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The ben1-1 brassinosteroid-catabolism mutation is unstable due to epigenetic modifications of the intronic T-DNA insertion.

Sandhu KS, Koirala PS, Neff MM - G3 (Bethesda) (2013)

Bottom Line: The previously functional NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) T-DNA marker gene (which encodes kanamycin resistance) was no longer functional in the recovered ben1-1 allele, though the length of the T-DNA insertion and the NPTII gene sequence did not change in the pretriple and posttriple ben1-1 mutants.Methylation analysis using both restriction endonuclease activity and bisulfite conversion followed by sequencing showed that the methylation status of the T-DNA is different between the original and the recovered ben1-1.These observations demonstrate that the recovered ben1-1 mutant is epigenetically different from the original ben1-1 allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop and Soil Sciences, Washington State University, Pullman, Washington 99164.

ABSTRACT
Loss-of-function genetic analysis plays a pivotal role in elucidating individual gene function as well as interactions among gene networks. The ease of gene tagging and cloning provided by transfer DNA (T-DNA) insertion mutants have led to their heavy use by the Arabidopsis research community. However, certain aspects of T-DNA alleles require caution, as highlighted in this study of an intronic insertion mutant (ben1-1) in the BEN1 (BRI1-5 ENHANCED 1) gene. As a part of our analysis of brassinosteroid catabolic enzymes, we generated a genetic triple-mutant from a cross between the bas1-2 sob7-1 double- (T-DNA exonic insertion mutants of phyB-4 ACTIVATION TAGGED SUPPRESSOR 1 and SUPPRESSOR OF phyB-4 7) and ben1-1. As previously described, the single ben1-1 line behaves as a transcript . However, in the triple-mutant background ben1-1 was reverted to a partial loss-of-function allele showing enhanced levels of the wild-type-spliced transcript. Interestingly, the enhanced expression of BEN1 remained stable when the ben1-1 single-mutant was reisolated from a cross with the wild type. In addition, the two genetically identical pretriple and posttriple ben1-1 mutants also differed phenotypically. The previously functional NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) T-DNA marker gene (which encodes kanamycin resistance) was no longer functional in the recovered ben1-1 allele, though the length of the T-DNA insertion and the NPTII gene sequence did not change in the pretriple and posttriple ben1-1 mutants. Methylation analysis using both restriction endonuclease activity and bisulfite conversion followed by sequencing showed that the methylation status of the T-DNA is different between the original and the recovered ben1-1. These observations demonstrate that the recovered ben1-1 mutant is epigenetically different from the original ben1-1 allele.

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The size and sequence of the T-DNA insertion remains unchanged in the pretriple and posttriple ben1-1 lines. (A) Graphic depiction of the location of the second intron flanking primer pair used for amplification in the ben1-1 allele. (B) The genomic PCR using the second intron flanking primers amplify equal size band from the original ben1-1 and the recovered ben1-1 line. (C) The original and the recovered ben1-1 alleles show identical restriction digestion pattern. The PCR amplification product from gel image (B) was gel purified and restriction digested with HindIII and KpnI.
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fig7: The size and sequence of the T-DNA insertion remains unchanged in the pretriple and posttriple ben1-1 lines. (A) Graphic depiction of the location of the second intron flanking primer pair used for amplification in the ben1-1 allele. (B) The genomic PCR using the second intron flanking primers amplify equal size band from the original ben1-1 and the recovered ben1-1 line. (C) The original and the recovered ben1-1 alleles show identical restriction digestion pattern. The PCR amplification product from gel image (B) was gel purified and restriction digested with HindIII and KpnI.

Mentions: In another scenario, a shortening of T-DNA insertion due to unequal recombination during the creation of the triple-mutant could also result in enhanced BEN1 transcript levels compared with the original ben1-1 line. To test this hypothesis, the inserted T-DNA was amplified from the original and the reisolated ben1-1 lines using primers anchored in the second and third exons of the BEN1 gene (Figure 7A). The PCR results show that, using wild-type DNA as a PCR template gave a band of the size expected in the case of no T-DNA insertion (Figure 7B). On the other hand, both the original and reisolated ben1-1 lines showed a large band of the same size, indicating the presence of approximately two T-DNA insertions in each case (Figure 7B). To further test the possibility of any re-arrangement of the T-DNA in original vs. the reisolated ben1-1, the large T-DNA containing amplification product of the extended PCR (Figure 7B) was purified and cut using two different REs. Identical restriction pattern of the two amplification products showed the absence of any T-DNA rearrangement between the original and the reisolated T-DNA insertions (Figure 7C). These experiments suggest that the T-DNA size and structure have not changed between the original and the reisolated ben1-1 lines.


