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Development of a high-throughput genome-wide method to assess Ty1 retrotransposon insertion upstream of tRNA genes in Saccharomyces cerevisiae Pattanshetti, Rutuja
Abstract
Transposable elements are DNA elements found in nearly all organisms that can change their location and abundance within a genome. Retrotransposons, a sub-class of transposable elements, mobilize via RNA intermediates. In the genome of the widely studied reference strain S288c of Saccharomyces cerevisiae (S. cerevisiae), Ty1 is the most abundant retrotransposon, with ~38 copies per genome. Ty1 typically integrates within a 1 kilobase (kb) window upstream of genes that are transcribed by RNA Polymerase III, such as transfer RNA (tRNA) genes. Because the structure and lifecycle of retrotransposons resemble those of retroviruses, studying Ty1 in yeast allows a better understanding of related human retrotransposons and retroviruses. Our aim is to develop and validate a high-throughput, genome-wide method to assess Ty1 insertion upstream of tRNA genes in S. cerevisiae. In a proof-of-principle study, a wildtype variant of S288C was transformed with a Ty1-donor plasmid containing a galactose-inducible Ty1 element, that allowed temporal control over Ty1 integration. We located the Ty1 insertion sites using Polymerase chain reaction (PCR) primers that recognize a synthetic barcode sequence within the long terminal repeat (LTR) of the Ty1 element. Amplicons derived from these PCR reactions were then sequenced by Next Generation Sequencing to identify their insertion sites. We also performed micrococcal nuclease (MNase) sequencing on strains before and after galactose-induced Ty1 transposition. This allowed us to define the chromatin architecture and nucleosome profile upstream of 10 tRNA genes. We then applied this method to well-characterized conditional alleles of essential genes predicted to influence Ty1 insertion. Specifically, we mapped the Ty1 insertion patterns and frequencies upstream of each of the 10 tRNA loci and successfully PCR multiplexed all 10 loci to identify differences in Ty1 insertion profiles between wildtype and temperature-sensitive mutant strains that show defects in DNA replication, chromosome division, RNA Pol III machinery and chromatin remodelling. Our sequencing results confirmed that the barcoded Ty1 transposons inserted upstream of the targeted tRNA genes, within nucleosome-occupied regions. We further found that all categories of mutants showed defects in Ty1 insertion with respect to the number of insertion sites, number of tRNA genes and the insertion frequencies.
Item Metadata
Title |
Development of a high-throughput genome-wide method to assess Ty1 retrotransposon insertion upstream of tRNA genes in Saccharomyces cerevisiae
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Creator | |
Supervisor | |
Publisher |
University of British Columbia
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Date Issued |
2024
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Description |
Transposable elements are DNA elements found in nearly all organisms that can change their location and abundance within a genome. Retrotransposons, a sub-class of transposable elements, mobilize via RNA intermediates. In the genome of the widely studied reference strain S288c of Saccharomyces cerevisiae (S. cerevisiae), Ty1 is the most abundant retrotransposon, with ~38 copies per genome. Ty1 typically integrates within a 1 kilobase (kb) window upstream of genes that are transcribed by RNA Polymerase III, such as transfer RNA (tRNA) genes. Because the structure and lifecycle of retrotransposons resemble those of retroviruses, studying Ty1 in yeast allows a better understanding of related human retrotransposons and retroviruses. Our aim is to develop and validate a high-throughput, genome-wide method to assess Ty1 insertion upstream of tRNA genes in S. cerevisiae. In a proof-of-principle study, a wildtype variant of S288C was transformed with a Ty1-donor plasmid containing a galactose-inducible Ty1 element, that allowed temporal control over Ty1 integration. We located the Ty1 insertion sites using Polymerase chain reaction (PCR) primers that recognize a synthetic barcode sequence within the long terminal repeat (LTR) of the Ty1 element. Amplicons derived from these PCR reactions were then sequenced by Next Generation Sequencing to identify their insertion sites. We also performed micrococcal nuclease (MNase) sequencing on strains before and after galactose-induced Ty1 transposition. This allowed us to define the chromatin architecture and nucleosome profile upstream of 10 tRNA genes. We then applied this method to well-characterized conditional alleles of essential genes predicted to influence Ty1 insertion. Specifically, we mapped the Ty1 insertion patterns and frequencies upstream of each of the 10 tRNA loci and successfully PCR multiplexed all 10 loci to identify differences in Ty1 insertion profiles between wildtype and temperature-sensitive mutant strains that show defects in DNA replication, chromosome division, RNA Pol III machinery and chromatin remodelling. Our sequencing results confirmed that the barcoded Ty1 transposons inserted upstream of the targeted tRNA genes, within nucleosome-occupied regions. We further found that all categories of mutants showed defects in Ty1 insertion with respect to the number of insertion sites, number of tRNA genes and the insertion frequencies.
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Genre | |
Type | |
Language |
eng
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Date Available |
2024-12-10
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Provider |
Vancouver : University of British Columbia Library
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Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
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DOI |
10.14288/1.0447444
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2025-05
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Campus | |
Scholarly Level |
Graduate
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Rights URI | |
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DSpace
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Rights
Attribution-NonCommercial-NoDerivatives 4.0 International