Abstract:
The present invention provides three insertion elements and transposases encoded by the insertion elements that are derived from the genome of Ralstonia solanacearum, which has been isolated with a transposon trap vector.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a transposable element isolated from the genome of Ralstonia solanacearum and a transposase encoded by the transposable element. 
     2. Description of the Related Art 
     It is known that transposable elements not only inactivate or activate genes before and after the transposition site by moving on the genome, but also cause various genome rearrangements such as deletion, inversion and duplication. It is also known that the transposable elements contribute to genome plasticity that is important for the evolution and the environmental adaptation of microorganisms (Arber et al., FEMS Microb. Ecol., 15: 5-14 (1994)). 
     Transposable elements can be classified into transposons and insertion sequence elements. Transposons are defined as genes involved in transposition and having phenotypic genes such as antibiotic resistances. On the other hand, insertion sequence elements (hereinafter, referred to as IS elements) are defined as genes that generally have a size of 2 kb or less and have no phenotypic genes other than the genes involved in gene transposition (Gay et al., J. Bacteriol., 164: 918-921 (1985)). 
     The IS element was first discovered in the late 1960&#39;s as an element that causes mutation of  E. coli . Thereafter, the IS elements of  E. coli  in particular were isolated and characterized. In the late 1980&#39;s, there were reports of isolation of the IS elements from bacteria other than the  E. coli  group. At present, about 500 IS elements are reported to have been isolated from medical bacteria, environmental bacteria and others (Mahillon et al., Microbiol. Mol. Bio. Rev., 62:725-774(1998)). 
     The isolated IS elements are compared with each other based on the base sequence and the deduced amino acid sequence. The IS elements that have been found so far are classified into 17 families. Among these families, the IS3 family and the IS5 family are large families, and IS elements belonging to these families are isolated from bacteria ranging widely from Gram-negative bacteria to Gram-positive bacteria (Mahillon et al., ibid.). 
     The structure of the IS elements in the IS3 and the IS5 families is characterized by the presence of inverted repeat sequences at their terminals and the inclusion of two open reading frames encoding a transposase. It seems that the terminal inverted repeat sequences play an important role in transposition, because recombination occurs in these portions. For example, in insertion, 2 to 13 base pairs in the site where insertion occurs are overlapped as same direct repeat sequences on both sides of the IS element (Galas and Chandler, Mobile DNA, 109-162, ASM Press (Washington, D.C.) 1989). 
     Transposase is an enzyme that catalyzes an insertion reaction of a gene. In the IS elements of the IS3 family and the IS5 family, the transposase is encoded by two open reading frames and is expressed as one protein in translation. For example, in the case of the IS3 family, two open reading frames overlap, and a frame shift occurs in the overlapping portion. Then, the two open reading frames are translated successively so that the transposase is expressed (Ohtsubo and Sekine, Transposable Elements, pages 1-26, Springer-Verlag Berlin Heidelberg, Germany (1996)). 
     Each of the IS elements obtained from various microorganisms has a sequence that is unique to the particular microorganism, although they are homologous. Utilizing the unique sequence, in the industrial fields of agriculture, foods, pharmacy and the like in which microorganisms are involved, the IS elements are used, for example, for identification of plant pathogenic bacteria, diagnosis of infectious diseases, determination of infection routes and isolation of useful genes. The IS element that is most common in practical use is IS6110, which is obtained from Mycobacterium tuberculosis and contributes to epidemiological surveys such as diagnosis of tuberculosis and determination of the infection route (Otal et al., J. Clin. Microbiol., 29:1252-1254 (1991)). Similar attempts have been conducted with various bacteria. 
     IS elements are characterized by being able to activate or inactivate genes before and after the inserted site by transposition. Utilizing this function positively, it is attempted to use the IS elements as a tool for isolating useful genes (Haas et al., Molecular Biology of Pseudomonas, 238-249, ASM Press (Washington D.C.) (1996)). 
     The isolation of the IS elements was not conducted strategically, but mostly was rather accidentally achieved by analysis of mutants. This is because, as seen from the definition, IS elements have no phenotype other than involvement in gene transposition, so that positive isolation was difficult. Various transposon trap vectors have been developed since positive selection of the transposable element (a method of utilizing activation of a detectable marker by transposition of the transposable element to a part of a structural gene) was successfully reported in 1985 (Gay et al., ibid.), and the IS elements have been isolated from bacteria such as Agrobacterium tumefaciens, Pseudomonas cepacia, Rhizobium meliloti, Rhizobium legumino sarum or the like by this method. 
     However, only 20 IS elements were isolated by this method, and this is a small number relative to the total number of the isolated IS elements. The isolation of new IS elements from various bacteria is expected to be accelerated as the positive selection is developed. 
     In regard to agriculturally important plant-related bacteria, Hasebe et al. isolated three kinds of IS elements from Pseudomonas glumae, which is a common pathogenic bacterium in rice plants in Japan, by utilizing a transposon trap vector pSHI1063 (Hasebe et al., Plasmid, 39:196-204 (1998)) (see Japanese Laid-Open Publication No. 10-248573). However, in reality, isolation of other IS elements has not been substantially researched. Isolation of IS elements of various plant pathogenic bacteria (microorganisms) makes it possible to counter the pathogenic bacteria, so that there is a demand for isolation and utilization of the IS elements in the field of agriculture. 
