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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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Related in: MedlinePlus

Sequence logo representation of sequences around the start sites of the top 20 HFSs. Sequences were plotted with WebLogo [23]. The height of the letter indicates the degree of conservation. Nucleotide 11 is the start site of HFSs.
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Figure 4: Sequence logo representation of sequences around the start sites of the top 20 HFSs. Sequences were plotted with WebLogo [23]. The height of the letter indicates the degree of conservation. Nucleotide 11 is the start site of HFSs.

Mentions: To characterize HFSs, various analysis measures were applied, including consensus sequence searches, homology searches, and local thermal stability (melting temperature) plotting. For consensus sequence analysis, the top 20 HFSs together with their upstream sequences (retrieved from the assembled genome) were plotted with Weblogo[23]. The results demonstrate an obvious consensus sequence around the cleavage breakpoint, with the major part of the consensus sequence not included in the HFS sequences, but in its upstream sequences (Figure 4). Of the top 20 HFSs, 16 (80%) have an identical cleavage site 5'-TTGGA↓G-3', indicating cleavage is highly sequence-preferred (not strictly sequence-specific). Since the forward and the reverse HFS sequences (relative to the genome orientation) have the same consensus cleavage sequence, it suggests that both 5' and 3' genome terminus cleavage share a common mechanism, which indicates that the packaging of genomic DNA is bidirectional (but the two ends of a particular genome are not cleaved with the same mechanism. The first cleavage is highly sequence-preferred and the second cleavage is probably random or nearly random, as discussed later in this paper). To extend the search for other sequence characteristics around the cleavage sites, the -100 to +100 nucleotides of the HFS cleavage sites were analyzed with Weblogo and Clustal software. The results did not reveal any additional consensus sequences (data not shown).


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)

Sequence logo representation of sequences around the start sites of the top 20 HFSs. Sequences were plotted with WebLogo [23]. The height of the letter indicates the degree of conservation. Nucleotide 11 is the start site of HFSs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Sequence logo representation of sequences around the start sites of the top 20 HFSs. Sequences were plotted with WebLogo [23]. The height of the letter indicates the degree of conservation. Nucleotide 11 is the start site of HFSs.
Mentions: To characterize HFSs, various analysis measures were applied, including consensus sequence searches, homology searches, and local thermal stability (melting temperature) plotting. For consensus sequence analysis, the top 20 HFSs together with their upstream sequences (retrieved from the assembled genome) were plotted with Weblogo[23]. The results demonstrate an obvious consensus sequence around the cleavage breakpoint, with the major part of the consensus sequence not included in the HFS sequences, but in its upstream sequences (Figure 4). Of the top 20 HFSs, 16 (80%) have an identical cleavage site 5'-TTGGA↓G-3', indicating cleavage is highly sequence-preferred (not strictly sequence-specific). Since the forward and the reverse HFS sequences (relative to the genome orientation) have the same consensus cleavage sequence, it suggests that both 5' and 3' genome terminus cleavage share a common mechanism, which indicates that the packaging of genomic DNA is bidirectional (but the two ends of a particular genome are not cleaved with the same mechanism. The first cleavage is highly sequence-preferred and the second cleavage is probably random or nearly random, as discussed later in this paper). To extend the search for other sequence characteristics around the cleavage sites, the -100 to +100 nucleotides of the HFS cleavage sites were analyzed with Weblogo and Clustal software. The results did not reveal any additional consensus sequences (data not shown).

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