Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids

Abstract

Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated ‘reference plasmids’), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these ‘reference plasmids’ revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).

Methylome Diversification through Changes in DNA Methyltransferase Sequence Specificity

“…However, this method cannot be applied to Type I systems because the corresponding restriction enzyme complex cleaves DNA at unpredictable positions outside the recognition sequence [12] . Their recognition sequences have been determined by transfer of labeled methyl groups by the methyltransferase [30] , [31] and transformation by plasmids with or without their candidates [32] .…”

Genetic Transformation of Geobacillus kaustophilus HTA426 by Conjugative Transfer of Host-Mimicking Plasmids

“…Culturing at 45 o C for 24 h had no effect on the N6m A ratios. The exact sites methylated by the type I R-M systems could not be determined because of the absence of a facile analytical method for type I methylation sites [2,11,16,23]. However, the methylation site obviously differed from the site methylated by GKP08, because the chromosome from E. coli IR27 harboring pIR408 was sensitive to DpnII and resistant to DpnI, similar to the chromosome from strain IR27 (Fig.…”

Host-Mimicking Strategies in DNA Methylation for Improved Bacterial Transformation

“…The sequences of recognition sites are asymmetric but not palindromic. Examples include 5′-AACN 6 GTGC-3′ and 5′-GCACN 6 GTT-3′ for EcoKI, 5′-TGAN 8 TGCT-3′ and 5′-AG-CAN 8 TCA-3′ for EcoBI, and 5′-TTAN 7 GTCY-3′ and 5′-RGACN 7 TAA-3′ for EcoDI (methylated adenine is underlined; R: A/G, Y: C/T, N: A/C/G/T) [29,30].…”

“…Recent epigenetic studies have developed many methods to analyze DNA methylation [29,30,[69][70][71][72][73][74][75][76]. Although most of these studies aimed to analyze 5-methylation of cytosine at spe-cific sites, or differential DNA methylation in chromosomes, there are a few methods that exhaustively determine methylated consensus sites in chromosomes as follows.…”