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Computational prediction and molecular confirmation of Helitron transposons in the maize genome.

Du C, Caronna J, He L, Dooner HK - BMC Genomics (2008)

Bottom Line: Four out of the five predicted Helitrons were confirmed by PCR assays and DNA sequencing in different maize inbred lines.Four out of five candidates were confirmed to be real by empirical methods, thus validating the predictions of HelitronFinder.Additional points to emerge from our study are that Helitrons do not always insert at an AT dinucleotide in the host sequences, that they can insert immediately adjacent to an existing Helitron, and that their movement may cause changes in the flanking region, such as deletions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept, of Biology & Molecular Biology, Montclair State University, Montclair, NJ 07043, USA. duc@mail.montclair.edu

ABSTRACT

Background: Helitrons represent a new class of transposable elements recently uncovered in plants and animals. One remarkable feature of Helitrons is their ability to capture gene sequences, which makes them of considerable potential evolutionary importance. However, because Helitrons lack the typical structural features of other DNA transposable elements, identifying them is a challenge. Currently, most researchers identify Helitrons manually by comparing sequences. With the maize whole genome sequencing project underway, an automated computational Helitron searching tool is needed. The characterization of Helitron activities in maize needs to be addressed in order to better understand the impact of Helitrons on the organization of the genome.

Results: We developed and implemented a heuristic searching algorithm in PERL for identifying Helitrons. Our HelitronFinder program will (i) take FASTA-formatted DNA sequences as input and identify the hairpin looping patterns, and (ii) exploit the consensus 5' and 3' end sequences of known Helitrons to identify putative ends. We randomly selected five predicted Helitrons from the program's high quality output for molecular verification. Four out of the five predicted Helitrons were confirmed by PCR assays and DNA sequencing in different maize inbred lines. The HelitronFinder program identified two head-to-head dissimilar Helitrons in a maize BAC sequence.

Conclusion: We have identified 140 new Helitron candidates in maize with our computational tool HelitronFinder by searching maize DNA sequences currently available in GenBank. Four out of five candidates were confirmed to be real by empirical methods, thus validating the predictions of HelitronFinder. Additional points to emerge from our study are that Helitrons do not always insert at an AT dinucleotide in the host sequences, that they can insert immediately adjacent to an existing Helitron, and that their movement may cause changes in the flanking region, such as deletions.

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Location of PCR primers flanking and internal to adjacent Helitrons identified in sequence AF466202. We designed four pairs of primers for these two Helitrons: F1/R1, F3/R3, F2/R4, and F4/R4. F and R represent forward and reverse primers, respectively.
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Figure 8: Location of PCR primers flanking and internal to adjacent Helitrons identified in sequence AF466202. We designed four pairs of primers for these two Helitrons: F1/R1, F3/R3, F2/R4, and F4/R4. F and R represent forward and reverse primers, respectively.

Mentions: We designed four pairs of primers for these two Helitrons, F1/R1, F3/R3, F2/R4, and F4/R4 (Fig. 8). F and R represent forward and reverse primers, respectively. According to the PCR products in Table 3, we detected both Helitrons in lines A636 and B73, only Helitron No.2 in lines McC, W22, and W23, and neither Helitron in lines A188, CML139, H99, Ki3, M14, or Mo17. This result lends itself to two interpretations. One possibility is that Helitron No.2 (left) inserted into the maize genome first and that Helitron No.1 (right) inserted subsequently, and noncanonically, at the GT dinucleotide found at the 3' end of Helitron No.2. An alternative is that Helitron No.1 inserted first and Helitron No.2 inserted subsequently, and canonically, at the AT dinucleotide created by the host A and the T at the 5' end of Helitron No.1. Following the formation of this head-to-tail configuration (found in lines B73 and A636), Helitron No.1 would have excised cleanly (see next section), leaving only Helitron No.2 at the insertion site (as in McC, W22, and W23).


