Computational capability of ecological dynamics.

Ecological dynamics is driven by complex ecological networks. Computational capabilities of artificial networks have been exploited for machine learning purposes, yet whether an ecological network possesses a computational capability and whether/how we can use it remain unclear. Here, we developed two new computational/empirical frameworks based on reservoir computing and show that ecological dynamics can be used as a computational resource. In silico ecological reservoir computing (ERC) reconstructs ecological dynamics from empirical time series and uses simulated system responses for information processing, which can predict near future of chaotic dynamics and emulate nonlinear dynamics. The real-time ERC uses real population dynamics of a unicellular organism, Tetrahymena thermophila. The temperature of the medium is an input signal and population dynamics is used as a computational resource. Intriguingly, the real-time ecological reservoir has necessary conditions for computing (e.g. synchronized dynamics in response to the same input sequences) and can make near-future predictions of empirical time series, showing the first empirical evidence that population-level phenomenon is capable of real-time computations. Our finding that ecological dynamics possess computational capability poses new research questions for computational science and ecology: how can we efficiently use it and how is it actually used, evolved and maintained in an ecosystem?

Digital PCR for determination of cytochrome P450 2D6 and sulfotransferase 1A1 gene copy number variations.

CYP2D6 and SULT1A1 occasionally show copy number variations (CNVs), with a larger number generally indicating greater enzymic activity. However, those variations are difficult to calculate using standard methods. With digital PCR, a recently introduced method for CNV analysis, DNA molecules are subjected to limited dilution and separated into nano-scale droplets prior to a PCR assay. Absolute quantitation of copy number can then be performed with high accuracy and sensitivity by determining the number of droplets showing an amplified signal for the target gene. This is the first report of analyses of CYP2D6 and SULT1A1 CNVs using a digital PCR method with blood sample from Japanese subject. Primers and probes were synthesized for the target and reference genes, and copy number calculation was performed using a QX200 Droplet Digital PCR System. Our results showed that the copy numbers in CYP2D6*5 hetero, non-CNV, and CYP2D6xN subjects were 1, 2, and 3 to 4, respectively. In addition, in non-CNV and multiplication subjects, the number of copies for SULT1A1 was 2 and 3 to 6, respectively. We found that the present digital PCR method was useful as well as accurate. In the future, a combined genotyping, allele distinction, and copy number calculation technique will be helpful for analysis of enzymic activity.

A set of external reference controls/probes that enable quality assurance between different microarray platforms.

RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration-response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT-PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT-PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.

Comprehensive expressional analyses of antisense transcripts in colon cancer tissues using artificial antisense probes.

BACKGROUND: Recent studies have identified thousands of sense-antisense gene pairs across different genomes by computational mapping of cDNA sequences. These studies have shown that approximately 25% of all transcriptional units in the human and mouse genomes are involved in cis-sense-antisense pairs. However, the number of known sense-antisense pairs remains limited because currently available cDNA sequences represent only a fraction of the total number of transcripts comprising the transcriptome of each cell type. METHODS: To discover novel antisense transcripts encoded in the antisense strand of important genes, such as cancer-related genes, we conducted expression analyses of antisense transcripts using our custom microarray platform along with 2376 probes designed specifically to detect the potential antisense transcripts of 501 well-known genes suitable for cancer research. RESULTS: Using colon cancer tissue and normal tissue surrounding the cancer tissue obtained from 6 patients, we found that antisense transcripts without poly(A) tails are expressed from approximately 80% of these well-known genes. This observation is consistent with our previous finding that many antisense transcripts expressed in a cell are poly(A)-. We also identified 101 and 71 antisense probes displaying a high level of expression specifically in normal and cancer tissues respectively. CONCLUSION: Our microarray analysis identified novel antisense transcripts with expression profiles specific to cancer tissue, some of which might play a role in the regulatory networks underlying oncogenesis and thus are potential targets for further experimental validation. Our microarray data are available at http://www.brc.riken.go.jp/ncrna2007/viewer-Saito-01/index.html.

