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The DNA methyltransferases of type I restriction-modification systems are trimeric enzymes composed of one DNA specificity (S) subunit and two modification (M) subunits. The S subunit contains two large regions, each of which recognizes one part of the split, asymmetrical DNA target sequence. Each M subunit contains an amino acid motif for binding the methyl group donor and cofactor, S-adenosyl methionine. The EcoKI methyltransferase has a strong preference for methylating a hemimethylated DNA target rather than an unmodified target. We have used partial proteolytic digestion of EcoKI methyltransferase to generate polypeptide domains that we have identified by amino acid sequencing. The S subunit was cut into two large, folded domains each containing one DNA binding region. Binding of DNA partially protected the S subunit from digestion. The M subunit was also cut into two large domains joined together by a short flexible loop, and a C-terminal tail region. The short loop contained part of the S-adenosyl methionine binding motif, and cofactor binding protected the loop and the two large domains from proteolysis. The C-terminal domain of M remained associated with the N-terminal domain of the S subunit even after the rest of the protein had been digested. The conformation of the tail region of the M subunit was sensitive to the methylation state of DNA in ternary complexes also containing S-adenosyl methionine, and could differentiate between unmethylated and hemimethylated DNA substrates.

Caulobacter crescentus was found to have a DNA methyltransferase, CcrM, that methylates the adenine base of the HinfI recognition sequence, GANTC. The ccrM gene was cloned, and DNA sequence analysis revealed that the predicted amino acid sequence has 49% identity with the Haemophilus influenzae methyltransferase HinfM. Expression of the ccrM gene was found to be restricted to the portion of the cell cycle immediately prior to cell division. At three separate chromosomal sites the CcrM recognition sequence is fully methylated in swarmer cells, becomes hemimethylated upon DNA replication in stalked cells, and does not become remethylated until just prior to cell division. The time of methyltransferase expression coincides with the time of methylation of these three chromosomal sites and of plasmid DNA in the predivisional cell. When ccrM gene expression is placed under control of a constitutive promoter, these chromosomal sites are fully methylated throughout the cell cycle. A high proportion of morphologically aberrant cells, and cells that have undergone an additional chromosome replication initiation, are found in this population. Thus, the temporal control of this methyltransferase appears to contribute to the accurate cell-cycle control of DNA replication and cellular morphology.

The BcgI restriction enzyme from Bacillus coagulans is unusual in that it cleaves on both sides of its recognition site, CGAN6TGC, releasing a fragment that includes the site and several bases on each side. We report the organization and nucleotide sequences of the genes for the BcgI restriction-modification system and the properties of the proteins that they encode. The system comprises two adjacent, similarly oriented genes. The proximal gene, bcgIA, codes for a 637-amino acid protein (molecular mass = 71.6 kDa) that resembles certain m6A-specific DNA-methyltransferases, particularly those that constitute the modification subunits of type I restriction-modification systems. The distal gene, bcgIB, codes for a 341-amino acid protein (molecular mass = 39.2 kDa) that resembles none of the sequences in the sequence data bases. The two genes overlap by several nucleotides. Alone, neither protein restricts or modifies DNA, but, together, they form a complex in the proportion A2B that does both. DNA binding assays showed that the DNA-protein complex can be formed only in the presence of both subunits, suggesting that the association of inactive subunits generates the active BcgI enzyme that can bind DNA and then either cleaves or methylates at target site.

Maximum likelihood linkage analyses were performed to test for linkage between serum apoB levels and several candidate gene markers including apolipoprotein B, lipoprotein lipase, hepatic lipase, cholesterol ester transfer protein, and apolipoprotein AI in a large pedigree. Parameters of general Mendelian inheritance derived from maximum likelihood segregation analysis of the serum apoB levels were used in the linkage analysis. The highest two-point lod score between the quantitative trait and a marker defined by a single restriction digest was 1.86 at recombination fraction (theta) = 0. This was observed for linkage between serum apoB levels and the presence or absence of a PvuII digestion site in the apoB gene. Linkage between serum apoB levels and polymorphisms of the apoB gene defined by the two restriction digests EcoR1 and PvuII was supported by a lod score of 3.30, while inclusion of VNTR typings led to a lod score of 2.33. None of the other candidate genes gave positive evidence of linkage.

The genes hsdM and hsdS for M. EcoKI modification methyltransferase and the complete set of hsdR, hsdM and hsdS genes coding for R. EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation in hsdS gene, were cloned in pBR322 plasmid and introduced into E. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. The hsdSts-1 mutation, mapped previously in the distal variable region of the hsdS gene with C1 245-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42 degrees C. In clones transformed with the whole hsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutant hsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of the hsdSts-1 mutation on restriction and modification.

