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Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network.

Kaboli S, Yamakawa T, Sunada K, Takagaki T, Sasano Y, Sugiyama M, Kaneko Y, Harashima S - Nucleic Acids Res. (2014)

Bottom Line: Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis.This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome.Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan.

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Construction of mini-chromosome in chromosomes III, IV, X, XV and XVI. (A) Illustration of the target chromosome, target region, length of artificial chromosomes and hybridization probe location. Mini-chromosomes were constructed by splitting chromosomes at the left- and right-hand side of each region using PCS technology. Each mini-chromosome harbored the CgURA3 marker (dark green box) and artificial CEN4 (yellow circle). The length of each mini-chromosome and other fragments is indicated. Red boxes represent probes corresponding to an internal sequence of the mini-chromosome. Blue box and black circle represent the CgHIS3 marker and natural CEN, respectively. (B) PFGE and Southern analysis showing the karyotype analysis of the parental haploid (P), strain after first splitting (designated 1st), and strain after second splitting (designated 2nd) harboring mini-chromosomes of regions Chr 3-4, Chr 4-5, Chr 10-7, Chr 15-2, Chr 15-4 and Chr 16-7. For all chromosomes, probes were prepared by PCR amplification of a 400-bp internal sequence of the constructed mini-chromosome.
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Figure 3: Construction of mini-chromosome in chromosomes III, IV, X, XV and XVI. (A) Illustration of the target chromosome, target region, length of artificial chromosomes and hybridization probe location. Mini-chromosomes were constructed by splitting chromosomes at the left- and right-hand side of each region using PCS technology. Each mini-chromosome harbored the CgURA3 marker (dark green box) and artificial CEN4 (yellow circle). The length of each mini-chromosome and other fragments is indicated. Red boxes represent probes corresponding to an internal sequence of the mini-chromosome. Blue box and black circle represent the CgHIS3 marker and natural CEN, respectively. (B) PFGE and Southern analysis showing the karyotype analysis of the parental haploid (P), strain after first splitting (designated 1st), and strain after second splitting (designated 2nd) harboring mini-chromosomes of regions Chr 3-4, Chr 4-5, Chr 10-7, Chr 15-2, Chr 15-4 and Chr 16-7. For all chromosomes, probes were prepared by PCR amplification of a 400-bp internal sequence of the constructed mini-chromosome.

Mentions: As described above, screening of the 110 regions by PCD identified 77 regions (red boxes in Figure 1) as undeletable (Table 3). To confirm whether these undeletable regions are in fact essential, we used the PCS method (13). PCS is a technology that can split a chromosome at any desired site. We converted 67 of the 77 potentially undeletable regions to mini-chromosomes marked with a URA3 gene by using PCS to split the chromosome at the left- and right-hand side of each region. Briefly, two modules consisting of the 400-bp upstream and downstream sequence of the target point, a CEN4/URA3 selective marker and six copies of the 5′-CCCCAA-3′ repeat sequence were prepared by PCR and introduced into cells by transformation. The target point then is thought to undergo homologous recombination at the right-hand end of the region to be deleted, resulting in splitting of the natural chromosome into two smaller chromosomes. Subsequently, a second splitting is conducted in a similar way at the left-hand end of the region. If splitting occurs at the correct site, a URA3-marked mini-chromosome of each region is created (Figure 3A). Figure 3B shows representative data from PFGE and Southern analysis demonstrating the expected generation of a mini-chromosome in strains generated to harbor a mini-chromosome of the 43-kb internal region of chromosome III, 30-kb internal region of chromosome IV, 39-kb internal region of chromosome X, 22-kb internal region of chromosome XV, 44-kb internal region of chromosome XV and 38-kb internal region of chromosome XVI.


Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network.

Kaboli S, Yamakawa T, Sunada K, Takagaki T, Sasano Y, Sugiyama M, Kaneko Y, Harashima S - Nucleic Acids Res. (2014)

Construction of mini-chromosome in chromosomes III, IV, X, XV and XVI. (A) Illustration of the target chromosome, target region, length of artificial chromosomes and hybridization probe location. Mini-chromosomes were constructed by splitting chromosomes at the left- and right-hand side of each region using PCS technology. Each mini-chromosome harbored the CgURA3 marker (dark green box) and artificial CEN4 (yellow circle). The length of each mini-chromosome and other fragments is indicated. Red boxes represent probes corresponding to an internal sequence of the mini-chromosome. Blue box and black circle represent the CgHIS3 marker and natural CEN, respectively. (B) PFGE and Southern analysis showing the karyotype analysis of the parental haploid (P), strain after first splitting (designated 1st), and strain after second splitting (designated 2nd) harboring mini-chromosomes of regions Chr 3-4, Chr 4-5, Chr 10-7, Chr 15-2, Chr 15-4 and Chr 16-7. For all chromosomes, probes were prepared by PCR amplification of a 400-bp internal sequence of the constructed mini-chromosome.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150759&req=5

Figure 3: Construction of mini-chromosome in chromosomes III, IV, X, XV and XVI. (A) Illustration of the target chromosome, target region, length of artificial chromosomes and hybridization probe location. Mini-chromosomes were constructed by splitting chromosomes at the left- and right-hand side of each region using PCS technology. Each mini-chromosome harbored the CgURA3 marker (dark green box) and artificial CEN4 (yellow circle). The length of each mini-chromosome and other fragments is indicated. Red boxes represent probes corresponding to an internal sequence of the mini-chromosome. Blue box and black circle represent the CgHIS3 marker and natural CEN, respectively. (B) PFGE and Southern analysis showing the karyotype analysis of the parental haploid (P), strain after first splitting (designated 1st), and strain after second splitting (designated 2nd) harboring mini-chromosomes of regions Chr 3-4, Chr 4-5, Chr 10-7, Chr 15-2, Chr 15-4 and Chr 16-7. For all chromosomes, probes were prepared by PCR amplification of a 400-bp internal sequence of the constructed mini-chromosome.
Mentions: As described above, screening of the 110 regions by PCD identified 77 regions (red boxes in Figure 1) as undeletable (Table 3). To confirm whether these undeletable regions are in fact essential, we used the PCS method (13). PCS is a technology that can split a chromosome at any desired site. We converted 67 of the 77 potentially undeletable regions to mini-chromosomes marked with a URA3 gene by using PCS to split the chromosome at the left- and right-hand side of each region. Briefly, two modules consisting of the 400-bp upstream and downstream sequence of the target point, a CEN4/URA3 selective marker and six copies of the 5′-CCCCAA-3′ repeat sequence were prepared by PCR and introduced into cells by transformation. The target point then is thought to undergo homologous recombination at the right-hand end of the region to be deleted, resulting in splitting of the natural chromosome into two smaller chromosomes. Subsequently, a second splitting is conducted in a similar way at the left-hand end of the region. If splitting occurs at the correct site, a URA3-marked mini-chromosome of each region is created (Figure 3A). Figure 3B shows representative data from PFGE and Southern analysis demonstrating the expected generation of a mini-chromosome in strains generated to harbor a mini-chromosome of the 43-kb internal region of chromosome III, 30-kb internal region of chromosome IV, 39-kb internal region of chromosome X, 22-kb internal region of chromosome XV, 44-kb internal region of chromosome XV and 38-kb internal region of chromosome XVI.

Bottom Line: Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis.This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome.Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan.

Show MeSH
Related in: MedlinePlus