Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes.

Deoxyribonucleic acid (DNA)-cytosine methylation specified by the wild-type Escherichia coli K 12 mec+ gene and by the N-3 drug resistance (R) factor was studied in vivo and in vitro. Phage lambda and fd were propagated in the presence of L-[methyl-3H]methionine in various host bacteria. The in vivo labeled DNA was isolated from purified phage and depurinated by formic acid-diphenylamine treatment. The resulting pyrimidine oligonucleotide tracts were separated according to size and base composition by chromatography on diethylaminoethyl-cellulose in 7 M urea at pH 5.5 and 3.5, respectively. The distribution of labeled 5-methylcytosine in DNA pyrimidine tracts was identical for phage grown in mec+ and mec minus (N-3) cells. For phage lambda the major 5-methylcytosine containing tract was the tripyrimidine, C2T; for both fd-mec minus (N-3) DNA and fd-mec+DNA, C2T was the sole 5-methylcytosine-containing tract. When various lambda DNAs were methylated to saturation in vitro by crude extracts from mec+ and mec minus (N-3) cells, the extent of cytosine methylation was the same. This is in contrast to in vivo methylation where lambda-mec minus (N-3) DNA contains twice as many 5-methylcytosines per genome as lambda-mec+ DNA. Therefore, we suggest that the K12 met+ cytosine methylase and the N-3 plasmid modification methylase are capable of recognizing the same nucleotide sequences, but that the in vivo methylation rate is lower in mec+ cells.     

Structure of animal mitochondrial DNA: nucleotide composition, pyrimidine clusters, and methylation character.

The nucleotide composition, relative concentration of pyrimidine clusters, and the degree of methylation of the mitochondrial and nuclear DNA's of various vertebrates and the protozoan Crithidia oncopelti have been studied. With respect to the relative concentration of GC pairs, the mtDNA of animals (bull, rat) does not differ from the corresponding nDNA. The relative concentration of GC pairs in the mtDNA of certain fish and birds is 1.5-2.5 mole% higher than in the respective nDNA. The kinetoplast DNA of the protozoan C. oncopelti (where the relative concentration of the GC pairs is 42.9 mole %) differs very sharply in composition from the nDNA (where the relative concentration of GC pairs is 51.3 mole %). The mtDNA's and kDNA's studied are distinguished from the respective nDNA'S by a lower degree of clustering of pyrimidine nucleotides. The proportion of mono- and dipyrimidine fragments in the mtDNA and kDNA is 30 mole %, while in the nDNA it does not exceed 23 mole %. The relative concentration of long pyrimidine clusters (hexapyrimidine clusters of larger) in the mtDNA is smaller than in the nDNA by a factor of 2-5. The low degree of clustering of the pyrimidine nucleotides is apparently characteristic of all the known mtDNA's and may support the fact that they have a single type of organization and are of a single origin. All the vertebrate mtDNA's studied contain 5-methylcytosine as a minor base (1.5-3.15 mole %), and their level of methylation is 1.5-2 times greater than that in the respective nDNA's. It has been shown that animals display species specificity with respect to the 5-methylcytosine content in the mtDNA. Its distribution among the pyrimidine clusters in the bovine heart mtDNA differs substantially from that in the nDNA. This suggests that the methylation specificities of nuclear and mitochondrial DNA are different. A DNA methylase, which effects the in vitro methylation of cytosine residues both in the homologous mtDNA and in different heterologous DNA's, has been found in rat liver and bovine heart mitochondria. The specificity of the in vitro methylation of the cytosine residues in the same heterologous Escherichia coli B DNA by the nuclear and mitochondrial enzymes is different: The mitochondrial enzyme methylates predominantly in monopyrimidine fragments, and the nuclear enzyme methylates mostly in di- and tripyrimidine fragments. They, therefore, recognize different nucleotide sequences.     

Restriction and modification in Bacillus subtilis: inducibility of a DNA methylating activity in nonmodifying cells.

The nonrestricting/nonmodifying strain Bacillus subtilis 222 (r-m-) can be induced to synthesize a DNA-modifying activity upon treatment with either mitomycin C (MC) or UV light. This is shown by the following facts. (i) Infection of MC-pretreated 222 cells with unmodified SPP1 phage yields about 3% modified phage that are resistant to restriction in B. subtilis R (r+m+). The induced modifying activity causes the production of a small fraction of fully modified phage in a minority class of MC-treated host cells. (ii) The MC-pretreated host cells contain a DNA cytosine methylating activity: both bacterial and phage DNAs have elevated levels of 5-methylcytosine. (iii) The MC-induced methylation of SPP1 DNA takes place at the recognition nucleotide sequences of restriction endonuclease R from B. subtilis R. (iv) Crude extracts of MC-pretreated 222 cells have enhanced DNA methyltransferase activities, with a substrate specificity similar to that found in modification enzymes present in (constitutively) modifying strains.     