Two different clonal groups of pathogenic Yersinia enterocolitica strains, American and non-American, have been recognized. These are distinguished by a number of criteria, including their virulence in a murine model of infection. However, genetic analysis of virulence in American strains has been hampered due to the severe restriction of transformed or electroporated DNA. Thus, we cloned the yenIMR locus from the American serotype strain 8081c, which encodes YenI, an isoschizomer of PstI. This clone encodes both the restriction endonuclease and methyltransferase. The location of the genes on the clone was determined and this information was used to construct a small deletion (400 bp) that results in an R-M+ phenotype. This mutation was recombined onto the Y. enterocolitica chromosome to give an R-M+ mutant which showed at least a 1000-fold increase in electroporation frequency compared to the wild-type strain. Southern analysis using a probe derived from yenIMR indicated that American serotype strains have this locus whereas non-American serotype strains do not.

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R-M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5' half site of the wild type site is repeated in inverse orientation. The additional base pair in the non-specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl-terminal DNA binding domain which recognizes the 3' half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino-terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R-M systems will be discussed.

The gene (bseCIM) encoding the BseCI DNA methyltransferase (MTase; M.BseCI) from a Bacillus stearothermophilus species was cloned and expressed in Escherichia coli using plasmid vector pBR322. Selection of transformants carrying bseCIM was based on the resistance of the modified plasmid to cleavage by BseCI. The MTase was purified to homogeneity and further characterized. Its size as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and size exclusion chromatography was 68 kDa, suggesting that the MTase exists as a monomer. When phage lambda DNA was used as a substrate, the optimum temperature for MTase activity was determined to be 50-55 degrees C and optimum pH approx. 7.4. M.BseCI is inhibited by concentrations of NaCl and KCl greater than 50 mM, and it does not require Mg2+ for activity. Finally, M.BseCI methylates the 3' adenine residue in the sequence, 5'-ATCGAT-3', similarly to its isoschizomer M.ClaI.

The adenine DNA methyltransferase M.CviRI (TGCmA) gene from chlorella virus XZ-6E was cloned into the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome and expressed in Spodoptera frugiperda insect cells under the control of two tandemly arranged viral promoters, the early ETL promoter and the late polyhedrin promoter. M.CviRI activity was first detected at 10 hr p.i and reached a maximum at 48 hr p.i. Viral DNA synthesized in insect cells infected with M.CviRI expressing virus (AcMTRI) was methylated at all TGCA sites. Unexpectedly, AcMTRI-infected cells lysed 48 hr earlier than wild-type AcMNPV-infected cells. Moreover, cellular DNA, but not viral DNA, from AcMTRI-infected cells was degraded to fragment sizes characteristic of apoptosis. These results suggest that M.CviRI methylation influences the onset of viral cytopathic effects and induces an apoptosis-like response.

The nucleotide (nt) sequence of a 2.7-kb HindIII-EcoRI DNA fragment encoding the bstVIR and bstVIM genes has been determined. The sequence predicts a restriction endonuclease of 224 amino acids (aa), M(r) 25,104, and a methyl-transferase of 561 aa, M(r0 65,702. Both genes are aligned in the same orientation and are separated by a 102-nt intergenic region. No homology was found between R.BstVI and M.BstVI when their deduced aa sequences were compared. Significant similarity at the aa level was found, however, when both enzymes were compared to their equivalents in the paeR7IRM system of Pseudomonas aeruginosa PAO303.

The 2.55 kb Staphylococcus aureus plasmid, pRJ5, confers constitutive resistance to macrolide-lincosamide-streptogramin B (MLS) antibiotics. pRJ5 is nearly identical to the inducible MLS resistance plasmid pT48, and has homology with the S. aureus plasmids pE194 and pSN2. The HindIII-C and/or Hind-B fragments were required for stable maintenance of the plasmid and probably carry palA. Plasmids pRJ5 and pT48 were shown to belong to the same incompatibility group, Inc12 (L). DNA sequencing showed that pRJ5 contains a 28 bp direct tandem duplication in the leader/attenuator region of ermC. This is likely to change the secondary structure of the methylase mRNA, allowing constitutive expression of ermC. The type of mutation found on plasmid pRJ5 is different from those observed in similar 2.5 kb constitutive MLS-resistance plasmids isolated from other Gram-positive bacteria, including staphylococci.

The StyLT1 restriction-modification (R-M) system of Salmonella typhimurium has recently been suggested to belong to the type-III R-M systems [De Backer and Colson, Gene 97 (1991) 103-107]. The nucleotide sequences of StyLT1 mod and res have been determined. Two closely adjacent open reading frames were found 12 bp apart with coding capacities of 651 (Mod) and 982 (Res) amino acids (aa), respectively. The genes, lying in the same direction of transcription in the mod-res order, are transcribed as distinct units. The deduced aa sequences reveal homologies with known type-III enzymes from the Escherichia coli P1 prophage, E. coli P15 plasmid and Bacillus cereus chromosome. In addition, the StyLT1 restriction endonuclease (ENase), like other type-I and type-III ENases, contains sequence motifs characteristic of superfamily-II helicases, which may be involved in DNA unwinding at the cleavage site.

High-resolution S1 nuclease mapping of mRNA synthesised in vivo, in vitro run-off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the SalI restriction-modification system of Streptomyces albus G. The salIR and salIM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal-pR1, a promoter located immediately upstream of salIR, with two possible minor promoters further upstream. Another promoter, sal-pM, is within the 3' end of the salIR coding region, and allows expression of the modification gene in the absence of sal-pR1. The sal-pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity. Sequences upstream of the apparent transcriptional start sites for sal-pR1 and sal-pM show similarity with the -10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered -35 regions are absent.

