Gene Codon Research Articles

A Comparative Analysis of Mitogenomes in Species of the Tapinoma nigerrimum Complex and Other Species of the Genus Tapinoma (Formicidae, Dolichoderinae)

Using next-generation sequencing data, the complete mitogenomes of six species from the genus Tapinoma were assembled. This study explores the mitochondrial genomes of Tapinoma species, among them the five species from the Tapinoma nigerrimum complex, comparing them with each other and with other species from Dolichoderinae subfamily to understand their evolutionary relationships and evolution. Tapinoma mitochondrial genomes contain the typical set of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNAs, and the A + T-rich control region. A phylogenetic analysis using the protein-coding gene sequences from available Dolichoderinae mitogenomes supports the monophyletic nature of the genus Tapinoma, with the T. nigerrimum complex forming a well-supported clade. Key findings include genetic traits unique to the T. nigerrimum complex, such as a start codon in the atp8 gene and a complete stop codon in cox1, distinguishing them from other Tapinoma species. Additionally, a gene rearrangement involving tRNA-Trp, tRNA-Cys, and tRNA-Tyr was found exclusively in the Tapinoma species, suggesting a potential phylogenetic marker for the genus.

Deep-amplicon sequencing of the complete beta-tubulin gene in Trichuris trichiura before and after albendazole treatment

Concerns about the emergence of benzimidazole resistance in soil-transmitted helminths (STH) infections, particularly against Trichuris trichiura, have arisen. Previous studies of veterinary nematodes have linked benzimidazole resistance to single-nucleotide polymorphisms (SNPs) at three specific codons in the beta-tubulin gene, but similar associations in STH have not been consistently observed. In this work, we screened the complete beta-tubulin gene previously linked to benzimidazole resistance in T. trichiura by deep-amplicon sequencing to identify genetic variants and associate levels of diversity with drug response to albendazole. We used 99 DNA samples extracted from T. trichiura pooled eggs, previously semi-purified from human stool samples collected in Manhiça district, Mozambique. We obtained a set of 39 amplicons of the complete gene by subjecting the pooled eggs to long-read PCR and subsequently sequencing them. Of those amplicons, 22 and 17 were obtained from stool samples collected before, and 21 days after albendazole treatment, respectively. We observed genetic variation across the whole gene sequence, in both exons and introns; however, none were associated with the previously proposed resistance-associated SNPs, and none were predicted to significantly affect protein function. No significant differences in genetic diversity were observed between pre- and post-treatment samples. Using publicly available genome-wide data, we also analysed a second beta-tubulin isotype in the T. trichiura genome. We focused on detecting the canonical SNPs and assessing for signatures of genetic selection around this second isotype gene. This analysis did not reveal evidence supporting this second isotype's role in anthelmintic resistance. Despite the limitations of our study, such as a small sample size, particularly paired pre- and post-treatment samples (n=6), or a restricted geographical area, we found no evidence linking either of the two beta-tubulin genes to benzimidazole resistance in T. trichiura, suggesting that genetic markers of drug resistance likely exist outside the beta-tubulin genes.

Comparative Analysis of Mitogenomes in Leafhopper Tribe Deltocephalini (Hemiptera: Cicadellidae: Deltocephalinae): Structural Conservatism and Phylogeny

ABSTRACTPrevious studies on the gene order and composition of leafhopper mitochondrial genomes have revealed a high level of conservation in overall genome structure. Some members of Deltocephalinae, the largest subfamily, exhibit tRNA gene rearrangements; however, few mitogenomes have been sequenced in this group and the degree of structural variation within tribes remains unclear. In this study, we sequenced the complete mitogenomes of 14 species belonging to four genera of tribe Deltocephalini from China and compared them with the two previously reported mitogenomes for this tribe. The studied mitogenomes showed a high degree of similarity to most other leafhopper mitogenomes in overall structure, mostly varying in the total length (14,961–15,416 bp) and number of non‐coding A + T‐rich regions. Gene size, order, arrangement, base composition, codon usage, and secondary structure of tRNAs in the newly sequenced mitogenomes were highly conserved in Deltocephalini, and variations in start/stop codon usage and tRNA secondary structure mostly matched those of other leafhoppers. Phylogenetic analysis of different combinations of protein‐coding and ribosomal genes using maximum likelihood and Bayesian inference under different models using either amino acid or nucleotide sequences were generally consistent and agreed with the previous nuclear and partial mitochondrial gene sequence data, indicating that complete mitochondrial genomes are phylogenetically informative at different levels of divergence within Deltocephalini and among different leafhoppers species. In addition to Deltocephalini, Deltocephalinae included members of Athysanini and Opsiini formed monophyletic groups. Maximum likelihood and Bayesian inference analyses consistently grouped Graminella nigrinota with Paralimnini, rendering Deltocephalini polyphyletic. The topology consistently divided Deltocephalini into two major branches, with Alobaldia tobae and Polyamia penistenuis forming a well‐supported sister group to the remaining species of the tribe.