     SUMMARY OF THE INVENTION 
     Therefore, with the foregoing in mind, it is an object of the present invention to provide new IS elements derived from Ralstonia solanacearum, which is one of the common pathogenic bacteria in vegetables. The Ralstonia solanacearum is a worldwide common soil infectious plant pathogenic bacterium that causes wilt vessel disease to more than 100 species of more than 30 families including Solanaceae plants such as tomatoes, tobacco and potatoes. It is another object of the present invention to provide transposases that are encoded by these IS elements. These IS elements and the transposases make it possible to prevent infection to Ralstonia solanacearum, for example, by effecting recombination so that the transposases are inactivated, which is very useful in the field of agriculture. 
     In order to achieve the above objects, the inventors of the present invention have succeeded in isolating new IS elements from Ralstonia solanacearum and realized the present invention. 
     The present invention provides an insertion sequence element or a functional equivalent thereof comprising: a base sequence of Sequence I.D. No.2 at the 5′ terminal and a base sequence of Sequence I.D. No.3 at the 3′ terminal as terminal inverted repeat sequences; and a base sequence encoding amino acid sequences of Sequence I.D. Nos.4 and 5 as open reading frames between the terminal inverted repeat sequences. Herein, the open reading frame can be present overlapped or independently. 
     The present invention further provides an insertion sequence element consisting of the base sequence of Sequence I.D. No.1. 
     Furthermore, the present invention provides an insertion sequence element or a functional equivalent thereof comprising: a base sequence of Sequence I.D. No.7 at the 5′ terminal and a base sequence of Sequence I.D. No.8 at the 3′ terminal as terminal inverted repeat sequences; and a base sequence encoding amino acid sequences of Sequence I.D. Nos.9 and 10 as open reading frames between the terminal inverted repeat sequences. Herein, the open reading frame can be present overlapped or independently. 
     The present invention further provides an insertion sequence element consisting of a base sequence of Sequence I.D. No.6. 
     Furthermore, the present invention provides an insertion sequence element or a functional equivalent thereof comprising: a base sequence of Sequence I.D. No.12 at the 5′ terminal and a base sequence of Sequence I.D. No.13 at the 3′ terminal as terminal inverted repeat sequences; and a base sequence encoding an amino acid sequence of Sequence I.D. No.14 as an open reading frame between the terminal inverted repeat sequences. 
     The present invention further provides an insertion sequence element consisting of a base sequence of Sequence I.D. No.11. 
     The present invention further provides a transposase or a functional equivalent thereof expressed from a base sequence of positions 56 to 855 of Sequence I.D. No.1. 
     The present invention further provides a transposase or a functional equivalent thereof expressed from a base sequence of positions 65 to 822 of Sequence I.D. No.6. 
     The present invention further provides a transposase or a functional equivalent thereof expressed from a base sequence of positions 44 to 865 of Sequence I.D. No.11. 
     It should be noted that “movable genetic elements” (transposable elements) such as transposons may be significantly involved in the evolution and the environmental adaptation of organisms as self-mechanism of self-alternation of the organism&#39;s genome. The gene of the present invention moves on the genome of microorganisms and has the nature of activating or inactivating the gene that is positioned downstream of the genome into which the gene of the present invention jumps. Utilizing this property, it is possible to isolate industrially useful genes efficiently. Furthermore, utilizing these genes, it is possible to determine the infection route and identify the bacteria in regard to Ralstonia solanacearum. Furthermore, the transposition function of the transposable element is promoted by utilizing the transposases of the present invention, so that it is possible to isolate industrially useful genes efficiently. In addition, it is expected that protectant effects such as promotion of a reduction in the pathogenicity caused by the mutation induction of Ralstonia solanacearum can be provided. 
     These and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a diagram showing insertion positions of insertion sequences of 26 clones. 
     FIG. 2 is a diagram showing the positional relationship between ISJsp104.2 and an incomplete inverted sequence, a targeted overlapping sequence, ORFA and ORFB (SEQ ID NO:1, with an additional TTA at each end, and SEQ ID NOS:4-5). 
     FIG. 3 is a diagram showing the positional relationship between ISmsp4.2 and an incomplete inverted sequence, a targeted overlapping sequence, ORFA and ORFB (SEQ ID NO:6, with an additional TAA at each end, and SEQ ID NOS:9-10). 
     FIG. 4 is a diagram showing the positional relationship between ISmsp101.3 and an incomplete inverted sequence, a targeted overlapping sequence and ORFA (SEQ ID NO:11, with an additional TAA at each end, and SEQ ID NO:14). 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     (1) Definitions 
     In the present invention, “IS element” refers to a gene unit that has a size of 2 kb or less, has no phenotypic gene other than genes involved in gene transposition, and includes one or two open reading frames encoding transposase and terminal inverted repeat sequences. 
     ISJsp104.2 is an IS element consisting of the base sequence of Sequence I.D. No.1. 
     ISmsp4.2 is an IS element consisting of the base sequence of Sequence I.D. No.6. 
     ISmsp.101.3 is an IS element consisting of the base sequence of Sequence I.D. No.11. 
