Are Overapping Genes Real?

Bacteria belonging to the Pseudomonas family are a perennial favorite among bacteriology instructors (and students) because of the curious ability of some of its members to produce pigments that fluoresce under an ultraviolet light. If you're unlucky enough to get an infected cut on the arm while working in the garden, it's possible your cut will fluoresce under a black light. That's enough of a diagnosis to pronounce the infectious agent. 

Fluorescent colonies of Pseudomonas.

Silby and Levy, investigating the adaptation of the bacterium Pseudomonas fluorescens to soil, uncovered the existence of at least ten antisense genes in P. fluorescens. They went on to demonstrate experimentally that one of the genes, cosA, produces not just antisense RNA but an associated protein. Tellingly, Silby and Levy commented:

These findings suggest that current genome annotations provide an incomplete view of the genetic potential of a given organism.

The implication is that additional antitranscriptome genes remain to be found, not only in Pseudomonas but in other organisms.

There's a good reason they haven't been found yet. Overlapping genes are automatically rejected by many of the annotation programs that are commonly used to find, identify, and label genes in genome sequences. (The oft-used freeware Glimmer 2 program allows you to set the overlap-rejection threshold.) Many yet-to-be-discovered antisense genes have been deliberately and systematically obscured in published genomes.

Still, once in a while such genes do surface. For example, in Pseudomonas stutzeri A1501, we find a pair of overlapping genes at an offset of 3035137 on the chromosome (see illustration below).

Overapping genes in Pseudomonas stutzeri.

The top gene is annotated merely as a "hypothetical protein," while the underlying gene on the opposite strand is an aspartyl-tRNA synthetase. One's normal inclination is to dismiss a hypothetical protein as being unimportant, but this may not be wise. Twenty percent or more of bacterial genes are annotated as hypothetical proteins; common sense says they can't all be unimportant. In fact, in "Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity" by Filiatrault et al. (2010), researchers found that 818 out of 1,646 protein genes in P. syringae annotated as "hypothetical proteins" were expressed under iron-limited conditions. Many (probably most) genes annotated as "hypothetical protein" are quite real and should probably be re-annotated as PUF: "protein of unknown function."

In this case, the "hypothetical protein" shown in yellow (above) turns up medium-strength protein-BLAST hits with other "hypothetical proteins" from other organisms, including a hit with an E-value of 3.0×10^-49 in Parasutterella excrementihominis YIT 11859 and a comparable hit on a predicted phosphatase/phosphohexomutase in Rothia mucilaginosa DY-18.

In this particular case, the hypothetical-protein gene lacks a strong upstream Shine Dalgarno sequence (a sequence preceding many genes that helps bind a ribosome to the mRNA). But so too does the gene on the opposite strand. (This is not unusual. The SD sequence is not required for translation and in fact, in about half of bacterial species, a Shine Dalgarno sequence is associated with fewer than 50% of genes.) Hence, the jury's out on whether the antigene is expressed. It could be that no protein is made from the top strand but the gene provides RNA-mediated control of the gene on the bottom strand. We won't know for sure until someone investigates.

In Pseudomonas aeruginosa strain PADK2_CF510, we find another instance of a bidirectional overlapping gene pair (see graphic below). In this case, the gene on the top strand (CF510_06030) encodes the large subunit of an isopropylmalate isomerase. The gene on the bottom strand (CF510_06025, shown in yellow) is annotated as "Flp pilus assembly protein TadG." It could very well be a misannotated non-gene. However, five genes away is FimV (CF510_06060), another pilus-assembly (motility) protein. Moreover, the gene marked TadG has a strong upstream SD sequence containing the canonical GGAGG motif. The gene above it has a weaker GGAAA motif.

P. aeruginosa has an overlap of an isopropylmalate isomerase gene and a gene for a motility protein. The latter is shown in yellow.

In previous posts, I've mentioned (and shown data for) the fact that in the overwhelming majority of protein-encoding genes (across every kind of genome), the first base of a codon tends to be purine-rich. One check of whether a bidi-overlap gene is "real" or not ought to be that the first codon base should be purine rich in both reading directions. This is, in fact, the case for the examples shown above. The aspartyl-tRNA synthetase gene for P. stutzeri has AG1 (1st base, purine) content averaging 59.8%, whereas its bidirectional partner gene ("hypothetical protein") has AG1 = 58.5%. The isopropylmalate isomerase of P. aeruginosa has AG1 = 65.9%, while its antisymmetric partner (TadG) has AG1 = 56.2%.

