Parallel molecular evolution of deletions and nonsense mutations in bacteriophage T7.
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Bacteriophage T7↗
Biomedical subjects
I J Molineux
Publications and source records attributed to I J Molineux.
Transcription-independent DNA translocation of bacteriophage T7 DNA into Escherichia coli.
Penetration of wild-type T7 DNA into the host cell occurs in two steps. The phage particle ejects a few hundred base pairs of the left end of the genome into the host. Translocation of the remainder of the DNA is then coupled to transcription. In a normal infection, transcription-coupled translocation of wild-type T7 DNA is initiated at the major A1, A2, and A3 promoters for Escherichia coli RNA polymerase. At 37 degrees C, various deletion mutants lacking these three promoters grow at the same efficiency as wild-type T7 because the minor B promoter is efficiently transferred from the phage head into the cell. As the temperature of the phage infection decreases, the latent periods of (A1, A2, A3)- phages increase relative to that of wild-type T7; nevertheless, (A1, A2, A3)- phages have normal plating efficiencies at reduced temperatures. Lengthening of the latent period at low temperatures is due to a delay in transferring the complete (A1, A2, A3)- genome into the host cell. The (A1, A2, A3)- phages eject the leading end of their genome into the host, but at low temperature, insufficient DNA is transferred into the cell to allow RNA polymerase immediate access the B promoter. However, by an inefficient transcription-independent process, the B promoter eventually translocates into the cell. Mutant derivatives of (A1, A2, A3)- phages that have growth profiles at low temperatures similar to that of wild-type T7 have been isolated. The mutations allow both (A1, A2, A3)- and (A1, A2, A3)+ phages to translocate their entire genomes into the cell by a transcription-independent mechanism. The mutations are located in gene 16, a gene that encodes a component of the internal virion core. We postulate that gp16 is directly involved with the process of DNA translocation from the virion into the cell.
Bacteriophage T7↗
Rate of translocation of bacteriophage T7 DNA across the membranes of Escherichia coli.
Translocation of bacteriophage T7 DNA from the capsid into the cell has been assayed by measuring the time after infection that each GATC site on the phage genome is methylated by cells containing high levels of DNA adenine methylase. Methylation at GATC sites on T7 DNA renders both the infecting genome and any newly synthesized molecules sensitive to the restriction enzyme DpnI. In a normal infection at 30 degrees C, translocation of the T7 genome into the cell takes between 9 and 12 min. In contrast, translocation of the entire phage lambda genome or of a T7 genome ejected from a lambda capsid can be detected within the first minute of infection. Entry of the leading end of the T7 genome occurs by a transcription-independent mechanism that brings both Escherichia coli and T7 promoters into the cell. Further translocation of the genome normally involves transcription by the RNA polymerases of both E. coli and T7; the rates of DNA translocation into the cell when catalyzed by each enzyme are comparable to the estimated rates of transcription of the respective enzymes. A GATC site located between the early E. coli promoters and the coding sequences of the first T7 protein made after infection is not methylated before the protein is synthesized, a result supporting the idea (B. A. Moffatt and F. W. Studier, J. Bacteriol. 170:2095-2105, 1988) that only certain proteins are permitted access to the entering T7 DNA. In the absence of transcription, the genomes of most T7 strains do not completely enter the cell. However, the entire genome of a mutant that lacks bp 3936 to 808 of T7 DNA enters the cell in a transcription-independent process at an average overall rate of 50 bp per s.
Bacteriophage T7↗
Incomplete entry of bacteriophage T7 DNA into F plasmid-containing Escherichia coli.
The penetration of bacteriophage T7 DNA into F plasmid-containing Escherichia coli cells was determined by measuring Dam methylation of the entering genome. T7 strains that cannot productively infect F-containing cells fail to completely translocate their DNA into the cell before the infection aborts. The entry of the first 44% of the genome occurs normally in an F-containing cell, but the entry of the remainder is aberrant. Bypassing the normal mode of entry of the T7 genome by transfecting naked DNA into competent cells fails to suppress F exclusion of phage development. However, overexpression of various nontoxic T7 1.2 alleles from a high-copy-number plasmid or expression of T3 1.2 from a T7 genome allows phage growth in the presence of F.
