Many proteins form complexes that function in reaction pathways. Therefore, to understand protein function, it is necessary to reconstitute complexes and pathways in vitro. However, it is not straightforward to achieve full activity in reconstituted systems. To address this problem, we present a yeast system named “in saccharo” analysis, which uses budding yeast for simultaneous expression and analysis of many kinds of non-host proteins, such as human proteins. For this purpose, vectors that can accommodate many genes are required. Here, we describe the construction of a chromosome vector by insertion of unique barcode sequences (BCs) into the ribosomal RNA gene repeat (rDNA). Each unit of the rDNA has a BC that is used as the target for integration of an external gene. Because rDNA is naturally capable of maintaining many repetitive copies, the vector is expected to retain the numerous external genes that may be required for reconstitution of functional protein complexes and reaction pathways. Consistent with this prediction, we were able to clone three human genes that form the RNA silencing pathway, which has no functional equivalent in budding yeast, and to demonstrate functionality in this in saccharo analysis system.

The tufted puffin Fratercula cirrhata (Charadriiformes: Alcidae) is distributed throughout the boreal and low Arctic areas of the North Pacific, from California, USA to Hokkaido, Japan. Few studies have investigated the genetic diversity of this species. Therefore, we analyzed the genetic diversity of two captive populations using nucleotide sequences of two mitochondrial loci (COX1 and D-loop) and one nuclear locus (RHBG). We sequenced these loci for birds from Tokyo Sea Life Park (Kasai Rinkai Suizokuen), originally from Alaska, and birds from Aqua World Oarai, originally from far eastern Russia. We found five COX1 haplotypes and 17 D-loop haplotypes for the mitochondrial loci, and obtained 14 predicted haplotypes for the nuclear RHBG locus. The major haplotypes of all three loci occurred in individuals from both populations. Thus, there were no clear genetic differences between the populations with respect to these three loci. Although the breeding range of the tufted puffin covers the boreal and low Arctic from California to Hokkaido, our results suggest that the species has not genetically diverged within its breeding range.

In various coastal areas of Japan, naturalized radish populations are observed. Radish is a cruciferous plant and exhibits self-incompatibility, involving a system controlled by a single locus with multiple S alleles. Although the S allele diversity of radish cultivars and wild radishes has been characterized, the S allele distribution in naturalized populations has not yet been analyzed in relation to the positions of the plants in situ. Here, we show the S allele distribution in naturalized radish populations of Yakushima, a small island in the East China Sea, with positions of the plants. Radish plants were sampled in coastal areas in Yakushima, and their S alleles were detected and characterized. Most of the S alleles had been previously identified in radish cultivars. However, four novel S alleles, which may be unique to Yakushima, were also found. Moreover, seeds in siliques from plants growing in the study areas were sampled, and S allele determination in DNA extracted from these seeds suggested that the plants had exchanged their pollen among their close neighbors. There was also a problem in that the PCR amplification of some SRK alleles was difficult because of their sequence diversity in the naturalized populations, as occurs in cultivars. Our results suggest that the exchange of S alleles between cultivars and naturalized populations occurs and that S alleles in naturalized populations are highly diverse. The methodology established in our study should be applicable to other self-incompatible species to dissect the diversity of S allele distribution in naturalized populations.

This study aimed to probe carcinogenic genes and pathways associated with Wilms’ tumor (WT) onset and malignancy progression. After screening, three datasets acquired from the Gene Expression Omnibus database were analyzed. Differentially expressed genes (DEGs) were identified and GO functional enrichment, KEGG pathway enrichment and protein–protein interaction (PPI) were analyzed. The DEGs with top fold change values or top protein interaction scores were used to analyze overall survival based on the TARGET WT dataset. Together, 866 up-regulated genes in GDS1791, 585 up-regulated genes in GDS2010, and 277 down-regulated genes in GDS4802 were found, from which 46 key DEGs were selected for further analysis. In the PPI network, hub positions included COL5A1, COL4A1, ARPP21, SPARCL1, CD86, LY96 and PPP1R12B. The top DEGs (ARPP21, SYNPO, PRRC2B, PPP1R12B, EFCAB2 and LY96) were selected for survival analysis, and they consistently showed a significantly positive correlation with poor survival. Together, five key carcinogenic genes (SYNPO, PRRC2B, PPP1R12B, EFCAB2 and LY96) were highly associated with WT onset and patient survival. These risk genes, interaction networks and enrichments should improve our understanding of the complex molecular mechanisms in WT development and help clinical applications.

ONSEN is a heat-activated LTR retrotransposon in Arabidopsis thaliana. Screens to identify transcriptional regulatory factors of ONSEN revealed a SWI/SNF-like chromatin remodeling protein, DRD1, which cooperates with plant-specific RNA polymerase and is involved in RNA-directed DNA methylation. ONSEN transcript level was increased in the drd1 mutant relative to wild-type under heat stress, indicating that DRD1 plays a significant role in the silencing of activated ONSEN under the stress condition. The transcript level of HsfA2, which is directly involved in transcriptional activation of ONSEN, was not higher in the drd1 mutant than in the wild-type. Interestingly, no transgenerational transposition of ONSEN was observed in the drd1 mutant, even though DNA methylation levels were significantly reduced and expression levels were increased compared to the wild-type. These results suggest that other factors are involved in the regulation of ONSEN transposition in addition to the transcript level of ONSEN.

Arnica mallotopus is a perennial herb endemic to the snowy regions of Japan. At the southern edge of its distribution, in Kyoto Prefecture, overgrazing by sika deer and decreased snowfall have resulted in the rapid decline of A. mallotopus populations. Therefore, there is an urgent need for a conservation genetic analysis of the remaining local populations. In this study, we first developed 13 EST-SSR markers to evaluate genetic variation in A. mallotopus. The average number of alleles per locus was 5.33. Genetic analysis using these markers showed that the investigated samples were classified into two groups corresponding to landscape structure. One group isolated from a tributary of the Yura River showed a strong population bottleneck signal, likely resulting from founder effects and subsequent drifts. On the other hand, the genetic diversity of the second group in the main distribution along the Yura River was higher and less inbred. Overall, our assessment suggested recognizing the two genetic groups as management units in conservation programs for the threatened populations.