Genes & genetic systems最新文献

We developed Huntington's disease (HD) modelling 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 modelling autosomal dominant diseases, our approach of employing biallelic modification may offer the distinct advantage enabling the investigation of the effects of specific genomic mutations with minimal interference from genetic background noise.

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.

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 the tree inference bias caused by the presence of long branches (defined as the "long-branch bias") increases with the accumulation of mutations, and influences all model tree or phylogenetic inference method. In other words, in Felsenstein tree models, methods that are highly sensitive to the 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 the long-branch bias always infer the same tree shape. Additionally, the 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.

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 the 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 ABL gene encodes a cytokinin receptor with histidine kinase activity. Our further analysis suggested that the abl1-d mutation is associated with a putative active form of histidine kinase and altered cytokinin signaling. However, it remains unclear whether abl1-d mutation indeed triggers aberrant cytokinin signal in rice plants, and how the abl1-d mutation affects developmental processes throughout the lifecycle 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 been observed to resemble two normal leaves fused together at their abaxial sides. The leaf with this particular phenotype exhibited enhanced photosynthetic efficiency under certain environmental conditions. Thus, the abl1-1d mutants, which express a putative active form of histidine receptor kinase, affect various developmental traits throughout the rice lifecycle probably due to altered cytokinin signaling.

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 a wheat-infecting 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 in 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 the Pyricularia fungus. We designated this element as the centromere-associated IR element (CenIR), 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 CG to TA transitions likely accelerate base substitutions in the centromeres of Pyricularia fungi.

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.

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.