The hsdR, hsdM and hsdS genes coding for R.EcoK restriction endonuclease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coli C3-6. The presence of the hsdSts-1 mutation has no effect on the R-M phenotype of this construct in bacteria grown at 42 degrees C. However, DNA sequencing indicates that the mutation is still present on the pBR322-hsdts-1 operon. The putative temperature-sensitive endonuclease was purified from bacteria carrying this plasmid and the ability to cleave and methylate plasmid DNA was investigated. The mutant endonuclease was found to show temperature-sensitivity for restriction. Modification was dramatically reduced at both the permissive and non-permissive temperatures. The wild type enzyme was found to cleave circular DNA in a manner which strongly suggests that only one endonuclease molecule is required per cleavage event. Circular and linear DNA appear to be cleaved using different mechanisms, and cleavage of linear DNA may require a second endonuclease molecule. The subunit composition of the purified endonucleases was investigated and compared to the level of subunit production in minicells. There is no evidence that HsdR is prevented from assembling with HsdM and HsdSts-1 to produce the mutant endonuclease. The data also suggests that the level of HsdR subunit may be limiting within the cell. We suggest that an excess of HsdM and HsdS may produce the methylase in vivo and that assembly of the endonuclease may be dependent upon the prior production of this methylase.

Nucleic acid amplification techniques have and are revolutionising the way research, academia, medicine, environmental and quality control issues are dealt with. In particular, the polymerase chain reaction, (PCR), originally mentioned in 1985 has greatly modified the ways in which scientists and clinicians go about nucleic acid sequence analysis and manipulation and has great implications for molecular biology in general and diagnostics in particular. This report attempts to review the use of PCR and isothermal nucleic acid amplification techniques in the rapid identification of microorganisms. Key methodologies have been noted, their strengths and weaknesses commented upon and comparisons have been drawn between isothermal and thermally cycled nucleic amplification techniques in rapid microbial identification.

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.BsuBI) consists of 1503 bp, encoding a protein of 501 amino acids with a calculated M(r) of 57.2 kD. The gene of the restriction endonuclease (R.BsuBI), comprising 948 bp, codes for a protein of 316 amino acids with a predicted M(r) of 36.2 kD. M.BsuBI modifies the adenine (A) residue of the BsuBI target site, thus representing the first A-N6-DNA methyltransferase identified in B. subtilis. Like R.PstI, R.BsuBI cleaves between the A residue and the 3' terminal G of the target site. Both enzymes of the BsuBI R/M system are, therefore, functionally identical with those of the PstI R/M system, encoded by the Gram negative species Providencia stuartii. This functional equivalence coincides with a pronounced similarity of the BsuBI/PstI DNA methyltransferases (41% amino acid identity) and restriction endonucleases (46% amino acid identity). Since the genes are also very similar (58% nucleotide identity), the BsuBI and PstI R/M systems apparently have a common evolutionary origin. In spite of the sequence conservation the gene organization is strikingly different in the two R/M systems. While the genes of the PstI R/M system are separated and transcribed divergently, the genes of the BsuBI R/M system are transcribed in the same direction, with the 3' end of the M gene overlapping the 5' end of the R gene by 17 bp.

A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco57I was cloned and expressed in E.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco57I endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7I, TaqI, CviBIII, PstI, BamHI and HincII methylases. Homologs of this sequence are also found within the sequences of the PaeR7I, PstI and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.

The interactions of an arsenic (III) reagent, (CH3)2AsSCH2CONH2, with two Escherichia coli RI methyltransferase mutants, W183F and C223S, have been studied by phosphorescence, optically detected magnetic resonance, and fluorescence spectroscopy. The phosphorescence spectrum of the W183F mutant containing only one tryptophan at position 225 reveals a single 0,0-band that is red-shifted by 9.8 nm upon binding of As(III). Fluorescence titration of W183F with (CH3)2AsSCH2CONH2 produces a large tryptophan fluorescence quenching. Analysis of the quenching data points to a single high-affinity As(III) binding site that is associated with the fluorescence quenching. Triplet-state kinetic measurements performed on the perturbed tryptophan show large reductions in the lifetimes of the triplet sublevels, especially that of the T chi sublevel. As(III) binding to the enzyme at a site very close to the Trp225 residue induces an external heavy-atom effect, showing that the perturber atom is in van der Waals contact with the indole chromophore. In the case of the C223S mutant, a single tryptophan 0,0-band also is observed in the phosphorescence spectrum, but no change occurs upon addition of the As(III) reagent. Fluorescence titration of C223S with As(III) shows essentially no quenching of tryptophan fluorescence, in contrast with W183F. These results, along with previous triplet-state and biochemical studies on the wild-type enzyme [Tsao, D. H.H., & Maki, A. H. (1991) Biochemistry 30, 4565-4572], show that As(III) binds with high affinity to the Cys223 residue and that the Trp225 side chain is located close enough to that of Cys223 to produce a heavy-atom perturbation when As(III) is bound.

