Hexagonally packed DNA within bacteriophage T7 stabilized by curvature stress

A continuum computation is proposed for the bending stress stabilizing DNA that is hexagonally packed within bacteriophage T7. Because the inner radius of the DNA spool is rather small, the stress of the curved DNA genome is strong enough to balance its electrostatic self-repulsion so as to form a stable hexagonal phase. The theory is in accord with the microscopically determined structure of bacteriophage T7 filled with DNA within the experimental margin of error.     

Overexpression of MerT, the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations

The small (116 amino acids) inner membrane protein MerT encoded by the transposon Tn501 has been overexpressed under the control of the bacteriophage T7 expression system. Random mutants of MerT were made and screened for loss of mercuric ion hypersensitivity. Several mutant merT genes were selected and sequenced: Cys24Arg and Cys25Tyr mutations abolish mercury resistance, as do charge-substitution mutations in the first predicted transmembrane helix (Gly14Arg, Gly15Arg, Gly27Arg, Ala18Asp), and the termination mutations Trp66Ter and Cys82Ter.     

Mechanism of bacteriophage SfII-mediated serotype conversion in Shigella flexneri

We have isolated the lysogenic bacteriophage SfII, which mediates glucosylation of Shigella flexneri O-antigen, resulting in expression of the type II antigen. SfII belongs to group A of the Bradley classification and has a genome size of 42.3kb. DNA sequencing of a 4 kb BamHI subclone identified four open reading frames (ORFs), of which only two were found to be necessary for serotype conversion. These genes were named bgt, which encodes a putative bactoprenol glucosyl transferase, and gtrII, encoding the putative type II antigen determining glucosyl transferase. These genes are adjacent to the integrase gene (int) and attachment site (attP), which are highly homologous to those of Salmonella bacteriophage P22. Another ORF encoded a highly hydrophobic protein of 120 amino acids with homologues in Escherichia coli, Salmonella bacteriophage P22 and S. flexneri. Previous studies identified gtrX, the glucosyl transferase gene, of bacteriophage SfX, which also glucosylates the O-antigen specifically. We determined that gtrX-mediated expression of the group 7,8 antigen also requires bgt. This allowed us to identify gtrII as being the serotype antigen II determining glucosyl transferase. Southern hybridization and polymerase chain reaction (PCR) analyses indicated that bgt homologues exist in the genomes of all S. flexneri serotypes and in E. coli K-12, whereas gtrII was only detected in strains of serotype 2. Transposon TnphoA-derived chromosomal mutations of bgt and gtrII in S. flexneri serotype 2a were isolated and characterized. [35S]-methionine labelling and the use of a T7 RNA polymerase expression system identified a protein of 34kDa corresponding to Bgt. However, GtrII, which has a predicted molecular weight of 55 kDa, was not detected. We propose that the function of Bgt is to transfer the glucose residues from the UDP-glucose onto bactoprenol and GtrII then transfers the glucose onto the O-antigen repeat unit at the rhamnose III position. The chromosomal organization of these serotype-converting genes, when compared with their homologues in E. coli K-12 chromosome and the P22 bacteriophage genome, were very similar. This suggests that the regions encode similar functions in these organisms and have a similar evolutionary origin.     

Assembly of productive T cell receptor delta variable region genes exhibits allelic inclusion

The generation of a productive "in-frame" T cell receptor beta (TCR beta), immunoglobulin (Ig) heavy (H) or Ig light (L) chain variable region gene can result in the cessation of rearrangement of the alternate allele, a process referred to as allelic exclusion. This process ensures that most alphabeta T cells express a single TCR beta chain and most B cells express single IgH and IgL chains. Assembly of TCR alpha and TCR gamma chain variable region genes exhibit allelic inclusion and alphabeta and gammadelta T cells can express two TCR alpha or TCR gamma chains, respectively. However, it was not known whether assembly of TCR delta variable regions genes is regulated in the context of allelic exclusion. To address this issue, we have analyzed TCR delta rearrangements in a panel of mouse splenic gammadelta T cell hybridomas. We find that, similar to TCR alpha and gamma variable region genes, assembly of TCR delta variable region genes exhibits properties of allelic inclusion. These findings are discussed in the context of gammadelta T cell development and regulation of rearrangement of TCR delta genes.     

