Bacteriophage T7 DNA packaging

Chung, Yeon-Bo; Nardone, Christopher; Hinkle, David C.

doi:10.1016/s0022-2836(99)80012-6

Cited by 33 publications

(10 citation statements)

References 25 publications

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“…7c). The repeat between the next genome-length and the one being packaged are duplicated so that each virion receives a chromosome with an identical repeat at each end [27–29]. The raw data for these sequences indicate that twice as many reads cover the exact DTR when compared with the rest of the genome since the exact DTR is found twice in each phage chromosome.…”

Section: Resultsmentioning

confidence: 99%

Software-based analysis of bacteriophage genomes, physical ends, and packaging strategies

et al. 2016

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BackgroundPhage genome analysis is a rapidly growing field. Recurrent obstacles include software access and usability, as well as genome sequences that vary in sequence orientation and/or start position. Here we describe modifications to the phage comparative genomics software program, Phamerator, provide public access to the code, and include instructions for creating custom Phamerator databases. We further report genomic analysis techniques to determine phage packaging strategies and identification of the physical ends of phage genomes.ResultsThe original Phamerator code can be successfully modified and custom databases can be generated using the instructions we provide. Results of genome map comparisons within a custom database reveal obstacles in performing the comparisons if a published genome has an incorrect complementarity or an incorrect location of the first base of the genome, which are common issues in GenBank-downloaded sequence files. To address these issues, we review phage packaging strategies and provide results that demonstrate identification of the genome start location and orientation using raw sequencing data and software programs such as PAUSE and Consed to establish the location of the physical ends of the genome. These results include determination of exact direct terminal repeats (DTRs) or cohesive ends, or whether phages may use a headful packaging strategy. Phylogenetic analysis using ClustalO and phamily circles in Phamerator demonstrate that the large terminase gene can be used to identify the phage packaging strategy and thereby aide in identifying the physical ends of the genome.ConclusionsUsing available online code, the Phamerator program can be customized and utilized to generate databases with individually selected genomes. These databases can then provide fruitful information in the comparative analysis of phages. Researchers can identify packaging strategies and physical ends of phage genomes using raw data from high-throughput sequencing in conjunction with phylogenetic analyses of large terminase proteins and the use of custom Phamerator databases. We promote publication of phage genomes in an orientation consistent with the physical structure of the phage chromosome and provide guidance for determining this structure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3018-2) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Software-based analysis of bacteriophage genomes, physical ends, and packaging strategies

et al. 2016

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“…9). Such a mechanism coupling gp55⅐gp45 to DNA packaging repair would be analogous to the role of the T7 RNA polymerase in DNA packaging coupled DNA synthesis (35,36). Further work is necessary to substantiate such a hypothetical DNA repair mechanism that is compatible with the densely interconnected late transcription, replication, and packaging pathways.…”

Section: Discussionmentioning

confidence: 99%

Mechanistic Coupling of Bacteriophage T4 DNA Packaging to Components of the Replication-dependent Late Transcription Machinery

