GATC-specific restriction and modification systems in treponemes

AIMS: To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp. METHODS: The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found. CONCLUSIONS: Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction-modification systems. SIGNIFICANCE: This is the first report on restriction activity in rumen treponemes.     

Underrepresentation of short palindromes in Selenomonas ruminantium DNA: evidence for horizontal gene transfer of restriction and modification systems?

Molecular analysis of isolates of the rumen bacterium Selenomonas ruminantium revealed a high variety and frequency of site-specific (restriction) endonucleases. While all known S. ruminantium restriction and modification systems recognize hexanucleotide sequences only, consistently low counts of both 6-bp and 4-bp palindromes were found in DNA sequences of S. ruminantium. Statistical analysis indicated that there is some correlation between the degree of underrepresentation of tetranucleotide words and the number of known restriction endonucleases for a given sequence. Control analysis showed the same correlation in lambda DNA but not in human adenovirus DNA. Based on the data presented, it could be proposed that there is a much higher historical occurrence of restriction and modification systems in S. ruminantium and (or) frequent horizontal gene transfer of restriction and modification gene complexes.     

Production of SacI and SacII isoschizomers by soil streptomycetes

Five fresh soil Streptomyces spp. strains were isolated, phylogenetically characterized on the basis of 16S rDNA sequences and analyzed for the presence of restriction modification systems. Three type II site-specific endonucleases were detected and partially purified. Two isolated enzymes were isoschizomers of SacI restriction endonuclease recognizing 5′-GAGCTC-3′ sequence; the third one recognised 5′-CCGCGG-3′ sequence and it was an isoschizomer of SacII. SacII like modification was observed in other two isolates having no detectable restriction activity. The lack of correlation between restriction and modification phenotypes and phylogenetic classification of the isolates indicates efficient gene transfer mechanism in the Streptomyces genus.     

Different restriction and modification phenotypes in ruminal lactate-utilizing bacteria

Analysis of restriction and modification activities in lactate-utilizing bacteria belonging to the Megasphaera elsdenii and Mitsuokella multiacida species revealed the presence of GATC-specific, MboI isospecific, restriction-modification (R-M) systems in all strains tested. While restriction endonucleases isolated from M. elsdenii strains were found to be sensitive to Dam methylation, enzymes from M. multiacida cleaved DNA irrespective of Dam methylation. The comparison of type II R-M systems specificities in three closely related lactate-utilizing ruminal bacterial species indicated complete lack of restriction and/or modification enzymes previously characterized from Selenomonas ruminantium in tested M. elsdenii and M. multiacida strains. R-M systems are believed to represent the main defense tool against phage infection. Based on the results of our experiments it could be assumed that M. elsdenii and M. multiacida use the different strategy for bacteriophage protection compared to S. ruminantium.     

Occurrence of restriction-modification systems in ruminal butyrate-producing bacteria

Thirty-five strains of ruminal bacteria belonging to the former Butyrivibrio fibrisolvens species were screened for the presence of site-specific restriction endonuclease and modification methyltransferase activities. Seven strains possessed endonuclease activities detectable in crude cell extracts. The recognition sequences and optimal reaction conditions for seven of them were determined. Five enzymes were found to be isoschizomers of type II endonucleases (EcoRV, NsiI, AseI (2x) and SauI), one was type IIS (FokI) and two remained unknown. The optimal reaction buffer was found to be a low ionic strength buffer and all enzymes possessed sufficient activity at 39 degrees C. The presence of DNA modification among all strains was also determined. Most of the methylation activities correlated with restriction activities, yet some strains possessed unaccompanied modification methyltransferases.     

Substrate specificity of restriction endonuclease Eco781

The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC-. There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends. Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence.     

