Supplementary Information for JB00834-04 Version 2 Nature of the Promoter Activated by C•PvuII, an Unusual Regulatory Protein Conserved among Restriction-Modification Systems Dieter Knowle, Robert E. Lintner, Yara M. Touma, and Robert M. Blumenthal* Department of Microbiology & Immunology and Program in Bioinformatics & Proteomics/Genomics Medical College of Ohio, Toledo, Ohio 43614-5806 *Corresponding author. Mailing address: Department of Microbiology & Immunology, Medical College of Ohio, 3055 Arlington Ave., Toledo, OH 43614-5806. Phone: (419) 383-5422. Fax: (419) 383-3002. E-mail: email@example.com. Promoter clones and their derivatives. Sources of C.PvuII or C.PvuIIEsp19. pDK74 and pDK80 were generated by excising the intact PvuII restriction-modification system from pPvuRM3.4, or its derivative containing a pvuIIC Esp19 null allele (2), using EcoRV and EcoRI and ligating into the ScaI and EcoRI sites of pACYC184. Derivatives were made defective for pvuIIM, by removing an SpeI-SpeI fragment, and pvuIIR, by excising a BaeI-EcoRI segment. The resulting plasmids were named pDK200 (pvuIIC+) and pDK201 (pvuIICEsp19). pvuIICR promoter plasmids. The longest promoter clones extended from –93 to +88 relative to the start codon of pvuIIC (see Fig. 1). The –93 position corresponds to a native SpeI site (ACTAGT) at nt 1341-1346 in the pPvu1 sequence (accession # gi11995221), while +88 is a native HindIII site (nt 1156-1161 in pPvu1). To construct plasmid pDK2, oligonucleotides DK1 and DK2 (Table S1) were hybridized, extended with Klenow DNA polymerase, cleaved at the ends with BamHI and HindIII, and ligated into BamHI- and HindIII-cut pKK232-8. Next, the region from -27 to +88 was PCR-amplified from pPvuRM3.4 using primers DK3 and DK4 (Table S1) and Taq DNA polymerase, digested with HindIII, and ligated into HindIII-cut pDK2 yielding pDK58. Both pDK2 and pDK58 were verified by DNA sequencing using primer DK5. pBH403 and derivatives. The lacZ gene was amplified as a promoterless 3107-bp fragment from E. coli W3110 chromosomal DNA, using primers B14 and B15 (Table S1) and PFU turbo DNA polymerase (Stratagene), and given TA-cloning overhangs with Taq polymerase. The PCR product was gel purified, subcloned into the TOPO TA 2.1 vector and excised as a HindIII fragment that was ligated into HindIII-cut pKK232-8. The promoterless lacZ and cat genes are transcribed in the same direction, with lacZ upstream of cat. The pvuIICR promoter (-59 to +88 relative to the initiation codon of pvuIIC) was amplified from pPvuRM3.4 using primers DK12 and DK13 and Taq polymerase. The PCR product was digested with SalI and ligated into SalI-digested pBH403. Similarly, the pvuIICR promoter (-93 to +88) was amplified using primers DK14 and DK15, digested with BamHI and SalI and ligated into BamHI- and SalI-digested pBH403. pvuIICR 3' deletions. The pvuIICR promoter region (-93 to +88 relative to the initiation codon of pvuIIC) was amplified from pDK58 using primers