title: Comparative whole genome sequencing reveals phenotypic tRNA gene duplication in spontaneous Schizosaccharomyces pombe La mutants.
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-null 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.
authors.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.
authors.forename: James R
authors.initials: JR
authors.lastname: Iben
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Epstein
authors.forename: Mark A
authors.initials: MA
authors.lastname: Bayfield
authors.forename: Monique W
authors.initials: MW
authors.lastname: Bruinsma
authors.forename: Samuel
authors.initials: S
authors.lastname: Hasson
authors.forename: Dagmar
authors.initials: D
authors.lastname: Bacikova
authors.forename: Daniel
authors.initials: D
authors.lastname: Ahmad
authors.forename: Denise
authors.initials: D
authors.lastname: Rockwell
authors.forename: Ellen L W
authors.initials: EL
authors.lastname: Kittler
authors.forename: Maria L
authors.initials: ML
authors.lastname: Zapp
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Maraia
mesh_terms: D000483:Alleles; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics; D004272:DNA, Mitochondrial / Q000737:chemistry; D020440:Gene Duplication; D005800:Genes, Fungal; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D012362:RNA, Transfer, Ser / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013489:Suppression, Genetic
chemical_list: D004272:DNA, Mitochondrial; D012362:RNA, Transfer, Ser; D016601:RNA-Binding Proteins; D029702:Schizosaccharomyces pombe Proteins; C513614:sla1 protein, S pombe