Leonard Mindich

Electron microscopy of cells infected with nonsense mutants of bacteriophage φ6

Abstract Cells infected with nonsense mutants of bacteriophage φ6 were examined by electron microscopy. Mutants of class 812, which are incapable of synthesizing protein P8, the major component of the viral nucleocapsid, directed the formation of particles that appear to contain RNA and have the appearance of nucleocapsids in sectioned preparations. These particles are designated as filled procapsids. Mutants of class 12 or 9511, which are incapable of forming viral membrane, directed the synthesis of nucleocapsids. These particles are found distributed throughout the infected cells, although they appear to be somewhat aggregated when compared to the particles formed by the class 812 mutants. Mutants of class 511, which do not synthesize protein P5 or P11 which are involved in cell lysis, form particles indistinguishable from wild-type virus.

Nucleotide sequence of the middle dsRNA segment of bacteriophage φ6: Placement of the genes of membrane-associated proteins

The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA. We have determined the nucleotide sequence of cDNA derived from the middle-size RNA segment. The coding sequences of three proteins on this segment were identified on the basis of size and the correlation of predicted N-terminal amino acid sequences with those found through the analysis of isolated proteins. In contrast to our results with the small phi 6 dsRNA segment, the open reading frames are not tightly clustered. The homologous terminal noncoding regions between the middle and small dsRNA segments are found to be more extensive than RNA sequencing had previously indicated.

The morphogenesis of bacteriophage φ6: Particles formed by nonsense mutants

Abstract φ6 is a bacteriophage containing a genome of three pieces of double-stranded RNA and an envelope composed of lipids and proteins. The particles formed during infection of a nonsuppressor host by wild-type virus and six classes of nonsense mutants were investigated in this study. Mutants defective in the synthesis of P5 and P1l formed particles with RNA and lipid and all virion proteins but P5 and P11. Mutants defective in the synthesis of P3 made particles with lipid and RNA but also lacked P6. Particles formed by mutants defective in P6 were similar and lacked P3. These particles do not adsorb to host cells, indicating a role for P3 and P6 in attachment. Particles formed by a mutant defective in the synthesis of P9, P5, and P11 contained RNA but no lipid and also lacked P10, P3, and P6, the other membrane proteins. Particles formed by a mutant unable to form P12, a nonstructural protein, also lacked P9, P10, P3, P6, P5, and P11, and contained no lipid. We conclude that the core of the virion can be formed without the membranous exterior and that membrane protein P9 and the nonstructural protein P12 are necessary for the formation of the lipid-containing structure.

The molecular weight of bacteriophage θ6 and its nucleocapsid

Abstract A diameter of 82 nm, a molecular weight of 99 × 106, and a refractive index increment of 0.152 g−1cm3 were obtained for θ6 bacteriophage from turbidity measurements made with an analytical ultracentrifuge and a conventional spectrophotometer. Turbidity was also used to obtain a diameter of 64 nm and a molecular weight of 40 × 106 for the θ6 nucleocapsid produced when virus solutions are made 2% Triton X-100. The RNA content found for the intact virus by the orcinol method was 10%- by weight, which corresponds to 10 million for the total molecular weight of the three double-stranded RNA molecules comprising the genome. The phospholipid content of the intact virus was found to be 20% by weight. The relative amounts of individual protein species were determined by gel electrophoretic analyses of radioactive virus. The results yielded estimates of the number of copies of each protein in the virion.

Bacteriophage ϕ6: a Unique Virus Having a Lipid-Containing Membrane and a Genome Composed of Three dsRNA Segments

Publisher Summary Bacteriophage Φ6 has two unique aspects that have resulted in its utility as a model system for processes in more complex systems. The virus contains a genome composed of three segments of double-stranded (ds) RNA. The replication of the genome appears to be similar to that of the dsRNA viruses that infect eukaryotic organisms. Φ6 is therefore a useful system for studying the basic mechanisms of dsRNA replication and genomic packaging. In addition, the viral nucleocapsid is enveloped by a lipid-containing membrane that is very simple in its protein composition. The assembly inside the infected cells offers a model for the study of membrane differentiation and translocation. The Φ6 system has proved to be amenable to analysis. The entire genome is cloned as cDNA and the nucleotide sequence of the cDNA is determined. In addition, it has been possible to isolate nonsense mutants for 9 of the 12 genes of Φ6 and temperature-sensitive (ts) or missense mutants for two others. The virion contains a very active transcriptase that is capable of directing the synthesis of full-length messages of the three genomic segments.

