Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule.

The structure of DNA from the temperate Bacillus subtilis phage phi105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis. The DNA contains six EcoRI cleavage sites. Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA. In neutral gradients, the majority of whole phi105 DNA sedimented as nicked circles and the remainder as oligomers. No unit-length linear structures were detected. The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease. On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, phi105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA.     

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

Restriction fragments hybridizing to phage HP1c1 DNA were identified in digests of DNA from lysogenic strains of Haemophilus influenzae. The results showed that the cohesive ends of the mature phage DNA were joined in lysogens and that the phage genome was covalently linked to the host DNA, indicating that lysogeny involves recombination between specific sites on the phage and host chromosomes. The site on the phage chromosome at which this recombination occurred was between 110 and 750 base pairs of the left end on the mature phage genome.     

SstI: a restriction endonuclease from Streptomyces sp. stanford.

A strain of Streptomyces has been isolated which is a convenient source of a new restriction endonuclease. The enzyme has been prepared from extracts of these cells and its cleavage sites localized on phage lambda DNA. The enzyme, termed SstI, produces cohesive ends and should be useful for molecular cloning experiments.     

Construction and transduction of a shuttle vector bearing the cos site of Streptomyces phage phi C31 and determination of its cohesive ends

A plasmid has been constructed which is a bifunctional vector for Escherichia coli and for streptomycetes, containing the cos site of Streptomyces phage phi C31. It can be efficiently transduced into S. lividans 66 and into several other Streptomyces species. The nucleotide sequence of a 311-bp DNA fragment containing the cohesive ends has been determined. The cohesive ends consist of 10 bases protruding at the 3'-end of the phage genome.     

Physical characterization of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

A generalized transducing bacteriophage of Myxococcus xanthus has been examined. The phage particle consists of an isometric head and a contractile tail. The genome of the phage is a linear DNA molecule of molecular weight 39 ± 2.1 × 10⁶, which contains the normal DNA bases 70% of which are guanosine + cytosine. No overall heterogeneity of base composition is present. The DNA does not carry easily detectable cohesive ends nor is it cyclically permuted. It does contain a large and somewhat variable terminal redundancy. Heating phage particles in the presence of EDTA causes tail sheath contraction and ejection of DNA, some of which remains attached to the tail. Digestion of tail-bound DNA with restriction enzymes shows that the phage tail can be attached to either end of the DNA. Thus the DNA probably contains recognition sites for the packaging of its DNA at both ends. These results suggest possible mechanisms for the genesis of transducing particles by phage MX4.     

Nucleotide sequence and properties of the cohesive DNA termini from bacteriophage HP1c1 of Haemophilus influenzae Rd

The termini of the mature DNA of phage HP1c1 of Haemophilus influenzae Rd have been characterized by DNA ligation, nucleotide sequencing, and deoxynucleotide incorporation experiments. A hybrid plasmid containing the joined phage termini (the cos site) inserted into pBR322 has been constructed. The phage DNA has cohesive termini composed of complementary 5' single-stranded extensions which are seven residues long. The left cohesive terminal extension consists only of pyrimidines and the right only of purines. When the ends of the phage are joined, the terminal sequences constitute the central 7 bp of an 11 bp sequence containing only purines on one strand and pyrimidines on the other strand. This oligopyrimidine/oligopurine sequence does not possess rotational symmetry. A 10-bp sequence and its inverted repeat are located approx. 20 bp to the left and right of the fused ends.     

Isolation and characterization of the Streptomyces cattleya temperate phage TG1

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.     

Initiation of Infection by Deoxyribonucleic Acid From Bacteriophage λ

Kinetic studies conducted on the early stages of infection of Escherichia coli K-12 by deoxyribonucleic acid (DNA) isolated from bacteriophage lambda indicate a rapid adsorption of the phage DNA to receptor sites at the bacterial surface prior to deoxyribonuclease-insensitive incorporation. A direct relationship found between the number of DNA molecules adsorbed per bacterium and the multiplicity of helper phage infection indicates a requirement for helper function during the attachment process. An apparent lack of attachment specificity with regard to the source of the DNA preparation, to the size of the inhibiting fragment, to the base ratio of the inhibiting DNA molecule, and to "cohesive" ends suggests a nonspecific interaction between the infectious DNA and the sites of helper phage attachment.     

Packaging of coliphage lambda DNA. I. The role of the cohesive end site and the gene A protein

The cohesive ends of the DNA of bacteriophage λ particles are normally formed by the action of a nuclease on the cohesive end sites (cos) of concatemeric λ DNA (reviewed by Hohn et al., 1977). The nuclease also cuts the cos site of an integrated prophage, and DNA located to the right is preferentially packaged into phage particles. This process occurs with approximately the same efficiency and rate in a single lysogen as in a tandem polylysogen. Thus, the rate of cos cutting does not increase when the number of cos sites per molecule increases, an hypothesis that has been proposed to explain why cohesive ends are not formed in circular monomers of λ DNA. We propose instead that the interaction of Ter with cos is influenced by the configuration of the DNA outside of cos during packaging, and that this configuration is different for circular monomers than for other forms of λ DNA. A model that gives rise to such a difference is described.We also found that missense mutations in the λ A gene changed the efficiency of packaging of phage relative to host DNA. This was not the case for missense mutations in several phage genes required for capsid formation. Thus, the product of gene A plays a role in determining packaging specificity, as expected if it is or is part of the nuclease that cuts λ DNA at cos.     

