Genes & genetic systems最新文献

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.

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.