Genome-wide analysis of expression modes and DNA methylation status at sense-antisense transcript loci in mouse.

The functionality of sense-antisense transcripts (SATs), although widespread throughout the mammalian genome, is largely unknown. Here, we analyzed the SATs expression and its associated promoter DNA methylation status by surveying 12 tissues of mice to gain insights into the relationship between expression and DNA methylation of SATs. We have found that sense and antisense expression positively correlate in most tissues. However, in some SATs with tissue-specific expression, the expression level of a transcript from a CpG island-bearing promoter is low when the promoter DNA methylation is present. In these circumstances, the expression level of its opposite-strand transcript, especially when it is poly(A)-negative was coincidentally higher. These observations suggest that, albeit the general tendency of sense-antisense simultaneous expression, some antisense transcripts have coordinated expression with its counterpart sense gene promoter methylation. This cross-strand relationship is not a privilege of imprinted genes but seems to occur widely in SATs.

Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes.

BACKGROUND: Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs). NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. RESULTS: First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard) confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation of cellular events and in pathological conditions. CONCLUSION: Our microarray platform targeting the complementary strand of annotated genes successfully identified novel NATs that could not be identified by publically available cDNA data, and as such could not be detected by the usual "sense-targeting" microarray approach. Differentially expressed NATs monitored by this platform may provide candidates for investigations of gene function. An advantage of our microarray platform is that it can be applied to any genes and target samples of interest.

Comparative expression analysis uncovers novel features of endogenous antisense transcription.

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.

Identification and characterization of a novel anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers: an enzyme that is phylogenetically separated from other anthocyanin acyltransferases.

Anthocyanin acyltransferases (AATs) catalyze a regiospecific acyl transfer from acyl-CoA to the glycosyl moiety of anthocyanins, thus playing an important role in flower coloration. The known AATs are subfamily members of an acyltransferase family, the BAHD family, which play important roles in secondary metabolism in plants. Here, we describe the purification, characterization, and cDNA cloning of a novel anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers. The purified enzyme (hereafter referred to as Ss5MaT2) is a monomeric 46-kDa protein that catalyzes the transfer of the malonyl group from malonyl-CoA to the 4"'-hydroxyl group of the 5-glucosyl moiety of anthocyanins. Thus, it is a malonyl-CoA:anthocyanin 5-glucoside 4"'-O-malonyltransferase. On the basis of the partial amino acid sequences of the purified enzyme, we isolated a cDNA that encodes an acyltransferase protein. The steady-state transcript level of the gene was the highest in recently opened, fully pigmented flowers and was also correlated with the trend observed for an AAT gene responsible for the first malonylation step during salvianin biosynthesis. Immunoprecipitation studies using antibodies against the recombinant acyltransferase protein corroborated the identity of this cDNA as that encoding Ss5MaT2. The deduced amino acid sequence of Ss5MaT2 showed a low similarity (22-24% identity) to those of AATs and lacked the AAT-specific signature sequence. A phylogenetic analysis suggested that Ss5MaT2 is more related to acetyl-CoA:benzylalcohol acetyltransferase (BEAT) rather than to AAT. This is another example in which enzymes with similar, although not identical, substrate evolved from different branches of the BAHD family.

Array-based analysis on tobacco plastid transcripts: preparation of a genomic microarray containing all genes and all intergenic regions.

The plastid genome of higher plants includes about 120 genes. We adopted genomic array technologies to the tobacco plastid genome. A microarray was constructed, consisting of 220 DNA fragments that cover the whole genome sequence. Each DNA fragment corresponds to a single known gene or an intergenic region. We evaluated reliability of this microarray by comparing the plastid RNA level in light- or dark-grown tobacco seedlings. The transcripts encoding photosynthetic subunits increased significantly in light-grown tissues as expected. Furthermore, we found unexpected signals in several intergenic regions, suggesting the existence of novel transcripts in tobacco plastids.