Modeling and measuring how codon usage modulates the relationship between burden and yield during protein overexpression in bacteria.

Excess utilization of translational resources is a critical source of burden on cells engineered to over-express exogenous proteins. To improve protein yields and genetic stability, researchers often use codon optimization strategies that improve translational efficiency by matching an exogenous gene's codon usage with that of the host organism's highly expressed genes. Despite empirical data that shows the benefits of codon optimization, little is known quantitatively about the relationship between codon usage bias and the burden imposed by protein overexpression. Here, we develop and experimentally evaluate a stochastic gene expression model that considers the impact of codon usage bias on the availability of ribosomes and different tRNAs in a cell. In agreement with other studies, our model shows that increasing exogenous protein expression decreases production of native cellular proteins in a linear fashion. We also find that the slope of this relationship is modulated by how well the codon usage bias of the exogenous gene and the host's genes match. Strikingly, we predict that an overoptimization domain exists where further increasing usage of optimal codons worsens yield and burden. We test our model by expressing sfGFP and mCherry2 from constructs that have a wide range of codon optimization levels in Escherichia coli . The results agree with our model, including for an mCherry2 gene sequence that appears to lose expression and genetic stability from codon overoptimization. Our findings can be leveraged by researchers to predict and design more optimal cellular systems through the use of more nuanced codon optimization strategies.

Survey for Activating Oncogenic Mutation Variants in Metazoan Germline Genes.

Most cancers present with mutations or amplifications in distinctive tumor promoter genes that activate principal cell-signaling cascades promoting cell proliferation, dedifferentiation, cell survival, and replicative immortality. Somatic mutations found in this these driver proto-oncogenes invariably result in constitutive activation of the encoded protein. A salient feature of the activating mutations observed throughout many thousands of clinical tumor specimens reveals these driver missense mutations are recurrent and restricted to just one or very few codons of the entire gene, suggesting they have been positively selected during the course of tumor development. The purpose of this study is to investigate whether these characteristic oncogenic driver mutations are observed in the germline genes of any metazoan species. Six well-known tumor promoter genes were chosen for this survey including BRAF, KRAS, JAK2, PIK3CA, EGFR, and IDH1/2. The sites of all driver mutations were found to occur in highly conserved regions of each gene comparing protein sequences throughout diverse phyla of metazoan species. None of the oncogenic missense mutations were found in germlines of any species of current genome and protein databases. Despite many tumors readily selecting these somatic mutations, the conclusion drawn from this study is that these variants are negatively rejected if encountered as a germline mutation. While cancer expansion ensues from dysregulated growth elicited by these mutations, this effect is likely detrimental to embryonic development and/or survival of multicellular organisms. Although all oncogenic mutations considered here are gain-of-function where five of the six increase activity of the encoded proteins, clonal advancement promotes tumor growth by these genomic changes without conferring selection advantages benefiting the organism or species.

Deciphering Codon Usage Patterns in the Mitochondrial Genome of the Oryza Species

Rice (Oryza) is a genus in the Gramineae family, which has grown widely all over the world and is a staple food source for people’s survival. The genetic information of rice has garnered significant attention in recent years, prompting numerous researchers to conduct extensive investigations in this field. But rice mitochondrial codon usage patterns have received little attention. The present study systematically analyzed the codon usage patterns and sources of variance in the mitochondrial genome sequences of five rice species by the CodonW and R software programs. Our results revealed that the GC content of codons in rice mitochondrial genome genes was determined to be 43.60%. Notably, the individual codon positions exhibited distinct GC contents: 48.00% for position 1, 42.65% for position 2, and 40.16% for position 3. These findings suggest the preference of the rice mitochondrial genome for codons ending in A or U. A weak codon bias was observed, with the effective number of codons (ENC) varying between 40.02 and 61.00, with an average value of 54.34. Subsequently, we identified 25 identical high-frequency codons in five rice mitochondrial genomes, with 11 codons ending in A and 12 codons ending in U. The regression lines in the neutrality plot exhibited slopes of less than 0.5 in five rice species, indicating a predominant role of natural selection, while mutation pressure remained relatively insignificant. In the PR2-plot analysis, most of the genes were located in the right half of the plot, indicating that the third base of the synonymous codon was preferred to end in G than C. Additionally, the ENC plot and ENC ratio analysis unveiled that codon preferences in the rice mitochondrial genome were predominantly influenced by natural selection rather than mutational pressure. The analysis of correspondence revealed distinct variations in the codon usage pattern across five rice mitochondrial genomes. Based on the RSCU values of species, a cluster tree was inconsistent with the mitochondrial genetic data, indicating that RSCU data could not be used as a basis for classification at the species level in the Oryza genus. These results will help decide the specific types of natural selection pressures influencing codon usage and improve the expression of exogenous genes in rice mitochondrial genomes by optimizing their codons.

