The Absence of Universally Conserved Protein-Coding Genes

P_n is the number of protein-coding genes in species n, whose coordinates are (X, Y). X is the sum of the proteins encoded by organisms 1 to n. Y_UNU and Y_ONO are the sum of the ONO or UNU, respectively, of these organisms.

Fig. 6. Change of the number of UNU, ONO, and all the protein-coding genes as new organisms were added. Each data point represents a species whose coordinates are (X, Y) and whose encoded protein number is Pn. X is the sum of the proteins encoded by all the organisms up to and including that species. Y is the sum of the UNU or ONO of these organisms.

Note that the number of UNU grew quickly initially with the addition of species, but the growth slowed down soon, and almost plateaued at around 3,300 genes (fig. 6A). Strikingly, the ONO number increased continuously, at a much greater speed than the initial, fastest, growth rate of the UNU number (fig. 6B, orange). Viewed at the same scale, the number of UNU genes appears to show a trend, or slope, of zero (that is, unchanging along the vertical axis) (fig. 6B, blue).

Identity of the Universal Genes

The numbers of different groups of genes are interesting and important to know but their identity is even more informative for our understanding of life. Here we will describe the identity of the universal genes, while that of the orphan genes will be described in other publications.

We first analyzed the homologs of the universal genes of our seventeen chosen organisms. We observed three phenomena. First, the vast majority of the homologs of a gene encode proteins performing the same function in different organisms, when functional data are available (S5 table). This indicates the specificity of homolog inference of OrthoInspector. The occasional out-of-place homologs, for example, the Escherichia coli lysine-tRNA ligase amongst the homologs of asparagine-tRNA ligase and the Caulobacter crescentus peptide chain release factor 3 in the mist of elongation factor G homologs, indicates the ability of OrthoInspector to recognize even very low similarity. For example, the E. coli lysine-tRNA ligase, though not an asparagine-tRNA ligase, belongs to the same subgroup (class IIb) of tRNA ligases as the asparagine-tRNA ligase, with the sole other member of this group being aspartate-tRNA ligase (Giege and Springer 2016). These three tRNA ligases recognize related anticodons: aspartate GUC, asparagine GUU, and lysine UUU triplets. The C. crescentus peptide chain release factor 3 belongs to the same subfamily of GTPases and probably has similar structure and binds to overlapping regions of the bacterial ribosomes as the elongation factor G (Burroughs and Aravind 2019; Graf et al. 2018; Leipe et al. 2002). The fact that most homologs of a gene encode proteins with the same characteristics and/or functions demonstrates that OrthoInspector is a reliable method to identify homologs. Second, the homolog of a universal gene is commonly not a universal gene. This is surprising. However, it explains why the number of universal genes is not an integer multiplication of the number of model species (317), though not why it is smaller than 317. Third, none of the universal genes in our seventeen chosen organisms kept their status as universal genes when checked against their in-domain non-model species in OrthoInspector (S5 table).

Next, we expanded our analysis to all the model species. Consistent with the observation of our seventeen chosen organisms, all universal genes lost their status as a universal gene when checked against their in-domain non-model species in OrthoInspector.

The protein characteristics of five universal genes (Q74MY3_NANEQ, R1E424_9ARCH, E4WXB9_ OIKDI, C4V6P4_NOSCE, A2ER26_TRIVA) were not clear. To solve this problem, we examined their homologs in our seventeen chosen organisms (S6 table). Assuming a gene’s homologs share its identity, we called Q74MY3_NANEQ and R1E424_9ARCH Obg-like ATPase 1, A2ER26_TRIVA and C4V6P4_ NOSCE elongation factor 2, and E4WXB9_OIKDI isoleucine-tRNA ligase.

With the new assignation for these five universal genes, there are a total of 261 universal genes in the 317 model species. These genes encode the following eight universal proteins: aspartate-tRNA ligase, phenylalanine-tRNA ligase alpha subunit, valine-tRNA ligase, isoleucine-tRNA ligase, elongation factor G (name according to bacteria, corresponding to the archaeal and eukaryotic elongation factor 2), elongation factor Tu (name according to bacteria, corresponding to the archaeal and eukaryotic elongation factor 1), DNA-directed RNA polymerase subunit beta, and Obg-like ATPase 1 (table 3 and S7 table). Five of these eight belong to the up-to-date bacterial core gene set (the two elongation factors and valine-tRNA ligase do not) (Na et al. 2018). Note that the aspartate-tRNA ligase (or any of the other seven of the eight universal proteins) of different species may differ in amino acid sequences and normally those differences are assumed to be the result of accumulated mutation in the history of life. However, the differences may be introduced by God when He created different organisms at the beginning.

