Identification and characterization of the gyrB gene from Treponema pallidum subsp. pallidum

The nucleotide sequence of a DNA gyrase B subunit gene (gyrB) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria. The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE). Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T. pallidum GyrB protein.     

Sequence specificity of the wild-type dam+) and mutant (damh) forms of bacteriophage T2 DNA adenine methylase

Non-glucosylated, non-methylated phage T2 DNA was methylated in vitro with partially purified wild-type (dam⁺) or mutant (dam^h) T2 DNA adenine methylase. The radioactively labeled methyladenine-containing DNA was enzymatically degraded and the resulting oligonucleotides were separated according to chain length by DEAE-cellulose chromatography. Following “fingerprinting” by two-dimensional electrophoresis, we determined the sequence for various di-, tri- and tetranucleotides containing radioactive N⁶-methyldeoxyadenosine. From this analysis we conclude that both T2 dam⁺ and T2 dam^h contain the sequence 5′…G-mA -Py…3′.     

Identification of the Treponema pallidum subsp. pallidum glycerophosphodiester phosphodiesterase homologue

To identify potential opsonic targets of Treponema pallidum subsp. pallidum, a treponemal genomic expression library was constructed and differentially screened with opsonic and non-opsonic T. pallidum antisera. This method identified an immunoreactive clone containing an open reading frame encoding a 356 residue protein. Nucleotide sequence analysis demonstrated the translated protein to be a homologue of glycerophosphodiester phosphodiesterase, a glycerol metabolizing enzyme previously identified in Haemophilus influenzae, Escherichia coli, Bacillus subtilis and Borrelia hermsii. Sequence alignment analyses revealed the T. pallidum and H. influenzae enzymes share a high degree of amino acid sequence similarity (72%), suggesting that in T. pallidum this molecule may be surface exposed and involved in IgD binding as is the case with its counterpart in H. influenzae.     

In vitro methylation of bacteriophage lambda DNA by wild type (dam+) and mutant (damh) forms of the phage T2 DNA adenine methylase.

The wild-type (dam⁺) and mutant (dam^h) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam⁺ and dam^h enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 dam^h enzyme methylated λ DNA to a higher level than did the dam⁺ enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 dam^h, but not T2 dam⁺ protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R·EcoP1. (3) T2 dam⁺ and dam^h were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ·dam₃ DNA (contains no N⁶-methyladonine) methylated with either enzyme was made resistant to cleavage by R·DpnII. In contrast, only the T2 dam^h modified DNA was resistant to further methylation by M·EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978b). (4) λ·dam₃ DNA was partially methylated to the same level with T2 dam⁺ or T2 dam^h; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam⁺ enzyme methylates G-A-C sequences less efficiently than the T2 dam^h methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes.     

Expression of a DNA methylation (dam) gene inEscherichia coli K-12

Plasmid pMQ3, carrying thedam gene ofEscherichia coli on a 6.1 Kb fragment, shows a tenfold increase in relative DNA adenine methylase activity, while plasmid pdam118, with a 1.14 Kbdam insert, shows only a twofold increase, although both plasmids were derived from plasmid pLC13–42. Since a copy number effect did not seem to be the cause of this difference, we have subcloned pMQ3 in order to determine whether the additional chromosomal DNA present in this plasmid is responsible for the enhancement of methylase activity. We show that the 346 base pairs upstream ofdam contain sequences necessary for expression. DNA sequence analysis has revealed that in pdam118 only the 118 bases 5-prime to thedam gene are present in other constructs and that the additional upstream material is pBR322 DNA. This shows that pdam118 carries a DNA duplication.     

The Cyanobacterium Synechocystis sp. Strain PCC 6803 Expresses a DNA Methyltransferase Specific for the Recognition Sequence of the Restriction Endonuclease PvuI

By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a dam-like site including N⁶-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain. slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C⁵-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in slr0214 were generated by the insertion of an aphII gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5′-CGATCG-3′, corresponding to the recognition sequence of PvuI. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis. The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro. We propose the designation SynMI (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.     

Molecular typing of Treponema pallidum: a systematic review and meta-analysis

Background

Syphilis is resurgent in many regions of the world. Molecular typing is a robust tool for investigating strain diversity and epidemiology. This study aimed to review original research on molecular typing of Treponema pallidum (T. pallidum) with three objectives: (1) to determine specimen types most suitable for molecular typing; (2) to determine T. pallidum subtype distribution across geographic areas; and (3) to summarize available information on subtypes associated with neurosyphilis and macrolide resistance.