We have cloned and sequenced the cvibIM gene from Chlorella virus NC-1A by selecting for the modification phenotype. The modification gene was cloned on a 7-kb BamHI fragment inserted into the BamHI site of the pUC13 plasmid. The cvibIM gene was localized at the 3' end of this fragment. Sequencing of this region revealed a large open reading frame that codes for methyltransferase (MTase; symbol M.) (predicting 260 amino acids). M.CviBI (GANTC) aa sequence is homologous to M.Dam(GATC), M.DpnII(GATC), and M.T4 (GATC), and not so to M.HinfI(GANTC), M.HhaII (GANTC), and M.DpnA(GATC). We also describe the use of the polymerase chain reaction technique to alter transcriptional and translational signals surrounding this gene so as to achieve overexpression in Escherichia coli. This construct yields M.CviBI at 2-3% of the total cellular protein. The MTase was purified by phosphocellulose, DEAE, and gel filtration chromatography. Its size by SDS-PAGE is approx. 28 kDa, in good agreement with that predicted from the nucleotide sequence.

A second DNA site-specific (restriction) endonuclease (R.CviAII) and its cognate adenine DNA methyltransferase (M.CviAII) were isolated from virus PBCV-1 infected Chlorella strain NC64A cells. R.CviAII, a heteroschizomer of the bacterial restriction endonuclease NlaIII, recognizes the sequence CATG, and does not cleave CmATG sequences. However, unlike NlaIII, which cleaves after the G and does not cleave either CmATG or mCATG sequences, CviAII cleaves between the C and A and is unaffected by mCATG methylation. The M.CviAII and R.CviAII genes were cloned and their DNA sequences were determined. These genes are tandemly arranged head-to-tail such that the TAA termination codon of the M.CviAII methyltransferase gene overlaps the ATG translational start site of R.CviAII endonuclease. R.CviAII is the first chlorella virus site-specific endonuclease gene to be cloned and sequenced.

EcoRI DNA methyltransferase contains tryptophans at positions 183 and 225. Tryptophan 225 is adjacent to residues previously implicated in S-adenosylmethionine (AdoMet) binding and to cysteine 223, previously shown to be the site of N-ethyl maleimide-mediated inactivation of the enzyme (Reich, N. O., and Everett, E. (1990) J. Biol. Chem. 265, 8929-8934; Everett, E. A., Falick, A. M., and Reich, N. O. (1990) J. Biol. Chem. 265, 17713-17719). The fluorescence spectra of the wild-type enzyme is centered at 338 nm indicating partial tryptophan solvent accessibility. Substitution of tryptophan 183 with phenylalanine results in a 45% drop in fluorescence intensity, but no shift in lambda max. DNA binding to the wild-type methyltransferase caused an increase in the fluorescence intensity, while binding to the tryptophan 183 mutant had a quenching effect, suggesting that DNA binding induces a conformational change near both tryptophans. Binding of AdoMet and various AdoMet analogs to the wild-type methyltransferase results in no change in the fluorescence spectrum when excitation occurs at 295 nm, suggesting that no conformational change occurs, and AdoMet does not interact with either tryptophan. In contrast, quenching was observed when excitation occurred at 280 nm, suggesting that AdoMet and its analogs may be quenching tyrosine to tryptophan energy transfer. Protein-ligand complexes were titrated with acrylamide, and the data also implicate conformational changes upon DNA binding but not upon AdoMet binding, consistent with previous limited proteolysis results (Reich, N. O., Maegley, K. A., Shoemaker, D.D., and Everett, E. (1991) Biochemistry 30, 2940-2946).

The gene (rsrIM) encoding the RsrI DNA methyltransferase (M.RsrI) from Rhodobacter sphaeroides was cloned and expressed in Escherichia coli. Under the control of a bacteriophage T7 promoter, 2% of the total protein in a crude extract was M.RsrI. This level of expression represents an approximately 50-fold increase over that present in the natural host. Chromatography using DNA cellulose and the S-adenosylmethionine analogue, sinefungin, was useful in purifying the enzyme to homogeneity. The purification yielded 100 times more enzyme than was obtained from the same quantity of R. sphaeroides cell paste. M.RsrI deposits one methyl group per productive DNA-binding event, as does its functional but sequence-nonhomologous analogue, M.EcoRI. Unlike M.EcoRI, the R. sphaeroides enzyme is a dimer at micromolar concentrations.

The gene coding for Escherichia coli dam methylase was isolated from a dam+ K12 strain by the PCR method. The gene was subcloned into an overexpression vector under the control of the strong lambda PL promoter. The resultant construct produced the dam methylase at about 20% of total cellular protein. Purification of the protein was achieved with two chromatography columns and yielded 6 mg of pure methylase per gram cell paste. The methylase readily methylates the synthetic dodecamer GACTGATCAGTC containing its recognition sequence (underlined). It also methylates a synthetic dodecamer containing the EcoRV recognition sequence GATATC. However, methyl transfer is to the second adenine in the EcoRV sequence.