Table 1. Numbers of orphan, nearly orphan, universal, and nearly universal genes in 17 chosen organisms.

Therefore, even though by definition, if there is one universal gene in the model species, we should have 317 such genes because it should have a homologous gene in each of the model species. And all of the 317 homologous genes should be the same, at least very similar. But we have only 261 universal genes and they are not all the same, although they all, except one (Obg-like ATPase 1), are involved in either gene transcription (the DNA-directed RNA polymerase subunit beta) or gene translation (the t-RNA ligases and elongation factors). Even Obg-like ATPase 1 may be involved in translation. For example, human Obg-like ATPase 1 can prevent eIF2 (eukaryotic initiation factor 2) ternary complex formation, leading to inhibition of protein synthesis and promotion of integrated stress response (Balasingam et al. 2019; Chen, Song et al. 2015).

Discussion

Our comparisons of the genetic profiles of 317 proteomes revealed that ONO genes are a common occurrence, while the UNU genes are very rare. The more organisms are included in the analysis, the more ONO genes are detected and the smaller the percentage of UNU gene becomes. Lastly, not a single universal gene remains its status as universal when enough organisms are included.

Universal vs Non-universal Genes

The continuous increase of the ONO numbers and the leveling off of the UNU numbers is consistent with earlier observations (Charlebois and Doolittle 2004; Graham et al. 2000; Khalturin et al. 2009; Koonin 2003; Lagesen, Ussery, and Wassenaar 2010; Mushegian and Koonin 1996; Prabh et al. 2018; Tautz and Domazet-Loso 2011; Wilson et al. 2005, 2007; Wissler et al. 2013). These analyses have led to a dramatic shrinking, or even vanishing, of the “universal,” or universally conserved, core set of genes and proteins— with a concomitant linear growth in the so-called “orphan” or “taxonomically restricted” sequences. “Accordingly,” notes Koonin, “the universal core of life has shrunk almost to the point of vanishing”(Koonin 2016). Indeed, after complete sequencing of the first two bacterial genomes, a comparison of the 1,727 protein-coding genes of Haemophilus influenza and the 468 Mycoplasma genitalium genes identified 240 homologous genes between the two (Mushegian and Koonin 1996). When the number of included prokaryotic genomes increased to 100, the number of universally conserved homologous genes decreased to 63 (Koonin 2003). With the inclusion of 1,000 genomes, the number of universally-conserved homologous genes became zero—not a single protein-coding gene was conserved across the 1,000 prokaryotes compared (Lagesen, Ussery, and Wassenaar 2010).

What is surprising, and counterintuitive, is that the homologs of universal genes normally are not universal genes. This results from how we define and detect homologs. Two genes are deemed homologous as long as part of their encoded proteins share some sequence similarity, normally an e-value of 10^-3 to 10^-5 in a BLASTp search. OrthoInspector uses an e-value cutoff of 1e^-9 (about 1.2 × 10^-4) (Linard et al. 2011). At this condition, the C. crescentus peptide chain release factor 3 and elongation factor G are detected as homologs because they share some sequence homology in their GTPase domains. With the normal e-value cutoffs, the well-known Drosophila orphan genes Jingwei and Zeus will not be recognized as orphan genes because their high sequence similarity with other wildly distributed genes (Bai et al. 2007; Chen et al. 2012; Chen, Krinsky, and Long 2013; Long et al. 2003; Long and Langley 1993; Wang et al. 2000; Zhang et al. 2004, 2010). For example, Drosophila melanogaster Zeus, though without homologs in the bacterial and archaeal model species, has homologs in 100 of Nevers’s 143 eukaryotic model species, including Saccharomyces cerevisiae, Caenorhabditis elegans, and Gallus gallus (Tan, unpublished observation).

To illustrate how the homologs of a universal gene can be non-universal genes, opposite to what one would expect by the definition of a universal gene, we made up three hypothetical proteins, A, B, and U (fig. 7). U shares parts 1, 2, and 3 with A and parts 1, 4, and 5 with B. Each part can be one or a group of amino acids. Under the criterion that two proteins are homologous if they share three parts, U and A are homologs, so are U and B. However, A and B only share part 1, thus are not homologous. U is a universal gene in this scenario, while A and B are not. How would the ancestor(s) of A, B, and U look like, the one on the bottom left, the one on the bottom right, or something else? How can we know?