Methodology/Principal Findings

Two researchers independently searched five databases from 1998 through 2010, assessed for eligibility and study quality, and extracted data. Search terms included “Treponema pallidum,” or “syphilis,” combined with the subject headings “molecular,” “subtyping,” “typing,” “genotype,” and “epidemiology.” Sixteen eligible studies were included. Publication bias was not statistically significant by the Begg rank correlation test. Medians, inter-quartile ranges, and 95% confidence intervals were determined for DNA extraction and full typing efficiency. A random-effects model was used to perform subgroup analyses to reduce obvious between-study heterogeneity. Primary and secondary lesions and ear lobe blood specimens had an average higher yield of T. pallidum DNA (83.0% vs. 28.2%, χ² = 247.6, p<0.001) and an average higher efficiency of full molecular typing (80.9% vs. 43.1%, χ² = 102.3, p<0.001) compared to plasma, whole blood, and cerebrospinal fluid. A pooled analysis of subtype distribution based on country location showed that 14d was the most common subtype, and subtype distribution varied across geographic areas. Subtype data associated with macrolide resistance and neurosyphilis were limited.

Conclusions/Significance

Primary lesion was a better specimen for obtaining T. pallidum DNA than blood. There was wide geographic variation in T. pallidum subtypes. More research is needed on the relationship between clinical presentation and subtype, and further validation of ear lobe blood for obtaining T. pallidum DNA would be useful for future molecular studies of syphilis.     

Genetic engineering of Treponema pallidum subsp. pallidum,the Syphilis Spirochete

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kan^R) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kan^R gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kan^R cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kan^R cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl₂-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kan^R cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kan^R message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprA^ko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.     

The Escherichia coli dam gene is expressed as a distal gene of a new operon

Summary DNA containing the Escherichia coli dam gene and sequences upstream from this gene were cloned from the Clarke-Carbon plasmids pLC29-47 and pLC13-42. Promoter activity was localized using pKO expression vectors and galactokinase assays to two regions, one 1650–2100 bp and the other beyon 2400 bp upstream of the dam gene. No promoter activity was detected immediately in front of this gene; plasmid pDam118, from which the nucleotide sequence of the dam gene was determined, is shown to contain the pBR322 promoter for the primer RNA from the pBR322 rep region present on a 76 bp Sau3A fragment inserted upstream of the dam gene in the correct orientation for dam expression. The nucleotide sequence upstream of dam has been determined. An open reading frame (ORF) is present between the nearest promoter region and the dam gene. Codon usage and base frequency analysis indicate that this is expressed as a protein of predicted size 46 kDa. A protein of size close to 46 kDa is expressed from this region, detected using minicell analysis. No function has been determined for this protein, and no significant homology exist between it and sequences in the PIR protein or GenBank DNA databases. This unidentified reading frame (URF) is termed urf-74.3, since it is an URF located at 74.3 min on the E. coli chromosome. Sequence comparisons between the regions upstream of urf-74.3 and the aroB gene show that the aroB gene is located immediately upstream of urf-74.3, and that the promoter activity nearest to dam is found within the aroB structural gene. This activity is relatively weak (about 15% of that of the E. coli gal operon promoter). The promoter activity detected beyond 2400 bp upstream of dam is likely to be that of the aroB gene, and is 3 to 4 times stronger than that found within the aroB gene. Three potential DnaA binding sites, each with homology of 8 of 9 bp, are present, two in the aroB promoter region and one just upstream of the dam gene. Expression through the site adjacent to the dam gene is enhanced 2-to 4-fold in dnaA mutants at 38°C. Restriction site comparisons map these regions precisely on the Clarke-Carbon plasmids pLC13-42 and pLC29-47, and show that the E. coli ponA (mrcA) gene resides about 6 kb upstream of aroB.     

Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.     

Adenine Methylation in Eukaryotic DNA

N⁶-Methyladenine (m⁶A) has been found in DNAs of various eukaryotes (algae, fungi, protozoa, and higher plants). Like bacterial DNA, DNAs of these organisms are subject to enzymatic modification (methylation) not only at cytosine, but also at adenine bases. There is indirect evidence that adenine methylation of the genome occurs in animals as well. In plants, m⁶A was detected in total, mitochondrial, and nuclear DNAs. It was observed that both adenines and cytosines can be methylated in one gene (DRM2). Open reading frames coding for homologs of bacterial adenine DNA methyltransferases were revealed in protozoan, yeast, higher plant, insect, nematode, and vertebrate genomes, suggesting the presence of adenine DNA methyltransferases in evolutionarily distant eukaryotes. The first higher-eukaryotic adenine DNA N⁶-methyltransferase (wad-mtase) was isolated from vacuolar vesicles of wheat coleoptiles. The enzyme depends on Mg²⁺ or Ca²⁺ and, in the presence of S-adenosyl-L-methionine, methylates de novo the first adenine of the sequence TGATCA in single- and double-stranded DNAs, preferring the former. Adenine methylation of eukaryotic DNA is probably involved in regulating gene expression and replication, including that of mitochondrial DNA; plays a role in controlling the persistence of foreign DNA in the cell; and acts as a component of a plant restriction— modification system. Thus, the eukaryotic cell has at least two different systems for enzymatic methylation of DNA (at adenines and at cytosines) and a special mechanism regulating the functions of genes via a combinatorial hierarchy of these interdependent modifications of the genome.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 557–566.Original Russian Text Copyright © 2005 by Vanyushin.To the memory of my teacher, Academician Andrei Nikolaevich Belozersky     

