We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.

SummaryEpiblast stem cells (EpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos, and thus provide a useful model for studying “primed” pluripotent states. Here, we devised a simple and robust technique to derive high-quality EpiSCs using an inhibitor of WNT secretion. Using this method, we readily established EpiSC lines with high efficiency and were able to use whole embryonic portions without having to separate the epiblast from the visceral endoderm (VE). Expression analyses revealed that these EpiSCs maintained a homogeneous, undifferentiated status, yet showed high potential for differentiation both in vitro and in teratomas. Unlike EpiSCs derived by the original protocol, new EpiSC lines required continuous treatment with the Wnt inhibitor, suggesting some intrinsic differences from the existing EpiSCs. The homogeneous properties of this new version of EpiSCs should facilitate studies on the establishment and maintenance of a “primed” pluripotent state, and directed differentiation from the primed state.

Increasing numbers of sense-antisense transcripts (SATs), which are transcribed from the same chromosomal location but in opposite directions, have been identified in various eukaryotic species, but the biological meanings of most SATs remain unclear. To improve understanding of natural sense-antisense transcription, we performed comparative expression profiling of SATs conserved among humans and mice. Using custom oligo-arrays loaded with probes that represented SATs with both protein-coding and non-protein-coding transcripts, we showed that 33% of the 291 conserved SATs displayed identical expression patterns in the two species. Among these SATs, expressional balance inversion of sense-antisense genes was mostly observed in testis at a tissue-specific manner. Northern analyses of the individual conserved SAT loci revealed that: (i) a smeary hybridization pattern was present in mice, but not in humans, and (2) small RNAs (about 60 to 80 nt) were detected from the exon-overlapping regions of SAT loci. In addition, further analyses showed marked alteration of sense-antisense expression balance throughout spermatogenesis in testis. These results suggest that conserved SAT loci are rich in potential regulatory roles that will help us understand this new class of transcripts underlying the mammalian genome.

Natural antisense transcripts and overlapping sense transcripts are expressed in a variety of tissues, including adult mouse brain. Here we show that a subset of mRNA-like sense-antisense transcript pairs are co-expressed within synaptoneurosomes of adult mouse forebrain, a subcellular fraction that is enriched in pinched-off dendritic spines of pyramidal neurons. Several of these pairs involve mRNAs that have been implicated in synaptic functions and in Alzheimer disease pathways. This study provides evidence that a new class of noncoding RNAs (natural antisense transcripts) are expressed near synapses, and encourages further studies of their roles in neuronal function.