Nucleotide clusters in deoxyribonucleic acids. Comparison of the sequences of the large pyrimidine oligonucleotides of bacteriophages S13 and phiX174 deoxyribonucleic acids.

The large pyrimidine oligonucleotides from the DNAs of the two related bacteriophages phiX174 and S13 have been sequenced. The largest pyrimidine oligonucleotide present is unique to S13 DNA and is the undecanucleotide C5T6, sequence C-T-T-C-C-T-C-T-T-C-T. Considerable sequence homology has been found between the pyrimidine oligonucleotides of the two phage DNAs. Out of 14 oligonucleotide sequences from S13 DNA (120 bases) at least ten are identical with sequences of oligonucleotides from phiX174 DNA (92 bases) and two are closely related (17 bases), the only difference being a single thymine to cytosine transition in each sequence (a total of 107 identical bases). The pyrimidine oligonucleotides of each phage DNA show extensive internal sequence homology among each other with up to eight bases identical in sequence in pairs of different oligonucleotides. Another interesting observation is the occurrence of symmetrical sequences (true palindromes) which read the same forwards as backwards. The longest symmetrical sequence is the nonanucleotide C4T5 sequence, C-T-C-T-T-T-C-T-C, present in both S13 and phiX174 DNAs. The extensive sequence homology observed between the pyrimidine oligonucleotides of S13 and phiX174 supports the close relationship of the two phages and provides further evidence that they were derived from recent common ancestors.     

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.     

A snapshot of viral evolution from genome analysis of the tectiviridae family

The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host.     

Relation between the Yersinia phage and bacteriophages isolated from the environment

The bacteriophage designated RD2 has been isolated from the sewage in Rostov-on-Don city and studied. The morphology of bacteriophage particles and the biological properties of the bacteriophage make it related to the plague bacteriophage isolated by D'Errel. The molecular masses of the compared bacteriophages are almost identical being 26.4 +/- 0.4 Md for RD2 and 24.7 +/- 0.2 Md for D'Errel bacteriophage. The DNAs of the bacteriophages share 80% of homology and possess 15 nonhomologous regions scattered along the genomes. The phages are serologically related. The DNAs of both bacteriophages give the similar pattern of hydrolysis by restriction endonuclease EcoRV, but have the different sensitivity to many other restriction endonucleases. The protein specter of bacteriophage RD2 contains 18 polypeptides (11 minor ones), while the one of D'Errel bacteriophage contains 7 polypeptides similar in molecular mass with the polypeptides of RD2. The bacteriophage RD2 cannot be considered one of the plague causative agents of bacteriophages since the region where it has been isolated has a long epidemiological and epizootical record of absence of plague.     

Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa

Seventeen virulent bacteriophages specific to Pseudomonas aeruginosa strains were isolated by screening various environmental samples. These isolated bacteriophages were grouped based on results obtained from restriction fragment analysis of phage genomes, random amplification of polymorphic DNA (RAPD) typing, morphology observations under transmission electron microscope, and host range analysis. All 17 bacteriophages are double-stranded DNA viruses and can be divided into 5 groups based on DNA restriction profiles. A set of 10-mer primers was used in RAPD typing of phages, and similar conclusions were obtained as for restriction fragment analysis. One phage was randomly selected from each of the 5 groups for morphology observations. Four of them had an icosahedral head with a long contractile tail, belonging to the Myoviridae family, and one phage had an icosahedral head with a short tail, thereby belonging to the Podoviridae family. Host range experiments were conducted on 7 laboratory strains and 12 clinical strains of P.?aeruginosa. The results showed that 13 phages had the same infection profile, killing 8 out of 19 tested P.?aeruginosa strains, and the remaining 4 phages had different and unique infection profiles. This study highlights the diversity of bacteriophages specific to P.?aeruginosa in the environment.     

