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New protein-coding genes can originate either through modification of existing genes or de novo. Recently, the importance of de novo origination has been recognized in eukaryotes, although eukaryotic genes originated de novo are relatively rare and difficult to identify. In contrast, viruses contain many de novo genes, namely those in which an existing gene has been "overprinted" by a new open reading frame, a process that generates a new protein-coding gene overlapping the ancestral gene. We analyzed the evolution of 12 experimentally validated viral genes that originated de novo and estimated their relative ages. We found that young de novo genes have a different codon usage from the rest of the genome. They evolve rapidly and are under positive or weak purifying selection. Thus, young de novo genes might have strain-specific functions, or no function, and would be difficult to detect using current genome annotation methods that rely on the sequence signature of purifying selection. In contrast to young de novo genes, older de novo genes have a codon usage that is similar to the rest of the genome. They evolve slowly and are under stronger purifying selection. Some of the oldest de novo genes evolve under stronger selection pressure than the ancestral gene they overlap, suggesting an evolutionary tug of war between the ancestral and the de novo gene.

An overlapping open reading frame (ORF) with a potential to encode a functional protein has been identified within the 3'-proximal ORF of Solenopsis invicta virus 1 (SINV-1) and three bee viruses. This ORF has been referred to as predicted overlapping gene (pog). Protein motif searches of POG revealed weak relationships precluding assignment of a potential function. Neither a transcript nor a protein encoded by the pog ORF has been detected. However, recently, a protein encoded by the corresponding +1 overlapping ORF (termed ORFx) in the Israeli acute paralysis virus (IAPV) was demonstrated by recombinant means as well as in IAPV-infected honey bees. The objective of our study was to attempt to provide empirical evidence for the presence of a pog-derived protein from SINV-1-infected fire ants. A number of different laboratory and field SINV-1-infected Solenopsis invicta preparations were examined by western blotting for the presence of a POG protein sequence. In every case, these preparations failed to yield any detectable bands when probed with a polyclonal antibody preparation raised to a portion of the pog predicted protein sequence. Although impossible to prove a negative result, proper controls used in these studies suggested that the pog ORF is not translated into a functional protein in SINV-1.

Protein-coding genes often contain long overlapping open-reading frames (ORFs), which may or may not be functional. Current methods that utilize the signature of purifying selection to detect functional overlapping genes are limited to the analysis of sequences from divergent species, thus rendering them inapplicable to genes found only in closely related sequences. Here, we present a method for the detection of selection signatures on overlapping reading frames by using closely related sequences, and apply the method to several known overlapping genes, and to an overlapping ORF on the negative strand of segment 8 of influenza A virus (NEG8), for which the suggestion has been made that it is functional. We find no evidence that NEG8 is under selection, suggesting that the intact reading frame might be non-functional, although we cannot fully exclude the possibility that the method is not sensitive enough to detect the signature of selection acting on this gene. We present the limitations of the method using known overlapping genes and suggest several approaches to improve it in future studies. Finally, we examine alternative explanations for the sequence conservation of NEG8 in the absence of selection. We show that overlap type and genomic context affect the conservation of intact overlapping ORFs and should therefore be considered in any attempt of estimating the signature of selection in overlapping genes.

As far as protein-coding genes are concerned, there is a non-zero probability that at least one of the five possible overlapping sequences of any gene will contain an open-reading frame (ORF) of a length that may be suitable for coding a functional protein. It is, however, very difficult to determine whether or not such an ORF is functional. Recently, we proposed a method that predicts functionality of an overlapping ORF if it can be shown that it has been subject to purifying selection during its evolution. Here, we use simulation to test this method under several conditions and compare it with the method of Firth and Brown. We found that under most conditions, our method detects functional overlapping genes with higher sensitivity than Firth and Brown's method, while maintaining high specificity. Further, we tested the hypothesis that the two aminoacyl tRNA synthetase classes have originated from a pair of overlapping genes. A central piece of evidence ostensibly supporting this hypothesis is the assertion that an overlapping ORF of a heat-shock protein-70 gene, which exhibits some similarity to class 2 aminoacyl tRNA synthetases, is functional. We found signature of purifying selection only in highly divergent sequences, suggesting that the method yields false-positives in high sequence divergence and that the overlapping ORF is not a functional gene. Finally, we examined three cases of overlap in the human genome. We find varying signatures of purifying selection acting on these overlaps, raising the possibility that two of the overlapping genes may not be functional.

The Israeli acute paralysis virus (IAPV) is a honeybee-infecting virus that was found to be associated with colony collapse disorder. The IAPV genome contains two genes encoding a structural and a nonstructural polyprotein. We applied a recently developed method for the estimation of selection in overlapping genes to detect purifying selection and, hence, functionality. We provide evolutionary evidence for the existence of a functional overlapping gene, which is translated in the +1 reading frame of the structural polyprotein gene. Conserved orthologs of this putative gene, which we provisionally call pog (predicted overlapping gene), were also found in the genomes of a monophyletic clade of dicistroviruses that includes IAPV, acute bee paralysis virus, Kashmir bee virus, and Solenopsis invicta (red imported fire ant) virus 1.

Background: Same-strand overlapping genes may occur in frameshifts of one (phase 1) or two nucleotides (phase 2). In previous studies of bacterial genomes, long phase-1 overlaps were found to be more numerous than long phase-2 overlaps. This bias was explained by either genomic location or an unspecified selection advantage. Models that focused on the ability of the two genes to evolve independently did not predict this phase bias. Here, we propose that a purely compositional model explains the phase bias in a more parsimonious manner. Same-strand overlapping genes may arise through either a mutation at the termination codon of the upstream gene or a mutation at the initiation codon of the downstream gene. We hypothesized that given these two scenarios, the frequencies of initiation and termination codons in the two phases may determine the number for overlapping genes., Results: We examined the frequencies of initiation- and termination-codons in the two phases, and found that termination codons do not significantly differ between the two phases, whereas initiation codons are more abundant in phase 1. We found that the primary factors explaining the phase inequality are the frequencies of amino acids whose codons may combine to form start codons in the two phases. We show that the frequencies of start codons in each of the two phases, and, hence, the potential for the creation of overlapping genes, are determined by a universal amino-acid frequency and species-specific codon usage, leading to a correlation between long phase-1 overlaps and genomic GC content., Conclusion: Our model explains the phase bias in same-strand overlapping genes by compositional factors without invoking selection. Therefore, it can be used as a null model of neutral evolution to test selection hypotheses concerning the evolution of overlapping genes.

Inferring the intensity of positive selection in protein-coding genes is important since it is used to shed light on the process of adaptation. Recently, it has been reported that overlapping genes, which are ubiquitous in all domains of life, seem to exhibit inordinate degrees of positive selection. Here, we present a new method for the simultaneous estimation of selection intensities in overlapping genes. We show that the appearance of positive selection is caused by assuming that selection operates independently on each gene in an overlapping pair, thereby ignoring the unique evolutionary constraints on overlapping coding regions. Our method uses an exact evolutionary model, thereby voiding the need for approximation or intensive computation. We test the method by simulating the evolution of overlapping genes of different types as well as under diverse evolutionary scenarios. Our results indicate that the independent estimation approach leads to the false appearance of positive selection even though the gene is in reality subject to negative selection. Finally, we use our method to estimate selection in two influenza A genes for which positive selection was previously inferred. We find no evidence for positive selection in both cases.