DNA methylation in lysogens of pathogenic Burkholderia spp. requires prophage induction and is restricted to excised phage DNA

Burkholderia mallei-specific phage PhiE125 encodes DNA methyltransferases in both the lysogenic and replication modules within its genome. Characterization of DNA methylation in recombinant systems, specifically in PhiE125 lysogenic strains of B. mallei and Burkholderia thailandensis, revealed that, upon induction, cytosine methylation was targeted specifically to the phage episome but not the phage provirus or the host chromosome.     

Properties of R plasmid R772 and the corresponding pilus-specific phage PR772.

R plasmid R772 was isolated from a strain of Proteus mirabilis and is a self-transmissible P-1 incompatibility group plasmid having a molecular weight of about 27 x 10(6). It renders bacterial hosts resistant to kanamycin. Phage PR772 was isolated as a phage dependent on the presence of R772 in bacterial hosts. It is hexagonal-shaped with a diameter of 53 nm, has a thick inner membrane and no tail. Vaguely defined appendages are sometimes apparent at some vertices and the phage possesses double-stranded DNA. The DNA has a guanine plus cytosine molar content of 48%. The phage is sensitive to chloroform and has a buoyant density of 1.26 g cm(-3). These observations suggested that the inner membrane of the phage could contain lipid. Phage PR772 differs in morphology from the double-stranded DNA plasmid-specific phages PR4 and PRR1 which adsorb to tips and sides, respectively, of sex pili coded for by P-1 incompatibility group plasmids. Phage PR772 formed clear plaques which varied in diameter. Serologically, phages PR772 and PR4 are possibly related though very distantly, but the two phages have identical host ranges. Phage PR772 adsorbed by one of its apices to tips of sex pili coded for by plasmid R772 in Escherichia coli. It also formed plaques on Salmonella typhimurium Proteus morganii and Providence strains harbouring this plasmid as well as strains of E. coli carrying plasmids of incompatibility groups N or W. The phage produced areas of partial clearing on lawns of P. mirabilis PM5006 harbouring plasmid R772, the P-1 incompatibility group plasmid RP4, the W group plasmid RSa or the N group plasmid N3, and on lawns of Providence strain P29 carrying plasmid RP4.     

The structure of animal mitochondrial DNA (base composition,pyrimidine clusters,character of methylation)

Summary Base composition, content of pyrimidine isopliths and the degree of methylation of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from various vertebrates and protozoonCrithidia oncopelti have been studied. MtDNAs from mammals (ox, rat) do not differ in fact in the GC content from the respective nDNA. The GC content in mtDNA from fishes (sheat fish) and birds (duck, chicken) is 1.5–2.5 mole % higher than in the respective nDNA. Kinetoplast DNA (kDNA) fromCrithidia oncopelti (GC = 42.9 mole %) differs significantly in base composition from nDNA (GC = 51.3 mole %). All the mtDNA and kDNA studied differ from the respective nDNA by a lower degree of pyrimidine clustering. Th amount of mono and dipyrimidine fragments in mtDNA is more than 30 mole %, whereas in nDNA it does not exceed 23 mole %. The quantity of long pyrimidine clusters (hexa and others) is 2–4 times lower in mtDNA than in nDNA. The lower degree of clustering of pyrimidine nucleotides seems to be a specific feature of all the mtDNA studied. This may be indicative of common traits in the organization and origin of mtDNA. All mtDNA of vertebrates contain 5-methylcytosine as a minor base (1.5–3.15 mole %) and surpass by 1.5–2 times the respective nDNA in the methylation degree. It has been found that in animals mtDNA is species specific as far as the 5-methyl-cytosine content is concerned. In mitochondria and nuclei of rat liver certain DNA methylase activity has been detected, which providesin vitro the methylation of cytosine residues both in homologous DNA and various heterologous DNAs. The specificity of methylationin vitro of cytosine residues in the same heterologous DNA fromE. coli B varies with the source of enzymes. The mitochondrial enzyme methylates cytosine as the lone monopyrimidine residue, whereas the nuclear enzyme methylates cytosine in the di- and tripyrimidine fragments.     

