Pro Thr Ile Val Thr Pro Phe Gln Arg Gln Ile Glu Ala Ala Thr 
                325                 330                 335 

Thr Gln Lys Glu Leu Arg Ala Ile Leu Asp Glu Leu Ala Ser Phe Gln 
            340                 345                 350 

Val Leu Asp Pro Ala Cys Gly Ser Gly Asn Phe Leu Tyr Val Ala Tyr 
        355                 360                 365 

Arg Glu Leu Arg Arg Leu Glu Ala Arg Ala Leu Leu Arg Leu Arg Asp 
    370                 375                 380 

Leu Ser Ala Pro Gly Thr Ala Leu Pro Pro Ala Arg Val Ser Ile Arg 
385                 390                 395                 400 

Gln Met His Gly Leu Glu Tyr Asp Pro Phe Gly Val Glu Leu Ala Lys 
                405                 410                 415 

Val Thr Leu Thr Leu Ala Lys Glu Leu Ala Ile Arg Glu Met His Asp 
            420                 425                 430 

Leu Leu Gly Asn Thr Gly Leu Asp Phe Asp Gln Pro Leu Pro Leu Asp 
        435                 440                 445 

Asn Leu Asp Asp Arg Ile Val Gln Gly Asp Ala Leu Phe Thr Pro Trp 
    450                 455                 460 

Pro Arg Val Asp Ala Ile Val Gly Asn Pro Pro Phe Gln Ser Lys Asn 
465                 470                 475                 480 

Lys Leu Gln Arg Glu Met Gly Ala Ala Tyr Val Lys Lys Leu Arg Ala 
                485                 490                 495 

His Tyr Pro Asp Val Pro Gly Arg Ala Asp Tyr Cys Val Tyr Trp Ile 
            500                 505                 510 

Arg Lys Ala His Asp Gln Leu Gly Ser Gly Gln Arg Ala Gly Leu Val 
        515                 520                 525 

Gly Thr Asn Thr Ile Arg Gln Asn Asp Ser Arg Val Gly Gly Leu Asp 
    530                 535                 540 

Tyr Val Val Gln His Gly Gly Thr Ile Thr Asp Ala Val Gly Thr Gln 
545                 550                 555                 560 

Val Trp Ser Gly Asp Ala Ala Val His Val Ser Ile Val Asn Trp Val 
                565                 570                 575 

Lys Gly Pro Ala Glu Gly Pro Lys His Leu Ala Trp Gln Val Gly Asp 
            580                 585                 590 

His Arg Thr Ser Pro Trp Gln Ser Thr Glu Leu Pro Val Ile Asn Ser 
        595                 600                 605 

Ala Leu Ser Ala Gly Thr Asp Val Thr Gln Ala Gln Lys Leu Arg Val 
    610                 615                 620 

Asn Met Asn Ser Gly Ala Cys Tyr Gln Gly Gln Thr His Gly His Lys 
625                 630                 635                 640 

Gly Phe Leu Leu Asp Gly Leu Glu Ala Gly Gln Met Leu Ser Ala Glu 
                645                 650                 655 

Arg Lys Asn Ala Glu Val Ile Phe Pro Tyr Leu Thr Gly Asp Glu Leu 
            660                 665                 670 

Leu Arg Thr Ser Pro Pro His Pro Thr Arg Tyr Val Ile Asp Phe Gln 
        675                 680                 685 

Pro Arg Asp Val Phe Gly Ala Arg Ala Tyr Lys Leu Pro Phe Ala Arg 
    690                 695                 700 

Ile Glu Arg Glu Val Leu Pro Thr Arg Gln Ala Ala Ala Ala Glu Glu 
705                 710                 715                 720 

Glu Ala Arg Asn Ala Glu Val Leu Ala Ala Asn Pro Lys Ala Lys Thr 
                725                 730                 735 

Asn Lys His His Arg Asn Phe Leu Asn Gln Trp Trp Ala Leu Ser Tyr 
            740                 745                 750 

Gly Arg Ser Glu Met Ile Glu Lys Ile Ser Ser Leu Ser Arg Tyr Ile 
        755                 760                 765 

Val Cys Ser Arg Val Thr Lys Arg Gln Val Phe Glu Phe Leu Asp Asn 
    770                 775                 780 

Gly Ile Arg Pro Ser Asp Gly Leu Gln Ile Phe Ala Phe Glu Asp Asp 
785                 790                 795                 800 

