Ancient dispersal events from the Korean Peninsula to the Japanese main islands of Honshu, Shikoku and Kyushu (HSK), and from the Eurasian continent to Hokkaido via Sakhalin, have played a critical role in shaping the mammalian diversity of the Japanese archipelago. However, the timing and dynamics of these events remain incompletely understood across different taxa. In addition, the 100,000-year climatic cycles of glacial and interglacial periods during the Middle and Late Quaternary likely influenced intraspecific genetic diversity, although the mechanisms driving these changes remain unclear. In this study, we analyzed mitochondrial cytochrome b gene sequences from Japanese shrews (Sorex and Crocidura) and other small mammals from HSK and Hokkaido. Using an evolutionary rate of 0.029 substitutions/site/million years, we inferred that ancestral lineages of HSK species diverged during critical periods in the early Quaternary, such as around 2.4 and 1.7 million years ago (Ma), potentially in response to major climatic transitions. Notably, dispersal events of the Laxmann’s shrew (S. caecutiens) and dark red-backed vole (Myodes rex) into Hokkaido around 1.7 Ma were also suggested. Regarding intraspecific mitochondrial DNA (mtDNA) lineages, species with broad distributions in HSK typically exhibit a north–south structure, characterized by two major lineages, as well as additional ancient lineages in surrounding offshore islands. Comparative analysis revealed that divergence among these lineages occurred at approximate intervals of 100,000 years. Our results indicate that the mtDNA genetic structure of HSK small mammals reflects geographic substructures shaped by climate-driven dispersal. Regions that facilitated rapid expansion during favorable climatic periods likely acted as centers of dispersal, from which haplotypes spread toward peripheral areas. As haplotypes radiated outward from these core regions, distinct mtDNA lineages became established across different geographic zones, giving rise to the spatial distribution patterns observed today.

Primula tibetica is an insect-pollinated, herbaceous, perennial plant belonging to the section Aleuritia (Primulaceae). The species exhibits the typical characteristics of heterostyly, with predominantly outcrossing populations comprising long-styled and short-styled floral morphs. Furthermore, significant variation occurs in floral morphology, categorised as homostyly, a phenomenon commonly associated with elevated selfing rates. Utilising next-generation sequencing, 25 microsatellite markers for P. tibetica were developed, with the objective of facilitating future investigations into the population genetics and mating patterns of the species. These markers were characterised by measuring polymorphism and genetic diversity in a sample of 36 individuals from three natural populations. The markers displayed relatively high polymorphism, with the number of observed alleles per locus ranging from two to 15 (mean = 7.26). The observed and expected heterozygosities ranged from 0.056 to 0.917 and 0.105 to 0.825, respectively. Furthermore, nineteen of these loci were also successfully amplified in P. pulchella. These microsatellite markers should serve as effective tools for investigating patterns of population genetic diversity and elucidating the evolutionary relationship between distyly and homostyly in P. tibetica.

In most eubacteria the initiator protein DnaA triggers chromosomal replication by forming an initiation complex at the origin of replication and also functions as a transcriptional regulator, coordinating gene expression with cell cycle progression. While genes regulated by DnaA are relatively well characterized in exponentially growing cells, its role in gene regulation during stationary phase remains insufficiently explored. Here, using the aquatic bacterium Caulobacter crescentus as a model, we show that C. crescentus DnaA (ccDnaA) acts as a repressor of the previously uncharacterized CCNA_00139 gene, which encodes a YifB family Mg chelatase-like AAA ATPase family protein of unknown function. Biochemical analyses reveal that ccDnaA forms multimers at this site, which may interfere with RNA polymerase access to the promoter by occupying overlapping binding sequences. Consistent with these findings, in exponentially growing C. crescentus cells the CCNA_00139 promoter is repressed in a ccDnaA-dependent manner. Notably, when cells enter stationary phase, CCNA_00139 promoter activity increases in parallel with ccDnaA clearance, supporting the idea that ccDnaA-mediated repression is relieved during this phase transition. Despite its regulated expression, deletion of CCNA_00139 did not result in any detectable growth, replication or DNA damage sensitivity phenotypes under the tested laboratory conditions, suggesting a possible role under specific environmental conditions. Given that this phase-dependent transcriptional switch may, in principle, apply to other uncharacterized ccDnaA-repressed genes, we infer that CCNA_00139, along with other such genes, form a regulatory network that supports quorum sensing or adaptation to growth phase transitions. We believe that these findings offer new insight into the potential role of bacterial DnaA in regulating gene expression in dormant or non-replicating cells across diverse bacterial species.