Bacteriophage Diversity in the North Sea

In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10⁴ to 10⁷ particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the γ subdivision of the class Proteobacteria.The marine bacterial community is responsible for a considerable portion of primary production and regeneration of nutrients in the microbial loop and is associated with a great variety of marine bacteriophages (5, 12). These phages are capable of infecting a large portion of the bacterioplankton (32, 34). It is assumed that as part of the marine food web, bacteriophages play important quantitative and qualitative roles in controlling marine bacterial populations (8, 24, 34, 39, 45). The phenotypic diversity and genotypic diversity of the phage populations are related to the interaction between phages and their host organisms, which provides a tool for understanding the interaction itself (13). To estimate the influence of marine bacteriophages on the diversity of bacterioplankton, we investigated phage diversity. The virus species concept proposed by Murphy et al. (37) delineates seven different families of bacteriophages based on morphological criteria and provides criteria for new phage species based on several traits, such as DNA homologies, serological data, protein profiles, and host ranges.In this paper, we describe the diversity and genetic relationships of marine phages based on investigations of 22 representatives from 85 phage-host systems (35, 36) collected between 1988 and 1992 from waters around an island, Helgoland, located in the North Sea. All of the phages were virulent and formed plaques on their host bacteria. We assigned the phages to different virus families, species, and strains based on morphology, DNA homology, and host range. Furthermore, we characterized the phenotypic and genotypic features of the host bacteria.     

Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4

Double-stranded DNA-packaging in icosahedral bacteriophages is believed to be driven by a packaging "machine" constituted by the portal protein and the two packaging/terminase proteins assembled at the unique portal vertex of the empty prohead shell. Although ATP hydrolysis is evidently the principal driving force, which component of the packaging machinery functions as the translocating ATPase has not been elucidated. Evidence suggests that the large packaging subunit is a strong candidate for the translocating ATPase. We have constructed new phage T4 terminase recombinants under the control of phage T7 promoter and overexpressed the packaging/terminase proteins gp16 and gp17 in various configurations. The hexahistidine-tagged-packaging proteins were purified to near homogeneity by Ni(2+)-agarose chromatography and were shown to be highly active for packaging DNA in vitro. The large packaging subunit gp17 but not the small subunit gp16 exhibited an ATPase activity. Although gp16 lacked ATPase activity, it enhanced the gp17-associated ATPase activity by >50-fold. The gp16 enhancement was specific and was due to an increased catalytic rate for ATP hydrolysis. A phosphorylated gp17 was demonstrated under conditions of low catalytic rates but not under high catalytic rates in the presence of gp16. The data are consistent with the hypothesis that a weak ATPase is transformed into a translocating ATPase of high catalytic capacity after assembly of the packaging machine.     

A leftward deletional α+ thalassemia found in East Sicily in conjunction with heterozygous β-thalassemia

Summary Two types of ⁺-antitrypsin thalassemia (-/) have been described, respectively termed leftward and rightward, which correspond to nonhomologous crossing-over in different homology zones X and Z within the -globin gene cluster. Up to now the leftward type has been described only in Asiatic populations, whereas the rightward type is universally distributed. We report here a first case of leftward deletion observed in a Sicilian male. This raises the question of an identical or not crossing-over event.     

Effect of nucleases on bacteria infected with bacteriophages

The effect of bacterial nucleases on bacteria infected by DNA- or RNA-containing bacteriophages with different serogroups was studied. Bacillary RNases have a strong inhibitory effect on RNA-containing bacteriophages. It was shown that nucleases suppressed the infection process of bacteria by bacteriophages M12, f2, PP7, and QB. The minimal inhibitory concentration ranged from 0.6 to 6 μg/mL. Bacterial ribonucleases have no impact on the development of DNA-containing bacteriophages PZ-A, PZ-B, P₃k, P118, and a lysogenic culture of Escherichia coli (λ) and Bacillus subtilis 168 (phi105). RNase from Bacillus pumilus did not inactivate bacteriophages Qβ and f2 in vitro and did not influence the adsorption on bacteriophages on the cell wall of the bacteria host E. coli AB301. The enzyme effect was shown at the level of bacteriophage infection of the host bacteria. Presumably, the phase between the adsorption and penetration of phage RNA into bacterial pili is the most sensitive to the effect of RNases.     