     In the present invention, “transposase” is an enzyme that catalyzes an insertion reaction of a gene. 
     A “functional equivalent” used in the present invention refers to an IS element or a transposase that substantially has the function or the activity of the original IS element or transposase, and has at least 90%, preferably at least 95% of homology in the base sequence or the amino sequence, respectively, when optimally aligned with the original IS element or transposase. 
     Such a functional equivalent of the IS element includes substitution, addition, deletion or insertion of at least one nucleotide, in addition to the original sequence in the terminal inverted repeat sequence or the open reading frame that is a functional site, and has at least functions or activities substantially equivalent to those of the original IS element or transposase. Examples of such a functional equivalent include IS elements having a nucleotide substitution that causes conservative substitution of the amino acid of the transposase to be encoded, and IS elements having an intervening nucleotide in the open reading frame. 
     Such a functional equivalent of transposase may include substitution of at least one amino acid (preferably conservative substitution), or additional amino acid (e.g., a reader sequence, a secretion sequence, and a sequence that would advantageously function in purification), in addition to the original sequence. It is appreciated that production of these functional equivalents is within a scope of technical knowledge that can be routinely obtained by those skilled in the art. 
     (2) Method for Searching a Transposable Element 
     In the search of a transposable element, molecular biological experiment techniques (electrophoresis of DNA, collection of electrophoresed DNA from a gel, digestion of restriction enzyme, PCR, labeling of DNA, hybridization, base sequencing and the like) can be used. Examples of these techniques include the techniques described in Sambrook et al., A Laboratory Manual, the second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) and other methods routinely used by those skilled in the art. 
     (a) Bacteria to be Used 
     In the present invention, Ralstonia solanacearum is used. A preferable strain is a Ralstonia solanacearum strain MAFF301556. This strain is a Ralstonia solanacearum isolated from a potato in Nagasaki in 1983. This strain is deposited as a distributable strain with the Gene Bank of the Ministry of Agriculture, Forestry and Fisheries (2-1-2, Kannondai, Tsukuba-shi, Ibaraki) and is available to anyone for test and research. 
     (b) Principle 
     A transposon trap vector is used as means for isolating a transposable element. In the present invention, pSHI1063 (lida et al., Abstract of Proceeding of The fourteenth Conference of Molecule Biology Society of Japan, p.216 (1991)) is used as the transposon trap vector. The trap vector pSHI1063 has a full length of 11.5 kb, and is a fusion plasmid of the plasmid pVS1 with wide host range of Pseudomonas aeruginosa and the plasmid pBR322 of  E. coli . The trap vector pSHI1063 can be grown in a wide range of Gram-negative bacteria such as  E. coli , bacteria of the Pseudomonas genus, the Agrobacterium genus, the Rhizobium genus and the like. The trap vector pSHI1063 has an ampicillin-resistant gene and a spectinomycin-resistant gene as selective marker genes. In addition, in order to trap a transposable element, this trap vector has a cI repressor gene of λ phage and a kanamycin-resistant gene (neo) connected to a P R  promoter that is under control of the cI repressor (this is called a trap cassette gene). After introducing the trap vector pSHI1063 to a bacterium, a transposable element such as an IS element of the bacterium transposes into the cI repressor gene in the trap vector pSHI1063. Then, the cI repressor in the trap vector is inactivated and the P R  promoter is activated, so that the kanamycin-resistant gene operates. Therefore, when the transposable element is present in the cI repressor gene of the trap vector pSHI1063, the bacterium is resistant to kanamycin, and therefore the transposable element can be selected efficiently. 
     (c) Acquisition of the Transposable Element 
     Based on the above principle, the trap vector pSHI1063 is introduced to a Ralstonia solanacearum strain MAFF301556, and a selected kanamycin-resistant strain is cultured, and boiled, for example, at 100° C. for 5 minutes so that the bacterial cells are lysed, and the DNA is extracted. Then, PCR is performed using the sequence of the trap cassette gene as the primer so as to amplify a DNA fragment having a transposable element (Sambrook et al., ibid.). 
     (d) Base Sequencing of the Transposable Element 
     Next, the base sequence of the amplified and collected insertion sequence is determined. First, an intended DNA fragment is prepared from a product of the PCR amplification including the transposable element obtained in (c), and is subcloned into an appropriate vector, for example, a pT7Blue vector. A plasmid DNA is prepared from the obtained positive clone, and is used as a template for base sequencing. Thus, this plasmid DNA is sequenced on both strands, for example, by primer walking method. For sequencing, an autosequencer (model 1377 manufactured by ABI Corp.) can be used for example. 
     (e) Homology Search of the Isolated Transposable Element 
     A database (DNA Information and Stock Center, is searched for the base sequence homology and the amino acid homology of the base sequenced transposable element, and a novel gene is characterized. In this manner, the transposable element is selected. 
     EXAMPLES 
     Hereinafter, the present invention will be described by way of examples. 
     Example 1 
     Search for a transposable element of a Ralstonia solanacearum strain MAFF301556 (hereinafter, referred to simply as “strain MAFF301556”) was performed. To search the transposable element, a transposon trap vector is required, and in the present invention, pSHI1063 was used. 