Site-specific restriction endonucleases in cyanobacteria

AIM: Planktic cyanobacteria were screened for endodeoxyribonucleases. Principal component analysis (PCA) was employed to demonstrate a potential relationship between certain enzymes and a group of cyanobacteria. The data were obtained from a data bank and this study. METHODS AND RESULTS: Enzymes were partially purified using column chromatography. Anabaena strains contained Asp83/1I (5'-TTCGAA-3'), Asp83/1II (5'-GGCC-3'), Asp90I (5'-ACRYGT-3') and five isoschizomeric enzymes (5'-ATCGAT-3'). Aphanizomenon and Microcystis strains contained ApcTR183I (5'-TGCGCA-3') and Msp199I (5'-CCGG-3'), respectively. Planktothrix strains possessed Psc2I (5'-GAANNNNTTC-3'), Psc27I and Psc28I (5'-TTCGAA-3'). PCA showed that the most common cyanobacterial endonuclease types were AvaII, AvaI and AsuII. CONCLUSIONS: All planktic cyanobacteria studied contained restriction endonucleases. The defined restriction endonucleases were isoschizomers of known enzymes. The Nostoc and the Spirulina genera had an association, while the majority of the genera had no association with certain endonuclease type(s). SIGNIFICANCE AND IMPACT OF THE STUDY: The defined enzymes in this study and the estimated trend in the endonuclease type distribution allow more efficient avoidance of cyanobacterial restriction barriers.     

Cloning and molecular characterization of the HgiCI restriction/modification system from Herpetosiphon giganteus Hpg9 reveals high similarity to BanI.

The genes coding for the GGYRCC specific restriction/modification system HgiCI from Herpetosiphon giganteus Hpg9 have been cloned in Escherichia coli in three steps. As an initial step, the methyltransferase gene could be obtained after heterologous in vitro selection of a plasmid gene bank by cleavage with the isoschizomeric restriction endonuclease BanI. The adjacent endonuclease gene was cloned following Southern blot analysis of flanking genomic regions. The two genes code for polypeptides of 420 amino acids (M.HgiCI) and 345 amino acids (R.HgiCI). Establishing a functional endonuclease gene could only be achieved using a tightly regulated expression system or by methylation of the genomic DNA prior to transformation of the endonuclease gene. The methyltransferase M.HgiCI shows significant similarities to the family of 5-methylcytidine methyltransferases. Striking similarities could be found with both the isoschizomeric endonuclease and methyltransferase of the BanI restriction/modification system from Bacillus aneurinolyticus.     

The MmeI family: type II restriction–modification enzymes that employ single-strand modification for host protection

The type II restriction endonucleases form one of the largest families of biochemically-characterized proteins. These endonucleases typically share little sequence similarity, except among isoschizomers that recognize the same sequence. MmeI is an unusual type II restriction endonuclease that combines endonuclease and methyltransferase activities in a single polypeptide. MmeI cuts DNA 20 bases from its recognition sequence and modifies just one DNA strand for host protection. Using MmeI as query we have identified numerous putative genes highly similar to MmeI in database sequences. We have cloned and characterized 20 of these MmeI homologs. Each cuts DNA at the same distance as MmeI and each modifies a conserved adenine on only one DNA strand for host protection. However each enzyme recognizes a unique DNA sequence, suggesting these enzymes are undergoing rapid evolution of DNA specificity. The MmeI family thus provides a rich source of novel endonucleases while affording an opportunity to observe the evolution of DNA specificity. Because the MmeI family enzymes employ modification of only one DNA strand for host protection, unlike previously described type II systems, we propose that such single-strand modification systems be classified as a new subgroup, the type IIL enzymes, for Lone strand DNA modification.     

Lack of DNA methylation in Schistosoma mansoni

Schistosoma mansoni genomic DNA from male and female adult worms was subjected to restriction by the isoschizomeric endonucleases HpaII and MspI, which display different sensitivities with respect to cytosine methylation. The digested DNA was hybridized with 13 S. mansoni probes. Southern blot analysis showed that there were no observable differences in the restriction patterns of the two isoschizomers and that the patterns were identical in male and female parasites. Adenine methylation was also ruled out since no differences were observed with DpnI, Sau3A1, or MboI restriction enzymes. The methylation-dependent restriction endonuclease McrBC, which cleaves DNA containing methylcytosine and will not cleave unmethylated DNA, did not digest S. mansoni genomic DNA. These results demonstrate that the genome of adult S. mansoni is not methylated.     