RNA sequencing analysis was performed to develop 16 novel expressed sequence tag–simple sequence repeat (EST–SSR) markers to evaluate genetic variation in the near-threatened halophyte Artemisia fukudo Makino, which inhabits riversides and tidal muds affected by brackish water at high tide. In the four populations examined, the total number of alleles at each locus ranged from two to 13, with an average of 4.3. The observed and expected heterozygosity ranged from 0.05 to 0.64 and 0.06 to 0.72, respectively. These newly developed EST–SSR markers will support the understanding of the population genetic structure of A. fukudo and contribute to the conservation of this species.

Maintenance DNA methylation is essential for the stable inheritance of epigenetic information in vertebrates. While DNMT1 has long been recognized as the principal maintenance methyltransferase, recent studies have shown that its activity critically depends on ubiquitin signaling. Specifically, the E3 ligase UHRF1 enables DNMT1 recruitment and activation at hemimethylated sites through dual monoubiquitylation of both replication-associated and histone substrates. These insights have revised classical models of maintenance methylation and revealed new layers of regulation involving chromatin context, histone modifications and nucleosome remodeling. In this review, we summarize the current understanding of the molecular mechanisms underlying DNMT1-mediated maintenance methylation, with a particular focus on ubiquitin-dependent pathways and their interplay with chromatin architecture.

DNA methylation is faithfully inherited during cell division, playing a crucial role in maintaining cellular identity. The process of DNA methylation maintenance relies on the DNA methyltransferase DNMT1 and the ubiquitin E3 ligase UHRF1. UHRF1 facilitates the ubiquitination of both the replication factor PAF15 and histone H3, with each ubiquitin signal regulating replication-coupled and -uncoupled DNA methylation maintenance, respectively. Over the past decades, advances in structural biology have significantly deepened our understanding of the molecular mechanisms governing DNA methylation maintenance. In particular, the emergence of cryo-electron microscopy—often referred to as the “resolution revolution”—has transformed many areas of biology, including epigenetics and chromatin biology. This review focuses on the structural mechanisms of DNA methylation maintenance, as revealed by the three-dimensional structures of key biomolecular complexes, and discusses the potential development of inhibitors targeting DNA methylation maintenance factors based on structural insights.

DNA methylation is essential for transcriptional regulation and the maintenance of chromosome stability, and its precise inheritance upon DNA replication is indispensable for cellular homeostasis. The DNMT1/UHRF1 complex is critical in copying DNA methylation with accessory proteins, including CDCA7 and HELLS. The DNMT1/UHRF1 complex is also crucial for maintaining DNA methylation at imprinting control regions during preimplantation development against genome-wide DNA demethylation, an essential process for early embryos to acquire totipotency. Pathogenic variants in the genes involved in the mechanism of DNA methylation maintenance result in immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, multilocus imprinting disturbance (MLID), autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCADN), neuropathy, hereditary sensory, type 1E (HSN1E), Kleefstra syndrome 1 (KLEFS1) and immunodeficiency 96 (IMD96). This review discusses recent progress in understanding the molecular pathogenesis of these diseases, with a particular focus on ICF syndrome and MLID.

