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.

Variations in the WNK1 gene modulates the effect of dietary intake of sodium and potassium on blood pressure determination.

WNK lysine-deficient protein kinase 1 (WNK1) is a member of the WNK family of serine/threonine kinases with no lysine (K), and these kinases have been implicated as important modulators of salt homeostasis in the kidney. It is well known that high dietary sodium and low dietary potassium have been implicated in the etiology of increased blood pressure. However, the blood pressure response to dietary sodium and potassium intake varies considerably among individuals. In this study, we have detected that the haplotypes of the WNK1 gene are associated with blood pressure variations in the general Japanese population. In addition, we investigated the interactions between the haplotypes of the WNK1 gene and dietary sodium and potassium intake for determining inter-individual variations in blood pressure. Our data support the hypothesis that part of the variation in blood pressure response to dietary sodium and potassium intake among individuals can be explained by variations in the WNK1 gene.

Comparative analysis of base correlations in 5' untranslated regions of various species.

Translational initiation signals, such as Shine-Dalgarno (SD) sequences in bacteria and Kozak consensus sequences in vertebrates, direct ribosomes to initiate protein synthesis from mRNAs. Investigating sequence characteristics of these signals is important, particularly to infer translational initiation mechanisms. Although various statistical analyses of translational initiation signals have been done, few have focused on base correlations that assess base dependencies in the signal sequences. We used relative entropy and mutual information to analyze base conservation and correlation, respectively, in the 5' UTRs of various species. In eukaryotes, we found peaks of relative entropy at -3 from the translational start site but no peak of mutual information at that position, indicating that the base at that position (known as the core base of the Kozak sequence) is well conserved but not correlated with neighboring bases and thus functions as a single base. We observed unexpected peaks of mutual information between positions -2 and -1 in most eukaryotes. Surprisingly these base correlation also occurred in some bacteria and archaea, although there were no base preferences at neither position. Various dinucleotide patterns existed at these positions, and the correlation between bases at -2 and -1 may be relevant to the context of translational initiation. Because dinucleotide patterns of correlated pairs of nucleotides at -2 and -1 were not unique within respective organisms, the correlation could not be found when analyzing single-nucleotide conservation. Therefore, mutual information allowed us to discover signals that were not found by simply analyzing base conservation.

Effects of Japanese torreya (Torreya nucifera) seed oil on lipid metabolism in rats.

OBJECTIVE: We investigated effects of Japanese torreya (Torreya nucifera) seed oil containing non-methylene-interrupted polyunsaturated fatty acid of all-cis-5,11,14-eicosatrienoic acid (sciadonic acid) on rat lipid metabolism. METHODS: Male Sprague-Dawley rats were fed the experimental diets based on AIN-93 containing 10% corn, soybean, or torreya oil for 4 wk. Blood and tissues were recovered from each rat, and concentrations of triacylglycerol, cholesterol, and phospholipid in plasma and liver were determined by enzymatic assays. Moreover, fatty acid composition was analyzed for triacylglycerol, cholesterol ester, and phospholipid isolated from plasma and liver lipids by gas liquid chromatography. RESULTS: Plasma triacylglycerol level in rats fed torreya oil was lower than that in rats fed corn or soybean oil, although there were no significant differences in plasma cholesterol and phospholipid levels in all rats. Liver triacylglycerol level was also lower in rats fed torreya oil, whereas liver cholesterol and phospholipid levers were same for all rats. omega-3 Polyunsaturated fatty acids such as 22:6 (omega-3) were lower in plasma and liver lipids of torreya and corn oil groups, whereas omega-6 polyunsaturated fatty acids such as 22:4 (omega-6) and 22:5 (omega-6) were higher. Considerable amounts of sciadonic acid were detected in cholesterol ester, triacylglycerol, and phospholipid in plasma and liver of rats fed torreya oil. CONCLUSION: These observations suggest that torreya seed oil can modify lipid metabolism, resulting in lower triacylglycerol levels in plasma and liver of rats.

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.

Effects of Japanese Torreya (Torreya nucifera) seed oil on the activities and mRNA expression of lipid metabolism-related enzymes in rats.

The mechanism for the plasma and liver triacylglycerol-reducing effects of Japanese torreya (Torreya nucifera) seed oil containing sciadonic acid (all-cis-5, 11, 14-eicosatrienoic acid) is reported. Male SD rats were fed experimental diets containing 10% torreya, corn, or soybean oil for 4 weeks, and the activities and mRNA expression of the enzymes involved in lipid metabolism were measured in the liver. The activities of some hepatic enzymes involved in fatty acid synthesis were lower in the rats fed torreya oil.

Improvement of binding activity of xylan-binding domain by amino acid substitution.

Xylanase J (XynJ) of alkaliphilic Bacillus sp. strain 41M-1 is a multi-domain enzyme and consists of a glycoside hydrolase (GH) family 11 catalytic domain and an additional xylan-binding domain (XBD) belonging to carbohydrate-binding module (CBM) family 36. Random mutations were introduced into the XBD gene and the repertoire was cloned for display on the surface of filamentous phage. The mutant XBD with amino acid substitution T316I (Thr317 was replaced by Ile) showed higher xylan-binding activity compared to the wild-type XBD. Furthermore, hydrolyzing activity of XynJ toward insoluble xylan was also improved by introducing the mutation T316I.