We have used the polymerase chain reaction to alter transcriptional and translational signals surrounding the hinfIM gene [encoding M.HinfI methyltransferase (MTase)] so as to achieve overexpression in Escherichia coli. The PCR-generated hinfIM gene was subcloned in a high-expression vector under control of the hybrid trp-lac promoter. In addition, the positive retroregulator stem-loop sequence derived from the crystal protein-encoding gene of Bacillus thuringiensis was inserted downstream from hinfIM. Using a similar approach, we have also constructed overproducer clones of a deletion mutant of M.HinfI MTase that has 97 amino acids from the C terminus deleted. The plasmid from the mutant clones is fully protected from HinfI restriction endonuclease digestion. It appears that the functional properties (the recognition and catalytic functions) are encoded within this mutant gene. The overproducer clones yield the wild type (wt) and the mutant enzymes to about 10% of total cellular protein upon induction with 1 mM IPTG. The wt M.HinfI and the mutant MTase were purified to near electrophoretic homogeneity by phosphocellulose, DEAE and gel chromatography. Their monomer sizes by SDS/polyacrylamide-gel electrophoresis are 43 kDa and 31 kDa, respectively, in good agreement with that predicted from the nucleotide sequence. DNA methylation experiments with purified enzymes using single-strand and double-strand M13mp18 DNA substrates indicate that while wt enzyme methylates both forms of DNA substrates, the mutant enzyme appears to preferentially methylate ss DNA substrate.

The nucleotide sequence of the genes encoding methyltransferase TaqI (M.TaqI) and restriction endonuclease TaqI (R.TaqI) with the recognition sequence, TCGA, were analyzed in clones isolated from independent libraries. The genes, originally reported as 363 and 236 codons long [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] were redetermined as 421 and 263 codons long, respectively. The C terminus of the taqIM gene overlaps the N terminus of the taqIR gene by 13 codons, as observed with the isoschizomeric TthHB8I restriction-modification system [Barany et al., Gene 112 (1992) 13-20]. Removal of the overlapping codons did not interfere with in vivo M.TaqI activity. We postulate the overlap plays a role in regulating taqIR expression.

Genes encoding the TthHB8I restriction and modification (R-M) system from Thermus thermophilus HB8 (recognition sequence T decreases CGA) were cloned in Escherichia coli. The genes have the same transcriptional orientation, with the last 13 codons of the methyltransferase (MTase) overlapping the first 13 codons of the endonuclease (ENase). Nucleotide sequence analysis of the TthHB8I ENase revealed a single chain of 263 amino acid (aa) residues that share a 77% identity with the corrected isoschizomeric TaqI ENase. Likewise, the Tth MTase (428 aa) shares a 79% identity with the corrected sequence of the TaqI MTase. This high degree of aa conservation suggests a common origin between the Taq and Tth R-M systems. However, codon usage and G+C content for the R-M genes differed markedly from that of other cloned Thermus genes. This suggests that these R-M genes were only recently introduced into the genus Thermus.

The genes from Haemophilus parainfluenzae encoding the HpaI restriction-modification system were cloned and expressed in Escherichia coli. From the DNA sequence, we predicted the HpaI endonuclease (R.HpaI) to have 254 amino acid residues (Mr 29,630) and the HpaI methyltransferase (M.HpaI) to have 314 amino acid residues (37,390). The R.HpaI and M.HpaI genes overlapped by 16 base pairs on the chromosomal DNA. The genes had the same orientation. The clone, named E. coli HB101-HPA2, overproduced R.HpaI. R.HpaI activity from the clone was 100-fold that from H. parainfluenzae. The amino acid sequence of M.HpaI was compared with those of other type II methyltransferases.

The KpnI restriction-modification (KpnI RM) system was previously cloned and expressed in E. coli. The nucleotide sequences of the KpnI endonuclease (R.KpnI) and methylase (M. KpnI) genes have now been determined. The sequence of the amino acid residues predicted from the endonuclease gene DNA sequence and the sequence of the first 12 NH2-terminal amino acids determined from the purified endonuclease protein were identical. The kpnIR gene specifies a protein of 218 amino acids (MW: 25,115), while the kpnIM gene codes for a protein of 417 amino acids (MW: 47,582). The two genes transcribe divergently with a intergeneic region of 167 nucleotides containing the putative promoter regions for both genes. No protein sequence similarity was detected between R.KpnI and M.KpnI. Comparison of the amino acid sequence of M.KpnI with sequences of various methylases revealed a significant homology to N6-adenine methylases, a partial homology to N4-cytosine methylases, and no homology to C5-methylases.

