With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes.

Genome-wide DNA rearrangements occur in many eukaryotes but are most exaggerated in ciliates, making them ideal model systems for epigenetic phenomena. During development of the somatic macronucleus, Oxytricha trifallax destroys 95% of its germ line, severely fragmenting its chromosomes, and then unscrambles hundreds of thousands of remaining fragments by permutation or inversion. Here we demonstrate that DNA or RNA templates can orchestrate these genome rearrangements in Oxytricha, supporting an epigenetic model for sequence-dependent comparison between germline and somatic genomes. A complete RNA cache of the maternal somatic genome may be available at a specific stage during development to provide a template for correct and precise DNA rearrangement. We show the existence of maternal RNA templates that could guide DNA assembly, and that disruption of specific RNA molecules disables rearrangement of the corresponding gene. Injection of artificial templates reprogrammes the DNA rearrangement pathway, suggesting that RNA molecules guide genome rearrangement.Parental RNA transcripts and microRNAs are critical for programming development in metazoa 1-4 , raising the possibility that altered RNA molecules can reprogramme patterning on a developmental or evolutionary timescale 5 . Despite the suggestion of template-directed events involving "an ancestral RNA-sequence cache" 6 there has been limited evidence for a direct role of RNA as a template of information across generations 7,8 . Information transfer from RNA to DNA usually involves polymerization 9 . Here we show that RNA molecules can also organize DNA rearrangements, expanding the epigenetic influence of RNA beyond gene expression and priming or directing DNA and RNA synthesis, editing, modification or repair 9-11 .O. trifallax is a unicellular eukaryote harbouring two kinds of nuclei: germline micronuclei and somatic macronuclei. Diploid micronuclei are transcriptionally inert during vegetative growth but they transmit the germline genome through subsequent generations. Effectively polyploid macronuclei provide all vegetative gene expression, but degrade after fertilization, when new micronuclei and macronuclei develop. DNA differentiation in ciliates such as Oxytricha (also called Sterkiella) involves massive chromosome fragmentation and deletion of transposons and internally eliminated sequences (IESs), accomplishing 95% genome Author Information TEBPα and TEBPβ macronucleus and micronucleus sequences have been submitted to GenBank under accession numbers EU047938-EU047941. Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to L. RNAi against putative templates disrupts rearrangementTo test the hypothesis that putative maternal RNA templates influence rearrangement, we induced RNA interference (RNAi) to target homologous RNA degradation. Oxytricha cells, before and during conjugation, were fed Escherichia coli producing double stranded RNA fragments of two m...

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4279fje; doi: 10.1096/fj.05-4279fje SPECIFIC AIMSOur current understanding of prion biology and disease is largely based on studies performed on mammals, yet basic questions about the physiological function of prion proteins (PrPs) and the molecular nature of prion disorders remain elusive. To facilitate the establishment of non-mammalian models for prion research, we characterized the sequence, structural, and genomic homology between fish and other vertebrate PrPs, and analyzed the distinct molecular mechanisms that shaped the evolution of vertebrate PrP domains. PRINCIPAL FINDINGS Teleost fish possess duplicated PrPs, which are the genetic, structural, and syntenic homologues of mammalian PrP and doppelWe provide new sequence data documenting the existence of two orthologous PrP loci in bony fish (PrP-1 and -2), which display extensive variation in their length and amino acid composition, and which are highly expressed in adult and developing fish brains. Despite the low sequence similarity with their mammalian counterparts, fish PrPs share all the expected PrP structural landmarks, such as an N-terminal repetitive region, a highly conserved hydrophobic domain, and a predicted C-terminal globular domain containing two ␤-strands, three ␣-helices, and a GPI-anchoring motif (Fig. 1A). Our genomic analysis in zebrafish and Fugu shows that PrP-1 and -2 map to different chromosomes which are mosaically syntenic to mammalian PrP chromosomes. Directly adjacent to each PrP there is a PrP-related locus (PrP-rel-1 and -2) encoding a short GPI-anchored polypeptide with the unique PrP hydrophobic domain (Fig. 1A). Cladistic analysis supports the idea that PrP and PrP-rels arose as tandem duplicates-a situation reminiscent of that of mammalian Prnp and Doppel (Dpl)-and later duplicated in block as the consequence of a large chromosomal/genome duplication in a teleost fish ancestor (Fig. 2). Due to the subsequent differential loss of paralogues in the fish genomes, the mosaic synteny is only detectable when both duplicated fish regions are considered together. Vertebrate PrP domains have evolved as modules following independent evolutionary dynamicsOur structural analyses across all vertebrate classes reveals that the N-and C-terminal protein moieties have evolved independently from one another following their own rules and different evolutionary pressures: while the former underwent differential (classspecific) expansion-degeneration cycles in its repetitive domains, the latter retained its basic globular structure despite high sequence divergence.

Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high-level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose-5phosphate ketol-isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 AE 0.41 mg l À1 in shake flasks, which were approximately 26-fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 AE 19.35 mg l À1 were accomplished in shake flasks. Furthermore, titres of 9.90 AE 0.06 g l À1 of tyrosol and 26.55 AE 0.43 g l À1 of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high valueadded aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae.

BackgroundProgrammed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20–30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development.Methodology/Principal FindingsTo understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways.Conclusions/Significance This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.