Analysis of the structure of Bacillus brevis neutral proteinase and its biosynthesis in Bacillus subtilis cells

Amino acid sequence of neutral metalloprotease from Bac. brevis has been compared with that of Bac. amyloloquefaciens, Bac. cereus, Bac. subtilis, Bac. stearothermophilis, Bac. thermoproteolyticus (thermolysine). A sequence region from N-40 to N-1 with a significant degree of homology allowed to predict the processing site of the propart of Bac. brevis enzyme. The sequence comparison allows to put Bac. brevis enzyme within the evolutionary branch of enzymes, which includes thermolysin and proteases of Bac. cereus and Bac. stearothermophilus. Using automated Edman degradation the N-terminal sequence of Bac. brevis protease has been determined. It does not differ from the sequence predicted from the nucleotide sequence of the gene. It was shown that, when Bac. brevis gene coding for thermostable protease is expressed on a plasmid vector in Bac. subtilis cells at 37 degrees C, enzyme forms possessing low activity are secreted. The enzyme may be significantly activated without an additional cleavage or processing and the activation includes numerous conformation transition states of the protein molecule.     

Salmonella typhimurium SA host specificity system is based on deoxyribonucleic acid-adenine methylation.

We have determined the nature of the deoxyribonucleic acid (DNA) modification governed by the SA host specificity system of Salmonella typhimurium. Two lines of evidence indicate that SA modification is based on methylation of DNA-adenine residues. (i) The SA+ locus of Salmonella was transferred into Escherichia coli B, a strain that does not contain 5-methylcytosine in its DNA; although the hybrid strain was able to confer SA modification, its DNA still did not contain 5-methylcytosine. (ii) the N6-methyladenine content of phage L DNA was measured after growth in various host strains; phage lacking SA modification contained fewer N6-methyladenine residues per DNA. We also investigated the possibility, suggested by others (32), that SA modification protects phage DNA against restriction by the RII host specificity system. Phages lambda, P3, and L were grown in various SA+ and SA- hosts and tested for their relative plating ability on strains containing or lacking RII restriction; the presence or absence of SA modification had no effect on RII restriation. In vitro studies revealed, however, that Salmonella DNA is protected against cleavage by purified RII restriction endonuclease (R-EcoRII). This protection is not dependent on SA modification; rather, it appears to be due to methylation by a DNA-cytosine methylase which has overlapping specificity with the RII modification enzyme, but which is not involved in any other known host specificity system.     

Oligopeptidase A is required for normal phage P22 development.

The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing.     

DNA protection with the DNA methylase M . BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII

BamHI fragments of the Bacillus brevis var. GB plasmid pAD1 have been cloned in Escherichia coli HB101 using pBR322 plasmid as a vector. The analysis of the recombinant plasmids showed that additional PstI sites had appeared in cloned fragments of pAD1. Methylation of the recombinant plasmids in vitro by enzymes from B. brevis GB cells blocks cleavage at these additional PstI sites of cloned pAD1 fragments and at the PstI site of pBR322. Among DNA methylases of B. brevis GB, the cytosine DNA methylase M . BbvI is the most likely agent modifying the recognition sequences of PstI. The methylase can modify cytosine residues in PstI or PvuII sites if these recognition sequences are linked to G at 5'- or to C at 3'-termini. In particular, in vitro methylation of the SV40 DNA by B. brevis GB methylases protects one of the two PstI sites and two of the three PvuII sites. The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.     

Lyophilization of the spores and vegetative cells of Bacillus brevis var. G-B.--producer of gramicidin S

Viability, antibiotic properties and variation of 4 variants of Bac. brevis var. G.-B. were studied after lyophilization and storage for a year in the lyophilized state. It was shown that the spores and vegetative cells of S and P- variants not synthesizing gramicidin S were somewhat more stable than the spores and cells of R and P+ variants producing the antibiotic. The latter dissociated by 10 per cent towards the cells producing and not producing gramicidin. The developmental rate of the lyophilized vegetative cells was higher than that of the lyophilized spores. Under analogous cultivation conditions they produced higher amounts of the biomass and antibiotic. The lyophilization method described may be recommended for the maintenance of viability and stability of the spores and vegetative cells of Bacillus brevis var. G.-B. producing gramicidin S.     

Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system.

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.     

DNA-methylase activities from animal mitochondria and nuclei: different specificity of DNA methylation]