The aquatic beetle Cybister rugosus has experienced ongoing habitat loss, a decline in population numbers and confirmed extinctions among insular populations in Japan. It has recently been classified on the Red List as endangered by the Japanese Ministry of Environment and has been designated a specified class II nationally rare species of wild fauna and flora. To design effective conservation strategies for this species, it is essential to compile data on its genetic variation to ascertain its genetic diversity and population structure. Previous studies found low levels of genetic variation in the COI gene among Japanese insular populations and failed to detect a fine population structure. Thus, we developed ten novel microsatellite markers for C. rugosus, using whole-genome shotgun sequencing. The degree of polymorphism for these markers was characterized using summary statistics describing the genetic variation in 49 individuals from populations in Cambodia and on the islands of the Ryukyu Archipelago. Microsatellite data indicated differentiation among the insular populations in Japan, which could not be clearly shown in the COI data, and some insular populations showed low levels of genetic diversity. The newly developed microsatellite markers will contribute to future ecological and evolutionary studies on this species and to conservation research.

Testicular differentiation of undifferentiated gonads is triggered by the SRY/Sry (sex-determining region of chromosome Y) gene on the Y chromosome in most mammals. SRY and NR5A1 (nuclear receptor subfamily 5, group A, member 1) proteins regulate transcription of the autosomal SOX9/Sox9 (SRY-box9) gene in XY embryonic gonads, inducing testicular differentiation. One exception, the Amami spiny rat (Tokudaia osimensis), lacks the Y chromosome and Sry. We previously reported that this species has a male-specific duplication upstream of Sox9, and an enhancer (tosEnh14) in the duplication regulates Sox9 transcription without Sry. However, tosEnh14 is not activated by NR5A1 alone, suggesting that another transcription factor(s) which binds to tosEnh14 is necessary. Because this species is endangered and heavily protected, it presents many challenges for genetic studies. Therefore, we explored novel transcription factors that regulate Sox9 via tosEnh14 using mouse samples. To detect proteins that bind to tosEnh14 DNA, Southwestern blotting analysis was performed using mouse embryonic gonad extracts. Bands of a similar molecular weight but prominent in males and faint in females were subjected to mass spectrometry analysis. Peptides derived from 174 genes were identified, and eight genes associated with gene ontology terms such as “DNA binding” and “regulation of transcription by RNA polymerase II” were selected. For further screening, the expression level of each gene was examined using single-cell RNA-sequencing data for mouse progenitor cells, which differentiate into Sertoli cells in mouse embryonic testes and granulosa cells in embryonic ovaries. Finally, five genes (Elf2, Etv6, Fiz1, Gtf2f1 and Trim27) encoding transcription factors, whose expression was confirmed in seminiferous tubules of E13.5 XY embryos by whole-mount in situ hybridization, were selected as candidates. Binding sites for ELF2 and ETV6 are present in the tosEnh14 DNA sequence. Our study contributes to understanding the molecular mechanisms underlying sex determination in mammals.

We developed Huntington’s disease (HD)-modeling induced pluripotent stem cells (iPSCs) by genome engineering of iPSCs from healthy donors. For this, we established a homologous-recombination-based biallelic substitution technique called the allele-specific universal knock-in system (asUKiS). asUKiS allows for scarless and allele-by-allele substitution of the entire region encompassing not only the polyQ repeat but also the associated genetic modifiers surrounding the repeat region, allowing us to generate five iPSC lines with identical genetic modifiers on both alleles, differing only in polyQ repeat numbers. All cell lines were validated by allele-specific genotyping to confirm the precise engineering of both alleles. Even for modeling autosomal dominant diseases, our approach of employing biallelic modification offers the distinct advantage of enabling investigation of the effects of specific genomic mutations with minimal interference from genetic background noise.