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How Antisense Genes Are Discovered

In the past ten years or so, a great deal of research has focused on antisense transcription of genes. Normally, RNA gets transcribed from one strand of DNA only. But it turns out, in many cases RNA also gets transcribed off the opposite strand of DNA (an antisense copy), either at the original gene (so-called cis transcription) or at a copy of the gene some distance away (trans transcription). The latter can be a pseudogene, or a normal copy of the gene.

Antisense transcripts occur very widely not only in human DNA but in bacteria, yeast, and (in fact) every place where scientists have looked, and places where they haven't looked. Some of the most interesting discoveries have happened when researchers weren't specifically looking for antisense transcripts but found them by accident. How does that happen? It happens in experiments involving IVET (in vivo expression technology), an important experimental technique for uncovering new genes.

IVET is a powerful gene manipulation strategy for discovering which genes in an organism (a pathogen, usually) are up-regulated or turned on during host infection. Let's say you're studying a new pathogen and you want to get an idea of which genes, in the pathogen, are turned on during the infection process. First, you need a strain of the organism that's disabled by virtue of lacking a working copy of a particular metabolic enzyme, say an enzyme needed for purine metabolism, e.g. purA. Secondly, you need a vector for inserting a promoterless copy of the working gene into the bacterium. What this usually means is, you need a plasmid (a small extra chromosome; many bacteria have them, and they can often be manipulated in the lab) on which to place a functional purA gene. The gene won't be expressed, however, if it lacks a suitable promoter region on the DNA upstream of the gene. That's good; that's what you want. You want to put a promoterless copy of the good gene on the plasmid, along with (this is crucial) a random chunk of DNA from the pathogen, inserted ahead of purA on the plasmid. In practice, it's easy to create a bunch of plasmids with this arrangement: a working copy of purA, and ahead of it, a random chunk of pathogen DNA. The idea is that you now attempt to infect a lab animal with the bacterium containing the plasmid. If the bacterium establishes infection in the animal, presumably it's because a random chunk of DNA happened to contain a promoter region (and associated downstream genes) that gets turned on during infection. If you now isolate the bacterium from the sick animal, you can look to see what kind(s) of genes got transduced into the bacterium.

IVET is a promoter trap technology for selecting bacterial genes that are specifically induced when bacteria infect a host organism. A plasmid vetor contains a random fragment of the chromosome of the pathogen (red) and a promoterless gene (selective marker, burgundy) that encodes an enzyme required for survival. Pooled plasmid-containing clones are inoculated into the mouse (B). Only those bacteria that contain the selective marker fused to a random gene that is transcriptionally active in the host are able to survive. After a suitable infection period, bacteria that express the marker are isolated from the spleen or other organs. The inclusion of a lacZY mutant gene (blue) allows post-selection screening for promoters that are active only in vivo. What you want are bacteria that are lac-positive only in the host environment, not "constitutive" (always-on).

Exactly this sort of technique was used by Silby, Rainey, and Levy to determine which genes were activated in Pseudomonas during colonization of soil. (The IVET technique can be adapted to any scenario in which an organism differentially expresses genes in its adaptation to a "host" environment, even if the environment is, in fact, a plant, or soil in this case, rather than a mouse.) They were looking to see which genes in Pseudomonas play an essential role in that organism's ability to thrive in soil, and they successfully identified more than 50 promoters (and associated fusions) that come alive during soil colonization. When they looked at 22 "soil genes" that got turned on, they found ten previously undescribed genes that were transcribed in the antisense direction from regions overlapping known genes. They called these ten genes "cryptic fusions" because of their un-annotated existence on the supposedly silent, antisense side of known genes.

Cryptic fusions discovered by Silby et al. are shown in grey, in their antisense orientation to known genes (darker grey).

It's not unusual to find that antisense transcripts are playing a regulatory role. When a gene gets transcribed in both directions, the resulting sense and antisense RNAs can combine (by Watson-Crick pairing) to form a double-stranded RNA product, preventing translation of the RNA into protein. But incredibly, sometimes an antisense RNA transcript encodes a legitimate protein (a protein that gets made off the antisense copy). Silby and Levy documented this for the previously unknown cosA gene in Pseudomonas. It seems likely additional antisense proteins await discovery. (Most studies stop at the level of identifying RNA products.)

