A 1.5 to 3 Mb microdeletion of chromosome 22q11.2 with loss of multiple genes including histone cell cycle regulator (HIRA) causes 22q11.2 deletion syndrome (22q11.2 DS), a common disorder with variable manifestations including congenital malformations affecting the heart, palate and kidneys in association with neurodevelopmental, psychiatric, endocrine and autoimmune abnormalities. The aim of this study was to develop a TaqMan based dosage analysis PCR (TaqMan qPCR) for use as a rapid, cost-effective test for clinically suspected patients fulfilling previously described criteria for molecular diagnosis of 22q11.2 DS in a lower middle-income country where the cost of testing limits its use in routine clinical practice. Nineteen patients were recruited with informed consent following ethical approval from the Ethics Review Committee (ERC), Lady Ridgway hospital, Colombo. Dosage analysis of extracted DNA was performed using a TaqMan qPCR assay by amplifying regions within the target (HIRA) and control [Testin LIM domain protein (TES)] genes of a suspected patient (P) and unaffected person (N). For detection of a deletion, the normalized values (HIRA / TES dosage) of a patient were compared with normalized values of an unaffected person. A ratio of P:N of 0.5 confirmed presence of a deletion while a ratio of 1.0 refuted this. Seven of 19 (37%) cases were confirmed to have a HIRA deletion, confirming the diagnosis of 22q11.2 DS, with these results being in complete agreement with those of fluorescence in-situ hybridization (FISH), (performed in 9/19 (47.3%) of recruited cases) and whole exome sequencing (WES) (all 19 samples tested). This TaqMan qPCR assay was able to reliably distinguish HIRA deleted cases from the non-deleted ones. The assay was both less expensive and faster compared to commercially available alternatives in our setting, including FISH and multiple ligation-dependent probe amplification (MLPA).

The mitochondrial cytochrome b gene (Cytb) of the Japanese field vole (Microtus montebelli), an herbivorous rodent, was subjected to an analysis of sequence variation with the objective of elucidating the population histories of this species. Construction of a phylogenetic tree revealed the existence of several region-specific lineages in Honshu and Kyushu, which were evenly separated from each other. In consideration of the documented time-dependent evolutionary rates of rodents, the estimated divergence times indicate that the region-specific lineages of M. montebelli emerged 160,000-300,000 years ago. In a haplotype network, the region-specific lineages from northern and central Honshu tended to show star-shaped clusters, with additional internal star-shaped clusters, indicative of two periods of population expansion. The onsets of these expansions were estimated to have occurred 15,000 and 10,000 years ago, respectively, suggestive of association with the two periods of rapid warming following the last glacial maximum (LGM). In contrast, such predicted post-LGM expansion events were less pronounced in the southern lineages, implying latitudinal dependence of the effect of the LGM on population dynamics. Sado Island haplotypes exhibited a network with a star-shaped pattern and a 10,000-year-old expansion signal, surrounded by a Honshu haplotype cluster with a 15,000-year-old expansion signal, suggesting that post-LGM expansion events contributed to the formation of the Sado population. A reanalysis of Cytb sequences of the Japanese hare (Lepus brachyurus), which has a similar geographic range to the voles, yielded results that were consistent with those of the vole analysis, confirming that the characteristics of the post-LGM expansion event were dependent on latitude, involved two successive expansion events, and enabled migration across deep straits. It seems reasonable to infer that the environmental changes that occurred during the warm periods following the LGM were a contributing factor in the expansion of the distribution range of newly emerged haplotype groups.

The sequencing of PCR fragments amplified from specific regions of genomes is a fundamental technique in molecular genetics. Sanger sequencing is commonly used for this analysis; however, amplicon sequencing utilizing next-generation sequencing has become widespread. In addition, long-read amplicon sequencing, using Nanopore or PacBio sequencers to analyze long PCR fragments, has emerged, although it is often more expensive than Sanger sequencing. Recently, low-cost commercial services for full-length plasmid DNA sequencing using Nanopore sequencers have been launched in several countries, including Japan. This study explored the potential of these services to sequence long PCR fragments without the need for cloning into plasmid DNA, as cloning long PCR fragments or blunt-end PCR fragments into plasmids is often challenging. PCR fragments of 4-11 kb, amplified from the DFR-B gene involved in the biosynthesis of anthocyanin, with or without Tpn1 transposons in Japanese morning glory (Ipomoea nil), were circularized using T4 DNA ligase and analyzed as templates. Although some inaccuracies in the length of homopolymer stretches were observed, the remaining sequences were obtained without significant errors. This method could potentially reduce the labor and costs associated with cloning, primer synthesis and sequence assembly, thus making it a viable option for the analysis of long PCR fragment sequences. Moreover, this study reconfirmed that Tpn1 transposons are major mutagens in I. nil and demonstrated their transposition in the Violet line, a long-used standard in plant physiology.