DK16 and DK13, digested with BamHI and SalI, and ligated into BamHI- and HindIII-cut pKK232-8, yielding pDK148. The insertion was sequence confirmed using primer DK5. Downstream of +88, BglII, Sac I and SalI sites were incorporated via the PCR primer. The Erase-a-Base system (Promega) was utilized to make nested 3' deletions from the BglII site after pDK148 was cut with BglII and SacI (the 3' overhang from SacI is resistant to exonuclease III digestion). The manufacturer's instructions were followed. Transformants were screened by colony PCR (1) using primers DK5 and DK16. The PCR products were resolved on a 6% polyacrylmide gel, and plasmid DNA was isolated from those colonies yielding PCR products smaller than the WT. These plasmids were sequenced using primer DK5. The resulting plasmids had a fixed upstream pvuIICR promoter end (-93), with downstream ends at +88 (pDK153), +68 (pDK155), +38 (pDK157), +23 (pDK150), +19 (pDK154), +18 (pDK152), -2 (pDK151) and -24 (pDK156), relative to the initiation codon of pvuIIC. Plasmids with shorter segments were prepared by PCR using primer DK39 and one of the primers DK40-43 (Table S1); the resulting plasmids had 1 (pDK525), 10 (pDK526), 16 (pDK527) or 24 (pDK528) bp downstream of the C boxes, corresponding to 3’ endpoints at –24, –15, –9, and –1 relative to the initiation codon of pvuIIC. Replacements in region downstream of C boxes. Oligonucleotides DK18 and DK19 were mixed, yielding a dsDNA fragment with 4-nt extensions at each end respectively compatible with cleaved SalI and HindIII sites. This fragment was ligated into SalI- and HindIII-cut pDK156, yielding plasmid pDK178 with a –41 to +1 insertion. This strategy was used with additional oligonucleotide pairs DK20 – 25 having replacements in the DSR sequence to produce the 5'- (pDK179), 3'- (pDK180) and 5+3'- (pDK181) substituted promoters. C box / –10 hexamer spacing variants. Self-complementary oligonucleotide DK26 was annealed, yielding dsDNA with 4-nt SalI-compatible extensions. This DNA was ligated into SalI-cut pDK151, resulting in a net insertion of 10 bp just downstream of the C boxes (pDK227). This strategy was also used, with pairs of complementary oligonucleotides DK27/28 and DK29/30, to produce the +15 and +20 spacing mutants (pDK228 and pDK229). The -4 spacing mutant was made by digesting pDK151 with SalI and treating with S1 nuclease before religation. The +4 spacing mutant was made by end-filling the SalI-cut pDK151with Klenow polymerase before religation. All spacing variants were sequence confirmed. Methyltransferase promoter plasmid. Oligonucleotides DK33 and DK34 were hybridized, yielding a duplex -61 to +5 relative to the start of transcription for pvuIIM promoter M82 (3). This was digested with BamHI and HindIII and ligated into BamHI- and HindIII-cut pKK232-8. The pvuIIM promoter together with the region between the C boxes and pvuIIC initiation codon (-61 to +46 relative to pM82) was PCR amplified from pPvuRM3.4 