The process of infection with bacteriophage φX174

Sinsheimer, Robert L.; Starman, Barbra J.; Nagler, Carolyn; Guthrie, Shirley C.

doi:10.1016/s0022-2836(62)80047-3

Cited by 361 publications

(58 citation statements)

References 18 publications

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“…3a) infect and enter the cell, forming double-stranded parental RF DNA molecules (Fig. 3b) (28), which are attached to the host cell membrane at an essential site (29). At some time early in infection (7), a single-strand break occurs or is introduced into one of the two parental genomes (11) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Recombinant DNA molecules of bacteriophage phi chi174.

Benbow¹,

Zuccarelli²,

Sinsheimer³

1975

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT4 X174 DNA structures containing two different parental genomes were detected genetically and examined by electron microscopy. These structures consisted of two monomeric double-stranded DNA molecules linked in a figure 8 configuration. Such DNA structures were observed to be formed preferentially in host recA+ cells or recA + cell-free systems. Since the host recA + allele is required for most 4OX174 recombinant formation, we conclude that the observed figure 8 molecules are intermediates in, or end products of, a 4X174 recombination event.We propose that recombinant figure 8 DNA molecules arise as a result of "single-strand aggression," are stabilized by double-strand "branch migration," and represent a specific example of a common intermediate in genetic recombination.Genetic recombination in bacteriophage OX174 (1,2) and in the closely related bacteriophage S13 (3) has been analyzed extensively by both genetic (4-9) and physical (10-16) methods. Most OX174 recombinants are formed by a major pathway, Tessman's primary mechanism (4, 5), which requires the host recA + allele (6) but apparently does not need any of the nine known OX174 gene products (7). Recombinant formation via this major pathway involves two parental replicative form (RF) DNA molecules (5, 7), and occurs very early in the infection process (10). Single recombination events via the major pathway usually generate only one parental genotype and one recombinant (7). The goal of this work was to identify OX174 DNA structures which were formed by this major pathway.To determine whether a particular DNA structure was recombinant, parental replicative form DNA molecules were isolated from cells infected with two parental genotypes (11). After purification and fractionation of the DNA molecules by velocity and equilibrium sedimentation procedures (12, 13), the frequency of recombinants associated with various DNA structures was examined genetically using a spheroplast assay in which further recombination could not occur (10). In addition, we were able to identify putative recombinant DNA molecules formed in mixed infections by using two parental genotypes which could be distinguished by electron microscopy (13).In this paper we present electron micrographs of OX174 DNA molecules which apparently are recombinant. These structures appear to be "figure 8" molecules (15,17) We propose a simple mechanism to generate figure 8 DNA molecules that are recombinant. It is based on two molecular processes-"single-strand aggression" (11) and "branch migration" (19-21). These were previously implicated in recombinant formation in OX174 (10,11,13,15) and in other organisms (20,22). MATERIALS AND METHODSReplicative form and multiple length DNA molecules were isolated from cells infected with two genotypes (9, 11, 13, 23; R. M. Benbow, M. Eisenberg, and R. L. Sinsheimer, to be published). The fractionated DNA molecules were examined for recombinants by carrying out spheroplast infections (24), after which the progeny phage were assayed for wild-type recombina...

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Section: Resultsmentioning

confidence: 99%

Recombinant DNA molecules of bacteriophage phi chi174.

Benbow¹,

Zuccarelli²,

Sinsheimer³

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…1, a small peak of 32p at the position of 60 % sucrose cushion (Fr. [4][5][6] was intact cells, which were confirmed by a colony forming ability. A small peak of 3H which appeared at a sucrose concentration of about 52 % (Fr.…”

Section: Assay Ofmentioning

confidence: 68%

DNA Polymerase Activity Associated with the Membrane Fraction ofE. coli

Komano

Fujiwara

Onodera

1972

Agricultural and Biological Chemistry

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“…Logarithmic phase Escherichia coli HF4704 (nonsuppressor) in TPG medium (12) was pretreated with mitomycin C at 300 ,g/ml [to prevent host protein synthesis and cell lysis (13)], washed once with TPG, suspended in pre-warmed TPG medium (4 X 108 cells per ml), and aerated for 10 min. This culture (0.5 ml) was incubated at 370 and infected with phage (multiplicity of infection = 5).…”

Section: Methodsmentioning

confidence: 99%

Bacteriophage phiX174: gene A overlaps gene B.