Polymorphism of bacteriophage T7

For viruses made of nucleic acid and protein, the structure of the protein outer shell has, in the past, been found to be uniquely determined by the viral genome. However, here, non-denaturing agarose gel electrophoresis of bacteriophage T7 reveals two states of the mature T7 capsid; the conditions of growth are found to alter the population by T7 of these two electrophoretically defined states. Both states have been previously observed for a genetically altered T7 and they are observed here for wild-type T7. The average electrical surface charge density of a bacteriophage particle (delta) determines its state; the delta of particles in both states is negative. For a given condition of growth, the population of these two states is influenced by the extent to which the major T7 outer shell protein, p10A, is accompanied by its minor readthrough variant, p10B. Comparison of the two electrophoretic states reveals the following. (1) No difference in radius is present in the outer shell (+/-2%). (2) As the pH of electrophoresis is either increased or decreased from neutrality, the state becomes more highly populated for which delta is greater in magnitude (state 1). By changing the pH, some T7 particles are made to change state. (3) Particles in state 1 adsorb less quickly to host cells than do the particles in the alternative state (state 2). This latter observation suggests the hypothesis that state 1 evolved to reduce the probability of re-initiating an infection when conditions are not favorable for growth. This hypothesis is supported by the observation that, as conditions of growth become apparently more unfavorable, progeny increasingly populate state 1.     

RAG reexpression and DNA recombination at T cell receptor loci in peripheral CD4+ T cells

Under most circumstances, allelic exclusion at the T cell receptor (TCR)beta locus is tightly regulated. Here, we describe a system in which TCRbeta allelic exclusion is overcome as a result of V(D)J recombination in peripheral CD4+ T cells. In TCRbeta chain transgenic mice, tolerogen-mediated chronic peripheral selection against cells expressing the transgene leads to surface expression of endogenous TCRbeta chains. Peripheral CD4+ T cells reexpress the recombination activating genes, RAG1 and RAG2, and contain signal end intermediates indicative of ongoing V(D)J recombination. The rescue from deletion of mature T cells expressing newly generated TCRbeta chains suggests that receptor revision plays a role in the maintenance of peripheral T cell tolerance.     

The interaction of acetoxy-dimethylnitrosamine, a proximate metabolite of the carcinogenic amine, and bacteriophages R17 and T7

The biological and physicochemical effects of reacting bacteriophages R17 and T7 with acetoxy-dimethylnitrosamine (ADMN) have been studied. The rate-determining step in the reactions appeared to be the loss of the acetoxy group by hydrolysis, the hydroxymethyl-methylnitrosamine generated decomposing rapidly to give a methyldiazonium ion and formaldehyde. In experiments with bacteriophage suspended in phosphate buffer the biological inactivation observed was the sum of the effects of the formaldehyde and of alkylation by the methylcarbonium ion produced from the diazonium ion. In experiments with bacteriophage suspended in Tris--HCl buffer the effects of formaldehyde were eliminated by its reaction with the buffer component. Alkylation by the carbonium ion produced unstable phosphotriesters in the bacteriophage RNA which on hydrolysis led to degradation of the molecule. In phosphate buffer the formaldehyde cross-linked the protein coat of the bacteriophage blocking the extraction of the RNA. Estimates of the mean lethal dose and of the extent of degradation of the RNA following reaction in Tris--HCl buffer were fairly close to those observed in experiments with N-methyl-N-nitrosourea (MNUA).     

Expression of HIV-1 epitopes included in particles formed by human hepatitis B virus nucleocapsid protein

Hybrids of the core protein of hepatitis B virus (HBcAg) have been designed which carry N-terminal insertions of B- and T-cell epitopes of HIV-1 an immunodominant B-epitope from gp41, a T-cell epitope from p34 pol, and a cluster of B- and T-cell epitopes from p17 gag. The hybrids have been synthesized using two expression systems-one based on the thermoinducible PR promoter of bacteriophage lambda and the other one based on phi 10 promoter of bacteriophage T7 with 3-5% and 7-14% yields, respectively. The hybrids have dual HBV and HIV-1 immunospecificity and are assembled into particles similar to those formed by the protein carrier HBcAg. Sandwich ELISA and immune electron microscopy revealed that HIV-1 epitopes are exposed on the surface of the particles.     