Bacteriophage T7↗
Streptomycin- and rifampin-resistant mutants of Escherichia coli perturb F exclusion of bacteriophage T7 by affecting synthesis of the F plasmid protein PifA.
Certain alleles of rpsL that confer resistance to the antibiotic streptomycin almost completely relieve F exclusion of bacteriophage T7. Introduction of a specific rpoB allele conferring resistance to rifampin into the rpsL strain restores the ability of the F-containing strain to exclude T7. This variation in the severity of F exclusion is reflected in the levels of the F-encoded inhibitor protein PifA: F'-containing cells that harbor specific rpsL alleles are phenotypically Pif-, but become Pif+ by the further acquisition of a specific rpoB allele. F-containing cells harboring the gyrA43(Ts) mutation also appear phenotypically Pif-, possibly because repression of the pif operon is enhanced by an altered DNA conformation in the gyrase mutant strain.
Bacterial Proteins↗
Experimental phylogenetics: generation of a known phylogeny.
Although methods of phylogenetic estimation are used routinely in comparative biology, direct tests of these methods are hampered by the lack of known phylogenies. Here a system based on serial propagation of bacteriophage T7 in the presence of a mutagen was used to create the first completely known phylogeny. Restriction-site maps of the terminal lineages were used to infer the evolutionary history of the experimental lines for comparison to the known history and actual ancestors. The five methods used to reconstruct branching pattern all predicted the correct topology but varied in their predictions of branch lengths; one method also predicts ancestral restriction maps and was found to be greater than 98 percent accurate.
Biological Evolution↗
Genes 1.2 and 10 of bacteriophages T3 and T7 determine the permeability lesions observed in infected cells of Escherichia coli expressing the F plasmid gene pifA.
Infections of F plasmid-containing strains of Escherichia coli by bacteriophage T7 result in membrane damage that allows nucleotides to exude from the infected cell into the culture medium. Only pifA of the F pif operon is necessary for "leakiness" of the T7-infected cell. Expression of either T7 gene 1.2 or gene 10 is sufficient to cause leakiness, since infections by phage containing null mutations in both of these genes do not result in permeability changes of the F-containing cell. Even in the absence of phage infection, expression from plasmids of either gene 1.2 or 10 can cause permeability changes, particularly of F plasmid-containing cells. In contrast, gene 1.2 of the related bacteriophage T3 prevents leakiness of the infected cell. In the absence of T3 gene 1.2 function, expression of gene 10 causes membrane damage that allows nucleotides to leak from the cell. Genes 1.2 and 10 of both T3 and T7 are the two genes involved in determining resistance or sensitivity to F exclusion; F exclusion and leakiness of the phage-infected cell are therefore closely related phenomena. However, since leakiness of the infected cell does not necessarily result in phage exclusion, it cannot be used as a predictor of an abortive infection.
Cell Membrane Permeability↗
Defective transcription of the right end of bacteriophage T7 DNA during an abortive infection of F plasmid-containing Escherichia coli.
Transcription of T7 and mutant T3 DNA during infections of F plasmid-containing cells has been analyzed by using Southern hybridization. A transcriptional defect is apparent in these abortively infected cells that is most severe in the class III region of the phage genome. In particular, RNAs that are initiated from the gene 13 promoter are not elongated to give full-length molecules. It is suggested that the transcription defect results from positive supercoiling of the template DNA and that torsional constraints may even prevent the complete entry of the phage genome into an abortively infected cell.
Blotting, Southern↗
Expression of gene 1.2 and gene 10 of bacteriophage T7 is lethal to F plasmid-containing Escherichia coli.
Plasmids expressing bacteriophage T7 gene 1.2 or gene 10 DNA transform F plasmid-containing strains of Escherichia coli only at low efficiency, though they transform plasmid-free strains normally. The gene products T7 gp1.2 and T7 gp10 appear to be the toxic agents, and their effects are directed towards the product of the F pifA gene, PifA. T7 gp1.2 and gp10 are also the two targets of the pif exclusion system of F, and their synthesis normally triggers the abortive infection of T7 in pifA+ hosts. The properties of plasmids containing T7 gene 1.2 or 10 suggest that they can be used to study the molecular mechanisms of phage exclusion in model systems that avoid the pleiotropic dysfunctions associated with an abortive infection.
