It should be noted that the scientific literature contains numerous examples of transposases having been domesticated to serve beneficial cellular functions – a process sometimes referred to as exaptation. In most of these cases, a single transposase copy has been co-opted, through mutational modification, for a beneficial variation on what was formerly a selfish proclivity – a bad habit transmuted into a useful skill. In fact, a growing body of evidence suggests that transposase domestication – exotic as it may seem – has been a key player in eukaryotic evolution.

”Evolution is, in its essence, an opportunistic process,” says King Jordan, a biologist at the Georgia Institute of Technology. The transposases “have sequences that can be of use to the host,” continues Jordan, who was not affiliated with the present study, “and that’s where the exaptation comes in. Evolution can take advantage of that fact and, fortuitously, those elements that once were strictly selfish can come to a more mutualistic state with the host genome.”

Most of the time, this involves site-specific DNA-binding by an exapted transposase as in, for example, a transcription factor. It may involve DNA cleavage, as in a recombinase. In the selfish context, these abilities allow the transposase to bind to the ends of its cognate transposon DNA sequence and catalyze cleavage. The results reported by Mariusz Nowacki, Brian Higgins, Genevieve Maquilan, Estienne Swart, Thomas Doak, and Laura Landweber, in last week’s online advance edition of Science, differ from these earlier examples with respect to scope - in that they describe the recruitment of thousands of transposases to function in concert on a genome-wide scale, and in the degree of mutualism involved in the arrangement.  

Oxytricha trifallax is a ciliated protozoan with an unorthodox genomic and cytological scheme. Like other ciliates, O. trifallax possesses two nuclei within a single cytoplasm: a macronucleus and – you guessed it – a micronucleus. The macronucleus contains the somatic genome, the transcription of which underlies the myriad cellular functions of a day in the life of O. trifallax. The micronucleus, on the other hand, is transcriptionally silent during vegetative growth, but houses the diploid germline genome, which undergoes meiosis and is transmitted in the act of conjugation – a unicellular form of sex.

The micronuclear genome is a sprawling 1 gigabase (Gb) hodgepodge in which the 30,000 O. trifallax genes are fragmented and the pieces shuffled and separated by intervening sequences of non-coding DNA. Additionally, the O. trifallax  micronuclear genome harbors thousands of transposons. By contrast, the macronuclear genome measures in at a svelte and gene-dense 50 megabase (Mb), devoid of transposons and intervening sequences.

In the act of conjugation, two O. trifallax of differing mating-type partially fuse, allowing a transfer of micronuclear-derived gametic nuclei. After zygosis, the newly constituted micronucleus is mitotically duplicated. Then, as the old macronucleus is degraded, one of the micronuclear genomes undergoes a radical restructuring, giving rise to a new macronucleus. During the genomic rearrangement the large, complex genome is minced into hundreds of thousands of pieces – averaging about 2 kilobases (kb) in length. Ninety-five percent of the starting sequence ends up in the recycle bin – including the thousands of transposon DNA sequences inhabiting the micronuclear genome. The remaining segments are arranged into nanochromosomes – most consisting of a single gene, a short regulatory sequence, and telomeres – with the help of an RNA template. The nanochromosomes are then duplicated to an average copy number of about one thousand – accounting for the tremendous size of the aptly named macronucleus.

The precise mechanism by which the micronuclear genome is cleaved and re-assembled in the process of macronuclear regeneration has been a mystery. Laura Landweber and colleagues hypothesized a role for the transposases encoded by the thousands of transposons within the micronuclear genome. To test their hypothesis, Landweber and her team employed several molecular biology techniques. First, they applied two distinct methods – Northern hybridization and RT-PCR - to the question of whether transposase genes are actively transcribed during macronuclear regeneration. Northern hybridization allows researchers to observe the presence of a particular mRNA transcript, within a complex mixture of total cellular RNA, by virtue of its binding affinity for a complementary nucleic acid probe.

In RT-PCR (Reverse Transcription Polymerase Chain Reaction), cellular RNA serves as template in the synthesis of complementary DNA (cDNA) through the activity of Reverse Transcriptase enzyme. The resultant cDNA in turn serves as template in a PCR reaction, using site-specific DNA primers designed to amplify only the DNA sequence of interest. Both methods revealed the presence of transposase transcripts after conjugation, but not during vegetative growth – confirming that transposase genes are expressed during, and only during, the post-conjugal period coinciding with genome rearrangement.

Of course, temporal overlap of transposase expression with macronuclear regeneration is hardly proof that the transposases play a role in genome rearrangement. To seek direct evidence of such a role, Landweber’s team applied another molecular technique – RNA interference (RNAi). RNAi involves the introduction of small double-stranded RNA (dsRNA) molecules into the cell. These sequences are then enzymatically cleaved by the cell, resulting in short single-stranded molecules which are bound to complementary mRNA transcripts, thereby blocking their translation into amino acid sequences. The result is post-transcriptional silencing of expression, or gene knockdown.

Landweber’s team applied RNAi using dsRNA sequences designed to target transposase transcripts. They found that microinjecting dsRNA twelve to fifteen hours after conjugation resulted in a low rate of survival and decreased rate of growth. They extracted total DNA from progeny cells and performed PCR, targeting portions of several genes. The amplification products of PCR reactions are typically stained and allowed to migrate through an agarose gel in response to an electrical current. The rate of migration is inversely proportional to the size of the amplified fragments.