The relationship between the severity of sleep apnea syndrome and 24-h blood pressure values in patients with obstructive sleep apnea

Analysis of the nucleotide sequence in the 5' flanking region of bacteriophage T4 gene 25 revealed three potential Shine and Dalgarno sequences, SD1, SD2 and SD3, with a spacing of 8, 17 and 27 nucleotides from the initiation codon of this gene, respectively. Results of our experiments in the bacteriophage T7 expression system clearly demonstrate that the SD3 sequence is required for efficient expression of gene 25. We propose the existence of a stem-loop structure that includes SD1 and SD2 sequences and brings the SD3 sequence to a favourable spacing with the initiation codon of gene 25. Since the predicted secondary structure in the translational initiation region of gene 25 is relatively unstable and the SD3 sequence, GAGG, is more typical than the SD1 sequence, GAG, we suggest that this structure could control the level of gene expression.     

rexB of bacteriophage lambda is an anti-cell death gene

In Escherichia coli, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of lambdarexB, one of the few genes expressed in the lysogenic state of bacteriophage lambda, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3',5'-bispyrophosphate (ppGpp) and thus by amino acid starvation. lambdaRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of lambdaRexB through its action on the ClpP proteolytic subunit. We also propose that the lambdarex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the "survival operon" of phage lambda.     

The use of synthetic oligodeoxynucleotide primers in cloning and sequencing segment of 8 influenza virus (A/PR/8/34)

Complete double-stranded DNA copies of the RNA genes of the human influenza virus A/PR/8/34 have been synthesized by using two synthetic oligodeoxynucleotide primers. The gene encoding the non-structural proteins NS1 and NS2, prepared with these primers, has been cloned into the bacteriophage M13mp7 and sequenced. The sequence is compared with that from another human strain and from an avian strain.     

Retention of replication fidelity by a DNA polymerase functioning in a distantly related environment

The primary structures of the replicative DNA polymerases (gp43s) of bacteriophage T4 and its distant phylogenetic relative RB69 are diverged, retaining only 61% identity and 74% similarity. Nevertheless, RB69 gp43 substitutes effectively for T4 gp43 in T4 DNA replication in vivo. We show here that RB69 gp43 replicates T4 genomes in vivo with a fidelity similar to that achieved by T4 gp43. Furthermore, replication by RB69 gp43 in the distantly related environment does not enhance the mutator activities of mutations in T4 genes that encode other components of the multienzyme DNA replicase. We also show that the fidelities of RB69 gp43 and T4 gp43 are both high in vitro and that they are similarly and sharply reduced in vivo by mutations that eliminate the 3'-exonucleolytic proofreading function. We conclude that gp43 interactions with the other replication proteins are probably nonessential for polymerase fidelity.     

Gene 4 helicase of bacteriophage T7 mediates strand transfer through pyrimidine dimers, mismatches, and nonhomologous regions

In bacteriophage T7 the gene 2.5 single-stranded DNA-binding protein and the gene 4 helicase together promote the annealing of homologous regions of two DNA partners to form a joint molecule and subsequent strand transfer. In this reaction T7 gene 2.5 protein is essential for joint molecule formation, but is not required for T7 gene 4 protein-mediated strand transfer. T7 gene 4 helicase alone is able to mediate strand transfer, provided that a joint molecule is available. The present paper shows that, in addition, strand transfer proceeds at a normal rate even when both DNA partners contain ultraviolet-induced pyrimidine dimers (0.6 dimer per 100 nt). An insert of a relatively long (842-nt) segment of nonhomologous DNA in the single-stranded DNA partner has no effect on strand transfer, whereas its presence in the double-stranded partner prevents strand transfer. A short insert (37 nt) can be tolerated in either partner. Thus, DNA helicase is able to participate in recombinational DNA repair through its role in strand exchange, providing a pathway distinct from nucleotide excision repair.     

Deletion between directly repeated DNA sequences measured in extracts of bacteriophage T7-infected Escherichia coli

An in vitro system based upon extracts of bacteriophage T7 infected Escherichia coli was used to study genetic deletions and to examine the importance of DNA replication in the deletion process. When T7 genomes with gene 1.3 inactivated by a 43-bp insert of random sequence DNA bracketed by 11-bp direct repeats were replicated in vitro the inserts were deleted with a frequency of about 10(-5) deletions per genome replication. Under conditions where deletion could take place only by recombination between direct repeats on distinct DNA molecules deletion frequency was at least an order of magnitude lower. These data demonstrate the utility of the in vitro system as a means for examining deletion mechanisms and underscore the importance of DNA replication in deletions.