Search Results (10)

Soft-furred tree mice (genus Typhlomys), which are native to southern China and northern Vietnam, are unique rodents capable of echolocation. Little is known about their taxonomy, ecology, and natural history. In this study, we generated the complete mitochondrial genomes of seven species/putative species of Typhlomys. We conducted a comprehensive comparative analysis of these mitochondrial genomes focusing on sequence length, A+T content, A/T bias, A+T-rich regions, overlapping and intergenic spacer regions, nucleotide composition, relative synonymous codon usage, ancestral distributions, and the non-synonymous/synonymous substitution ratio (Ka/Ks). Additionally, we analyzed the phylogeny and adaptive evolution of these species/putative species. The mitogenomes of Typhlomys ranged from 16,487 to 17,380 bp in length, encoding the complete set of 37 genes typically found in mammalian mitogenomes. The base composition exhibited an A+T bias. The most frequently used codons were CUA (Leu), AGC (Ser), GGA (Gly) and UUA (Leu), UUG, CUG, CGU and GCG were the less frequently used codons. Ancestral distribution reconstruction suggests that Typhlomys originated in Central or Southwestern China. Notably, we found that the Ka/Ks ratio of the ND5 gene in T. huangshanensis was greater than 1, indicating that this gene has undergone positive selection for efficient respiration in higher elevations and colder climates. Full article

In the standard genetic code, the CUG triplet is translated as leucine. The pathogenic yeast Candida albicans and other CTG-clade yeasts contain tRNA_CAG, which is recognized by both leucine- and serine-tRNA synthetases. The CUG codon in these yeasts is translated most often as serine, and only in 3–5% of cases as leucine. Therefore, CTG Candida species have unstable proteomes. The effect of serine–leucine exchange on the structure and function of proteins has only been experimentally examined for a few cases. In C. albicans, CUG codons occur even in genes deemed to be essential. This means that serine–leucine ambiguity either does not affect the structure and function of the respective proteins, or that the presence of these amino acids at specific positions is associated with meaningful alteration of the proteins’ function. This study employed AlphaFold2 to evaluate the potential effects of serine-to-leucine exchange in 12 proteins encoded by essential genes lacking orthologs in other yeasts and human genomes. The low homology with known proteins allowed us to make only low-confidence predictions. The analyzed proteins could be grouped into subsets based on the structural outcomes. Structural changes were observed only in four proteins. The remaining eight proteins showed no significant differences between serine and leucine variants. Full article

The halotolerant yeast Debaryomyces hansenii belongs to the CTG-Ser1 clade of fungal species that use the CUG codon to translate as leucine or serine. The ambiguous decoding of the CUG codon is relevant for expanding protein diversity, but little is known about the role of leucine–serine ambiguity in cellular adaptations to extreme environments. Here, we examine sequences and structures of tRNA_CAG from the CTG-Ser1 clade yeasts, finding that D. hansenii conserves the elements to translate ambiguously. Then, we show that D. hansenii has tolerance to conditions of salinity, acidity, alkalinity, and oxidative stress associated with phenotypic and ultrastructural changes. In these conditions, we found differential expression in both the logarithmic and stationary growth phases of tRNA^Ser, tRNA^Leu, tRNA_CAG, LeuRS, and SerRS genes that could be involved in the adaptive process of this yeast. Finally, we compare the proteomic isoelectric points and hydropathy profiles, detecting that the most important variations among the physicochemical characteristics of D. hansenii proteins are in their hydrophobic and hydrophilic interactions with the medium. We propose that the ambiguous translation, i.e., leucylation or serynation, on translation of the CUG-encoded residues, could be linked to adaptation processes in extreme environments. Full article