Refined molecular weights for phage, viral and ribosomal RNA.

The RNAs of the Escherichia coli bacteriophages MS2 and Qbeta as well as E. coli 16S ribosomal RNA were examined under identical conditions by electron microscopy using the protein-free benzyldimethylalkylammonium chloride (BAC) spreading technique. From the contour length ratios of the RNAs and the known number of nucleotides for MS2, the chain lengths for Qbeta RNA and 16S RNA were found to be 4790 +/- 150 and 1645 +/- 55 nucleotides. Correcting for the base composition of Qbeta RNA the molecular weight of the Na salt of this RNA is (1.64 +/- 0.06) . 10(6) daltons. Since published values on the relative lengths of Qbeta RNA and several other homogeneous RNAs (E. coli 23S rRNA, E. Coli bacteriophage R17 and f2 RNAs, Pseudomonas aeruginosa phage PP7 RNA and Newcastle disease virus RNA) are available, we are able to calculate the approximate number of nucleotides for these useful standards.     

Protection of foreign DNA against host-controlled restriction in bacterial cells

Summary Foreign Flac plasmid DNA which is introduced into potentially restricting E. coli recipient cells can be protected from restriction by preinfecting the recipient cells with UV-inactivated T3 or T7 bacteriophages which express the ocr gene function. The recipient cells survive and are able to replicate themselves as well as the newly acquired plasmid.     

Characterization of Bacteriophage S13 suN15 Single-Strand and Replicative-Form Deoxyribonucleic Acid

The deoxyribonucleic acid (DNA) of bacteriophage S13 was shown to be single-stranded by the criteria of reactivity with formaldehyde, dependence of optical density on ionic strength, broad temperature-absorbance profile, and lack of molar equivalence of the purine and pyrimidine bases. The DNA has a molecular weight of 1.8 × 10⁶ daltons, an S°₂₀ of 24.6 in SSC (0.15 m NaCl plus 0.015 m sodium citrate), and a buoyant density of 1.726 g/cc in CsCl. Electron microscopy showed the molecule to be circular. S13 replicative-form DNA was shown to be a double-stranded, circular molecule with a molecular weight of 3.5 × 10⁶ daltons, an S_[ill] of 20.7 in SSC, and a buoyant density in CsCl of 1.710 g/cc. The finding that S13 DNA is slightly more pyrimidine-rich than X174 DNA but is indistinguishable by all other parameters supports the close genetic relationship between the two bacteriophages.     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3 extended DNAs, with the following sequence: 5-GTTAGGCTT-3 3-CAATCCGAA-5. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3 extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5 extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.     

Cyclic parathyroid hormone related protein antagonists: lysine 13 to aspartic acid 17 [i to (i + 4)] side chain to side chain lactamization.

Cyclization of parathyroid hormone related protein (7-34)amide [PTHrP(7-34)NH2] via covalent bond formation between the epsilon-amino of Lys13 and the beta-carboxyl of Asp17 yielded a 20-membered ring lactam. This analogue, [Lys13,Asp17]PTHrP(7-34)NH2, was 5-10-fold more potent than the linear parent peptide (Kb = 15 and 18 nM in PTH receptor binding assays, and Ki = 130 and 17 nM in PTH-stimulated adenylate cyclase assays in bovine renal cortical membrane and in human bone derived B10 cells, respectively). In contrast, a linear analogue in which charges in positions 13 and 17 were eliminated and other stereoisomers of the above-mentioned lactam in which either Lys13 and/or Asp17 were replaced by the corresponding D-amino acids were much less potent with regard to antagonist bioactivity than the parent peptide. The rationale for the design of the lactam as well as the conformational implications for the PTHrP sequence in light of reported models suggested for the 1-34 peptide are described. The potential use of conformationally constrained analogues for elucidating the "bioactive conformation" of antagonists and for the design of substantially simplified molecular structures for antagonists is discussed.     

