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Stable Positional Cloning of Long Continuous DNA in the Bacillus subtilis Genome Vector

Stable Positional Cloning of Long Continuous DNA in the Bacillus subtilis Genome Vector Abstract Direct cloning of a long continuous genome segment in a Bacillus subtilis genome vector was demonstrated for the first time. Two small DNA fragments had to be installed in the vector prior to cloning. The DNA between these two fragments was cloned via homologous recombination. The efficiency of cloning was estimated using the 3,573-kb genome of a cyanobacterium, Synechocystis sp. PCC 6803. Recombinants were selected using the internal selection system of the Bacillus genome vector or with the antibiotic resistance marker in the cyanobacterial genome. Designated genomic segments as large as 77-kb were cloned by means of a single procedure. Cloning efficiency is affected by the molecular weight of the donor DNA and the size of the DNA to be cloned. The method is suitable for direct target cloning of large-sized DNA. Key words: genome vector, homologous recombination, positional cloning, transformation. Abbreviation: CHEF, contour-clamped homogeneous electric field. Received June 17, 2003; accepted July 14, 2003 DNA cloning technology emerged in the late 70s and has led to great successes in DNA manipulation (1, 2). The complete sequences of a number of genomes, of from bacteria to eukaryotes, have unveiled much about genome structure as well as the functions of gene products. Given that all the ORF information has been presented, the handling of a large number of genes as one set becomes important (3, 4). As regards the size of clonable DNA, Escherichiacoli vectors such as BAC (5) and PAC (6) can be used for handling DNA larger than 100 kb. The YAC vector in Saccharomyces cerevisiae can harbor larger DNA segments, however, it is associated with a high degree of chimera formation and clonal instability (7). There are few methods, however, for precise positional cloning for DNA above the limit of PCR-mediated amplification. A novel Bacillus subtilis genome vector was developed to fill this technical gap (8, 9). The cloning principle of the genome vector differs from that of the prevailing plasmid-born vectors. B. subtilis develops natural competency by which DNA outside the cell is taken up into the cytoplasm (10, 11). If a homologous sequence is present in the genome, the DNA taken up is integrated into the genome through the recA-dependent homologous recombination pathway (12, 13). According to this protocol, two short DNA fragments that flank the target region are required (Fig. 1). These short DNA, or LPSs, standing for Landing Pad Sequences (9), are prepared in the E. coli plasmid pBR322 and installed in the pBR322 sequence of the B. subtilis genome (12). The intervening DNA segment delineated precisely by the two LPSs is integrated via homologous recombination at both LPSs. The B. subtilis strain is called a BGM vector, standing for BacillusGenoMe vector (9), and the cloning procedure for the BGM vector itself is equivalent to positional cloning. A positive selection system developed previously facilitates the cloning process (14). Two examples have been reported, one includes a 48.5-kb E.coli bacteriophage lambda DNA (8), and the other mouse genomic DNA ranging 100–140 kb in size (9). These cloned segments were stably maintained in the BGM vector regardless of the highly repetitive nature of the mouse genome. Cloning in the BGM vector in the two latter cases, however, resulted in the transfer of DNA previously cloned by means of a different technology (8, 9). Cloning directly from genomic DNA has not been examined. Direct cloning of the designated region of a cyanobacterium genome was attempted in this study and it was demonstrated that continuous genomic DNA of up to 77 kb could be successfully cloned in the BGM vector. Factors affecting the cloning efficiency were quantitatively measured. This genome vector has high potential to provide properly manipulated DNA through direct cloning of submega-sized DNA. MATERIALS AND METHODS 1.1. Bacterial Strains and Plasmids B. subtilis 168 trpC2, from the Bacillus Genetic Stock Center (Ohio, USA), and E. coli JA221 are routinely used as hosts for molecular cloning in this laboratory (15). Competent B. subtilis cells were prepared (10) and stored at –70°C in the presence of 20% (v/v) glycerol before use (13). 1.2. B. Subtilis Genome Vector Two DNA sequences characterize the B. subtilis genome vector (9). They are a 4.3-kb pBR322 sequence inserted in the NotI site of the proB gene (12) and the neomycin resistance gene [Pr-neo] integrated into the NotI site in the yvfC-yveP gene (14). The integrated pBR322 is referred to hereafter as the genomic pBR and is divided into two halves, the 2.4 kb amp-half including a β-lactamase gene and the 1.9 kb tet-half including a tetracycline resistance determinant gene. DNA is cloned between these two-halves, as illustrated in Fig. 1. Expression of the neo gene is regulated by a Pr promoter to which the cI repressor protein binds, which represses it (14). 1.3. Synechocystis Strains The genome of unicellular cyanobacterium Synechocystis sp. strain PCC (Pasteur Culture Collection) 6803 has been sequenced (16). This photosynthetic bacterium was chosen because its genome sequence is known (http://www.kazusa.or.jp/cyano/) and it is non-pathogenic. Cyanobacteria are unique organisms that perform oxygenic photosynthesis like chloroplasts of plants. Systematic analysis of gene expression has been carried out with many tools such as DNA microarrays (17). BG11 medium (18) was used to cultivate Synechocystis strains in liquid and on agar plates. The SynechocystisrnhB mutant was derived from the wild type strain of PCC 6803. The gene encoding ribonuclease H [EC. 3.1.26.4] chosen in this study is related to another target of our laboratory (19). The SynechocystisrnhB gene was replaced by the mutated gene rnhB::spc of pBRSYNrnhBS1 according to the method developed by Sugita and Sugiura (20). Colonies formed after 3 days on BG11 plates containing spectinomycin at 20 µg/ml under illumination at 50 µmol photons m–2 s–2 at 30°C. They were transferred to fresh BG11 medium containing spectinomycin (20 µg/ml) and then incubated for 4 weeks with occasional dilution, allowing them to pass through 11 generations. As the multiplicity of the Synechocystis genome is 12 (21), the mutant can be validated by the ratio of Southern band intensities of the mutant and wild type alleles. BUSY1001 contained 94.9% mutant rnhB::spc allele. The incomplete replacement by the mutant allele together with no associated phenotype indicates that the rnhB gene may be essential for the strain. 1.4. Preparation of High Molecular Weight Synechocystis Genome DNA Genomic DNA prepared according to the reported method (21, 22) included little DNA above 50 kb, as shown in Fig. 2A, and gave no recombinants. The extraction protocol in reference 21 was modified to obtain high-molecular weight DNA. A 50-ml Synechocystis culture was treated three times by freeze-thawing in dry ice and a water bath before harvesting. The cell pellet was washed with 5 ml of a NaCl/EDTA solution (120 mM/50 mM, pH 8.0), and then suspended in 4 ml of NaCl/EDTA and 1 ml of a saturated sodium iodide (NaI) solution. After incubation at 37°C for 30 min, 5 ml of a 25% sucrose TES solution (100 mM Tris-HCl [pH 7.6], 10 mM NaCl, and 1 mM EDTA) supplemented with 10 mg of lysozyme ml–1 was added, followed by incubation for 2 h at 37°C. After incubation for 17 hours with proteinase K at 250 µg ml–1 and 1.0% sodium dodesyl sulfate (w/v), a 5.75 ml sample was shaken with an equal amount of a phenol/chloroform mixture, followed by centrifugation at 3,500 ×g for 15 min. The upper phase, approximately 5 ml, was transferred to a fresh 50 ml tube (Falcon, 2070), and then three times the volume of Ethanol was added to precipitate the genomic DNA. The DNA collected by centrifugation at 3,500 ×g for 15 min was rinsed in 10 ml of 70% ethanol. The concentration of the DNA dissolved in 0.2 ml was determined by UV absorption at 260 nm using GeneQuantII (Pharmacia Biotech). This process yielded 5.69 mg/ml for PCC6803 and 3.6 mg/ml for BUSY1001. These DNA preparations included high molecular weight DNA (above 100 kb), as shown in Fig. 2A, and are used throughout this work. 1.5. Preparation of B. Subtilis Genome DNA Intact unsheared DNA for CHEF gel electrophoretic analysis was prepared in agarose plugs as described previously (23). Liquid DNA for conventional Southern analysis was prepared by