Fig. 7 A possible scenario of how a homolog of a universal gene could be a non-universal gene.

Different Levels of Homology

The puzzle that homologs of a universal gene are normally non-universal genes necessitates distinguishing different levels of homology. The concept of gene homology and the identification of homologous genes among different species are foundational to our study and numerous other comparative genomic studies. We think that it will be fruitful to define and distinguish different levels of homology. Here we propose the following levels of homology: 1) protein-domains (or partial sequence homology, or regional homology), 2) full length proteins, 3) full length genes, including the 5’- and 3’- untranslated regions and introns, 4) signal transduction pathways, 5) tissues, 6) organs, 7) body parts. The first three levels are at the molecular level and concern protein-coding genes. The higher the level of homology two genes share, the more likely they will perform the same function. Each level has its own value, even the lowest level, the level one. For instance, if two proteins both have a kinase domain, then they will be a kinase of some sort. Their other non-homologous regions may determine their substrates and their cellular locations of functioning. If these two proteins share homology throughout their entire length, then we can predict that they possess the same substrate specificity and function in the same cell compartment.

Currently, it is a general practice to call two proteins homologous as long as they share level one homology and be put into the same protein family. This can cause unnecessary challenges for functional annotation of genes because thus assigned family members may perform opposite or unrelated functions. This can be confusing, even misleading, especially when thus-identified “homologous” genes are given the same or similar names. For example, Frizzled, a seven-transmembrane protein, and FrzB (also known as soluble or secreted frizzled-related proteins), a protein that is similar to the amino-terminal cysteine rich domain of Frizzled but has no transmembrane segments, are included in the same protein family (InterPro: https://www.ebi.ac.uk/interpro/entry/InterPro/IPR015526/, PANTHER: http://www.pantherdb.org/panther/ family.do?clsAccession=PTHR11309, UniProtKB: https://www.uniprot.org/uniprot/Q92765). However, the former is a Wnt receptor necessary for Wnt signaling, while the latter inhibits Wnt-signaling. A similar example is the C. crescentus peptide chain release factor 3 and the elongation factor G discussed earlier. They are put into the same subfamily of GTPases but perform different functions. Many bacterial and archaeal genes are called globins because they share partial sequence similarity with hemoglobins, but instead of carrying oxygen around like a hemoglobin, a bacterial globin does not bind oxygen, instead may function as a nitrogen monoxide detoxifier (Vinogradov et al. 2006).

We propose to provide the following information when declaring two genes homologous: 1) level of homology, or homology coverage, 2) degree of homology, 3) e-value cutoff, and 4) a visual representation of the homology. Homology coverage should indicate whether the homology level is of protein-domain, full-length protein, or full-length DNA sequence. For example, we may divide every protein into four quarters, 1 to 4 from the N-terminal to the C-terminal. The homology between FrzB and Frizzled can be described as FrzB-p1-4/Frizzled-p1, “p” for protein. That is, the full length FrzB protein is homologous to the first quarter of the Frizzled protein. Homology degree can be indicated with the percent identity of a BLASTp search. A visual representation of the homology should include both the regions that can be aligned and those that cannot be aligned. Fig. 8 depicts a visual representation of homology between two subunits, Rpb A’ and A”, of archaea Pyrococcus furiosus RNA polymerase and the largest subunit Rpb1 of S. cerevisiae RNA polymerase II. A’ aligns with the first two quarters of Rpb1. A” aligns with the third quarter of Rpb1. The C-terminal quarter of Rpb1 is unique to eukaryotes and is essential for eukaryotic gene transcription initiation, elongation, termination and intron splicing (Tan 2017). Specific information about the levels at which proteins are homologous will not only avoid making incorrect connection of protein functions but also facilitate understanding of true relationship of genes.

Fig. 8. A comparison of S. cerevisiae Rpb1 (gray, bottom) with P. furiosus Rpb A’ and A” (green, top). Segment locations are based on amino acid positions of Rpb1 protein. Segments present in Rpb1 but not in Rpb A’ and A” are indicated with trapezoids and negative numbers, while those segments absent in Rpb1 but present in Rpb A’ and A” are indicated with inverted triangles and positive numbers. The values of the numbers, which correlate with the sizes of the triangles and trapezoids, represent the numbers of amino acids of the corresponding regions that are present in only one of the proteins compared. The C-terminal tail of Rpb1 that is missing in P. furiosus RNA polymerase is highlighted with a red dash-lined box. The comparison is from Fig. 1I of (Tan 2017).

