Nucleotide sequence of thePaeR7 restriction/modification system and partial characterization of its protein products

Abstract

Bal31 deletion experiments on clones of the PaeR7 restriction-modification system from Pseudomonas aeruginosa demonstrate that it is arranged as an operon, with the methylase gene preceding the endonuclease gene. The DNA sequence of this operon agrees with in vitro transcription-translation assays which predict proteins of 532 amino acids, Mr = 59,260 daltons, and 246 amino acids, Mr = 27,280 daltons, coincident with the methylase and endonuclease genes, respectively. These predicted values coincide with the measured molecular weights of the purified, denatured PaeR7 endonuclease and methylase proteins. The first twenty amino acids from the amino-terminus of the purified endonuclease exactly match those predicted from the DNA sequence. Finally, potential regulatory mechanisms for the expression of phage restriction are described based on the properties of several PaeR7 subclones.

Cloning and Nucleotide Sequence ofApaLI Restriction-modification System fromAcetobacter pasteurianusIFO 13753†

“…14) The two genes in the Apa LI restriction-modification system are in the same orientation with the methylase gene preceding, and separated by two bp. This arrangement is similar to those of the NgoMJ15) and PaeR7I 16 ) systems but separated by three bp. The Accl system was separated by two bp but in a reverse order on two genes.…”

Regulation of the BamHI restriction-modification system by a small intergenic open reading frame, bamHIC, in both Escherichia coli and Bacillus subtilis

“…In crude extracts and during methylase purification, aliquots were assayed by using ligated BamHI linkers [d(pCGGATCCG)] as described previously (8). Methylase units were quantified by using a protection assay (35) in which 1 ,g of treated A DNA was challenged with 25 U of R.BamHI for 20 min. One unit of M.BamHI is defined as the amount of enzyme which, in a 50-,ul reaction volume after 1 h at 37°C, is sufficient to completely protect 1 ,ug of A DNA (containing five BamHI sites) against R.BamHI cleavage (8).…”

nfi, the gene for endonuclease V in Escherichia coli K-12

“…Of the proteins of known sequence, the one to which endonuclease V has the greatest similarity is an N 6 adenine-specific DNA methyl transferase encoded by a plasmid of Pseudomonas aeruginosa (35). The aligned sequences displayed 28% identity and 41% similarity over a length of 111 amino acids.…”