the method of Saito and Miura (24). Agarose gel (1.0% w/v) in TBE solution (45 mM Tris-borate, [pH8.0], 1.0 mM EDTA) was used for CHEF gel electrophoresis at a constant voltage of 3 or 4 V cm–1 at 14°C. The pulse and running times are specified in the legends to the figures. Agarose gel (1.0% v/v) in a TAE solution (50 mM Tris-acetate [pH 8.0] and 1.0 mM EDTA) was used for conventional gel electrophoresis at room temperature. The DNA in the gels after electrophoresis was stained with an ethidium bromide solution (60 µg/ml) for 15 min, followed by photography. The Southern hybridization experiment was performed according to the protocol for a DNA labeling and detection kit (Roche, USA) RESULTS 2.1. Cloning with an Indirect Selection Marker As Synechocystis has no appropriate selection markers, the use of the internal selection system of the BGM vector (14) allows cloning of any genomic loci. Cloning from three genomic loci, the 16-kb region A (0882–0899), the 29-kb region B (1696–1725), and the 33-kb region C (2558–2591), is illustrated in Fig. 1. The LPS plasmids and the plasmids combined with the two LPSs for each region were prepared in E. coli as listed in Table 1. The latter plasmid is referred to as an LPA plasmid, standing for LPS Array plasmid. B. subtilis strains having LPA in the genomic pBR were constructed separately aiming at different target regions; BEST7166 for region A, BEST7332 for region B, and BEST8135 for region C (Table 1). For region A, 271 colonies were selected using neomycin at 3 µg/ml after the transformation of BEST7166 with PCC6803 genome DNA. Nine clones sensitive to spectinomycin at 50 µg/ml were subjected to colony PCR screening. The internal 4,650-bp of the 16 kb was amplified from two clones. Both carry the same 27–kb DNA generated by I-PpoI-digestion. The I-PpoI fragment produced from BEST7171 was resolved by pulsed-field gel electrophoresis (Fig. 2B). The primer set used to amplify the internal 4,650-bp segment of 16 kb was 5′-GTGGCAGAGTCGGTATTGGCTC-3′ (0898890F) and 5′-CCGGGTATTTATGGATCCCTAACC-3′ (0903540R). Similarly, the 29-kb segment of region B was cloned in BEST7336. Only this strain was isolated from 15 neomycin-resistant and spectinomycin-sensitive candidates via colony PCR screening using 5′-GGGATCAACTACAGTGCCCGG-3′ (1724984F) and 5′-TGTGGATCCTTGGATTTCATCAGG-3′ (1729678R) to detect the internal 4,694-bp segment. Cloning of the 33-kb region C resulted in only one strain, BEST8155, on screening for the 55 neomycin-resistant and spectinomycin-sensitive clones by colony PCR screening. The primer set for the internal 726-bp segment of 33 kb was 5′-CAATTCCCTCAGTCCCGACG-3′ (2591470F) and 5′-GGTCCTGGGCGTTAAAGGC-3′ (2592196R). The I-PpoI segments from these strains were confirmed, as shown in Fig. 2B. Southern analysis of BamHI and HindIII digests using Synechocystis genomic DNA as a probe verified that these cloned DNA were identical with those predicted from the sequences in the database (data not shown). These results indicated that target cloning with an internal positive selection system proceeds through the mechanism illustrated in Fig. 1. 2.2. Cloning with a Direct Selection Marker Despite the effectiveness of the internal marker system, the cloning of DNA segments longer than 50 kb was unsuccessful (data not shown). The number of true recombinants seemed underestimated due to the large number of background neomycin for resistant colonies for a poorly understood reason (9, 14). a direct selection method was used for quantitative measurements of size-dependent efficiency. A Synechocystis derivative, strain BUSY1001, that has a spectinomycin resistance gene in the rnhB gene was constructed. As the spectinomycin resistance gene functions for selection of B. subtilis, only recombinants containing the resistance gene are selected directly. Four B. subtilis strains for cloning of rnhB::spc segments of various sizes, from 14 kb to 77 kb, were constructed. Initially, the number of spectinomycin-resistant colonies varied among the experiments but the number became reproducible when the outgrowth period after DNA uptake was extended from 1 hour to 2 hours. It is likely that appropriate expression from the spectinomycin resistance gene requires prolonged incubation. This condition was employed throughout this study. Recombinants selected using spectinomycin at 50 µg/ml were all sensitive to erythromycin at 5 µg/ml. Six representative clones for 14-kb, 4 for 28-kb, 4 for 42-kb, and 3 for 77-kb were analyzed by Southern hybridization using Synechocystis genome DNA as a probe. As shown in Fig. 3, the numbers and sizes of the NotI and BglII Southern bands were consistent with those predicted from the sequence information. The lack of unexpected bands indicated high structural stability of the cloned segment in the BGM vector. No structural alteration of the B. subtilis genome part was detected on SfiI and NotI fragment analysis (data not shown). These results demonstrated that all colonies selected with spectinomycin integrated the rnhB::spc segment replacing the erm gene between the two LPSs, as shown in Fig. 1. 2.3. Size-Dependent Efficiency of Cloning Experiments were performed in triplicate with two BUSY1001 DNA concentrations, 1.80 and 6.05 µg/ml. The average number of spectinomycin- resistant transformants was plotted with standard deviations (Fig. 4). The number decreased as the size of the clone increased from 14 kb to 77 kb at both DNA concentrations. Saturation by Synechocystis DNA was not clear under the present conditions. This is consistent with the observation that the frequency of segment transfer between B. subtilis genomes appeared to be inversely proportional to the segment size (13). The degrees of competency of the four parental strains determined by a standard method (13) did not differ significantly, as indicated in Fig. 4. The cloning efficiency of cyanobacterial fragments was normalized by comparison with the degree of competency: 3.1% for 14 kb, 0.85% for 28 kb, 0.42% for 42 kb, and 0.09% for 77 kb. The rate-determining step may be the integration process, as discussed previously (13). 2.4. Cloned Segment as Part of the BGM Vector These recombinants showed no apparent reduction in the rate of growth, as measured in antibiotic-free LB medium at three temperatures, 25, 35, and 45°C (data not shown). Although the expression profiles of genes in the cloned region were not investigated, the cloned Synechocystis segment likely replicates as part of the B. subtilis genome. This suggests that cloned Synechocystis DNA functions similarly with respect to transformation. The 77 kb Synechocystis–originated DNA of BEST7019 was examined for transformation of rnhB::spc of BUSY1001. As shown in Fig. 5, BEST7021 was derived from BEST7019 through conversion of rnhB::spc to rnhB. This conversion was carried out by gene-directed mutagenesis using leuB::tet as a catalyst gene (25). Transformation of BEST7021 with the BUSY1001(rnhB::spc) DNA gave a substantial number of spectinomycin-resistant colonies. The number, 2.83 × 102, i.e. 43.3% of the relevant transfer of leuB::tet→leuB::cat, 6.55 × 102, by BEST4110, indicated that the 90-kb cyano segment and other loci in the B. subtilis genome are not discriminatory in terms of genetic transformation. The slightly lower frequency may be accounted for by the limited length for homologous recombination, only 10 kb, as indicated in Fig. 5. DISCUSSION The present results strongly indicate that the target DNA of the B. subtilis genome vector basically can be of any kind. The preparation of two flanking segments prior to BGM construction is unavoidable but allows for the positional cloning of large DNA beyond the limit of PCR technology. Cloning in the B. subtilis genome vector has several advantages compared with cloning in plasmids. The DNA integrated in the genome exhibited high genetic stability and did not segregate even in antibiotic-free LB-medium. It was proven that the cloned DNA becomes indistinguishable from the rest of the B. subtilis genome with respect to genetic transformation. This finding raised the possibility of unlimited manipulation against the cloned segment in the BGM vector (15, 26). Precise positional cloning depends on effective double homologous recombination. The cloning process starts with the incorporation of DNA by competent cells. According to the proposed mechanism (11), the double- stranded DNA is converted to single-stranded DNA by a competent complex formed in the cell wall. About 20–30 kb fragments enter the competent B. subtilis cells effectively during transformation (27), and a single-stranded