Functional genomics of genes with small open reading frames (sORFs) in S. cerevisiae.
ABSTRACT Genes with small open reading frames (sORFs; <100 amino acids) represent an untapped source of important biology. sORFs largely escaped analysis because they were difficult to predict computationally and less likely to be targeted by genetic screens. Thus, the substantial number of sORFs and their potential importance have only recently become clear. To investigate sORF function, we undertook the first functional studies of sORFs in any system, using the model eukaryote Saccharomyces cerevisiae. Based on independent experimental approaches and computational analyses, evidence exists for 299 sORFs in the S. cerevisiae genome, representing approximately 5% of the annotated ORFs. We determined that a similar percentage of sORFs are annotated in other eukaryotes, including humans, and 184 of the S. cerevisiae sORFs exhibit similarity with ORFs in other organisms. To investigate sORF function, we constructed a collection of gene-deletion mutants of 140 newly identified sORFs, each of which contains a strain-specific "molecular barcode," bringing the total number of sORF deletion strains to 247. Phenotypic analyses of the new gene-deletion strains identified 22 sORFs required for haploid growth, growth at high temperature, growth in the presence of a nonfermentable carbon source, or growth in the presence of DNA damage and replication-arrest agents. We provide a collection of sORF deletion strains that can be integrated into the existing deletion collection as a resource for the yeast community for elucidating gene function. Moreover, our analyses of the S. cerevisiae sORFs establish that sORFs are conserved across eukaryotes and have important biological functions.
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Functional genomics of genes with small open
reading frames (sORFs) in S. cerevisiae
James P. Kastenmayer,1,6Li Ni,2,6Angela Chu,3Lauren E. Kitchen,1Wei-Chun Au,1
Hui Yang,2Carole D. Carter,1David Wheeler,4Ronald W. Davis,3Jef D. Boeke,5
Michael A. Snyder,2and Munira A. Basrai1,7
1Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
20889, USA;2Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520,
USA;3Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA;4National Center for
Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA;
5Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205, USA
Genes with small open reading frames (sORFs; <100 amino acids) represent an untapped source of important
biology. sORFs largely escaped analysis because they were difficult to predict computationally and less likely to be
targeted by genetic screens. Thus, the substantial number of sORFs and their potential importance have only
recently become clear. To investigate sORF function, we undertook the first functional studies of sORFs in any
system, using the model eukaryote Saccharomyces cerevisiae. Based on independent experimental approaches and
computational analyses, evidence exists for 299 sORFs in the S. cerevisiae genome, representing ∼5% of the annotated
ORFs. We determined that a similar percentage of sORFs are annotated in other eukaryotes, including humans, and
184 of the S. cerevisiae sORFs exhibit similarity with ORFs in other organisms. To investigate sORF function, we
constructed a collection of gene-deletion mutants of 140 newly identified sORFs, each of which contains a
strain-specific “molecular barcode,” bringing the total number of sORF deletion strains to 247. Phenotypic analyses
of the new gene-deletion strains identified 22 sORFs required for haploid growth, growth at high temperature,
growth in the presence of a nonfermentable carbon source, or growth in the presence of DNA damage and
replication-arrest agents. We provide a collection of sORF deletion strains that can be integrated into the existing
deletion collection as a resource for the yeast community for elucidating gene function. Moreover, our analyses of
the S. cerevisiae sORFs establish that sORFs are conserved across eukaryotes and have important biological functions.
[Supplemental material is available online at www.genome.org.]
The initial Saccharomyces cerevisiae genome sequencing effort an-
notated all ORFs of at least 100 contiguous codons (including the
first ATG) not contained entirely within a longer ORF (Goffeau et
al. 1996). Knowledge of sORF (small open reading frame; <100
amino acids) function is limited compared to that of larger genes,
although small proteins include members of important classes
such as mating pheromones, proteins involved in energy me-
tabolism, proteolipids, chaperonins, stress proteins, transporters,
transcriptional regulators, nucleases, ribosomal proteins, thiore-
doxins, and metal ion chelators (for review, see Basrai et al.
1997). Computational discovery of sORFs is difficult because they
are “buried” in an enormous pile of meaningless short ORFs that
arise by chance. In addition, sORFs are not favorable targets for
random mutagenesis. Similar challenges plague attempts to iden-
tify non-coding RNAs (ncRNAs), transcripts that function at the
level of RNA rather than as templates for translation (for review,
see Eddy 2001). Despite the challenges of sORF identification,
reports since the publication of the S. cerevisiae genome indicate
that sORFs are quite numerous in S. cerevisiae and many are evo-
lutionarily conserved from distantly related fungi to humans.
