We have analyzed a total of 84,086 independent yeast transformants, each of which contains a random fragment of the yeast genome placed under the control of a doxycyclin-repressible promoter (Figure 1a,1b). Effects on growth or survival were monitored by spotting serial dilutions of the transformants in the presence and absence of doxycyclin (uninduced and overexpression conditions respectively, Figure 1c). Phenotypes were recorded using numerical values from 0 to 3 (Figure 2): value 3 was assigned to normal growth (similar to non-toxic control), 2 and 1 were assigned to intermediate growth levels (less abundant and/or smaller-sized colonies), and 0 was assigned to complete or almost complete absence of colonies (comparable to the toxic control on the same plate). We have retained 714 clones (0.85% of total) that show impaired growth in overexpression conditions (Table 1). Among these, 112 also show a slight or severe growth reduction (level 2 for 77 cases, or level 1 for 35 cases, respectively) in unexpressed conditions. Proof that the observed growth defects were caused by the presence of the plasmid rather than an accidental mutation in the clone was directly demonstrated by the recovery of the wild-type phenotype after plasmid loss using selection for resistance to 5-fluoroorotic acid (5-FOA) (Figure 2).

Inserts of the selected clones were identified by DNA sequencing (Materials and methods). The complete list of inserts is described in Additional file 1 and 2, and results are summarized in Table 1. A majority of inserts (493, or 69% of total) carry in-frame portions of annotated open reading frames (ORFs), excluding Ty and Y' ORFs. In addition, a significant number of inserts (162 (23%)) correspond to fragments of ORFs cloned either in antiparallel orientation or out-of-frame with respect to the initiator ATG codon or to intergenic regions. The 59 remaining cases (8% of total) correspond to fragments of transposable elements (17 clones) and subtelomeric Y' elements (9 clones), to RNA-coding genes (4 clones), and to non-chromosomal replicons such as the 2 μm plasmid and mitochondrial DNA (mtDNA) (29 clones). If any random fragment of the yeast genome were capable of generating a toxic phenotype, in-frame ORF fusions would represent only around 10-12% of the selected inserts (around 70% of the genome correspond to coding regions, and only one frame out of six corresponds to the natural frame). The fact that the toxic inserts correspond principally to in-frame portions of natural ORFs suggests that the coding part of the genome is the most prone to confer toxicity when overexpressed.

The 493 inserts corresponding to in-frame ORF fragments represent 454 distinct annotated ORFs (see Materials and methods), which are randomly distributed throughout the 16 chromosomes of S. cerevisiae (see Additional file 1). In our screening, 32 ORFs were found twice, two ORFs were found three times and one ORF (YHR056c in the CUP1 region) was found four times, the cloned fragments being either overlapping (22 ORFs) or non-overlapping (13 ORFs). Mean size of the coding region of inserts is 659 bp. The chosen cloning strategy favors recovery of central-or carboxy-terminal coding parts of the natural yeast genes, whereas the amino-terminal coding regions are rare 7. In our work, the cloned insert encompasses the entire gene in only six cases (additional file 3, column 20 to 23). In 154 additional cases, the insert corresponds to the carboxy-terminal portion of the natural protein (the stop codon is present). In 10 cases, the inserts start upstream of the natural ATG initiator codons, lengthening the natural peptides by reading in-frame through the untranslated region. Other cases correspond to the central coding region of natural genes.

To find possible common characteristics, we have compared between themselves all the peptides encoded by in-frame ORF fragments. BLASTP analysis was combined with detection of characterized conserved domains, of COG patterns (clusters of predicted orthologous groups of proteins 32), and of transmembrane spans (TMS) to identify toxic inserts similar to each other (see Materials and methods). Out of the 493 in-frame ORF fragments, a total of 170 were divided up into 57 distinct groups of similarity, containing from two to 12 inserts, including overlapping fragments of the same ORF (see Additional file 4). It is expected that several ORFs from a same paralogous gene family are found in a same group. Note that in 16 out of 57 groups, the inserts contain transport-specific domains and/or transmembrane spans.

As well as comparing inserts to each other, we also analyzed the totality of the conserved domains present in all peptides encoded by the 493 toxic inserts (see Materials and methods). Characterized domains are found, at least partially, in a total of 281 inserts (see additional file 1 and 3). Of a total of 183 distinct domains, 46 are represented more than once. We have compared the frequency of these 46 domains among the toxic inserts versus their frequency among the 5,803 ORF-encoded proteins of the entire genome (Table 2). We find that 37 domains are significantly over-represented compared to a random expectation, suggesting that we have screened specific domains.