Escherichia coli↗
Host-parasite interactions: recent developments in the genetics of abortive phage infections.
Recent progress towards the elucidation of the mechanisms of phage exclusion systems that lead to abortive infections is described. Abortive infections constitute model systems for the study of host-parasite interactions, but until the 1980s such studies were confined largely to descriptions of a plethora of physiological dysfunctions. The use of cloning vectors containing the genes involved in the exclusion phenomena have helped to reduce the complexity of the abortive infection while maintaining many of its natural biological characteristics. The use of these vectors has also led to the development of a convenient assay for the identification of the targets of an exclusion system. Plasmids expressing a target gene are frequently found to be unable to transform a cell that contains the exclusion system. This article focuses on two classic abortive infections, the exclusion of T4 rII mutants by lambda rex+ lysogens and of wild-type T7 by cells containing the plasmid F. In addition, some less well understood phage exclusion systems are described, since they share a number of features with the T4 and T7 systems. Elucidation of the normal functions of the components involved in some abortive infections is also helping our understanding of the underlying mechanisms, and it seems likely that one or more of these model systems will soon be fully described.
Bacteriophage lambda↗
Sequence of bacteriophage T3 DNA from gene 2.5 through gene 9.
The nucleotide sequence of bacteriophage T3 DNA, from gene 2.5 through gene 9 has been determined. In addition to regulatory sites, the sequence predicts 19 close-packed genes plus two genes that overlap, in a different reading frame, another gene. The majority of these genes are highly homologous to those in the corresponding region of bacteriophage T7. However, there are some genes that are present in one, but not the other, phage. These apparent deletions are almost exactly gene size and thus the close-packed organization of genes remains the same in T3 as in T7. The varying levels of homology between T3 and T7 DNAs, first noted by Davis and Hyman in their study of DNA heteroduplexes, are also demonstrated here by a comparison of T3 and T7 nucleotide sequences. Many regions of extremely high homology immediately abut sequences that have no apparent homology. These data suggest that bacteriophages T3 and T7 have recombined, both with each other and with other members of a pool of T7-like phages, during their co-evolution.
Amino Acid Sequence↗
Synthesis of the capsid protein inhibits development of bacteriophage T3 mutants that abortively infect F plasmid-containing cells.
Mutants of bacteriophage T3 that lack gene 1.2 resemble wild-type phage T7 in that they are unable productively to infect F plasmid-containing cells of Escherichia coli. Pseudorevertants of a T3 gene 1.2 deletion mutant that have regained the ability to plate efficiently on male cells have been isolated and characterized. At least two mutations in the gene for the major capsid protein are necessary for these phages to bypass F-mediated restriction. One mutation serves to reduce the rate of synthesis of the capsid protein; a second mutation apparently alters an unknown property that is intrinsic to the free, or unassembled form of the protein. During the abortive infection of an F-containing host, synthesis of the wild-type capsid protein directly inhibits further phage development.
Capsid↗
Nucleotide sequence and complementation studies of the gene 10 region of bacteriophage T3.
The nucleotide sequence of bacteriophage T3 gene 10 and surrounding regulatory elements has been determined and compared to the analogous region of bacteriophage T7. T3 genes 9, 10 and 11 have been shown to complement T7 mutants. The DNA sequences of T3 and T7 gene 10A are homologous, as are the amino acid sequences of the respective products. The translational shift to the -1 frame is predicted to occur at the same position in gene 10 of T3 and T7, though different nucleotide sequences are probably responsible. The resulting gp10B products have completely different C termini.
Base Sequence↗
Mutants of bacteriophage T7 that escape F restriction.
