Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments.

Insertion of foreign DNA into an established mammalian genome can extensively alter the patterns of cellular DNA methylation. Adenovirus type 12 (Ad12)-transformed hamster cells, Ad12-induced hamster tumor cells, or hamster cells carrying integrated DNA of bacteriophage lambda were used as model systems. DNA methylation levels were examined by cleaving cellular DNA with Hpa II, Msp I, or Hha I, followed by Southern blot hybridization with 32P-labeled, randomly selected cellular DNA probes. For several, but not all, cellular DNA segments investigated, extensive increases in DNA methylation were found in comparison with the methylation patterns in BHK21 or primary Syrian hamster cells. In eight different Ad12-induced hamster tumors, moderate increases in DNA methylation were seen. Increased methylation of cellular genes was also documented in two hamster cell lines with integrated Ad12 DNA without the Ad12-transformed phenotype, in one cloned BHK21 cell line with integrated plasmid DNA, and in at least three cloned BHK21 cell lines with integrated lambda DNA. By fluorescent in situ hybridization, the cellular hybridization probes were located to different hamster chromosomes. The endogenous intracisternal A particle genomes showed a striking distribution on many hamster chromosomes, frequently on their short arms. When BHK21 hamster cells were abortively infected with Ad12, increases in cellular DNA methylation were not seen. Thus, Ad12 early gene products were not directly involved in increasing cellular DNA methylation. We attribute the alterations in cellular DNA methylation, at least in part, to the insertion of foreign DNA. Can alterations in the methylation profiles of hamster cellular DNA contribute to the generation of the oncogenic phenotype?

Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.

Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, three-dimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Transcription of downstream genes in the early operons of phage lambda requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of lambda N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.

Bistability and switching in the lysis/lysogeny genetic regulatory network of bacteriophage lambda

Bistability and switching are two important aspects of the genetic regulatory network of phage. Positive and negative feedbacks are key regulatory mechanisms in this network. By the introduction of threshold values, the developmental pathway of A phage is divided into different stages. If the protein level reaches a threshold value, positive or negative feedback will be effective and regulate the process of development. Using this regulatory mechanism, we present a quantitative model to realize bistability and switching of phage based on experimental data. This model gives descriptions of decisive mechanisms for different pathways in induction. A stochastic model is also introduced for describing statistical properties of switching in induction. A stochastic degradation rate is used to represent intrinsic noise in induction for switching the system from the lysogenic pathway to the lysis pathway. The approach in this paper represents an attempt to describe the regulatory mechanism in genetic regulatory network under the influence of intrinsic noise in the framework of continuous models. (C) 2003 Elsevier Ltd. All rights reserved.

Forced retroevolution of an RNA bacteriophage

The operator hairpin ahead of the replicase gene in RNA bacteriophage MS2 contains overlapping signals for binding the coat protein and ribosomes. Coat protein binding inhibits further translation of the gene and forms the first step in capsid formation. The hairpin sequence was partially randomized to assess the importance of this structure element for the bacteriophage and to monitor alternative solutions that would evolve on the passaging of mutant phages. The evolutionary reconstruction of the operator failed in the majority of mutants. Instead, a poor imitation developed containing only some of the recognition signals for the coat protein. Three mutants were of particular interest in that they contained double nonsense codons in the lysis reading frame that runs through the operator hairpin. The simultaneous reversion of two stop codons into sense codons has a very low probability of occurring. Therefore the phage solved the problem by deleting the nonsense signals and, in fact, the complete operator, except for the initiation codon of the replicase gene. Several revertants were isolated with activities ranging from 1% to 20% of wild type. The operator, long thought to be a critical regulator, now appears to be a dispensable element. In addition, the results indicate how RNA viruses can be forced to step back to an attenuated form.

The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition

Bacteriophage T7 DNA primase recognizes 5'-GTC-3' in single-stranded DNA. The primase contains a single Cys4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys4 motif contacts cytosine of 5'-GTC-3' and may also contribute to thymine recognition. Residues His33 and Asp31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5'-GTC-3', by base pairing. Our deduced mechanism for recognition of ssDNA by Cys4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys2His2 zinc fingers.

