Escherichia coli traD(Ts) mutant temperature sensitive for assembly of RNA bacteriophage MS2.

We report here a study on the temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39, carrying a traD(Ts) mutation, which is also temperature sensitive for group I RNA phages (MS2, f2, and R17). It is shown that, when the mutant was infected with MS2 at 42 degrees C, phage RNA replicated; a 27S MS2 RNA and phage proteins were synthesized. However, neither PFU nor physical MS2 particles were formed, showing that phage assembly was inhibited. In addition, the high temperature affected the membranes of the host mutant: the mutant was hypersensitive to chemicals, and the electrophoretic pattern of the membranal proteins was modified. We suggest that the pleiotropic effects of the traD mutation on MS2 assembly and DNA transfer during conjugation were a result of the changes in the membrane of the mutant.     

An electron microscopic analysis of pathways for bacteriophage T4 DNA recombination

The interaction between ribosomes of Bacillus stearothermophilus and the RNA genomes of R17 and Qβ bacteriophage has been studied. Whereas Escherichia coli ribosomes can initiate the synthesis of all three RNA phage-specific proteins in vitro, ribosomes of B. stearothermophilus were previously shown to recognize only the A (or maturation) protein initiation site of f2 or R17 RNA. Under these same conditions, a Qβ region is bound and protected from nuclease digestion. Qβ RNA, however, does not direct the synthesis of any formylmethionyl dipeptide in the presence of B. stearothermophilus ribosomes, nor does the binding of either this Qβ region or the R17 A protein initiation site to these ribosomes show the same fMet-tRNA requirement for recognition of initiator regions as that previously established with E. coli ribosomes. Analysis of a 38-nucleotide sequence in the protected Qβ region reveals no AUG or GUG initiator codon. These observations suggest that messenger RNA may be recognized and bound by B. stearothermophilus ribosomes quite independently of polypeptide chain initiation.Binding experiments using R17 RNA and mixtures of components from B. stearothermophilus and E. coli ribosomes confirm the conclusion drawn by Lodish (1970a) that specificity in the selection of authentic phage initiator regions by the two species resides in the ribosomal subunit(s). However, anomalous attachment of B. stearothermophilus ribosomes to R17 RNA, which is observed upon lowering the incubation temperature of the binding reaction, is clearly a property of the initiation factor fraction. The results are discussed with respect to current ideas on the role of ribosomes and initiation factors in determining the specificity of polypeptide chain initiation.     

Complete nucleotide sequence of the group I RNA bacteriophage fr

We report the complete nucleotide sequence of the group I RNA bacteriophage fr. The entire genome consists of 3575 nucleotides, six nucleotides more than the only other sequenced group I representative, MS2. The greatest divergence between these phages occurs in the 5' terminal region of the A gene, while the lysis-replicase gene overlap, the coat gene and the central region of the replicase gene are highly conserved. Overall sequence homology between fr and MS2 is 77%. Here, we present a general comparison between the two phages. In the accompanying paper we use phylogenetic sequence comparison between MS2 and fr to deduce the secondary structure at the 3' untranslated region.     

A mathematical model for RNA bacteriophage production in batch culture

The interaction between male-specific RNA phages and bacterial cells as well as the complete life cycle of RNA phages in the host cells are complicated phenomena. In this study, a mathematical model is proposed to describe the kinetics of RNA phage production in batch culture. The model consists of several important considerations: (1) adsorption and desorption of phages on cell pili, (2) injection and transport of viral RNA, (3) viral protein synthesis, (4) phage maturation, and (5) cell lysis. Experimental data of MS2 RNA phage production in E. coli C 300o bacteria culture were used to evaiuate the model parameters. Reasonably good fit was obtained between the model and one set of data. However, simulation study based on the estimated parameter values revealed a discrepancy between experimental observation and model prediction. It seems that variation both in F-piliation and in the competence of cells to be infected by phages through different phasae of growth must be taken into account in order to make the model useful.     

