A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes.

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

Determination of methylation site of DNA-methyltransferase NlaX by a hybrid method.

Using a new method based on a combination of bisulfite reaction, the repair enzyme uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the methylation site of DNA-methyltransferase NlaX (M.NlaX) from Neisseria lactamica was established to be the inner cytosine in the double-stranded pentanucleotide recognition sequence 5'-CCNGG-3' (where N = any nucleoside). 5-Methylcytosine (m5C) type modification by M-N1aX was confirmed by the use of oligonucleotide substrates that contain 5-fluoro-2'-deoxycytidine.

Determination of a non-methylated deoxycytidine residue in the recognition site of DNA-methyltransferases.

A method for determination of a non-methylated deoxycytidine (dC) residue in the recognition site of 5-cytosine DNA-methyltransferases is suggested. The method is based on treatment of methylated DNA by sodium bisulfite and successive reaction of the thus modified DNA with a repair enzyme, uracil-DNA glycosylase. This method was successfully applied to identify NlaX methyltransferase specificity.

Characterization of the type IV restriction modification system BspLU11III from Bacillus sp. LU11.

We report the characterization and cloning of the genes for an unusual type IV restriction-modification system, BspLU11III, from Bacillus sp. LU11. The system consists of two methyltransferases and one endonuclease, which also possesses methyltransferase activity.

Shape and DNA packaging activity of bacteriophage SPP1 procapsid: protein components and interactions during assembly.

The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7. The protein stoichiometry suggests a T=7 symmetry for the SPP1 procapsid. Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures.

Structure of the 13-fold symmetric portal protein of bacteriophage SPP1.

We have determined the three-dimensional structure of bacteriophage SPP1 portal protein (gp6) using electron microscopy at liquid-helium temperatures and angular reconstitution. The 13-fold symmetric gp6 oligomer is a turbine-shaped structure with three distinct regions: a conical stem with a central channel; the turbine wings region; and a fringe of small 'tentacles' at the end of the channel exposed to the viral head interior. The tentacle region appears flexible and may be associated with a particular function - sensing when the correct amount of DNA has been packaged.

M.(phi)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme.

In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target-recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes.

Masc2, a C5-DNA-methyltransferase from Ascobolus immersus with similarity to methyltransferases of higher organisms.

The filamentous fungus Ascobolus immersus represents an eukaryotic model organism to study genetic phenomena linked to DNA methylation. Following our previous characterization of a gene, masc1 from A. immersus, encoding the 'de novo' C5-DNA-methyltransferase (MTase), we report here the identification of a second MTase gene, masc2.

The complete nucleotide sequence and functional organization of Bacillus subtilis bacteriophage SPP1.

The complete nucleotide sequence of the B. subtilis bacteriophage SPP1 is described. The genome is 44,007 bp in size and has a base composition of 43.

A gene essential for de novo methylation and development in Ascobolus reveals a novel type of eukaryotic DNA methyltransferase structure.

Molecular mechanisms determining methylation patterns in eukaryotic genomes still remain unresolved. We have characterized, in Ascobolus, a gene for de novo methylation. This novel eukaryotic gene, masc1, encodes a protein that has all motifs of the catalytic domain of eukaryotic C5-DNA-methyltransferases but is unique in that it lacks a regulatory N-terminal domain.

Head morphogenesis genes of the Bacillus subtilis bacteriophage SPP1.

We have identified and characterized the phage cistrons required for assembly of SPP1 heads. A DNA fragment containing most of the head morphogenesis genes was cloned and sequenced. The 3'-end of a previously identified gene (gene 6) and eight complete open reading frames (7 to 15) were predicted.

Sequential headful packaging and fate of the cleaved DNA ends in bacteriophage SPP1.

The virulent Bacillus subtilis bacteriophage SPP1 packages its DNA from a precursor concatemer by a headful mechanism. Following disruption of mature virions with chelating agents the chromosome end produced by the headful cut remains stably bound to the phage tail. Cleavage of this tail-chromosome complex with restriction endonucleases that recognize single asymmetric positions within the SPP1 genome yields several distinct classes of DNA molecules whose size reflects the packaging cycle they were generated from.