In our study, we aimed to identify new mutants resulting from ONSEN transposition in Arabidopsis thaliana by subjecting nrpd1 mutants to heat stress. We isolated a mutant with a significantly elongated hypocotyl, named "Long hypocotyl in ONSEN inserted line 1" (HYO1). This phenotype was heritable, with progeny consistently displaying longer hypocotyls than the wild type. Genetic analysis revealed that this trait was due to a single recessive mutation. Further mapping and sequencing identified the insertion of ONSEN in the HY2 gene, a crucial regulator of hypocotyl elongation. The insertion disrupted HY2 transcription, as confirmed by quantitative PCR, leading to the observed phenotype. To assess the influence of the nrpd1 background, we generated lines backcrossed twice to wild-type Col-0, and the results were consistent with those observed in the original mutant lines. Furthermore, we examined the effect of HY2 and HYO1 mutations on flowering time by analyzing the expression levels of FT. The hyo1 mutant exhibited earlier flowering compared to both the wild type and nrpd1 mutants, with increased FT expression levels. This research underscores the significant impact of ONSEN transposition on gene function and phenotypic variation in Arabidopsis thaliana, providing new insights into the mutagenic potential of transposons and their role in shaping plant traits.

Next-generation RNA sequencing analysis was performed to develop 13 novel expressed sequence tag-simple sequence repeat markers to evaluate the genetic variation in the near-threatened halophyte Limonium tetragonum (Thunb.) A. A. Bullock. In the four populations examined, the total number of alleles at each locus ranged from two to seven, with an average of 3.1. The average observed and expected heterozygosity ranged from 0.00 to 0.13 and 0.28 to 0.78, respectively. Three of the 13 loci had the same homozygous alleles within populations, but different alleles among populations. Compared to other halophytes, relatively low genetic diversity was observed in this species. Further studies are necessary to determine the population demography of L. tetragonum and to clarify the cause of its low genetic diversity.

Strict control of the expression levels of heterologously introduced protein-coding genes is important for the functional analysis of the protein of interest and its effective use in new situations. For this purpose, various promoters with different expression strengths, codon optimization, and expression stimulation by low molecular weight compounds are commonly used. However, methods to control protein expression levels by combining regulation of translation efficiency have not been studied in detail. We previously observed relatively high basal expression of Cre, when it was heterologously expressed in fission yeast. Here, we used a fission yeast strain that is susceptible to centromere disruption and thus highly sensitive to Cre levels and report successful fine-tuning of heterologous Cre expression by modulating the Cre translation efficiency. To inhibit Cre translation initiation, we generated two mutations in the 5' untranslated region of the Cre mRNAs which both interfered with the scanning process of start codon recognition, mediated by the specialized ribosomal subunits. These mutations successfully reduced the levels of exogenously expressed Cre to different degrees in fission yeast. Combining them with different promoter strengths allowed us to conduct centromere-disruption experiments in fission yeast. Our data indicate that modification of translational control is an additional tool in heterologous gene expression.

Nucleosomes are complexes of DNA and histone proteins that form the basis of eukaryotic chromatin. Eukaryotic histones are descended from Archaean homologs; however, how this occurred remains unclear. Our previous genetic analysis on the budding yeast nucleosome identified 26 histone residues conserved between S. cerevisiae and T. brucei; 15 that are lethal when mutated and 11 that are synthetically lethal with deletion of the FEN1 nuclease. These residues are partially conserved in nucleosomes of a variety of giant viruses, allowing us to follow the route by which they were established in the LECA (Last Eukaryote Common Ancestor). We analyzed yeast nucleosome genetic data to generate a model for the emergence of the eukaryotic nucleosome. In our model, histone H2B-H2A and H4-H3 doublets found in giant virus nucleosomes facilitated the formation of the acidic patch surface and nucleosome entry sites of the eukaryotic nucleosome, respectively. Splitting of the H2B-H2A doublet resulted in the H2A variant, H2A.Z., and subsequent splitting of the H4-H3 doublet led to a eukaryotic specific domain required for chromatin binding of H2A.Z. We propose that the LECA emerged when the newly-split H3 N-terminal horizontally acquired a common N-tail found in extinct pre-LECA lineages and some extant giant viruses. This hypothesis predicts that the emergence of the H3 variant CENP-A and establishment of CENP-A-dependent chromosome segregation occurred after the emergence of the LECA, implying that the root of all eukaryotes is assigned within Euglenida.