Characterization of bacteriophage N3 DNA.

The DNA from Haemophilus influenzae temperate phage N3 was characterized by centrifugation and by electrophoresis after nuclease digestion. The double-stranded DNA, with a mass of 25.8 X 10(6) daltons, had single-strand cohesive ends. Strand association through cohesion was reduced by heat and removed by S1 nuclease digestion. N3 DNA contained five EcoRI, one KpnI, two SacI, six XbaI, and four XhoI cleavage sites. The cohesive end segments were identified by heating the digests before electrophoresis. This was the first step in the construction of the physical maps of this DNA.     

Bacteriophage lambda derivatives carrying two copies of the cohesive end site

A spontaneously arising tandem duplication derivative of bacteriophage lambda has been isolated, which carries two copies of the site where the cohesive ends are formed (designated cos). Its structure has been determined by electron microscopy of DNA heteroduplexes. These heteroduplexes reveal that the duplication is usually, but not always, carried on the left end of the chromosome. A second duplication phage having two copies of cos, constructed by Feiss &; Campbell (1974), has also been studied by electron microscopy and is found to have a similar property.Unlike most tandem duplication derivatives of phage λ, the mutant studied here is not stable during growth in the absence of generalized recombination, but segregates both the triplication and the parental phage. This verifies that both cos sites are functional. The triplication does not arise as a result of end-to-end aggregation of phage chromosomes or site-specific recombination catalyzed by the chromosome maturation system at cos. It must therefore result from the cutting of mature ι chromosomes from concatemeric replication intermediates. The pattern of cutting observed shows that the λ cohesive ends are not created by a free nuclease acting on unpackaged DNA. The cutting appears to be influenced by the amount of DNA previously packaged into a phage head. A model for λ packaging is presented which explains the results.The duplication phage of Feiss &; Campbell (1974) carries a novel addition containing self-complementary sequences.     

Processing of bacteriophage lambda DNA during its assembly into heads

DNA purified from bacteriophage λ added to a cell-free extract derived from induced λ lysogens can be packaged into infectious phage particles (Kaiser & Masuda, 1973). In this paper the structure of the DNA which is the substrate for in vitro packaging and head assembly is described. The active precursor is a multichromosomal polymer that contains covalently closed cohesive end sites. Neither circular or linear DNA monomers nor polymers with unsealed cohesive ends are packaged efficiently into heads. The unit length monomer is packaged when it is either contained in the interior of a polymer (both of its ends are in cos sites) or when it has a free left end and a cos site on its right. The monomer unit with a free right end is not a substrate for packaging.A procedure is given for the purification of λ DNA fragments that contain either the left or the right cohesive end. The fragments are produced by digesting λ DNA with the site-specific Escherichia coli R₁ endonuclease; the left and right ends are separated by sedimentation through a sucrose gradient. These fragments are used to construct small polymers that have a unit length λ monomer with (1) a free left end and a closed right end, (2) a free right end and a closed left end, or (3) both ends closed in cos sites.     

Sequence analysis of cohesive ends of the actinophage RP3 genome and construction of a transducible shuttle vector

Abstract The sequence of the cohesive ends of actinophage RP3 DNA has been determined. As with all other phages of Gram-positive bacteria that have been studied sofar, RP3 DNA has 3'-protruding ends. A shuttle cosmid has been constructed containing this cos area which can be efficiently transduced by phage RP3 to host cells of Streptomyces rimosus .     

Serotype-converting bacteriophage D3 of Pseudomonas aeruginosa: vegetative and prophage restriction maps

D3 is a temperate serotype-converting bacteriophage of Pseudomonas aeruginosa. A restriction map, based upon BamHI, PstI, PvuI, HindIII and SmaI sites, indicates that the phage genome is 56.4 kb long, and that it possesses cohesive ends. The prophage map suggests a unique insertion site in the strain AK1380 genome. Phage DNA integration occurs upon the circularization of D3 genome with the integration point approximately equidistant from the two ends.     

Construction and characterization of a cosmid of Streptomyces lividans

Summary We constructed a plasmid pR4C1 in which a DNA fragment containing the cohesive ends of an actinophage, R4 was inserted into the ClaI site of plasmid pIJ365. The plasmid pR4C1 was packaged efficiently into R4 phage particles in vivo and therefore transferred by transduction. Southern hybridization analysis of DNA from pR4C1-transducing R4 phage particles indicated that the plasmid DNA was encapsidated as head-to-tail concatemers with the cohesive ends in the termini. We designated the pR4C1 plasmid a cosmid, following the termination of Collins and Hohn (1978).     