Upstream open reading frame (uORF)-mediated translational control has emerged as an important regulatory mechanism in human health and disease. However, a systematic search for cancer-associated somatic uORF mutations has not been performed. Here, we analyzed the genetic variability at canonical (uAUG) and alternative translational initiation sites (aTISs), as well as the associated upstream termination codons (uStops) in 3394 whole-exome-sequencing datasets from patient samples of breast, colon, lung, prostate, and skin cancer and of acute myeloid leukemia, provided by The Cancer Genome Atlas research network. We found that 66.5% of patient samples were affected by at least one of 5277 recurrent uORF-associated somatic single nucleotide variants altering 446 uAUG, 347 uStop, and 4733 aTIS codons. While twelve uORF variants were detected in all entities, 17 variants occurred in all five types of solid cancer analyzed here. Highest frequencies of individual somatic variants in the TLSs of NBPF20 and CHCHD2 reached 10.1% among LAML and 8.1% among skin cancer patients, respectively. Functional evaluation by dual luciferase reporter assays identified 19 uORF variants causing significant translational deregulation of the associated main coding sequence, ranging from 1.73-fold induction for an AUG.1 > UUG variant in SETD4 to 0.006-fold repression for a CUG.6 > GUG variant in HLA-DRB1. These data suggest that somatic uORF mutations are highly prevalent in human malignancies and that defective translational regulation of protein expression may contribute to the onset or progression of cancer. Full article

α-amylase which catalyzes the hydrolysis of α-1,4-glycosidic bonds in starch have frequently been cloned into various microbial workhorses to yield a higher recombinant titer. A thermostable SR74 α-amylase from Geobacillus stearothermophilus was found to have a huge potential in detergent industries due to its thermostability properties. The gene was cloned into a CTG-clade yeast Meyerozyma guilliermondii strain SO. However, the CUG ambiguity present in the strain SO has possibly altered the amino acid residues in SR74 amylase wild type (WT) encoded by CUG the codon from the leucine to serine. From the multiple sequence alignment, six mutations were found in recombinant SR74 α-amylase (rc). Their effects on SR74 α-amylase structure and function remain unknown. Herein, we predicted the structures of the SR74 amylases (WT and rc) using the template 6ag0.1.A (PDB ID: 6ag0). We sought to decipher the possible effects of CUG ambiguity in strain SO via in silico analysis. They are structurally identical, and the metal triad (CaI–CaIII) might contribute to the thermostability while CaIV was attributed to substrate specificity. Since the pairwise root mean square deviation (RMSD) between the WT and rc SR74 α-amylase was lower than the template, we suggest that the biochemical properties of rc SR74 α-amylase were better deduced from its WT, especially its thermostability. Full article

Pus1-dependent pseudouridylation occurs in many tRNAs and at multiple positions, yet the functional impact of this modification is incompletely understood. We analyzed the consequences of PUS1 deletion on the essential decoding of CAG (Gln) codons by tRNA^GlnCUG in yeast. Synthetic lethality was observed upon combining the modification defect with destabilized variants of tRNA^GlnCUG, pointing to a severe CAG-decoding defect of the hypomodified tRNA. In addition, we demonstrated that misreading of UAG stop codons by a tRNA^GlnCUG variant is positively affected by Pus1. Genetic approaches further indicated that mildly elevated temperature decreases the decoding efficiency of CAG and UAG via destabilized tRNA^GlnCAG variants. We also determined the misreading of CGC (Arg) codons by tRNA^HisGUG, where the CGC decoder tRNA^ArgICG contains Pus1-dependent pseudouridine, but not the mistranslating tRNA^His. We found that the absence of Pus1 increased CGC misreading by tRNA^His, demonstrating a positive role of the modification in the competition against non-synonymous near-cognate tRNA. Part of the in vivo decoding defects and phenotypes in pus1 mutants and strains carrying destabilized tRNA^GlnCAG were suppressible by additional deletion of the rapid tRNA decay (RTD)-relevant MET22, suggesting the involvement of RTD-mediated tRNA destabilization. Full article

