Microarray Analysis of Gene Expression during Bacteriophage T4 Infection

Luke, Kimberly; Radek, Agnes; Liu, Xiu Ping; Campbell, John W.; Uzan, Marc; Haselkorn, Robert; Kogan, Yakov

doi:10.1006/viro.2002.1409

Cited by 59 publications

(71 citation statements)

References 33 publications

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“…To add to the complexity, ϳ30 transcripts were detected early, disappeared, and then reappeared as late transcripts (these CDSs are marked in Supplement S2 in the supplemental material). A similar phenomenon has been reported in transcriptional studies with the bacteriophage T4 (18) and Red Sea bream iridovirus (17). In addition to the early/late CDSs described in the preceding section, the most striking feature of this early/late class was the presence of many genes encoding proteins associated with genome integration, including five GIY-YIG endonucleases (A134L, A287R, A315L, A495R, and A651L) and four HNH endonucleases (A354R, A422R, A478L, and A490L).…”

Section: Resultssupporting

confidence: 83%

“…Interestingly, these small regions are also clustered and conserved in another sequenced chlorovirus (NY-2A) that infects the same host chlorella (results not shown). Unlike the phage T4 genome, where most late CDSs are located in contiguous regions on the same DNA strand (18), PBCV-1 expression did not show a strong strand-specific bias. In addition, there is no relationship between time of expression and GϩC content of the genome (Fig.…”

Section: Resultsmentioning

confidence: 61%

“…The reference sample, for the time course experiments, consisted of a pool of transcripts obtained by mixing equal amounts of total RNA from each time point. Competitive hybridization experiments were conducted for each sample against the reference sample (15,18,41). For the aphidicolin experiments, a direct comparison was carried out with each treated sample versus the corresponding untreated infected control.…”

Section: Methodsmentioning

confidence: 99%

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Microarray Analysis of Paramecium bursaria Chlorella Virus 1 Transcription

Yanai-Balser

Duncan

Eudy

et al. 2010

J Virol

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Paramecium bursaria chlorella virus 1 (PBCV-1), the prototype of the genus Chlorovirus (family Phycodnaviridae), is a large, icosahedral (190 nm in diameter), plaque-forming virus that infects the unicellular, eukaryotic green alga Chlorella sp. strain NC64A. The PBCV-1 virion has a lipid membrane located inside an outer glycoprotein capsid. The 330-kb genome is a linear, nonpermutated, double-stranded DNA (dsDNA) molecule with covalently closed hairpin ends that has approximately 365 protein encoding genes (CDSs), as well as 11 tRNA encoding genes (reviewed in references 34, 39, and 40). The CDSs are evenly distributed on both strands and intergenic space is minimal (typically fewer than 100 nucleotides); the exception is a 1,788-bp sequence in the middle of the genome that encodes the tRNA genes. Approximately 35% of the 365 PBCV-1 gene products resemble proteins in the public databases.PBCV-1 initiates infection by attaching rapidly and specifically to the cell wall of its host (22), probably at a unique virus vertex (4, 26). Attachment is immediately followed by host cell wall degradation by a virus-packaged enzyme(s) at the point of contact. After wall degradation, the viral internal membrane presumably fuses with the host membrane, causing host membrane depolarization (9), potassium ion efflux (25), and an increase in the cytoplasm pH (2). These events are predicted to facilitate entry of the viral DNA and virion-associated proteins into the cell. PBCV-1 lacks a gene encoding a recognizable RNA polymerase or a subunit of it, and RNA polymerase activity is not detected in PBCV-1 virions. Therefore, viral DNA and virion-associated proteins are predicted to migrate to the nucleus, and early viral transcription is detected 5 to 10 min postinfection (p.i.), presumably by commandeering a host RNA polymerase(s) (possibly RNA polymerase II) (14, 29). Virus DNA synthesis begins 60 to 90 min p.i., followed by virus assembly at 3 to 5 h p.i. in localized regions of the cytoplasm, called virus assembly centers (21). At 6 to 8 h p.i., virusinduced host cell lysis occurs resulting in release of progeny virions (ϳ1,000 viruses/cell, ϳ25% of which are infectious). These events are depicted in Fig. 1.To initiate PBCV-1 transcription, the host RNA polymerase(s), possibly in combination with a virus transcription factor(s), must recognize virus DNA promoter sequences. Recently, three short nucleotide sequences were identified in putative virus promoter regions (150 bp upstream and 50 bp downstream of the ATG translation site) that are conserved in PBCV-1 and other Chlorovirus members (7). PBCV-1 CDSs are not spatially clustered on the genome by either temporal or functional class, suggesting that transcription regulation must occur via cis-and possible trans-acting regulatory elements.To understand the dynamics of PBCV-1 global gene expression during virus replication, we constructed a microarray containing 50-mer probes to each of the 365 PBCV-1 CDSs.

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 61%

Section: Methodsmentioning

confidence: 99%

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Microarray Analysis of Paramecium bursaria Chlorella Virus 1 Transcription

Yanai-Balser

Duncan

Eudy

et al. 2010

J Virol

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“…In a recent report (672), the expression profile of the entire T4 genome was evaluated by mRNA hybridization microarray analysis. RNA samples were obtained from 0 to 25 min during a T4 infection cycle at 30°C.…”

Section: Microarray Analysis Of T4 Transcriptionmentioning

confidence: 99%

Bacteriophage T4 Genome

Miller

Kutter

Mosig

et al. 2003

Microbiol Mol Biol Rev

686

801

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SUMMARY Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth.

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“…For example, the researcher may simply be interested in when phage capsid proteins are synthesized, in which case it would be sensible to monitor the expression of the major capsid protein. For T4, the expression profiles of most genes has been determined (2,9) and proves a useful starting point when evaluating gene expression of under-studied T4 type phages infecting other bacteria.…”

Section: Target Gene Selectionmentioning

confidence: 99%

Quantification of Host and Phage mRNA Expression During Infection Using Real-Time PCR

Clokie

2009

Methods in Molecular Biology

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Real-time, or quantitative PCR is a valuable technique useful in bacteriophage research to quantify the abundance of phage or host gene transcripts. It can be used during the infection cycle both to monitor the expression of individual viral transcripts and to compare relative gene expression levels throughout the infection cycle. It is fairly economical to conduct and is useful in bacteria-phage systems where obtaining high yields of RNA is problematic. To perform real-time PCR it is simply necessary to know the DNA sequence of the genes to be monitored, to have accurately quantified mRNA and good quality cDNA, and access to a light-cycler. Although this chapter briefly reviews the basic principles of real-time PCR, the emphasis is on aspects of technique that are specific to the study of bacteriophage transcriptomics. These include (1) the 2 selection of the target gene, (2) the choice of calibrator and reference genes, and (3) RNA isolation for cDNA synthesis and (4) subsequent analysis of samples. This chapter should also be useful to those wishing to amplify genes from other types of templates such as metagenomic DNA or RNA extracted either from filtered samples or from agarose gels.

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Microarray Analysis of Gene Expression during Bacteriophage T4 Infection

Cited by 59 publications

References 33 publications

Microarray Analysis of Paramecium bursaria Chlorella Virus 1 Transcription

Microarray Analysis of Paramecium bursaria Chlorella Virus 1 Transcription

Bacteriophage T4 Genome

Quantification of Host and Phage mRNA Expression During Infection Using Real-Time PCR

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