Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing. We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified. We report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.

The ciliated protozoan Tetrahymena thermophila undergoes extensive programmed DNA rearrangements during the development of a somatic macronucleus from the germ line micronucleus in its sexual cycle. To investigate the relationship between programmed DNA rearrangements and transposable elements, we identified several members of a family of non-long terminal repeat (LTR) retrotransposons (retroposons) in T. thermophila , the first characterized in the ciliated protozoa. This multiple-copy retrotransposon family is restricted to the micronucleus of T. thermophila . The REP ( Tetrahymena non-LTR retroposon) elements encode an ORF2 typical of non-LTR elements that contains apurinic/apyrimidinic endonuclease (APE) and reverse transcriptase (RT) domains. Phylogenetic analysis of the RT and APE domains indicates that the element forms a deep-branching clade within the non-LTR retrotransposon family. Northern analysis with a probe to the conserved RT domain indicates that transcripts from the element are small and heterogeneous in length during early macronuclear development. The presence of a repeated transposable element in the genome is consistent with the model that programmed DNA deletion in T. thermophila evolved as a method of eliminating deleterious transposons from the somatic macronucleus.

NASP is an essential protein in mammals that functions in histone transport pathways and maintenance of a soluble reservoir of histones H3/H4. NASP has been studied exclusively in Opisthokonta lineages where some functional diversity has been reported. In humans, growing evidence implicates NASP miss-regulation in the development of a variety of cancers. Although a comprehensive phylogenetic analysis is lacking, NASP-family proteins that possess four TPR motifs are thought to be widely distributed across eukaryotes.

Darwin's assertion that “it is mere rubbish thinking, at present, of origin of life” is no longer valid. By synthesizing origin of life (OoL) research from its inception to recent findings, with a focus on (i) proof-of-principle prebiotically plausible syntheses and (ii) molecular relics of the ancient RNA World, we present a comprehensive up-to-date description of science's understanding of the OoL and the RNA World hypothesis. Based on these observations, we solidify the consensus that RNA evolved before coded proteins and DNA genomes, such that the biosphere began with an RNA core where much of the translation apparatus and related RNA architecture arose before RNA transcription and DNA replication. This supports the conclusion that the OoL was a gradual process of chemical evolution involving a series of transitional forms between prebiotic chemistry and the last universal common ancestor (LUCA) during which RNA played a central role, and that many of the events and their relative order of occurrence along this pathway are known. The integrative nature of this synthesis also extends previous descriptions and concepts and should help inform future questions and experiments about the ancient RNA World and the OoL.