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Transposition-mediated DNA re-replication in maize.

Zhang J, Zuo T, Wang D, Peterson T - Elife (2014)

Bottom Line: Every DNA segment in a eukaryotic genome normally replicates once and only once per cell cycle to maintain genome stability.The DNA re-replication can spontaneously abort to generate double-strand breaks, which can be repaired to generate Composite Insertions composed of transposon termini flanking segmental duplications of various lengths.These results show how alternative transposition coupled with DNA replication and repair can significantly alter genome structure and may have contributed to rapid genome evolution in maize and possibly other eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States.

ABSTRACT
Every DNA segment in a eukaryotic genome normally replicates once and only once per cell cycle to maintain genome stability. We show here that this restriction can be bypassed through alternative transposition, a transposition reaction that utilizes the termini of two separate, nearby transposable elements (TEs). Our results suggest that alternative transposition during S phase can induce re-replication of the TEs and their flanking sequences. The DNA re-replication can spontaneously abort to generate double-strand breaks, which can be repaired to generate Composite Insertions composed of transposon termini flanking segmental duplications of various lengths. These results show how alternative transposition coupled with DNA replication and repair can significantly alter genome structure and may have contributed to rapid genome evolution in maize and possibly other eukaryotes.

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PCR screening and DNA gel blotting of candidate TDDCI alleles.(A) Detailed structures of P1-ovov454(progenitor) and RET-generated P1-rr-twin/p1-ww-twin(TDDCI/Deletion) alleles deduced from Figure1. The horizontal blue lines are p1 gene sequencewhile the green lines are p1 proximal sequences, includingthe p2 gene sequence (a p1 paralog,∼70 kb proximal to p1); the blue and green boxes areexons 1, 2, and 3 (right to left) of p1 andp2, respectively. The small horizontal arrows indicatethe orientation and the approximate position of the PCR primers. The grayboxes indicate probe 8B used in DNA gel blot analysis, the short verticalblack lines are SacI sites, and the numbers between theSacI sites indicate the lengths of those fragmentsdetected by probe 8B. The hatched boxes represent the distal (black) andproximal (green) 5248 bp repeats flanking the p1 locus.These repeats are identical except for six SNPs, indicated by short redvertical lines inside the green hatched box (SNPs 3 and 4 are only 43 bpapart). Other symbols have the same meaning as in Figure 1. (B) PCR products obtained usingprimers 1 + Ac5 (upper) or 2 + Ac3 (lower). Lane 1, 1 kb DNAladder; lane 2, P1-ovov454; lane 3,P1-rr-T22; 4, p1-ww-T22; lane 5,P1-ovov454; lane 6, P1-rr-T24; 7,p1-ww-T24; lane 8, P1-ovov454; lane 9,P1-rr-E17; lane10, P1-ovov454; lane 11,P1-rr-E340; lane 12, P1-ovov454; lane13, P1-rr-T21; 14, p1-ww-T21; lane 15,P1-ovov454; lane 16, P1-rr-E5; lane 17,P1-ovov454; lane 18, P1-rr-E311. Note:the sequences of primers 1 and 2 are specific for each allele.(C) DNA gel blot analysis of the TDDCI/deletion alleles.Genomic DNA was digested with SacI and the blot washybridized with probe 8B (see Figure2A for the position of the probe). Lane 1:p1-ww[4Co63], lane 2:P1-ovov454/p1-ww[4Co63], lane 3:P1-rr-T22/p1-ww[4Co63], lane 4:p1-ww-T22/p1-ww[4Co63], lane 5:P1-rr-T24/p1-ww[4Co63], lane 6:p1-ww-T24/p1-ww[4Co63], lane 7:P1-rr-E17/p1-ww[4Co63], lane 8:P1-rr-E340/p1-ww[4Co63], lane 9:P1-rr-T21/p1-ww[4Co63], lane 10:p1-ww-T21/p1-ww[4Co63], lane 11:P1-rr-E311/p1-ww[4Co63], lane 12:P1-rr-E5/p1-ww[4Co63].DOI:http://dx.doi.org/10.7554/eLife.03724.006
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fig2: PCR screening and DNA gel blotting of candidate TDDCI alleles.(A) Detailed structures of P1-ovov454(progenitor) and RET-generated P1-rr-twin/p1-ww-twin(TDDCI/Deletion) alleles deduced from Figure1. The horizontal blue lines are p1 gene sequencewhile the green lines are p1 proximal sequences, includingthe p2 gene sequence (a p1 paralog,∼70 kb proximal to p1); the blue and green boxes areexons 1, 2, and 3 (right to left) of p1 andp2, respectively. The small horizontal arrows indicatethe orientation and the approximate position of the PCR primers. The grayboxes indicate probe 8B used in DNA gel blot analysis, the short verticalblack lines are SacI sites, and the numbers between theSacI sites indicate the lengths of those fragmentsdetected by probe 8B. The hatched boxes represent the distal (black) andproximal (green) 5248 bp repeats flanking the p1 locus.These repeats are identical except for six SNPs, indicated by short redvertical lines inside the green hatched box (SNPs 3 and 4 are only 43 bpapart). Other symbols have the same meaning as in Figure 1. (B) PCR products obtained usingprimers 1 + Ac5 (upper) or 2 + Ac3 (lower). Lane 1, 1 kb DNAladder; lane 2, P1-ovov454; lane 3,P1-rr-T22; 4, p1-ww-T22; lane 5,P1-ovov454; lane 6, P1-rr-T24; 7,p1-ww-T24; lane 8, P1-ovov454; lane 9,P1-rr-E17; lane10, P1-ovov454; lane 11,P1-rr-E340; lane 12, P1-ovov454; lane13, P1-rr-T21; 14, p1-ww-T21; lane 15,P1-ovov454; lane 16, P1-rr-E5; lane 17,P1-ovov454; lane 18, P1-rr-E311. Note:the sequences of primers 1 and 2 are specific for each allele.(C) DNA gel blot analysis of the TDDCI/deletion alleles.Genomic DNA was digested with SacI and the blot washybridized with probe 8B (see Figure2A for the position of the probe). Lane 1:p1-ww[4Co63], lane 2:P1-ovov454/p1-ww[4Co63], lane 3:P1-rr-T22/p1-ww[4Co63], lane 4:p1-ww-T22/p1-ww[4Co63], lane 5:P1-rr-T24/p1-ww[4Co63], lane 6:p1-ww-T24/p1-ww[4Co63], lane 7:P1-rr-E17/p1-ww[4Co63], lane 8:P1-rr-E340/p1-ww[4Co63], lane 9:P1-rr-T21/p1-ww[4Co63], lane 10:p1-ww-T21/p1-ww[4Co63], lane 11:P1-rr-E311/p1-ww[4Co63], lane 12:P1-rr-E5/p1-ww[4Co63].DOI:http://dx.doi.org/10.7554/eLife.03724.006