Many S. cerevisiae sORFs were discovered through expres-
sion-based analyses. Velculescu and colleagues used serial analy-
sis of gene expression (SAGE) to identify, quantitate, and com-
pare global gene expression patterns in S. cerevisiae (Velculescu et
al. 1995, 1997; Basrai and Hieter 2002). The SAGE technique is
based on two principles: (1) a 9–10-bp sequence tag derived from
a defined region in any poly(A)+transcript that uniquely identi-
fies the transcript; and (2) multiple sequence tags that are con-
catenated and sequenced in a single sequencing lane. In addition
to confirming expression of annotated genes, the SAGE study
provided the first evidence that hundreds of non-annotated read-
ing frames (NORFs), including many sORFs, are transcribed in S.
cerevisiae. We subsequently characterized one of these sORFs,
NORF5/HUG1, and determined that it is a downstream target of
the MEC1-mediated pathway for DNA damage and replication
arrest (Basrai et al. 1999). These results validated the functional
significance of sORFs found through systems biology approaches
and suggested that other sORFs may have important functions.
Since the SAGE study, additional studies provided expres-
sion-based evidence for sORFs. Transcripts for potential sORFs or
ncRNAs from intergenic regions were detected by Northern blot-
ting (Olivas et al. 1997). A combined microarray and proteomics
6These authors contributed equally to this work.
E-mail email@example.com; fax (301) 480-0380.
Article and publication are at http://www.genome.org/cgi/doi/10.1101/
16:365–373 ©2006 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/06; www.genome.org
approach confirmed transcription of many sORFs discovered by
SAGE and detected peptides corresponding to numerous sORFs,
including some not reported by SAGE (Oshiro et al. 2002). Ad-
ditional sORFs were discovered using a gene-trap strategy based
on genomic integration of a modified bacterial transposon, and
their expression was confirmed by strand-specific oligonucleo-
tide dot-blot arrays (Kumar et al. 2002). Interestingly, some of the
sORFs discovered by gene-trap are antisense to coding genes (Ku-
mar et al. 2002).
Potential sORF homologs were identified for many of the
sORFs discovered in the expression-based studies, and recent
comparative genomic studies have expanded the number of
sORFs with potential orthologs. Conserved sORFs were reported
from comparisons of the S. cerevisiae genome to partial genome
sequences from 13 hemiascomycetes and the complete genome
sequences from distantly and closely related fungi (Blandin et al.
2000; Brachat et al. 2003; Cliften et al. 2003; Kellis et al. 2003). A
recent study that combined homology searching with RT-PCR
identified conserved sORFs whose expression was detected at the
level of RNA (Kessler et al. 2003).
Based on the published literature, at least 299 genes in S.
cerevisiae likely encode sORFs. We discovered that a similar per-
centage of sORFs are annotated in multiple eukaryotes and that
many of the S. cerevisiae sORFs have potential orthologs in other
eukaryotes. We constructed gene-deletion strains for 140 sORFs,
bringing the total number of sORF deletion strains to 247. We
analyzed these 140 new sORF deletion strains for growth pheno-
types and identified sORFs that are essential for haploid growth
and for growth at high temperature. We also identified sORFs
required for growth under genotoxic conditions including expo-
sure to hydroxyurea (HU), bleomycin, methyl methane sulfonate
(MMS), or ultraviolet (UV) radiation. These data highlight the
value of expression analyses and comparative genomics to iden-
tify sORFs and the advantages of S. cerevisiae genetics in investi-
gating sORF function.
Results and Discussion
Evidence of S. cerevisiae sORFs
The S. cerevisiae genome has 299 annotated sORFs (Saccharomyces
Genome Database; http://www.yeastgenome.org/) (Fig. 1A;
Supplemental Table A). By comparing the sORFs reported since
the publication of the S. cerevisiae genome, we determined that
the majority of sORFs (170) were discovered in the gene expres-
sion and homology studies mentioned above, while the remain-
der were previously reported in the literature (Fig. 1A, “129 pre-
viously known”). We analyzed the literature for reports of tran-
scription, translation, or homology for the 170 new sORFs. Those
that were reported by SAGE (Velculescu et al. 1997), microarrays
(Kumar et al. 2002; Oshiro et al. 2002), RT-PCR (Kessler et al.
2003), Northern blot (Olivas et al. 1997), or gene-trap (Kumar et
al. 2002) were considered transcribed. The sORFs detected by
gene-trapping were considered transcribed and translated be-
cause the ?-galactosidase assays used to detect integration require
transcription and translation. The mass-spectrometry study also
identified sORFs with evidence of translation (Oshiro et al. 2002).
Finally, sORFs reported in homology searches were classified as
supported by homology (Velculescu et al. 1997; Blandin et al.
2000; Kumar et al. 2002; Oshiro et al. 2002; Brachat et al. 2003;
Cliften et al. 2003; Kessler et al. 2003).