Microglia contribute to the production of the amyloidogenic ABri peptide in familial British dementia

Mutations in ITM2B cause familial British, Danish, Chinese, and Korean dementias. In familial British dementia (FBD), a mutation in the stop codon of the ITM2B gene (also known as BRI2) causes a C-terminal cleavage fragment of the ITM2B/BRI2 protein to be extended by 11 amino acids. This fragment, termed amyloid-Bri (ABri), is highly insoluble and forms extracellular plaques in the brain. ABri plaques are accompanied by tau pathology, neuronal cell death and progressive dementia, with striking parallels to the aetiology and pathogenesis of Alzheimer’s disease. The molecular mechanisms underpinning FBD are ill-defined. Using patient-derived induced pluripotent stem cells, we show that expression of ITM2B/BRI2 is 34-fold higher in microglia than neurons and 15-fold higher in microglia compared with astrocytes. This cell-specific enrichment is supported by expression data from both mouse and human brain tissue. ITM2B/BRI2 protein levels are higher in iPSC-microglia compared with neurons and astrocytes. The ABri peptide was detected in patient iPSC-derived microglial lysates and conditioned media but was undetectable in patient-derived neurons and control microglia. The pathological examination of post-mortem tissue supports the presence of ABri in microglia that are in proximity to pre-amyloid deposits. Finally, gene co-expression analysis supports a role for ITM2B/BRI2 in disease-associated microglial responses. These data demonstrate that microglia are major contributors to the production of amyloid forming peptides in FBD, potentially acting as instigators of neurodegeneration. Additionally, these data also suggest ITM2B/BRI2 may be part of a microglial response to disease, motivating further investigations of its role in microglial activation. These data have implications for our understanding of the role of microglia and the innate immune response in the pathogenesis of FBD and other neurodegenerative dementias including Alzheimer’s disease.

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Genetics and bone mineral density predict the fractures in adults with osteogenesis imperfecta: a prospective study.

Osteogenesis imperfecta (OI) is a rare genetic bone disorder characterized by recurrent fractures. In adults, the value of bone mineral density (BMD) in fracture risk is unknown. We prospectively investigated changes in BMD over time and analysed the determinants of fracture in OI. Among 106 individuals with grade 1 and 4 OI in the Reference Centre of Rare Bone Diseases in Paris, we included those with BMD measurements at one or more skeletal sites (hip, lumbar spine, radius) from 2000 to 2022. For 71 individuals with reliable measurements (44 women, 8 postmenopausal; mean age 41.4 ± 13.7 years), baseline BMD was low at the lumbar spine only (mean Z-score -2.3±1.5), affecting mainly men (mean Z-score -3±1.6). Longitudinal changes were assessed for a median follow-up of 5.1 years (interquartile range 3.2-8.8). On adjustment for age, sex and body mass index, BMD did not significantly change at any site. Logistic regression analysis revealed a high probability of fracture with baseline BMD Z-score <-2 SD versus ≥-2 SD (odds ratio 4.38, 95% CI 1.10-21.75, p=0.048) and harbouring splicing, stop codon and frameshift variants of COL1 gene (odds ratio 29.8, 95% CI 2.56-1503, p=0.024). our OI cohort showed low BMD at the lumbar spine but no significant change at any site after a median of 5.0 years of follow-up. The probability of fracture was associated with baseline BMD Z-score <-2 SD versus ≥-2 SD and harbouring COL1 splicing, stop codon and frameshift variants.

Phylogenetic and divergence analysis of Pentatomidae, with a comparison of the mitochondrial genomes of two related species (Hemiptera, Pentatomidae).