The finding of antisense transcripts in IVET experiments is common. One of the authors of the Pseudomonas study (Rainey) had previously published a study of rhizosphere-induced genes in Pseudomonas but had not published the fact that 20% of genes found this way were in an antisense orientation to normal genes. Likewise, a 1996 study of Pseudomonas aeruginosa infection in the mouse (Pseudomonas is an opportunistic pathogen) found antisense activity. In fact, the first-ever paper on IVET (by Mahan et al., 1993) described finding antiscript products.

IVET has uncovered a previously unknown "antitranscriptome" world hidden inside living cells. Until we explore this world fully, we won't know how much undiscovered biology we've left on the table.

The Pathogen's Playbook

When comparing pathogenic bacteria with non-pathogenic species of the same genus or family, we often find a common pattern. In the pathogen:

• The genome is often reduced in size (particularly in endosymbionts, but also in others).
• The genome is often shifted in the direction of higher A+T content (lower G+C content).
• Many pseudogenes are present.
• Often, the pathogen is a slow-grower in pure culture (if it can be cultured at all).
• The pathogen has special nutritional needs.

An extreme case that illustrates all of these points is Mycobacterium leprae, the leprosy bacterium. It has fewer genes than its cousin, M. tuberculosis (which in turn has fewer genes than non-pathogenic Mycobacteria); its genomic G+C content is 8% lower than most other Mycobacteria; it contains over 1100 pseudogenes; it has a doubling time of two weeks; and it cannot be grown in pure culture (presumably because of fastidious nutritional requirements).

M. tuberculosis can be grown in the laboratory, but it, and its M. avium-group cousins, are very slow growers, taking anywhere from four days to two weeks to develop colonies on solid media.

It seems likely that some pathogens (certainly members of the Mycobacteria, but also the tiny Tenericutes, e.g. Mycoplasma, among many others) have evolved slow growth as a survival strategy. Certainly, organisms that have evolved an intracellular parasitic lifestyle need to be careful not to out-grow the host, if the relationship is to be a long one.

All of the factors listed above suggest a certain scenario, a "pathogen's playbook," if you will, which can be summarized as follows:

1. The organism invades a warm-booded host.
2. Phagocytes (white blood cells) ingest the organism.
3. The phagocytes undergo a respiratory burst, flooding the microbe(s) with peroxides, hypochlorites, nitrous oxide, and other noxious oxidants.
4. The flood of reactive oxygenated species triggers an SOS response in the microbe.
5. The microbe's DNA undergoes massive damage. 
6. Any surviving microbial cells are now pathogenic.

The SOS response is known to trigger mutagenicity. In Mycobacterium, for example, peroxides (as well as UV light) can induce up-regulation of dnaE, an error-prone polymerase. Since Mycobacteria are known to lack a MutS mismatch repair system, SOS-induced errors in DNA replication will almost certainly include uncorrected frameshift errors leading to the creation of pseudogenes. But that's a good thing, if you're a Mycobacterium interested in forming a longterm relationship with a host cell. The loss of certain genes (as long as they're not essential!) will likely slow your metabolism and make you dependent on host nutrients. Truly non-essential pseudogenes will simply be jettisoned over time, reducing the footprint of the remaining genome. Any pseudogenes that survive will likely have done so because they're now playing an essential gene-silencing role.

Let's expand on that last part. Take the dnaE gene, for example. M leprae has two copies of this gene, only one of which is functional. Suppose both copies were functional at the time of the massive pseudogenization event that converted so many of M. leprae's genes to pseudogenes 9 to 20 million years ago. After the pseudogenization event (probably a phagocytic respiratory burst), one copy of dnaE became a pseudogene. But continued transcription of the pseudogene in the forward direction means the pseudo-mRNA competes with the "normal" dnaE transcript for ribosomal attention. Transcription of the antisense strand of the disabled gene would, of course, create a messenger RNA product that could silence the normal transcript by doublestranded interaction. Either way, once the pseudogenization event is over, dnaE expression is attenuated—as it should be, once pathogenicity has been established.

