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Nucleosomes affect local transformation efficiency.

ABSTRACT: Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today's recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation efficiency. We investigated the landscape of transformation efficiency at various positions in the Saccharomyces cerevisiae genome and correlated these measurements with nucleosome positioning. We find that transformation efficiency shows a highly significant inverse correlation with relative nucleosome density. This correlation was lost when the nucleosome pattern, but not the underlying sequence was changed. Together, our results demonstrate a novel role for nucleosomes and also allow researchers to predict transformation efficiency of a target region and select spots in the genome that are likely to yield higher transformation efficiency.

SUBMITTER: Aslankoohi E

PROVIDER: S-EPMC3479212 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Nucleosomes affect local transformation efficiency.

Aslankoohi Elham E Voordeckers Karin K Sun Hong H Sanchez-Rodriguez Aminael A van der Zande Elisa E Marchal Kathleen K Verstrepen Kevin J KJ

Nucleic acids research 20120816 19

Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today's recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation effici ...[more]

PMID: 22904077

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Project description:A cornerstone of bacterial molecular biology is the ability to genetically manipulate the microbe under study. Many bacteria are difficult to manipulate genetically, a phenotype due in part to robust removal of newly acquired DNA, for example, by restriction-modification (R-M) systems. Here, we report approaches that dramatically improve bacterial transformation efficiency, piloted using a microbe that is challenging to transform due to expression of many R-M systems, Helicobacter pylori. Initially, we identified conditions that dampened expression of several R-M systems and concomitantly enhanced transformation efficiency. We then identified an approach that would broadly protect newly acquired DNA. We computationally predicted under-represented short DNA sequences in the H. pylori genome, with the idea that these sequences reflect targets of sequence-based surveillance such as R-M systems. We then used this information to modify and eliminate such sites in antibiotic resistance cassettes, creating a "stealth" version. Modifying antibiotic resistance cassettes in this way resulted in significantly higher transformation efficiency compared to non-modified cassettes, a response that was genomic loci independent. Our results suggest that avoiding R-M systems, via modification of under-represented DNA sequences or transformation conditions, is a powerful method to enhance DNA transformation. Our approach to identify under-represented sequences is applicable to any microbe with a sequenced genome.IMPORTANCEManipulating the genomes of bacteria is critical to many fields. Such manipulations are made by genetic engineering, which often requires new pieces of DNA to be added to the genome. Bacteria have robust systems for identifying and degrading new DNA, some of which rely on restriction enzymes. These enzymes cut DNA at specific sequences. We identified a set of DNA sequences that are missing normally from a bacterium's genome, more than would be expected by chance. Eliminating these sequences from a new piece of DNA allowed it to be incorporated into the bacterial genome at a higher frequency than new DNA containing the sequences. Removing such sequences appears to allow the new DNA to fly under the bacterial radar in "stealth" mode. This transformation improvement approach is straightforward to apply and likely broadly applicable.

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Nucleosomes affect local transformation efficiency.

Publications

Nucleosomes affect local transformation efficiency.

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