The Nul subunit of bacteriophage lambda terminase binds to specific sites in cos DNA.

The maturation and packaging of bacteriophage lambda DNA are under the control of the multifunctional viral terminase enzyme, which is composed of the protein products of Nu1 and A, the two most leftward genes of the phage chromosome. Terminase binds selectively to the cohesive end site (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12-base single-stranded cohesive ends of the mature phage genome. The purified gpNu1 subunit of terminase forms specific complexes with cos lambda DNA. DNase I footprinting experiments showed that gpNu1 bound to three distinct regions near the extreme left end of the lambda chromosome. These regions coincided with two 16-base-pair sequences (CTGTCGTTTCCTTTCT) that were in inverted orientation, as well as a truncated version of this sequence. Bear et al. (J. Virol. 52:966-972,1984) isolated a mutant phage which contained a CG to TA transition at the 10th position of the rightmost 16-base-pair sequence, and this phage (termed lambda cos 154) exhibits a defect in DNA maturation when it replicates in Escherichia coli which is deficient in integration host factor. Footprinting experiments with cos 154 DNA showed that gpNu1 could not bind to the site which contained the mutation but could protect the other two sites. Since the DNA-packaging specificity of terminase resides in the gpNu1 subunit, these studies suggest that terminase uses these three sites as recognition sequences for specific binding to cos lambda.     

Restriction endonuclease mapping of bacteriophage phi105 and closely related temperate Bacillus subtilis bacteriophages rho10 and rho14.

Cleavage maps of the three similar Bacillus subtilis temperate bacteriophages, phi105, rho10, and rho14, were constructed by partial digestion analysis utilizing the restriction endonuclease EcoRI. Comparison of the topography of these maps indicates that all phage DNAs posses cohesive ends and a number of EcoRI restriction sites; the fragments are conserved, and the estimated base substitution/nucleotide divergence between these phages is 0.03 to 0.07 based on conserved fragments or between 0.03 and 0.11 based on conserved cleavage sites. These lines of evidence indicate that phi105, rho10, and rho14 are closely related. Double-enzyme digestion analysis reveals that rho14 DNA has unique SalGI and BglII restriction sites and phi105 DNA has a unique SalGI restriction site, making these phages possible cloning vectors for B. subtilis.     

The interaction of Escherichia coli integration host factor with the cohesive end sites of phages lambda and 21

The interaction of E. coli integration host factor (IHF) with the cohesive end sites (cos's) of phages lambda and 21 has been studied by the DNAase I footprinting technique. Six potential sites in cos lambda differ from the consensus IHF binding sequence by 1 to 3 base pairs. Of the six, one site, I1, binds IHF strongly. The I1 segment protected by IHF contains two sequences that closely match the IHF consensus binding sequence. Another site, I2, binds IHF moderately well, and three sites: 10', 13 and 14 bind IHF very weakly. The 10 site does not bind IHF under the conditions used here. In phage 21 the DNA segment extending to the right from the cohesive ends, which contains three potential IHF binding sites, was examined. Two sites bind IHF well; I1, the 21 analogue of one of the lambda I1 sites, and I0, a site not analogous to a lambda site. The third 21 site, I2, binds IHF moderately well, as does the analogous I2 site in lambda. The significance of the results for lambda DNA packaging is discussed.     

Characterization of the genome of the Rhodococcus rhodochrous bacteriophage NJL.

Temperate bacteriophage NJL of Rhodococcus rhodochrous has a 49-kb linear double-stranded DNA with cohesive ends (cos). NJL DNA has unique target sites for HindIII and SspI, two target sites each for NheI and ScaI, and no cleavage site for AxyI, DraI, EcoRI, SacI, and SphI. The single-stranded regions of cos ends were ligated to each other with T4 DNA ligase, removed with mung bean nuclease, or blunted with the Klenow large fragment of DNA polymerase I; then the sequences of the cos ends were determined. Comparison of these sequences revealed that the single-stranded regions are complementary and 18 bases long and protrude at the 3' ends; they have the following sequences: 5'-TTGGCACCGTGGGAGGAG-3' and 3'-AACCGTGGCAC CCTCCTC-5'. A physical map of NJL was constructed by a cos mapping method based on information about the structure of the cohesive ends and multiple digestions with restriction endonucleases.     

Bacteriophage of Haemophilus influenzae III. Morphology, DNA Homology, and Immunity Properties of HP1c1, S2, and the Defective Bacteriophage from Strain Rd

The phages HP1c1 and S2 and a defective phage of Haemophilus influenzae have been compared. The morphology of the phages and the mol wt of their DNAs are similar, although the defective phage appears to have a different tail plate region. Electron microscope observation indicates that the defective phage does not attach to the cell surface, and its DNA appears to lack cohesive ends. The homology of the DNAs of the phages has been measured by hydridization. DNA from the defective phage shows little or no homology with the other phage DNAs. HP1c1 and S2 DNAs show a high level of homology. Each of these phages can form plaques on lawns of the lysogen of the other phage but at reduced plating efficiencies, suggesting that the two phages have related but not identical immunity systems.