These 37 domains correspond predominantly to various transporter domains (11 cases), such as amino-acid permeases and mitochondrial carrier protein domains. The toxicity of these domains is probably due to the presence of transmembrane spans. Indeed, 132 out of the 493 toxic peptides contain at least two transmembrane spans, including cases where one span is putative (see Materials and methods). Among these, 63 contain three or more predicted spans and 26 have five spans or more. Putative spans were also recognized in 84 other ORF fragments (seven with at least three spans, 15 with two spans, and 62 with one span) (see Additional file 1 and 3).

RNA-and DNA-binding domains (nine cases) involved in replication, transcription or translation functions, such as PUF, KH and rrm, are also much more represented than expected (Table 2). The PUF domain is also involved in recruitment of proteins into a complex that controls mRNA translation (see 33 for review).

Other important domains for interactions with polypeptides, phospholipids or small molecules (nine cases) are also over-represented. The WD40 motif, a propeller-like platform for stable or reversible binding of proteins in eukaryotes, has been found in inserts of 12 distinct ORFs (see additional data file 3). The 12 ORFs code for proteins having interactions with other proteins in complexes related to RNA processing or transcription 10, and nine have at least one partner also selected during our screening (see Discussion). Other interacting domains were found, such as dynamin, MRS6, and adaptin_N domains, which have roles in the dynamics of proteins, membranes and cytoskeleton, and PBD, a small domain which binds small GTPases and inhibits transcription activation. The PH domain, which binds phosphoinositides or other ligands and is involved in signal transduction, was found in inserts of three distinct ORFs involved in different functions: metabolism, cell fate, transcription (see Additional data file 3). Finally, other over-represented domains are related to metabolism and other functions (eight cases), of which several may be involved in interactions with other domains.

The serine/threonine protein kinase domain (S_TKc) is significantly under-represented in our screen. Among the 10 toxic inserts whose cognate genes code for protein kinases (PK), only four contain this domain (Additional data file 3). In these four cases, the S_TKc domain is either truncated (Additional data file 4), or flanked by a coiled-coil region and/or a low-complexity segment. Two other inserts contain the PBD (and PH) domains, and the four remaining inserts contain no characterized domain to date. As it is known that overexpression of some protein kinases is deleterious for cells (see 24 and references therein), our results suggest that a domain different from the catalytic domain is responsible for the toxicity of these proteins, and that the fragments selected in our screen have a role in binding ligands such as substrates or regulators of protein kinase activity, or of proteins involved in the signaling cascades. Three other genes coding for protein kinases of the phosphatidylinositol 3-kinase (PI kinase) family are also represented in our screen by four toxic inserts, none of which contained the kinase domain (see Discussion).

The remaining 137 domains (out of 183) were found only once each. Many correspond to functional categories described above, such as transport, metabolism, and interactions with nucleotides, other proteins or other ligands. Seven domains associated with ubiquitination functions were also found (see Additional data file 3 and 5). Several of the domains encountered have also been isolated as mammalian genetic suppressor elements (GSEs), which are cDNA fragments that inhibit cell growth (see 34 and references therein).

In addition to the domains described above, we found toxic inserts coding for natural peptides without recognizable domains but containing regions of low complexity (56 cases). A number of these peptides are highly charged, either negatively or positively (see Additional data file 3). Such charged peptides might interact in an artifactual way with other charged domains of proteins or nucleic acids or with small molecules. Interestingly, the prion-like (Q+N)-rich domain was found in eight of the natural peptides having low-complexity regions.

We have seen above that 493/714 toxic inserts are in-frame fragments of protein-coding genes. The complete list of the 454 genes corresponding to these toxic inserts is given in Additional data files 1 and 2. Their sizes range between 282 bp and 14,733 bp. The mean size of this distribution is 2,401 bp (standard deviation (SD) 1,671 bp), to be compared with a mean size of 1,444 bp (SD 1,094 bp) for the entire set of 5,803 ORFs of the yeast genome. The bias towards longer ORFs is expected from our cloning strategy (see above). Note that the 35 ORFs that we found more than once are nearly randomly distributed in various size classes.