     First, the strain MAFF301556 was grown on PTYG agar medium (0.25 g of bactopeptone, 0.25 g of bactotryptone, 0.5 g of bacto-yeast extract, 0.5 g of glucose, 30 mM of MgSO 4 .7H 2 O, 3.5 mg of CaCl 2 . 2H 2 O, and 15 g of agar powder in 1000 ml of distilled water) at 28° C. The grown colony was inoculated into a 5 ml of a PTYG liquid medium and was cultured with shaking at 28° C. for 2 days. 
     Competent cells were made from this culture solution. Four ml of the culture solution were added to 200 ml of the PTYG medium, and cultivation was performed with shaking at 28° C. until the OD 500  reaches about 0.2. After cultivation, the culture solution was placed in ice for 20 minutes and then centrifuged (3000 rpm/10 min/4° C.) so as to collect bacterial pellets. The pellets were suspended in 10 ml of an ice-cold 1 mM HEPES. NaOH buffer (pH 7.0). Then, the suspension was centrifuged again to collect bacterial pellets, and the pellets were suspended in 10 ml of an ice-cold 1 mM HEPES. NaOH buffer (pH 7.0). Then, the bacterial pellets were collected by centrifugation (8000 rpm/10 min/4° C.), and were suspended in 2 ml of an ice-cold 10% glycerol solution, so that competent cells were prepared. 
     Then, 40 μl of the obtained competent cells were mixed with about 0.36 pg of a transposon trap vector pSHI1063 (a liquid volume of 1 μl) dissolved in a TE buffer (10 mM Tris. HCl (pH8.0)−1 mM EDTA) and the mixture was transferred to a cuvette that was previously cooled to 4° C. Electric pulses were applied to this cuvette with a high voltage pulse generator, “Gene Pulser” manufactured by Biorad Co. The parameters for the electric pulse were an electrical resistance of 200 Ω, an electrical capacity of 25 μFD, and an electric field of 6.25 KV/cm. 
     After applying pulses, a cell/DNA mixture was immediately transferred to 2 ml of the PTYG medium, and was cultured with shaking at 28° C. for one hour, followed by centrifugation (8000 rpm/10 min/4° C.). Then, bacterial pellets were suspended in 1 ml of sterilized distilled water. The suspension was serial diluted by a factor of 10 with sterilized distilled water sequentially, and diluted solutions with bacterial cells of up to 10 −4  were obtained. Then, 100 μl of the diluted solutions were plated onto PTYG agar plates containing 100 μg/ml of spectinomycin. Further, the same number of bacterial cells was plated onto a PTYG agar plate that did not contain any antibodies as the control to check the transformation frequency, followed by culturing at 280° C. for 2 days. The transformed colonies resistant to spectinomycin that appeared were applied to a PTYG plate containing spectinomycin again with a platinum loop, and were cultured at 280° C. for 2 days, so that a single colony was formed. The transformation frequency of pSHI1063 to the Ralstonia solanacearum strain MAFF 301556 was 5×10 −4 . 
     Next, the spectinomycin-resistant colonies that formed a single colony were picked up one by one with a platinum loop and inoculated into 5 ml of a PTYG medium containing 100 μg/ml of spectinomycin, followed by culturing with shaking at 28° C. for 2 days. After cultivation, the suspension was serially diluted by a factor of 10 with sterilized distilled water. Then, 100 μl of the diluted solutions with the bacterial cells ranging from an undiluted solution of the suspension to a 10 −6  diluted solution were applied onto PTYG agar plates containing 50 μg/ml of kanamycin and 100 μg/ml of spectinomycin. Further, the same number of bacterial cells was plated onto a PTYG agar plate containing 100 μg/ml of spectinomycin as the control to check the appearance frequency of mutants, followed by culturing at 28° C. for 2 days. The obtained transformed colonies resistant to kanamycin were applied to a PTYG plate containing 50 μg/ml of kanamycin and 100 μg/ml of spectinomycin again with a platinum loop, so that a single colony was formed. 
     The appearance frequency of mutants from the obtained transformants ranged widely from about 100% to 6×10 −7 , and was significantly different depending on the transformed clone. Ninety-seven mutants obtained from these transformants were used for the following examinations. 
     With respect to the 97 mutants, the DNA fragments of the insertion sequence were amplified by a PCR technique using various primers prepared based on the trap cassette gene sequence of pSHI1063 by routine procedures. For amplification, using a DNA amplifier (PC800 manufactured by ASTEC Inc.), a cycle of DNA denaturation at 94° C. for 1 minute, annealing at 55° C. for 2 minutes, and DNA elongation at 72° C. for 3 minute was repeated 25 times. After a PCR reaction, 1 μl of the reaction solution was used for agarose electrophoresis (1.5 % agarose) to confirm the DNA amplification. 
     As a result, it was confirmed that DNA insertion was not observed in the plasmids of 71 kanamycin resistant mutants, whereas DNA fragments were inserted in the trap vectors pSHI1063 in the plasmids obtained from 26 mutants. The trap cassette gene of pSHI1063 was divided into 6 regions from the upstream to the downstream (divided into I to VI regions from the uppermost stream region of the cI gene to the upstream of the neo gene), and it was investigated into which region the DNA was inserted. The results were region I for 3 mutants, region II for 14 mutants, region III for 2 mutants, region IV for 6 mutants, region V for 1 mutant, and region VI for none (FIG.  1 ). 
     Next, the base sequences of the plasmids obtained from the 26 mutants were determined. The PCR reaction products were subcloned into pT7Blue vectors. Plasmid DNAs were prepared from the obtained positive clones and used as templates for base sequencing. These plasmid DNAs were sequenced on both strands, for example by primer walking with an autosequencer (model 1377 manufactured by ABI Corp.) (Sambrook et al., ibid). 
     With respect to the sequenced DNA fragments, a public (DNA Information and Stock Center, was searched for base sequence homology and amino acid sequence homology, and the genes were characterized. Table 1 shows the results. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 The characteristics of DNA inserted in the pSHI1063 trap cassette 
               
