Robert L. Sinsheimer

A single-stranded deoxyribonucleic acid from bacteriophage φX174†

The deoxyribonucleic acid (DNA) of bacteriophage φX174 can be extracted by phenolic denaturation of the virus protein. The DNA thus obtained has a molecular weight of 1·7 × 10^6, indicating that there is one molecule per virus particle. This φX DNA does not have a complementary nucleotide composition. The ultraviolet absorption of this DNA is strongly dependent upon temperature in the range 20° to 60°C, and upon NaCl concentration in the range 10^(−3) to 10^0 M. This DNA reacts with formaldehyde at 37°C and is precipitated by plumbous ions. This evidence is interpreted to mean that the purine and pyrimidine rings are not involved in a tightly hydrogen-bonded complementary structure. Light scattering studies indicate that this DNA is highly flexible and that its configuration is strongly dependent upon the ionic strength of the solution. Upon treatment with pancreatic deoxyribonuclease, the weight-average molecular weight decreases in accordance with the function expected for a single-stranded molecule. It is concluded that the DNA of bacteriophage φX174 is single-stranded.

Purification and Properties of Bacteriophage MS2 and of its Ribonucleic Acid

Methods are described for the isolation in pure form of bacteriophage MS2 and of its RNA. The virus has a particle weight of 3·6 × 10^6, and from phosphorus content is estimated to be 32% RNA by weight. The particle weight of the isolated RNA is 1·05 ± 0·1 × 10^6, indicating that there is one molecule of RNA per virus particle. The RNA is shown to have several unusual properties. In 0·2 M-NaCl it has a very small radius of gyration and a large sedimentation coefficient for its molecular weight, indicating a very compact structure. Upon heating the RNA in 0·14 M-NaCl solutions, the ultraviolet absorbancy transition has a midpoint at 76°C, suggesting a high degree of hydrogen bonding. In lower salt concentrations the RNA aggregates; in 0·02 M-NaCl the observed molecular weight is twice that in 0·2 M-NaCl, and in 0·003 M-tris it is 3 times this value.

The process of infection with bacteriophage φX174: I. Evidence for a “Replicative form”†

Procedures are described for the preparation of density- and radioactivity-labelled φX174 particles. The process of bacterial infection with such particles has been studied by density-gradient analysis of the contents of the infected cells prior to normal lysis. Upon entry into a susceptible cell, the single-stranded DNA of φX174 is converted to an altered “replicative form” (RF). The RF, which is infective to bacterial protoplasts, is then multiplied manyfold. Conversion to RF and multiplication of RF can take place in the presence of chloramphenicol. At no time during the infective process is there a pool of free single-stranded DNA. In normal infection single-stranded progeny DNA appears in mature progeny virus particles as early as eight minutes after infection. In chloramphenicol neither progeny virus particles nor single-stranded DNA are formed. Several properties of the RF are similar to those of double-stranded DNA. It is lighter in density than the single-stranded DNA. The density of the RF particles containing the density-labelled parental DNA is approximately that which would be expected if these molecules had been converted to a double-stranded complementary form. Upon heating, the RF appears to denature in a manner similar to double-stranded DNA. The RF is considerably more resistant to inactivation by ultraviolet radiation than is the single-stranded DNA; this resistance can be melted out by heating of the RF.Procedures are described for the preparation of density- and radioactivity-labelled φX174 particles. The process of bacterial infection with such particles has been studied by density-gradient analysis of the contents of the infected cells prior to normal lysis. Upon entry into a susceptible cell, the single-stranded DNA of φX174 is converted to an altered “replicative form” (RF). The RF, which is infective to bacterial protoplasts, is then multiplied manyfold. Conversion to RF and multiplication of RF can take place in the presence of chloramphenicol. At no time during the infective process is there a pool of free single-stranded DNA. In normal infection single-stranded progeny DNA appears in mature progeny virus particles as early as eight minutes after infection. In chloramphenicol neither progeny virus particles nor single-stranded DNA are formed. Several properties of the RF are similar to those of double-stranded DNA. It is lighter in density than the single-stranded DNA. The density of the RF particles containing the density-labelled parental DNA is approximately that which would be expected if these molecules had been converted to a double-stranded complementary form. Upon heating, the RF appears to denature in a manner similar to double-stranded DNA. The RF is considerably more resistant to inactivation by ultraviolet radiation than is the single-stranded DNA; this resistance can be melted out by heating of the RF.

Observations on the infection of bacterial protoplasts with the deoxyribonucleic acid of bacteriophage ΦX174

An improved procedure for preparation and infection of protoplasts with ΦX DNA, resulting in a higher efficiency of infection and a linear relation between infective centers produced and DNA concentration, is described. The effects of various ionic conditions and of various interfering agents upon this procedure are discussed. Studies of the fate of the viral DNA using this procedure indicate that at least 70% remains free, and largely still potentially infective, while at least 10% attaches to the protoplasts. Most of this attached fraction is, potentially, infective, but only a few molecules per thousand succeed in iniating an infection. Studies of the combined effects of DNA concentration and protoplast concentration reveal that over a wide range of conditions [infected protoplasts] = 10−12 [DNA molecules] [protoplasts] This result is interpreted to mean that the limitations upon infection are primarily kinetic, and do not represent limitations imposed by the proportions of infective DNA or of infectable protoplasts.