DNA of approximately 7 kb is found in the cells (11). The actual size of the DNA incorporated has been controversial. In our previous study, it was demonstrated that continuous DNA of longer than 50 kb actually enters a competent cell and recombines with the genome (13). This size was nearly doubled in the present study, leaving the argument unresolved. The efficiency of integration apparently decreased as the size of the DNA to be cloned increased at all concentrations. Although LPS was empirically employed as 5 to 10% of the target DNA length (9), no significant difference in cloning efficiency between the smallest [LPS3] (4.17 kb) and largest [LPS5] (5.11 kb) was observed. Above all, high molecular weight donor DNA is critical for effective cloning as well as for determination of the maximum DNA size entering a competent cell. The internal selection system facilitates regional-specific cloning from not only bacterial but also eukaryote genomes, as long as high molecular weight DNA is prepared. In addition to the present Synechocystis genome, whose G+C content is 45% (16), which is close to that (43%) of B. subtilis (28), cloning of DNA with a higher or lower G+C content is underway to exploit the use of the BGM vector. It is likely that larger DNA could be harbored if the internal selection marker system is used repeatedly (Itaya and Fujita, unpublished experiment). Manipulation of the cloned DNA with a combination of efficient recovery tools (15, 27) makes the BGM system prominent. We wish to thank K. Matsui for her technical help. We also thank Drs. H. Yoshikawa and H. Yanagawa for the helpful discussions. * To whom correspondence should be addressed. Tel: +81-427-24-6254, Fax: +81-427-24-6316, E-mail: [email protected] View largeDownload slide Fig. 1. Positional cloning of the Synechocystis genome in the B. subtilis genome (BGM) vector. BEST6016 and BEST7003 are genome vectors for direct and indirect selection. The structure of the intermediate genome is shown in parentheses. X indicates homologous recombination. The genomic pBR322 sequence is presented in the yellow (amp-half) and blue (tet-half) hatched boxes divided by the cloning site. Antibiotic resistance genes are indicated by closed circles (chloramphenicol), open circles (erythromycin), closed triangles (tetracycline), and closed diamonds (spectinomycin). DNA fragment sizes are not drawn to scale. A twisted arrow indicates suppression of the Pr-promoter by the CI gene product. [I] for BEST7003 indicates the site for I-PpoI. Screening of the bottom recombinants is described in the text. View largeDownload slide Fig. 1. Positional cloning of the Synechocystis genome in the B. subtilis genome (BGM) vector. BEST6016 and BEST7003 are genome vectors for direct and indirect selection. The structure of the intermediate genome is shown in parentheses. X indicates homologous recombination. The genomic pBR322 sequence is presented in the yellow (amp-half) and blue (tet-half) hatched boxes divided by the cloning site. Antibiotic resistance genes are indicated by closed circles (chloramphenicol), open circles (erythromycin), closed triangles (tetracycline), and closed diamonds (spectinomycin). DNA fragment sizes are not drawn to scale. A twisted arrow indicates suppression of the Pr-promoter by the CI gene product. [I] for BEST7003 indicates the site for I-PpoI. Screening of the bottom recombinants is described in the text. View largeDownload slide Fig. 2. A. High molecular weight genomic DNA of Synechocystis. BUSY1001 DNA was prepared by the standard method (lane 1) or by the modified method, as described under materials and methods (lane 2). The discrete bands are plasmids of this bacterium (16). Lambda oligomers plus HindIII digests with their sizes are given on the left. B. I-PpoI fragments resolved by CHEF. The Synechocystis DNA of 27, 39 and 44 kb was from BEST7171, BEST7336, and BEST8155. These sequences include LPSs and a Cm (1 kb) or Em (1.2 kb) resistance gene. M includes Lambda oligomers plus HindIII digests, with their sizes on the right. View largeDownload slide Fig. 2. A. High molecular weight genomic DNA of Synechocystis. BUSY1001 DNA was prepared by the standard method (lane 1) or by the modified method, as described under materials and methods (lane 2). The discrete bands are plasmids of this bacterium (16). Lambda oligomers plus HindIII digests with their sizes are given on the left. B. I-PpoI fragments resolved by CHEF. The Synechocystis DNA of 27, 39 and 44 kb was from BEST7171, BEST7336, and BEST8155. These sequences include LPSs and a Cm (1 kb) or Em (1.2 kb) resistance gene. M includes Lambda oligomers plus HindIII digests, with their sizes on the right. View largeDownload slide Fig. 3. The cloned cyanobacterial segment in the B. subtilis genome vector. Genomic DNA of the indicated strains digested with NotI (left) or BglII (right) was run. The running conditions for CHEF are shown. The probe was prepared using BUSY1001 genomic DNA after complete digestion with HindIII. The Southern band indicated by the circled number corresponds to the Synechocystis genomic NotI and BglII restriction map of this region. The end fragments were altered due to the cloning in the BGM vector. Fragment 7 of BglII is too small to be seen under these conditions. View largeDownload slide Fig. 3. The cloned cyanobacterial segment in the B. subtilis genome vector. Genomic DNA of the indicated strains digested with NotI (left) or BglII (right) was run. The running conditions for CHEF are shown. The probe was prepared using BUSY1001 genomic DNA after complete digestion with HindIII. The Southern band indicated by the circled number corresponds to the Synechocystis genomic NotI and BglII restriction map of this region. The end fragments were altered due to the cloning in the BGM vector. Fragment 7 of BglII is too small to be seen under these conditions. View largeDownload slide Fig. 4. Size-dependent efficiency of cloning. The number of spectinomycin-resistant transformants is plotted against the size of the cloned segment. Two concentrations of Synechocystis genomic DNA, 1.80 µg/ml (closed diamonds) and 6.05 µg/ml (closed triangles), were used. The degrees of competency of the four strains measured as the number of tetracycline-resistant transformants DNA (closed box) were nearly identical. Standard deviation (SD) is indicated by vertical bars. View largeDownload slide Fig. 4. Size-dependent efficiency of cloning. The number of spectinomycin-resistant transformants is plotted against the size of the cloned segment. Two concentrations of Synechocystis genomic DNA, 1.80 µg/ml (closed diamonds) and 6.05 µg/ml (closed triangles), were used. The degrees of competency of the four strains measured as the number of tetracycline-resistant transformants DNA (closed box) were nearly identical. Standard deviation (SD) is indicated by vertical bars. View largeDownload slide Fig. 5. Genetic transformation within the new genome region. X indicates homologous recombination. Cognate genomic transformation was measured using BEST4110 DNA. Other symbols are the same as in Fig. 1. BEST7021 supplies only 10 kb of Synechocystis DNA of the left end instead of the sequence longer than 70 kb of the right. View largeDownload slide Fig. 5. Genetic transformation within the new genome region. X indicates homologous recombination. Cognate genomic transformation was measured using BEST4110 DNA. Other symbols are the same as in Fig. 1. BEST7021 supplies only 10 kb of Synechocystis DNA of the left end instead of the sequence longer than 70 kb of the right. Table 1. Bacterial strains and plasmids for indirect selection. Bacteria  Genotypes  Sources or references    Synechocystis sp. PCC6803    (Institut Pasteur, France)    BUSY1001  rnhB::spc  pBRSYNrnhBS1 × PCC6803    Bacillus subtilis (1A1)  trpC2  (BGSC, Ohio, USA)    RM125  argleu  (9)    BEST6016  proB::pBRTc  (13)    BEST7003  proB::pBRTc, yah::pr-neo  this study    BEST4110  leuB::cat  this study            LPA strains and recombinants  BEST7166  proB::pC[0878/0903]  CmR, SpcR, NmS  pC[0878/0903] × BEST7003  BEST7171  proB::pC[26 kb]  NmR, SpcS, CmR  PCC6803 × BEST7166  BEST7332  proB::pC[1691/1729]  CmR, SpcR, NmS  pC[1691/1729] × BEST7003  BEST7336  proB::pC[38 kb]  NmR, SpcS, CmR  PCC6803 × BEST7166  BEST8135  proB::pE[2553/2596]   EmR, SpcR, NmS  pE[2553/2596] × BEST7003  BEST8155  proB::pE[43 kb]  NmR, SpcS, EmR  PCC6803 × BEST8135  Plasmids  Construction or features  References    pBRSYNrnhB  rnhB (PCC6803)  gene amplified cloned in pBR322    pBRSYNrnhBS1  rnhB::spc  1.3kb/SmaI into pBRSYNrnhB/AatI    pBMAP105TT  leuB::tet  (13)            LPS clones isolated from PCC6803 and derivatives  LPS  Plasmid  Segment size (kb)  Region or vector  [0878]   pCR[0878–0882]¶  4.67  878,001 to 882,669  [0903]  pCR[0899–0903]¶  4.53  899,015 to 903,540  [1691]  pCR[1691–1696]¶  4.74  1,691,653 to 1,696,392  [1729]  pCR[1725–1729]¶  4.54  1,725,139 to 1,729,678  [2553]  pCR[2553–2558]¶  4.91  2,553,209 to 2,558,117  [2596]  pCR[2591–2596]¶  4.96  2,591,742 to 2,596,704  pC0878  pCR[0878–0882]/BamHI    pCISP310B/BamHI†  pC1691  pCR[1691–1696]/BamHI    pCISP310B/BamHI†  pE2553  pCR[2553–2558]/BamHI    pCISP311B/BamHI†          LPA plasmids for integration into BEST7003  pC[0878/0903]  pCR[0899–0903]/EcoRI  pC0878/EcoRI    pC[1691/1729]  pCR[1725–1729]/EcoRI  pC1691/EcoRI    pE[2553/2596]  pCR[2591–2596]/EcoRI  pE2553/EcoRI    Bacteria  Genotypes  Sources or references    Synechocystis sp. PCC6803    (Institut Pasteur, France)    BUSY1001  rnhB::spc  pBRSYNrnhBS1 × PCC6803    Bacillus subtilis (1A1)  trpC2  (BGSC, Ohio, USA)    RM125  argleu  (9)    BEST6016  proB::pBRTc  (13)    BEST7003  proB::pBRTc, yah::pr-neo  this study    BEST4110  leuB::cat  this study            LPA strains and recombinants  BEST7166  proB::pC[0878/0903]  CmR, SpcR, NmS  pC[0878/0903] × BEST7003  BEST7171  proB::pC[26 kb]  NmR, SpcS, CmR  PCC6803 × BEST7166  BEST7332  proB::pC[1691/1729]  CmR, SpcR, NmS  pC[1691/1729] × BEST7003  BEST7336  proB::pC[38 kb]  NmR, SpcS, CmR  PCC6803 × BEST7166  BEST8135  proB::pE[2553/2596]   EmR, SpcR, NmS  pE[2553/2596] × BEST7003  BEST8155  proB::pE[43 kb]  NmR, SpcS, EmR  PCC6803 × BEST8135  Plasmids  Construction or features  References    pBRSYNrnhB  rnhB (PCC6803)  gene amplified cloned in pBR322    pBRSYNrnhBS1  rnhB::spc  1.3kb/SmaI into pBRSYNrnhB/AatI    pBMAP105TT  leuB::tet  (13)            LPS clones isolated from PCC6803 and derivatives  LPS  Plasmid  Segment size (kb)  Region or vector  [0878]   pCR[0878–0882]¶  4.67  878,001 to 882,669  [0903]  pCR[0899–0903]¶  4.53  899,015 to 903,540  [1691]  pCR[1691–1696]¶  4.74  1,691,653 to 1,696,392  [1729]  pCR[1725–1729]¶  4.54  1,725,139 to 1,729,678  [2553]  pCR[2553–2558]¶  4.91  2,553,209 to 2,558,117  [2596]  pCR[2591–2596]¶  4.96  2,591,742 to 2,596,704  pC0878  pCR[0878–0882]/BamHI    pCISP310B/BamHI†  pC1691  pCR[1691–1696]/BamHI    pCISP310B/BamHI†  pE2553  pCR[2553–2558]/BamHI    pCISP311B/BamHI†          LPA plasmids for integration into BEST7003  pC[0878/0903]  pCR[0899–0903]/EcoRI  pC0878/EcoRI    pC[1691/1729]  pCR[1725–1729]/EcoRI  pC1691/EcoRI    pE[2553/2596]  pCR[2591–2596]/EcoRI  pE2553/EcoRI    Cm, chloramphenicol; Sp, spectinomycin; Nm, neomycin; Em, erythromycin; R, resistance; S, sensitive. ¶Cloned in pCR-XL-TOPO vector. †pCISP310B and pCISP311B are reported in Ref. 9. View Large Table 2. Bacterial strains and plasmids for direct selection. Bacteria  Genotypes  Antibiotic markers  Sources or references  BEST7004  proB::p[LPS1/Em/LPS2]  EmR  p[LPS1/Em/LPS2] × BEST6016  BEST7016  proB::pBR[16.5 kb (89554–115223)]  SpR, EmS  BUSY1001 × BEST7016  BEST7012  proB::p[LPS1/Em/LPS3]  EmR  p[LPS1/Em/LPS3] × BEST6016  BEST7017  proB::pBR[42kb (75564–115223)]   SpR, EmS  BUSY1001 × BEST7016  BEST7008  proB::p[LPS1/Em/LPS4]  EmR  p[LPS1/Em/LPS4] × BEST6016  BEST7018  proB::pBR[50 kb (61054–115223)]  SpR, EmS  BUSY1001 × BEST7008  BEST7015  proB::p[LPS1/Em/LPS5]  EmR  p[LPS1/Em/LPS5] × BEST6016  BEST7019  proB::pBR[90 kb (25603–115223)]  SpR, EmS  BUSY1001 × BEST7015  BEST7021  proB::pBR[90 kb (25603–115223)]  leuB::tet, SpS  see the text and Fig. 5  LPS clones isolated from PCC6803 and derivatives  Plasmid  Segment size (kb)  Region  Vector  [LPS1] pSYNrnhB2′  7.48  107755–115223  HindIII/pBR322  [LPS2] pCRNHBOT  4.18  89554–93735  pT7BlueT  [LPS3] pCRNHB-101  4.17  75564–79734  BamHI/pBR322  [LPS4] pSYN3-TO  5.01  61054–66059  pCR-XL-TOPO  [LPS5] pCRNHB-203  5.11  25603–30713  EcoRI/pBRcI′.  LPA plasmids  Donor  Vector    p[LPS1/LPS2]  pSYNrnhB2′/BamHI  pCRNHBOT/BamHI    p[LPS1/LPS3]  pSYNrnhB2′/BamHI  pCRNHB-101/BamHI    p[LPS1/LPS4]  pSYNrnhB2′/BamHI  pSYN3-TO/BamHI    p[LPS1/LPS5]  pSYNrnhB2′/BamHI  pCRNHB-203/BamHI    LPA plasmids for integration into BEST6016§  p[LPS1/Em/LPS2]  p[LPS1/LPS2]/EcoRV      p[LPS1/Em/LPS3]  p[LPS1/LPS3]/EcoRV      p[LPS1/Em/LPS4]  p[LPS1/LPS4]/EcoRV      p[LPS1/Em/LPS5]  p[LPS1/LPS5]/NheI-T4DNApolymerase      Bacteria  Genotypes  Antibiotic markers  Sources or references  BEST7004  proB::p[LPS1/Em/LPS2]  EmR  p[LPS1/Em/LPS2] × BEST6016  BEST7016  proB::pBR[16.5 kb (89554–115223)]  SpR, EmS  BUSY1001 × BEST7016  BEST7012  proB::p[LPS1/Em/LPS3]  EmR  p[LPS1/Em/LPS3] × BEST6016  BEST7017  proB::pBR[42kb (75564–115223)]   SpR, EmS  BUSY1001 × BEST7016  BEST7008  proB::p[LPS1/Em/LPS4]  EmR  p[LPS1/Em/LPS4] × BEST6016  BEST7018  proB::pBR[50 kb (61054–115223)]  SpR, EmS  BUSY1001 × BEST7008  BEST7015  proB::p[LPS1/Em/LPS5]  EmR  p[LPS1/Em/LPS5] × BEST6016  BEST7019  proB::pBR[90 kb (25603–115223)]  SpR, EmS  BUSY1001 × BEST7015  BEST7021  proB::pBR[90 kb (25603–115223)]  leuB::tet, SpS  see the text and Fig. 5  LPS clones isolated from PCC6803 and derivatives  Plasmid  Segment size (kb)  Region  Vector  [LPS1] pSYNrnhB2′  7.48  107755–115223  HindIII/pBR322  [LPS2] pCRNHBOT  4.18  89554–93735  pT7BlueT  [LPS3] pCRNHB-101  4.17  75564–79734  BamHI/pBR322  [LPS4] pSYN3-TO  5.01  61054–66059  pCR-XL-TOPO  [LPS5] pCRNHB-203  5.11  25603–30713  EcoRI/pBRcI′.  LPA plasmids  Donor  Vector    p[LPS1/LPS2]  pSYNrnhB2′/BamHI  pCRNHBOT/BamHI    p[LPS1/LPS3]  pSYNrnhB2′/BamHI  pCRNHB-101/BamHI    p[LPS1/LPS4]  pSYNrnhB2′/BamHI  pSYN3-TO/BamHI    p[LPS1/LPS5]  pSYNrnhB2′/BamHI  pCRNHB-203/BamHI    LPA plasmids for integration into BEST6016§  p[LPS1/Em/LPS2]  p[LPS1/LPS2]/EcoRV      p[LPS1/Em/LPS3]  p[LPS1/LPS3]/EcoRV      p[LPS1/Em/LPS4]  p[LPS1/LPS4]/EcoRV      p[LPS1/Em/LPS5]  p[LPS1/LPS5]/NheI-T4DNApolymerase      †http://www.kazusa.or.jp/cyano/.§The erm cassette prepared from pBEST701 (9) with SmaI was inserted into the site indicated between the two LPSs. A 1.3-kb fragment containing the rnhB gene was obtained by PCR-mediated amplification from genomic DNA of BUSY101 using primers rnhB, 5′-CCTAATCAAAACGGAGTACG-3′ and 5′-AATTGCCATTGAAGTGGCGG-3′. The primer sets used for amplification were: [LPS5] pCRNHB-203: SYNrnhB-20F 5′-GGGACCAAGTACAACAACTC-3′ and SYNrnhB-20R 5′-GGGGGAGGAAATTATCAGCG-3′; [LPS4] pSYN3-TO: SYN3F 5′-GCCGAATTCCGGGCTGGATCCCCT-3′ and SYN3R 5′-TATGGATCCTAACAACGACTTCCCC-3′; [LPS3] pCRNHB-101: SYNrnhB1F 5′-GGAGGAAGTCTGTTGCTCGG-3′ and SYNrnhB1R 5′-GTTGGGGTTTACAGGCGGTG-3′; [LPS2]: CRNHBOF 5′-TCAGGATCCGCCACCTTTTGACCGTTG-3′ and CRNHBOR 5′-TCAGGATCCCGGTCCAGAAGCATGG-3′; [LPS1]: SYNrnhB2F 5′-GGGGTTAAACGAATGACAGCGG-3′ and SYNrnhB2R 5′-AGGGACTGGAGGGGACAATG-3′. View Large References 1. Sambrook, J., Fritsch, E.F., and Maniatis, T. ( 1989) Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Google Scholar 2. Cutting, S. and Horn, P.B.V. ( 1990) Gene cloning techniques in Molecular Biological Methods for Bacillus (Harwood, C.R. and Cutting, S., eds.) pp. 27–74, John Wiley and Sons, Chichester, England Google Scholar 3. Hutchison, C.A., Peterson, S.N., Gill, S.R., Cline, R.T., White, O., Fraser, C.M., Smith, H.O., and Venter, J.C. ( 1999) Global transposon mutagenesis and a minimal Mycoplasma genome. Science  286, 2165–2169 Google Scholar 4. Kobayashi, K., Ehrlich, S.D., Albertini, A., Amati, G., Anderson, K., Arnaud, M., Asai, K., Ashikaga, S., Aymerich, S., Bessieres, P. et al. ( 2003) Essential Bacillus subtilis genes. Proc. Natl Acad. Sci. USA  100, 4678–4683 Google Scholar 5. Shizuya, H., Birren, B., Kim, U.J., Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M. ( 1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F- factor-based vector. Proc. Natl Acad. Sci. USA  89, 8794–8797 Google Scholar 6. Pierce, J. and Sternberg, N.L. ( 1992) Using bacteriophage P1 system to clone high molecular weight genomic DNA. Methods Enzymol.  216, pp. 549–574 Google Scholar 7. Burke, D.T., Carle, G.F., and Olson, M.V. ( 1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science , 236, 806–812 Google Scholar 8. Itaya, M. ( 1995) Toward a bacterial genome technology: Integration of the Escherichia coli prophage lambda genome into the Bacillus subtilis 168 chromosome. Mol. Gen. Genet.  248, 9–16 Google Scholar 9. Itaya, M., Shiroishi, T., Nagata, T., Fujita, K., and Tsuge, K. ( 2000) Efficient cloning and engineering of giant DNAs in a novel Bacillus subtilis genome vector. J. Biochem.  128, 869–875 Google Scholar 10. Spizizen, J. ( 1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl Acad. Sci. USA  44, 1072–1078 Google Scholar 11. Dubnau, D. ( 1999) DNA uptake in Bacteria. Annu. Rev. 