Is it realistic to think M. leprae transcribes antisense strands of its pseudogenes? Given that E. coli has been found to contain ~1000 antisense transcripts, and given that we know M. leprae transcribes many of its pseudogenes, I think the answer has to be yes.

So the pattern is: infection, respiratory burst, massive mutation, silencing of many genes, and (oh by the way) creation of many brand-new gene products, some of them no doubt quite toxic to the host, as the result of gene truncation and pseudogene expression.

An Example of Antisense Proteogenesis?

The question of how organisms develop entirely new genes is one of the most important open questions in biology. One possibility is that new genes often develop through accidental translation of antisense strands of DNA.

An example of this can be seen with the S1 protein of the 30S bacterial ribosome. If you take the amino-acid sequence for an S1 gene and use it as the query sequence in a blast-p (protein blast), you'll mostly get back hits on other S1 proteins, but you'll also get minor (low-fidelity) hits on polynucleotide phosphorylase. Why? When you do a blast search, the search engine, by default, looks at both DNA strands of target genes (sense and antisense strands) to see if there's a potential sequence match with the query. If there's a match on the antisense strand, it will be reported along with "sense" matches. In the case of the S1 protein, blast-p searches often report weak antisense hits on polynucleotide phosphorylase in addition to strong sense hits on ribosomal S1.

Ribosomal proteins are, of course, among the most highly conserved proteins in nature. It turns out that polynucleotide phosphorylase (PNPase) is very highly conserved as well. It's an enzyme that occurs in every life form (bacteria, fungi, plants, animals), absent only in a scant handful of microbial endosymbionts that have lost the majority of their genes through deletions. While the chemical function of PNPase is well understood (it catalyzes the interconversion of nucleoside diphosphates to RNA), its physiologic purpose is not well understood, although recent research shows that PNPase-knockout mutants of E. coli exhibit lower mutation rates. (Hence, PNPase may actually be involved in generating mutations.)

The bacterium Rothia mucilaginosa, strain DY18, has a (putative) PNPase gene at a genome offset of 1277514. When this gene is used as the query for a blast-p search, the hits that come back include many strong matches for the S1 ribosomal proteins of various organisms. By "strong match," I mean better than 80% sequence identity coupled with an E-value (expectation value) of zero. (Recall that the E-value represents the approximate odds of the match in question happening due to random chance.

If we use the Genome Viewer at genomevolution.org to look at the PNPase gene of Rothia mucilaginosa, we see something extraordinarily peculiar (look carefully at the graphic below). Click to enlarge the following image, or better yet, to see this genome view for yourself, go to this link.

Notice the presence of overlapping sense and antisense open reading frames on a portion of DNA from Rothia mucilaginosa. The top reading frame contains the gene for polynucleotide phosphorylase. The lower (-1 strand) reading frame contains ribosomal S1. To see this in your own browser, go to this link.

Notice that there are overlapping genes. On the top strand is the gene for PNPase; on the bottom strand, in the same location, is a gene for ribosomal S1. These are bidirectionally overlapping open reading frames, something occasionally encountered in virus nucleic acids but rarely seen in bacterial or other genomes.

How do we explain this anomaly? It could be just that: an anomaly, two open reading frames that happen to overlap (but that aren't necessarily translated in vivo). Or it could be that at some point, many millions of years ago, the ribosomal S1 gene of a Rothia ancestor was erroneously translated via the antisense strand, producing a protein with PNPase characteristics. We don't know why PNPase confers survival value (its physiologic purpose is not fully understood), but we do know, with a fair degree of certainty, that PNPase does, in fact, confer survival value—because every organism, at every level of the tree of life, has at least one copy of PNPase. Once Rothia's ancestor, through whatever process, opened up a reading frame on the antisense strand of ribosomal S1, the reading frame stayed open, because it conferred survival value. In this way, the first Rothia PNPase was born. (Arguably.)

At some point in its history, Rothia duplicated its PNPase gene and placed a new copy at genome offset 1650959. Over time, this second copy diverged from the original copy, becoming more like E. coli PNPase (which is also to say, less S1-like). Rothia's second PNPase shows a blast-p similarity of 45% (in terms of AA identities) to E. coli PNPase, with E-value 4.0e-147. It shows a blast-p similarity of 26% (AA identities) with E. coli ribosomal S1 (E-value: 4.0e-17). Neither E. coli PNPase nor Rothia PNPase-2 overlaps an S1 gene. However, both are colocated with the ribosomal S15 protein gene. And you'll find (if you look at lots of bacterial genomes) that PNPase is almost always located immediately next to an S15 ribosomal gene.