The mammalian sex-determining gene SRY is highly conserved across species, with only a few exceptions. The Japanese rodent genus Tokudaia is known for its unique sex chromosome evolution. The Okinawa spiny rat T. muenninki (TMU) acquired neo-sex chromosomes with multiple Sry copies by sex chromosome–autosome fusions. All SRY copies in TMU have a substitution from alanine to serine at position 21 in the high-mobility group (HMG) box, a critical DNA-binding domain, suggesting that they are nonfunctional. However, the sex determination system in TMU remains unclear, in part because the species is endangered and it is therefore extremely difficult to obtain experimental samples. In this study, we performed in silico and in vitro analyses to investigate the molecular properties and function of SRY using recently obtained whole-genome sequence and RNA-seq data. A comparison of SRY sequences from 225 species showed that TMU is the only species with a substitution at the 21st position. This result highlights the rarity and specificity of this substitution. Structural predictions, DNA docking simulations, electrophoretic mobility shift assays and fluorescence anisotropy showed that although the affinity was slightly lower than that of the mouse homolog, DNA-binding ability was retained. However, Sry expression was not detected in the testis, liver or brain in adult TMU. The complete absence of Sry expression in the adult tissues, despite an intact sequence, strongly indicates a loss of regulatory function. These findings provide insight into the unique evolution of the Sry gene in this species.

YchF is a universally conserved unconventional G protein. It is known to be involved in the translation of leaderless mRNA. However, leaderless mRNA is rare in Escherichia coli under normal culture conditions, so we analyzed E. coli YchF to clarify its function in vivo. First, bioinformatics analysis was performed, and then the growth and survival of ychF mutants were investigated. The results suggest that the functional domains and important amino acid residues of YchF are conserved. We next found that the ychF mutants exhibited delayed re-growth in late stationary phase in the presence of oxidative stress. Moreover, the growth inhibition by catalase overexpression was suggested to be caused by oxidase activity. We found that the E. coli ychF mutants exhibited reduced growth in early stationary phase that was associated with a decreased level of ribosomal 70S subunit. In the ychF mutants, we also found that overproduction of the ribosomal protein S18 inhibited growth, which was further suppressed by overproduction of S11. YchF of E. coli is involved in the regulation of ribosomal 70S levels possibly through interaction with ribosomal proteins S18 and S11 as well as IF-3, suggesting that YchF is important for growth and survival in the early and late stationary phase of growth.

A hemizygous 1.5–3.0-Mb microdeletion of human chromosome 22q11.2 with the 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, Lady Ridgeway Hospital for Children, 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 suspected patient (P) and unaffected person (N) samples. For detection of a deletion, the normalized value (HIRA/TES dosage) of a P sample was compared with that of an N sample. A ratio of P:N of 0.5 confirmed the presence of a deletion while a ratio of 1.0 refuted this. Seven of the 19 patients were found 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 nine of the 19 cases) and whole-exome sequencing (all 19 samples tested). This TaqMan qPCR assay was able to reliably distinguish HIRA-deleted cases from non-deleted ones. The assay was both cheaper and faster compared to commercially available alternatives in our setting, including FISH and multiple ligation-dependent probe amplification.

Sex-specific DNA markers are effective tools for sex identification and sex-controlled breeding of dioecious organisms. The seeds of the dioecious Herpetospermum pedunculosum are utilized in traditional Chinese medicine, and the development of sex-linked markers for seedlings is crucial for enhancing the number of female plants. In this study, we screened sex-specific markers based on whole-genome resequencing of 20 male and 24 female H. pedunculosum individuals, and validated a male-specific DNA fragment of 505 bp among 80 individuals from four populations using simple PCR. The findings provide a reliable male-specific marker for the sex identification of H. pedunculosum seedlings.

