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Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling
The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural...
Autores principales: | , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352883/ https://www.ncbi.nlm.nih.gov/pubmed/25654978 http://dx.doi.org/10.1101/gr.182477.114 |
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author | Suzuki, Yo Assad-Garcia, Nacyra Kostylev, Maxim Noskov, Vladimir N. Wise, Kim S. Karas, Bogumil J. Stam, Jason Montague, Michael G. Hanly, Timothy J. Enriquez, Nico J. Ramon, Adi Goldgof, Gregory M. Richter, R. Alexander Vashee, Sanjay Chuang, Ray-Yuan Winzeler, Elizabeth A. Hutchison, Clyde A. Gibson, Daniel G. Smith, Hamilton O. Glass, John I. Venter, J. Craig |
author_facet | Suzuki, Yo Assad-Garcia, Nacyra Kostylev, Maxim Noskov, Vladimir N. Wise, Kim S. Karas, Bogumil J. Stam, Jason Montague, Michael G. Hanly, Timothy J. Enriquez, Nico J. Ramon, Adi Goldgof, Gregory M. Richter, R. Alexander Vashee, Sanjay Chuang, Ray-Yuan Winzeler, Elizabeth A. Hutchison, Clyde A. Gibson, Daniel G. Smith, Hamilton O. Glass, John I. Venter, J. Craig |
author_sort | Suzuki, Yo |
collection | PubMed |
description | The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes. |
format | Online Article Text |
id | pubmed-4352883 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-43528832015-09-01 Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling Suzuki, Yo Assad-Garcia, Nacyra Kostylev, Maxim Noskov, Vladimir N. Wise, Kim S. Karas, Bogumil J. Stam, Jason Montague, Michael G. Hanly, Timothy J. Enriquez, Nico J. Ramon, Adi Goldgof, Gregory M. Richter, R. Alexander Vashee, Sanjay Chuang, Ray-Yuan Winzeler, Elizabeth A. Hutchison, Clyde A. Gibson, Daniel G. Smith, Hamilton O. Glass, John I. Venter, J. Craig Genome Res Method The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes. Cold Spring Harbor Laboratory Press 2015-03 /pmc/articles/PMC4352883/ /pubmed/25654978 http://dx.doi.org/10.1101/gr.182477.114 Text en © 2015 Suzuki et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Method Suzuki, Yo Assad-Garcia, Nacyra Kostylev, Maxim Noskov, Vladimir N. Wise, Kim S. Karas, Bogumil J. Stam, Jason Montague, Michael G. Hanly, Timothy J. Enriquez, Nico J. Ramon, Adi Goldgof, Gregory M. Richter, R. Alexander Vashee, Sanjay Chuang, Ray-Yuan Winzeler, Elizabeth A. Hutchison, Clyde A. Gibson, Daniel G. Smith, Hamilton O. Glass, John I. Venter, J. Craig Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling |
title | Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling |
title_full | Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling |
title_fullStr | Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling |
title_full_unstemmed | Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling |
title_short | Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling |
title_sort | bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352883/ https://www.ncbi.nlm.nih.gov/pubmed/25654978 http://dx.doi.org/10.1101/gr.182477.114 |
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