De novo gene birth is the process by which new genes evolve from non-coding DNA. De novo genes represent a subset of novel genes, and may be protein-coding or instead act as RNA genes. The processes that govern de novo gene birth are not well understood, although several models exist that describe possible mechanisms by which de novo gene birth may occur.
Ancient de novo gene birth events are difficult to detect. Most studies of de novo genes to date have thus focused on young genes, typically taxonomically restricted genes (TRGs) that are present in a single species or lineage, including so-called orphan genes, defined as genes that lack any identifiable homolog.
Not all orphan genes arise de novo, but rather through fairly well characterized mechanisms such as gene duplication (including retroposition) or horizontal gene transfer followed by sequence divergence, or by gene fission/fusion.
Although de novo gene birth was once viewed as a highly unlikely occurrence, thousands of examples have now been described, and some researchers speculate that de novo gene birth could play a major role in evolutionary innovation, morphological specification, and adaptation, probably promoted by their low level of pleiotropy.
Conversely, criticism of the methodology and interpretation used to identify de novo gene candidates has appeared in the literature, including arguments that some reported cases may reflect artefacts of homology detection and annotation rather than genuine novelty. Rapid evolutionary divergence, along with the multiple and parallel loss of orthologs in some lineages, can give the false appearance of the emergence of a novel gene from scratch. Recent syntheses highlight both rapid progress and ongoing debates in de novo gene research, including challenges in identification, functional validation, and links to microproteins and random-peptide experiments.
History As early as the 1930s, J. B. S. Haldane and others suggested that copies of existing genes may lead to new genes with novel functions. In 1970, Susumu Ohno published the seminal text Evolution by Gene Duplication.Ohno S (1970) Evolution by Gene Duplication Allen & Unwin; Springer-Verlag For some time subsequently, the consensus view was that virtually all genes were derived from ancestral genes, with François Jacob famously remarking in a 1977 essay that "the probability that a functional protein would appear de novo by random association of amino acids is practically zero."
In the same year, however, Pierre-Paul Grassé coined the term "overprinting" to describe the emergence of genes through the expression of alternative open reading frames (ORFs) that overlap preexisting genes.Grassé P-P (1977) Evolution of living organisms: evidence for a new theory of transformation Academic Press These new ORFs may be out of frame with or antisense to the preexisting gene. They may also be in frame with the existing ORF, creating a truncated version of the original gene, or represent 3' extensions of an existing ORF into a nearby ORF. The first two types of overprinting may be thought of as a particular subtype of de novo gene birth; although overlapping with a previously coding region of the genome, the primary amino-acid sequence of the new protein is entirely novel and derived from a frame that did not previously contain a gene. The first examples of this phenomenon in bacteriophages were reported in a series of studies from 1976 to 1978, and since then numerous other examples have been identified in viruses, bacteria, and several eukaryotic species.
The phenomenon of exonization also represents a special case of de novo gene birth, in which, for example, often-repetitive intronic sequences acquire splice sites through mutation, leading to de novo exons. This was first described in 1994 in the context of Alu sequences found in the coding regions of primate mRNAs. Interestingly, such de novo exons are frequently found in minor splice variants, which may allow the evolutionary "testing" of novel sequences while retaining the functionality of the major splice variant(s).
Still, it was thought by some that most or all eukaryotic proteins were constructed from a constrained pool of "starter type" exons. Using the sequence data available at the time, a 1991 review estimated the number of unique, ancestral eukaryotic exons to be < 60,000, while in 1992 a piece was published estimating that the vast majority of proteins belonged to no more than 1,000 families. Around the same time, however, the sequence of chromosome III of the budding yeast Saccharomyces cerevisiae was released, representing the first time an entire chromosome from any eukaryotic organism had been sequenced. Sequencing of the entire yeast nuclear genome was then completed by early 1996 through a massive, collaborative international effort. In his review of the yeast genome project, Bernard Dujon noted that the unexpected abundance of genes lacking any known homologs was perhaps the most striking finding of the entire project.
In 2006 and 2007, a series of studies provided arguably the first documented examples of de novo gene birth that did not involve overprinting. These studies were conducted using the accessory gland transcriptomes of Drosophila yakuba and Drosophila erecta and they identified 20 putative lineage-restricted genes that appeared unlikely to have resulted from gene duplication. Levine and colleagues identified and confirmed five de novo candidate genes specific to Drosophila melanogaster and/or the closely related Drosophila simulans through a rigorous approach that combined bioinformatic and experimental techniques.