Subcloning of DNA fragments of the simian adenovirus SA7 oncogene in bacteriophage M13

The XmaI/PstI and XmaI DNA fragments of adenovirus SA7 oncogene and the adjacent region (16.7% of the physical map of SA7 left end DNA) were recloned in M13 bacteriophages mp8 and mp9 in order to obtain the singlestranded fragments EIa and EIb from the DNA region of monkey adenovirus SA7 located on the recombinant plasmid pASP carrying the DNA APstI fragment including the adenovirus SA7 oncogene.     

The alpha-glucosyltransferases of bacteriophages T2, T4 and T6. A comparison of their primary structures

Summary With the aim of comparing the primary structures of gene products coded for by T-even bacteriophages we constructed clone libraries of the DNAs of bacteriophages T2 and T6. Using hybrid M13 phages carrying the gene for the T4-coded -glucosyl transferase (gt) we isolated corresponding T2 and T6 clones. The nucleotide sequences of the three gt genes and the amino acid sequences derived were compared. The differences between the genes and their products are discussed in terms of structure, function and evolutionary aspects.Abbreviations bp base pair - gt glucosyl transferase - HMC 5-hydroxymethyl cytosine - orf open reading frame - Xgal 5-bromo-4-chloro-3-indolyl--d-galactoside     

Modeling Virus Adsorption in Batch and Column Experiments

Experiments with batch suspensions, recirculating columns and flow-through columns have been carried out involving a sandy soil and five bacteriophages: MS2, PRD1, X174, Q and PM2. In batch and recirculating column experiments, attachment and detachment rate coefficients were determined by fitting a two-parameter (attachment and detachment) model. In general, attachment and detachment rate coefficients were not found to be significantly different between the two kinds of experiments. There was one exception, however: MS2 appeared to detach faster in the presence of strong advective flow. In the case of flow-through column experiments, it is shown that a two-site model, with adsorption to equilibrium and kinetic sites, fits the breakthrough curves of all the phages, except PM2, satisfactorily. A one-site kinetic model was found to be appropriate for phage PM2. A small proportion of bacteriophages MS2, PRD1, and Q adsorbed to equilibrium sites, whereas a large proportion of X174 adsorbed to equilibrium sites. Such a distinction between adsorption to equilibrium and kinetic sites cannot be made in the case of batch or recirculating column experiments. Kinetic attachment rate coefficients were found to be significantly higher for the bacteriophages with presumably stronger negative charge. This may be ascribed to the presence of multivalent cations. Under these conditions, bacteriophage X174 appears to behave more conservatively than more negatively charged viruses, and may then be a better choice as a relatively conservative tracer for virus transport through the subsurface.     

Effect of Nalidixic Acid on the Growth of Deoxyribonucleic Acid Bacteriophages

The effect of nalidixic acid on the growth of various deoxyribonucleic acid (DNA) bacteriophages has been investigated by one-step growth experiments. The Escherichia coli bacteriophages T5, lambda, T7 and phiR are strongly inhibited by nalidixic acid, whereas T4 and T2 are only partially inhibited. The Bacillus subtilis bacteriophages SP82, SP50, and phi29 are relatively unaffected by nalidixic acid. There is no correlation between those bacteriophages which can grow in the presence of nalidixic acid and the presence of an unusual base in the phage DNA.     

Active protection by bacteriophages T3 and T7 againstE. coli B-and K-specific restriction of their DNA

Summary The bacteriophages T3 and T7 are not modified and restricted byE. coli strains with different host specificity (E. coli B, K, O) in vivo. The phages code for a gene product with the ability toovercomeclassicalrestriction (ocr):ocr ^– mutants are subject to modification and restriction via DNA methylation vs cleavage. The T3 genome possesses recognition sites for the restriction endonuclease R.EcoB which, unless the DNA is B-specifically modified, trigger 5–7 DNA cleavages. Theocr gene function of T3 and T7 is located within the gene 0.3 region of these phages and is not identical with thesam (SAMase) function of T3. The mechanisms ofocr protection remains unclear, while it is certain that this protection by the gene 0.3 protein is exerted in the infected cell and not through over-all modification in the preceding growth cycle of the phage.