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Bacteriophage Mu integration in yeast and mammalian genomes.

Paatero AO, Turakainen H, Happonen LJ, Olsson C, Palomäki T, Pajunen MI, Meng X, Otonkoski T, Tuuri T, Berry C, Malani N, Frilander MJ, Bushman FD, Savilahti H - Nucleic Acids Res. (2008)

Bottom Line: In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption.In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers.These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Viikki Biocenter, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

ABSTRACT
Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed 'transpososomes'. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.

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Analysis of integration site distributions in murine cells. The experimentally determined Mu integration sites reported here were compared to previously reported integration sites for MLV and HIV in the murine genome. In each figure, the proportion of Mu integration sites in each category is divided by the proportion in matched random controls—a bar below the line at 1.0 indicates disfavored Mu integration compared to random, while a bar above the line indicates favored integration. (A) Mu integration frequency in transcription units (defined as RefGenes). Comparison of Mu to random achieves P = 5.9e-4. (B) Mu integration frequency within 5 kb of the center of a CpG island. Comparison of Mu to random achieves P = 4.22e-7. (C) Analysis of Mu integration frequency in gene-dense regions. The murine genome was partitioned into 10 bins of increasing gene density (analyzed over four megabase regions), then the proportion of integration quantified in each bin and divided by random. Comparison of Mu to random achieved P = 3.58e-10. (D) Analysis of Mu integration as a function of transcriptional intensity. Affymetrix microarray data for murine ES cells was used to quantify transcriptional intensity. Transcriptional intensity was measured exactly as for gene density described above, but only genes in more highly expressed upper half of all genes queried on the microarray were scored. Comparison of Mu to random achieved P = 6.43e-8. (E) Mu integration frequency as a function of G/C content. The G/C content was measured over 5-kb intervals. Comparison of Mu to random achieves P = 5.13e-14.
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Figure 6: Analysis of integration site distributions in murine cells. The experimentally determined Mu integration sites reported here were compared to previously reported integration sites for MLV and HIV in the murine genome. In each figure, the proportion of Mu integration sites in each category is divided by the proportion in matched random controls—a bar below the line at 1.0 indicates disfavored Mu integration compared to random, while a bar above the line indicates favored integration. (A) Mu integration frequency in transcription units (defined as RefGenes). Comparison of Mu to random achieves P = 5.9e-4. (B) Mu integration frequency within 5 kb of the center of a CpG island. Comparison of Mu to random achieves P = 4.22e-7. (C) Analysis of Mu integration frequency in gene-dense regions. The murine genome was partitioned into 10 bins of increasing gene density (analyzed over four megabase regions), then the proportion of integration quantified in each bin and divided by random. Comparison of Mu to random achieved P = 3.58e-10. (D) Analysis of Mu integration as a function of transcriptional intensity. Affymetrix microarray data for murine ES cells was used to quantify transcriptional intensity. Transcriptional intensity was measured exactly as for gene density described above, but only genes in more highly expressed upper half of all genes queried on the microarray were scored. Comparison of Mu to random achieved P = 6.43e-8. (E) Mu integration frequency as a function of G/C content. The G/C content was measured over 5-kb intervals. Comparison of Mu to random achieves P = 5.13e-14.

Mentions: A collection of 214 Mu integration sites in mice was available for statistical analyses (Table 5). This collection was compared to a randomly generated group of control integration sites (Supplementary Text 1). All chromosomes except Y hosted at least four Mu integration events (Supplementary Figure 7). Integration in the mouse genome has also been extensively characterized for murine leukaemia (MLV) and human immunodeficiency virus (HIV), so data sets for these retroviruses (Table 5) were included for comparison in the analysis (Figure 6).Figure 6.


Bacteriophage Mu integration in yeast and mammalian genomes.

Paatero AO, Turakainen H, Happonen LJ, Olsson C, Palomäki T, Pajunen MI, Meng X, Otonkoski T, Tuuri T, Berry C, Malani N, Frilander MJ, Bushman FD, Savilahti H - Nucleic Acids Res. (2008)

Analysis of integration site distributions in murine cells. The experimentally determined Mu integration sites reported here were compared to previously reported integration sites for MLV and HIV in the murine genome. In each figure, the proportion of Mu integration sites in each category is divided by the proportion in matched random controls—a bar below the line at 1.0 indicates disfavored Mu integration compared to random, while a bar above the line indicates favored integration. (A) Mu integration frequency in transcription units (defined as RefGenes). Comparison of Mu to random achieves P = 5.9e-4. (B) Mu integration frequency within 5 kb of the center of a CpG island. Comparison of Mu to random achieves P = 4.22e-7. (C) Analysis of Mu integration frequency in gene-dense regions. The murine genome was partitioned into 10 bins of increasing gene density (analyzed over four megabase regions), then the proportion of integration quantified in each bin and divided by random. Comparison of Mu to random achieved P = 3.58e-10. (D) Analysis of Mu integration as a function of transcriptional intensity. Affymetrix microarray data for murine ES cells was used to quantify transcriptional intensity. Transcriptional intensity was measured exactly as for gene density described above, but only genes in more highly expressed upper half of all genes queried on the microarray were scored. Comparison of Mu to random achieved P = 6.43e-8. (E) Mu integration frequency as a function of G/C content. The G/C content was measured over 5-kb intervals. Comparison of Mu to random achieves P = 5.13e-14.
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Figure 6: Analysis of integration site distributions in murine cells. The experimentally determined Mu integration sites reported here were compared to previously reported integration sites for MLV and HIV in the murine genome. In each figure, the proportion of Mu integration sites in each category is divided by the proportion in matched random controls—a bar below the line at 1.0 indicates disfavored Mu integration compared to random, while a bar above the line indicates favored integration. (A) Mu integration frequency in transcription units (defined as RefGenes). Comparison of Mu to random achieves P = 5.9e-4. (B) Mu integration frequency within 5 kb of the center of a CpG island. Comparison of Mu to random achieves P = 4.22e-7. (C) Analysis of Mu integration frequency in gene-dense regions. The murine genome was partitioned into 10 bins of increasing gene density (analyzed over four megabase regions), then the proportion of integration quantified in each bin and divided by random. Comparison of Mu to random achieved P = 3.58e-10. (D) Analysis of Mu integration as a function of transcriptional intensity. Affymetrix microarray data for murine ES cells was used to quantify transcriptional intensity. Transcriptional intensity was measured exactly as for gene density described above, but only genes in more highly expressed upper half of all genes queried on the microarray were scored. Comparison of Mu to random achieved P = 6.43e-8. (E) Mu integration frequency as a function of G/C content. The G/C content was measured over 5-kb intervals. Comparison of Mu to random achieves P = 5.13e-14.
Mentions: A collection of 214 Mu integration sites in mice was available for statistical analyses (Table 5). This collection was compared to a randomly generated group of control integration sites (Supplementary Text 1). All chromosomes except Y hosted at least four Mu integration events (Supplementary Figure 7). Integration in the mouse genome has also been extensively characterized for murine leukaemia (MLV) and human immunodeficiency virus (HIV), so data sets for these retroviruses (Table 5) were included for comparison in the analysis (Figure 6).Figure 6.

Bottom Line: In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption.In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers.These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Viikki Biocenter, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

ABSTRACT
Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed 'transpososomes'. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.

Show MeSH
Related in: MedlinePlus