Grouping of RNA Phages by a Millipore Filtration Method

Twenty-five strains of RNA phages were tested for their filtration and elution patterns (F-E patterns) by a millipore filtration method. These strains, including MS2, f2 and R17, were separated in three groups in this aspect. We named these groups as group I, II and III. According to this grouping, MS2, f2 and R17 belonged to group I and Qβ belonged to group III. Furthermore, it was shown that group III was further divided in two sub-groups (IIIa and IIIb) by this method. Grouping based on the F-E patterns was in extremely good accordance with the grouping based on the serological properties. This grouping was also supported by the results of chemical and physical analyses of these RNA phages, that is, RNA phages which belonged to the same group had several common properties. Basic Information on the millipore filtration method was also presented in this paper.     

Discriminative effect of rifampin of RNA replication of various RNA bacteriophages.

Rifampin interferes exclusively with RNA replication in vivo of the group I phages MS2, f2, and R17, whereas QbetaRNA replication is unaffected by the drug. In addition, rifampin has a discriminative effect of group I phage RNA replication. In the experimental system employed by us the antibiotic differentially interferes with the synthesis of minus RNA strands in f2, whereas it has almost no effect on the synthesis of progeny plus strands. In MS2, the drug differentially arrests the synthesis of progeny plus strands and almost fails to affect the synthesis of minus RNA strands. In R17 both steps of its RNA replication are affected by rifampin, although each step is only partially (approximately 50%) inhibited. The relation of the present results to the possible role of bacterial proteins and tertiary structure of phage RNA in the process of template recognition is discussed.     

Possible Origin of a Minor Virus Specific Protein (A<Subscript>1</Subscript>) in Qβ Particles

RNA phages such as R17, MS2 and f2 (group I of Watanabe¹) consist of a single-stranded RNA, about 180 molecules of coat protein and one of A (or maturation) protein, a component required for the functional integrity of the viral particle, possibly for its adsorption to the host^2–4. The phage genome comprises three cistrons, accounting for the two proteins mentioned above as well as for a component of the viral RNA replicase^5,6.     

A new family of interspersed repetitive DNA sequences in the mouse genome

Repetitive DNA sequences near immunoglobulin genes in the mouse genome (Steinmetz et al., 1980a,b) were characterized by restriction mapping and hybridization. Six sequences were determined that turned out to belong to a new family of dispersed repetitive DNA. From the sequences, which are called R1 to R6, a 475 base-pair consensus sequence was derived. The R family is clearly distinct from the mouse B1 family (Krayev et al., 1980). According to saturation hybridization experiments, there are about 100,000 R sequences per haploid genome, and they are probably distributed throughout the genome. The individual R sequences have an average divergence from the consensus sequence of 12.5%, which is largely due to point mutations and, among those, to transitions. Some R sequences are severly truncated. The R sequences extend into A-rich sequences and are flanked by short direct repeats. Also, two large insertions in the R2 sequence are flanked by direct repeats. In the neighbourhood of and within R sequences, stretches of DNA have been identified that are homologous to parts of small nuclear RNA sequences. Mouse satellite DNA-like sequences and members of the B1 family were also found in close proximity to the R sequences. The dispersion of R sequences within the mouse genome may be a consequence of transposition events. The possible role of the R sequences in recombination and/or gene conversion processes is discussed.     

Construction and characterization of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA.

An apparently full-length complementary DNA copy of in vitro polyadenylated MS2 RNA was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. After the MS2 RNA template was removed from the complementary DNA strand with T₁ and pancreatic RNase digestion, the complementary DNA became a good template for the synthesis of double-stranded MS2 DNA with Escherichia coli DNA polymerase I. We then constructed molecular chimeras by inserting the double-stranded MS2 DNA into the PstI restriction endonuclease cleavage site of the E. coli plasmid pBR322 by means of the poly(dA)· poly(dT) tailing procedure. An E. coli transformant carrying a plasmid with a nearly full-length MS2 DNA insertion, called pMS2-7, was chosen for further study. Correlation between the restriction cleavage site map of pMS2-7 DNA and the cleavage map predicted from the primary structure of MS2 RNA, and nucleotide sequence analysis of the 5′ and 3′ end regions of the MS2 DNA insertion, showed that the entire MS2 RNA had been faithfully copied, and that, except for 14 nucleotides corresponding to the 5′-terminal sequence of MS2 RNA, the fulllength DNA copy of the viral genetic information had been inserted into the plasmid. Restriction endonuclease analysis of the chimera plasmid DNA also revealed the presence of an extra DNA insertion which was identified as the translocatable element IS1³ (see following paper).     