GATC-specific restriction-modification systems in ruminal bacteria

The GATC-specific restriction and modification activities were analyzed in 11 major bacterial representatives of ruminal microflora. Modification phenotype was observed in 13 out of 40 ruminal strains. MboI isoschizomeric restriction endonucleases were detected in 10 bacterial strains tested; three strains lacked any detectable corresponding endonuclease activity. The only examined strain of Mitsuokella multi-acida was found to possess a different type of endonuclease activity. This is the first report on restriction activity in ruminal treponemes M. multiacida and Megasphaera elsdenii.     

Sequence of the 3''-noncoding and adjacent coding regions of human gamma-globin mRNA.

In cloning human fetal globin cDNA in bacterial plasmids, we obtained a recombinant which contained a fragment of gammg-globin cDNA corresponding to the region from amino acid 99 to the poly A. We determined a sequence of 169 nucleotides which included the complete 3' non-coding region of the gamma-globin mRNA. The codon for amino acid 136 was GCA, indicating that this cloned fragment was derived from the Agamma-globin gene. In conjunction with the surrounding sequences, the GCA codon provides the Agamma-species with a unique CTGCAG hexanucleotide that is recognized by the restriction enzyme Pst I. The 3'-untranslated region of the gamma-globin mRNA consists of 90 nucleotides, and shares little homology with that of the human beta-globin mRNA. As in other mammalian mRNAs, a symmetrical sequence and the hexanucleotide AAUAAA are present.     

A method for detecting new strains producing DNA modification and restriction enzymes--isoschizomers and isomethylomers of known restrictases and methylases

The paper describes a technique for the detection of new strains producing enzymes which mediate DNA modification and restriction, and isoschizomers and isomethylomers of the known restriction endonucleases and methylases. Three Bacillus subtilis strains whose DNA carries a BamH1 modification have been found. Two of these strains exert the restrictase activity with an R BamH1 specificity.     

Sequence-specific endonucleases in strains of Anabaena and Nostoc

The complements of restriction endonucleases of 12 strains of cyanobacteria were determined in cell-free extracts, and were compared with the complements of restriction activities assessed by measuring the relative efficiencies of plating of cyanophages on those cyanobacteria. The hosts which were susceptible to all of the phages contained endo R · AvaI and endo R · AvaII, and in several cases probably endo R · AvaIII, or isoschizomers of these enzymes. Three hosts which were lysed by only a subset (1 or 3) of the phages contained different restriction endonuclease. Anabaena sp. PCC 7120 showed apparent phenotypic restriction of phage An-22 grown in hosts with (isoschizomers of) AvaI, II and III, but no corresponding endonuclease has yet been detected in vitro. Nostoc sp. ATCC 29131 (PCC 6705) was found to contain a restriction enzyme, NspBII, with hitherot unknown specificity, C(A/C)GC(T/G)G.     

The SalI (SalGI) restriction-modification system of Streptomyces albus G

The salIR and salM genes of Streptomyces albus G specify the SalGI (SalI) restriction enzyme and its cognate methyltransferase, respectively. These enzymes are responsible for restriction and modification of bacteriophages. Some phages carry genes that interfere with SalI-specific modification. The sal genes have been cloned in a Streptomyces host-vector system. Use of the cloned DNA as a hybridization probe reveals that sal mutants frequently arise from transposition of a DNA segment of approx. 1 kb into the sal genes. Some, but not all, other bacteria that produce SalGI isoschizomers contain nucleotide sequences that hybridize with sal DNA.     

Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids

Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated 'reference plasmids'), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these 'reference plasmids' revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).