             
          
           
               
                   
                   
                   
                 Insertion 
                   
               
               
                 No. 
                 Clone 
                 Deduced size 
                 region 
                 IS element 
               
               
                   
               
             
          
           
               
                 1 
                 Jsp104.2 
                  864bp 
                 I 
                 ISJsp104.2 
               
               
                 2 
                 Jsp105.1 
                 1.2kb 
                 I 
                 iso-IS1420 
               
               
                 3 
                 Jsp101.5 
                 1.2kb 
                 I 
                 iso-IS1420 
               
               
                 4 
                 msp101.3 
                  884bp 
                 II 
                 ISmsp101.3 
               
               
                 5 
                 Jsp104.1 
                 1.3kb 
                 II 
               
               
                 6 
                 Jsp107.3 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 7 
                 Jsp107.4 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 8 
                 Jsp105.5 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 9 
                 Jsp120.5 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 10 
                 Jsp120.1 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 11 
                 Jsp101.2 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 12 
                 Jsp102.3 
                 1.0kb 
                 II 
               
               
                 13 
                 Jsp101.4 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 14 
                 Jsp105.3 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 15 
                 Jsp120.2 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 16 
                 Jsp120.3 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 17 
                 Jsp120.4 
                 1.2kb 
                 II 
                 iso-IS1420 
               
               
                 18 
                 msp4.4 
                 0.8kb 
                 III 
               
               
                 19 
                 Jsp105.2 
                 0.8kb 
                 III 
               
               
                 20 
                 msp101.5 
                 1131bp 
                 IV 
                 IS1420 
               
               
                 21 
                 msp4.2 
                  842bp 
                 IV 
                 ISmsp4.2 
               
               
                 22 
                 Jsp102.1 
                 1.2kb 
                 IV 
                 iso-IS1420 
               
               
                 23 
                 Jsp107.1 
                 1.2kb 
                 IV 
                 iso-IS1420 
               
               
                 24 
                 Jsp104.3 
                 0.2kb 
                 IV 
               
               
                 25 
                 Jsp105.4 
                 1.2kb 
                 IV 
                 iso-IS1420 
               
               
                 26 
                 Jsp107.2 
                 1.2kb 
                 V 
                 iso-IS1420 
               
               
                   
               
             
          
         
       
     
     As a result, three novel IS elements (ISJsp104.2, ISmsp4.2 and ISmsp101.3) were isolated as the transposable elements. Their properties are as follows. 
     (a) ISJsp104.2 
     ISJsp104.2 is a base sequence with a full length of 864 bp composed of the base sequence of Sequence I.D. No.1, and has incomplete inverted repeat sequences (19 bp) at its terminals (the underlined arrow portion of FIG. 2, Sequence I.D. Nos. 2 and 3). Targeted overlapping sequences of 3 bp are coupled to both terminals of ISJsp104.2, and the sequence was TTA (the squared portions in FIG.  2 ). In comparison with the homology of the base sequence, ISJsp104.2 has a high homology of 74.8% with IS1418 (Burkholderia glumae), 70.9% with ISB111 (Ralstonia solanacearum) and 59.5% with IS402 (Burkholderia cepacia), which are IS elements belonging to the IS427 subgroup of the IS5 family (Mahillon et al., ibid.). Therefore, it seems that ISJsp104.2 is a novel IS element obtained from Ralstonia solanacearum that belongs to the IS427 subgroup of the IS5 family. 
     (Transposase Encoded by ISJsp104.2) 
     The base sequence analysis and the amino acid sequence homology analysis of ISJsp104.2 were performed and it was confirmed that this IS element encoded transposase, as other IS elements. ISJsp104.2 has two open reading frames (ORFA, ORFB) that are believed to encode a transposase (transposable enzyme), as generally seen in the IS3 and the IS5 families (Mahillon et al., ibid.). These open reading frames partly overlap, and are frame shifted (FIG.  2 ). Furthermore, there is a characteristic motif (C 5 G 6 ) that is estimated to be involved in the frame shift in the base sequence in the overlapped portion (the underlined portion in FIG. 2) (Iversen et al., Plasmid 32:46-54 (1994)). ORFA is composed of 134 amino acids (Sequence I.D. No. 4) and ORFB is composed of 211 amino acids (Sequence I.D. No. 5). The ORFA and the ORFB of ISJsp104.2 have homologies of at least 70% in the amino acid sequences with the ORFA and the ORFB of IS 1418, respectively. 
     (b) ISmsp4.2 
     ISmsp4.2 is a base sequence with a full length of 842 bp composed of Sequence I.D. No.6, and has incomplete inverted repeat sequences (18 bp) at its terminals (the underlined arrow portion of FIG. 3, Sequence I.D. Nos. 7 and 8). Targeted overlapping sequences of 3 bp are coupled to both terminals of ISmsp4.2, and the sequence is TAA (the squared portions in FIG.  3 ). In comparison with the homology in the base sequence, ISmsp4.2 has a high homology of 56.7% with IS427 (Agrobacterium tumefaciens) and 54.9% with IS298 (Caulobacter crescentus), which are IS elements belonging to the IS427 subgroup of the IS5 family (Mahillon et al., ibid.). Therefore, it seems that ISmsp4.2 is a novel IS element obtained from Ralstonia solanacearum that belongs to the IS427 subgroup of the IS5 family. 
     Both the ISmsp4.2 and the ISmsp104.2 belong to the IS427 subgroup of the IS5 family, but have a homology as low as 50% or less to each other. 
     (Transposase Encoded by ISmsp4.2) 
     Also ISmsp4.2 has two open reading frames, ORFA (116 amino acids) (Sequence I.D. No.9) and ORFB (159 amino acids) (Sequence I.D. No.10) that are believed to encode a transposase. As other IS elements that belong to the IS427 subgroup of the IS5 family, the ORFA and the ORFB partly overlap, and are frame shifted (FIG. 
     3). Furthermore, there is a characteristic motif (A 6 G) that is estimated to be involved in the frame shift in the base sequence in the overlapped portion (the underlined portion in FIG. 3) (Ohtsubo and Sekine, ibid.). The ORFA and the ORFB do not have a high homology in the amino acid sequence with other IS elements, and the homologies are 40% or less in any cases. 
     (c) ISmsp101.3 
     ISmsp101.3 is a base sequence with a full length of 884 bp composed of Sequence I.D. No.11, and has incomplete inverted repeat sequences (18 bp) at its terminals (the underlined arrow portion of FIG. 4, Sequence I.D. Nos.12 and 13). Targeted overlapping sequences of 3 bp are coupled to both terminals of ISmsp101.3, and the sequence is TAA (the squared portions in FIG.  4 ). In comparison with the homology in the base sequence, ISmsp101.3 has homologies of 67.6% with IS12528 (Gluconobacter suboxydans), 56.6% with ISR1F7-2 (Rhizobium leguminosarum), 56.5% with ISRm220-12-1 (Sinorhizobium meliloti) and 54.6% with IS1031 (Acetobacter xylinum), which are IS elements belonging to the IS1031 subgroup of the IS5 family (Mahillon et al., ibid.). Therefore, it is believed that ISmsp101.3 is a novel IS element obtained from Ralstonia solanacearum that belongs to the IS1031 subgroup of the IS5 family. 
     (Transposase Encoded by ISmsp101.3) 
     Also ISmsp101.3 has an open reading frame, ORFA (274 amino acids) (Sequence I.D. No.14) that is believed to encode a transposase, and has a high homology in the amino acid sequence of 71.1% with the ORFA274 of IS12528, which belongs to the IS1031 subgroup of the IS5 family. 
     