The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein

Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor. Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis. Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis. Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function. Expression of other cistrons in addition to the A2 cistron does not enhance host lysis. Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis. This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.     

EcoRI restriction endonuclease cleavage site map of bacteriophage P22DNA.

The F plasmid is able to co-transfer (mobilize) the small, chimeric R plasmid pBR322 during conjugation only at a very low frequency (Bolivar et al., 1977). Mobilization has been found here to be invariably (> 99%) associated with a structural alteration of pBR322. The alteration was shown, by restriction endonuclease analysis and electron microscopy, to be an insertion of the F attachment sequence λδ (2.8 to 8.5F). λδ is, therefore, an insertion sequence.     

Organization of chimeras between filamentous bacteriophage f1 and plasmid pSC101

Two recombinants formed in vivo between the filamentous phage f1 and the tetracycline-resistance-conferring plasmid pSC101 are capable of transducing sensitive cells to Tet^r. These chimeric filamentous phage, VO-1 and VO-2, were previously shown to contain the entire f1 and pSC101 genomes (Vovis et al., 1977; Ohsumi et al., 1978). The genomes of VO-1 and VO-2 are unstable in vivo; VO-1 breaks down to yield a molecule similar to pSC101 and an f1-like species, f1′. f1′ was previously shown to differ from f1 by the presence of 209 additional nucleotides inserted in the carboxy-terminal portion of gene IV (Ravetch et al., 1979). We have found by hybridization analysis and direct DNA sequencing that this 209-nucleotide segment is present in one copy in pSC101, and that it has properties similar to known transposable elements. Therefore, we have called this sequence IS101. We have characterized the structures of both VO-1 and VO-2 in greater detail by restriction mapping and DNA sequence analysis. Both chimeras contain two copies of IS101, which are present as direct repeats and form the junctions between the f1 and pSC101 genomes. The IS101 elements in VO-1 and VO-2 are flanked by a five-base direct repeat of f1 sequence that is not repeated in wild-type f1. The junction between f1 and pSC101 in VO-1 is located at the same point as the IS101 element in f1′, while in VO-2 the junction between the two genomes is at a point in f1 located between the promoter and ribosome binding site for gene VIII. The pSC101-like molecules derived from the breakdown of VO-1 in vivo are identical to the original pSC101 in the region of IS101. The IS101 elements in the original and derived pSC101 plasmids are not flanked by any repeated sequence. Attempts to regenerate VO-1 from f1′ and pSC101, both of which contain one IS101 element, indicate that the breakdown of VO-1 is irreversible. These results are discussed in terms of current models for transposition, which postulate structures similar to VO-1 and VO-2 as intermediates in transposition.     

Coat proteins of strains of two RNA viruses: comparison of their amino acid sequences

Summary The amino acid sequences of four strains of tobacco mosaic virus isolated in different parts of the world are compared. The differences between the strains are discussed with respect to special proteinchemical features (such as beginning of the chain, deletion of amino acids, number of different amino acids, sizes and distribution of regions with invariable amino acids) and with respect to the possibility of deducing the most probable nucleotide sequence for the coat protein cistron of tobacco mosaic virus.The complete amino acid sequences of the two RNA bacteriophage strains fr and f₂ are compared. According to their coat proteins three groups of phages can be formed: 1) MS 2, f₂ M 12 and R 17, 2) fr and 3) Q.     

Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E₃ from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.     

Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). I. Initial characterization