Mentions: Both TDD and TDDCI alleles contain similar duplication structures and should exhibitsimilar phenotypes. Therefore, we screened maize ears as described previously tovisually identify putative TDD-containing alleles (Zhang et al., 2013). We identified 25 candidate alleles, and cloned andsequenced the duplication/Ac junctions (the green segment flankingthe Ac 5′ end in Figure1E and Figure 2A) from 16 of the 25TDD/TDDCI candidates via Ac casting (Singh et al., 2003; Wang andPeterson, 2013) or inverse PCR (iPCR) (See Zhang et al. (2013) for detailed screening and cloningmethods). To identify the TDDCI alleles, we designed PCR primers that flank theprogenitor insertion target sites for each allele (Figure 2A, primers 1 and 2). Primers 1 + Ac5 can amplify a productfrom both TDD and TDDCI while primers 2 + Ac3 can amplify a product only fromTDDCI since the latter contains an additional CI (Figure 2A). As expected, PCR using primers 1 + Ac5 produced bands ofthe expected sizes in all the 16 alleles (Figure2B, upper panel; seven examples are shown here). Whereas, primers 2 +Ac3 produced bands with expected sizes from only seven alleles (Figure 2B, lower panel). Sequencing of the PCR products obtainedfrom primers 1 + Ac5 and 2 + Ac3 revealed that these seven TDDCI candidateshave duplication/insertion breakpoints located from 13,392 bp to 1.7 Mb proximal tothe p1 locus on chromosome 1 (Table 1). Importantly, the Ac termini are flanked by 8-bptarget site duplications (TSDs; green/black triangles in Figure 1E) as predicted by the model in Figure 1 (See Supplementary file 1 for sequences containing TSDs).10.7554/eLife.03724.006Figure 2.PCR screening and DNA gel blotting of candidate TDDCI alleles.