Rothia PNPase is an example of an enzyme that may very well have started out as an antisense copy of another protein (the S1 ribosomal protein). Of course, the mere presence of bidirectionally overlapping open reading frames doesn't prove that both frames are actually transcribed and translated in vivo. But the fact that blast-p searches using PNPase as the query almost always turn up faint S1 echoes (in a wide variety of organisms) is highly suggestive of an ancestral relationship between the two proteins.

Evolution and Antisense Translation of DNA

Yesterday I offered a theory for new gene creation which might be called the Erroneous Translation Theory. Basically, I proposed that new proteins arise through frameshifted and/or reversed translation of nucleic acids (translation of antisense strands of DNA).

Erroneous translation of DNA offers interesting possibilities for gain of function. (Recall that most point mutations result in loss of function, and one of the major criticisms of Darwinian theory is that evolution based on accumulation of point mutations cannot account for gain-of-function events.) Wholesale mistranslation via frameshift errors and/or wrong-strand transcription allow for the sudden emergence of entirely new classes of proteins. The unit of change is no longer the single base-pair polymorphism but the functional domain or motif.

An important aspect of antisense-strand translation has to do with stop codons. In DNA, the sequences TCA, TTA, and CTA specify amino acids serine, leucine, and leucine, respectively. But when these three codons are complemented, then read in 5'-to-3' direction—in other words, when they're antisense-translated—they form the stop codons TGA, TAA, and TAG, which tell the cell's protein-making machinery to terminate the production of the current polypeptide. Thus, if a typical gene containing codons TCA, TTA, and CTA is translated "backwards," translation will end prematurely: It will end as soon as a stop codon is encountered.

How important a consideration is this in the real world? Consider the following DNA sequence, which represents the gene for the cytidine deaminase enzyme of Clostridium botulinum:

>Clostridium botulinum A strain ATCC 19397(v1, unmasked), Name: ABS32549.1, CLB_0040, Type: CDS, Feature Location: (Chr: 1, 37028..37465) Genomic Location: 37028-37465 ATGAATGATTATATAGAATATGCAATAATTGAAGCAAAAAAAGCATTAGCAATAGGAGAAGTACCTGTTGGAGCTATTATAGTTAAAGAAAATAAAATTATAGCAAAAAGTCATAATTTAAAAGAGTCATTGAAGGATCCAACAGCTCATGCAGAGATATTAGCTATAAAAGAAGCTTGCAATACAATACATAATTGGAGATTAAAAGGATGTAAGATGTATGTAACATTAGAACCATGTGCTATGTGTGCTAGTGCAATAATTCAATCTAGAATAAGTGAATTGCATATAGGAACCTTTGATCCAGTGGGAGGGGCTTGTGGATCAGTAGTAAATATAACAAATAATAGTTATTTAAAAAATAATTTAAATATTAAATGGTTATATGATGATGAATGTAGTAGAATAATAACAAATTTTTTTAAAAATATTAGATAA

The above sequence is the "sense" strand of the DNA, in 5'-to-3' direction. The sequence below is the corresponding 3'-to-5' complementary sequence (in other words, what's on the antisense strand of DNA):

TACTTACTAATATATCTTATACGTTATTAACTTCGTTTTTTTCGTAATCGTTATCCTCTTCATGGACAACCTCGATAATATCAATTTCTTTTATTTTAATATCGTTTTTCAGTATTAAATTTTCTCAGTAACTTCCTAGGTTGTCGAGTACGTCTCTATAATCGATATTTTCTTCGAACGTTATGTTATGTATTAACCTCTAATTTTCCTACATTCTACATACATTGTAATCTTGGTACACGATACACACGATCACGTTATTAAGTTAGATCTTATTCACTTAACGTATATCCTTGGAAACTAGGTCACCCTCCCCGAACACCTAGTCATCATTTATATTGTTTATTATCAATAAATTTTTTATTAAATTTATAATTTACCAATATACTACTACTTACATCATCTTATTATTGTTTAAAAAAATTTTTATAATCTATT