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Tomioka, N., Shinozaki, K., and Sugiura, M. ( 1981) Molecular cloning and characterization of Ribosomal Genes from a blue-green alga, Anacystis nidulans. Mol. Gen. Genet.  184, 359–363 Google Scholar 23. Itaya, M. and Tanaka, T. ( 1991) Complete physical map of the Bacillus subtilis 168 chromosome constructed by a gene-directed mutagenesis method. J. Mol. Biol.  220, 631–648 Google Scholar 24. Saito, H. and Miura, K. ( 1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta  72, 619–629 Google Scholar 25. Itaya, M. and Tanaka, T. ( 1990) Gene-directed mutagenesis on the chromosome of Bacillus subtilis. Mol. Gen. Genet.  223, 268–272 Google Scholar 26. Tsuge, K. and Itaya, M. ( 2001) Recombinational transfer of 100-kb genomic DNA to plasmid in Bacillus subtilis 168. J. Bacteriol.  183, 5453–5458 Google Scholar 27. Henner, J.D. and Hoch, J.A. ( 1980) The Bacillus subtilis chromosome in Microbiol. Rev.  Vol. 44, pp. 57–82 Google Scholar 28. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V., Bertero, M.G., Bessieres, P., Bolotin, A., Borchert, S., et al. ( 1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature  390, 249–256 Google Scholar Author notes 1Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194-8511; and 2Department of Life Sciences (Biology), University of Tokyo, Komaba, Meguro, Tokyo 153-8902 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Stable Positional Cloning of Long Continuous DNA in the Bacillus subtilis Genome Vector

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Publisher
Oxford University Press
ISSN
0021-924X
eISSN
1756-2651
DOI
10.1093/jb/mvg168
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Abstract

Abstract Direct cloning of a long continuous genome segment in a Bacillus subtilis genome vector was demonstrated for the first time. Two small DNA fragments had to be installed in the vector prior to cloning. The DNA between these two fragments was cloned via homologous recombination. The efficiency of cloning was estimated using the 3,573-kb genome of a cyanobacterium, Synechocystis sp. PCC 6803. Recombinants were selected using the internal selection system of the Bacillus genome vector or with the antibiotic resistance marker in the cyanobacterial genome. Designated genomic segments as large as 77-kb were cloned by means of a single procedure. Cloning efficiency is affected by the molecular weight of the donor DNA and the size of the DNA to be cloned. The method is suitable for direct target cloning of large-sized DNA. Key words: genome vector, homologous recombination, positional cloning, transformation. Abbreviation: CHEF, contour-clamped homogeneous electric field. Received June 17, 2003; accepted July 14, 2003 DNA cloning technology emerged in the late 70s and has led to great successes in DNA manipulation (1, 2). The complete sequences of a number of genomes, of from bacteria to eukaryotes, have unveiled much about genome structure as well as the functions of gene products. Given that all the ORF information has been presented, the handling of a large number of genes as one set becomes important (3, 4). As regards the size of clonable DNA, Escherichiacoli vectors such as BAC (5) and PAC (6) can be used for handling DNA larger than 100 kb. The YAC vector in Saccharomyces cerevisiae can harbor larger DNA segments, however, it is associated with a high degree of chimera formation and clonal instability (7). There are few methods, however, for precise positional cloning for DNA above the limit of PCR-mediated amplification. A novel Bacillus subtilis genome vector was developed to fill this technical gap (8, 9). The cloning principle of the genome vector differs from that of the prevailing plasmid-born vectors. B. subtilis develops natural competency by which DNA outside the cell is taken up into the cytoplasm (10, 11). If a homologous sequence is present in the genome, the DNA taken up is integrated into the genome through the recA-dependent homologous recombination pathway (12, 13). According to this protocol, two short DNA fragments that flank the target region are required (Fig. 1). These short DNA, or LPSs, standing for Landing Pad Sequences (9), are prepared in the E. coli plasmid pBR322 and installed in the pBR322 sequence of the B. subtilis genome (12). The intervening DNA segment delineated precisely by the two LPSs is integrated via homologous recombination at both LPSs. The B. subtilis strain is called a BGM vector, standing for BacillusGenoMe vector (9), and the cloning procedure for the BGM vector itself is equivalent to positional cloning. A positive selection system developed previously facilitates the cloning process (14). Two examples have been reported, one includes a 48.5-kb E.coli bacteriophage lambda DNA (8), and the other mouse genomic DNA ranging 100–140 kb in size (9). These cloned segments were stably maintained in the BGM vector regardless of the highly repetitive nature of the mouse genome. Cloning in the BGM vector in the two latter cases, however, resulted in the transfer of DNA previously cloned by means of a different technology (8, 9). Cloning directly from genomic DNA has not been examined. Direct cloning of the designated region of a cyanobacterium genome was attempted in this study and it was demonstrated that continuous genomic DNA of up to 77 kb could be successfully cloned in the BGM vector. Factors affecting the cloning efficiency were quantitatively measured. This genome vector has high potential to provide properly manipulated DNA through direct cloning of submega-sized DNA. MATERIALS AND METHODS 1.1. Bacterial Strains and Plasmids B. subtilis 168 trpC2, from the Bacillus Genetic Stock Center (Ohio, USA), and E. coli JA221 are routinely used as hosts for molecular cloning in this laboratory (15). Competent B. subtilis cells were prepared (10) and stored at –70°C in the presence of 20% (v/v) glycerol before use (13). 1.2. B. Subtilis Genome Vector Two DNA sequences characterize the B. subtilis genome vector (9). They are a 4.3-kb pBR322 sequence inserted in the NotI site of the proB gene (12) and the neomycin resistance gene [Pr-neo] integrated into the NotI site in the yvfC-yveP gene (14). The integrated pBR322 is referred to hereafter as the genomic pBR and is divided into two halves, the 2.4 kb amp-half including a β-lactamase gene and the 1.9 kb tet-half including a tetracycline resistance determinant gene. DNA is cloned between these two-halves, as illustrated in Fig. 1. Expression of the neo gene is regulated by a Pr promoter to which the cI repressor protein binds, which represses it (14). 1.3. Synechocystis Strains The genome of unicellular cyanobacterium Synechocystis sp. strain PCC (Pasteur Culture Collection) 6803 has been sequenced (16). This photosynthetic bacterium was chosen because its genome sequence is known (http://www.kazusa.or.jp/cyano/) and it is non-pathogenic. Cyanobacteria are unique organisms that perform oxygenic photosynthesis like chloroplasts of plants. Systematic analysis of gene expression has been carried out with many tools such as DNA microarrays (17). BG11 medium (18) was used to cultivate Synechocystis strains in liquid and on agar plates. The SynechocystisrnhB mutant was derived from the wild type strain of PCC 6803. The gene encoding ribonuclease H [EC. 3.1.26.4] chosen in this study is related to another target of our laboratory (19). The SynechocystisrnhB gene was replaced by the mutated gene rnhB::spc of pBRSYNrnhBS1 according to the method developed by Sugita and Sugiura (20). Colonies formed after 3 days on BG11 plates containing spectinomycin at 20 µg/ml under illumination at 50 µmol photons m–2 s–2 at 30°C. They were transferred to fresh BG11 medium containing spectinomycin (20 µg/ml) and then incubated for 4 weeks with occasional dilution, allowing them to pass through 11 generations. As the multiplicity of the Synechocystis genome is 12 (21), the mutant can be validated by the ratio of Southern band intensities of the mutant and wild type alleles. BUSY1001 contained 94.9% mutant rnhB::spc allele. The incomplete replacement by the mutant allele together with no associated phenotype indicates that the rnhB gene may be essential for the strain. 