S-1 Nuclease Protection Analysis

S-l nuclease protection analysis was performed essentially as described by Mullet et al.［16］.The source of plasmid DNA for purification of restriction fragments was a series of specific subclones of plasmid pJWl［15］in the plasmid pUC19.［1］Restriction fragments were eluted from 5%polyacrylamide gels and purified by chromatography over DE52 prior to ethanol precipitation［17］.Individual restriction fragments were dephosphorylated with bacterial alkaline phosphatase prior to 5'-end labeling with［γ-32 P］ATP and T4 polynucleotide kinase［17］.The［32P］DNA was denatured，and single complementary strands were separated on 5%polyacrylamide gels［17］.For hybridization，the purified 5'-end-labeled single-stranded［32P］DNA（5，000—20，000 cpm/assay）and carrier yeast tRNA（10 μg/assay）were resuspended in 80%formamide（10μl/assay），boiled for 5 min，and then added to a dry pellet containing R.sphaeroides RNA（50 μg/assay）and S-l hybrdization buffer to give a final concentration of 400 mmol/L NaCl，40 mmol/L Pipes-NaOH，pH 6.4，1 mmol/L EDTA.Hybridization was performed at 37℃for 4 h.The sample was then diluted 1∶10 with a mixture containing 250 mmol/L NaCl，30 mmol/L NaOAc（pH 4.6），1 mmol/L ZnSO4，20 μg/ml denatured salmon sperm DNA，and 25 units of S-l nuclease were added.The mixture was incubated at 30℃for 40 min.After phenol chloroform extraction，the S-l-protected nucleic acids were analyzed on either a 5%polyacrylamide gel or a 6%polyacrylamide，8.3 mol/L urea DNA-sequencing gel relative to Hinfl-or AluI-generated pBR322 molecular weight standards［17］.For precise mapping of 5'-ends，DNA sequence ladders were generated by Maxam and Gilbert sequencing of the appropriate end-labeled restriction fragment［17］.(https://www.daowen.com)