The Rsr1 protein of Saccharomyces cerevisiae has been shown to be essential for bud site selection (Bender, A., and Pringle, J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9976-9980). This protein of 272 amino acids shares approximately 50% sequence identity with both Ras and Rap GTPases. However, neither GTP binding nor GTPase activity of the Rsr1 protein has been reported. The Rsr1 protein shares with human Rap1 GTPases the four specific motifs, i.e. Gly-12, residues 32-40, Ala-59, and residues 64-70, that are required for GAP3-dependent activation of the Rap1 GTPases. In this paper we demonstrate that the intrinsic GTPase activity of the Rsr1 protein is stimulated by GAP3 purified from bovine brain cytosol. The Rsr1 GTPase is not activated by either GAP1 or GAP2 which are specific for the Ras and Rho GTPases, respectively. Thus, it appears that the Rsr1 GTPase is a new member of the Rap1 GTPase family. Replacement of Gly-12 by Val in the Rsr1 GTPase completely abolishes the GAP3-dependent activation. The chimeric GTPases, Ras(1-60)/Rsr1(61-168) and Rsr1(1-65)/Ras(66-189), are activated by GAP3 but not by GAP1. Replacement of Thr-65 by Ser in the latter chimeric GTPase completely abolishes the GAP3-dependent activation, indicating that Thr-65 is required for distinguishing GAP3 from GAP1. We have previously shown that Gln-61 and Ser-65 are sufficient to determine the GAP1 specificity. Replacement of Thr-35 by Ala in the common effector domain (residues 32-40) of the chimeric Ras/Rsr1 GTPases completely abolishes GAP3-dependent activation.

A gene from the periodontal organism Porphyromonas gingivalis has been identified as encoding a DNA methylase. The gene, referred to as pgiIM, has been sequenced and found to contain a reading frame of 864 basepairs. The putative amino acid sequence of the encoded methylase was 288 amino acids, and shared 47% and 31% homology with the Streptococcus pneumoniae DpnII and E. coli Dam methylases, respectively. The activity and specificity of the pgi methylase (M.PgiI) was confirmed by cloning the gene into a dam- strain of E. coli (JM110) and performing a restriction analysis on the isolated DNA with enzymes whose activities depended upon the methylation state of the DNA. The data indicated that M.PgiI, like DpnII and Dam, methylated the adenine residue within the sequence 5'-GATC-3'.

The conjugative plasmid pTR2030 confers bacteriophage resistance to lactococci by two independent mechanisms, an abortive infection mechanism (Hsp+) and a restriction and modification system (R+/M+). pTR2030 transconjugants of lactococcal strains are used in the dairy industry to prolong the usefulness of mesophilic starter cultures. One bacteriophage which has emerged against a pTR2030 transconjugant is not susceptible to either of the two defense systems encoded by the plasmid. Phage nck202.50 (phi 50) is completely resistant to restriction by pTR2030. A region of homology between pTR2030 and phi 50 was subcloned, physically mapped, and sequenced. A region of 1,273 bp was identical in both plasmid and phage, suggesting that the fragment had recently been transferred between the two genomes. Sequence analysis confirmed that the transferred region encoded greater than 55% of the amino domain of the structural gene for a type II methylase designated LlaI. The LlaI gene is 1,869 bp in length and shows organizational similarities to the type II A methylase FokI. In addition to the amino domain, upstream sequences, possibly containing the expression signals, were present on the phage genome. The phage phi 50 fragment containing the methylase amino domain, designated LlaPI, when cloned onto the shuttle vector pSA3 was capable of modifying another phage genome in trans. This is the first report of the genetic exchange between a bacterium and a phage which confers a selective advantage on the phage. Definition of the LlaI system on pTR2030 provides the first evidence that type II systems contribute to restriction and modification phenotypes during host-dependent replication of phages in lactococci.

The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.

The genes encoding the restriction-modification system StyLTI of Salmonella typhimurium were inserted in vivo into the conjugative plasmid pULB21. This allowed us to transfer the StyLTI genes at a very high frequency and to monitor the fate of recipient cells after mating. Transfer of the StyLTI restriction and modification genes into a modificationless recipient was lethal and resulted in degradation of the cell's DNA. This indicates that, in contrast to any other known restriction-modification systems, StyLTI cannot be established after horizontal transfer into a naive host.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.

We constructed a semisynthetic gene encoding a DNA-adenine-methyltransferase (Dam) that codes for the same amino acid sequence as the wild type (wt) Escherichia coli dam gene. Since for unknown reasons the entire wt sequence, from the start codon to the end of the gene, could not be cloned, a gene was constructed consisting of a chemically synthesized 5' portion and a 3' portion from the E. coli chromosome. Introduction of this semisynthetic gene into a suitable vector allows overproduction of E. coli Dam in mg amounts per liter E. coli culture, with optimum expression of the gene in the vector pJLA503. This plasmid places the target gene under control of the strong, tandemly arranged pR pL promoters from bacteriophage lambda, regulated by a temperature-sensitive lambda repressor. A rapid, two-column purification protocol is described that allows for very fast purification of the protein. The 32-kDa recombinant protein methylates the sequence GATC.

The gene encoding the DNA methyltransferase M.CviRI from Chlorella virus XZ-6E was cloned and expressed in Escherichia coli. M.CviRI methylates adenine in TGCA sequences. DNA containing the M.CviRI gene was sequenced and a single open reading frame of 1137 bp was identified which could code for a polypeptide of 379 amino acids with a predicted molecular weight of 42,814. Comparison of the M.CviRI predicted amino acid sequence with another Chlorella virus and 14 bacterial adenine methyltransferases revealed extensive similarity to the other Chlorella virus enzyme.

