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The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease. The macronuclear genome of the ciliate Oxytricha trifallax, contained in its somatic nucleus, has a unique genome architecture. Unlike its diploid germline genome, which is transcriptionally inactive during normal cellular growth, the macronuclear genome is fragmented into at least 16,000 tiny (∼3.2 kb mean length) chromosomes, most of which encode single actively transcribed genes and are differentially amplified to a few thousand copies each. The smallest chromosome is just 469 bp, while the largest is 66 kb and encodes a single enormous protein. We found considerable variation in the genome, including frequent alternative fragmentation patterns, generating chromosome isoforms with shared sequence. We also found limited variation in chromosome amplification levels, though insufficient to explain mRNA transcript level variation. Another remarkable feature of Oxytricha's macronuclear genome is its inordinate fondness for telomeres. In conjunction with its possession of tens of millions of chromosome-ending telomeres per macronucleus, we show that Oxytricha has evolved multiple putative telomere-binding proteins. In addition, we identified two new domesticated transposase-like protein classes that we propose may participate in the process of genome rearrangement. The macronuclear genome now provides a crucial resource for ongoing studies of genome rearrangement processes that use Oxytricha as an experimental or comparative model.

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ∼45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated. Ciliates are unicellular eukaryotes that rearrange their genomes at every sexual generation when a new somatic macronucleus, responsible for gene expression, develops from a copy of the germline micronucleus. In Paramecium, assembly of a functional somatic genome requires precise excision of interstitial DNA segments, the Internal Eliminated Sequences (IES), involving a domesticated piggyBac transposase, PiggyMac. To study IES origin and evolution, we sequenced germline DNA and identified 45,000 IESs. We found that at least some of these unique-copy elements are decayed Tc1/mariner transposons and that IES insertion is likely an ongoing process. After insertion, elements decay rapidly by accumulation of deletions and substitutions. The 93% of IESs shorter than 150 bp display a remarkable size distribution with a periodicity of 10 bp, the helical repeat of double-stranded DNA, consistent with the idea that evolution has only retained IESs that can form a double-stranded DNA loop during assembly of an excision complex. We propose that the ancient domestication of a piggyBac transposase, which provided a precise excision mechanism, enabled transposons to subsequently invade Paramecium coding sequences, a fraction of the genome that does not usually tolerate parasitic DNA.

The prevailing view of the nuclear genetic code is that it is largely frozen and unambiguous. Flexibility in the nuclear genetic code has been demonstrated in ciliates that reassign standard stop codons to amino acids, resulting in seven variant genetic codes, including three previously undescribed ones reported here. Surprisingly, in two of these species, we find efficient translation of all 64 codons as standard amino acids and recognition of either one or all three stop codons. How, therefore, does the translation machinery interpret a "stop" codon? We provide evidence, based on ribosomal profiling and "stop" codon depletion shortly before coding sequence ends, that mRNA 3' ends may contribute to distinguishing stop from sense in a context-dependent manner. We further propose that such context-dependent termination/readthrough suppression near transcript ends enables genetic code evolution.