Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

Estimation of the source-by-source effect of autorepression on genetic noise.

Transcriptional autorepression has been thought to be one of the simplest control circuits to attenuate fluctuations in gene expression. Here, we explored the effect of autorepression on fluctuations from different noise sources. We theoretically represent the fluctuations in the copy number of proteins as the sum of several terms, each of which is related to a specific noise source and expressed as the product of the source-specific fluctuations under no autorepression (path gain) and the effect of autorepression on them (loop gain). Inspection of each term demonstrates the source-independent noise-attenuating effect of autorepression as well as its source-dependent efficiency. Our experiments using a synthetic autorepression module revealed that autorepression attenuates fluctuations of various noise compositions. These findings indicate that the noise-attenuating effect of autorepression is robust against variation in noise compositions. We also experimentally estimated the loop gain for mRNA noise, demonstrating that loop gains are measurable parameters. Decomposition of fluctuations followed by experimental estimation of path and loop gains would help us to understand the noise-related feature of design principles underlying loop-containing biological networks.

Reconstructing the single-cell-level behavior of a toggle switch from population-level measurements.

Single-cell-level behaviors of cells are typically inferred from ensemble measurements. However, such inferences implicitly assume a biological version of ergodicity: the percentage of cells in a state is identical to the probability to find a cell in that state. While the ergodicity does not always hold, it has been rarely tested. Here, we reveal that the ergodicity does not necessarily hold even for simple toggle switches and that apparent stabilities of the switches are due to a balance between single-cell-level biased stabilities and growth rates differences. Therefore, verification of the ergodicity and reconstructing single-cell-level behaviors are crucial for understanding intracellular systems.

CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor.

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3'-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.

Expression of Nogo protein by growing axons in the developing nervous system.

We produced monoclonal antibody NG1 that strongly binds growing axons in the developing nervous system of mice. This antibody intensely labeled the growth cone of cultured neurons. Although these immunostaining patterns suggested the association of growing axons with the antigen recognized by this antibody, the antigen was identified as Nogo protein, an axonal repulsive factor isolated from the myelin. On the basis of this unexpected finding, we discuss the possible functions of Nogo in the developing nervous system.

An attempt to reveal a role of a transcription/translation feedback loop in the cyanobacterial KaiC protein-based circadian system by using a semi-synthetic method.

The use of synthetic biology to design artificial gene circuits is an important approach for understanding the principles underlying the complicated dynamic behaviours of biomolecular networks, such as genetic switching and biological rhythms. The synthetic approach is also useful in systems biology in that it can be used to create artificial bypasses for processes related to cellular phenomena of interest for their easier analysis. To validate the role of transcription feedback in the cyanobacterial circadian system, we propose an experimental design for a 'semi-synthetic' approach that involves transplantation of the kaiABC genes into Escherichia coli and the construction of chimeric transcriptional outputs. The design principle and preliminary results are discussed.

Theoretical consideration of olfactory axon projection with an activity-dependent neural network model.

Olfactory sensory neurons (OSNs) that express a given odorant receptor (OR) target their axons onto a few specific glomeruli in the olfactory bulb. Although the odorant receptor plays an indispensable role in olfactory axon targeting, the mechanisms underlying this guidance are largely unknown. In particular, there is much controversy regarding the involvement of activity-dependent mechanism in the targeting process. In this study, we developed an activity-dependent self-organization model of the glomerular layer in the olfactory bulb and simulated the targeting of olfactory axons onto the layer. Our model successfully constructed discrete glomeruli that received olfactory axons expressing a common odorant receptor through odorant-evoked neural activities. Furthermore, our model explained the perplexing experimental results that have been reported in olfactory axon targeting. For example, dispersion of olfactory axons in knockout mice for the odorant receptor gene was reasonably reproduced in the simulation. The segregated projection of the axons that express the same odorant receptor transcribed from the different alleles was also successfully simulated if the genetically modified allele was assumed to express a smaller amount of the receptor protein. The activity-dependent model even explained the inconsistent effects of disruption of the activity-evoking ion channel on axons expressing different odorant receptors, although some of these results were regarded as evidence for activity-independency of the olfactory targeting. Taken together, the activity-dependent targeting of olfactory axons seems to be a simple probable mechanism that can provide a unified explanation of glomerular formation.