Product info: Glu I


Name
Glu I
Cat. #E519E520
Package, u.a.50250
Concentration, u.a./ml10001000

Recognition site
G(5mC)↑NG(5mC)
(5mC)GN↓(5mC)G
SourceGlacial ice bacterium GL24
Substrate specificityThe enzyme cleaves C5-methylated DNA and does not cut unmodified DNA [1].

Optimal recognition site (100% activity ):
5'-G(5mC)↑NG(5mC)-3'/3'-(5mC)GN↓(5mC)G-5`
Assayed onDNA pFsp4HI3/DriI is a linearized plasmid pFsp4HI3, which carries a gene of DNA-methyltransferase M.Fsp4HI and includes a unique GluI site: 5`-G(5mC)NG(5mC)-3`/3`-(5mC)GN(5mC)G-5` [2].
Unit definitionOne unit is defined as the amount of enzyme
required to hydrolyze completely a unique site:
5`-G(5mC)NG(5mC)-3`/3`-(5mC)GN(5mC)G-5`
in 1 μg of linearized plasmid pFsp4HI3/DriI in 1 hour at 37°C in a total reaction volume of 50 μl. As a result a linearized plasmid pFsp4HI3 disappears (see run 4 in the figure). GluI digestion of recognition sequences with three 5-methylcytosines results in additional bands appearance (runs 4-6 in the figure).
GluI activity assay on DNA pFsp4HI3/DriI

Lanes:
1 and 6 1 Kb SE DNA-markers.
2 Control DNA pFsp4HI3/DriI,
3 0.5 μl Glu I
4 1 μl Glu I
5 2 μl Glu I

Products were separated in 1,4% agarose gel in Buffer TAE.
GluI activity assay on DNA pFsp4HI3/DriI
Optimal SE-bufferY (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.)
Enzyme activity (%)
BGOWYRose
75 - 10075 - 10025 - 5050 - 75100100
Reaction bufferSE-buffer Y, (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.)
Optimal temperature
37oC
Storage conditions10 mM KH2PO4 (pH 7.45); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg/ml BSA, 50% glycerol; Store at -20°C.
Non-specific hydrolisisNo detectable degradation of 1μg of Lambda DNA was observed after incubation with 1 units of enzyme for 16 hours at 37°C in a total reaction volume of 50 μl.
Reagents Supplied with Enzyme 10 X SE-buffer Y
Methylation sensitivityThe enzyme cleaves C5-methylated DNA and does not cut unmodified DNA [1].
Inactivation 20 minutes under
80oC
NotesWhen using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
MSDS:Download MSDS as PDF
References:1. Chernukhin V.A., Chmuzh E.V., Tomilova J.E., Nayakshina T.N., Dedkov V.S., Degtyarev S.Kh. Bacterial strain Glacial ice bacterium - producer of GluI site specific endonuclease. // Russian Federation patent RU 2322492 C1 (2006).
2.
 V.A. Chernukhin, E.V. Chmuzh, Yu.E. Tomilova, T.N. Nayakshina, D.A. Gonchar, V.S. Dedkov, S.Kh. Degtyarev A novel site-specific endonuclease GluI recognizes methylated DNA sequence 5-G(5mC)^NG(5mC)-3/3-(5mC)GN^(5mC)G. // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.3, No 2, pp 13-17, 2007

Application:
 Chernukhin V.A, Abdurashitov M.A., Tomilov V.N., Gonchar D.A., Degtyarev S.Kh. Comparative analysis of mouse chromosomal DNA digestion with restriction endonucleases in vitro and in silico // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.3, No 4, pp 19-27, 2007
Kravets AP, Mousseau TA, Litvinchuk AV, Ostermiller Sh, Vengen GS, Grodzinski DM. Changes in wheat DNA methylation pattern after chronic seed gamma-irradiation.// Tsitol Genet. 2010 Sep-Oct;44(5):18-22. Russian.