A Novel Index of Gene Diversity

Our birth-event-weighted gene distribution method can be used as a reasonable indication of the diversity of genes, or different types of genes. Since homologous genes (correctly defined as discussed above) tend to be similar, a non-discriminating counting would inflate the total gene number count encoded by all species on earth. The weighting approach corrects this, and thus can be used as an index of the number of gene types or gene diversity. Moreover, weighting is logically required if indeed homologous genes share a common ancestral gene. But this makes the explanation of the origin of ONO genes more acutely mysterious and makes it more important to study their functions, which we will address in the future. We propose to use the birth-event-weighted distribution of genes as an indication of gene diversity, even though the weighting makes it more difficult to explain the origin of genes by the common belief that genes were generated via duplication and diversification because of the greatly enlarged portion of the ONO genes.

Limitation of This Study

The number and identity of genes in each of our orphan, o+1, o+2 . . . and universal homologous groups may be different using a different method and/or a different e-value cutoff for homology calling or when different species are included, though the trend of the differential growth of ONO and UNU genes will not change. It will be interesting to study how the phylogenetic profiles of proteins will change with a change of the criteria of homolog calling, such as the percentage of gene length covered, percentage of identity, alignment gap penalty, e-value cutoff, and calculation models (alignment algorithms) for these parameters. Furthermore, how the phylogenetic profiles would change when different species are included.

Recent years have witnessed a growth of interests in orphan genes (Arendsee, Li, and Wurtele 2014; Bianchi et al. 1999; Bowling, Schultheis, and Strome 2016; Johnson 2018; Johnson and Tsutsui 2011; Khalturin et al. 2009; McLysaght and Hurst 2016; Palmieri, Kosiol, and Schlotterer 2014; Prabh and Rodelsperger 2016, 2019; Schmitz and Bornberg- Bauer 2017; Tan 2015; Tautz and Domazet-Loso 2011; Toll-Riera et al. 2009; Van Oss and Carvunis 2019; Verster et al. 2017; Wilson et al. 2005; 2007; Wissler et al. 2013; Wu and Knudson 2018; Zhang et al. 2019). However, the increase of interests is incomparable to the increase of orphan gene number. Most of the orphans have unknown functions and will be a rich soil for discovery of new enzymes and/ or unknown substrates of known enzymes (Hanson et al. 2009) or new genotype-phenotype connections. The broad existence of orphan genes calls for a greater attention to them from the biological community.

Conclusions

Our in-depth analysis of phylogenetic profiles of 317 proteomes across the three domains of life shows that ONO genes are a common occurrence in the sense that each organism has a significant number of them, while UNU genes are very rare. Most organisms, especially eukaryotes, do not have any UNU genes. Furthermore, the sum total of UNU genes almost plateaued while the number of ONO genes grew continuously when the number of organisms being analyzed increased. More importantly, every universal gene lost its status as a universal gene when the sampled number of organisms is increased. These results revealed a great challenge to explain not only the origin of genes but also the origin of life and the origin of biodiversity. Finally, we have introduced the birth-event-weighted distribution of genes and propose to use it as an indication of gene diversity, even though the weighting makes it more difficult to explain the origin of genes by the common belief that genes were generated via duplication and diversification because of the greatly enlarged portion of the ONO genes.

Acknowledgments

The author thanks Andrew Jones for help with calculating the numbers of homologs, Rob Sadler and Paul Nelson for comments on the manuscript, Alan Marshall and Nicholas Valentine for help with computers.

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Supplementary Materials

S1 fig. (Part A and Part B) Change of numbers of genes in different gene groups in 12 additional species

S2 table. Number of genes in different gene groups for all NMS

S3 table. Distribution of orphan, nearly-orphan, universal, and nearly-universal genes of all NMS. The 17 chosen species were highlighted with red font.

S4 table. Weighted distribution of orphan, nearly-orphan, universal, and nearly-universal genes of all NMS

S5 table. Analysis of universal genes of selected organisms. Reference genes are highlighted with red font. The seemingly out-of-place homologs are shaded with yellow.

S6 table. Analysis of universal genes of unknown identity. Reference genes are highlighted with red font.

S7 table. Analysis of the identity of universal genes of all NMS.