Restriction endonucleases from Selenomonas ruminantium which recognize and cleave 5′-AT/TAAT-3′

Two natural isolates from fallow-deer rumen identified as Selenomonas ruminantium were found to produce a restriction endonuclease which we called Sru4DI. This enzyme was isolated from cell extracts by phosphocellulose chromatography. Analysis of the Sru4DI recognition site showed that Sru4DI recognizes the hexanucleotide sequence 5-AT/TAAT-3 generating 5 dinucleotide protruding ends upon cleavage and thus is a true isoschizomer of VspI, a restriction enzyme isolated from Vibrio sp.     

SruI restriction endonuclease from Selenomonas ruminantium

Abstract Sru I, specific restriction endonuclease, has been characterized from Selenomonas ruminantium isolated from the rumen of fallow deer. Results from the study demonstrate that S. ruminantium 18D possesses a type II restriction endonuclease, which recognizes the sequence 5'-TTT↓AAA-3'. The recognition sequence of Sru I was identified using digestions on pBR322, pBR328, pUC18, M13mp18RF, pACYC184 and λDNA. The cleavage patterns obtained were compared with computer-derived data. Sru I recognises the palindromic hexanucleotide sequence and cleaves DNA after the third T in the sequence, producing blunt ends. The purification and characterization of restriction endonuclease Sru I presented here is the first described for Selenomonas ruminantium spp. and demonstrates that this microorganism pocesses a DNA-cleaving enzyme with the same specificity as Dra I or Aha III.     

Bme585 I [5'-CCCGC(4/6)-3'], a new isoschizomer of restriction endonuclease Fau I, isolated from a strain of Bacillus mesentericus

Bme585 I is a new member of the restriction endonuclease type IIS family. It was partially purified from the heterothrophic, mesophilic bacterial strain Bacillus mesentericus 585 by ammonium sulphate precipitation and phosphocellulose column chromatography. Bme585 I is a monomeric protein with a molecular mass of 62 kD. The enzyme is active over a broad pH range from 7.0 to 8.8, has a temperature optimum of 37 degrees C and tolerance of NaCl in reaction buffer from 0 to 400 mM. Bme585 I recognizes the asymmetric sequence 5'-CCCGC(4/6)-3' and is therefore an isoschizomer of restriction endonuclease Fau I.     

Purification and characterization of restriction endonuclease BcoI from a soil isolate of Bacillus coagulans

Abstract A soil identified as Bacillus coagulans is found to produce Bco I, an isoschizomer of Ava I and with the same cleavage site. This thermal stable enzyme, Bco I, is produced at high level and can be isolated by passing the crude bacterial lysate through a DEAE-cellulose column.     

Different in vivo localization of the Escherichia coli proteins CspD and CspA

Neisseria cuniculi produces the restriction enzyme NcuI which is an isoschizomer of MboII. We have demonstrated that NcuI recognizes a pentanucleotide sequence (5'-GAAGA-3'/3'-CTTCT-5'), and cleaves the DNA 8 and 7 nucleotides downstream from the recognition site leaving a single 3'-protruding nucleotide. We have purified this enzyme to electrophoretic homogeneity using a four-step chromatographic procedure. NcuI endonuclease is a monomeric protein with a M(r)=48,000+/-1000 under denaturing conditions. The properties of NcuI are consistent with those for MboII, the position of the cleavage site being identical and the pH profile and divalent cation requirements being similar. Moreover, NcuI cross-reacts strongly with anti-MboII serum suggesting the presence of similar antigenic determinants. We have determined the sequence of 20 N-terminal amino acids for NcuI and concluded that this sequence is identical to the N-terminal portion of the MboII enzyme.     

Characterization of restriction endonuclease AjoI from Acinetobacter johnsonii

Abstract A new type II restriction endonuclease, named Ajo I, was detected in Acinetobacter johnsonii . The enzyme Ajo I, an isoschizomer of Pst I, recognized the hexanucleotide sequence [5'-CTGCA/G-3'], with a cleavage site generating fragments of DNA with protruding cohesive 3' termini.     

Mesophilic cyanobacteria producing thermophilic restriction endonucleases

When searching for the site-specific endonucleases in several strains of Phormidium we made the following observations. Among the 16 strains that originated from 15 species of Phormidium, 12 produced one or more restriction enzymes, of which two produced the highly thermophilic restriction endonucleases PtaI and PpaAII with their optimum activity at 65-80 degrees C, which is far above the lethal temperature for the host microorganism (40 degrees C). These two temperature-resistant enzymes are isoschizomers of known BspMII and TaqI endonucleases, respectively. The presence of the thermophilic TaqI isoschizomer does not seem to play any role in the mesophilic host microorganism, which does not even contain an active cognate methyltransferase. Among the remaining 10 strains, six produced isoschizomers of endonucleases which we first described in cyanobacteria, namely: PfaAII (NdeI), PinBII and PtaI (BspMII), PlaAII (RsalI), PpaAII, PpeI (ApaI). Two enzymes, PauAII (AhaIII) and PfaAII (NdeI), belong to a group of a very rarely occurring isoschizomers. Out of 21 cyanobacterial endonucleases investigated by us, four were active in a wide range of temperatures (from 15 to 60 degrees C) which also extended the optimal growth temperature of the hosts. We assume that our observation on the presence of temperature-resistant restriction enzymes in mesophilic hosts supports the idea of horizontal gene transfer. Restriction modification systems may be an excellent tool for investigation of that phenomenon.     

Two site-specific endonucleases BinSI and BinSII from Bifidobacterium infantis

Two site-specific endonucleases, BinSI and BinSII, were isolated from Bifidobacterium infantis S76e. BinSI was found to be an isoschizomer of EcoRII, while BinSII was shown to have the same sequence and cutting specificity as BbeI, 5′-GGCGCC-3′. Both BinSII- and BbeI-generated DNA fragments could be ligated with HaeII-generated DNA fragments.     

Isolation of charaterization of two sequence-specific endonucleases from the cyanobacterium Synechocystis sp. PCC 6308

The first two restriction endonucleases to be characterized in the cyanobacterium Synechocystis sp. PCC 6308 are described. SynI, an AvaII isoschizomer, recognizes the base sequence 5-GG[AT]CC-3. SynII, an XmnI isoschizomer, recognizes the sequence 5-GAANNNNTTC-3.