A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7

Two colinear bacteriophage T7 gene 4 proteins provide helicase and primase functions in vivo. T7 primase differs from T7 helicase by an additional 63 residues at the amino terminus. This terminal domain contains a zinc-binding motif which mediates an interaction with the basic primase recognition sequence 3'-CTG-5'. We have generated a chimeric primase in which the 81 amino-terminal residues are derived from the primase of phage T3 and the 484 carboxyl-terminal residues are those of phage T7 helicase. The amino-terminal domain of T3 primase is 50% homologous with that of T7 primase. The resulting T3/T7 chimeric protein is a functional primase in vivo. While the primase activity of the purified protein is about one-third that of T7 primase, the recognition sites used and the oligoribonucleotides synthesized from these sites are identical. We conclude that the residues responsible for the interaction with the sequence 3'-CTG-5' are conserved between the chimeric and T7 proteins.

Bacteriophage Can Prevent Encrustation and Blockage of Urinary Catheters by Proteus mirabilis.

Proteus mirabilis forms dense crystalline biofilms on catheter surfaces that occlude urine flow, leading to serious clinical complications in long-term catheterized patients, but there are presently no truly effective approaches to control catheter blockage by this organism. This study evaluated the potential for bacteriophage therapy to control P. mirabilis infection and prevent catheter blockage. Representative in vitro models of the catheterized urinary tract, simulating a complete closed drainage system as used in clinical practice, were employed to evaluate the performance of phage therapy in preventing blockage. Models mimicking either an established infection or early colonization of the catheterized urinary tract were treated with a single dose of a 3-phage cocktail, and the impact on time taken for catheters to block, as well as levels of crystalline biofilm formation, was measured. In models of established infection, phage treatment significantly increased time taken for catheters to block (∼ 3-fold) compared to untreated controls. However, in models simulating early-stage infection, phage treatment eradicated P. mirabilis and prevented blockage entirely. Analysis of catheters from models of established infection 10 h after phage application demonstrated that phage significantly reduced crystalline biofilm formation but did not significantly reduce the level of planktonic cells in the residual bladder urine. Taken together, these results show that bacteriophage constitute a promising strategy for the prevention of catheter blockage but that methods to deliver phage in sufficient numbers and within a key therapeutic window (early infection) will also be important to the successful application of phage to this problem.

Culturable Bacterial Diversity From A Feed Water Of A Reverse Osmosis System, Evaluation Of Biofilm Formation And Biocontrol Using Phages.

Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this study the planktonic culturable bacterial community was characterized from a feed water of a RO system and their capacities were evaluated to form biofilm in vitro. Bacterial motility and biofilm control were also analysed using phages. As results, diverse Protobacteria, Actinobacteria and Bacteroidetes were identified. Alphaproteobacteria was the predominant group and Brevundimonas, Pseudomonas and Mycobacterium the most abundant genera. Among the 30 isolates, 11 showed at least one type of motility and 11 were classified as good biofilm formers. Additionally, the influence of non-specific bacteriophage in the bacterial biofilms formed in vitro was investigated by action of phages enzymes or phage infection. The vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation of most tested bacteria and may represent a good alternative in biofilm control. These findings provide important information about the bacterial community from the feed water of a RO system that may be used for the development of strategies for biofilm prevention and control in such systems.

Role of the gp85/Trans-Sialidases in Trypanosoma cruzi Tissue Tropism: Preferential Binding of a Conserved Peptide Motif to the Vasculature In Vivo

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. Findings and Conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase

in Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 [DnaG(434-581)] that interact with the hexameric DnaB helicase. Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E coli strain. This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage gimel-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR. A TOCSY spectrum of a 2 mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579. This was consistent with sequence conservation among most-closely related primases. Linewidths in a NOESY spectrum of a 0.5 mM sample in 10 mM phosphate, pH 6.05, 0.1 M NaCl, recorded at 36 degreesC, indicated the protein to be monomeric. Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a = b 142.2 Angstrom, c = 192.1 Angstrom, and diffracted beyond 2.7 Angstrom resolution with synchrotron radiation. (C) 2003 Elsevier Inc. All rights reserved.

Significance in replication of the terminal nucleotides of the Flavivirus genome

Point mutations that resulted in a substitution of the conserved 3'-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3'-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3'-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of phi6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3'-penultimate cytidines and a 3'-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3'-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.

Construction of an innocuous Salmonella phage

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