The gene coding for the ATCGAT specific BanIII DNA methyltransferase (M-BanIII) of Bacillus aneurinolyticus was cloned and its nucleotides sequenced. The coding region was assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequence and molecular weight of the enzyme. The M-BanIII gene coded for a protein of 580 amino acid residues (MW 66,344). Comparison with other methylases indicated that the M-BanIII sequence contained a segment of tetra-amino acids, NPPY, characteristic of N6-adenine methylases. In addition, some homologous regions were found in the sequences of type II adenine methylases PaeR7I(CTCGAG), TaqI(TCGA) and PstI(CTGCAG), containing TCGA within the recognition sequences.

The gene encoding the Neisseria lactamica III DNA methyltransferase (M.NlaIII) which recognizes the sequence CATG has been cloned and expressed in Escherichia coli. DNA sequencing of a 3.125 kb EcoRI-PstI fragment localizes the M. NlaIII gene to a 334 codon open reading frame (ORF) and identifies, 468 bp downstream, a second ORF of 313 amino acids, which is referred to as M.NlaX. Both proteins are detectable in the E. coli coupled in vitro transcription-translation system; they are apparently expressed from separate N. lactamica promoters. The N-terminal half of the previously characterized M.FokI, which methylates adenine in one of the DNA strands with its asymmetric recognition sequence (GGATG), is found to have 41% sequence identity and a further 11.7% sequence similarity with M.NlaIII. Among the conserved amino acids is the wellknown DPPY sequence motif. With one exception, analysis of the nucleotides coding for the DP dipeptide in all known DPPY sequences shows the presence of an inherent DNA adenine methylation (dam) recognition site of GATC. A low level of expression of M.NlaX in E. coli prevents the elucidation of its sequence recognition specificity. Sequence analysis of M.NlaX shows that it is closely related to the group of monospecific 5-methylcytosine DNA methyltransferases (M.EcoRII, Dcm, M.HpaII and M.HhaI) which all have a modified cytosine at the second position of the recognition sequences. Both M.EcoRII and Dcm amino acid sequences are about 50% identical with M.NlaX; a considerable degree of sequence identity is found in the so-called variable region which is believed to be responsible for sequence recognition specificity. M.NlaX is probably the counterpart to the E. coli Dcm in N. lactamica.

We have developed an assay that allows analysis of the activity of EcoRI restriction endonuclease (ENase) and its mutants in vivo. This assay is based on the fact that wild type (wt) EcoRI ENase is toxic for Escherichia coli cells not expressing the EcoRI methyltransferase (MTase). The viability factor defined by the ratio of the viable counts of E. coli cultures having or not having expressed the ecoRIR gene for a defined time is 10(-6) for wt EcoRI ENase and close to one for a totally inactive EcoRI ENase mutant. While the EcoRI MTase (M.EcoRI) provides substantial protection against the toxic effects of the wt EcoRI ENase and several of the mutants, some mutants become more toxic in the presence of M.EcoRI. Twenty-four different DNA-binding-site mutants of EcoRI ENase were characterized in their activity in vivo with this assay. The results obtained allow us to conclude that the structural integrity of the region at and around aa 200 seems to be very critical for the enzymatic function of EcoRI ENase: nonconservative replacements there lead to viability factors of 1-10(-2). While our results indicate that the region around aa 144 and 145 is also involved in the EcoRI ENase-catalyzed reaction, it is also evident that the effects of mutation there are not as large: viability factors of approx. 10(-3) are obtained even for drastic replacements. These results are discussed in the light of the x-ray structure analysis of an EcoRI ENase-DNA recognition complex.

The gene coding for the GGTNACC specific Ecal DNA methyltransferase (M.Ecal) has been cloned in E. coli from Enterobacter cloacae and its nucleotide sequence has been determined. The ecalM gene codes for a protein of 452 amino acids (Mr: 51,111). It was determined that M.Ecal is an adenine methyltransferase. M.Ecal shows limited amino acid sequence similarity to other adenine methyltransferases. A clone that expresses Ecal methyltransferase at high level was constructed.

The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.

Various plasmids that contain the Escherichia coli ksgA gene, which encodes a 16S rRNA adenosine dimethyltransferase (methylase), were constructed. In one of these plasmids, the DNA encoding the N-terminal part of the methylase was fused to the lacZ gene, and in another construct, the ksgA gene contained a deletion which resulted in a truncated version of the methylase. When a cell contained one plasmid directing the synthesis of the intact, active methylase and another plasmid encoding the methylase-beta-galactosidase protein, production of the latter product became strongly reduced. Likewise, synthesis of the truncated version of the methylase was diminished when the cell at the same time contained a plasmid producing the complete enzyme. These results were partly substantiated by in vitro experiments with a coupled transcription-translation assay system. By using a recently developed gel electrophoresis system for measuring protein-nucleic acid interactions, a specific binding of the ksgA methylase with its own mRNA could be established. Our results demonstrate that the expression of the ksgA gene can be, at least partly, autogenously controlled at the level of translation.