Many of the new sORFs were detected by more than one
approach (Fig. 1B; Supplemental Table A). For example, a large
number of sORFs were discovered as both transcribed and trans-
lated (43 sORFs) or transcribed and with potential orthologs (67
sORFs), while several (15 sORFs) show evidence of transcription,
translation, and homology. sORFs discovered only by transcrip-
tion-based assays (18 sORFs) may represent ncRNAs, rather than
protein-coding genes. sORFs detected at the level of RNA and
homology may also be ncRNAs rather than protein-coding genes
if the homology is the result of conservation of an RNA rather
than protein-coding sequence. The sORFs discovered only by ho-
mology (21 sORFs) may represent genes expressed under certain
conditions not used in the gene expression studies or could rep-
resent conserved sequences such as regulatory elements that are
not expressed (Cliften et al. 2003). Most of the sORFs were de-
tected by two or more techniques and likely represent bona fide
Small proteins constitute a significant percentage of annotated
proteins in eukaryotes
The 299 sORFs constitute ∼5% of the 5865 genes annotated for S.
cerevisiae in the NCBI RefSeq database (http://www.ncbi.nlm.
nih.gov/RefSeq/) (Fig. 2; Pruitt et al. 2005). We determined the
percentage of annotated small proteins from additional eukary-
otes in the NCBI RefSeq database (see Methods). We selected
representative eukaryotes including another fungus (Schizosac-
charomyces pombe), worms (Caenorhabditis elegans), plants (Arabi-
analyses and homology searching reveal 170 potential sORFs, bringing
the total number of annotated sORFs in S. cerevisiae to 299. Reports in the
literature provided empirical evidence of transcription derived from
SAGE, microarray, RT-PCR, Northern blot, and gene-trap experiments,
while empirical evidence of translation was derived from reports of mass-
spectrometry and gene-trap experiments. Comparative genomic studies
provided evidence of homology. (B) The bar graph depicts the subcat-
egorization of the evidence for the 170 new sORFs, showing that the
largest number of sORFs were identified based on evidence of transcrip-
tion followed by evidence based on translation and homology.
Evidence of S. cerevisiae sORFs. (A) Gene expression-based
Kastenmayer et al.
dopsis thaliana), insects (Drosophila melanogaster), and mammals
(Mus musculus and Homo sapiens). Interestingly, a similar percent-
age of sORFs are annotated for these organisms (∼5%), including
multicellular eukaryotes that have much larger genomes and a
greater number of ORFs (Fig. 2). These results suggest that sORFs
are not favored in single-celled eukaryotes or in those with
smaller genomes and fewer genes. However, the evidence for the
sORFs of S. cerevisiae comes from multiple analyses that may not
have been used for all the representative eukaryotes (Fig. 2), and
future experiments may reveal additional sORFs in these and
other systems. Nevertheless, sORFs represent hundreds and in
some cases >1000 ORFs in eukaryotes, and likely contribute sig-
nificantly to the biology of eukaryotes.
sORFs are evolutionarily conserved
Many of the new sORFs were discovered based on homology (103
of 170 sORFs) (Fig. 1B), indicating that sORFs likely have funda-
mental functions across eukaryotes. However, the databases used
to search for sORF orthologs differed between reports (e.g.,
Kessler et al. 2003 used the NCBI fungi sequences, while Oshiro
et al. 2002 used the nonredundant sequences from all species),
and a search for orthologs of the complete set of 299 sORFs had
not been reported. We conducted two searches using the entire
set of 299 sORFs. First, we conducted BLAST analyses to examine
the conservation of the sORFs in the representative eukaryotes
(Fig. 2). Second, to examine sORF conservation more broadly, we
examined the data on the sORFs in the HomoloGene database,
which was built with genome sequences from a wide variety
of eukaryotes (http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?DB=homologene) (Wheeler et al. 2005).
For our BLAST analyses, we compared the sORFs to the an-
notated proteins from the representative eukaryotes (Fig. 2) and
to a database derived from genomic and EST sequences of these
organisms, the UniGene database (http://www.ncbi.nlm.nih.
gov/entrez/query.fcgi?db=unigene) (Pontius et al. 2003; see
Supplemental material). We discovered that 46 of the sORFs ex-
hibit significant alignments with annotated proteins from two or
more of the representative eukaryotes, with BLAST bit scores
ranging from 48 to 147 (Table 1). In the representative eukary-
otes, ∼60% of the proteins that are similar to the S. cerevisiae
sORFs are less than ∼100 amino acids (data not shown). We also
discovered 44 sORFs that align with transcripts from two or more
representative eukaryotes in the UniGene database (Supplemen-
tal Table B; see Supplemental material).