Genetic studies of temperature-sensitive and nonsense mutants of bacteriophage φ6

Abstract Forty-six temperature-sensitive mutants of bacteriophage φ6 were isolated. Crosses between these mutants yielded a pattern consistent with three segregation groups for the mutant genes. Nonsense mutants were also isolated and these fell into eight classes on the basis of proteins missing during infection of nonsuppressor cells. Crosses between these mutants also showed three classes of segregation groups and these classes were shown to be the same as those found for the temperature-sensitive mutants. The three sets of segregation groups were as follows: Set A included mutants affecting polymerase factors PI and P2. Set B included mutants affecting adsorption proteins P3 and P6 as well as the host range marker hl. Set C included mutants affecting the synthesis of proteins P8, P12, P9, P5, and Pll. The three segregation groups probably correspond to the three segments of dsRNA found in virions and infected cells. On the basis of the size of proteins Pl and P2, we conclude that set A must be localized on the largest segment of dsRNA.

Membrane synthesis in Bacillus subtilis. II. Integration of membrane proteins in the absence of lipid synthesis.

Abstract Net lipid synthesis stops when glycerol is removed from a glycerol auxotroph of Bacillus subtilis. Under these conditions, the incorporation of proteins into cell membranes continues. The amount of protein in the membrane fraction and the total activity of succinic dehydrogenase both increase in the membrane fraction of the deprived cells. Membranes isolated from cells deprived of glycerol for one hour have approximately 12% lipid as compared to 21% lipid in normal cells. The membranes of deprived cells exhibit a greater buoyant density (1.19 g/ml.) than those from normal cells (1.17 g/ml.). When protein synthesis is inhibited by the growth of cells in the presence of chloramphenicol, isolated membranes have a 15 to 30% higher lipid content than that of normal cells. These findings are discussed in relation to the regulation of membrane composition.

In vitro packaging of the bacteriophage φ6 ssRNA genomic precursors

Bacteriophage phi 6 contains three segments of double-stranded RNA within a nucleocapsid. Plasmids containing cDNA copies of the large genomic segment direct the synthesis of viral proteins that assemble into procapsids in Escherichia coli or Pseudomonas phaseolicola. These structures are dodecahedral assemblages of proteins P1, P2, P4, and P7. We report in this paper that these particles are capable of packaging viral single-stranded plus-sense RNA in vitro. The packaging reaction requires the presence of ATP or dATP. Synthesis of minus strands takes place within this filled procapsid in the presence of all four nucleoside triphosphates. Packaged ssRNA is found to be protected from added ribonuclease.

The characterization of a 120 S particle formed during θ6 infection

Abstract A particle is formed in cells infected with wild-type and mutant bacteriophage θ that could be a precursor of the nucleocapsid. This particle is composed of proteins P1, P2, P4 and P7, has no RNA, and gives the appearance in negatively stained electron micrographs of a hexagon or a star.

Membrane synthesis in Bacillus subtilis: I. Isolation and properties of strains bearing mutations in glycerol metabolism

Abstract An experimental system for the study of membrane synthesis was constructed with mutant strains of Bacillus subtilis . Mutants were selected for glycerol auxotrophy and the inability to use glycerol as a source of carbon. Cells containing both mutations incorporated glycerol almost entirely into lipid. When cells were deprived of glycerol under otherwise normal growth conditions, the following results were obtained. The deprived cells underwent one slow division and remained viable for at least four hours. Net phospholipid synthesis stopped immediately, fatty acid synthesis was reduced to about 25% of the supplemented control, protein and DNA synthesis continued for about 90 minutes and net RNA synthesis ceased after about 30 minutes. Messenger RNA synthesis continued during the period in which net RNA synthesis was stopped, as indicated by continued protein synthesis and the inducibility of several enzymes. DNA synthesis did not stop at the end of a round of replication. Cell wall synthesis continued at the same rate as protein synthesis. The incorporation of labeled amino acids into membrane protein continued in the absence of lipid synthesis, indicating that a close co-ordination of membrane protein and lipid synthesis does not occur.