| Name | | Gla I |  |
| | Cat. # | E493 | E494 | | Package, u.a. | 100 | 500 | | Concentration, u.a./ml | 8000 | 8000 |
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| Recognition site | Pu(5mC)↑GPy
PyG↓(5mC)Pu
| | Source | Glacial ice bacterium GL29 | | Substrate specifity | The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA
and DNA with N4-methylcytosines [1].
The enzyme activity depends on number and position of methylated nucleotides in the recognition sequence [3]:
Optimal substrate (100% activity )
5`-G(5mC)G(mC)-3`/3`-(5mC)G(5mC)G-5`
Good substrates ( > 25% activity)
5`-R(5mC)G(5mC)-3`/3`-YG(5mC)G-5`
5`-A(5mC)GT-3`/3`-TG(5mC)A-5`
Medium substrates ( > 6% activity)
5`-G(5mC)R(5mC)-3`/3`-(5mC)GYG-5`
5`-G(5mC)GT-3`/3`-CG(5mC)A-5`
Bad substrates (6% activity)
5`-G(5mC)GC-3`/3`-CG(5mC)G-5` | | Assayed on | DNA pHspAI2/GsaI is a linearized plasmid pHspAI2, which carries a gene of DNA-methyltransferase M.HspAI (recognition sequence 5`-GCGC-3`) and includes a unique GlaI recognition site
5`-G(5mC)G(5mC)-3`/3`-(5mC)G(5mC)G-5` [2].
| | Unit | One unit is defined as the amount of enzyme
required to hydrolyze completely a unique
5`-G(5mC)G(5mC)-3`/3`-(5mC)G(5mC)G-5`
site in 1 μg of pHspAI2 plasmid DNA, which is linearized with GsaI, in 1 hour at 30°C in a total reaction volume of 50 μl. As a result of this site hydrolysis two DNA fragments are produced (see run 3 in the figure). GlaI digestion of recognition sequences with three and two 5-methylcytosines results in additional bands appearance (runs 4-6 in the figure).
| GlaI activity assay on DNA pHspAI2/GsaI
Lanes:
2 – Control DNA pHspAI2/GsaI,
3 – 0.5 μl Gla I (1/10)
4 – 1 μl Gla I (1/10)
5 – 2 μl Gla I (1/10)
6 – 1 μl of undiluted GlaI
1 and 7 – 1 Kb SE DNA-markers.
Products were separated in 1% agarose gel in Buffer TAE. |
 | | Reaction buffer | SE-buffer GlaI, (10 mM Tris-HCl (pH 8,5 at 25°C); 5 mM MgCl2; 10 mM NaCl; 1 mM 2-mercaptoethanol.) | | Optimal temperature | 30oC | | Storage conditions | 10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 0,05% Triton X-100, 0.1 mg/ml BSA, 50% glycerol; Store at -20°C. | | Non-specific hydrolisis | No detectable degradation of 1μg of Lambda DNA was observed after incubation with 8 units of enzyme for 16 hours at 30°C in a total reaction volume of 50 μl.
| | Reagents Supplied with Enzyme |
10 X SE-buffer GlaI, pHspAI2/GsaI DNA | | Methylation sensitivity | The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA
and DNA with N4-methylcytosines [1]. | | Inactivation 20 minutes under | 65oC | | MSDS: | Download MSDS as PDF | | References: | 1. Chernukhin V.A., Nayakshina T.N., Tomilova J.E., Mezentseva N.V., Dedkov V.S., Degtyarev S.Kh. Bacterial strain Glacial ice bacterium I - producer of GlaI restriction endonuclease. // Russian Federation patent RU 2287012 C1 (2006).
2.  Valery A. Chernukhin, Tatyana N. Najakshina, Murat A. Abdurashitov, Julia E. Tomilova, Nina V. Mezentzeva, Vladimir S. Dedkov, Natalya A. Mikhnenkova, Danila A. Gonchar, Sergei Kh. Degtyarev A novel restriction endonuclease GlaI recognizes methylated sequence 5’-G(m5C)^GC-3’ // Biotechnologia (russ.). 2006. N 4. P. 31-35
3. G. V. Tarasova, T. N. Nayakshina, S. Kh. Degtyarev Substrate specificity of new methyl-directed DNA endonuclease GlaI // BMC Molecular Biology 2008, 9:7
4. Abdurashitov M.A., Chernukhin V.A, Gonchar D.A., Degtyarev S.Kh.
GlaI digestion of mouse γ-satellite DNA: study of primary structure and ACGT sites methylation // BMC Genomics 2009, 10:322.
5. 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
6. Tomilova J.E., Chernukhin V.A., Degtyarev S.Kh. Dependence of site-specific endonuclease GlaI activity on quantity and location of methylcytosines in the recognition sequence 5’-GCGC-3’. // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.2, No 1, pp 30-39 (2006) (Russian)
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