Weisbeek¹,

Borrias²,

Langeveld³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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AI3STRACT The map position of several 4X174 mutations in the genes A and B was determined by marker rescue with DNA fragments produced by the restriction enzymes Hha I, HindII, Hae III, and Alu I. All the gene B mutants were found to be located within In a previous paper (6) we described an amber mutant (to8) that was mapped physically within gene A, whereas it did not behave like an A mutant. This result indicated either a complex genetic organization of this region or that the number of phage genes had to be increased. In this paper we describe a mote detailed analysis of this part of the genome. The results show that (i) the gene A extends itself far into (or even over) the gene B and (ii) the overlapping DNA sequence is used in two different reading frames, one frame for the synthesis of the A and A* protein and another frame for the B protein. MATERIALS AND METHODSMedia, methods, and bacterial strains for phage growth, titration, spheroplast infection, and preparation of single-stranded and double-stranded viral DNA have been described (1).Phage Mutants. am33 (A), amS5 (A), aml8 (A), aml6 (B), ts6 (B), ts116 (B), och6 (C), and am6 were given to us by R. L. Sinsheimer (2); amS29 (A), tsS6 (A), ts173 (B), and amH210 (B) were gifts from M. Hayashi (7); to8 (B), tsR5-2 (B), and amR8-1 were isolated in this laboratory (6).Isolation of DNA Fragments. The preparation of the restriction enzymes together with their conditions for DNA digestion has been described for HindII from Haemophilus influenzae Rd (R fragments), Hae III from Haemophilus aegyptius (Z fragments), and Alu I from Arthrobacter luteus (A fragments) by Vereyken et al. (8) and for Hha I from Haemophilus haemolyticus (H fragments) by Baas et al. (9). The RFI digests were fractionated on slab gels. The gels were autoradiographed and the fragments were excised and eluted (8).Fragment Assay. Restriction fragments were dissolved in 50 mM Tris.HCI (pH 7.0), the circular single-stranded mutant DNA in 0.60 M NaCl/0.06 M sodium citrate, pH 7 (4 X SSC).A 10-Al sample of fragments (A260 = 0.05) was added to'10 Al of circular ssDNA (A260 = 0.5), heated for 2 min in boiling water, and incubated for 20 min in a 64°water bath. After cooling of the mixture in ice, 0.18 ml distilled water was added. This solution was used in the spheroplast assay.Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis. Electrophoresis of cell extracts was performed in 12.5 or 15% slab gels that had a 3% stacking gel (10). Bisacrylamide was used as a crosslinker. Tritium-labeled proteins were made visible by scintillation autography (11); 35S-labeled proteins, by direct autoradiography of dried gels.Preparation of Infected Cell Extracts. Logarithmic phase Escherichia coli HF4704 (nonsuppressor) in TPG medium (12) was pretreated with mitomycin C at 300 ,g/ml [to prevent host protein synthesis and cell lysis (13)], washed once with TPG, suspended in pre-warmed TPG medium (4 X 108 cells per ml), and aerated for 10 min. This culture (0.5 ml) was incubated at 370 and infected with phag...

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The process of infection with bacteriophage φX174

Cited by 361 publications

References 18 publications

Recombinant DNA molecules of bacteriophage phi chi174.

Recombinant DNA molecules of bacteriophage phi chi174.

DNA Polymerase Activity Associated with the Membrane Fraction ofE. coli

Bacteriophage phiX174: gene A overlaps gene B.

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