Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

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Background: GABA A receptor ␥2 and ␦ subunits are thought to be responsible for synaptic and extrasynaptic targeting. Results: We demonstrate here that ␣2 and ␣6 subunits can target ␦/␥2 chimeras to synaptic and extrasynaptic sites. Conclusion:The ␣ subunits play a direct role in GABA A receptor targeting. Significance: Different subunits of GABA A receptors encode intrinsic signals to control subcellular targeting.

Brain structure and function experience dramatic changes from embryonic to postnatal development. Microarray analyses have detected differential gene expression at different stages and in disease models, but gene expression information during early brain development is limited. We have generated >27 million reads to identify mRNAs from the mouse cortex for >16,000 genes at either embryonic day 18 (E18) or postnatal day 7 (P7), a period of significant synaptogenesis for neural circuit formation. In addition, we devised strategies to detect alternative splice forms and uncovered more splice variants. We observed differential expression of 3,758 genes between the 2 stages, many with known functions or predicted to be important for neural development. Neurogenesis-related genes, such as those encoding Sox4, Sox11, and zinc-finger proteins, were more highly expressed at E18 than at P7. In contrast, the genes encoding synaptic proteins such as synaptotagmin, complexin 2, and syntaxin were up-regulated from E18 to P7. We also found that several neurological disorder-related genes were highly expressed at E18. Our transcriptome analysis may serve as a blueprint for gene expression pattern and provide functional clues of previously unknown genes and disease-related genes during early brain development.E18 ͉ P7 ͉ brain ͉ transcription factors ͉ neural diseases M ammalian brain development can be largely divided into 2 periods: embryonic and postnatal. Embryonic mouse brain development starts Ϸ10-11 days after gestation (E10-E11) with massive neuronal production from neural stem cells. The development of rodent cerebral cortex is a well-studied model system, where the initial neurons form the subplate layer, although subsequent neurons migrate in an inside-out pattern to form the multilayer cortical structure (1, 2). By embryonic day 18 (E18), neurons start to send out axons and dendrites to be poised for synaptic connections. After birth, the first week of postnatal brain development is characterized by elevated production of astrocytes, which are crucial for neuronal synaptogenesis (3, 4). By postnatal day 7, many neurons start to establish synaptic connections with other neurons, forming a primitive neural circuit. Early brain development is precisely controlled by transcription factors, cell adhesion molecules, receptors and channels, synaptic proteins, and other effectors. A single misstep might result in a severe deformation of the brain circuit. For example, loss of Otx2 function results in the absence of early brain development (5). Since many psychiatric disorders such as autism and mental retardation are closely associated with early brain development, understanding the gene expression profile will facilitate the search for an optimal treatment for these disorders. Previous surveys of early brain development have focused on a small number of genes. More recently, microarray studies and others have revealed differential expression of groups of genes in specific brain regions or associated with brain disorders (6-1...

Genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200-nucleotide resolution. By quantifying BrdU incorporation along pulse-chased replication intermediates from Saccharomyces cerevisiae, we orient 58,651 replication tracks reproducing population-based replication directionality profiles and map 4964 and 4485 individual initiation and termination events, respectively. Although most events cluster at known origins and fork merging zones, 9% and 18% of initiation and termination events, respectively, occur at many locations previously missed. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.