We examined the distribution of these genes according to different criteria, such as function, subcellular localization, viability and phylogeny (Table 3) and compared it to the distribution of the genes of S. cerevisiae.

Among the 454 ORFs identified, 91 are unclassified, and function is not yet clear for six others (see Additional data file 3). The remaining ORFs represent a variety of functional classes (Table 3). Distribution of the 454 ORFs shows statistically significant deviations for eight out of the 15 functional classes, taking into account biases due to mean size of genes in each class. Globally, there is a deficit of genes involved in protein synthesis and of unclassified genes, and an excess of genes involved in transport facilitation and cellular transport (echoing the fact that we found many inserts containing transporter domains and transmembrane spans), in cell fate, in transcription and, to a lesser extent, in cell cycle/DNA processing and in homeostasis (regulation of/interaction with the environment).

As seen above, many toxic inserts contain multiple predicted TMS. Such inserts correspond most often to genes coding for transporters or for non-transporter membrane proteins 35. We have selected a total of 96 transporters (see Additional data file 3) of which 18 belong to the class of putative uncharacterized transporters, whose toxic inserts contain several TMS. Fourteen others belong to the class of transporters of unknown classification, including 13 genes of the nuclear-pore complex family, whereas there is a total of 58 genes in this family in the whole genome. On the other hand, 24 genes coding for non-transporter membrane proteins were also selected. Taken together, 120 transporters and non-transporter membrane proteins are represented in our screen, twice as many as expected (61 expected), as 782/5,803 ORFs are known or predicted as coding for such proteins 35.

The distribution of the proteins encoded by these genes in the cell is strongly biased in favour of the plasma membrane and against the cytoplasm, and, to a lesser extent, in favour of nucleus and cytoskeleton (Table 3).

Although the majority of inserts originate from non-essential genes, we have found 96 essential genes (21%) among the selected ORFs. This is a significantly higher percentage than in the whole genome, where 939/5,803 genes (16.2%) are essential (Table 3).

Using the classification from Malpertuy et al. 36 and additional updating (Génolevures 37), we find that the majority of genes yielding toxic fragments in this work are conserved (336/454 (74%)) between S. cerevisiae and other sequenced organisms, whereas 106 (23%) are ascomycete-specific and 10 (2.2%) are orphan genes. This distribution is significantly different from the distribution among the 5,803 genes of S. cerevisiae, where 64% of protein-coding genes are conserved (see Table 3). The under-representation of orphan genes in our screen is already apparent in the under-representation of functionally unclassified genes, as a high rate of orphans of the whole genome (79%) are also unclassified (data from Génolevures 37 and Munich Information Center for Protein Sequences (MIPS) 38).

To compare the phenotypes conferred by overexpression of the entire gene and of the gene fragment, we have cloned the cognate entire genes of 13 in-frame toxic inserts into the vector pCMha191 (see Materials and methods). One criterion for the choice of the genes was the absence of a mutant phenotype of the corresponding gene disruption at the time this work was started, except for the NOP4 gene whose disruption is lethal. Six of these genes are singletons; three others have a paralog already known as toxic upon overexpression. Six out of the 13 still have no known function to date (Table 4). Expression at the protein level of both entire gene and gene fragment was verified by western-blot analysis, using an anti-hemagglutinin (HA) antibody (data not shown). As seen in Table 4 and Figure 3, we found that overexpression of 10 genes was as toxic or more toxic than overexpression of the gene fragments. One gene, YGR149w, was less toxic in its entire version than in the truncated form, which was weakly toxic. Finally, we found that two genes, YML128c/MSC1 and YDL112w/TRM3, showed no toxicity when overexpressed, whereas the cloned inserts were strongly toxic. In these two cases, the immunolocalization of overexpressed products was examined, and the cytoplasmic localization of the fragment agreed with the location of the natural gene product (data not shown), indicating that the toxic effect is not the result of mislocalization of the overexpressed fragment. The gene MSC1 had already been screened 24 as a toxic fragment in overexpression conditions, the region concerned being the same as in our screening. This gene has low similarity to a stress protein of Schizosaccharomyces pombe and has a role in meiotic recombination. The TRM3 gene contains a carboxy-terminal domain responsible for tRNA methyltransferase activity 39, which is absent from our insert. The protein is a member of a complex probably involved in signaling 10.