Mutants of bacteriophage T7 that escape F restriction have been isolated. Two mutations in gene 10, which codes for the capsid protein, and one mutation in gene 1.2 are required for these phages to grow on F-containing strains. The products of these two genes are the two targets of the exclusion system; the presence of either wild-type product results in an abortive infection. Phages that grow normally in male hosts still lead to membrane dysfunction and nucleotide efflux from the infected cell. This type of membrane damage and the abortive infection are therefore separable phenomena.
DNA, Viral↗
Sequence of a conditionally essential region of bacteriophage T3, including the primary origin of DNA replication.
The 3526 base-pair nucleotide sequence from near the end of bacteriophage T3 gene 1 to within the coding sequence of gene 2.5 is given. It includes the complete coding sequences for nine known or presumptive proteins, most of which are only conditionally essential for phage growth. The sequence includes five promoters for the phage RNA polymerase, the terminator for early (host enzyme-catalyzed) transcription, and two recognition sites for RNAase III. The primary origin of T3 DNA replication that is utilized by the phage in vivo has been localized to a 142 base-pair region. It has several features in common with the phage T7 origin of DNA replication, and exhibits considerable homology to recognition sites for the mRNA processing enzyme RNAase III. It is proposed that the primary origin of T3 DNA replication may have evolved directly from an RNAase III recognition site. The deletions present in a number of T3 mutant strains and the location of the nucleotide changes in several T3 strains that are defective in their ability to grow on F+-containing strains or on optA mutant hosts have been determined. We discuss how T3 may have become genetically isolated from its relatives in the T7-T3 group and simultaneously acquired novel biological and biochemical properties.
Amino Acid Sequence↗
DNA sequence specificity of the pyrrolo[1,4]benzodiazepine antitumor antibiotics. Methidiumpropyl-EDTA-iron(II) footprinting analysis of DNA binding sites for anthramycin and related drugs.
Anthramycin, tomaymycin, and sibiromycin are members of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. These drugs bind covalently through N2 of guanine and lie within the minor groove of DNA [Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981) Biochemistry 20, 1111-1119]. The DNA sequence specificity of the P(1,4)B antibiotics has been determined by a footprinting method using methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)], and the results show that each of the drugs has a two to three base pair sequence specificity that includes the covalently modified guanine residue. While 5'PuGPu is the most preferred binding sequence for the P(1,4)Bs, 5'PyGPy is the least preferred sequence. Footprinting analysis by MPE.Fe(II) reveals a minimum of a three to four base pair footprint size for each of the drugs on DNA with a larger than expected offset (two to three base pairs) on opposite strands to that observed in previous analyses of noncovalently bound small molecules. There is an extremely large enhancement of MPE.Fe(II) cleavage between drug binding sites in AT rich regions, probably indicating a drug-induced change in the conformational features of DNA which encourages interaction with MPE.Fe(II). In the presence of sibiromycin or tomaymycin the normally guanine-specific methylene blue reaction used in Maxam and Gilbert sequencing cleaves at other bases in defined positions relative to the drug binding sites. Finally, modeling studies are used to rationalize the differences and similarities in sequence specificities between the various drugs in the P(1,4)B group and their reactions with DNA.
Aminoglycosides↗
Expression of the unassembled capsid protein during infection of Shigella sonnei by bacteriophage T7 results in DNA damage that is repairable by bacteriophage T3, but not T7, DNA ligase.
The abortive infection of bacteriophage T7 in Shigella sonnei D2 371-48 is characterized by a premature inhibition of phage DNA replication and nucleolytic breakdown of all phage DNA. Mutations in T7 gene 10 which are recessive to the presence of the wild-type allele can alleviate the restriction of phage growth. Phage T3 productively infects S. sonnei D2 371-48, as does a T7-T3 hybrid phage that contains, in particular, a gene 10 of T7 origin. It is the presence of T3 DNA ligase that allows phage growth on S. sonnei D2 371-48, and this enzyme can also rescue wild-type T7 from the abortive infection. T7+ is therefore functionally ligase deficient during the infection of S. sonnei D2 371-48; this deficiency is a result of the expression of the phage capsid protein, but it is independent of the assembly of the protein into a procapsid or other morphogenetic structure.
Capsid↗