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Sequence characteristics of T4-like bacteriophage IME08 benome termini revealed by high throughput sequencing.

Jiang X, Jiang H, Li C, Wang S, Mi Z, An X, Chen J, Tong Y - Virol. J. (2011)

Bottom Line: The literature indicates that T4-like phage genomes have permuted terminal sequences, and are generated by a DNA terminase in a sequence-independent manner; genomic DNA of T4-like bacteriophage IME08 was subjected to high throughput sequencing, and the read sequences with extraordinarily high occurrences were analyzed; we demonstrate that both the 5' and 3' termini of the IME08 genome starts with base G or A.The presence of a consensus sequence TTGGA/G around the breakpoint of the high frequency read sequences suggests that the terminase cuts the branched pre-genome in a sequence-preferred manner.Our analysis also shows that terminal cleavage is asymmetric, with one end cut at a consensus sequence, and the other end generated randomly.

View Article: PubMed Central - HTML - PubMed

Affiliation: Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.

ABSTRACT

Background: T4 phage is a model species that has contributed broadly to our understanding of molecular biology. T4 DNA replication and packaging share various mechanisms with human double-stranded DNA viruses such as herpes virus. The literature indicates that T4-like phage genomes have permuted terminal sequences, and are generated by a DNA terminase in a sequence-independent manner;

Methods: genomic DNA of T4-like bacteriophage IME08 was subjected to high throughput sequencing, and the read sequences with extraordinarily high occurrences were analyzed;

Results: we demonstrate that both the 5' and 3' termini of the IME08 genome starts with base G or A. The presence of a consensus sequence TTGGA/G around the breakpoint of the high frequency read sequences suggests that the terminase cuts the branched pre-genome in a sequence-preferred manner. Our analysis also shows that terminal cleavage is asymmetric, with one end cut at a consensus sequence, and the other end generated randomly. The sequence-preferred cleavage may produce sticky-ends, but with each end being packaged with different efficiencies;

Conclusions: this study illustrates how high throughput sequencing can be used to probe replication and packaging mechanisms in bacteriophages and/or viruses.

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Distribution of top 50 forward and reverse HFSs on the IME08 genome.
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Figure 7: Distribution of top 50 forward and reverse HFSs on the IME08 genome.

Mentions: It is assumed that both ends of T4-like phage genomes are processed by the terminase. Our results suggest that both the forward and the reverse termini have similar terminal consensus sequences. Since T4-like phage have a fixed genome length (though circularly permutated), which is about 102% of its genome size[20], if both ends of a genome are flanked by HFS sites, then HFS sites should exist as pairs along the genome with the forward HFS sites located about 2% of the genome (about 3.4 kb) upstream of the reverse HFS sites. However, genome distribution analysis of the HFSs did not reveal a position correlation between the forward and the reverse HFSs (Figure 7 and additional file 3). In some regions, HFSs of only one orientation were observed (e.g. only forward HFSs exist in regions 70-90k, 164-2k, and only reverse HFSs exist in 150-164k). The unpaired distribution pattern thus contradicts the assumption that both ends of a genome are generated with the same cleavage mechanism, and suggests that for a particular genome, one end is generated by a sequence-preferred mechanism, whereas the other end is produced randomly, with a headful size-dependent cleavage. In this hypothetic packaging mechanism, the terminase will first recognize and cleave the consensus sequence, and insert one end into the virion prehead. The package process continues until a headful length of genomic DNA is loaded into the prehead when the terminase executes a second cleavage to terminate the DNA package process. This second cleavage of the genome may be less sequence-preferred or even random.


Sequence characteristics of T4-like bacteriophage IME08 benome termini revealed by high throughput sequencing.

Jiang X, Jiang H, Li C, Wang S, Mi Z, An X, Chen J, Tong Y - Virol. J. (2011)

Distribution of top 50 forward and reverse HFSs on the IME08 genome.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105952&req=5

Figure 7: Distribution of top 50 forward and reverse HFSs on the IME08 genome.
Mentions: It is assumed that both ends of T4-like phage genomes are processed by the terminase. Our results suggest that both the forward and the reverse termini have similar terminal consensus sequences. Since T4-like phage have a fixed genome length (though circularly permutated), which is about 102% of its genome size[20], if both ends of a genome are flanked by HFS sites, then HFS sites should exist as pairs along the genome with the forward HFS sites located about 2% of the genome (about 3.4 kb) upstream of the reverse HFS sites. However, genome distribution analysis of the HFSs did not reveal a position correlation between the forward and the reverse HFSs (Figure 7 and additional file 3). In some regions, HFSs of only one orientation were observed (e.g. only forward HFSs exist in regions 70-90k, 164-2k, and only reverse HFSs exist in 150-164k). The unpaired distribution pattern thus contradicts the assumption that both ends of a genome are generated with the same cleavage mechanism, and suggests that for a particular genome, one end is generated by a sequence-preferred mechanism, whereas the other end is produced randomly, with a headful size-dependent cleavage. In this hypothetic packaging mechanism, the terminase will first recognize and cleave the consensus sequence, and insert one end into the virion prehead. The package process continues until a headful length of genomic DNA is loaded into the prehead when the terminase executes a second cleavage to terminate the DNA package process. This second cleavage of the genome may be less sequence-preferred or even random.

Bottom Line: The literature indicates that T4-like phage genomes have permuted terminal sequences, and are generated by a DNA terminase in a sequence-independent manner; genomic DNA of T4-like bacteriophage IME08 was subjected to high throughput sequencing, and the read sequences with extraordinarily high occurrences were analyzed; we demonstrate that both the 5' and 3' termini of the IME08 genome starts with base G or A.The presence of a consensus sequence TTGGA/G around the breakpoint of the high frequency read sequences suggests that the terminase cuts the branched pre-genome in a sequence-preferred manner.Our analysis also shows that terminal cleavage is asymmetric, with one end cut at a consensus sequence, and the other end generated randomly.

View Article: PubMed Central - HTML - PubMed

Affiliation: Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.

ABSTRACT

Background: T4 phage is a model species that has contributed broadly to our understanding of molecular biology. T4 DNA replication and packaging share various mechanisms with human double-stranded DNA viruses such as herpes virus. The literature indicates that T4-like phage genomes have permuted terminal sequences, and are generated by a DNA terminase in a sequence-independent manner;

Methods: genomic DNA of T4-like bacteriophage IME08 was subjected to high throughput sequencing, and the read sequences with extraordinarily high occurrences were analyzed;

Results: we demonstrate that both the 5' and 3' termini of the IME08 genome starts with base G or A. The presence of a consensus sequence TTGGA/G around the breakpoint of the high frequency read sequences suggests that the terminase cuts the branched pre-genome in a sequence-preferred manner. Our analysis also shows that terminal cleavage is asymmetric, with one end cut at a consensus sequence, and the other end generated randomly. The sequence-preferred cleavage may produce sticky-ends, but with each end being packaged with different efficiencies;

Conclusions: this study illustrates how high throughput sequencing can be used to probe replication and packaging mechanisms in bacteriophages and/or viruses.

Show MeSH
Related in: MedlinePlus