       
         
           
             14 
           
           
             1 
             864 
             DNA 
             Ralstonia solanacearum 
           
            1
gggccgctaa caaaaccaag tcatcgaacg caggtggttg agcgttgttg ttggcatggc     60
acgaaagaag atcagcaatg aactgtggaa ggcgttgcaa ccgctgctgc cggttgtgga    120
gccttcgacc aaaggcggtc gtccgcgcgt ggatgatcgg gcggcgctga acggcatcct    180
gtttgttctg cataccggta tcccgtggga agacctgcct aaagaactgg gctttggcag    240
cggcatgacg tgctggcgtc gcctgcggga gtggcaggcc aacggcgttt gggagcggct    300
gcatttggct ctgctcaagc gcctgcgcga acacgaccag atcgactgga gccgagccag    360
tgtcgacggt gcaacggtgg ccagcccccg gggggcgagc agacggggcc gaatccaacg    420
gatcgtggca agctcggtag caagcgccat ctcgtcgtag atcggcgcgg cgtgccgttg    480
gcgctgatgg tcaccggtgc caatcgtcac gactcggtgg tgttcgaggt gctcgttgac    540
gccatcccga gcgtgcccgg actcgatggc cgcccgcgat gccgccccga caagcttcac    600
gcggataagg gatacgactt cgcgcgatgc cgccggcatc tgcgcaagcg gggcatgact    660
ccccggatcg ctcgccgtgg catcgagaag aacgaccggc tcggcaagca tcgctgggtt    720
gtcgagcgca cccatgcctg gcttgctggc ttcggcaagt tgcgcattcg tttcgagcgt    780
tctcttcaga ctcatctcgc tttgctcacc ctggcttgcg ccgtcatctg cgggcgattt    840
gttgatcggt tttgttagcg actc                                           864
 
           
             2 
             19 
             DNA 
             Ralstonia solanacearum 
           
            2
gggccgctaa caaaaccaa                                                  19
 
           
             3 
             19 
             DNA 
             Ralstonia solanacearum 
           
            3
tcggttttgt tagcgactc                                                  19
 
           
             4 
             134 
             PRT 
             Ralstonia solanacearum 
           
            4
Met Ala Arg Lys Lys Ile Ser Asn Glu Leu Trp Lys Ala Leu Gln Pro
1               5                   10                  15
Leu Leu Pro Val Val Glu Pro Ser Thr Lys Gly Gly Arg Pro Arg Val
            20                  25                  30
Asp Asp Arg Ala Ala Leu Asn Gly Ile Leu Phe Val Leu His Thr Gly
        35                  40                  45
Ile Pro Trp Glu Asp Leu Pro Lys Glu Leu Gly Phe Gly Ser Gly Met
    50                  55                  60
Thr Cys Trp Arg Arg Leu Arg Glu Trp Gln Ala Asn Gly Val Trp Glu
65                  70                  75                  80
Arg Leu His Leu Ala Leu Leu Lys Arg Leu Arg Glu His Asp Gln Ile
                85                  90                  95
Asp Trp Ser Arg Ala Ser Val Asp Gly Ala Thr Val Ala Ser Pro Arg
            100                 105                 110
Gly Ala Ser Arg Arg Gly Arg Ile Gln Arg Ile Val Ala Ser Ser Val
        115                 120                 125
Ala Ser Ala Ile Ser Ser
    130
 
