Research work performed during this study was aimed to discover new type II restriction endonucleases (Reases) and clone interesting restriction-modification (R-M) system (s) into Escherichia coli to study the expression and primary structure of restricting and modifying proteins.
During this study thirty bacterial strains, collected from different ecological environments of Pakistan, were screened for the presence of type II specificity REase. Protein extracts from eleven strains were found positive for the presence of endonucleolytic activity.
Eleven enzymes wee highly purified by a combination of gel filtration, ion-exchange and affinity chromatography. Purified enzymes were characterized for their target recognition sequence and cleavage site. Ten enzymes were proved isoschizomers of already known specificities. One enzyme BseRI isolated from Bacillus species R (CAMB 2669) was recognized as a novel specificity (Mushtaq et al., 1993). Type IIS, BseRI was characterized to recognize the GAGGAG asymmetric sequence and found to cleave double stranded DNA ten nucleotides beyond this sequence on the top strand and eight nucleotides beyond the sequence on the complimentary strand to produce a two nucleotide 3’ extension.
In addition, reaction conditions for pH optima, temperature and requirements of mono and divalent cations were also established for the isolated enzymes.
Two R-M systems Ec/X1, isoschizomer of Xmal/Cfr91/XcyI, and SauMI isoschizomer of Sau3AI were selected for detailed study. Ec/XI R-M system was cloned in pUC19 vector (shot gun cloning). Positive clones were selected on the basis of methytransferase (MTase) gene expression, resulted self modifying plasmids which became resistant against digestion with cognate REase Ec/XI (Lunnen et al., 1988). Two clones carrying the MTase gene were also examined for the presence of REase gene. This was done because REase and MTase genes are usually linked and more than 100 cloned R-M systems have restricting and modifying activities lying next to one another (Wilson, 1991). Approximate location of R-M genes was determined by restriction enzyme mapping of the clone pRM-1.1, carrying a 3.5kb insert. In addition, mutants of pRM-1.1 were partially sequenced. Nucleotide (nt.) sequence was transcribed into three reading frames and a search made for the conserved motifs, characteristic of N4-methyl cytosines (Klimasauskas et al., 1989, 1990, 1991). There was presence of TSPPYWGLRD and KPISAFGSGDVQI sequence motifs in the ex/XIM gene and SGCG-HIPHH sequence motif in the ex/XIR gene. Such a sequence SGTDVSNIHN for XcyI and SGNT-TNIGS for EcoRV REases has also been reported by Withers et a. (1992).
Bacterial DNA MTases that catalyze the formation of m5C.posses related structure. Similar motifs containing invariant position, are found in the same order in almost all the known MTase sequences (Posfai et al., 1989). On the basis of the above information it was hypothesized that m5C generating sauMIM gene should also hae the highly conserved amino acid sequences. Using the thirteen m5C gene sequence reported by Posfai et al. (1989) a strategy was devised to amplify unknown m5C sauMIM gene. Keeping in view the conserved motif PCQ (motif IV) and FNV (motif VIII) a set of degenerate oligonucleotide primers was designed. The amplified PCR prduct was cloned into the M13 mp 19 vector and DNA sequence determined. 238 nt. Sequence was transcribed and compared to the protein sequences of already studied MTases. The cloned sauMIM gene fragment was characterized to contain the PCQ (motif IV) and FNV (motif VIII) motifs along with the ENV (motif VI) motif existing in-between them.