1.4. Preparation of High Molecular Weight Synechocystis Genome DNA Genomic DNA prepared according to the reported method (21, 22) included little DNA above 50 kb, as shown in Fig. 2A, and gave no recombinants. The extraction protocol in reference 21 was modified to obtain high-molecular weight DNA. A 50-ml Synechocystis culture was treated three times by freeze-thawing in dry ice and a water bath before harvesting. The cell pellet was washed with 5 ml of a NaCl/EDTA solution (120 mM/50 mM, pH 8.0), and then suspended in 4 ml of NaCl/EDTA and 1 ml of a saturated sodium iodide (NaI) solution. After incubation at 37°C for 30 min, 5 ml of a 25% sucrose TES solution (100 mM Tris-HCl [pH 7.6], 10 mM NaCl, and 1 mM EDTA) supplemented with 10 mg of lysozyme ml–1 was added, followed by incubation for 2 h at 37°C. After incubation for 17 hours with proteinase K at 250 µg ml–1 and 1.0% sodium dodesyl sulfate (w/v), a 5.75 ml sample was shaken with an equal amount of a phenol/chloroform mixture, followed by centrifugation at 3,500 ×g for 15 min. The upper phase, approximately 5 ml, was transferred to a fresh 50 ml tube (Falcon, 2070), and then three times the volume of Ethanol was added to precipitate the genomic DNA. The DNA collected by centrifugation at 3,500 ×g for 15 min was rinsed in 10 ml of 70% ethanol. The concentration of the DNA dissolved in 0.2 ml was determined by UV absorption at 260 nm using GeneQuantII (Pharmacia Biotech). This process yielded 5.69 mg/ml for PCC6803 and 3.6 mg/ml for BUSY1001. These DNA preparations included high molecular weight DNA (above 100 kb), as shown in Fig. 2A, and are used throughout this work. 1.5. Preparation of B. Subtilis Genome DNA Intact unsheared DNA for CHEF gel electrophoretic analysis was prepared in agarose plugs as described previously (23). Liquid DNA for conventional Southern analysis was prepared by the method of Saito and Miura (24). Agarose gel (1.0% w/v) in TBE solution (45 mM Tris-borate, [pH8.0], 1.0 mM EDTA) was used for CHEF gel electrophoresis at a constant voltage of 3 or 4 V cm–1 at 14°C. The pulse and running times are specified in the legends to the figures. Agarose gel (1.0% v/v) in a TAE solution (50 mM Tris-acetate [pH 8.0] and 1.0 mM EDTA) was used for conventional gel electrophoresis at room temperature. The DNA in the gels after electrophoresis was stained with an ethidium bromide solution (60 µg/ml) for 15 min, followed by photography. The Southern hybridization experiment was performed according to the protocol for a DNA labeling and detection kit (Roche, USA) RESULTS 2.1. Cloning with an Indirect Selection Marker As Synechocystis has no appropriate selection markers, the use of the internal selection system of the BGM vector (14) allows cloning of any genomic loci. Cloning from three genomic loci, the 16-kb region A (0882–0899), the 29-kb region B (1696–1725), and the 33-kb region C (2558–2591), is illustrated in Fig. 1. The LPS plasmids and the plasmids combined with the two LPSs for each region were prepared in E. coli as listed in Table 1. The latter plasmid is referred to as an LPA plasmid, standing for LPS Array plasmid. B. subtilis strains having LPA in the genomic pBR were constructed separately aiming at different target regions; BEST7166 for region A, BEST7332 for region B, and BEST8135 for region C (Table 1). For region A, 271 colonies were selected using neomycin at 3 µg/ml after the transformation of BEST7166 with PCC6803 genome DNA. Nine clones sensitive to spectinomycin at 50 µg/ml were subjected to colony PCR screening. The internal 4,650-bp of the 16 kb was amplified from two clones. Both carry the same 27–kb DNA generated by I-PpoI-digestion. The I-PpoI fragment produced from BEST7171 was resolved by pulsed-field gel electrophoresis (Fig. 2B). The primer set used to amplify the internal 4,650-bp segment of 16 kb was 5′-GTGGCAGAGTCGGTATTGGCTC-3′ (0898890F) and 5′-CCGGGTATTTATGGATCCCTAACC-3′ (0903540R). Similarly, the 29-kb segment of region B was cloned in BEST7336. Only this strain was isolated from 15 neomycin-resistant and spectinomycin-sensitive candidates via colony PCR screening using 5′-GGGATCAACTACAGTGCCCGG-3′ (1724984F) and 5′-TGTGGATCCTTGGATTTCATCAGG-3′ (1729678R) to detect the internal 4,694-bp segment. Cloning of the 33-kb region C resulted in only one strain, BEST8155, on screening for the 55 neomycin-resistant and spectinomycin-sensitive clones by colony PCR screening. The primer set for the internal 726-bp segment of 33 kb was 5′-CAATTCCCTCAGTCCCGACG-3′ (2591470F) and 5′-GGTCCTGGGCGTTAAAGGC-3′ (2592196R). The I-PpoI segments from these strains were confirmed, as shown in Fig. 2B. Southern analysis of BamHI and HindIII digests using Synechocystis genomic DNA as a probe verified that these cloned DNA were identical with those predicted from the sequences in the database (data not shown). These results indicated that target cloning with an internal positive selection system proceeds through the mechanism illustrated in Fig. 1. 2.2. Cloning with a Direct Selection Marker Despite the effectiveness of the internal marker system, the cloning of DNA segments longer than 50 kb was unsuccessful (data not shown). The number of true recombinants seemed underestimated due to the large number of background neomycin for resistant colonies for a poorly understood reason (9, 14). a direct selection method was used for quantitative measurements of size-dependent efficiency. A Synechocystis derivative, strain BUSY1001, that has a spectinomycin resistance gene in the rnhB gene was constructed. As the spectinomycin resistance gene functions for selection of B. subtilis, only recombinants containing the resistance gene are selected directly. Four B. subtilis strains for cloning of rnhB::spc segments of various sizes, from 14 kb to 77 kb, were constructed. Initially, the number of spectinomycin-resistant colonies varied among the experiments but the number became reproducible when the outgrowth period after DNA uptake was extended from 1 hour to 2 hours. It is likely that appropriate expression from the spectinomycin resistance gene requires prolonged incubation. This condition was employed throughout this study. Recombinants selected using spectinomycin at 50 µg/ml were all sensitive to erythromycin at 5 µg/ml. Six representative clones for 14-kb, 4 for 28-kb, 4 for 42-kb, and 3 for 77-kb were analyzed by Southern hybridization using Synechocystis genome DNA as a probe. As shown in Fig. 3, the numbers and sizes of the NotI and BglII Southern bands were consistent with those predicted from the sequence information. The lack of unexpected bands indicated high structural stability of the cloned segment in the BGM vector. No structural alteration of the B. subtilis genome part was detected on SfiI and NotI fragment analysis (data not shown). These results demonstrated that all colonies selected with spectinomycin integrated the rnhB::spc segment replacing the erm gene between the two LPSs, as shown in Fig. 1. 2.3. Size-Dependent Efficiency of Cloning Experiments were performed in triplicate with two BUSY1001 DNA concentrations, 1.80 and 6.05 µg/ml. The average number of spectinomycin- resistant transformants was plotted with standard deviations (Fig. 4). The number decreased as the size of the clone increased from 14 kb to 77 kb at both DNA concentrations. Saturation by Synechocystis DNA was not clear under the present conditions. This is consistent with the observation that the frequency of segment transfer between B. subtilis genomes appeared to be inversely proportional to the segment size (13). The degrees of competency of the four parental strains determined by a standard method (13) did not differ significantly, as indicated in Fig. 4. The cloning efficiency of cyanobacterial fragments was normalized by comparison with the degree of competency: 3.1% for 14 kb, 0.85% for 28 kb, 0.42% for 42 kb, and 0.09% for 77 kb. The rate-determining step may be the integration process, as discussed previously (13). 