|ชื่อเรื่อง||:||การจำแนกและการทำบริสุทธิ์ของ Restriction Endonucleases จาก Streptomyces|
|คำค้น||:||DNA cleavage mechanism. , nicking endonuclease , Streptomyces , Type Iis restriction endonucleases|
|อ้างอิง||:||http://elibrary.trf.or.th/project_content.asp?PJID=PDF4480041 , http://research.trf.or.th/node/2331|
Forty strains of unidentified Streptomyces isolated from soil sample collected in a deciduous dipterocarpe forest in Thailand were screened for novel restriction endonuclease. Four of unidentified strain of Streptomyces , designated as DC1 , DC12 , DC13 and DC81 , were detected to contain specific endonucleases. In search of novel restriction endonuclease , specific endonucleases from Streptomyces DC12 and DC13 were partially purified and characterized. Two different restriction endonucleases had been purified from 200 grams of Streptomyces DC12. Purification of these endonucleases involved removal of nucleic acids by addition of streptomycin sulfate , ammonium sulfate fractionation, DEAE-cellulose and CM – cellulose chromatography. With DEAE-cellulose chromatography, the enzymes resolved into two active fractions, designated as SdcAI and SdcAII, respectively. The specific activities of the purified SdacAI and SdcAII from Streptomyces DC12 were 50 and 190 units/mg, respectively. Similarly , two different restriction endonucleases had purified from Streptomyces DC13. When loaded onto DEAE-cellulose, the restriction endonucleases also resolved into two active fractions , designated as SdcBI and SdcBII, respectively. The specific activities of the purified SdcAI and SdcAII from Streptomyces DC13 were 267 and 288 units/mg, respectively. Analysis of these purified proteins on SDS-PAGE showed that the enzymes still contaminated with other Substrate specified and kinetic cleavage of plasmid by SdAI , SdcAII , SdcBI , and SdcBII were characterized. All these enzymes required Mg2+ for catalytic activity with the optimum temperature at 37? C and cleaved super-colled pBluescript, pUC118 , pET-15b and pET-26b only once. However ,unlike type it restriction enzyme EcoRI, cleavage of these super-colled plasmlds resulted in formation of mostly cirular product rather than linear product. When linear pBluescript was used as substrate for SdcAI and SdcBI, no cleavage activity was observed. This result suggested two possibilities. First, the cleavage sites of SdcAI and SdcBI are located in close proximity to the cleavage site of EcoRI. Second SdcAI and SdcBI nicked the linear pBluescript that cannot be resolved from the non-nicked linear pBluescript on agarose gel. A computer-aided search of homologous nucleotide sequences of all four plasmids revealed at least four possible nucleotide sequences with 6 bp in length, which located either inside or within the origin of replication of the plasmid. PCR generated 500 bp, 1000 bp, and 1500 bp fragments that contained these possible sequences were used as substrates for SdcAI and SdcBI. The result suggested 500 bp, 1000 bp , and 1,500 bp PCR fragments were nicked instead of cleaved on both strand by these enzymes. The exact cleavage sites of SdcAI , SdcAII , SdcBI and SdcBII are remained to be determined. Kinetic cleavage of pBluescript by SdcAI , SdcAII , SdcBI , and SdcBII was compared to that of EcoRI, a type II restrication endonuclease. With EcoRI , cleavage of super-colled pBluescript generated mostly linear product, with transient accumulation of cicular product at state. Such cleavage pattern is consistent with a concerted mechanism. With SdcAI , SdcAII , SdcBI . and SdcBII , cleavage of super-colled pBluescript produced mostly circular product, with minor linear product. This data indicated that these enzymes rapidly nicked one strand of super-coiled DNA and converted it into the open circular form, which was subsequently converted to linear form at slower rate. Such DNA cleavage pattern is consistent with a step-wise mechanism that had been proposed for the type Iis nicking restriction endonuclease, N.BstNBI. It appears proposed that SdcAI , SdcAII , SdcBi and SdcBII are novel type Iis nicking restriction endonucleases that had never been isolated before until this study. It is proposed that the nicking endonucleases purified in this study also existed in solution as monomer and bind to their recognition site as monomer. Unlike typical type II restriction endonucleases such as EcoRI, the nicking endonucleases purified in this study cannot dimierize due to mutation in their dimerization domains. Thus , cleavage of the super-colled plasmid resulted in the formation of mostly circular product rather than linear product.
สมชัย พรบันลือลาภ . (2554). การจำแนกและการทำบริสุทธิ์ของ Restriction Endonucleases จาก Streptomyces.
กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย.
สมชัย พรบันลือลาภ . 2554. "การจำแนกและการทำบริสุทธิ์ของ Restriction Endonucleases จาก Streptomyces".
กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย.
สมชัย พรบันลือลาภ . "การจำแนกและการทำบริสุทธิ์ของ Restriction Endonucleases จาก Streptomyces."
กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย, 2554. Print.
สมชัย พรบันลือลาภ . การจำแนกและการทำบริสุทธิ์ของ Restriction Endonucleases จาก Streptomyces. กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย; 2554.