Tyr Ser Phe Gly Val Ile Gln Ser Ser Val His Trp Gln Trp Leu Ile 
                805                 810                 815 

Ala Arg Gly Gly Thr Leu Thr Ala Arg Leu Met Tyr Thr Ser Asp Thr 
            820                 825                 830 

Val Phe Asp Thr Phe Pro Trp Pro Asp Pro Thr Leu Ala Gln Val Arg 
        835                 840                 845 

Ala Val Ala Ala Ala Ala Val Lys Leu Arg Glu Leu Arg Asn Lys Val 
    850                 855                 860 

Met Arg Glu Gln Gly Trp Ser Leu Arg Asp Leu Tyr Arg Thr Leu Asp 
865                 870                 875                 880 

Met Pro Gly Lys Asn Pro Leu Arg Asp Ala Gln Glu Arg Leu Asp Ala 
                885                 890                 895 

Ala Val Ser Ala Ala Tyr Gly Leu Pro Ala Gly Ala Asp Met Leu Asp 
            900                 905                 910 

Phe Leu Leu Ala Leu Asn Ala Xaa Val Ala Ala Ala Glu Ala Arg Gly 
        915                 920                 925 

Ala Ala Val Thr Gly Pro Gly Leu Pro Ala Gly Leu Asn Thr Ala Asp 
    930                 935                 940 

Phe Val Thr Ala Asp Ala Val Arg Pro Leu Gly 
945                 950                 955 

 
           
             15  
             14  
             PRT  
             unknown  
             
               first 14 amino terminal residues of MmeI  
             
           
            15 

Ala Leu Ser Trp Asn Glu Ile Arg Arg Lys Ala Ile Glu Phe 
1               5                   10 

 
           
             16  
             29  
             PRT  
             unknown  
             
               first 29 residues of the 25kD peptide  
             
           
            16 

Met Lys Ile Ser Asp Glu Phe Gly Asn Tyr Phe Ala Arg Ile Pro Leu 
1               5                   10                  15 

Lys Ser Thr Xaa Xaa Ile Xaa Glu Xaa Asn Ala Leu Gln 
            20                  25 

 
           
             17  
             40  
             PRT  
             unknown  
             
               first 40 amino acid residues obtained from the 
      14 kD fragment  
             
           
            17 

Met Asp Ala Lys Lys Arg Arg Asn Leu Gly Ala His Tyr Thr Ser Glu 
1               5                   10                  15 

Ala Asn Ile Leu Lys Leu Ile Lys Pro Leu Leu Leu Asp Glu Leu Trp 
            20                  25                  30 

Val Val Phe Xaa Lys Val Lys Asn 
        35                  40 

 
           
             18  
             25  
             PRT  
             unknown  
             
               first 25 residues of the 7.5 kD peptide  
             
           
            18 

Met Lys Ser Arg Gly Lys Asp Leu Asp Lys Ala Tyr Asp Gln Ala Leu 
1               5                   10                  15 

Asp Tyr Phe Ser Gly Ile Ala Glu Arg 
            20                  25 

 
           
             19  
             8  
             PRT  
             unknown  
             
               25 kD fragment primer  
             
           
            19 

Asp Glu Phe Gly Asn Tyr Phe Ala 
1               5 

 
           
             20  
             20  
             DNA  
             unknown  
             
               forward primer  
             
           
            20 

garttyggna aytayttygc                                                 20 

 
           
             21  
             20  
             DNA  
             unknown  
             
               reverse primer  
             
           
            21 

aartarttnc craaytcrtc                                                 20 

 
           
             22  
             6  
             PRT  
             unknown  
             
               14 kD fragment primer  
             
           
            22 

Met Asp Ala Lys Lys Arg 
1               5 

 
           
             23  
             17  
             DNA  
             unknown  
             
               forward primer  
             
           
            23 

atggaygcna araarcg                                                    17 

 
           
             24  
             17  
             DNA  
             unknown  
             
               reverse primer  
             
           
            24 

atggaygcna araarag                                                    17 

 
           
             25  
             20  
             DNA  
             unknown  
             
               reverse primer  
             
           
            25 

cgncgyttyt tngcrtccat                                                 20 

 
           
             26  
             7  
             PRT  
             unknown  
             
               7.5 kD fragment primer  
             
           
            26 

Asp Lys Ala Tyr Asp Gln Ala 
1               5 

 
           