Our analysis of HomoloGene revealed additional conserved
sORFs. HomoloGene is a system that automatically detects ho-
mologs among the annotated genes of several completely se-
quenced eukaryotic genomes including H. sapiens and M. muscu-
lus (Supplemental material). Seventy-one sORFs were found in
HomoloGene clusters conserved at several taxonomic levels, and
55 of the clusters have an assignment from the Conserved Do-
main Database (Marchler-Bauer et al. 2005), a collection of mul-
tiple sequence alignments for ancient domains and full-length
proteins (Supplemental Table B). The conserved domains cover a
broad spectrum; however, a conserved domain derived from
SMART domain 00651, annotated as “small nuclear ribonucleo-
protein particles (snRNPs) involved in pre-mRNA splicing,” was
represented most frequently, occurring five times. Zinc-finger,
representative eukaryotes. The percentage of sORFs for S. cerevisiae and
representative eukaryotes was calculated and is depicted in the bar
graph. The genome size (megabases) and the number of RefSeq ORFs for
each eukaryote are displayed below the graph.
sORFs constitute a similar percentage of annotated ORFs in
significant alignments with annotated proteins from
BLAST bit scores of S. cerevisiae sORFs that exhibit
The BLAST bit scores obtained from a comparison of the sORFs to anno-
tated proteins present in NCBI RefSeq are shown. A higher number in-
dicates a more significant match. The BLAST bit score is a normalized
version of the raw BLAST alignment score that is given in units of bits. Bit
scores are independent of the scoring system used, so that, in addition to
the score itself, only the size of the search space is needed to calculate
their statistical significance. (S.p.) Schizosaccharomyces pombe; (C.e.) Cae-
norhabditis elegans; (A.t.) Arabidopsis thaliana; (D.m.) Drosophila melano-
gaster; (M.m.) Mus musculus; (H.s.) Homo sapiens.
S. cerevisiae small ORFs
ubiquitin-like, and ribosomal protein domains were also encoun-
tered multiple times.
In summary, our results, combined with previously pub-
lished reports, establish that 184 of the S. cerevisiae sORFs may
have potential orthologs in other organisms (Supplemental Table
B), including distantly related organisms, such as humans, and
∼60% of these orthologs may themselves be sORFs (data not
shown). Therefore, functional analysis of the S. cerevisiae sORFs
has the potential to yield insight into the functions of the S.
cerevisiae sORFs and those of other eukaryotes.
Generation of sORF deletion strains
Gene-deletion strain collections of S. cerevisiae have revolution-
ized functional analyses of genes (e.g., Winzeler et al. 1999).
Since only 106 of the 299 sORFs are represented in the previous
collection (version 1.0), we attempted to construct gene-deletion
strains of the remaining sORFs in the same genetic background as
described for the initial yeast gene-deletion strains (Winzeler et
al. 1999; Supplemental Fig. 1) (see Methods).
Using homologous recombination, we constructed indi-
vidual strains in which sequences from the start codon to the
stop codon of the sORF were replaced by a kanMX cassette in
a diploid strain (Methods) (Supplemental Fig. 1). Each sORF
gene-deletion mutant is publicly available either as haploids
(MATa and MAT?) or as diploids (homozygous or heterozygous;
project/deletions3.html). The gene-deletion strains con-
tain “molecular barcodes” that will facilitate rapid identifica-
tion and analysis of genes in genome-wide approaches to analyze
gene function (Winzeler et al. 1999; for review, see Pan et al.
2004). We determined that the molecular barcodes correspond-
ing to the sORF deletions are detectable in microarray ex-
periments using the Tag3 arrays (Affymetrix) (data not shown).
In total, we constructed 140 sORF heterozygous deletion strains
(∼93% of the 151 attempted), bringing the total number of
sORF deletion strains available to the yeast community to 247
(Supplemental Table A). The remaining sORF deletion strains
were not constructed because of technical problems including
the inability to design gene-specific primers or to recover trans-
Identification of essential sORFs
Sporulation of eight of the 140 new sORF heterozygous dele-
tion strains resulted in two viable (genticin-sensitive) and
two inviable (sORF deletion) spores, indicating that the
corresponding sORFs are essential for viability (Table 2). Three
of these genes were previously uncharacterized—YLR099W-A,
YNL024C-A, and YNL138W-A—while the remaining five sORFs
were previously shown to be essential, which we confirmed
in the gene-deletion strain background. These sORFs are required
for functions such as kinetochore or spindle integrity (Cheese-
man et al. 2002; Li et al. 2005), ER to golgi transport (Heidt-
man et al. 2005), and pseudouridine biosynthesis (Henras et al.
1998). Combined with the results from Version 1.0, 21 of the
sORFs are essential, representing ∼8% of sORFs analyzed. The
percentage of essential sORFs differs from that of larger ORFs in
which ∼20% of ORFs tested are essential (Winzeler et al. 1999).
This difference may reflect redundancy in sORF function, or in-
dicate that sORFs have regulatory roles rather than essential func-
Phenotypic analyses of haploid sORF deletion strains
Six of the new haploid sORF deletion strains exhibit slow-growth
phenotypes when grown at 30°C (Table 2), including strains de-
leted for YBL071W-A/KTI11 and YPL096C-A/ERI1, which are
known to exhibit slow growth (Fichtner and Schaffrath 2002;
Sobering et al. 2003). We further analyzed the growth of the new
haploid sORF deletion strains in pilot screens under the follow-
ing conditions: (1) at high (37°C) or low (11°C) temperatures; (2)
in the presence of a sole nonfermentable carbon source; and (3)
in the presence of the replication-arrest agent HU and DNA-
damaging agents including MMS, bleomycin, and UV radiation.
