Specificity of deoxyribonucleic acid transmethylase induced by bacteriophage T2. I. Nucleotide sequences isolated from tmicrococcus luteus DNA methylated in vitro.

(Deoxyribonucleic acid from Micrococcus luteus was methylated in vitro in the presence of S-adenosyl-(14C methyl)methionine with a DNA methyltransferase purified from extracts of te. coli infected with bacteriophage T2. The labelled DNA was degraded by enzymatic and specific chemical methods and the resulting short oligonucleotides were separated and characterized. tthe analytical data permit the conclusion that the tdna transmethylase reacts specifically with N-G-A-T-C-N sequences in which it converts adenine to a 6-methyl-aminopurine residue.     

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.     

Glucosylated nucleotide sequences from T-even bacteriophage deoxyribonucleic acids

1. The frequencies of various pyrimidine nucleotide sequences in phage-T2VG111 DNA and phage-T6 DNA have been measured. The hydroxymethylcytosine nucleotides that bear glucosyl groups in phage-T2VG111 DNA and gentiobiosyl groups in phage-T6 DNA are not randomly distributed. 2. Sequences in which two or three hydroxymethylcytosine nucleotides are terminated at both ends by purine nucleotides bear only one sugar substituent and this is attached to the first hydroxymethylcytosine when the sequences are written in the conventional notation; i.e. with the 5′-phosphate before and the 3′-phosphate after each nucleoside residue. This resembles the distribution of glucosyl residues previously found in the corresponding sequences from phage-T2 DNA. Sequences in which one hydroxymethylcytosine nucleotide is attached through the 3′-position to a purine nucleotide are more fully glucosylated in phage-T2VG111 DNA and phage-T6 DNA than in the DNA of phage T2. 3. Pyrimidine sequences from the DNA of phage T6(S) have been found to contain glucosyl but not gentiobiosyl residues. The relative amounts of the sequences isolated indicate that the distribution of the glucosyl residues in this DNA resembles that of the gentiobiosyl groups in phage-T6 DNA. 4. It is suggested that the distributions of glycosyl substituents in these T-even-phage deoxyribonucleic acids reflect the specificity requirements of the α-glucosyltransferases induced by the different phages. The results obtained with the DNA of phage T6(S) suggest that this phage induces the usual phage-T6 α-glucosyltransferase but not the phage-T6 β-glucosyltransferase.     

Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.     

Transformation of a Bacillus subtilis L-form with bacteriophage deoxyribonucleic acid.

A stable L-form, sal-1, of Bacillus subtilis was transformed with deoxyribonucleic acid (DNA) from bacteriophages phi 25 and phi 29 to determine whether exogenous DNA can be introduced into this organism. The viral transformation (transfection) was successful with the use of polyethylene glycol. In the presence of the fusogen, bacteriophage phi 25 DNA initiated a single cycle of infection. When compared with transfection of competent cells of Bacillus subtilis, the appearance of viral particles was delayed and their production occurred over a longer time period. L-form cells were best able to support intracellular replication of phi 25 viral particles when in balanced growth in a rich medium. The addition of polyethylene glycol also induced infection of sal-1 with whole bacteriophage phi 25 particles which could not otherwise infect the L-form and enhanced infection by intact phi 29 particles. Primary recombination was shown to be required for polyethylene glycol-mediated phi 25 transfection, but not phi 29 transfection or for whole bacteriophage phi 25 infection mediated by polyethylene glycol. Successful transfection of sal-1 suggests that the L-form may be amenable to genetic modification with exogenous DNA.     