Next-generation sequencing (NGS) has become widely available and is routinely used in basic research and clinical practice. The reference genome sequence is an essential resource for NGS analysis, and several population-specific reference genomes have recently been constructed to provide a choice to deal with the vast genetic diversity of human samples. However, resources supporting population-specific references are insufficient, and it is burdensome to perform analysis using these reference genomes. Here, we constructed a set of resources to support NGS analysis using the Japanese reference genome, JG. We created resources for variant calling, variant effect prediction, gene and repeat element annotations, read mappability and RNA-seq analysis. We also provide a resource for reference coordinate conversion for further annotation enrichment. We then provide a variant calling protocol with JG. Our resources provide a guide to prepare sufficient resources for the use of population-specific reference genomes and can facilitate the migration of reference genomes.

β-sitosterol is a natural plant steroidal compound with anti-cancer properties against various tumors. This work attempts to explore the inhibitory effect of β-sitosterol on the progression of lung adenocarcinoma (LUAD) and further analyze its targets.

To explore the oncogenic mechanism of FOXM1 in the tumor microenvironment (TME) regarding triple negative breast cancer (TNBC) promotion. The mRNA and protein levels of target genes in TNBC cells and their exosomes were detected by RT-qPCR and western blot. Co-culture models of TNBC cells and THP-1/M0 macrophages was established to detect the impact of co-culture on FOXM1 expression and macrophage polarization direction. The bioinformatics website was used to predict the binding sites between the FOXM1 and IDO1 promoter, which were further validated using dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. Finally, after erastin-induced ferroptosis, Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and other experiments were conducted to investigate whether the FOXM1/IDO1 axis regulates M2 macrophage polarization through ferroptosis. It was found that FOXM1 was highly expressed in exosomes derived from TNBC cells, and TNBC cells upregulated FOXM1 expression in THP-1 cells through exosomes to promote M2 macrophage polarization. Furthermore, FOXM1 upregulated IDO1 in M2-type TAMs by regulating transcription. Lastly, FOXM1/IDO1 inhibited ferroptosis, promoting M2 macrophage polarization, thereby advancing TNBC progression. In conclusions, FOXM1 derived from TNBC cell-derived exosomes activated IDO1 transcription in TAMs to inhibit ferroptosis, promoting TAMs' M2 polarization and exerting carcinogenic effects.

Mycoplasmas, autonomously culturable bacteria with the smallest genome, are an important organism to understand the minimal form of life. Mutagenesis using mutagens is a useful methodology for understanding the essential regions of genomic information. Ultraviolet light (UV) and trimethyl psoralen (TMP) are mutagens known to induce various mutations; the latter is reported to specifically induce deletions in nematodes. However, their mutagenic effects on mycoplasma are not known. Here, we exposed Metamycoplasma salivarium to UV-C light or TMP and UV-A as mutagens, and analyzed the mutational pattern after serial cultivation ranging from 34 to 56 rounds for different lineages. Our results showed that more deletions, but fewer point mutations, were induced with TMP and UV-A than with UV-C, indicating the usefulness of TMP in inducing deletions. In addition, we compared our results with mutational data from other studies, which suggested that the combination of TMP and UV-A or UV-C exposure both induced point mutations that were highly biased toward C→T and G→A transitions. These data provide useful basic knowledge for mutational studies on M. salivarium.

Response regulators (RRs) are implicated in various developmental processes as well as environmental responses by acting as either positive or negative regulators, and are crucial components of cytokinin signaling in plants. We characterized 36 RRs in rice (Oryza sativa L.; Os) using in silico analysis of publicly available data. A comprehensive analysis of OsRR family members covered their physicochemical properties, chromosomal distribution, subcellular localization, phylogeny, gene structure, distribution of conserved motifs and domains, and gene duplication events. Gene Ontology analysis indicated that 22 OsRR genes contribute mainly to the cytokinin response and signal transduction. Predicted cis-elements in RR promoter sequences related to phytohormones and abiotic stresses indicated that RRs are involved in hormonal and environmental responses, supporting previous studies. MicroRNA (miRNA) target analysis showed that 148 miRNAs target 29 OsRR genes. In some cases, multiple RRs are targets of the same miRNA group, and may be controlled by common stimulus responses. Based on the analysis of publicly available gene expression data, OsRR4, OsRR6, OsRR9, OsRR10, OsRR22, OsPRR73 and OsPRR95 were found to be involved in responses to abiotic stresses. Using quantitative reverse transcription polymerase chain reaction we confirmed that six of these RRs, namely OsRR4, OsRR6, OsRR9, OsRR10, OsRR22 and OsPRR73, are involved in the response to salinity, osmotic, alkaline and wounding stresses, and can potentially be used as models to understand molecular mechanisms underlying stress responsiveness.