To confirm the results of the pilot screen, we sporulated hetero-
zygous strains corresponding to the haploid strains with pheno-
types and characterized the meiotic progeny. For each strain, we
analyzed three independent sORF deletion spores and confirmed
nonfermentable carbon source (petite phenotype). (A) sORFs required for
growth at 37°C. Growth assays of 3 µL of fivefold serial dilutions of
logarithmic-phase cells of the sORF deletion strains spotted on YPD plates
and incubated at 30°C or 37°C for 2 to 3 d. The wild-type strain BY4741
and the temperature-sensitive strain ndc10-1 (JK421) served as controls.
(B) Growth assays of 3 µL of fivefold serial dilutions of logarithmic-phase
cells of the sORF deletion strains spotted on YP Dextrose or YP Glycerol
plates and incubated at 30°C for 2 to 3 d.
sORFs required for growth at 37°C (Ts) or in the presence of
when deleted in haploid strains
sORFs that result in a lethal or a slow-growth phenotype
aSize = size of protein encoded by sORF in amino acids.
Kastenmayer et al.
that the phenotype was linked to the sORF deletion. Upon veri-
fication of the phenotypes, we confirmed chromosomal deletion
of the sORF by PCR and sequence analysis of the genomic locus
at the site of integration of the kanMX cassette (see Methods).
We observed that three of the sORF deletion strains are tem-
perature-sensitive (Ts) for growth at 37°C (Fig. 3A), while none of
the sORF deletion strains showed a cold-sensitive growth pheno-
type at 11°C (data not shown). A Ts allele of the kinetochore
mutant (ndc10-1) served as a control (Goh and Kilmartin 1993).
We discovered a new gene required for growth at the nonpermis-
sive temperature of 37°C, YKL096C-B, and confirmed the previ-
ously reported Ts growth phenotypes of strains with a mutation
in sORFs YBR058C-A/TSC3 and YDR079C-A/TFB5, which are in-
volved in sphingolipid biosynthesis and transcription regulation,
respectively (Fig. 3A; Gable et al. 2000; Ranish et al. 2004).
We tested the sORF deletion strains for a “petite” pheno-
type, which refers to an inability to grow in the presence of a
nonfermentable carbon source and is an attribute of several mu-
tants including mitochondrial mutants (for review, see Chen and
Clark-Walker 2000). We determined that three sORF deletion
strains exhibit a “petite” phenotype (Fig. 3B; Table 3). Our results
confirm the role of YEL059C-A/SOM1 for mitochondrial function
(Esser et al. 1996) and suggest a similar function for the previ-
ously uncharacterized sORFs YJL062W-A and YPL189C-A. Con-
sistent with a role in mitochondrial function, Yjl062w-ap fused to
GFP has been localized to the mitochondrion (Huh et al. 2003).
To investigate the potential role of sORFs in response to
genotoxic stress, we assayed the sORF deletion strains for sensi-
tivity to the replication-arrest agent HU and to DNA-damaging
agents bleomycin, MMS, and UV radiation. Sensitivity to these
genotoxic agents can provide important clues about the roles of
the genes in replication, transcription, cell-cycle progression, and
chromosome segregation (Chang et al. 2002; Aouida et al. 2004;
Parsons et al. 2004). In addition, many S. cerevisiae genes required
for responding to DNA damage and replication arrest have hu-
man orthologs, mutations in which lead to human diseases (for
review, see Zhou and Elledge 2000).
For these studies, strains grown to logarithmic phase were
serially diluted, spotted on medium containing the appropriate
drug, or exposed to UV, and incubated for 2–3 d at 30°C. The S.
cerevisiae checkpoint mutant mec1? sml1?, which exhibits sensi-
tivity to HU, bleomycin, MMS, and UV radiation, served as a
control (Kiser and Weinert 1996). HU inhibits ribonucleotide re-
ductase, an enzyme that is required for synthesis of dNTPs in S.
cerevisiae and other systems, and leads to an arrest in S-phase of
the cell cycle (Elledge et al. 1993). As shown in Figure 4A, three
sORF deletion strains exhibit varying degrees of sensitivity to
growth on HU-containing media, with ybr058c-a?/tsc3? being
the most sensitive. Our results suggest new roles for Tsc3p, a
sphingolipid biosynthetic enzyme (Gable et al. 2000); Sus1p, a
component of the SAGA and Sac3p–Tthp1p complexes (Rod-
riguez-Navarro et al. 2004); and the uncharacterized YBR196C-A,
in responding to replication arrest.
Next, we tested bleomycin, a radiomimetic drug that leads
to both single- and double-stranded DNA damage (Chen and
Stubbe 2005), and discovered that four sORF deletion strains are
sensitive to bleomycin. sORF deletion strains lacking YBR058C-
A/TSC3 showed the most sensitivity, while ykl096c-b? and
ydr524w-c? strains were only moderately sensitive to bleomycin
(10 mU/mL) (Fig. 4B). Our results extend the role of Tsc3p in
responding to replication arrest caused by HU (Fig. 4A) to an addi-
tional role in responding to DNA damage caused by bleomycin.