The ben1-1 brassinosteroid-catabolism mutation is unstable due to epigenetic modifications of the intronic T-DNA insertion.

Sandhu KS, Koirala PS, Neff MM - G3 (Bethesda) (2013)

The size and sequence of the T-DNA insertion remains unchanged in the pretriple and posttriple ben1-1 lines. (A) Graphic depiction of the location of the second intron flanking primer pair used for amplification in the ben1-1 allele. (B) The genomic PCR using the second intron flanking primers amplify equal size band from the original ben1-1 and the recovered ben1-1 line. (C) The original and the recovered ben1-1 alleles show identical restriction digestion pattern. The PCR amplification product from gel image (B) was gel purified and restriction digested with HindIII and KpnI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3755919&req=5

fig7: The size and sequence of the T-DNA insertion remains unchanged in the pretriple and posttriple ben1-1 lines. (A) Graphic depiction of the location of the second intron flanking primer pair used for amplification in the ben1-1 allele. (B) The genomic PCR using the second intron flanking primers amplify equal size band from the original ben1-1 and the recovered ben1-1 line. (C) The original and the recovered ben1-1 alleles show identical restriction digestion pattern. The PCR amplification product from gel image (B) was gel purified and restriction digested with HindIII and KpnI.
Mentions: In another scenario, a shortening of T-DNA insertion due to unequal recombination during the creation of the triple-mutant could also result in enhanced BEN1 transcript levels compared with the original ben1-1 line. To test this hypothesis, the inserted T-DNA was amplified from the original and the reisolated ben1-1 lines using primers anchored in the second and third exons of the BEN1 gene (Figure 7A). The PCR results show that, using wild-type DNA as a PCR template gave a band of the size expected in the case of no T-DNA insertion (Figure 7B). On the other hand, both the original and reisolated ben1-1 lines showed a large band of the same size, indicating the presence of approximately two T-DNA insertions in each case (Figure 7B). To further test the possibility of any re-arrangement of the T-DNA in original vs. the reisolated ben1-1, the large T-DNA containing amplification product of the extended PCR (Figure 7B) was purified and cut using two different REs. Identical restriction pattern of the two amplification products showed the absence of any T-DNA rearrangement between the original and the reisolated T-DNA insertions (Figure 7C). These experiments suggest that the T-DNA size and structure have not changed between the original and the reisolated ben1-1 lines.

Bottom Line: The previously functional NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) T-DNA marker gene (which encodes kanamycin resistance) was no longer functional in the recovered ben1-1 allele, though the length of the T-DNA insertion and the NPTII gene sequence did not change in the pretriple and posttriple ben1-1 mutants.Methylation analysis using both restriction endonuclease activity and bisulfite conversion followed by sequencing showed that the methylation status of the T-DNA is different between the original and the recovered ben1-1.These observations demonstrate that the recovered ben1-1 mutant is epigenetically different from the original ben1-1 allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop and Soil Sciences, Washington State University, Pullman, Washington 99164.

ABSTRACT
Loss-of-function genetic analysis plays a pivotal role in elucidating individual gene function as well as interactions among gene networks. The ease of gene tagging and cloning provided by transfer DNA (T-DNA) insertion mutants have led to their heavy use by the Arabidopsis research community. However, certain aspects of T-DNA alleles require caution, as highlighted in this study of an intronic insertion mutant (ben1-1) in the BEN1 (BRI1-5 ENHANCED 1) gene. As a part of our analysis of brassinosteroid catabolic enzymes, we generated a genetic triple-mutant from a cross between the bas1-2 sob7-1 double- (T-DNA exonic insertion mutants of phyB-4 ACTIVATION TAGGED SUPPRESSOR 1 and SUPPRESSOR OF phyB-4 7) and ben1-1. As previously described, the single ben1-1 line behaves as a transcript . However, in the triple-mutant background ben1-1 was reverted to a partial loss-of-function allele showing enhanced levels of the wild-type-spliced transcript. Interestingly, the enhanced expression of BEN1 remained stable when the ben1-1 single-mutant was reisolated from a cross with the wild type. In addition, the two genetically identical pretriple and posttriple ben1-1 mutants also differed phenotypically. The previously functional NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) T-DNA marker gene (which encodes kanamycin resistance) was no longer functional in the recovered ben1-1 allele, though the length of the T-DNA insertion and the NPTII gene sequence did not change in the pretriple and posttriple ben1-1 mutants. Methylation analysis using both restriction endonuclease activity and bisulfite conversion followed by sequencing showed that the methylation status of the T-DNA is different between the original and the recovered ben1-1. These observations demonstrate that the recovered ben1-1 mutant is epigenetically different from the original ben1-1 allele.

Show MeSH
Related in: MedlinePlus