The repressor of bacteriophage P1, encoded by the c1 gene, is responsible for maintaining the P1 prophage in the lysogenic state. Previously, 11 c1 repressor binding sites or operators scattered over the whole genome of P1 have been found. From sequence analysis an asymmetric, 17-base pair consensus sequence, ATTGCTCTAATAAATTT, was derived. Using a synthetic 15-base-long oligodeoxyribonucleotide as operator probe, we have identified three additional operators. We have mapped the operators at the positions 21,68, and 88 of the P1 genome and determined their sequence. These operators are controlled by c1 because corresponding P1 DNA fragments (i) require c1 repressor in vivo in order to be clonable in multicopy plasmids, (ii) exhibit a c1-repressible promoter activity, (iii) are retarded by c1 repressor protein during electrophoresis, and (iv) contain the 17-base pair consensus sequence with one mismatch base each. Furthermore, we suggest that expression of the DNA adenine methylase (dam) encoded by P1 is controlled via Op68.

Vibrio cholerae mutants sensitive to 2-aminopurine (2AP) but with DNA adenine methylase activity similar to parental cells have been isolated. The mutant strains were sensitive to ultraviolet light (UV), methyl methane sulphonate (MMS) and 9-aminoacridine. The spontaneous mutation frequency of the mutants were not significantly affected. Attempts to isolate dam V. cholerae cells by screening 2AP sensitive cells have not been successful. All the mutant phenotypes could be suppressed by introducing the plasmid pRB103 carrying the dam gene of Escherichia coli into the mutant cells.

The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside results in overproduction of the methyltransferase to about 3% of total cellular protein. The methyltransferase was purified to near electrophoretic homogeneity by phosphocellulose, DEAE, and gel chromatography. Its monomer Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25 kDa, in good agreement with that predicted from the nucleotide sequence. Crystals of the methyltransferase were obtained in the presence of a two-fold molar excess of the duplex oligodeoxynucleotide substrate 5'd-GGACTCC.CCTGAGG.

The sequences of the genes coding for M.CviBIII (from virus NC-1A which infects a eukaryotic alga) [Narva et al., Nucleic Acids Res. 15 (1987) 9807-9823] and M.TaqI (from the bacterium Thermus aquaticus) [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] have been determined recently. Both enzymes methylate adenine in the sequence TCGA. We have compared the predicted amino acid sequences of these two methyltransferases (MTases), with each other and with ten other N6 A-MTases and find regions of similarity. M.CviBIII and M.TaqI were most closely related followed by M.PaeR7, whose recognition sequence (CTCGAG) contains the M.TaqI/M.CviBIII recognition sequence TCGA, and M.PstI, whose recognition sequence is CTGCAG. All of the N6-MTases contain the sequence Asp/Asn-Pro-Pro-Tyr (B-P-P-Y) referred to by Hattman et al. [J. Bacteriol. 164 (1985) 932-937] as region IV. The predicted secondary structure of this region forms a finger-like structure ('beta finger') containing a beta-pleated sheet (...XXXB), two beta-turns (P-P) followed by another beta-pleated sheet [Y/FXXX...].

The two DNA-adenine methylases encoded by the Dpn II restriction gene cassette were purified, and their activities were compared on various DNA substrates. DpnA was able to methylate single-strand DNA and double-strand DNA, whereas DpnM methylated only double-strand DNA. Although both enzymes act at 5'-GATC-3' in DNA, DpnA can also methylate sequences altered in the guanine position, but at a lower rate. A deletion mutation in the dpnA gene was constructed and transferred to the chromosome. Transmission by way of the transformation pathway of methylated and unmethylated plasmids to dpnA mutant and wild-type recipients was examined. The mutant cells restricted unmethylated donor plasmid establishment much more strongly than did wild-type cells. In the wild type, the single strands of donor plasmid DNA that enter by the transformation pathway are apparently methylated by DpnA prior to conversion of the plasmid to a double-strand form, in which the plasmid would be susceptible to the Dpn II endonuclease. The biological function of DpnA may, therefore, be the enhancement of plasmid transfer to Dpn II-containing strains of Streptococcus pneumoniae.

Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam) which recognizes primarily the sequence GATC in both cytosine- and hydroxymethylcytosine-containing DNA. Hypermethylating mutants, damh, exhibit a relaxation in sequence specificity, that is, they are readily able to methylate non-canonical sites. We have determined that the damh mutation produces a single amino acid change (Pro126 to Ser126) in a region of homology (III) shared by three DNA-adenine methyltransferases; viz, T4 Dam, Escherichia coli Dam, and the DpnII modification enzyme of Streptococcus pneumoniae. We also describe another mutant, damc, which methylates GATC in cytosine-containing DNA, but not in hydroxymethylcytosine-containing DNA. This mutation also alters a single amino acid (Phe127 to Val127). These results implicate homology region III as a domain involved in DNA sequence recognition. The effect of several different amino acids at residue 126 was examined by creating a polypeptide chain terminating codon at that position and comparing the methylation capability of partially purified enzymes produced in the presence of various suppressors. No enzyme activity is detected when phenylalanine, glutamic acid, or histidine is inserted at position 126. However, insertion of alanine, cysteine, or glycine at residue 126 produces enzymatic activity similar to Damh.