We also discovered a new sORF required for growth in the
presence of MMS. MMS is a DNA-alkylating agent that primarily
methylates DNA on N7-deoxyguanine and N3-deoxyadenine
(Pegg 1984). Resistance to MMS requires genes from the bypass,
post-replication, recombination, base excision repair, and/or
checkpoint pathways (Weinert et al. 1994; Xiao et al. 1996; Ter-
cero and Diffley 2001). The sORF deletion strain ybr111w-a?/
sus1? is sensitive to growth on MMS medium (Fig. 4C), a phe-
notype that, to our knowledge, has not been previously reported
for strains deleted for YBR111W-A/SUS1. Ybr111w-ap/Sus1p is a
component of the SAGA complex and the Sac3p–Tthp1p mRNA
export complex (Rodriguez-Navarro et al. 2004). These results,
combined with earlier results (Fig. 4A), suggest a novel role for
Sus1p in response to DNA damage induced by MMS and replica-
tion arrest induced by HU. Finally, we confirmed a UV-sensitivity
phenotype previously reported for the ydr079c-a?/tfb5? strain in
a different genetic background (Fig. 4D; Ranish et al. 2004).
The sORF deletion strains exhibit overlapping and distinct
Taken together, we observed conditional phenotypes for nine
sORF deletion strains (Table 3). Not surprisingly, we observed
sORFs with phenotypes when deleted
Phenotypes of sORF deletion strains
Reported (Gable et al. 2000)
Reported (Rodriguez-Navarro et al. 2004)
Reported (Ranish et al. 2004)
Reported (Jan et al. 2000)
aSize = size of protein encoded by sORF in amino acids.
bTS = temperature-sensitive.
cGlycerol = petite phenotype when grown on media containing glycerol and ethanol as sole carbon source.
dND = not done.
eS = sensitive to high temperature (37°C) or to genotoxic agents.
S. cerevisiae small ORFs
that several of the sORF deletion strains exhibit overlapping phe-
notypes when subjected to DNA damage or replication arrest, an
observation made with other ORF deletion strains (Chang et al.
2002; Table 3). For example, two of the HU-sensitive strains also
exhibit sensitivity to bleomycin and MMS. Interestingly, all three
Ts sORF deletion strains are also sensitive to DNA-damage or
replication-arrest agents. These results may suggest that the role
of these genes in response to DNA damage and replication arrest
may be essential for haploid growth at the nonpermissive tem-
perature of 37°C.
Phenotypic analyses of deletion strains for genes flanking
Six of the sORFs that exhibited phenotypes distinct from wild
type when deleted (YBR058C-A/TSC3, YBR111W-A/SUS1,
YDR079C-A/TFB5, YEL059C-A/SOM1, YJL062W-A, and YKL096C-
B) are within 300 bp of larger ORFs. The phenotypes we observed
may be due to altered expression of the neighboring ORFs caused
by disruptions in their promoters or 5?- or 3?-untranslated re-
gions rather than loss of function of the deleted sORFs. We there-
fore examined the phenotypes of strains with deletions of genes
that are within 300 bp of the sORFs, a conservative approach, as
∼60% of ORFs, both large and small, are within 300 bp of another
ORF. In all but two cases (YBR111W-A/SUS1, YJL062W-A), dele-
tion of the neighboring genes did not produce the phenotypes
we observed for the sorf? strain (Supplemental Table C). For these
two deletions strains, their phenotypes could be due to interfer-
ence of expression of a neighboring ORF, loss of the sORF, or
We determined that the deletion strain for YGR271C-A
showed slow growth, Ts, and an HU-sensitivity phenotype and
that a deletion strain for YGR272C, which is 51 bp away from
YGR271C-A, also exhibits such phenotypes (Fig. 5A). Sequence
analysis of the genomic locus revealed that YGR271C-A is con-
tiguous with YGR272C, forming a single ORF, consistent with the
similarity of these two predicted ORFs to a single ORF
(PABR143C) from Ashbya gossypii (Fig. 5B; Brachat et al. 2003).
We constructed a new gene-deletion strain for the larger ORF,
which we denote as ygr271c-a?/ygr272c?, and determined that
this strain showed a more severe slow growth, Ts, and HU-
sensitivity phenotype compared to the ygr271c-a? or ygr272c?
strains (Fig. 5A). Further analysis in a cell cycle arrest–release
experiment showed that the ygr271c-a?/ygr272c? strain exhibits
a significant delay of at least 40 min in exiting from the G1phase
of the cell cycle after an arrest with ?-factor (Fig. 5C). Our results,
combined with the analysis of protein expression described be-
low, establish that YGR271C-A and YGR272C constitute a single
ORF, which we have named EFG1 (Exit from G1).