Since these initial studies, many groups have identified specific cases of de novo gene birth events in diverse organisms. The first de novo gene identified in yeast, BSC4 gene was identified in S. cerevisiae in 2008. This gene shows evidence of purifying selection, is expressed at both the mRNA and protein levels, and when deleted is synthetically lethal with two other yeast genes, all of which indicate a functional role for the BSC4 gene product. Historically, one argument against the notion of widespread de novo gene birth is the evolved complexity of protein folding. Interestingly, Bsc4 was later shown to adopt a partially folded state that combines properties of native and non-native protein folding. In plants, the first de novo gene to be functionally characterized was QQS, an Arabidopsis thaliana gene identified in 2009 that regulates carbon and nitrogen metabolism. The first functionally characterized de novo gene identified in mice, a noncoding RNA gene, was also described in 2009. In primates, a 2008 informatic analysis estimated that 15/270 primate orphan genes had been formed de novo. A 2009 report identified the first three de novo human genes, one of which is a therapeutic target in chronic lymphocytic leukemia. This was, however, shown to lack sufficient evidence for translation and it is regarded as a lncrna. Since this time, a plethora of genome-level studies have identified large numbers of orphan genes in many organisms, although the extent to which they arose de novo, and the degree to which they can be deemed functional, remain debated.
Novel genes
A key caveat is that orphan (taxonomically restricted) genes are heterogeneous in origin and age—lack of detectable homology can reflect multiple processes (including horizontal transfer, transposable element domestication, overprinting, or extreme divergence) and is not, by itself, evidence of de novo origin.
Identification
Identification of de novo emerging sequences There are two major approaches to the systematic identification of novel genes: genomic phylostratigraphy and synteny-based methods. Both approaches are widely used, individually or in a complementary fashion. To standardise terminology for translated non-canonical ORFs (often implicated in de novo gene studies), a community proposal introduced the term "translons" to denote all translated regions detected by approaches such as ribosome profiling.
Genomic phylostratigraphy Genomic phylostratigraphy involves examining each gene in a focal, or reference, species and inferring the presence or absence of ancestral homologs through the use of the BLAST sequence alignment algorithms or related tools. Each gene in the focal species can be assigned an age (aka "conservation level" or "genomic phylostratum") that is based on a predetermined phylogeny, with the age corresponding to the most distantly related species in which a homolog is detected. When a gene lacks any detectable homolog outside of its own genome, or close relatives, it is said to be a novel, taxonomically restricted or orphan gene.
Phylostratigraphy is limited by the set of closely related genomes that are available, and results are dependent on BLAST search criteria. In addition, it is often difficult to determine based on lack of observed sequence similarity whether a novel gene has emerged de novo or has diverged from an ancestral gene beyond recognition, for instance following a duplication event. This was pointed out by a study that simulated the evolution of genes of equal age and found that distant orthologs can be undetectable for rapidly evolving genes. On the other hand, when accounting for changes in the rate of evolution in young regions of genes, a phylostratigraphic approach was more accurate at assigning gene ages in simulated data. Subsequent studies using simulated evolution found that phylostratigraphy failed to detect an ortholog in the most distantly related species for 13.9% of D. melanogaster genes and 11.4% of S. cerevisiae genes. However, a reanalysis of studies that used phylostratigraphy in yeast, fruit flies and humans found that even when accounting for such error rates and excluding difficult-to-stratify genes from the analyses, the qualitative conclusions were unaffected. The impact of phylostratigraphic bias on studies examining various features of de novo genes remains debated. Because some "orphan" genes may be ancient but diverged beyond recognition, machine-learning classifiers trained on patterns of sub-threshold similarity-search hits have been proposed to estimate which orphans are more consistent with extreme divergence rather than true de novo origin.
Synteny-based approaches Synteny-based approaches use order and relative positioning of genes (or other features) to identify the potential ancestors of candidate de novo genes. Syntenic alignments are anchored by conserved "markers." Genes are the most common marker in defining syntenic blocks, although k-mers and exons are also used. Confirmation that the syntenic region lacks coding potential in outgroup species allows a de novo origin to be asserted with higher confidence. The strongest possible evidence for de novo emergence is the inference of the specific "enabling" mutation(s) that created coding potential, typically through the analysis of smaller sequence regions, termed microsyntenic regions, of closely related species.