Both cDNA tag-based and DNA chip hybridization assays have revealed widespread transcriptional activity across mammalian genomes, providing a rich source of novel protein-coding and non-coding transcripts. Annotation and functional evaluation of this undefined transcriptome space represents a major step towards the comprehensive definition of biomolecules regulating the properties of living cells, including embryonic stem cells (ESCs) and their derivatives. In this study I analysed 87 rare mRNA transcripts from human ESCs that mapped uniquely to the human genome, in regions lacking evidence for known genes or transcripts. In addition, the transcripts appeared enriched in the hESC transcriptome as enumerated by serial analysis of gene expression (SAGE). Full-length transcripts corresponding to twelve novel LongSAGE tags were recovered and evaluated with respect to gene structure, protein-coding potential, and gene regulatory features. In addition, transcript abundance was compared between RNA isolated from undifferentiated hESCs and differentiated cells. Analysis of full-length transcripts revealed that the novel ORFs did not exceed a size of129 amino acids and no matches were observed to well characterized protein domains. Interesting protein level predictions included small disulfide-bonded proteins, known members of which are important in a variety of biological processes. Transcripts evaluated for differential expression by real-time RT-qPCR (Reverse Transcription followed by real-time quantitative Polymerase Chain Reaction) were found to be variably expressed (0.2- to 4.5-fold) in Day-2 orDay-4 retinoic acid-induced differentiation cultures compared to undifferentiated hESCs. Relative quantitation using a universal reference RNA (derived from pooled adult tissues)showed large differences in novel transcript levels (0.002- to 35-fold) compared to hESCs. Collectively, these results provide a detailed analysis of a set of novel hESC transcripts and their abundance in early and adult differentiated cell types, both of which may advance our understanding of the transcriptional events governing stem cell behavior.

The origin of new coding sequence is a major puzzle in biology. The evolutionary pressures on new sequences are largely unknown, but structural constraints are thought to play a role. Previously, 3' untranslated region (UTR) conversion to open reading frame (ORF) was observed in Saccharomyces. We sought to identify genes that were polymorphic for stop codon position in S. cerevisiae and S. paradoxus. Using strain sequence data from the Saccharomyces Genome Resequencing Project, we found 1336 genes that had evidence of stop codon polymorphism. Of those, we found 62 genes that had evidence of addition to ancestral sequence that represented the conversion of ancestral 3' UTR to derived ORF. Two of these genes, YGL058W and YNL034W-A, are prime candidates for structural studies as they are short proteins with long additions and known structures. In future studies, they will be used to infer any structural constraints on newly evolving proteins.