Protein expression analysis of the sORFs
Recent evidence of expression at the protein level for sORFs has
come from genome-wide TAP- and GFP-tagging experiments
(Ghaemmaghami et al. 2003; Huh et al. 2003; Supplemental
Table A). Protein expression for some of the sORFs detected in
our screens has been reported in these (Supplemental Table A)
and other studies (Table 3). We epitope-tagged a subset of sORFs
identified in our phenotypic analyses by introducing a haemag-
glutinin epitope (HA) at the C-terminus in their chromosomal
context and examined expression of the tagged protein by West-
ern blot analysis. We detected expression of proteins from strains
expressing HA-tagged YJL062W-A, YPL189C-A, and YDR524W-C
(Fig. 6, lanes 1,2,4). We also detected a band of expected size for
EFG1, further confirming that YGR271C-A and YGR272C consti-
tute a single ORF (Fig. 6, lane 3).
In the past, the function of sORFs has been elusive owing to
inherent difficulties in identifying them based on genetic, bio-
chemical, or solely computational approaches. S. cerevisiae repre-
sents one of the few systems with a wealth of data derived from
several functional genomic and comparative genomic studies.
Using the strengths of S. cerevisiae as a model, we provide the first
systematic investigation of sORF function in any system. Our
analysis of the literature combined with our genetic analyses for
sORF function presents a comprehensive database for the 299
sORFs in S. cerevisiae. Of the S. cerevisiae sORFs, 184 are related to
dilutions of logarithmic-phase cells of the sORF deletion strains spotted on YPD plates (control) or spotted on YPD plates containing (A) 200 mM HU,
(B) 10 mU/mL bleomycin, or (C) 0.02% MMS. For sensitivity to UV, we irradiated strains spotted on YPD with (D) 20 mJ/m2of UV radiation. All plates
were incubated at 30°C for 2 to 3 d. The checkpoint mutant (mec1? sml1?, U953–61D) and the wild-type strain (BY4741) served as controls.
sORFs required for growth on media containing replication-arrest and DNA-damaging agents. Growth assays of 3 µL of fivefold serial
Kastenmayer et al.
sequences in other eukaryotes, suggesting the evolutionary con-
servation of the structure and perhaps function of these sORFs.
Although relatively little is known about sORF functions, they
have been implicated in key cellular processes including trans-
port, intermediary metabolism, chromosome segregation, ge-
nome stability, and other functions. The sORF gene-deletion col-
lection should lead to the discovery of additional functions for
sORFs in S. cerevisiae. Moreover, our results, which emphasize the
biological significance of sORFs in S. cerevisiae that are conserved
across eukaryotes, should provide an impetus for the identifica-
tion and characterization of sORFs in other systems, including
Analysis of sORF percentage in representative eukaryotes
The number of sORFs coding for proteins of 100 amino acids
in length or less, annotated on the transcripts of model organ-
isms in the NCBI RefSeq database, was determined using a query
of the Entrez Protein database of the form: srcdb refseq[prop]
AND homo sapiens[orgn] AND 0:100[slen]. The total number of
ORFs in each set was counted using a query of the form: srcdb
refseq[prop] AND homo sapiens[orgn]. The version of RefSeq
used was that present in Entrez on 3/15/2005 corresponding to
RefSeq release 10, available on 3/6/2005 with updates from
3/6/2005 to 3/15/2005.
For this study, HomoloGene build 38.1, dating from November
23, 2004, was used (ftp://ftp.ncbi.nih.gov/pub/HomoloGene/).
BLAST of sORFs with annotated proteins and UniGene
Single sequence representatives of the UniGene clusters, the
“seq-uniques” described in Supplemental material, were down-
loaded for each organism from the NCBI FTP site (ftp://ftp.ncbi.
nih.gov/) and compared to the sORFs using BLAST (see Supple-
mental material; Altschul et al. 1997). The best BLAST hit was
extracted for each sORF only if the hit spanned at least one-third
of the translated ORF with an amino acid identity of at least 40%;
otherwise, no hit was extracted. The results are summarized in
Supplemental Table B.
Media and yeast strains
The media and methodology for yeast growth were as described
(Gietz et al. 1992, 1995; Adams et al. 1997; Brachmann et al.
1998). The deletion strain was generated in diploid strain BY4743
(MATa/? his3?1/his3?1 leu2?0/leu2?0 lys2?0/LYS2 MET15/
met15?0 ura3?0/ura3?0), and the haploid spores isogenic with
progression. (A) The ygr271c-a?, ygr272c? and ygr271c-a?/ygr272c? strains exhibit slow growth, Ts, and HU-sensitive phenotypes. Growth assays were
carried out as in Figures 3 and 4. (B) YGR271C-A and YGR272C exhibit similarity to the A. gossypii ORF PABR143C (Brachat et al. 2003). By sequencing
the genomic locus in S. cerevisiae, we determined that YGR271C-A and YGR272C constitute a single larger ORF. (C) Cell cycle analysis was done by
arresting wild-type (BY4741) and ygr271c-a?/ygr272c? strains with ?-factor (0 min) and then releasing them into pheromone-free media. Samples were
analyzed for DNA content at 20-min intervals as indicated by flow cytometry. The 1C and 2C peaks denote cells with a 1N and 2N DNA content
corresponding to cells in G1or G2/M phase of the cell cycle, respectively.