Transposition-mediated DNA re-replication in maize.

Zhang J, Zuo T, Wang D, Peterson T - Elife (2014)

PCR screening and DNA gel blotting of candidate TDDCI alleles.(A) Detailed structures of P1-ovov454(progenitor) and RET-generated P1-rr-twin/p1-ww-twin(TDDCI/Deletion) alleles deduced from Figure1. The horizontal blue lines are p1 gene sequencewhile the green lines are p1 proximal sequences, includingthe p2 gene sequence (a p1 paralog,∼70 kb proximal to p1); the blue and green boxes areexons 1, 2, and 3 (right to left) of p1 andp2, respectively. The small horizontal arrows indicatethe orientation and the approximate position of the PCR primers. The grayboxes indicate probe 8B used in DNA gel blot analysis, the short verticalblack lines are SacI sites, and the numbers between theSacI sites indicate the lengths of those fragmentsdetected by probe 8B. The hatched boxes represent the distal (black) andproximal (green) 5248 bp repeats flanking the p1 locus.These repeats are identical except for six SNPs, indicated by short redvertical lines inside the green hatched box (SNPs 3 and 4 are only 43 bpapart). Other symbols have the same meaning as in Figure 1. (B) PCR products obtained usingprimers 1 + Ac5 (upper) or 2 + Ac3 (lower). Lane 1, 1 kb DNAladder; lane 2, P1-ovov454; lane 3,P1-rr-T22; 4, p1-ww-T22; lane 5,P1-ovov454; lane 6, P1-rr-T24; 7,p1-ww-T24; lane 8, P1-ovov454; lane 9,P1-rr-E17; lane10, P1-ovov454; lane 11,P1-rr-E340; lane 12, P1-ovov454; lane13, P1-rr-T21; 14, p1-ww-T21; lane 15,P1-ovov454; lane 16, P1-rr-E5; lane 17,P1-ovov454; lane 18, P1-rr-E311. Note:the sequences of primers 1 and 2 are specific for each allele.(C) DNA gel blot analysis of the TDDCI/deletion alleles.Genomic DNA was digested with SacI and the blot washybridized with probe 8B (see Figure2A for the position of the probe). Lane 1:p1-ww[4Co63], lane 2:P1-ovov454/p1-ww[4Co63], lane 3:P1-rr-T22/p1-ww[4Co63], lane 4:p1-ww-T22/p1-ww[4Co63], lane 5:P1-rr-T24/p1-ww[4Co63], lane 6:p1-ww-T24/p1-ww[4Co63], lane 7:P1-rr-E17/p1-ww[4Co63], lane 8:P1-rr-E340/p1-ww[4Co63], lane 9:P1-rr-T21/p1-ww[4Co63], lane 10:p1-ww-T21/p1-ww[4Co63], lane 11:P1-rr-E311/p1-ww[4Co63], lane 12:P1-rr-E5/p1-ww[4Co63].DOI:http://dx.doi.org/10.7554/eLife.03724.006
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fig2: PCR screening and DNA gel blotting of candidate TDDCI alleles.(A) Detailed structures of P1-ovov454(progenitor) and RET-generated P1-rr-twin/p1-ww-twin(TDDCI/Deletion) alleles deduced from Figure1. The horizontal blue lines are p1 gene sequencewhile the green lines are p1 proximal sequences, includingthe p2 gene sequence (a p1 paralog,∼70 kb proximal to p1); the blue and green boxes areexons 1, 2, and 3 (right to left) of p1 andp2, respectively. The small horizontal arrows indicatethe orientation and the approximate position of the PCR primers. The grayboxes indicate probe 8B used in DNA gel blot analysis, the short verticalblack lines are SacI sites, and the numbers between theSacI sites indicate the lengths of those fragmentsdetected by probe 8B. The hatched boxes represent the distal (black) andproximal (green) 5248 bp repeats flanking the p1 locus.These repeats are identical except for six SNPs, indicated by short redvertical lines inside the green hatched box (SNPs 3 and 4 are only 43 bpapart). Other symbols have the same meaning as in Figure 1. (B) PCR products obtained usingprimers 1 + Ac5 (upper) or 2 + Ac3 (lower). Lane 1, 1 kb DNAladder; lane 2, P1-ovov454; lane 3,P1-rr-T22; 4, p1-ww-T22; lane 5,P1-ovov454; lane 6, P1-rr-T24; 7,p1-ww-T24; lane 8, P1-ovov454; lane 9,P1-rr-E17; lane10, P1-ovov454; lane 11,P1-rr-E340; lane 12, P1-ovov454; lane13, P1-rr-T21; 14, p1-ww-T21; lane 15,P1-ovov454; lane 16, P1-rr-E5; lane 17,P1-ovov454; lane 18, P1-rr-E311. Note:the sequences of primers 1 and 2 are specific for each allele.