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. A co-culture model of TNBC cells and THP-1/M0 macrophages was established to detect the impact of co-culture on FOXM1 expression and the direction of macrophage polarization. A bioinformatics website was used to predict FOXM1 binding sites in the IDO1 promoter, which were further validated using dual-luciferase reporter and chromatin immunoprecipitation assays. Next, after erastin-induced ferroptosis, we conducted cell viability assays, apoptosis assays and other experiments to investigate whether the FOXM1/IDO1 axis regulates M2 macrophage polarization through ferroptosis. We found that FOXM1 was abundant in exosomes derived from TNBC cells, and that TNBC cells upregulated FOXM1 expression in THP-1 cells through exosomes to promote M2 macrophage polarization. Furthermore, FOXM1 upregulated IDO1 in M2-type tumor-associated macrophages (TAMs) by stimulating its transcription. Finally, FOXM1/IDO1 inhibited ferroptosis, promoting M2 macrophage polarization, thereby advancing TNBC progression. In conclusion, FOXM1 carried by TNBC cell-derived exosomes activated IDO1 transcription in TAMs to inhibit ferroptosis, promoting M2 polarization of TAMs and exerting carcinogenic effects.

β-sitosterol is a natural plant steroidal compound with anti-cancer properties against various tumors. This work explored the inhibitory effect of β-sitosterol on the progression of lung adenocarcinoma (LUAD) and further analyzed its targets. We applied network pharmacology to obtain the components and targets of Ganoderma spore powder. The biological functions of β-sitosterol and CHRM2 were studied using the homograft mouse model and a series of in vitro experiments involving quantitative reverse transcription polymerase chain reaction, western blot, CCK-8, flow cytometry, immunohistochemistry and immunofluorescence. The regulatory influence of β-sitosterol on the glycolysis pathway was validated by measuring glucose consumption and lactate production, as well as the extracellular acidification rate and oxygen consumption rate. We found that CHRM2 binds directly to β-sitosterol. In vitro, CHRM2 overexpression repressed the apoptosis rate and expression of apoptosis-related proteins in LUAD cells, and promoted glycolysis, while the addition of lonidamine attenuated the apoptosis-inhibiting effect conferred by CHRM2 overexpression. Furthermore, β-sitosterol hindered glycolysis as well as the growth of tumors in vitro and in vivo. CHRM2 overexpression reversed the effect of β-sitosterol on the biological behavior of LUAD cells. Our results emphasize that CHRM2 is a direct target of β-sitosterol in LUAD cells. β-sitosterol can repress the glycolysis pathway, exerting an anti-tumor effect. These findings provide new support for the use of β-sitosterol as a therapeutic agent for LUAD.

Centromeres are essential for chromosome segregation, yet they are among the most rapidly evolving regions of the genome. The mechanisms driving this rapid evolution of centromeric sequences are still not well understood. In this study, we identified the centromeric sequences of the wheat-infecting fungus Pyricularia oryzae (strain Br48) using CENP-A chromatin immunoprecipitation followed by high-throughput sequencing. The Br48 centromeres range from 71 kb to 101 kb in length and are highly AT-rich (72.1–75.5%) and repeat-rich (63.4–85.0%). These regions are also enriched for H3K9me3 and 5-methylcytosine but depleted of H3K4me2 and H3K27me3. During the analysis of repetitive sequences in the Br48 centromere, we identified a stretch of approximately 530 bp that is tightly associated with centromeres in P. oryzae. We named this element the CenIR (centromere-associated IR element), as it often forms inverted repeat structures with two elements adjacent in reverse orientation. A comparison of putative centromere sequences across phylogenetically distinct P. oryzae strains suggests that changes in centromeric sequences are non-uniform across chromosomes and do not always align with the fungal phylogenetic relationships. Repeat-induced point mutation (RIP)-like C:G to T:A transitions likely accelerate base substitutions in the centromeres of Pyricularia fungi.

