ABSTRACT
DNA ligases catalyze the sealing of 5'-phosphate and 3'-hydroxyl termini at single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome in DNA metabolism. An ATP-dependent DNA ligase from the archaeon Thermococcus sp. 1519 was overexpressed, purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion method employing 35%(v/v) Tacsimate pH 7.0 as a precipitant and diffracted X-rays to 3.09 A resolution. They belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 79.7, c = 182.6 A.
Subject(s)
DNA Ligases/chemistry , DNA Ligases/isolation & purification , Temperature , Thermococcus/enzymology , Crystallization , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
The DNA ligase gene from thermophilic archaea of the genus Thermococcus (strain 1519) was identified and sequenced in the polymerase chain reaction. The recombinant enzyme LigTh1519 was expressed in Escherichia coli, purified, and characterized. LigTh1519 was capable of ligating the cohesive ends and single-strand breaks in double-stranded DNA (ATP as a cofactor). The optimum conditions for the ligase reaction appeared as follows: 100 mM NaCl, 50 mM MgCl2, pH 7.0-10.5, and temperature 70 degrees C. More than 50% Lig1519 activity were preserved after incubation of the enzyme at 80 degrees C for 30 min. New thermostable DNA ligase LihTh1519 may be used for basic and applied researches in molecular biology and genetic engineering.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/isolation & purification , DNA Ligases/chemistry , DNA Ligases/isolation & purification , Thermococcus/enzymology , Archaeal Proteins/genetics , DNA Breaks, Single-Stranded , DNA Ligase ATP , DNA Ligases/genetics , Gene Expression , Hot Temperature , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Thermococcus/geneticsABSTRACT
Suprapyloric transversal resection of the stomach is one of relatively sparing methods of surgical treatment for ulcerative disease. In Medical Centre of "VAZ", suprapyloric resection of the stomach was carried out as a method of treatment of gastric ulcer in 181 patients (study group) and 139 patients underwent resection 2/3 of the stomach by Billroth-1 (control group). The ulcer located in cardial and mediogastric regions (1 type by Johnson). Among the patients of study group postoperative lethality made up 1.1%. Follow-up results of operative treatment was quite favourable in 96.3% of cases and unsatisfactory--in 3.7% (persistent pylorospasm). In control group follow-up results of surgical treatment in 86.4% were appraised as excellent and good, in 13.6%--satisfactory due to postoperative development of damping-syndrome. Comparison of results of follow-up examination in two groups of operated patients has evidenced of the best functional results after performance of suprapyloric resection of the stomach.
