Systematic discovery of new genes in the Saccharomyces cerevisiae genome

Abstract

We used genome-wide comparative analysis of predicted protein sequences to identify many novel small genes, named smORFs for small open reading frames, within the budding yeast genome. Further analysis of 117 of these new genes showed that 84 are transcribed. We extended our analysis of one smORF conserved from yeast to human. This investigation provides an updated and comprehensive annotation of the yeast genome, validates additional concepts in the study of genomes in silico, and increases the expected numbers of coding sequences in a genome with the corresponding impact on future functional genomics and proteomics studies.

Figure 1.

Overall strategy for smORF identification. Computational method used to identify new ORFs not identified by conventional methods.

Figure 2.

Experimental validation of the S. cerevisiae smORFs. (A) Primers specific for the yeast ACT1 gene as well as the three smORFs were used for PCR amplification using no template (lanes 1,5,9,13), 50 ng genomic DNA (lanes 2,6,10,14), 500 ng total RNA from cells grown in rich media (lanes 3,7,11,15), and 500 ng total RNA from cells grown in minimal media (lanes 4,8,12,16) .(B) Primers specific for the yeast ACT1 gene as well as the three smORFs were used for RT-PCR amplification using no template (lanes 1,4,7,10), 500 ng total RNA from cells grown in rich media (lanes 2,5,8,11), and 500 ng total RNA from cells grown in minimal media (lanes 3,6,9,12). PCR and RT-PCR products were fractionated on a 1% agarose gel with DNA size markers and visualized after ethidium bromide staining. Sizes of DNA fragments in bp are indicated. (C) Two-step orientation-specific RT-PCR. Primers whose sequence is complementary to the predicted mRNAs of smORF2, 8, and 31 were used for first-strand cDNA synthesis. After heat inactivation of the reverse transcriptase, PCR amplification was carried out with both smORF-specific primers (lanes 5,6,11,12,17,18). As control, the experiment was repeated using primers with the same sequence as the mRNA for first-strand cDNA synthesis (lanes 3,4,9,10,15,16). (D) Examples of RT-PCR results with various smORFs indicated on top. RT-PCR detection of transcripts from the annotated smORFs YLL018C-A and YHR132W-A are shown. smORFs for which RT-PCR reactions resulted in no products are indicated (*) as are those for which the product is not of the expected size (^∧). These smORFs were not included in Table 1. Sizes of DNA fragments in bp are indicated. (E) Transcript size determination by Northern analysis of the ACT1 (lane 3), smORF2 (lane 5), and smORF31 (lane 7). Unlabeled RNA markers stained with methylene blue (lane 1) and labeled RNA markers (lanes 2,4,6) were fractionated together with yeast poly (A)+ RNA. Sizes are shown in nucleotides.

Figure 2.

Experimental validation of the S. cerevisiae smORFs. (A) Primers specific for the yeast ACT1 gene as well as the three smORFs were used for PCR amplification using no template (lanes 1,5,9,13), 50 ng genomic DNA (lanes 2,6,10,14), 500 ng total RNA from cells grown in rich media (lanes 3,7,11,15), and 500 ng total RNA from cells grown in minimal media (lanes 4,8,12,16) .(B) Primers specific for the yeast ACT1 gene as well as the three smORFs were used for RT-PCR amplification using no template (lanes 1,4,7,10), 500 ng total RNA from cells grown in rich media (lanes 2,5,8,11), and 500 ng total RNA from cells grown in minimal media (lanes 3,6,9,12). PCR and RT-PCR products were fractionated on a 1% agarose gel with DNA size markers and visualized after ethidium bromide staining. Sizes of DNA fragments in bp are indicated. (C) Two-step orientation-specific RT-PCR. Primers whose sequence is complementary to the predicted mRNAs of smORF2, 8, and 31 were used for first-strand cDNA synthesis. After heat inactivation of the reverse transcriptase, PCR amplification was carried out with both smORF-specific primers (lanes 5,6,11,12,17,18). As control, the experiment was repeated using primers with the same sequence as the mRNA for first-strand cDNA synthesis (lanes 3,4,9,10,15,16). (D) Examples of RT-PCR results with various smORFs indicated on top. RT-PCR detection of transcripts from the annotated smORFs YLL018C-A and YHR132W-A are shown. smORFs for which RT-PCR reactions resulted in no products are indicated (*) as are those for which the product is not of the expected size (^∧). These smORFs were not included in Table 1. Sizes of DNA fragments in bp are indicated. (E) Transcript size determination by Northern analysis of the ACT1 (lane 3), smORF2 (lane 5), and smORF31 (lane 7). Unlabeled RNA markers stained with methylene blue (lane 1) and labeled RNA markers (lanes 2,4,6) were fractionated together with yeast poly (A)+ RNA. Sizes are shown in nucleotides.

Figure 2.