(C) DNA gel blot analysis of the TDDCI/deletion alleles.Genomic DNA was digested with SacI and the blot washybridized with probe 8B (see Figure2A for the position of the probe). Lane 1:p1-ww[4Co63], lane 2:P1-ovov454/p1-ww[4Co63], lane 3:P1-rr-T22/p1-ww[4Co63], lane 4:p1-ww-T22/p1-ww[4Co63], lane 5:P1-rr-T24/p1-ww[4Co63], lane 6:p1-ww-T24/p1-ww[4Co63], lane 7:P1-rr-E17/p1-ww[4Co63], lane 8:P1-rr-E340/p1-ww[4Co63], lane 9:P1-rr-T21/p1-ww[4Co63], lane 10:p1-ww-T21/p1-ww[4Co63], lane 11:P1-rr-E311/p1-ww[4Co63], lane 12:P1-rr-E5/p1-ww[4Co63].DOI:http://dx.doi.org/10.7554/eLife.03724.006
Mentions: Both TDD and TDDCI alleles contain similar duplication structures and should exhibitsimilar phenotypes. Therefore, we screened maize ears as described previously tovisually identify putative TDD-containing alleles (Zhang et al., 2013). We identified 25 candidate alleles, and cloned andsequenced the duplication/Ac junctions (the green segment flankingthe Ac 5′ end in Figure1E and Figure 2A) from 16 of the 25TDD/TDDCI candidates via Ac casting (Singh et al., 2003; Wang andPeterson, 2013) or inverse PCR (iPCR) (See Zhang et al. (2013) for detailed screening and cloningmethods). To identify the TDDCI alleles, we designed PCR primers that flank theprogenitor insertion target sites for each allele (Figure 2A, primers 1 and 2). Primers 1 + Ac5 can amplify a productfrom both TDD and TDDCI while primers 2 + Ac3 can amplify a product only fromTDDCI since the latter contains an additional CI (Figure 2A). As expected, PCR using primers 1 + Ac5 produced bands ofthe expected sizes in all the 16 alleles (Figure2B, upper panel; seven examples are shown here). Whereas, primers 2 +Ac3 produced bands with expected sizes from only seven alleles (Figure 2B, lower panel). Sequencing of the PCR products obtainedfrom primers 1 + Ac5 and 2 + Ac3 revealed that these seven TDDCI candidateshave duplication/insertion breakpoints located from 13,392 bp to 1.7 Mb proximal tothe p1 locus on chromosome 1 (Table 1). Importantly, the Ac termini are flanked by 8-bptarget site duplications (TSDs; green/black triangles in Figure 1E) as predicted by the model in Figure 1 (See Supplementary file 1 for sequences containing TSDs).10.7554/eLife.03724.006Figure 2.PCR screening and DNA gel blotting of candidate TDDCI alleles.

Bottom Line: Every DNA segment in a eukaryotic genome normally replicates once and only once per cell cycle to maintain genome stability.The DNA re-replication can spontaneously abort to generate double-strand breaks, which can be repaired to generate Composite Insertions composed of transposon termini flanking segmental duplications of various lengths.These results show how alternative transposition coupled with DNA replication and repair can significantly alter genome structure and may have contributed to rapid genome evolution in maize and possibly other eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States.

ABSTRACT
Every DNA segment in a eukaryotic genome normally replicates once and only once per cell cycle to maintain genome stability. We show here that this restriction can be bypassed through alternative transposition, a transposition reaction that utilizes the termini of two separate, nearby transposable elements (TEs). Our results suggest that alternative transposition during S phase can induce re-replication of the TEs and their flanking sequences. The DNA re-replication can spontaneously abort to generate double-strand breaks, which can be repaired to generate Composite Insertions composed of transposon termini flanking segmental duplications of various lengths. These results show how alternative transposition coupled with DNA replication and repair can significantly alter genome structure and may have contributed to rapid genome evolution in maize and possibly other eukaryotes.

Show MeSH
Related in: MedlinePlus