Purification and properties of bacteriophage φX174

Procedures are described for the isolation in pure form of the bacterial virus φX174. This virus is shown to have a particle weight of 6·2 × 10^6 and to contain 25 % by weight of DNA. At neutral pH, the purified virus forms a discrete aggregate, probably a tetra-mer. This aggregate dissociates either upon dilution or at alkaline pH. Lysates produced by φX174 contain a second particle, antigenically related to the virus, but not infective and of lower sedimentation rate and lesser DNA content. The DNA of φX174 appears to have an unusual structure in that it reacts with formaldehyde (even before extraction from the virus) and in that the atomic efficiency of inactivation of the virus by decay of incorporated phosphorus-32, calculated by combining our data and that of Tessman, is 1·0.

Lysis of Escherichia coli with a neutral detergent.

1. When Escherichia coli cells are treated with lysozyme and versene for 30–45 sec in an ice-bath, they become susceptible to lysis by the neutral detergent Brij-58. 2. The rate of lysis is dependent upon the concentration of the detergent. Brij-58 is approx. 1000-fold less active in lysis of the lysozyme-versene-treated cells than is deoxycholic acid or sodium docecylsulphate. 3. The extent of cell disruption is dependent upon the Mg^(2+) concentration and the ionic strength at the time of lysis. In 70 mM Mg^(2+), only the low molecular weight soluble RNA and soluble protein are released from the cell. In 40 mM Mg^(2+), the 70-S monosomes and the ribosome subunits are released together with the soluble material. At still lower Mg^(2+) concentrations, the polyribosomes are released. 4. Unless the Mg^(2+) concentration is reduced below 5 mM, the cellular DNA, after lysis, may be almost completely sedimented by centrifugation at 3000 × g for 5 min. 5. All strains of E. coli tested were susceptible to lysis by this method.

The process of infection with bacteriophage ΦX174: X. Mutations in a ΦX lysis gene†

The ability of conditional lethal mutants of phage ΦX174 to induce host cell lysis during infection under restrictive conditions has been studied. We have found amber (am) and temperature-sensitive (ts) mutants which present a variety of alterations in the normal lytic process. In particular, there is a class of am mutants which do not produce cell lysis but otherwise replicate normally in the restrictive host. These mutants constitute a single complementation group. The existence of these mutants implicates a phage-coded protein in the lytic process. This protein is not an essential structural component of the phage, since normal phage particles are produced in the absence of lysis.

The structure of the DNA of bacteriophage φX174: III. Ultracentrifugal evidence for a ring structure*

By velocity sedimentation, in appropriate solvents, the presence of two discrete components can be demonstrated in preparations of the DNA of bacteriophage φX174. The major, faster-moving S_1 and the slower S_2 are present under conditions which exclude the possibility of hydrogen-bond formation. It can be shown, either by treatment with pancreatic deoxyribonuclease or by thermal inactivation, that S_2 is the first degradation product of S_1, formed by scission of S_1, without significant decrease in molecular weight. A subsequent chain scission in S_2, which occurs with equal likelihood, results in random fragmentation. These results are interpreted to mean that the S_1 component is a covalently linked ring structure and the S_2 component is the corresponding open-chain degradation product. Under certain conditions the S_2 component can be selectively degraded by E. coli phosphodiesterase. The digestion is not complete and there appears to be a single discontinuity, resistant to phosphodiesterase, present in the φX-DNA ring.

Infection of Protoplasts of Escherichia coli by Subviral Particles of Bacteriophage φX174

Infection of protoplasts of various strains of Escherichia coli may be obtained with purified DNA preparations from bacteriophage φX174 made by two distinctly different methods. Infection of such protoplasts can also be obtained with nucleoprotein particles made from φX174 by heat treatment. None of the particles, infective to protoplasts, are infective to whole cells and all are infective to protoplasts of strains of bacteria usually resistant to whole φX174 virus. The protoplast-infective agents are in all cases destroyed by enzymatic treatment with DNase. The infective DNA is not inactivated by treatment with enzymatic amounts of trypsin but is by treatment with stoichiometric amounts, suggesting the formation of an inactive DNA-trypsin complex. Studies of the properties of protoplast infection indicate certain general characteristics which distinguish it from the more familiar virus infection of whole bacterial cells.

The process of infection with bacteriophage ΦX174: VII. Ultracentrifugal analysis of the replicative form

A procedure is described for the preparation of a purified ΦX174 replicative form DNA. Ultracentrifugal analyses, zone sedimentation and density gradient of this DNA, both when native and when denatured, are described. At neutral pH, two major sedimentation components in the purified replicative form of 21·2 s (I) and 16·2 s (II) (as the sodium salts) are observed. At alkaline pH, in high ionic strength medium, or at neutral pH in formamide, (I) gives rise primarily to a rapidly sedimenting component, the denatured double-stranded DNA ring. Under the same conditions, (II) gives rise to infective single-stranded DNA rings. It is concluded that (I) is the closed double-strand DNA ring, whereas (II) is composed of double-stranded DNA rings in which one strand is open.