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 Tokudaia 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, and 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 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 E. 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 the ychF mutant were investigated. The results suggest that the functional domains and important amino acid residues of YchF are conserved. We next found that the E. coli ychF mutant exhibits delayed re-growth in late stationary phase in the presence of oxidative stress. And the growth inhibition by catalase overexpression was suggested to be caused by oxidase activity. We found that the E. coli ychF mutant exhibits reduced growth in early stationary phase and that is associated with decreased ribosomal 70S subunit. In the ychF mutant, 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.

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.

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).