Pentatomidae, the most diverse family of Pentatomoidea, is found worldwide. Currently, the phylogenetic relationships among Pentatomidae tribes remain unstable, and subfamily divergence has not been estimated. Here, we sequenced and analyzed the complete mitochondrial genomes of two species of Lelia, and studied the phylogenetic relationships among Pentatominae tribes. We also selected three available fossil as the calibration points in the family, and preliminarily discussed the divergence time of Pentatomidae. Trees of Pentatomidae were reconstructed using the Bayesian inference method. Divergence times of Pentatominae were estimated based on the nucleotide sequences of protein-coding genes with a relaxed clock log-normal model in BEASTv.1.8.2. The results showed that the gene arrangements, nucleotide composition, and codon preferences were highly conserved in Lelia. Further, a phylogenetic analysis recovered Eysarcorini, Strachiini, Phyllocephalini, and Menidini as monophyletic with strong support, however, the monophyly of Antestiini, Nezarini, Carpocorini, Pentatomini and Cappaeini were rejected. Moreover, Pentatominae diverged from Pentatomidae soon after the origin of the Cretaceous Period, at approximately 110.38 Ma. This study enriches the mitochondrial genome database of Pentatomidae and provides a reference for further phylogenetic studies, and provides a more accurate estimate of divergence time.

Carbamoly-phosphate synthetase 1 (CPS1) deficiency: A tertiary center retrospective cohort study and literature review

BackgroundProtein metabolism and urea production maintain protein and amino acid homeostasis in normal status. Ammonia results from amino acid turnover and is produced by intestinal urease-positive bacteria. Ammonia must be detoxified, and the urea cycle converts ammonia into urea. CPS1 is an enzyme in the urea cycle that catalyzes ammonia and bicarbonate condensation. CPS1 deficiency presents in the neonatal period with hyperammonemia, resulting in death or neurological sequelae if patients survive. Objectives/aimsTo share the experience of patients with CPS1 deficiency from Tawam Hospital and to shed light on the spectrum of variants found in those patients. MethodsA retrospective chart review was done. All patients with CPS1 deficiency admitted to Tawam Hospital from 2010 to 2023 were included. Collected data included age and ammonia level at presentation, the time needed to drop ammonia level below 100 μmol/L, acute management modality provided, long-term neurological sequelae, sequence variants, severity, and duration of hyperammonemia encephalopathy, age at last follow-up, and, if applicable, survival for at least six months. ResultsOnly five patients with CPS1 deficiency over 13 years were found; two males and three females. Three patients are doing relatively well at 18 months, 7, and 9 years of age. The presented age was in the neonatal period except in one patient. One patient was found to have frameshift, resulting in a premature stop codon in the CPS1 gene, had a devastating course that ended with death. One patient had recurrent hyperammonemia episodes in her first year of life, which led to microcephaly and global developmental delay. One patient underwent hemodialysis, and one patient underwent peritoneal dialysis. All patients except one were on Carglumic acid which could contribute to their survival and disease control. All variants reported here are novel except one. ConclusionAlthough the presentation was different in severity, three patients are doing relatively well and approaching their developmental milestones. Thus, early recognition, prompt actions to drop high ammonia level, and good follow-up plans are emphasized. Further studies are needed to correlate the genotype-phenotype of reported variants here, which can help predict the severity of CPS1 deficiency.

Non-canonical start codons confer context-dependent advantages in carbohydrate utilization for commensal E. coli in the murine gut.

Resource competition is a driver of gut microbiota composition. Bacteria can outcompete metabolically similar rivals through the limitation of shared growth-fuelling nutrients. The mechanisms underlying this remain unclear for bacteria with identical sets of metabolic genes. Here we analysed the lactose utilization operon in the murine commensal Escherichia coli 8178. Using in vitro and in vivo approaches, we showed that translation of the lactose utilization repressor gene lacI from its native non-canonical GTG start codon increases the basal expression of the lactose utilization cluster, enhancing adaptation to lactose consumption. Consequently, a strain carrying the wild type lacI GTG start codon outperformed the lacI ATG start codon mutant in the mouse intestine. This advantage was attenuated upon limiting host lactose intake through diet shift or altering the mutant frequency, emphasizing the context-dependent effect of a single nucleotide change on the bacterial fitness of a common member of the gut microbiota. Coupled with a genomic analysis highlighting the selection of non-ATG start codons in sugar utilization regulator genes across the Enterobacteriaceae family, our data exposed an unsuspected function of non-canonical start codons in metabolic competition.

Temporal Characterization of Prion Shedding in Secreta of White-Tailed Deer in Longitudinal Study of Chronic Wasting Disease, United States.

Chronic wasting disease (CWD) affects cervids in North America, Asia, and Scandinavia. CWD is unique in its efficient spread, partially because of contact with infectious prions shed in secreta. To assess temporal profiles of CWD prion shedding, we collected saliva, urine, and feces from white-tailed deer for 66 months after exposure to low oral doses of CWD-positive brain tissue or saliva. We analyzed prion seeding activity by using modified amyloid amplification assays incorporating iron oxide bead extraction, which improved CWD detection and reduced false positives. CWD prions were detected in feces, urine, and saliva as early as 6 months postinfection. More frequent and consistent shedding was observed in deer homozygous for glycine at prion protein gene codon 96 than in deer expressing alternate genotypes. Our findings demonstrate that improved amplification methods can be used to identify early antemortem CWD prion shedding, which might aid in disease surveillance of cervids.