Intraspecific variation in specialized metabolites plays a crucial role in the adaptive response to diverse environments. Two major subspecies, japonica and indica, are observed in Asian cultivated rice (Oryza sativa L.). Previously, we identified (3R)-β-tyrosine, a novel nonproteinogenic β-amino acid in plants, along with the enzyme tyrosine aminomutase (TAM1), which is required for β-tyrosine biosynthesis, in the japonica cultivar Nipponbare. Notably, TAM1 and β-tyrosine were preferentially distributed in japonica cultivars compared with indica cultivars. Considering its phytotoxicity and antimicrobial activity, intraspecific variation in β-tyrosine may contribute to the defensive potential of japonica rice. Investigation of the evolutionary trajectory of TAM1 and β-tyrosine should enhance our understanding of the evolution of rice defense. However, their distribution patterns in O. rufipogon, the direct ancestor of O. sativa, remain unclear. Therefore, in this study, we extensively examined TAM1 presence/absence and β-tyrosine content in 110 genetically and geographically diverse O. rufipogon accessions and revealed that they are characteristically observed in the ancestral subpopulation of japonica rice, while being absent or slightly accumulated in other subpopulations. Thus, we conclude that TAM1 and β-tyrosine in japonica rice are likely derived from its ancestral subpopulation. Furthermore, the high and low TAM1 possession rates and β-tyrosine content in japonica and indica rice, respectively, could be attributed to distribution patterns of TAM1 and β-tyrosine in their ancestral subpopulations. This study provides fundamental insights into the evolution of rice defense.

We investigated the variation and geographical distribution of the Pseudo-regulator response 37 (Setaria italica PRR37; SiPRR37) gene, which is involved in heading time (photoperiodism) in foxtail millet. An allele of the SiPRR37 gene, in which an approximately 4.9-kb transposable element (designated TE1) is inserted (a loss-of-function or reduction-of-function type), is distributed sporadically in East Asia and broadly in Southeast Asia and South Asia, implying that this gene is important in latitudinal adaptation. In addition, we found a new allele of SiPRR37 with an insertion of a 360-bp TE (TE2) at this locus and investigated the geographical distribution of this new type. This SiPRR37 allele with TE2 is distributed in Japan, Korea, Nepal, Iran and Turkey. Both TE1 and TE2 are useful markers for tracing foxtail millet dispersal pathways in Asia.

Homeostasis is essential for muscle repair and regeneration after skeletal muscle exercise. This study investigated the role of methyltransferase-like 21C (METTL21C) in skeletal muscle of mice after exercise and the potential mechanism. First, muscle samples were collected at 2, 4 and 6 weeks after exercise, and liver glycogen, muscle glycogen, blood lactic acid and triglyceride were assessed. Moreover, the expression levels of autophagy markers and METTL21C in skeletal muscle were analyzed. The results showed that the expression levels of METTL21C and MYH7 in the gastrocnemius muscle of mice in the exercise group were significantly higher after exercise than those in the control group, which suggested that long-term exercise promoted the formation of slow-twitch muscle fibers in mouse skeletal muscle. Likewise, the autophagy capacity was enhanced with the prolongation of exercise in muscles. The findings were confirmed in mouse C2C12 cells. We discovered that knockdown of Mettl21c reduced the expression of MYH7 and the autophagy level in mouse myoblasts. These findings indicate that METTL21C promotes skeletal muscle homeostasis after exercise by enhancing autophagy, and also contributes to myogenic differentiation and the formation of slow muscle fibers.

Primula secundiflora is an insect-pollinated, perennial herb belonging to the section Proliferae (Primulaceae) that exhibits considerable variation in its mating system, with predominantly outcrossing populations comprising long-styled and short-styled floral morphs and selfing populations comprising only homostyles. To facilitate future investigations of the population genetics and mating patterns of this species, we developed 25 microsatellite markers from P. secundiflora using next-generation sequencing and measured polymorphism and genetic diversity in a sample of 30 individuals from three natural populations. The markers displayed high polymorphism, with the number of observed alleles per locus ranging from three to 16 (mean = 8.36). The observed and expected heterozygosities ranged from 0.100 to 1.000 and 0.145 to 0.843, respectively. Twenty-one of the loci were also successfully amplified in P. denticulata. These microsatellite markers should provide powerful tools for investigating patterns of population genetic diversity and the evolutionary relationships between distyly and homostyly in P. secundiflora.

