Journal: Genes & Development
Article Title: Long palindromes formed in Streptomyces by nonrecombinational intra-strand annealing
Figure Lengend Snippet: Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.
Article Snippet: Aliquots of DNA were incubated with 100 units of E. coli exonuclease III or 10 units of bacteriophage λ exonuclease (either purchased from Life Technologies, Inc. or a gift of Drs. Deb Chatterjee and Per Harbury) at 37°C for 1 hr and the completeness of their digestion was confirmed by gel electrophoresis.
Techniques: Southern Blot, Derivative Assay, Isolation, Agarose Gel Electrophoresis, Electrophoresis, Migration, Neutralization