Interspersion of mouse satellite deoxyribonucleic acid sequences

DNA sequences with homology to the major (A + T)-rich mouse satellite component were localized in CsCl gradients by hybridization with a labeled satellite cRNA probe. Although, as expected, most of the hybridization was to DNA in the satellite-rich shoulder, substantial radioactive cRNA hybridized with DNA from denser regions of the gradient. Further examination revealed that hybridization to main-band DNA was not due to physical trapping of satellite DNA in the gradient, and melting experiments argue that the associated radioactivity was due to true RNA/DNA hybridization. Nearest-neighbor analysis of hybridized [alpha-32P]CTP-labeled l-strand cRNA indicates that hybridization to main-band DNA is by the satellite cRNA and not a contaminant. Together, these data argue that mouse satellite-like sequences are interspersed within the main-band fraction of DNA. For the support of this contention, total mouse DNA, purified main-band DNA, and purified satellite DNA were digested with EcoRI, sedimented in a sucrose gradient, and hybridized with labeled satellite cRNA. Mouse satellite DNA is not cleaved with EcoRI, so that purified EcoRI-digested satellite DNA sediments as a high molecular weight component. When total mouse DNA is digested with EcoRI, the majority of satellite-like sequences remain as high molecular weight DNA; however, significant amounts of satellite-like sequences sediment with the bulk of the lower molecular weight digested DNA, lending further credence to the argument that satellite-like sequences are interspersed with main-band DNA.     

Nucleotide sequences at the sites of action of the deoxyribonucleic acid modification enzyme of bacteriophage P1

Labelled oligonucleotides have been fractionated from DNAase digests of phage λ DNA that had been methylated with the phage P1 modification enzyme and S-[methyl-¹⁴C]adenosyl-l-methionine. The longest sequences established are the tetranucleotides pG-ǎ?-T-C4 and p?-T-C-T, which, together with the other sequences determined, particularly pA-G-?, show that the modification enzyme, M.EcoP1, methylates adenine residues within defined sequences and suggest that the oligonucleotide sequence recognized by this enzyme is the hexanucleotide pA-G-A-T-C-T. The duplex formed by base-pairing this hexanucleotide with its complementary sequence resembles the recognition sequence for several restriction enzymes in being bisected by an axis of 2-fold rotational symmetry.     

The deoxyribonucleic acid modification enzyme of bacteriophage P1. Purification and properties

The stereochemistry of dl-glycerol 3-phosphate was studied by X-ray-crystallographic techniques. All the bond lengths and angles are within normally accepted limits except the ester bond, which is one of the largest yet noted, being 0.1637nm. The conformation of the molecule is such that an intramolecular hydrogen bond is formed between the hydroxyl group on the beta-carbon atom and the phosphate group. The crystal, which was grown by alcohol diffusion into an aqueous solution, is held together by sodium co-ordination and a complex system of hydrogen bonds. A table of the observed and calculated structure factors, F(obs.) and F(calc.), has been deposited as Supplementary Publication 50010 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.     

Escherichia coli heat-labile enterotoxin genes are flanked by repeated deoxyribonucleic acid sequences.

The enterotoxin regions of the heat-labile and heat-stable enterotoxin (LT+ ST+) plasmid, pJY11, originating in a clinically isolated Escherichia coli strain, have been isolated as various-sized deoxyribonucleic acid (DNA) fragments by using cloning vehicles. The structure of the LT+ region and its neighboring DNA regions was studied by utilizing these recombinant plasmids. The LT+ region consisted of at least two genes, toxA and toxB, which could complement each other in trans. The toxA- and toxB-encoded polypeptides (LT subunits A and B, respectively) were identified by their immunological cross-reactivity with Vibrio cholerae enterotoxin subunit A or B. These tox genes and the promoter(s) were localized with respect to the restriction endonuclease cleavage map. The LT+ region was flanked by repeated DNA sequences (designated as beta). Another tox gen(s), encoding ST (designated as toxS), which was also flanked by inverted, repeated DNA sequences (designated as alpha), was located between one of the beta sequences and the LT+ region. These novel DNA structures (beta-alpha-toxS-alpha-toxA-toxB-beta) suggest the possibility that the LT+ region is on a transposon containing an ST transposon within the structure.     

The deoxyribonucleic acid modification enzyme of bacteriophage P1. Subunit structure

The bacteriophage P1 modification enzyme was purified 1400-fold from induced lysogens of a thermoinducible mutant of bacteriophage P1. The most purified fraction, when analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate, showed two principal stained bands. The two bands co-sedimented in a glycerol gradient with the modification activity, at a rate which, when compared with the rate of sedimentation of marker proteins, corresponds to a sedimentation coefficient in water of 6S. The mobilities of the bands on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis corresponded to polypeptides of molecular weight 70000 and 45000 and they were present in equimolar amounts. It was concluded that the 6S species of the enzyme is a dimer of unlike subunits.