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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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Deliberate introduction of an additional chromosomal copy of the suppressor tRNASerUCA-C47:6U gene into yMWB3-15 is sufficient to produce the suppressed phenotype with increased tRNASerUCA-C47:6U levels. (A) Suppression phenotypes of parent and mutant strains as compared to parent transformed with either the integrated vector without a suppressor tRNASerUCA-C47:6U gene (Parent + empty vector, 3 out of 3 isolates shown), or the integrated vector with the suppressor tRNASerUCA-C47:6U gene insert (Parent + suppressor-tRNA, 5 out of 5 isolates shown) (see text). (B) Northern blot of RNA purified from various strains and probed for mature suppressor tRNASerUCA-C47:6U (upper panel). Parent, mutant and one each of the ‘parent + empty vector' and ‘parent + suppressor-tRNA’ strains from (A) are shown; yAS99 is a negative control strain that does not carry a suppressor tRNASerUCA gene. Lower panel shows the same blot probed for U5 snRNA used as a loading control for normalizing quantitation; the ratio of tRNA/U5 snRNA is indicated below the lanes. (C) Semiquantitative PCR of the suppressor tRNASerUCA-C47:6U gene and the sla1+ locus used here as a single copy gene control. After ensuring that amplification was in the quantitative linear range of the assay (data not shown), duplex PCR was performed under linear quantitative conditions. Band densities were quantitated and normalized to parent, and indicated under the lanes; numbers in parentheses reflect the range obtained from quadruplicate experiments.
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Figure 3: Deliberate introduction of an additional chromosomal copy of the suppressor tRNASerUCA-C47:6U gene into yMWB3-15 is sufficient to produce the suppressed phenotype with increased tRNASerUCA-C47:6U levels. (A) Suppression phenotypes of parent and mutant strains as compared to parent transformed with either the integrated vector without a suppressor tRNASerUCA-C47:6U gene (Parent + empty vector, 3 out of 3 isolates shown), or the integrated vector with the suppressor tRNASerUCA-C47:6U gene insert (Parent + suppressor-tRNA, 5 out of 5 isolates shown) (see text). (B) Northern blot of RNA purified from various strains and probed for mature suppressor tRNASerUCA-C47:6U (upper panel). Parent, mutant and one each of the ‘parent + empty vector' and ‘parent + suppressor-tRNA’ strains from (A) are shown; yAS99 is a negative control strain that does not carry a suppressor tRNASerUCA gene. Lower panel shows the same blot probed for U5 snRNA used as a loading control for normalizing quantitation; the ratio of tRNA/U5 snRNA is indicated below the lanes. (C) Semiquantitative PCR of the suppressor tRNASerUCA-C47:6U gene and the sla1+ locus used here as a single copy gene control. After ensuring that amplification was in the quantitative linear range of the assay (data not shown), duplex PCR was performed under linear quantitative conditions. Band densities were quantitated and normalized to parent, and indicated under the lanes; numbers in parentheses reflect the range obtained from quadruplicate experiments.

Mentions: Since two analytical approaches suggested duplication of the suppressor tRNASerUCA-C47:6U gene, we tested if introduction of an additional copy of this gene would result in the suppression phenotype. The parent strain was transformed by chromosomal integration of linearized kanR-containing pFA6A vector harboring either a suppressor tRNASerUCA-C47:6U insert of ∼300 bp, or no insert. Transformed cells were plated on YES, then replica plated onto YES + G418. G418-resistant colonies were randomly picked from both the no-insert and the suppressor tRNASerUCA-C47:6U-insert transformants, streak purified and transferred to limiting adenine for comparison to each other, parent and mutant, for TMS phenotype. None of the three analyzed transformants derived from the empty vector showed suppression (Figure 3A, row 3). By contrast, all five analyzed transformants derived from the suppressor tRNASerUCA-C47:6U gene clearly demonstrated suppression greater than in the parent strain yMWB3-15 (Figure 3A, compare rows 1 and 4) and to a similar degree as in the spontaneous mutant yDA317 (Figure 3A, row 2). This demonstrated that introduction of a suppressor tRNASerUCA-C47:6U gene into our parent strain results in a phenocopy of the mutant phenotype. Moreover, although yDA317 contained an extra tRNASerUCA-C47:6U gene as well as associated pJK148 vector DNA, the present result isolated the phenotypic segment to the extra tRNASerUCA-C47:6U gene. It also showed that the synonymous erg3-SNP in yDA317 was not required for the suppression phenotype. The data argue that the suppressor tRNASerUCA-C47:6U gene duplication detected by whole genome sequencing is responsible for the suppression phenotype in the yDA317 mutant.Figure 3.