M.BssHII, a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties.

A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.BssHII, was identified in Bacillus stearothermophilus H3. The M.

Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5' part of their DNA target.

In previous work on DNA-(cytosine-C5)-methyltransferases (C5-MTases), domains had been identified which are responsible for the sequence specificity of the different enzymes (target-recognizing domains, TRDs). Here we have analyzed the DNA methylation patterns of two C5-MTases containing reciprocal chimeric TRDs, consisting of the N- and C-terminal parts derived from two different parental TRDs specifying the recognition of 5'-CC(A/T)GG-3' and 5'-GCNGC-3'. Sequences recognized by these engineered MTases were non-symmetrical and degenerate, but contained at their 5' part a consensus sequence which was very similar to the 5' part of the target recognized by the parental TRD which contributed the N-terminal moiety of the chimeric TRD.

Exact size and organization of DNA target-recognizing domains of multispecific DNA-(cytosine-C5)-methyltransferases.

A large portion of the sequences of type II DNA-(cytosine-C5)-methyltransferases (C5-MTases) represent highly conserved blocks of amino acids. General steps in the methylation reaction performed by C5-MTases have been found to be mediated by some of these domains. C5-MTases carry, in addition at the same relative location, a region variable in size and amino acid composition, part of which is associated with the capacity of each C5-MTase to recognize its characteristic target.

BsuCI, a type-I restriction-modification system in Bacillus subtilis.

A type-I R-M system was identified in B. subtilis. The genes comprising the system have striking similarity to type-I R-M systems observed in Enterobacteriaceae.

Altered sequence recognition specificity of a C5-DNA methyltransferase carrying a chimeric 'target recognizing domain'.

A MTase with a chimeric TRD with the N-terminal half derived from a TRD recognizing GCNGC, the C-terminal half from one with CCWGG recognition, was constructed. Its target specificity is reported.

M.BssHII: a new multispecific C5-DNA-methyltransferase.

M.BssHII is a new multispecific C5-DNA-methyltransferase recognizing five different targets. As the enzyme has been isolated from a thermophilic Bacillus, the protein should show enhanced intrinsic thermostability and therefore be a promising candidate for crystallizing a multispecific MTase.

M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases.

The temperate B.subtilis phages phi 3T and rho 11s code, in addition to the multispecific DNA (cytosine-C5) methyltransferases (C5-MTases) M. phi 3TI and M.

M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases.

The temperate B.subtilis phages phi 3T and rho 11s code, in addition to the multispecific DNA (cytosine-C5) methyltransferases (C5-MTases) M.phi 3TI and M.

Sequence analysis of the left end of the Bacillus subtilis bacteriophage SPP1 genome.

The left end of the genome of Bacillus subtilis bacteriophage SPP1 is represented by EcoRI DNA fragments 12 and 1 (EcoRI-12 and EcoRI-1). A number of different deletions were identified in EcoRI-1. A detailed physical and genetic map of EcoRI-1 from wild-type (wt) phage and SPP1 deletion mutants was constructed.

BsuBI--an isospecific restriction and modification system of PstI: characterization of the BsuBI genes and enzymes.

The enzymes of the Bacillus subtilis BsuBI restriction/modification (R/M) system recognize the target sequence 5'CTGCAG. The genes of the BsuBI R/M system have been cloned and sequenced and their products have been characterized following overexpression and purification. The gene of the BsuBI DNA methyltransferase (M.

Intramolecular homologous recombination in Bacillus subtilis 168.

Plasmid resolution from a phage::plasmid chimera was used to measure directly intramolecular recombination in Bacillus subtilis. The system is based on a sigma-replicating plasmid (pC194) cloned into a dispensable region of the lytic bacteriophage SPP1. The plasmid, which confers chloramphenicol resistance, is resolved when SPP1::pC194 phages infect B.