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Genomic subtraction recovers rye-specific DNA elements enriched in the rye genome.
Mol Biotechnol 2009; 42(2):160-7MB

Abstract

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3-1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.

Authors+Show Affiliations

Tottori University, Koyama-cho, Japan. tomita@muses.tottori-u.ac.jpNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19288229

Citation

Tomita, Motonori, et al. "Genomic Subtraction Recovers Rye-specific DNA Elements Enriched in the Rye Genome." Molecular Biotechnology, vol. 42, no. 2, 2009, pp. 160-7.
Tomita M, Akai K, Morimoto T. Genomic subtraction recovers rye-specific DNA elements enriched in the rye genome. Mol Biotechnol. 2009;42(2):160-7.
Tomita, M., Akai, K., & Morimoto, T. (2009). Genomic subtraction recovers rye-specific DNA elements enriched in the rye genome. Molecular Biotechnology, 42(2), pp. 160-7. doi:10.1007/s12033-009-9151-2.
Tomita M, Akai K, Morimoto T. Genomic Subtraction Recovers Rye-specific DNA Elements Enriched in the Rye Genome. Mol Biotechnol. 2009;42(2):160-7. PubMed PMID: 19288229.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Genomic subtraction recovers rye-specific DNA elements enriched in the rye genome. AU - Tomita,Motonori, AU - Akai,Keiichi, AU - Morimoto,Takayoshi, Y1 - 2009/03/14/ PY - 2008/10/23/received PY - 2009/01/22/accepted PY - 2009/3/17/entrez PY - 2009/3/17/pubmed PY - 2009/8/28/medline SP - 160 EP - 7 JF - Molecular biotechnology JO - Mol. Biotechnol. VL - 42 IS - 2 N2 - Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3-1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species. SN - 1073-6085 UR - https://www.unboundmedicine.com/medline/citation/19288229/Genomic_subtraction_recovers_rye_specific_DNA_elements_enriched_in_the_rye_genome_ L2 - https://dx.doi.org/10.1007/s12033-009-9151-2 DB - PRIME DP - Unbound Medicine ER -