The evolutionary origin of a complex scrambled gene

Abstract

Some species of ciliates undergo massive DNA elimination and genome rearrangement to construct gene-sized ''chromosomes'' in their somatic nucleus. An example is the extensively scrambled DNA polymerase ␣ gene that is broken into 48 pieces and distributed over two unlinked loci in Stylonychia. To understand the emergence of this complex phenomenon during evolution, we examined DNA polymerase ␣ genes in several earlier diverging species, representing evolutionary intermediates. Mapping these data onto an evolutionary tree suggests that this gene became extensively fragmented and scrambled over evolutionary time through a series of steps, each leading to greater complexity. Our results also suggest a possible mechanism for intron loss by deletion of intron sequences as DNA during development of the somatic nucleus.ciliate ͉ DNA polymerase ͉ hypotrich ͉ intron loss ͉ spirotrich C iliates comprise a diverse group of microbial eukaryotes, or protists. These organisms contain two types of nuclei: a somatic macronucleus that is active during vegetative growth and a germ-line micronucleus that is quiescent except during sexual recombination (1). During sexual conjugation, the two mating cells exchange haploid micronuclei, after which the old macronucleus degrades, and the new diploid micronucleus develops into the new macronucleus. Extensive loss of DNA from the long micronuclear chromosomes, coupled to DNA rearrangement, removes transposons and most intergenic spacer DNA between genes, as well as intragenic spacer DNA, known as internal eliminated segments (IESs). In spirotrichous (formerly hypotrichous) ciliates, the remaining DNA sequences, known as macronuclear destined segments (MDSs), can occupy as little as 2-5% of the germ-line genome. These mostly coding regions assemble together to form Ϸ24,000 types of DNA molecules in the somatic nucleus. Because their average size is just 2.2 kbp and they usually contain only one gene, they are sometimes called ''nano-chromosomes.'' Short telomeres are added to both ends before these molecules are amplified several-thousand-fold (1-3).Furthermore, the germ-line order of the coding regions in 20-30% of the genes in stichotrichous ciliates (a subset of spirotrichous ciliates) are permuted, or scrambled, relative to their linear order in the macronucleus. Although the mechanism of reordering these segments is not well understood, short direct repeats, called pointers, may guide the unscrambling process (1, 4). Invariably, one copy of a repeated word pair is present at the 3Ј end of germ-line segment n and at the 5Ј end of segment n ϩ 1, providing the information to link them together, with only one copy of the repeated word retained in the final DNA molecule (1). Although fragmented genes do occur in other organisms [examples include V(D)J recombination in the vertebrate immune system and split ribosomal RNA genes in the mitochondria of fungi (5) and protists (6, 7)], rarely are they sewn back together at the DNA level.Three different scrambled genes, actin I (8-10), ␣ telomer...

Transcription-Independent Functions of an RNA Polymerase II Subunit, Rpb2, During Genome Rearrangement in the Ciliate, Oxytricha trifallax

“…Ciliates include three oligohymenophoreans with completely sequenced somatic genomes (Ichthyophthirius multifiliis, Paramecium tetraurelia, and Tetrahymena thermophila), a spirotrich (Emeus crassus), and seven stichotrichs (Urostyla sp., Paraurostyla sp., Laurentiella sp., O. nova, Sterkiella histriomuscorum, Stylonychia lemnae, and O. trifallax). With the exception of I. multifiliis, the gene duplication appears specific to stichotrich ciliates, a group that shares germline genome scrambling (Chang et al 2005). The Rpb2 gene duplication in I. multifiliis appears to be an independent duplication event, and its two paralogs could not be classified as Rpb2-a and -b, as defined by the corresponding stichotrich orthologs, based on the sequence alignment.…”

Complexity Measures for Gene Assembly

“…As a matter of fact, these are the shortest DNA molecules known in Nature, see [20]! On the other hand, micronuclear genome is organized on very long chromosomes (about 120 chromosomes, each with about 10 7 bp in S.nova, see [19]), with coding sequences occupying as little as 2 -5% of the genome, see, e.g., [3]. During the process of sexual reproduction, ciliates destroy the old macronuclei and transform a micronucleus into a new macronucleus.…”

“…The macronuclear genes are very short molecules, e.g., ranging in the S.nova organisms between 200bp and 3700bp, with an average of 2200 bp in length, see [22], [19], [3], [4]. As a matter of fact, these are the shortest DNA molecules known in Nature, see [20]!…”

Three models for gene assembly in ciliates: a comparison

“…Macronuclear molecules consist mainly of coding sequences. On the other hand, coding sequences occupy as little as 2 -5 % of the micronuclear molecules of the length about 10 7 bp (in Sterkiella nova, see [5,23]). …”

“…This shuffling and inversion of MDSs is especially visible in a species of ciliates called stichotrichs. Macronuclear molecules are known to be the shortest DNA in Nature, ranging in the Sterkiella nova organisms between 200bp and 3700bp with an average of 2200 bp in length (see [11,5,6,23,24,27]). Macronuclear molecules consist mainly of coding sequences.…”