DNA-methylase activities which methylate cytosine residues in homo- and heterologous DNA were detected in mitochondria and nuclei from rat liver and beef heart. Adenine modifying DNA-methylases in mitochondria and nuclei were not found. DNA from mitochondria and nuclei differ significantly in the methylation degree and in the pattern of the 5-methyl-cytosine distribution by pyrimidine isostichs as DNA in vivo and in vitro being methylated. Mitochondrial DNA methylase has the maximum activity at 30 degrees and pH 7.8 this enzyme(s) differ(s) from the nuclear one(s) in the pH dependence of its activity. After exhaustive in vitro methylation of various DNA by the nuclear enzyme DNA-methylase from mitochondria additionally introduces CH3 groups from S-adenosylmethionine into these DNA (about 3 times more CH3 groups than nuclear enzyme). Nuclear DNA-methylase also methylates DNA which is previously fully-methylated by the mitochondrial enzyme, but to a lesser degree. In conditions of exhaustive DNA methylation mitochondrial enzyme introduces into E. coli B DNA about four times more methyl groups as compared to the nuclear one. After the methylation of E. coli B DNA by mitochondrial enzyme the label (3H-methyl) was detected predominantly in mono-, and in case of nuclear enzyme--in di- and tripyrimidine fragments. Mitochondrial DNA-methylase differs from the nuclear one in the nature of recognized DNA sequences; these enzymes seems to be represented by different proteins. The mitochondrial enzyme methylates shorter nucleotide sequences in DNA as compared to the nuclear DNA-methylase. All these data suggest there exist organoid specificity of genome methylation in animal cell and the modification-restriction systems in animal nucleus and mitochondria are different in character.     

Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine.

Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.     

Dark Repair of Ultraviolet-Irradiated Deoxyribonucleic Acid by Bacteriophage T4: Purification and Characterization of a Dimer-Specific Phage-Induced Endonuclease

The purification and properties of an ultraviolet (UV) repair endonuclease are described. The enzyme is induced by infection of cells of Escherichia coli with phage T4 and is missing from extracts of cells infected with the UV-sensitive and excision-defective mutant T4V(1). The enzyme attacks UV-irradiated deoxyribonucleic acid (DNA) containing either hydroxymethylcytosine or cytosine, but does not affect native DNA. The specific substrate in UV-irradiated DNA appears to be pyrimidine dimer sites. The purified enzyme alone does not excise pyrimidine dimers from UV-irradiated DNA. However, dimer excision does occur in the presence of the purified endonuclease plus crude extract of cells infected with the mutant T4V(1).     

Relative levels of methylation in human growth hormone and chorionic somatomammotropin genes in expressing and non-expressing tissues.

It has been shown that the extent of methylation of cytosine in vertebrate DNA is inversely correlated with gene expression. We studied cytosine methylation in and around the homologous human growth hormone (GH) and chorionic somatomammotropin (CS) genes to determine if these genes are undermethylated in DNA from tissues in which they are expressed (pituitary and placenta, respectively) compared to other tissues. Hpa II and Hha I (which cleave only unmethylated 5' CCGG 3' and 5' GCGC 3' respectively) and Msp I (which cleaves CCGG and CmeCGG) were used to digest DNA samples followed by gel electrophoresis, Southern transfer and hybridization with a GH cDNA probe. The extent of methylation of Hpa II and Hha I sites in the GH and CS genes was leukocyte much greater than pituitary greater than placenta = hydatidiform mole. Taken as a whole, our data support the hypothesis that undermethylation is a necessary but not sufficient condition for gene expression since placental and pituitary DNAs are less methylated than leukocyte DNA in this region. However, the correlation between gene expression and undermethylation is imperfect since (1) hydatiform mole DNA has a very similar methylation pattern compared to placental DNA even though moles make little or no CS and (2) the level of methylation of the GH gene compared to the CS gene does not vary in a tissue-specific manner.     

Nucleotide clusters in deoxyribonucleic acids. Comparison of the sequences of the large pyrimidine oligonucleotides of bacteriophages S13 and phiX174 deoxyribonucleic acids.

The large pyrimidine oligonucleotides from the DNAs of the two related bacteriophages phiX174 and S13 have been sequenced. The largest pyrimidine oligonucleotide present is unique to S13 DNA and is the undecanucleotide C5T6, sequence C-T-T-C-C-T-C-T-T-C-T. Considerable sequence homology has been found between the pyrimidine oligonucleotides of the two phage DNAs. Out of 14 oligonucleotide sequences from S13 DNA (120 bases) at least ten are identical with sequences of oligonucleotides from phiX174 DNA (92 bases) and two are closely related (17 bases), the only difference being a single thymine to cytosine transition in each sequence (a total of 107 identical bases). The pyrimidine oligonucleotides of each phage DNA show extensive internal sequence homology among each other with up to eight bases identical in sequence in pairs of different oligonucleotides. Another interesting observation is the occurrence of symmetrical sequences (true palindromes) which read the same forwards as backwards. The longest symmetrical sequence is the nonanucleotide C4T5 sequence, C-T-C-T-T-T-C-T-C, present in both S13 and phiX174 DNAs. The extensive sequence homology observed between the pyrimidine oligonucleotides of S13 and phiX174 supports the close relationship of the two phages and provides further evidence that they were derived from recent common ancestors.