When the antisense sequence is translated in the normal 5'-to-3' direction, the following amino acid sequence results:

LSNIFKKICYYSTTFIII*PFNI*IIF*ITIICYIYY*STSPSHWIKGSYMQFTYSRLNYCTSTHSTWF*CYIHLTSF*SPIMYCIASFFYS*YLCMSCWILQ*LF*IMTFCYNFIFFNYNSSNRYFSYC*CFFCFNYCIFYIIIH

This sequence of 146 amino acids (shown here using standard one-letter amino-acid abbreviations) contains 10 stop codons (depicted as asterisks). Any attempt to translate the antisense strand of the C. botulinum cytidine deaminase gene will result in (at best) a series of short oligopeptides.

It's tempting to conclude that this is nature's ingenious way of preventing the occurrence of nonsense proteins. Translate the wrong strand of DNA by mistake, and translation quickly terminates. (In the above example, a stop codon occurs every 14 amino acids, on average.) But before you jump to that conclusion, consider the cytidine deaminase gene of Anaeromyxobacter dehalogenans strain 2CP-C:

GTGGACGAGCGCGAGGCGATGCAGGAGGCGCTGGGGCTGGCGCGCGAGGCGGCGGCCCGCGGCGAGGTGCCGGTCGGCGCGGTGGCGCTGTTCGAGGGCCGCGTGGTCGGCCGCGGCGCGAACGCCCGCGAGGCGGCGCGCGATCCCACCGCGCACGCGGAGCTCCTCGCGATCCAGGAGGCGGCGCGCACCCTCGGGCGCTGGCGCCTCACCGGCGTCACGCTGGTGGTGACGCTCGAGCCCTGCGCCATGTGCGCCGGCGCCATGGTGCTCGCCCGCATCGACCGGCTCGTCTACGGGGCGAGCGATCCCAAGGCCGGCTGCACCGGCTCCCTCCAGGACCTGTCGGCGGACCCCCGGCTGAACCACCGGTTCCCGGTGGAGCGCGGCCTGCTGGCCGAGGAGTCCGGCGAGCTCCTCCGGGCCTTCTTCCGGGCCCGCCGGGGCGCCGGGAACGGAAACGGCAACGGCGGCGAGGGTTAG

The translation of the antisense version of this gene is:

LTLAAVAVSVPGAPAGPEEGPEELAGLLGQQAALHREPVVQPGVRRQVLEGAGAAGLGIARPVDEPVDAGEHHGAGAHGAGLERHHQRDAGEAPAPEGARRLLDREELRVRGGIARRLAGVRAAADHAALEQRHRADRHLAAGRRLARQPQRLLHRLALVH

Which contains no stop codons! Why does one version of the gene give ten stop codons when anti-translated, whereas the other version gives zero stop codons? Clostridium botulinum has a genome G+C content of 28% whereas the DNA of Anaeromyxobacter dehalogenans has a G+C content of 74%. The two organisms favor entirely different codons. Anaeromyxobacter uses codons TCA, TTA, and CTA only 0.03%, 0%, and 0.02% of the time, respectively. Clostridium uses the same codons 1.72%, 5.62%, and 4.67% of the time—over 200 times more often than Anaeromyxobacter.

Bottom line: Almost any gene in Anaeromyxobacter (or any high-GC organism, it turns out) can be antisense-translated without generating stop codons. Stop codons occur in antisense genes in inverse proportion to the amount of G+C in the gene.  

If it's true that antisense-strand translation is (or has been) an important source of new proteins in nature, the foregoing observation is tremendously relevant, because it means successful reverse translation has likely occurred far more often in high-GC organisms than in low-GC organisms. It suggests that bacteria with high G+C content in their genomes may, in fact, have been the incubators of early proteins. It implies a "GC Eden" scenario in which early life forms had predominantly high-GC genomes. Low-GC organisms then arose through continuous "AT pressure," from large numbers of accumulated GC-to-AT transition mutations. (We know that GC-to-AT transition mutations occur at a much higher rate than AT-to-GC transitions; this fact is not in dispute.)

Even so, we have to ask: What is the evidence for reverse (antisense-strand) translation having occurred in nature? Is there any such evidence?

More on this subject tomorrow.