Fitness Landscape of the Fission Yeast Genome

The relationship between DNA sequence, biochemical function and molecular evolution is relatively well-described for protein-coding regions of genomes, but far less clear in non-coding regions, particularly in eukaryote genomes. In part, this is because we lack a complete description of the essential non-coding elements in a eukaryote genome. To contribute to this challenge, we used saturating transposon mutagenesis to interrogate the Schizosaccharomyces pombe genome. We generated 31 million transposon insertions, a theoretical coverage of 2.4 insertions per genomic site. We applied a five-state hidden Markov model (HMM) to distinguish insertion-depleted regions from insertion biases. Both raw insertion-density and HMM-defined fitness estimates showed significant quantitative relationships to gene knockout fitness, genetic diversity, divergence and expected functional regions based on transcription and gene annotations. Through several analyses, we conclude that transposon insertions produced fitness effects in 66-90% of the genome, including substantial portions of the non-coding regions. Based on the HMM, we estimate that 10% of the insertion depleted sites in the genome showed no signal of conservation between species and were weakly transcribed, demonstrating limitations of comparative genomics and transcriptomics to detect functional units. In this species, 3' and 5' untranslated regions were the most prominent insertion-depleted regions that were not represented in measures of constraint from comparative genomics. We conclude that the combination of transposon mutagenesis, evolutionary and biochemical data can provide new insights into the relationship between genome function and molecular evolution

Human proton/oligopeptide transporter (POT) genes: Identification of putative human genes using bioinformatics

Purpose: The proton-dependent oligopeptide transporters (POT) gene family currently consists of ∼70 cloned cDNAs derived from diverse organisms. In mammals, two genes encoding peptide transporters, PepT1 and PepT2 have been cloned in several species including humans, in addition to a rat histidine/peptide transporter (rPHT1). Because the Candida elegans genome contains five putative POT genes, we searched the available protein and nucleic acid databases for additional mammalian/human POT genes, using iterative BLAST runs and the human expressed sequence tags (EST) database. The apparent human orthologue of rPHT1 (expression largely confined to rat brain and retina) was represented by numerous ESTs originating from many tissues. Assembly of these ESTs resulted in a contiguous sequence covering ∼95% of the suspected coding region. The contig sequences and analyses revealed the presence of several possible splice variants of hPHT1. A second closely related human EST-contig displayed high identity to a recently cloned mouse cDNA encoding cyclic adenosine monophosphate (cAMP)-inducible 1 protein (gi:4580995). This contig served to identify a PAC clone containing deduced exons and introns of the likely human orthologue (termed hPHT2). Northern analyses with EST clones indicated that hPHT1 is primarily expressed in skeletal muscle and spleen, whereas hPHT2 is found in spleen, placenta, lung, leukocytes, and heart. These results suggest considerable complexity of the human POT gene family, with relevance to the absorption and distribution of cephalosporins and other peptoid drugs