2.4. Cloned Segment as Part of the BGM Vector These recombinants showed no apparent reduction in the rate of growth, as measured in antibiotic-free LB medium at three temperatures, 25, 35, and 45°C (data not shown). Although the expression profiles of genes in the cloned region were not investigated, the cloned Synechocystis segment likely replicates as part of the B. subtilis genome. This suggests that cloned Synechocystis DNA functions similarly with respect to transformation. The 77 kb Synechocystis–originated DNA of BEST7019 was examined for transformation of rnhB::spc of BUSY1001. As shown in Fig. 5, BEST7021 was derived from BEST7019 through conversion of rnhB::spc to rnhB. This conversion was carried out by gene-directed mutagenesis using leuB::tet as a catalyst gene (25). Transformation of BEST7021 with the BUSY1001(rnhB::spc) DNA gave a substantial number of spectinomycin-resistant colonies. The number, 2.83 × 102, i.e. 43.3% of the relevant transfer of leuB::tet→leuB::cat, 6.55 × 102, by BEST4110, indicated that the 90-kb cyano segment and other loci in the B. subtilis genome are not discriminatory in terms of genetic transformation. The slightly lower frequency may be accounted for by the limited length for homologous recombination, only 10 kb, as indicated in Fig. 5. DISCUSSION The present results strongly indicate that the target DNA of the B. subtilis genome vector basically can be of any kind. The preparation of two flanking segments prior to BGM construction is unavoidable but allows for the positional cloning of large DNA beyond the limit of PCR technology. Cloning in the B. subtilis genome vector has several advantages compared with cloning in plasmids. The DNA integrated in the genome exhibited high genetic stability and did not segregate even in antibiotic-free LB-medium. It was proven that the cloned DNA becomes indistinguishable from the rest of the B. subtilis genome with respect to genetic transformation. This finding raised the possibility of unlimited manipulation against the cloned segment in the BGM vector (15, 26). Precise positional cloning depends on effective double homologous recombination. The cloning process starts with the incorporation of DNA by competent cells. According to the proposed mechanism (11), the double- stranded DNA is converted to single-stranded DNA by a competent complex formed in the cell wall. About 20–30 kb fragments enter the competent B. subtilis cells effectively during transformation (27), and a single-stranded DNA of approximately 7 kb is found in the cells (11). The actual size of the DNA incorporated has been controversial. In our previous study, it was demonstrated that continuous DNA of longer than 50 kb actually enters a competent cell and recombines with the genome (13). This size was nearly doubled in the present study, leaving the argument unresolved. The efficiency of integration apparently decreased as the size of the DNA to be cloned increased at all concentrations. Although LPS was empirically employed as 5 to 10% of the target DNA length (9), no significant difference in cloning efficiency between the smallest [LPS3] (4.17 kb) and largest [LPS5] (5.11 kb) was observed. Above all, high molecular weight donor DNA is critical for effective cloning as well as for determination of the maximum DNA size entering a competent cell. The internal selection system facilitates regional-specific cloning from not only bacterial but also eukaryote genomes, as long as high molecular weight DNA is prepared. In addition to the present Synechocystis genome, whose G+C content is 45% (16), which is close to that (43%) of B. subtilis (28), cloning of DNA with a higher or lower G+C content is underway to exploit the use of the BGM vector. It is likely that larger DNA could be harbored if the internal selection marker system is used repeatedly (Itaya and Fujita, unpublished experiment). Manipulation of the cloned DNA with a combination of efficient recovery tools (15, 27) makes the BGM system prominent. We wish to thank K. Matsui for her technical help. We also thank Drs. H. Yoshikawa and H. Yanagawa for the helpful discussions. * To whom correspondence should be addressed. Tel: +81-427-24-6254, Fax: +81-427-24-6316, E-mail: [email protected] View largeDownload slide Fig. 1. Positional cloning of the Synechocystis genome in the B. subtilis genome (BGM) vector. BEST6016 and BEST7003 are genome vectors for direct and indirect selection. The structure of the intermediate genome is shown in parentheses. X indicates homologous recombination. The genomic pBR322 sequence is presented in the yellow (amp-half) and blue (tet-half) hatched boxes divided by the cloning site. Antibiotic resistance genes are indicated by closed circles (chloramphenicol), open circles (erythromycin), closed triangles (tetracycline), and closed diamonds (spectinomycin). DNA fragment sizes are not drawn to scale. A twisted arrow indicates suppression of the Pr-promoter by the CI gene product. [I] for BEST7003 indicates the site for I-PpoI. Screening of the bottom recombinants is described in the text. View largeDownload slide Fig. 1. Positional cloning of the Synechocystis genome in the B. subtilis genome (BGM) vector. BEST6016 and BEST7003 are genome vectors for direct and indirect selection. The structure of the intermediate genome is shown in parentheses. X indicates homologous recombination. The genomic pBR322 sequence is presented in the yellow (amp-half) and blue (tet-half) hatched boxes divided by the cloning site. Antibiotic resistance genes are indicated by closed circles (chloramphenicol), open circles (erythromycin), closed triangles (tetracycline), and closed diamonds (spectinomycin). DNA fragment sizes are not drawn to scale. A twisted arrow indicates suppression of the Pr-promoter by the CI gene product. [I] for BEST7003 indicates the site for I-PpoI. Screening of the bottom recombinants is described in the text. View largeDownload slide Fig. 2. A. High molecular weight genomic DNA of Synechocystis. BUSY1001 DNA was prepared by the standard method (lane 1) or by the modified method, as described under materials and methods (lane 2). The discrete bands are plasmids of this bacterium (16). Lambda oligomers plus HindIII digests with their sizes are given on the left. B. I-PpoI fragments resolved by CHEF. The Synechocystis DNA of 27, 39 and 44 kb was from BEST7171, BEST7336, and BEST8155. These sequences include LPSs and a Cm (1 kb) or Em (1.2 kb) resistance gene. M includes Lambda oligomers plus HindIII digests, with their sizes on the right. View largeDownload slide Fig. 2. A. High molecular weight genomic DNA of Synechocystis. BUSY1001 DNA was prepared by the standard method (lane 1) or by the modified method, as described under materials and methods (lane 2). The discrete bands are plasmids of this bacterium (16). Lambda oligomers plus HindIII digests with their sizes are given on the left. B. I-PpoI fragments resolved by CHEF. The Synechocystis DNA of 27, 39 and 44 kb was from BEST7171, BEST7336, and BEST8155. These sequences include LPSs and a Cm (1 kb) or Em (1.2 kb) resistance gene. M includes Lambda oligomers plus HindIII digests, with their sizes on the right. View largeDownload slide Fig. 3. The cloned cyanobacterial segment in the B. subtilis genome vector. Genomic DNA of the indicated strains digested with NotI (left) or BglII (right) was run. The running conditions for CHEF are shown. The probe was prepared using BUSY1001 genomic DNA after complete digestion with HindIII. The Southern band indicated by the circled number corresponds to the Synechocystis genomic NotI and BglII restriction map of this region. The end fragments were altered due to the cloning in the BGM vector. Fragment 7 of BglII is too small to be seen under these conditions. View largeDownload slide Fig. 3. The cloned cyanobacterial segment in the B. subtilis genome vector. Genomic DNA of the indicated strains digested with NotI (left) or BglII (right) was run. The running conditions for CHEF are shown. The probe was prepared using BUSY1001 genomic DNA after complete digestion with HindIII. The Southern band indicated by the circled number corresponds to the Synechocystis genomic NotI and BglII restriction map of this region. The end fragments were altered due to the cloning in the BGM vector. Fragment 7 of BglII is too small to be seen under these conditions. View largeDownload slide Fig. 4. Size-dependent efficiency of cloning. The number of spectinomycin-resistant transformants is plotted against the size of the cloned segment. Two concentrations of Synechocystis genomic DNA, 1.80 µg/ml (closed diamonds) and 6.05 µg/ml (closed triangles), were used. The degrees of competency of the four strains measured as the number of tetracycline-resistant transformants DNA (closed box) were nearly identical. Standard deviation (SD) is indicated by vertical bars. View largeDownload slide Fig. 4. Size-dependent efficiency of cloning. The number of spectinomycin-resistant transformants is plotted against the size of the cloned segment. Two concentrations of Synechocystis genomic DNA, 1.80 µg/ml (closed diamonds) and 6.05 µg/ml (closed triangles), were used. The degrees of competency of the four strains measured as the number of tetracycline-resistant transformants DNA (closed box) were nearly identical. Standard deviation (SD) is indicated by vertical bars. View largeDownload slide Fig. 5. Genetic transformation within the new genome region. X indicates homologous recombination. Cognate genomic transformation was measured using BEST4110 DNA. Other symbols