Rapid Treponema pallidum Clearance from Blood and Ulcer Samples following Single Dose Benzathine Penicillin Treatment of Early Syphilis

Currently, the efficacy of syphilis treatment is measured with anti-lipid antibody tests. These can take months to indicate cure and, as a result, syphilis treatment trials require long periods of follow-up. The causative organism, Treponema pallidum (T. pallidum), is detectable in the infectious lesions of early syphilis using DNA amplification. Bacteraemia can likewise be identified, typically in more active disease. We hypothesise that bacterial clearance from blood and ulcers will predict early the standard serology-measured treatment response and have developed a qPCR assay that could monitor this clearance directly in patients with infectious syphilis. Patients with early syphilis were given an intramuscular dose of benzathine penicillin. To investigate the appropriate sampling timeframe samples of blood and ulcer exudate were collected intensively for T. pallidum DNA (tpp047 gene) and RNA (16S rRNA) quantification. Sampling ended when two consecutive PCRs were negative. Four males were recruited. The mean peak level of T. pallidum DNA was 1626 copies/ml whole blood and the mean clearance half-life was 5.7 hours (std. dev. 0.53). The mean peak of 16S rRNA was 8879 copies/ml whole blood with a clearance half-life of 3.9 hours (std. dev. 0.84). From an ulcer, pre-treatment, 67,400 T. pallidum DNA copies and 7.08x107 16S rRNA copies were detected per absorbance strip and the clearance half-lives were 3.2 and 4.1 hours, respectively. Overall, T. pallidum nucleic acids were not detected in any sample collected more than 56 hours (range 20–56) after treatment. All patients achieved serologic cure. In patients with active early syphilis, measuring T. pallidum levels in blood and ulcer exudate may be a useful measure of treatment success in therapeutic trials. These laboratory findings need confirmation on a larger scale and in patients receiving different therapies.     

Complete nucleotide sequence of the glutamate dehydrogenase gene from Escherichia coli K-12

A 2.3-kb PstI-ClaI chromosomal DNA segment, carrying the complete coding region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, has been sequenced. The complete amino acid sequence (447 residues) of the GDH monomer has been deduced, and comparisons are made with reported amino acid sequences of GDH from other organisms.     

Treponema pallidum Infection in the Wild Baboons of East Africa: Distribution and Genetic Characterization of the Strains Responsible

It has been known for decades that wild baboons are naturally infected with Treponema pallidum, the bacterium that causes the diseases syphilis (subsp. pallidum), yaws (subsp. pertenue), and bejel (subsp. endemicum) in humans. Recently, a form of T. pallidum infection associated with severe genital lesions has been described in wild baboons at Lake Manyara National Park in Tanzania. In this study, we investigated ten additional sites in Tanzania and Kenya using a combination of macroscopic observation and serology, in order to determine whether the infection was present in each area. In addition, we obtained genetic sequence data from six polymorphic regions using T. pallidum strains collected from baboons at two different Tanzanian sites. We report that lesions consistent with T. pallidum infection were present at four of the five Tanzanian sites examined, and serology was used to confirm treponemal infection at three of these. By contrast, no signs of treponemal infection were observed at the six Kenyan sites, and serology indicated T. pallidum was present at only one of them. A survey of sexually mature baboons at Lake Manyara National Park in 2006 carried out as part of this study indicated that roughly ten percent displayed T. pallidum-associated lesions severe enough to cause major structural damage to the genitalia. Finally, we found that T. pallidum strains from Lake Manyara National Park and Serengeti National Park were genetically distinct, and a phylogeny suggested that baboon strains may have diverged prior to the clade containing human strains. We conclude that T. pallidum infection associated with genital lesions appears to be common in the wild baboons of the regions studied in Tanzania. Further study is needed to elucidate the infection''s transmission mode, its associated morbidity and mortality, and the relationship between baboon and human strains.     