So, by comparison to the rate of migration of a standard of known size, researchers can determine the approximate size of the amplified sequence. Due to the genomic rearrangements associated with macronuclear regeneration, the expected PCR products from amplification of the macronuclear versions of these sequences differ considerably in size from the expected amplification products of the micronuclear versions.

In the Princeton experiments, DNA extracted from normal cells produced observable PCR products of a size consistent with amplification of only the rearranged macronuclear sequences. This is expected because, under normal cellular conditions, following conjugation the newly regenerated macronuclear nanochromosomes are amplified to an average copy number of roughly one thousand. The relatively rare micronuclear sequences are much more difficult to detect. However, DNA extracted from cells in which transposase genes were silenced by RNAi did, in fact, produce an additional PCR product of a size consistent with amplification of unprocessed micronuclear DNA – for each of the genes considered. This accumulation of micronuclear precursors suggests a breakdown in the cells’ normal genomic rearrangement.

Additionally, the researchers sequenced PCR products for one of the amplified genes – TEBPβ. Examination of the nucleotide sequence revealed various examples of aberrant rearrangement - exhibiting improper linear order of gene segments, retention of intervening sequences, and/or cryptic junctions between gene segments.

Going farther, Landweber’s group verified an increased abundance of large DNA molecules (> 12kb) in transposase-knockdown cells relative to normal cells – by two methods: gel mobility assay and DNA hybridization. Recall that the normal macronuclear genome comprises nanochromosomes roughly 1 kb in length – much smaller than the micronuclear chromosomes. The accumulation of larger DNA molecules in knockdown cells is consistent with stalled genome rearrangement in the absence of transposase. Furthermore, the hybridization experiments utilized DNA probes selected to bind transposase genes. The increased abundance of hybridization products in knockdown cells likewise reflects the inability of these cells to excise transposon DNA in the absence of transposase.

Taken together, these results convincingly demonstrate that active transposases play an integral role in the removal of transposon DNA from the macronuclear genome – as part of the normal developmental cycle of O. trifallax.  This is a dramatic example of the remarkable ingenuity of evolution. The transposon literature contains numerous examples of the domestication of transposases for cellular functions. This recent work by Nowacki et al, however, marks the first documentation of a large body of transposases recruited en masse to serve a global function throughout a genome. Additionally, the O. trifallax case appears to be unique with regard to its degree of mutualism.

Under the standard model of transposase exaptation, the transposase is said to be domesticated – it is tamed, as a result of mutation – and stripped of its normal selfish behavior: namely, the mobilization of transposon DNA. In the O. trifallax case, by contrast, the transposases appear to maintain their normal selfish function – and it is precisely this innate activity which has been harnessed by the host.

“Here’s an example, in biology,” Landweber explains, “where the junk DNA – the transposons – have actually been recruited. It’s almost like there’s an ad: ‘Available for Residence’… if they provide, for their rent, the transposase protein. And rent has to be paid at a very precise time in the life cycle of the organism, when it needs it.” In this symbiotic relationship, the host genome gets the benefit of the transposases’ cutting and pasting skills for its own genomic renovation project; in return the transposases get, as Landweber puts it, “squatters’ rights” – a home, sweet home of their own in the micronucleus.

In the early days of the genomic era, it was widely noted that significant portions of many eukaryotic genomes, including our own, comprise so called “junk DNA” – including transposons. Indeed, less than 2% of the human genome actually encodes cellular proteins. More recently, however, what is already being referred to as the post-genomic era is coming to be characterized by an increasing awareness that more than meets the eye may be going on in the vast, sprawling dark matter of the genome. Domesticated transposases, small non-coding RNAs, anti-sense transcription, ultraconserved elements, all suggest layers of genomic subtlety and complexity unimaginable only a few years ago. Landweber and company’s findings reveal yet another layer – and add an important chapter to that discussion.

“This is obviously one of the most striking examples telling us that transposable elements can really be easily co-opted by the host,” says Claudio Casola, a biologist at Indiana University. When asked how these findings bear on the broader question of transposons as junk DNA, Casola – who was not affiliated with the present work - chooses his words carefully. “I wouldn’t say that most TEs are friends of the host genome – not at all. In most cases they’re still dangerous. But just calling them junk? It’s so…” He chuckles softly, repressing the impulse to cringe, and pauses, as if trying to think of a suitable non-expletive to complete the thought. “It’s such a bad name,” he begins again, “that I really don’t like it at all.” He continues, with an almost Steinbeckian empathy, “Because even if they’re just doing their job – they’re just, you know, spreading around in the genome – it’s not junk. It’s dependent on the point of view. If you’re a transposable element, you’re just trying to survive. So, in that case, the rest of the genome is junk – from your point of view.”

It is appropriate that in this Year of Darwin we behold the humble ciliate, and the much-maligned transposon, and the assorted “junk” so intimately woven into the fabric of our being and recognize that all around us, among us, above us, and below us, “endless forms most beautiful and most wonderful have been, and are being evolved.”

REFERENCE: 

Mariusz Nowacki, Brian P. Higgins, Genevieve M. Maquilan, Estienne C. Swart, Thomas G. Doak, and Laura F. Landweber, “A Functional Role for Transposases in a Large Eukaryotic Genome”, Science, 16 April 2009 (10.1126/science.1170023)

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