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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

Occurrence of the reverse sequences around the top 50 forward and reverse HFSs. The frequencies of the reverse sequences starting from a particular position around the first base of the HFSs were calculated. The frequency profile of the forward and reverse HFSs are near identical, with position +2 reverse sequences (R+2) having a remarkablely higher occurrence than the others.
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Figure 5: Occurrence of the reverse sequences around the top 50 forward and reverse HFSs. The frequencies of the reverse sequences starting from a particular position around the first base of the HFSs were calculated. The frequency profile of the forward and reverse HFSs are near identical, with position +2 reverse sequences (R+2) having a remarkablely higher occurrence than the others.

Mentions: To test if both ends generated from the same terminase cut can be simultaneously packaged into two viral heads, occurrence of the reverse sequences located around the HFSs were counted. The results highlighted an interesting phenomenon. For most of the HFSs, the reverse sequences starting from position +2 (R+2) occurred at a striking higher frequency than those from any other position (Figure 5 and additional file 2). The high frequency of these R+2 sequences indicates that such sequences should also be the genome termini, suggesting that both ends of a single terminase cleavage could be packaged. Consensus sequence analysis showed that these paired HFSs contained a core consensus sequence TTGNA/GCT (Figure 6) which is slightly different from the above defined top HFS consensus sequence. The forward and reverse HFS pairs have an overlap of two base pairs, indicating terminase cleavage probably generated sticky ends with a 2 nucleotide 5' overhang (mostly GC) (Figure 6). Although it has been shown that T4 genomic DNA can be directly ligated[25], it does not necessarily mean that the termini are blunt-ended, since sticky ends can be ligated with high efficiency.


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)

Occurrence of the reverse sequences around the top 50 forward and reverse HFSs. The frequencies of the reverse sequences starting from a particular position around the first base of the HFSs were calculated. The frequency profile of the forward and reverse HFSs are near identical, with position +2 reverse sequences (R+2) having a remarkablely higher occurrence than the others.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Occurrence of the reverse sequences around the top 50 forward and reverse HFSs. The frequencies of the reverse sequences starting from a particular position around the first base of the HFSs were calculated. The frequency profile of the forward and reverse HFSs are near identical, with position +2 reverse sequences (R+2) having a remarkablely higher occurrence than the others.
Mentions: To test if both ends generated from the same terminase cut can be simultaneously packaged into two viral heads, occurrence of the reverse sequences located around the HFSs were counted. The results highlighted an interesting phenomenon. For most of the HFSs, the reverse sequences starting from position +2 (R+2) occurred at a striking higher frequency than those from any other position (Figure 5 and additional file 2). The high frequency of these R+2 sequences indicates that such sequences should also be the genome termini, suggesting that both ends of a single terminase cleavage could be packaged. Consensus sequence analysis showed that these paired HFSs contained a core consensus sequence TTGNA/GCT (Figure 6) which is slightly different from the above defined top HFS consensus sequence. The forward and reverse HFS pairs have an overlap of two base pairs, indicating terminase cleavage probably generated sticky ends with a 2 nucleotide 5' overhang (mostly GC) (Figure 6). Although it has been shown that T4 genomic DNA can be directly ligated[25], it does not necessarily mean that the termini are blunt-ended, since sticky ends can be ligated with high efficiency.

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