YGR271C-A and YGR272C constitute a contiguous ORF required for growth at 37°C, growth on HU-containing media and cell cycle
analysis of protein extracts prepared from strains expressing HA-tagged
ORFs (YJL062W-A, YPL189C-A, YGR271C-A/YGR272C, YDR524W-C) and
the wild-type strain (BY4741) not expressing a HA-tagged protein (no HA
tag). Proteins of the expected size were detected (ORF-HA), and Tub2p
served as a loading control (Tub2).
Protein expression analysis of HA-tagged sORFs. Western blot
S. cerevisiae small ORFs
BY4741 (MATa his3?1 leu2?0 met15?0 ura3?0) and BY4742
(MAT? his3?1 leu2?0 lys2?0 ura3?0) were identified (Winzeler et
al. 1999). Other strains include the temperature-sensitive control
strain JK421 (MATa ade2-1 ura3-1 his3-11,1 trp1-1 leu2-3,112
can1-100 ndc10-1) (Goh and Kilmartin 1993) and the checkpoint
mutant U953–61D (MATa leu2-3,112 ade2-1 can1-100 his3-11,15
ura3-1 trp1-1 RAD5 mec1??TRP1 sml1??HIS3) (Zhao et al. 1998).
Gene-deletion strain construction and confirmation
A PCR-generated (Baudin et al. 1993; Wach et al. 1994) deletion
strategy was used to systematically replace each sORF from its
start to its stop codon with a kanMX module and two unique
20-mer molecular barcodes as done previously for the gene-
deletion strain collection (Winzeler et al. 1999; Giaever et al.
2002; Supplemental Fig. 1; Supplemental material; barcode se-
quences are given in Supplemental Table A). Each sORF gene-
deletion mutant is publicly available either as haploids (MATa
and MAT?) or as diploids (homozygous or heterozygous; see
Phenotypic analyses of sORF deletion strains
For sensitivity to HU, MMS, bleomycin, UV, and nonpermissive
growth temperatures, we assayed serial dilutions of the sORF
strains on YPD or YPD containing 200 mM hydroxyurea (HU;
H8627; Sigma), 0.02% methane methylsulfonate (MMS; 64294;
Fluka Chemika), or 10 mU/mL bleomycin (BLM; 3154-01; Bristol-
Myers Squibb Co.). For sensitivity to UV-radiation, we irradiated
strains spotted on YPD with 20 mJ/m2using a Stratalinker (Strata-
gene). For growth at the nonpermissive temperatures, we incu-
bated plates at either 11°C or 37°C. A “petite” phenotype was
determined by plating strains on a modified YPD medium in
which dextrose was substituted with 2% glycerol and 2% etha-
Protein expression analysis of HA-tagged ORFs
ORFs were fused in-frame at the genomic locus with three copies
of the HA epitope at their C-terminus as previously described
(Longtine et al. 1998; Supplemental material). Protein extracts of
ORF-HA-expressing strains were analyzed by Western blot analy-
sis as described previously (Crotti and Basrai 2004). The primary
antibody was anti-HA (clone 12CA5-Roche) or anti-Tub2p (poly-
clonal antibody, Basrai lab), and the secondary antibody was
HRP-conjugated sheep anti-mouse IgG (NA931V; Amersham).
?-Factor arrest/release experiments
Strains were grown overnight at 30°C in YPD medium and then
diluted into fresh medium to obtain a logarithmic-phase culture.
Cells were arrested in the presence of 3 µM ?-factor (T-6901;
Sigma) at 30°C for 90 min, washed twice with water, and resus-
pended in fresh YPD medium and incubated at 30°C. DNA con-
tent was assayed every 20 min after release from the ?-factor
arrest for a total of 3 h as described previously (Doheny et al.
1993; Basrai et al. 1996) using a Becton-Dickinson FACSort flow
cytometer and CellQuest software (BD Biosciences).
The authors thank Anand Sethuraman and Mike Cherry for help
with compiling the sequences of the sORFs; Anuj Kumar for shar-
ing unpublished data; Lucy Liu and Xiuquiong Zhou for tetrad
dissections; Keith Anderson, Ana Aparicio, and Mike Jensen of
the SGTC (Stanford Genome Technology Center) for assistance
with the A.M.O.S. primers; and Mark Johnston for advice and
support of this work. This work was supported in part by NIH
grant R01-HG02432 to J.D.B. and by the Intramural Research
Program of the NIH and NCI.
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Received June 28, 2005; accepted in revised form October 6, 2005.
S. cerevisiae small ORFs