Black

Peng

2006

Journal of Biological Chemistry

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Regulation of the terminal stage of viral DNA development, DNA packaging, is poorly understood. A new phage T4 in vitro DNA packaging assay employed purified proheads, terminase (gp17 ؉ gp16), and ATP to encapsidate DNA resistant to nuclease. Mature phage T4 DNA and linearized plasmid DNAs containing or lacking a cloned T4 gene were packaged with high (ϳ10%) efficiency. Supercoiled, relaxed covalently closed, and nicked circular plasmid DNAs were packaged inefficiently, if at all, by these components. However, efficient packaging is achieved for nicked circular plasmid DNA, but not covalently closed plasmid DNA, upon addition to packaging mixtures of the purified T4 late transcription-replication machinery proteins: gp45 (sliding clamp), gp44/gp62 (clamp loader complex), gp55 (late -factor), and gp33 (transcriptional co-activator). The small terminase subunit (gp16) is inhibitory for packaging linear DNAs, but enhances the transcription-replication protein packaging of nicked plasmid DNA. Taken together with genetic and biochemical evidence of a requirement for gp55 for concatemer packaging to assemble active wild-type phage particles (1), the plasmid packaging results show that initiation of phage T4 packaging on "endless" concatemeric DNA in vivo by terminase depends upon interaction with the DNA loaded gp45 coupled late transcription-replication machinery. The results suggest a close mechanistic connection in vivo between DNA packaging and developmentally concurrent replication-dependent late transcription.Phage T4 replicates its DNA autonomously with a full set of phage-encoded replication proteins (2). Replication culminates in many fold amplification of injected linear ϳ170-kb mature phage DNA to form concatemeric DNA. This "endless" DNA is the product of multiple replication and recombination pathways (3). Concatemeric DNA is the in vivo substrate for DNA packaging into proheads and for the generation of mature phage DNA by terminase (4).Phage T4 employs Escherichia coli RNA polymerase to carry out a complex program of early, middle and late transcription by synthesizing numerous proteins that modify RNA polymerase (RNAP) 2 promoter recognition. In the late stage of its development, concurrent T4 phage DNA replication and late transcription are intimately connected (5). A unique mechanism accounts for the following interesting features of late phage T4 development: phage T4 late transcription depends upon: (i) concurrent DNA replication; (ii) activation of RNAP by late transcription proteins (gp55, late -factor; gp33, transcription co-activator); and (iii) direct action of the DNA polymerase processivity factor gp45. The mechanism of late transcription at short (Ϫ10 TATAAATA, no Ϫ35) late T4 promoters has been shown to require tracking along the DNA of the gp45 sliding clamp to which gp55 and gp33 attach and through which contact is made with RNAP (6). In fact, these two transcription factors and DNA polymerase (gp43) share similar C-terminal peptide sequences that account for gp45 binding (7). In additio...

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“…IL-1 β is a proinflammatory cytokine known to upregulate a plethora of several inflammatory mediators, including IL-1 β itself, TNF, inducible nitric oxide synthase, and chemokines [22–25]. IL-1 β elicits responses in cells only through the activation of IL-1 type I receptor (IL-1RI) although it can also bind to IL-1 type II receptor (IL-1RII), a decoy receptor.…”

Section: Introductionmentioning

confidence: 99%

Elevated Glucose and Interleukin-1β Differentially Affect Retinal Microglial Cell Proliferation

Baptista

Aveleira

Castilho

et al. 2017

Mediators of Inflammation

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Diabetic retinopathy is considered a neurovascular disorder, hyperglycemia being considered the main risk factor for this pathology. Diabetic retinopathy also presents features of a low-grade chronic inflammatory disease, including increased levels of cytokines in the retina, such as interleukin-1 beta (IL-1β). However, how high glucose and IL-1β affect the different retinal cell types remains to be clarified. In retinal neural cell cultures, we found that IL-1β and IL-1RI are present in microglia, macroglia, and neurons. Exposure of retinal neural cell cultures to high glucose upregulated both mRNA and protein levels of IL-1β. High glucose decreased microglial and macroglial cell proliferation, whereas IL-1β increased their proliferation. Interestingly, under high glucose condition, although the number of microglial cells decreased, they showed a less ramified morphology, suggesting a more activated state, as supported by the upregulation of the levels of ED-1, a marker of microglia activation. In conclusion, IL-1β might play a key role in diabetic retinopathy, affecting microglial and macroglial cells and ultimately contributing to neural changes observed in diabetic patients. Particularly, since IL-1β has an important role in retinal microglia activation and proliferation under diabetes, limiting IL-1β-triggered inflammatory processes may provide a new therapeutic strategy to prevent the progression of diabetic retinopathy.

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Bacteriophage T7 DNA packaging

Cited by 33 publications

References 25 publications

Software-based analysis of bacteriophage genomes, physical ends, and packaging strategies

Software-based analysis of bacteriophage genomes, physical ends, and packaging strategies

Mechanistic Coupling of Bacteriophage T4 DNA Packaging to Components of the Replication-dependent Late Transcription Machinery

Elevated Glucose and Interleukin-1β Differentially Affect Retinal Microglial Cell Proliferation

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