Three mutant strains of Escherichia coli were independently isolated based upon their inability to propagate bacteriophage λ. The strain most extensively studied, NS-1, has a pleiotropic temperature sensitive alteration that affects cell growth, stable RNA synthesis and λ propagation. Labeling experiments and colorimetric determinations of total RNA carried out in this strain demonstrate that within the first five minutes after raising the temperature to 44.5 °C the rate of total RNA accumulation is reduced to a level that is about 15% that of the control, while protein and DNA synthesis continue at nearly normal rates for at least 30 min. This effect is either due to a very rapid degradation of stable RNA species or a reduced synthesis of RNA. Although the accumulation of all stable RNA species (23, 16 and 4 S RNAs) is reduced co-ordinately to levels ranging from 12 to 16% that of the control, the synthesis of messenger RNA is affected to a lesser degree, if at all. The defect in RNA accumulation can be partially reversed by the addition of chloramphenicol at the moment of temperature shift.In addition to phage λ these strains are unable to propagate RNA phage R17 and lambdoid phages φ80, 21 and 434 at elevated temperatures. The growth of phages T4, T7, P1 and P2 is normal.A genetic analysis of strain NS-1 indicates that all of its temperature sensitive properties depend on a mutation, designated groE-1, which co-transduces with a mel (melibiose) marker. However, the expression of the RNA synthesis defect requires, in addition, a second mutation which does not co-transduce with mel.     

Membrane-like protein involved in phage adsorption associated with phage-sensitivity in the bloom-forming cyanobacterium Microcystis aeruginosa

The cyanobacterium, Microcystis aeruginosa, contains a large number of defense genes (Makarova et al., 2011); thus, it is a good model to study the co-evolution of phage and bacteria. Here, we isolated and characterized two phage-resistant M. aeruginosa mutants that came from a phage intermediate-sensitive culture. To determine the mutation conferring resistance, a protein expression pattern analysis was performed comparing phage-sensitive and -resistant sub-strains using SDS-PAGE. There were no apparent differences in expression patterns in the soluble fraction; however, a ∼90 kDa protein in the hydrophobic fraction from the phage-sensitive sub-strain was observed. Using a successive thermal asymmetric interlaced-PCR, the entire sequence encoding the protein, assigned ISP90, as well as its neighboring regions (ca. 7.8 kb) was determined. ISP90 contained no conserved domains and was predicted to be a membrane-associated protein. No mutations were detected in the nucleotide sequences coding ISP90 and diversification of ISP90 regions within this species were observed. Diversification of ISP90 regions within this species suggests a possible genomic island that may be subjected to selective pressures from phages. The ISP90 sequence involving phage resistance/sensitivity contributes to the understanding of co-evolution between M. aeruginosa and phages.     

Escherichia coli mutant temperature sensitive for group I RNA bacteriophages.

The temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39, carrying a traD(Ts) mutation, is herein described as also being temperature sensitive for group I RNA phages (MS2, f2, and R17) but not for Q beta. Temperature shift experiments showed that the growth of group I phage MS2 in the mutant could be inhibited by a post-penetration event at high temperature. A possible role for the traD cistron of sex factor F in the intracellular development of MS2 is suggested.     

Isolation and sequencing of the 5′ end of the rat microtubule-associated protein (MAP1B)-encoding cDNA

We have isolated and sequenced the 5′ end of the cDNA encoding the rat microtubule-associated protein 1B (MAP1B). We found that this region is highly homologous to the corresponding regions of the human [Lien et al., 22 (1994) 273–280] and mouse [Noble et al., J. Cell Biol. 109 (1989) 3367–3376] MAPIB genes. The combination of the sequence that we are presenting with the previously published sequence [Zauner et al., Eur. J. Cell Biol. 57 (1992) 66–74], represents the complete rat MAP1B cDNA coding sequence.     

Superinfection exclusion reveals heteroimmunity between Pseudomonas aeruginosa temperate phages

Temperate siphophages (MP29, MP42, and MP48) were isolated from the culture supernatant of clinical Pseudomonas aeruginosa isolates. The complete nucleotide sequences and annotation of the phage genomes revealed the overall synteny to the known temperate P. aeruginosa phages such as MP22, D3112, and DMS3. Genome-level sequence analysis showed the conservation of both ends of the linear genome and the divergence at the previously identified dissimilarity regions (R1 to R9). Protein sequence alignment of the c repressor (ORF1) of each phage enabled us to divide the six phages into two groups: D3112 group (D3112, MP29, MP42, and MP48) and MP22 group (MP22 and DMS3). Superinfection exclusion was observed between the phages belonging to the same group, which was mediated by the specific interaction between the c repressor and the cognate operator. Based on these, we suggest that the temperate siphophages prevalent in the clinical strains of P. aeruginosa represent at least two distinct heteroimmunity groups.