using primers DK3 and DK4, digested with HindIII, and ligated into HindIII-cut pKK232-8. The resulting clones were analyzed using primer DK5 to verify their sequence and orientation relative to cat. TABLE S1. Oligonucleotides used in this study a B14 TCCCCCCGGGAAGCTTAAATAAATAAAACACAGGAAACAGCTATGACCATGATTACG B15 TCCCCCCGGGAAGCTTTTATTTTTGACACCAGACCAACTGGTAATGG DK1 TGGGGATCCTTGTGTAGGCAGGTTTTTTTCCAAAATTCAACATATCATTGCGACT CATAGTCTGTAGACTCAAAGTCAAAGCTTGGGGG DK2 CCCCCAAGCTTTGA DK3 CCCCCAAGCTTCATCATTATCCCGTCTATGAGCAGAA DK4 CCCCCAAGCTTTCTTGGGATAGACCTAGCTCGC DK5 CAACGGTGGTATATCCAGT DK6 TGGGGATCCTTGTGTAGGCAGGTTTTTTTCCAAAATTCAACATATCATTGCGACT CATCTGATGTAGACTCAAAGTCAAAGCTTGGGGG DK7 TGGGGATCCTTGTGTAGGCAGGTTTTTTTCCAAAATTCAACATATCATTGCGACT CATAGTCTGTAGACTCAACTGAAAAGCTTGGGGG DK8 TGGGGATCCTTGTGTAGGCAGGTTTTTTTCCAAAATTCAACATATCATTGCGACT CATCTGATGTAGACTCAACTGAAAAGCTTGGGGG DK9 TGGGGATCCTTGTGTAGGCAGGTTTTTTTCCAAAATTCAACATATCATTGCGACT CATATTCTGTAGACTCAAAGTCAAAGCTTGGGGG DK10 TGGGGATCCTTGTGTAGGCAGGTTTTTTTCCAAAATTCAACATATCATTGCGACT CATAGTCTGTAGACTCAAATTCAAAGCTTGGGGG DK11 TGGGGATCCTTGTGTAGGCAGGTTTTTTTCCAAAATTCAACATATCATTGCGACT CATATTCTGTAGACTCAAATTCAAAGCTTGGGGG DK12 ACGCTGTCGACATCATTGCTACTCATAGT DK13 ACGCGTCGACGAGCTCAGATCTTCTTGGGATAGACCTAGCTCG DK14 CGCGGATCCACTAGTTGTGTAGGCAGGTTTT DK15 ACGCGTCGACGTCTCAGCTAAGCTTTCTTGGGATAGA DK16 CGCGGATCCTTGTGTAGGCAGGTTTTTTTC DK17 AACAGCAACTGATGGAAAC DK18 TCGACCCATCATTATCCCGTCTC DK19 AGCTGAGACGGGATAATGATGGG DK20 TCGACCGGGCCCTATCCCGTCTC DK21 AGCTGAGACGGGATAGGGCCCGG DK22 TCGACCCATCATCCCGGGGTCTC DK23 AGCTGAGACCCCGGGATGATGGG DK24 TCGACCTTGCGGCCGCTTGTCTC DK25 AGCTGAGACAAGCGGCCGCAAGG DK26 TCGAACGCGT DK27 TCGAACTAGTACGCG DK28 TCGACGCGTACTAGT DK29 TCGACACTAGTACGCGTAGA DK30 TCGATCTACGCGTACTAGTG DK31 GGGGTCGACCNNNNNNNNNCCCGCGAAGCTTTTT (N= mix of A, C, G, and T) DK32 AAAAAGCTTCGCGGG DK33 GGGCGGATCCTAAGCTCTCTTGGGATAGACCTAGCTCGCCTCGCATTTTTTTTA CATTCTTTGCAAGGGTTAATCGGGCAAGCTTTTTT DK34 AAAAAAGCTTGCCCGATTAACCCTTGCAAAGAATGTAAAAAAAATGCGAGGCG AGCTAGGTCTATCCCAAGAGAGCTTAGGATCCGCCC DK35 CATTCTTTGCAAGGGTTAATCGGGCT DK36 AGACCTAGCT DK37 GGCTGATAAAGGATTTGTATTTCTGCTCATAGACGGGATAATGATGGTATGATGAC TTTGAATCTACAGACTATGAG DK38 GGGATAATGATGGTATGATGACTTTGAATCTACAGACTATGAGTAGCAATG DK39 CGCGGATCCACTAGTTGTGTAGGCAGG DK40 ACGCGTCGACTGACTTTGAATCTACAGAC DK41 ACGCGTCGACATGGTATGATGACTTTGAATC DK42 ACGCGTCGACATAATGATGGTATGATGAC DK43 ACGCGTCGACTAGACGGGATAATGATGGTATG RV1 TCGGGCTGATAAAGGATTT From (3) a All are shown 5’ 3’ REFERENCES 1. Dallas-Yang, Q., G. Jiang, and F. M. Sladek. 1998. Avoiding false positives in colony PCR. Biotechniques 24:580-2. 2. Tao, T., J. C. Bourne, and R. M. Blumenthal. 1991. A family of regulatory genes associated with type II restriction- modification systems. J Bacteriol 173:1367-75. 3. Vijesurier, R. M., L. Carlock, R. M. Blumenthal, and J. C. Dunbar. 2000. Role and mechanism of action of C.PvuII, a regulatory protein conserved among restriction- modification systems. J Bacteriol 182:477-487.
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