The mutK Gene of Vibrio cholerae: a New Gene Involved in DNA Mismatch Repair

A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants. The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V. cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product. The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E. coli vsr gene. ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V. cholerae strain from which the gene was isolated (10).     

Complementary action of restriction enzymes endo R-DpnI and Endo R-DpnII on bacteriophage f1 DNA

Bacteriophage f1 duplex DNA was isolated from Escherichia coli strains containing different DNA methylases and assayed for its sensitivity to endonucleolytic cleavage by the enzymes endo R · DpnI and endo R · DpnII. The former enzyme is specific for methylated DNA, the latter for unmethylated DNA (Lacks &; Greenberg, 1975). The E. coli dam methylase was found to be responsible for making f1 resistant to endo R · DpnII and sensitive to endo R · DpnI. Endo R · DpnI cleaved f 1 DNA from dam⁺ cells at four sites. Additional methylation by enzymes other than the dam methylase gave no further cleavage. Endo R · DpnII cleaved f1 DNA from dam^? cells also at four sites to give restriction fragments identical to those obtained with endo R · DpnI cleavage. Thus, the two enzymes are complementary in that they recognize and cleave within the same DNA sequence, one if the DNA is methylated, the other if it is unmethylated. DNA duplexes containing one methylated strand (dam ⁺) and one unmethylated strand (dam^?) were prepared in vitro. These methylated hybrids were refractory to endonucleolytic cleavage by both endo R · DpnI and endo R · DpnII. Neither enzyme, therefore, appears to make even a single strand break at a methylated/unmethylated hybrid site.     

Cloning and nucleotide sequence of the gene encoding the Ecal DNA methyltransferase.

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.     

Methyl directed DNA mismatch repair inVibrio cholerae

Mismatches in DNA occur either due to replication error or during recombination between homologous but non-identical DNA sequences or due to chemical modification of bases. The mismatch in DNA, if not repaired, result in high spontaneous mutation frequency. The repair has to be in the newly synthesized strand of the DNA molecule, otherwise the error will be fixed permanently. Three distinct mechanisms have been proposed for the repair of mismatches in DNA in prokaryotic cells and gene functions involved in these repair processes have been identified. The methyl-directed DNA mismatch repair has been examined inVibrio cholerae, a highly pathogenic gram negative bacterium and the causative agent of the diarrhoeal disease cholera. The DNA adenine methyltransferase encoding gene (dam) of this organism which is involved in strand discrimination during the repair process has been cloned and the complete nucleotide sequence has been determined.Vibrio cholerae dam gene codes for a 21.5 kDa protein and can substitute for theEscherichia coli enzyme. Overproduction ofVibrio cholerae Dam protein is neither hypermutable nor lethal both in Escherichia coli andVibrio cholerae. WhileEscherichia coli dam mutants are sensitive to 2-aminopurine,Vibrio cholerae 2-aminopurine sensitive mutants have been isolated with intact GATC methylation activity. The mutator genesmutS andmutL involved in the recognition of mismatch have been cloned, nucleotide sequence determined and their products characterized. Mutants ofmutS andmutL ofVibrio cholerae have been isolated and show high rate of spontaneous mutation frequency. ThemutU gene ofVibrio cholerae, the product of which is a DNA helicase II, codes for a 70 kDa protein. The deduced amino acid sequence of themutU gene hs all the consensus helicase motifs. The DNA cytosine methyltransferase encoding gene (dam) ofVibrio cholerae has also been cloned. Thedcm gene codes for a 53 kDa protein. This gene product might be involved in very short patch (VSP) repair of DNA mismatches. The vsr gene which is directly involved in VSP repair process codes for a 23 kDa protein. Using these information, the status of DNA mismatch repair inVibrio cholerae will be discussed.     

Sequence specificity of the P1 modification methylase (M.Eco P1) and the DNA methylase (M.Eco dam) controlled by the Escherichia coli dam gene.

Labeled oligonucleotides have been fractionated from pancreatic DNase digests of DNA that had been methylated in vitro with the P1 modification enzyme (M·Eco P1) or with the DNA-adenine methylase (M·Eco dam) controlled by the Escherichia coli dam gene. The sequences of methylated oligonucleotides were established for M·Eco dam modification of calf thymus DNA. The results show that M·Eco dam inethylates adenine residues contained in the twofold symmetrical sequence, 5′ … G-A-T-C … 3′. The sequence for the site methylated by M·Eco P1 has also been deduced; we propose that M·Eco P1 modification produces the following methylated pentameric sequence: 5′ … A-G-A¹-C-Py … 3′ (where $\text{A}{}^1\text{= N}{}^6$ methyladenine and Py is C or T).