Experimental validation of the S. cerevisiae smORFs. (A) Primers specific for the yeast ACT1 gene as well as the three smORFs were used for PCR amplification using no template (lanes 1,5,9,13), 50 ng genomic DNA (lanes 2,6,10,14), 500 ng total RNA from cells grown in rich media (lanes 3,7,11,15), and 500 ng total RNA from cells grown in minimal media (lanes 4,8,12,16) .(B) Primers specific for the yeast ACT1 gene as well as the three smORFs were used for RT-PCR amplification using no template (lanes 1,4,7,10), 500 ng total RNA from cells grown in rich media (lanes 2,5,8,11), and 500 ng total RNA from cells grown in minimal media (lanes 3,6,9,12). PCR and RT-PCR products were fractionated on a 1% agarose gel with DNA size markers and visualized after ethidium bromide staining. Sizes of DNA fragments in bp are indicated. (C) Two-step orientation-specific RT-PCR. Primers whose sequence is complementary to the predicted mRNAs of smORF2, 8, and 31 were used for first-strand cDNA synthesis. After heat inactivation of the reverse transcriptase, PCR amplification was carried out with both smORF-specific primers (lanes 5,6,11,12,17,18). As control, the experiment was repeated using primers with the same sequence as the mRNA for first-strand cDNA synthesis (lanes 3,4,9,10,15,16). (D) Examples of RT-PCR results with various smORFs indicated on top. RT-PCR detection of transcripts from the annotated smORFs YLL018C-A and YHR132W-A are shown. smORFs for which RT-PCR reactions resulted in no products are indicated (*) as are those for which the product is not of the expected size (^∧). These smORFs were not included in Table 1. Sizes of DNA fragments in bp are indicated. (E) Transcript size determination by Northern analysis of the ACT1 (lane 3), smORF2 (lane 5), and smORF31 (lane 7). Unlabeled RNA markers stained with methylene blue (lane 1) and labeled RNA markers (lanes 2,4,6) were fractionated together with yeast poly (A)+ RNA. Sizes are shown in nucleotides.

Figure 2.

Experimental validation of the S. cerevisiae smORFs. (A) Primers specific for the yeast ACT1 gene as well as the three smORFs were used for PCR amplification using no template (lanes 1,5,9,13), 50 ng genomic DNA (lanes 2,6,10,14), 500 ng total RNA from cells grown in rich media (lanes 3,7,11,15), and 500 ng total RNA from cells grown in minimal media (lanes 4,8,12,16) .(B) Primers specific for the yeast ACT1 gene as well as the three smORFs were used for RT-PCR amplification using no template (lanes 1,4,7,10), 500 ng total RNA from cells grown in rich media (lanes 2,5,8,11), and 500 ng total RNA from cells grown in minimal media (lanes 3,6,9,12). PCR and RT-PCR products were fractionated on a 1% agarose gel with DNA size markers and visualized after ethidium bromide staining. Sizes of DNA fragments in bp are indicated. (C) Two-step orientation-specific RT-PCR. Primers whose sequence is complementary to the predicted mRNAs of smORF2, 8, and 31 were used for first-strand cDNA synthesis. After heat inactivation of the reverse transcriptase, PCR amplification was carried out with both smORF-specific primers (lanes 5,6,11,12,17,18). As control, the experiment was repeated using primers with the same sequence as the mRNA for first-strand cDNA synthesis (lanes 3,4,9,10,15,16). (D) Examples of RT-PCR results with various smORFs indicated on top. RT-PCR detection of transcripts from the annotated smORFs YLL018C-A and YHR132W-A are shown. smORFs for which RT-PCR reactions resulted in no products are indicated (*) as are those for which the product is not of the expected size (^∧). These smORFs were not included in Table 1. Sizes of DNA fragments in bp are indicated. (E) Transcript size determination by Northern analysis of the ACT1 (lane 3), smORF2 (lane 5), and smORF31 (lane 7). Unlabeled RNA markers stained with methylene blue (lane 1) and labeled RNA markers (lanes 2,4,6) were fractionated together with yeast poly (A)+ RNA. Sizes are shown in nucleotides.

Figure 3.

Multiple sequence alignments for smORF2p. smORF2p has highly conserved homologs in other fungi and in mammalian species. Abbreviations: Dm, Drosophila melanogaster; Hs, Homo sapiens; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; Ca, Candida albicans; Af, Aspergillus fumigatus; An, Aspergillus nidulans; Sp, Schizosaccharomyces pombe; Bt, Bos taurus; Mm, Mus musculus. Residues that are identical or similar in all protein homologs are shaded in black, and those identical or similar in two or more, but not all, proteins in the alignment are shaded in gray. Homology shading was done with GeneDoc (Nicholas et al. 1997).

Figure 4.

smORF2 is expressed in yeast. A triple HA tag was fused to the C-terminal end of smORF2 using PCR, and the wild-type smORF2 gene was replaced by the tagged smORF2 gene by allele replacement into the chromosome. Soluble extracts were prepared and analyzed in a Western blot probed with monoclonal antibodies that recognize the HA epitope. Extracts from wild-type cells (lane 2) and extracts from two separate isolates carrying the HA-tagged smORF2 (lanes 3,4).

Figure 5.

Human smORF2 complementation of the temperature-sensitive phenotype of the smorf2Δ strain. A yeast strain with a deleted smORF2 (smorf2Δ) was transformed with plasmids carrying the wild-type yeast smORF2, human smORF2 under the control of the GAL1 promoter, or empty vector. Transformants were obtained at 30°C, and individual colonies were streaked and then incubated at 30°C and 37°C.