Computational prediction and molecular confirmation of Helitron transposons in the maize genome.

Du C, Caronna J, He L, Dooner HK - BMC Genomics (2008)

Location of PCR primers flanking and internal to adjacent Helitrons identified in sequence AF466202. We designed four pairs of primers for these two Helitrons: F1/R1, F3/R3, F2/R4, and F4/R4. F and R represent forward and reverse primers, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267711&req=5

Figure 8: Location of PCR primers flanking and internal to adjacent Helitrons identified in sequence AF466202. We designed four pairs of primers for these two Helitrons: F1/R1, F3/R3, F2/R4, and F4/R4. F and R represent forward and reverse primers, respectively.
Mentions: We designed four pairs of primers for these two Helitrons, F1/R1, F3/R3, F2/R4, and F4/R4 (Fig. 8). F and R represent forward and reverse primers, respectively. According to the PCR products in Table 3, we detected both Helitrons in lines A636 and B73, only Helitron No.2 in lines McC, W22, and W23, and neither Helitron in lines A188, CML139, H99, Ki3, M14, or Mo17. This result lends itself to two interpretations. One possibility is that Helitron No.2 (left) inserted into the maize genome first and that Helitron No.1 (right) inserted subsequently, and noncanonically, at the GT dinucleotide found at the 3' end of Helitron No.2. An alternative is that Helitron No.1 inserted first and Helitron No.2 inserted subsequently, and canonically, at the AT dinucleotide created by the host A and the T at the 5' end of Helitron No.1. Following the formation of this head-to-tail configuration (found in lines B73 and A636), Helitron No.1 would have excised cleanly (see next section), leaving only Helitron No.2 at the insertion site (as in McC, W22, and W23).

Bottom Line: Four out of the five predicted Helitrons were confirmed by PCR assays and DNA sequencing in different maize inbred lines.Four out of five candidates were confirmed to be real by empirical methods, thus validating the predictions of HelitronFinder.Additional points to emerge from our study are that Helitrons do not always insert at an AT dinucleotide in the host sequences, that they can insert immediately adjacent to an existing Helitron, and that their movement may cause changes in the flanking region, such as deletions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept, of Biology & Molecular Biology, Montclair State University, Montclair, NJ 07043, USA. duc@mail.montclair.edu

ABSTRACT

Background: Helitrons represent a new class of transposable elements recently uncovered in plants and animals. One remarkable feature of Helitrons is their ability to capture gene sequences, which makes them of considerable potential evolutionary importance. However, because Helitrons lack the typical structural features of other DNA transposable elements, identifying them is a challenge. Currently, most researchers identify Helitrons manually by comparing sequences. With the maize whole genome sequencing project underway, an automated computational Helitron searching tool is needed. The characterization of Helitron activities in maize needs to be addressed in order to better understand the impact of Helitrons on the organization of the genome.

Results: We developed and implemented a heuristic searching algorithm in PERL for identifying Helitrons. Our HelitronFinder program will (i) take FASTA-formatted DNA sequences as input and identify the hairpin looping patterns, and (ii) exploit the consensus 5' and 3' end sequences of known Helitrons to identify putative ends. We randomly selected five predicted Helitrons from the program's high quality output for molecular verification. Four out of the five predicted Helitrons were confirmed by PCR assays and DNA sequencing in different maize inbred lines. The HelitronFinder program identified two head-to-head dissimilar Helitrons in a maize BAC sequence.

Conclusion: We have identified 140 new Helitron candidates in maize with our computational tool HelitronFinder by searching maize DNA sequences currently available in GenBank. Four out of five candidates were confirmed to be real by empirical methods, thus validating the predictions of HelitronFinder. Additional points to emerge from our study are that Helitrons do not always insert at an AT dinucleotide in the host sequences, that they can insert immediately adjacent to an existing Helitron, and that their movement may cause changes in the flanking region, such as deletions.

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