Hepatitis B virus (HBV) is endemic in Rwanda and is a major etiologic agent for chronic liver disease in the country. In a previous analysis of HBV strains from Rwanda, the S genes of most strains segregated into one single clade of subgenotype, A1. More than half (55%) of the anti-HBe positive individuals were viremic. In this study, 23 complete HBV genomes and the core promoter region (CP) from 18 additional strains were sequenced. Phylogenetic analysis of complete genomes confirmed that most Rwandan strain formed a single unique clade, within subgenotype A1. Strains from 17 of 22 (77%) anti-HBe positive HBV carriers had either mutated the precore start codon (9 strains with either CUG, ACG, UUG, or AAG) or mutations in the Kozak sequence preceding the pre-core start codon (8 strains). These mutually exclusive mutations were also identified in subgenotypes A1 (70/266; 26%), A2 (12/255; 5%), and A3 (26/49; 53%) sequences from the GenBank. The results showed that previous, rarely described HBV variants, expressing little or no HBeAg, are selected in anti-HBe positive subgenotype Al carriers from Rwanda and that mutations reducing HBeAg synthesis might be unique for a particular HBV clade, not just for a specific genotype or subgenotype. Full article

Yeasts belonging to the Metschnikowia genus are particularly interesting for the unusual formation of only two needle-shaped ascospores during their mating cycle. Presently, the meiotic process that can lead to only two spores from a diploid zygote is poorly understood. The expression of fluorescent nuclear proteins should allow the meiotic process to be visualized in vivo; however, no large-spored species of Metschnikowia has ever been transformed. Accordingly, we aimed to develop a transformation method for Metschnikowia borealis, a particularly large-spored species of Metschnikowia, with the goal of enabling the genetic manipulations required to study biological processes in detail. Genetic analyses confirmed that M. borealis, and many other Metschnikowia species, are CUG-Ser yeasts. Codon-optimized selectable markers lacking CUG codons were used to successfully transform M. borealis by electroporation and lithium acetate, and transformants appeared to be the result of random integration. Mating experiments confirmed that transformed-strains were capable of generating large asci and undergoing recombination. Finally, random integration was used to transform an additional 21 yeast strains, and all attempts successfully generated transformants. The results provide a simple method to transform many yeasts from an array of different clades and can be used to study or develop many species for various applications. Full article

Blunt snout bream (Megalobrama amblycephala) is an important fish species for its delicacy and high economic value in China. Codon usage analysis could be helpful to understand its codon biology, mRNA translation and vertebrate evolution. Based on RNA-Seq data for M. amblycephala, high-frequency codons (CUG, AGA, GUG, CAG and GAG), as well as low-frequency ones (NUA and NCG codons) were identified. A total of 724 high-frequency codon pairs were observed. Meanwhile, 14 preferred and 199 avoided neighboring codon pairs were also identified, but bias was almost not shown with one or more intervening codons inserted between the same pairs. Codon usage bias in the regions close to start and stop codons indicated apparent heterogeneity, which even occurs in the flanking nucleotide sequence. Codon usage bias (RSCU and SCUO) was related to GC₃ (GC content of 3rd nucleotide in codon) bias. Six GO (Gene ontology) categories and the number of methylation targets were influenced by GC₃. Codon usage patterns comparison among 23 vertebrates showed species specificities by using GC contents, codon usage and codon context analysis. This work provided new insights into fish biology and new information for breeding projects. Full article

Lipases from microorganisms have multi-faceted properties and play an important role in ever-growing modern biotechnology and, consequently, it is of great significance to develop new ones. In the present work, a lipase gene from Candida albicans (CaLIP10) was cloned and two non-unusual CUG serine codons were mutated into universal codons, and its expression in Pichia pastoris performed optimally, as shown by response surface methodology. Optimal conditions were: initial pH of culture 6.86, temperature 25.53 °C, 3.48% of glucose and 1.32% of yeast extract. The corresponding maximal lipolytic activity of CaLIP10 was 8.06 U/mL. The purified CaLIP10 showed maximal activity at pH 8.0 and 25 °C, and a good resistance to non-ionic surfactants and polar organic solvent was noticed. CaLIP10 could effectively hydrolyze coconut oil, but exhibited no obvious preference to the fatty acids with different carbon length, and diacylglycerol was accumulated in the reaction products, suggesting that CaLIP10 is a potential lipase for the oil industry. Full article