           
             5 
             211 
             PRT 
             Ralstonia solanacearum 
           
            5
Arg Thr Gly Leu Trp Gln Arg His Asp Val Leu Ala Ser Pro Ala Gly
1               5                   10                  15
Val Ala Gly Gln Arg Arg Leu Gly Ala Ala Ala Phe Gly Ser Ala Gln
            20                  25                  30
Ala Pro Ala Arg Thr Arg Pro Asp Arg Leu Glu Pro Ser Gln Cys Arg
        35                  40                  45
Arg Cys Asn Gly Gly Gln Pro Pro Gly Gly Glu Gln Thr Gly Pro Asn
    50                  55                  60
Pro Thr Asp Arg Gly Lys Leu Gly Ser Lys Arg His Leu Val Val Asp
65                  70                  75                  80
Arg Arg Gly Val Pro Leu Ala Leu Met Val Thr Gly Ala Asn Arg His
                85                  90                  95
Asp Ser Val Val Phe Glu Val Leu Val Asp Ala Ile Pro Ser Val Pro
            100                 105                 110
Gly Leu Asp Gly Arg Pro Arg Cys Arg Pro Asp Lys Leu His Ala Asp
        115                 120                 125
Lys Gly Tyr Asp Phe Ala Arg Cys Arg Arg His Leu Arg Lys Arg Gly
    130                 135                 140
Met Thr Pro Arg Ile Ala Arg Arg Gly Ile Glu Lys Asn Asp Arg Leu
145                 150                 155                 160
Gly Lys His Arg Trp Val Val Glu Arg Thr His Ala Trp Leu Ala Gly
                165                 170                 175
Phe Gly Lys Leu Arg Ile Arg Phe Glu Arg Ser Leu Gln Thr His Leu
            180                 185                 190
Ala Leu Leu Thr Leu Ala Cys Ala Val Ile Cys Gly Arg Phe Val Asp
        195                 200                 205
Arg Phe Cys
    210
 
           
             6 
             842 
             DNA 
             Ralstonia solanacearum 
             
               CDS 
               (65)...(822) 
             
           
            6
gggtcaggac ccattgattt gaattgacgg ctatgattca gacgggcgga taggagcctg     60
actgatgagt aatttgttct ggctgactaa cgagcaaatg gctcgtcttc agccctattt    120
ccccaagagc catggccgcc agcgtgtcga tgatcggcgt gtgctgagcg gcatcatttt    180
cgtcaatcgc aacgggctcc ggtggtgcga tgcgccgaag gaatatggcc cggcgaaaac    240
gctgtataac cgctggaaac gctggagcga caagggcatc tttatccaga tgatggacgg    300
cctggctgtg cctgaagctg cagaacacca gaccgtcatg attgatgcaa cctatctcaa    360
ggcccaccgc acggcttcga gcctgcgggt aaaaaagggg gcgcgggtcg cctgattgga    420
cgcacgaaag gcgggatgaa caccaagctt catgccgtga cggatgcgag tggtcgcccg    480
atcagtttct tcataacggc cggtcaaatc agcgattaca ccggtgctgc cgccttgctt    540
gatgaacttc ccaaggccaa atggctactg gccgaccgtg gctatgatgc cgactggtat    600
cgtgacgcgt tacaggcgaa ggggatcact ccctgcattc ccggtcggaa atcccggacc    660
acgaccatca aatacgacaa acgccgctat aaacggcgca accgaataga gatcatgttc    720
gggcgtctca aggattggcg acgtgtcgct acgcgctatg acaggtgccc aatggctttt    780
ctttccgcca tctctctcgc tgcaaccgtt atcttctggc tctgatcaac gagtcctgac    840
cc                                                                   842
 
           
             7 
             17 
             DNA 
             Ralstonia solanacearum 
           
            7
gggtcaggac ccattga                                                    17
 
           
             8 
             17 
             DNA 
             Ralstonia solanacearum 
           
            8
tcaacgagtc ctgaccc                                                    17
 
           
             9 
             116 
             PRT 
             Ralstonia solanacearum 
           
            9
Met Ser Asn Leu Phe Trp Leu Thr Asn Glu Gln Met Ala Arg Leu Gln
1               5                   10                  15
Pro Tyr Phe Pro Lys Ser His Gly Arg Gln Arg Val Asp Asp Arg Arg
            20                  25                  30
Val Leu Ser Gly Ile Ile Phe Val Asn Arg Asn Gly Leu Arg Trp Cys
        35                  40                  45
Asp Ala Pro Lys Glu Tyr Gly Pro Ala Lys Thr Leu Tyr Asn Arg Trp
    50                  55                  60
Lys Arg Trp Ser Asp Lys Gly Ile Phe Ile Gln Met Met Asp Gly Leu
65                  70                  75                  80
Ala Val Pro Glu Ala Ala Glu His Gln Thr Val Met Ile Asp Ala Thr
                85                  90                  95
Tyr Leu Lys Ala His Arg Thr Ala Ser Ser Leu Arg Val Lys Lys Gly
            100                 105                 110
Ala Arg Val Ala
        115
 
