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Comparative whole genome sequencing reveals phenotypic tRNA gene duplication in spontaneous Schizosaccharomyces pombe La mutants.

Iben JR, Epstein JA, Bayfield MA, Bruinsma MW, Hasson S, Bacikova D, Ahmad D, Rockwell D, Kittler EL, Zapp ML, Maraia RJ - Nucleic Acids Res. (2011)

Bottom Line: Further sequence analyses found a duplication of the tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype.By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a 'reference' mtDNA, providing the first identification of these S. pombe mtDNA discrepancies.Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.

View Article: PubMed Central - PubMed

Affiliation: Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, University of Massachusetts Medical School, Worcester, MA, USA.

ABSTRACT
We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNA(Ser)UCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNA(Ser)UCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNA(Ser)UCA-C47:6U levels in sla1-rrm but not sla1- cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a 'reference' mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.

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A simple statistical distribution of annotated gene copy number as estimated from mapped read depth. A Z-score of zero represents a 1:1 ratio of wild type, (WT, yMWB3-15) and mutant (Mut, yDA317) read depth at an annotated genome feature. A negative Z-score reflects greater read depth or copy number in the mutant strain, and vice versa. The downward arrow indicates the suppressor tRNASerUCA-C47:6U gene, representing a 2-fold greater copy number in the mutant strain.
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Figure 2: A simple statistical distribution of annotated gene copy number as estimated from mapped read depth. A Z-score of zero represents a 1:1 ratio of wild type, (WT, yMWB3-15) and mutant (Mut, yDA317) read depth at an annotated genome feature. A negative Z-score reflects greater read depth or copy number in the mutant strain, and vice versa. The downward arrow indicates the suppressor tRNASerUCA-C47:6U gene, representing a 2-fold greater copy number in the mutant strain.

Mentions: The next approach was to determine if a change in copy number of a gene or region had occurred, and, for this, two strategies were employed. At the positions of known and predicted genes, the mapped read depth was averaged across the entire length of the annotated feature, as an annotation context-dependent approach. These averages led to ratios of mapped read depth for the parent and mutant at each known transcription/annotation unit. These ratios were corrected slightly to reflect the difference in average read depth for the entire genomes of the strains, 40 for the parent and 45 for the mutant. When the ratios were plotted in a simple statistical distribution, the copy number varied along a standard bell curve at a mean of a 1-to-1 ratio (Figure 2). One significant outlier, however, was in the number of reads mapping to the suppressor tRNASerUCA-C47:6U sequence, suggesting gene duplication. The position on the bell curve of this copy number variation was more than 4 SD away from the median, toward increased copy number in the mutant strain (Figure 2).


Comparative whole genome sequencing reveals phenotypic tRNA gene duplication in spontaneous Schizosaccharomyces pombe La mutants.

Iben JR, Epstein JA, Bayfield MA, Bruinsma MW, Hasson S, Bacikova D, Ahmad D, Rockwell D, Kittler EL, Zapp ML, Maraia RJ - Nucleic Acids Res. (2011)

A simple statistical distribution of annotated gene copy number as estimated from mapped read depth. A Z-score of zero represents a 1:1 ratio of wild type, (WT, yMWB3-15) and mutant (Mut, yDA317) read depth at an annotated genome feature. A negative Z-score reflects greater read depth or copy number in the mutant strain, and vice versa. The downward arrow indicates the suppressor tRNASerUCA-C47:6U gene, representing a 2-fold greater copy number in the mutant strain.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113579&req=5

Figure 2: A simple statistical distribution of annotated gene copy number as estimated from mapped read depth. A Z-score of zero represents a 1:1 ratio of wild type, (WT, yMWB3-15) and mutant (Mut, yDA317) read depth at an annotated genome feature. A negative Z-score reflects greater read depth or copy number in the mutant strain, and vice versa. The downward arrow indicates the suppressor tRNASerUCA-C47:6U gene, representing a 2-fold greater copy number in the mutant strain.
Mentions: The next approach was to determine if a change in copy number of a gene or region had occurred, and, for this, two strategies were employed. At the positions of known and predicted genes, the mapped read depth was averaged across the entire length of the annotated feature, as an annotation context-dependent approach. These averages led to ratios of mapped read depth for the parent and mutant at each known transcription/annotation unit. These ratios were corrected slightly to reflect the difference in average read depth for the entire genomes of the strains, 40 for the parent and 45 for the mutant. When the ratios were plotted in a simple statistical distribution, the copy number varied along a standard bell curve at a mean of a 1-to-1 ratio (Figure 2). One significant outlier, however, was in the number of reads mapping to the suppressor tRNASerUCA-C47:6U sequence, suggesting gene duplication. The position on the bell curve of this copy number variation was more than 4 SD away from the median, toward increased copy number in the mutant strain (Figure 2).

Bottom Line: Further sequence analyses found a duplication of the tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype.By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a 'reference' mtDNA, providing the first identification of these S. pombe mtDNA discrepancies.Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.

View Article: PubMed Central - PubMed

Affiliation: Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, University of Massachusetts Medical School, Worcester, MA, USA.

ABSTRACT
We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNA(Ser)UCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNA(Ser)UCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNA(Ser)UCA-C47:6U levels in sla1-rrm but not sla1- cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a 'reference' mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.

Show MeSH
Related in: MedlinePlus