The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying chromatin regulation with high-resolution genome-wide analyses. Since newly generated genome-wide data are often compared with publicly available datasets, expanding our dataset repertoire will be beneficial for the field. Information on transcription start sites (TSSs) determined at base pair resolution is essential for elucidating mechanisms of transcription and related chromatin regulation, yet no datasets that cover two different cell types are available. Here, we present a CAGE (cap analysis of gene expression) dataset for a-cells and α-cells grown in defined and rich media. Cell type-specific genes were differentially expressed as expected, ensuring the reliability of the data. Some of the differentially expressed TSSs were medium-specific or detected due to unrecognized chromosome rearrangement. By comparing the CAGE data with a high-resolution nucleosome map, major TSSs were primarily found in +1 nucleosomes, with a peak approximately 30 bp from the promoter-proximal end of the nucleosome. The dataset is available at DDBJ/GEA.

In Saccharomyces cerevisiae, boundaries formed by DNA sequence-dependent or -independent histone modifications stop the spread of the heterochromatin region formed via the Sir complex. However, it is unclear whether the histone modifiers that control DNA sequence-independent boundaries function in a chromosome-specific or -nonspecific manner. In this study, we evaluated the effects of the SAGA complex, a histone acetyltransferase (HAT) complex, and its relationship with other histone-modifying enzymes to clarify the mechanism underlying boundary regulation of the IMD2 gene on the right subtelomere of chromosome VIII. We found that Spt8, a component of the SAGA complex, is important for boundary formation in this region and that the inclusion of Spt8 in the SAGA complex is more important than its interaction with TATA-binding protein and TFIIS. In addition to SAGA, various HAT-related factors, such as NuA4 and Rtt109, also functioned in this region. In particular, the SAGA complex induced weak IMD2 expression throughout the cell, whereas NuA4 induced strong expression. These results indicate that multiple HATs contribute to the regulation of boundary formation and IMD2 expression on the right subtelomere of chromosome VIII and that IMD2 expression is determined by the balance between these factors.

Lack of pigmentation in seed coats of soybean is caused by natural RNA silencing of chalcone synthase (CHS) genes. This phenomenon is an evolutionary consequence of structural changes in DNA that resulted in the production of double-stranded RNAs (dsRNAs) that trigger RNA degradation. Here we determined that a mutant with pigmented seed coats derived from a cultivar that lacked the pigmentation had a deletion between DNA regions ICHS1 and a cytochrome P450 gene; the deletion included GmIRCHS, a candidate gene that triggers CHS RNA silencing via production of CHS dsRNAs. We also characterized CHS short interfering RNAs (siRNAs) produced in the wild-type seed coats that had CHS RNA silencing. Phased 21-nt CHS siRNAs were detected in all 21 phases and were widely distributed in exon 2 of CHS7, which indicates commonality in the pattern of RNA degradation in natural CHS RNA silencing between distantly related species. These results with the similarities in the rearrangements found in spontaneous mutants suggest that the structural organization that generates dsRNAs that trigger phased siRNA production is vulnerable to further structural changes, which eventually abolish the induction of RNA silencing.

The importance of the parent-progeny relationship tracking technique in single-cell analysis has grown with the passage of time. In this study, fundamental image-processing techniques were combined to develop software capable of inferring cell cycle alterations in fission yeast cells, which exhibit equipartition during division. These methods, exclusively relying on bright-field images as input, could track parent-progeny relationships after cell division by assessing the temporal morphological transformation of these cells. In the application of this technique, the software was employed for calculating intracellular fluorescent dots during every stage of the cell cycle, using a yeast strain expressing EGFP-fused Swi6, which binds to chromatin. The results obtained with this software were consistent with those of previous studies. This software facilitated single-cell-level tracking of parent-progeny relationships in cells exhibiting equipartition during division and enabled the monitoring of spatial fluctuations in a cell cycle-dependent protein. This method, expediting the analysis of extensive datasets, may also empower large-scale screening experiments that cannot be conducted manually.

The ClinVar accession number on p. 176 (SCV002817173) should be replaced with the correct number, SCV002817373. The PDF file for DOI: https://doi.org/10.1266/ggs.22-00138 has been replaced with the corrected version as of November 6, 2024.