Cytokinin plays a major role in the regulation of plant development. It is perceived by receptors with histidine kinase activity to regulate the expression of various transcription factors. In a previous study, we reported a semi-dominant mutant, named adaxial-abaxial bipolar leaf1 (abl1)-d, which exhibited a characteristic feature in the fourth leaf of rice, and that the ABL1 gene encodes a cytokinin receptor with histidine kinase activity. Our further analysis suggested that the abl1-d mutation is associated with an active form of histidine kinase and altered cytokinin signaling. However, it remained unclear whether the abl1-d mutation indeed triggers aberrant cytokinin signaling in rice plants, and how the abl1-d mutation affects developmental processes throughout the life cycle of rice. In the present study, we found that homozygous abl1-1d calli have the capacity to regenerate shoots in the absence of cytokinin, suggesting that the abl1-1d homozygous mutation is associated with constitutive cytokinin signaling in rice. We next examined morphological characteristics of both homozygous and heterozygous abl1-1d plants from the post-germination vegetative phase through to reproduction. The results showed that homozygous abl1-1d plants had a reduced number of panicles and were completely sterile, and that leaf size and the midrib structure were altered. Furthermore, the adaxial–abaxial bipolar leaf, a phenotype that is characteristic of the abl1-1d mutant, has previously been observed to resemble two normal leaves fused together at their abaxial sides. Leaves with this particular phenotype exhibited enhanced photosynthetic efficiency under certain environmental conditions. Thus, the abl1-1d mutation, which results in a putative active form of receptor histidine kinase, affects various developmental traits throughout the rice life cycle, probably due to altered cytokinin signaling.

We aimed to identify new mutants resulting from ONSEN transposition in Arabidopsis thaliana by subjecting nrpd1 mutant seedlings 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 into 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 any 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 wild type and the nrpd1 mutant, with increased FT expression levels. This research highlights the impact of ONSEN transposition on gene function and phenotypic variation in A. thaliana, providing new insights into the mutagenic potential of transposons and their role in shaping plant traits.

Nucleosomes are complexes of DNA and histone proteins that form the basis of eukaryotic chromatin. Eukaryotic histones are descended from archaeal homologs; however, how this occurred remains unclear. Our previous genetic analysis of the budding yeast nucleosome identified 26 histone residues conserved between Saccharomyces cerevisiae and Trypanosoma 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 eukaryotic 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 eukaryote-specific domain required for chromatin binding of H2A.Z. We propose that the LECA emerged when the newly split H3 N-terminus 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 the 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

In phylogenetic analysis, long-branch attraction (LBA) occurs when two distantly related species with longer branches are mistakenly grouped as the most closely related species. Previous research addressing this issue has focused on phylogenetic trees with four operational taxonomic units and three topologies, using two models: the Felsenstein model tree, which has two long branches that are not closely related, and the Farris tree, which has two long branches that are most closely related. For the Felsenstein model, the maximum parsimony method is more prone to estimating incorrect tree shapes compared to the maximum likelihood (ML) method, whereas in the Farris model, the opposite tendency is observed. However, the underlying reason for these differences remains unclear. Therefore, we inferred phylogenetic trees using sequence data from molecular evolution simulations of model phylogenetic trees with different long-branch lengths and measured the tree shapes and branch lengths of the obtained phylogenetic trees. Our findings revealed that tree inference bias caused by the presence of long branches (defined as ‘long-branch bias’) increases with the accumulation of mutations, and influences all model trees or phylogenetic inference methods. In other words, in Felsenstein tree models, methods that are highly sensitive to long-branch bias tend to cause LBA, and in Farris tree models, the methods tend to infer apparently correct phylogenetic trees because of this influence. Thus, methods sensitive to long-branch bias always infer the same tree shape. Additionally, long-branch bias causes similar misestimations of branch lengths in both Felsenstein and Farris trees inferred by neighbor-joining or ML. This insight into long-branch bias will lead to a more reliable interpretation of phylogenetic trees, such as the shift of branching points, improving the accuracy of future research in molecular evolution.

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, both of which interfered with the scanning process of start codon recognition, mediated by specialized ribosomal subunits. These mutations successfully reduced the levels of exogenously expressed Cre to different degrees in fission yeast. Combining them with promoters of different 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.

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.