             27  
             20  
             DNA  
             unknown  
             
               forward primer  
             
           
            27 

gayaargcnt aygaycargc                                                 20 

 
           
             28  
             20  
             DNA  
             unknown  
             
               reverse primer  
             
           
            28 

gcytgrtcrt angcyttrtc                                                 20 

 
           
             29  
             26  
             DNA  
             unknown  
             
               primer IP 1  
             
           
            29 

gttggatccc gcacagattg ctcagg                                          26 

 
           
             30  
             30  
             DNA  
             unknown  
             
               primer IP 2  
             
           
            30 

gttggatcct acgttaatct gaataagatg                                      30 

 
           
             31  
             28  
             DNA  
             unknown  
             
               primer IP 3  
             
           
            31 

gttggatcct gttaatctga aacgctgg                                        28 

 
           
             32  
             29  
             DNA  
             unknown  
             
               primer IP 4  
             
           
            32 

gttggatcct tataccaaaa tgtgaggtc                                       29 

 
           
             33  
             20  
             DNA  
             unknown  
             
               primer IP 5  
             
           
            33 

ttcagaaata cgagcgatgc                                                 20 

 
           
             34  
             20  
             DNA  
             unknown  
             
               primer IP 6  
             
           
            34 

gtcaagccat aaacaccatc                                                 20 

 
           
             35  
             20  
             DNA  
             unknown  
             
               primer IP 7  
             
           
            35 

gagggtcaga aaggaagctg                                                 20 

 
           
             36  
             20  
             DNA  
             unknown  
             
               primer IP 8  
             
           
            36 

gtccaactaa ccctttatgg                                                 20 

 
           
             37  
             20  
             DNA  
             unknown  
             
               primer IP 9  
             
           
            37 

ttcctagtgc tgaacctttg                                                 20 

 
           
             38  
             20  
             DNA  
             unknown  
             
               primer IP 10  
             
           
            38 

gttgcgttac ttgaaatgac                                                 20 

 
           
             39  
             20  
             DNA  
             unknown  
             
               primer IP 11  
             
           
            39 

ccaaaatgga acttgtttcg                                                 20 

 
           
             40  
             20  
             DNA  
             unknown  
             
               primer IP 12  
             
           
            40 

gtgagtgcgc cctgaattag                                                 20 

 
           
             41  
             21  
             DNA  
             unknown  
             
               primer S1  
             
           
            41 

gcttcatttc atcctctgtg c                                               21 

 
           
             42  
             21  
             DNA  
             unknown  
             
               primer S2  
             
           
            42 

taaccgccaa aattaatcgt g                                               21 

 
           
             43  
             20  
             DNA  
             unknown  
             
               primer S3  
             
           
            43 

ccactattca ttacaacacc                                                 20 

 
           
             44  
             43  
             DNA  
             unknown  
             
               20 nucleotides that matched the M. 
      methyltrophus DNA sequence  
             
           
            44 

gttctgcagt taaggataac atatggcttt aagctggaac gag                       43 

 
           
             45  
             37  
             DNA  
             unknown  
             
               22 nucleotides that matched the M. 
      methylotrophus DNA sequence  
             
           
            45 

gttggatccg tcgacattaa ttaatttttg cccttag                              37 

 
           
             46  
             48  
             DNA  
             unknown  
             
               oligonucleotide 1  
             
           
            46 

gtttgaagac tccgacgcga tggccagcga tcggcgcctc agcttttg                  48 

 
           
             47  
             48  
             DNA  
             unknown  
             
               oligonucleotide 2  
             
           
            47 

caaaagctga ggcgccgatc gctggccatc gcgtcggagt cttcaaac                  48 

 
           
             48  
             48  
             DNA  
             unknown  
             
               oligonucleotide 3  
             
           
            48 

gtttgaagac tccgacgcga tggccagcga tcggcgcctc agcttttg                  48 

 
           
             49  
             48  
             DNA  
             unknown  
             
               oligonucleotide 4  
             
           
            49 

caaaagctga ggcgccgatc gctggccatc gcgtcggagt cttcaaac                  48 

 
           
             50  
             8  
             PRT  
             unknown  
             
               single internal CnBr digestion fragment  
             
           
            50 

Gly Arg Gly Arg Gly Val Gly Val 
1               5