           
             10 
             159 
             PRT 
             Ralstonia solanacearum 
           
            10
Cys Asn Leu Ser Gln Gly Pro Pro His Gly Phe Glu Pro Ala Gly Lys
1               5                   10                  15
Lys Gly Gly Ala Gly Arg Leu Ile Gly Arg Thr Lys Gly Gly Met Asn
            20                  25                  30
Thr Lys Leu His Ala Val Thr Asp Ala Ser Gly Arg Pro Ile Ser Phe
        35                  40                  45
Phe Ile Thr Ala Gly Gln Ile Ser Asp Tyr Thr Gly Ala Ala Ala Leu
    50                  55                  60
Leu Asp Glu Leu Pro Lys Ala Lys Trp Leu Leu Ala Asp Arg Gly Tyr
65                  70                  75                  80
Asp Ala Asp Trp Tyr Arg Asp Ala Leu Gln Ala Lys Gly Ile Thr Pro
                85                  90                  95
Cys Ile Pro Gly Arg Lys Ser Arg Thr Thr Thr Ile Lys Tyr Asp Lys
            100                 105                 110
Arg Arg Tyr Lys Arg Arg Asn Arg Ile Glu Ile Met Phe Gly Arg Leu
        115                 120                 125
Lys Asp Trp Arg Arg Val Ala Thr Arg Tyr Asp Arg Cys Pro Met Ala
    130                 135                 140
Phe Leu Ser Ala Ile Ser Leu Ala Ala Thr Val Ile Phe Trp Leu
145                 150                 155
 
           
             11 
             884 
             DNA 
             Ralstonia solanacearum 
             
               CDS 
               (44)...(865) 
             
           
            11
gagcccgttt gaaaattccc cgccgttgtg gtgtaaaggc gggatgtgga aaaaagaaga     60
tcgagagcgt gaggcgaagc tggctcggaa gaccaagcgt tacccgagcg acctgacgga    120
tatcgaatgg gccgctgtgc agccgctgct gccacgcgcg gccgtgcgag gccggcgtcg    180
ggagtgcgac ttgagggagg tggtcaacgc cttgcgctat ctggtgcgag cgggctgcgg    240
ttggcgcatg ctgccgcacg acttcccgcc ttggcaaacc gtgtattggt ggtttcgtcg    300
gctcatgcgt cgcttcctgt tccgcacgct gcacgacgtg gtgctgatgt tggaccggga    360
gttggctggg cgccagccgt gcccgagtgc gggcgtcatc gacagccaga cagtcaaagc    420
gccctcggcc gacaagcgtg gctacgacgc ggccaagaaa atcgtcgggc gcaagcggca    480
tatcgcggtg gacacggatg gacggctgct gatggtgaac ctgacaccgg cggacattgc    540
cgatagcacg ggtgcgctgg cggtgctgga ggcggtgaag aagcgctggc caggcataaa    600
acacctgttc gctgacggtg cgtatgaccg cacaacgctg atggacaagg catcgaccct    660
cgacttcgtg gttgaggtgg tgcgccggca cgagcagcaa acgggctttg ccgttctgcc    720
gcgccgctgg gtggtcgagc gcacctttgg gtggatggtt cgctggcgcc gactcgtacg    780
cgactacgag cagcgcgcgg acgtctcgga agccatgatt catatcgcga tgagcggctt    840
gctactgcgc agaatcgctc atccttgaat ttccaaacgg gctc                     884
 
           
             12 
             18 
             DNA 
             Ralstonia solanacearum 
           
            12
gagcccgttt gaaaattc                                                   18
 
           
             13 
             18 
             DNA 
             Ralstonia solanacearum 
           
            13
gaatttccaa acgggctc                                                   18
 
           
             14 
             274 
             PRT 
             Ralstonia solanacearum 
           
            14
Met Trp Lys Lys Glu Asp Arg Glu Arg Glu Ala Lys Leu Ala Arg Lys
1               5                   10                  15
Thr Lys Arg Tyr Pro Ser Asp Leu Thr Asp Ile Glu Trp Ala Ala Val
            20                  25                  30
Gln Pro Leu Leu Pro Arg Ala Ala Val Arg Gly Arg Arg Arg Glu Cys
        35                  40                  45
Asp Leu Arg Glu Val Val Asn Ala Leu Arg Tyr Leu Val Arg Ala Gly
    50                  55                  60
Cys Gly Trp Arg Met Leu Pro His Asp Phe Pro Pro Trp Gln Thr Val
65                  70                  75                  80
Tyr Trp Trp Phe Arg Arg Leu Met Arg Arg Phe Leu Phe Arg Thr Leu
                85                  90                  95
His Asp Val Val Leu Met Leu Asp Arg Glu Leu Ala Gly Arg Gln Pro
            100                 105                 110
Cys Pro Ser Ala Gly Val Ile Asp Ser Gln Thr Val Lys Ala Pro Ser
        115                 120                 125
Ala Asp Lys Arg Gly Tyr Asp Ala Ala Lys Lys Ile Val Gly Arg Lys
    130                 135                 140
Arg His Ile Ala Val Asp Thr Asp Gly Arg Leu Leu Met Val Asn Leu
145                 150                 155                 160
Thr Pro Ala Asp Ile Ala Asp Ser Thr Gly Ala Leu Ala Val Leu Glu
                165                 170                 175
Ala Val Lys Lys Arg Trp Pro Gly Ile Lys His Leu Phe Ala Asp Gly
            180                 185                 190
Ala Tyr Asp Arg Thr Thr Leu Met Asp Lys Ala Ser Thr Leu Asp Phe
        195                 200                 205
Val Val Glu Val Val Arg Arg His Glu Gln Gln Thr Gly Phe Ala Val
    210                 215                 220
Leu Pro Arg Arg Trp Val Val Glu Arg Thr Phe Gly Trp Met Val Arg
225                 230                 235                 240
Trp Arg Arg Leu Val Arg Asp Tyr Glu Gln Arg Ala Asp Val Ser Glu
                245                 250                 255
Ala Met Ile His Ile Ala Met Ser Gly Leu Leu Leu Arg Arg Ile Ala
            260                 265                 270
His Pro