scholarly journals Two thermostable type II restriction endonucleases from Icelandic strains of the genus Thermus: Tsp4C I (ACN/GT), a novel type II restriction endonuclease, and Tsp8E I, an isoschizomer of the mesophilic enzyme Bgl I (GCCNNNN/NGGC)

1995 ◽  
Vol 309 (2) ◽  
pp. 595-599 ◽  
Author(s):  
S G Welch ◽  
R A D Williams
Keyword(s):  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Thermophilic Bacteria ◽  
Hot Water ◽  
Restriction Endonucleases ◽  
Type Ii ◽  
Endonuclease Activity ◽  
Lambda Phage ◽  
Recognition Sequence ◽  
Phosphodiester Bond

Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction endonucleases. Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA. Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined. Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA. Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3′-OH overhang. Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) [Lee, Clanton and Chirikjiam (1979) Fed. Proc. 28, 294], generating fragments with a three base 3′-OH overhang. However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C. Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction endonucleases.

scholarly journals A rapid and simple method for detection of type II restriction endonucleases in cells of bacteria with high activity of nonspecific nucleases.

2012 ◽  
Vol 59 (4) ◽  
Author(s):  
Beata Podgórska ◽  
Grażyna Kujawska ◽  
Michał Skurzewski ◽  
Olesya Batsko ◽  
Tadeusz Kaczorowski
Keyword(s):  
Restriction Endonuclease ◽  
High Activity ◽  
Restriction Endonucleases ◽  
Type Ii ◽  
Endonuclease Activity ◽  
Simple Method ◽  
In Cells

In this work we describe a novel, rapid and simple microscale procedure for identification of restriction endonuclease activity in bacteria lysates, which contain high levels of non-specific DNA nucleases.

scholarly journals Two different isoschizomers of the type-II restriction endonuclease Taq I (T/CGA) within the same Thermus isolate: Tsp32 I, an enzyme with similar heat stability properties to the prototype enzyme Taq I, and Tsp32 II, a hyperthermostable isoschizomer of Taq I

1995 ◽  
Vol 312 (2) ◽  
pp. 505-510 ◽  
Author(s):  
S G Welch ◽  
R A D Williams
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Cleavage Site ◽  
Heat Stability ◽  
Hot Water ◽  
Heat Inactivation ◽  
Type Ii ◽  
Magnesium Ions ◽  
Water Springs

We have recently screened 112 separate isolates of the genus Thermus, collected from neutral and alkaline hot water springs on four continents, for the presence of the Type-II restriction endonuclease Taq I (T/CGA). One particular isolate from the Azores (strain 32) was found to contain high levels of a restriction endonuclease with the same recognition and cleavage site as Taq I. Initial studies revealed that the partially purified enzyme from strain 32 was considerably more resistant to heat inactivation than the prototype enzyme Taq I, being able to withstand temperatures at least 10 degrees C higher than Taq I, before showing evidence of heat inactivation. Subsequently it became clear that the partially purified extract from strain 32 contains two separate enzymes, both of which are isoschizomers of Taq I. One of the enzymes, Tsp32 I, has similar thermal stability characteristics to Taq I, whereas the second Taq I isoschizomer, Tsp32 II, found in the same Thermus isolate as Tsp32 I, is considerably more thermostable than Taq I, retaining full enzyme activity up to a temperature of 85 degrees C. Tsp32 I and Tsp32 II were further distinguished by virtue of their different requirements for magnesium ions.

Type II restriction endonucleases fromHelicobacter pyloriinclude an enzyme with a novel recognition sequence

1999 ◽  
Vol 179 (1) ◽  
pp. 175-180 ◽  
Author(s):  
Ana Ivic ◽  
Kenneth J Jakeman ◽  
Charles W Penn ◽  
Nigel L Brown
Keyword(s):  
Restriction Endonucleases ◽  
Type Ii ◽  
Recognition Sequence

scholarly journals The reactions of the EcoRi and other restriction endonucleases

1979 ◽  
Vol 179 (2) ◽  
pp. 353-365 ◽  
Author(s):  
S E Halford ◽  
N P Johnson ◽  
J Grinsted
Keyword(s):  
Restriction Endonuclease ◽  
Dna Sequences ◽  
Recognition Site ◽  
Single Strand ◽  
Restriction Endonucleases ◽  
Dna Duplex ◽  
Phosphodiester Bond ◽  
Linear Dna ◽  
Ecori Restriction ◽  
Dna Substrates

The reaction of the EcoRI restriction endonuclease was studied with both the plasmid pMB9 and DNA from bacteriophage lambda as the substrates. With both circular and linear DNA molecules, the only reaction catalysed by the EcoRI restriction endonuclease was the hydrolysis of the phosphodiester bond within one strand of the recognition site on the DNA duplex. The cleavage of both strands of the duplex was achieved only after two independent reactions, each involving a single-strand scission. The reactivity of the enzyme for single-strand scissions was the same for both the first and the second cleavage within its recognition site. No differences were observed between the mechanism of action on supercoiled and linear DNA substrates. Other restriction endonucleases were tested against plasmid pMB9. The HindIII restriction endonuclease cleaved DNA in the same manner as the EcoRI enzyme. However, in contrast with EcoRI, the Sa/I and the BamHI restriction endonucleases appeared to cleave both strands of the DNA duplex almost simultaneously. The function of symmetrical DNA sequences and the conformation of the DNA involved in these DNA–protein interactions are discussed in the light of these observations. The fact that the same reactions were observed on both supercoiled and linear DNA substrates implies that these interactions do not involve the unwinding of the duplex before catalysis.

scholarly journals The SalGI restriction endonuclease. Purification and properties

1982 ◽  
Vol 203 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Anthony Maxwell ◽  
Stephen E. Halford
Keyword(s):  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Final Stage ◽  
Enzyme Stability ◽  
Type Ii ◽  
Optimal Conditions ◽  
Purification And Properties

The type II restriction endonuclease SalGI has been purified to near homogeneity. At least 80% of the protein remaining after the final stage of the preparation is SalGI restriction endonuclease; no contaminating nucleases remain detectable. The principal form of the protein under both native and denaturing conditions is a monomer of Mr about 29000. The optimal conditions for both enzyme stability and enzyme activity have been determined.

scholarly journals Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorella-like green alga.

1986 ◽  
Vol 6 (5) ◽  
pp. 1430-1439 ◽  
Author(s):  
Y N Xia ◽  
D E Burbank ◽  
L Uher ◽  
D Rabussay ◽  
J L Van Etten
Keyword(s):  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Green Alga ◽  
Nuclear Dna ◽  
De Novo ◽  
Dna Degradation ◽  
Endonuclease Activity ◽  
Modification System

An enzyme was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5' to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1-induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.

scholarly journals Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorella-like green alga

1986 ◽  
Vol 6 (5) ◽  
pp. 1430-1439
Author(s):  
Y N Xia ◽  
D E Burbank ◽  
L Uher ◽  
D Rabussay ◽  
J L Van Etten
Keyword(s):  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Green Alga ◽  
Nuclear Dna ◽  
De Novo ◽  
Dna Degradation ◽  
Endonuclease Activity ◽  
Modification System

An enzyme was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5' to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1-induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.

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