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)

Deliberate introduction of an additional chromosomal copy of the suppressor tRNASerUCA-C47:6U gene into yMWB3-15 is sufficient to produce the suppressed phenotype with increased tRNASerUCA-C47:6U levels. (A) Suppression phenotypes of parent and mutant strains as compared to parent transformed with either the integrated vector without a suppressor tRNASerUCA-C47:6U gene (Parent + empty vector, 3 out of 3 isolates shown), or the integrated vector with the suppressor tRNASerUCA-C47:6U gene insert (Parent + suppressor-tRNA, 5 out of 5 isolates shown) (see text). (B) Northern blot of RNA purified from various strains and probed for mature suppressor tRNASerUCA-C47:6U (upper panel). Parent, mutant and one each of the ‘parent + empty vector' and ‘parent + suppressor-tRNA’ strains from (A) are shown; yAS99 is a negative control strain that does not carry a suppressor tRNASerUCA gene. Lower panel shows the same blot probed for U5 snRNA used as a loading control for normalizing quantitation; the ratio of tRNA/U5 snRNA is indicated below the lanes. (C) Semiquantitative PCR of the suppressor tRNASerUCA-C47:6U gene and the sla1+ locus used here as a single copy gene control. After ensuring that amplification was in the quantitative linear range of the assay (data not shown), duplex PCR was performed under linear quantitative conditions. Band densities were quantitated and normalized to parent, and indicated under the lanes; numbers in parentheses reflect the range obtained from quadruplicate experiments.
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Related In: Results  -  Collection

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Figure 3: Deliberate introduction of an additional chromosomal copy of the suppressor tRNASerUCA-C47:6U gene into yMWB3-15 is sufficient to produce the suppressed phenotype with increased tRNASerUCA-C47:6U levels. (A) Suppression phenotypes of parent and mutant strains as compared to parent transformed with either the integrated vector without a suppressor tRNASerUCA-C47:6U gene (Parent + empty vector, 3 out of 3 isolates shown), or the integrated vector with the suppressor tRNASerUCA-C47:6U gene insert (Parent + suppressor-tRNA, 5 out of 5 isolates shown) (see text). (B) Northern blot of RNA purified from various strains and probed for mature suppressor tRNASerUCA-C47:6U (upper panel). Parent, mutant and one each of the ‘parent + empty vector' and ‘parent + suppressor-tRNA’ strains from (A) are shown; yAS99 is a negative control strain that does not carry a suppressor tRNASerUCA gene. Lower panel shows the same blot probed for U5 snRNA used as a loading control for normalizing quantitation; the ratio of tRNA/U5 snRNA is indicated below the lanes. (C) Semiquantitative PCR of the suppressor tRNASerUCA-C47:6U gene and the sla1+ locus used here as a single copy gene control. After ensuring that amplification was in the quantitative linear range of the assay (data not shown), duplex PCR was performed under linear quantitative conditions. Band densities were quantitated and normalized to parent, and indicated under the lanes; numbers in parentheses reflect the range obtained from quadruplicate experiments.
Mentions: Since two analytical approaches suggested duplication of the suppressor tRNASerUCA-C47:6U gene, we tested if introduction of an additional copy of this gene would result in the suppression phenotype. The parent strain was transformed by chromosomal integration of linearized kanR-containing pFA6A vector harboring either a suppressor tRNASerUCA-C47:6U insert of ∼300 bp, or no insert. Transformed cells were plated on YES, then replica plated onto YES + G418. G418-resistant colonies were randomly picked from both the no-insert and the suppressor tRNASerUCA-C47:6U-insert transformants, streak purified and transferred to limiting adenine for comparison to each other, parent and mutant, for TMS phenotype. None of the three analyzed transformants derived from the empty vector showed suppression (Figure 3A, row 3). By contrast, all five analyzed transformants derived from the suppressor tRNASerUCA-C47:6U gene clearly demonstrated suppression greater than in the parent strain yMWB3-15 (Figure 3A, compare rows 1 and 4) and to a similar degree as in the spontaneous mutant yDA317 (Figure 3A, row 2). This demonstrated that introduction of a suppressor tRNASerUCA-C47:6U gene into our parent strain results in a phenocopy of the mutant phenotype. Moreover, although yDA317 contained an extra tRNASerUCA-C47:6U gene as well as associated pJK148 vector DNA, the present result isolated the phenotypic segment to the extra tRNASerUCA-C47:6U gene. It also showed that the synonymous erg3-SNP in yDA317 was not required for the suppression phenotype. The data argue that the suppressor tRNASerUCA-C47:6U gene duplication detected by whole genome sequencing is responsible for the suppression phenotype in the yDA317 mutant.Figure 3.

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