are the same as in Fig. 1. BEST7021 supplies only 10 kb of Synechocystis DNA of the left end instead of the sequence longer than 70 kb of the right. View largeDownload slide Fig. 5. Genetic transformation within the new genome region. X indicates homologous recombination. Cognate genomic transformation was measured using BEST4110 DNA. Other symbols are the same as in Fig. 1. BEST7021 supplies only 10 kb of Synechocystis DNA of the left end instead of the sequence longer than 70 kb of the right. Table 1. Bacterial strains and plasmids for indirect selection. Bacteria  Genotypes  Sources or references    Synechocystis sp. PCC6803    (Institut Pasteur, France)    BUSY1001  rnhB::spc  pBRSYNrnhBS1 × PCC6803    Bacillus subtilis (1A1)  trpC2  (BGSC, Ohio, USA)    RM125  argleu  (9)    BEST6016  proB::pBRTc  (13)    BEST7003  proB::pBRTc, yah::pr-neo  this study    BEST4110  leuB::cat  this study            LPA strains and recombinants  BEST7166  proB::pC[0878/0903]  CmR, SpcR, NmS  pC[0878/0903] × BEST7003  BEST7171  proB::pC[26 kb]  NmR, SpcS, CmR  PCC6803 × BEST7166  BEST7332  proB::pC[1691/1729]  CmR, SpcR, NmS  pC[1691/1729] × BEST7003  BEST7336  proB::pC[38 kb]  NmR, SpcS, CmR  PCC6803 × BEST7166  BEST8135  proB::pE[2553/2596]   EmR, SpcR, NmS  pE[2553/2596] × BEST7003  BEST8155  proB::pE[43 kb]  NmR, SpcS, EmR  PCC6803 × BEST8135  Plasmids  Construction or features  References    pBRSYNrnhB  rnhB (PCC6803)  gene amplified cloned in pBR322    pBRSYNrnhBS1  rnhB::spc  1.3kb/SmaI into pBRSYNrnhB/AatI    pBMAP105TT  leuB::tet  (13)            LPS clones isolated from PCC6803 and derivatives  LPS  Plasmid  Segment size (kb)  Region or vector  [0878]   pCR[0878–0882]¶  4.67  878,001 to 882,669  [0903]  pCR[0899–0903]¶  4.53  899,015 to 903,540  [1691]  pCR[1691–1696]¶  4.74  1,691,653 to 1,696,392  [1729]  pCR[1725–1729]¶  4.54  1,725,139 to 1,729,678  [2553]  pCR[2553–2558]¶  4.91  2,553,209 to 2,558,117  [2596]  pCR[2591–2596]¶  4.96  2,591,742 to 2,596,704  pC0878  pCR[0878–0882]/BamHI    pCISP310B/BamHI†  pC1691  pCR[1691–1696]/BamHI    pCISP310B/BamHI†  pE2553  pCR[2553–2558]/BamHI    pCISP311B/BamHI†          LPA plasmids for integration into BEST7003  pC[0878/0903]  pCR[0899–0903]/EcoRI  pC0878/EcoRI    pC[1691/1729]  pCR[1725–1729]/EcoRI  pC1691/EcoRI    pE[2553/2596]  pCR[2591–2596]/EcoRI  pE2553/EcoRI    Bacteria  Genotypes  Sources or references    Synechocystis sp. PCC6803    (Institut Pasteur, France)    BUSY1001  rnhB::spc  pBRSYNrnhBS1 × PCC6803    Bacillus subtilis (1A1)  trpC2  (BGSC, Ohio, USA)    RM125  argleu  (9)    BEST6016  proB::pBRTc  (13)    BEST7003  proB::pBRTc, yah::pr-neo  this study    BEST4110  leuB::cat  this study            LPA strains and recombinants  BEST7166  proB::pC[0878/0903]  CmR, SpcR, NmS  pC[0878/0903] × BEST7003  BEST7171  proB::pC[26 kb]  NmR, SpcS, CmR  PCC6803 × BEST7166  BEST7332  proB::pC[1691/1729]  CmR, SpcR, NmS  pC[1691/1729] × BEST7003  BEST7336  proB::pC[38 kb]  NmR, SpcS, CmR  PCC6803 × BEST7166  BEST8135  proB::pE[2553/2596]   EmR, SpcR, NmS  pE[2553/2596] × BEST7003  BEST8155  proB::pE[43 kb]  NmR, SpcS, EmR  PCC6803 × BEST8135  Plasmids  Construction or features  References    pBRSYNrnhB  rnhB (PCC6803)  gene amplified cloned in pBR322    pBRSYNrnhBS1  rnhB::spc  1.3kb/SmaI into pBRSYNrnhB/AatI    pBMAP105TT  leuB::tet  (13)            LPS clones isolated from PCC6803 and derivatives  LPS  Plasmid  Segment size (kb)  Region or vector  [0878]   pCR[0878–0882]¶  4.67  878,001 to 882,669  [0903]  pCR[0899–0903]¶  4.53  899,015 to 903,540  [1691]  pCR[1691–1696]¶  4.74  1,691,653 to 1,696,392  [1729]  pCR[1725–1729]¶  4.54  1,725,139 to 1,729,678  [2553]  pCR[2553–2558]¶  4.91  2,553,209 to 2,558,117  [2596]  pCR[2591–2596]¶  4.96  2,591,742 to 2,596,704  pC0878  pCR[0878–0882]/BamHI    pCISP310B/BamHI†  pC1691  pCR[1691–1696]/BamHI    pCISP310B/BamHI†  pE2553  pCR[2553–2558]/BamHI    pCISP311B/BamHI†          LPA plasmids for integration into BEST7003  pC[0878/0903]  pCR[0899–0903]/EcoRI  pC0878/EcoRI    pC[1691/1729]  pCR[1725–1729]/EcoRI  pC1691/EcoRI    pE[2553/2596]  pCR[2591–2596]/EcoRI  pE2553/EcoRI    Cm, chloramphenicol; Sp, spectinomycin; Nm, neomycin; Em, erythromycin; R, resistance; S, sensitive. ¶Cloned in pCR-XL-TOPO vector. †pCISP310B and pCISP311B are reported in Ref. 9. View Large Table 2. Bacterial strains and plasmids for direct selection. Bacteria  Genotypes  Antibiotic markers  Sources or references  BEST7004  proB::p[LPS1/Em/LPS2]  EmR  p[LPS1/Em/LPS2] × BEST6016  BEST7016  proB::pBR[16.5 kb (89554–115223)]  SpR, EmS  BUSY1001 × BEST7016  BEST7012  proB::p[LPS1/Em/LPS3]  EmR  p[LPS1/Em/LPS3] × BEST6016  BEST7017  proB::pBR[42kb (75564–115223)]   SpR, EmS  BUSY1001 × BEST7016  BEST7008  proB::p[LPS1/Em/LPS4]  EmR  p[LPS1/Em/LPS4] × BEST6016  BEST7018  proB::pBR[50 kb (61054–115223)]  SpR, EmS  BUSY1001 × BEST7008  BEST7015  proB::p[LPS1/Em/LPS5]  EmR  p[LPS1/Em/LPS5] × BEST6016  BEST7019  proB::pBR[90 kb (25603–115223)]  SpR, EmS  BUSY1001 × BEST7015  BEST7021  proB::pBR[90 kb (25603–115223)]  leuB::tet, SpS  see the text and Fig. 5  LPS clones isolated from PCC6803 and derivatives  Plasmid  Segment size (kb)  Region  Vector  [LPS1] pSYNrnhB2′  7.48  107755–115223  HindIII/pBR322  [LPS2] pCRNHBOT  4.18  89554–93735  pT7BlueT  [LPS3] pCRNHB-101  4.17  75564–79734  BamHI/pBR322  [LPS4] pSYN3-TO  5.01  61054–66059  pCR-XL-TOPO  [LPS5] pCRNHB-203  5.11  25603–30713  EcoRI/pBRcI′.  LPA plasmids  Donor  Vector    p[LPS1/LPS2]  pSYNrnhB2′/BamHI  pCRNHBOT/BamHI    p[LPS1/LPS3]  pSYNrnhB2′/BamHI  pCRNHB-101/BamHI    p[LPS1/LPS4]  pSYNrnhB2′/BamHI  pSYN3-TO/BamHI    p[LPS1/LPS5]  pSYNrnhB2′/BamHI  pCRNHB-203/BamHI    LPA plasmids for integration into BEST6016§  p[LPS1/Em/LPS2]  p[LPS1/LPS2]/EcoRV      p[LPS1/Em/LPS3]  p[LPS1/LPS3]/EcoRV      p[LPS1/Em/LPS4]  p[LPS1/LPS4]/EcoRV      p[LPS1/Em/LPS5]  p[LPS1/LPS5]/NheI-T4DNApolymerase      Bacteria  Genotypes  Antibiotic markers  Sources or references  BEST7004  proB::p[LPS1/Em/LPS2]  EmR  p[LPS1/Em/LPS2] × BEST6016  BEST7016  proB::pBR[16.5 kb (89554–115223)]  SpR, EmS  BUSY1001 × BEST7016  BEST7012  proB::p[LPS1/Em/LPS3]  EmR  p[LPS1/Em/LPS3] × BEST6016  BEST7017  proB::pBR[42kb (75564–115223)]   SpR, EmS  BUSY1001 × BEST7016  BEST7008  proB::p[LPS1/Em/LPS4]  EmR  p[LPS1/Em/LPS4] × BEST6016  BEST7018  proB::pBR[50 kb (61054–115223)]  SpR, EmS  BUSY1001 × BEST7008  BEST7015  proB::p[LPS1/Em/LPS5]  EmR  p[LPS1/Em/LPS5] × BEST6016  BEST7019  proB::pBR[90 kb (25603–115223)]  SpR, EmS  BUSY1001 × BEST7015  BEST7021  proB::pBR[90 kb (25603–115223)]  leuB::tet, SpS  see the text and Fig. 5  LPS clones isolated from PCC6803 and derivatives  Plasmid  Segment size (kb)  Region  Vector  [LPS1] pSYNrnhB2′  7.48  107755–115223  HindIII/pBR322  [LPS2] pCRNHBOT  4.18  89554–93735  pT7BlueT  [LPS3] pCRNHB-101  4.17  75564–79734  BamHI/pBR322  [LPS4] pSYN3-TO  5.01  61054–66059  pCR-XL-TOPO  [LPS5] pCRNHB-203  5.11  25603–30713  EcoRI/pBRcI′.  LPA plasmids  Donor  Vector    p[LPS1/LPS2]  pSYNrnhB2′/BamHI  pCRNHBOT/BamHI    p[LPS1/LPS3]  pSYNrnhB2′/BamHI  pCRNHB-101/BamHI    p[LPS1/LPS4]  pSYNrnhB2′/BamHI  pSYN3-TO/BamHI    p[LPS1/LPS5]  pSYNrnhB2′/BamHI  pCRNHB-203/BamHI    LPA plasmids for integration into BEST6016§  p[LPS1/Em/LPS2]  p[LPS1/LPS2]/EcoRV      p[LPS1/Em/LPS3]  p[LPS1/LPS3]/EcoRV      p[LPS1/Em/LPS4]  p[LPS1/LPS4]/EcoRV      p[LPS1/Em/LPS5]  p[LPS1/LPS5]/NheI-T4DNApolymerase      †http://www.kazusa.or.jp/cyano/.§The erm cassette prepared from pBEST701 (9) with SmaI was inserted into the site indicated between the two LPSs. A 1.3-kb fragment containing the rnhB gene was obtained by PCR-mediated amplification from genomic DNA of BUSY101 using primers rnhB, 5′-CCTAATCAAAACGGAGTACG-3′ and 5′-AATTGCCATTGAAGTGGCGG-3′. The primer sets used for amplification were: [LPS5] pCRNHB-203: SYNrnhB-20F 5′-GGGACCAAGTACAACAACTC-3′ and SYNrnhB-20R 5′-GGGGGAGGAAATTATCAGCG-3′; [LPS4] pSYN3-TO: SYN3F 5′-GCCGAATTCCGGGCTGGATCCCCT-3′ and SYN3R 5′-TATGGATCCTAACAACGACTTCCCC-3′; [LPS3] pCRNHB-101: SYNrnhB1F 5′-GGAGGAAGTCTGTTGCTCGG-3′ and SYNrnhB1R 5′-GTTGGGGTTTACAGGCGGTG-3′; [LPS2]: CRNHBOF 5′-TCAGGATCCGCCACCTTTTGACCGTTG-3′ and CRNHBOR 5′-TCAGGATCCCGGTCCAGAAGCATGG-3′; [LPS1]: SYNrnhB2F 5′-GGGGTTAAACGAATGACAGCGG-3′ and SYNrnhB2R 5′-AGGGACTGGAGGGGACAATG-3′. View Large References 1. Sambrook, J., Fritsch, E.F., and Maniatis, T. ( 1989) Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Google Scholar 2. Cutting, S. and Horn, P.B.V. 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Journal

The Journal of BiochemistryOxford University Press

Published: Oct 1, 2003

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