Comparative mitochondrial genomics of Terniopsis yongtaiensis in Malpighiales: structural, sequential, and phylogenetic perspectives

BackgroundTerniopsis yongtaiensis, a member of the Podostemaceae family, is an aquatic flowering plant displaying remarkable adaptive traits that enable survival in submerged, turbulent habitats. Despite the progressive expansion of chloroplast genomic information within this family, mitochondrial genome sequences have yet to be reported.ResultsIn current study, the mitochondrial genome of the T. yongtaiensis was characterized by a circular genome of 426,928 bp encoding 31 protein-coding genes (PCGs), 18 tRNAs, and 3 rRNA genes. Our comprehensive analysis focused on gene content, repeat sequences, RNA editing processes, intracellular gene transfer, phylogeny, and codon usage bias. Numerous repeat sequences were identified, including 130 simple sequence repeats, 22 tandem repeats, and 220 dispersed repeats. Phylogenetic analysis positioned T. yongtaiensis (Podostemaceae) within the Malpighiales order, showing a close relationship with the Calophyllaceae family, which was consistent with the APG IV classification. A comparative analysis with nine other Malpighiales species revealed both variable and conserved regions, providing insights into the genomic evolution within this order. Notably, the GC content of T. yongtaiensis was distinctively lower compared to other Malpighilales, primarily due to variations in non-coding regions and specific protein-coding genes, particularly the nad genes. Remarkably, the number of RNA editing sites was low (276), distributed unevenly across 27 PCGs. The dN/dS analysis showed only the ccmB gene of T. yongtaiensis was positively selected, which plays a crucial role in cytochrome c biosynthesis. Additionally, there were 13 gene-containing homologous regions between the mitochondrial and chloroplast genomes of T. yongtaiensis, suggesting the gene transfer events between these organellar genomes.ConclusionsThis study assembled and annotated the first mitochondrial genome of the Podostemaceae family. The comparison results of mitochondrial gene composition, GC content, and RNA editing sites provided novel insights into the adaptive traits and genetic reprogramming of this aquatic eudicot group and offered a foundation for future research on the genomic evolution and adaptive mechanisms of Podostemaceae and related plant families in the Malpighiales order.

Effectiveness of benzimidazole treatments against Haemonchus contortus in sheep and goats – Do they produce similar responses?

The primary aim of this study was to compare the in vivo responses to orally administered doses of albendazole (5 mg/kg body weight) between experimentally infected sheep and goats. Fifty-four Improved Valachian lambs and 54 Saanen goat kids were split into six groups of nine animals. The sheep and goats were infected with larvae of the gastrointestinal nematode parasite Haemonchus contortus containing 10, 20, 30, 40, 60, and 80 % of the isotype-1 β-tubulin gene codon 200 alleles previously shown to be associated with benzimidazole (BZ)-resistance. All groups of goats generally had higher mean eggs per gram (EPG) before treatment, which was significant (p<0.05) only for the group with 80 % resistance alleles. An in vivo faecal egg reduction test (FECRT) was used to determine the efficacy of albendazole (ABZ) eight days after treatment. Anthelmintic treatment significantly reduced the EPGs in the groups with 10, 20, and 80 % resistance alleles in sheep and with 10, 20, 30, and 40 % resistance alleles in goats. Differences in efficacy between the sheep and goats after the application of doses of ABZ recommended for sheep mostly ranged from 2 % to 10 %. The largest variation was in the group infected with worms containing 60 % resistance alleles, where the efficacy was 13 % higher in goats. Our secondary aims were to evaluate the data obtained from an in vitro egg hatch test (EHT) in sheep and goats and to compare these data with the results from the isotype-1 β-tubulin gene codon 200 pyrosequencing and the FECRT. The percentages of the BZ-resistance alleles were comparable with the mean hatching obtained in the EHT and were also supported by the FECRT data for all groups. The results of the in vivo tests should be verified in the future using in vivo surveys conducted in mixed breeds and infections in multiple species.

Real-world data analysis of next-generation sequencing and corresponding clinical characteristics in thyroid tumor.

Next-generation sequencing (NGS) is of great benefit to clinical practice in terms of identifying genetic alterations. This study aims to clarify the gene background and its influence on thyroid tumors in the Chinese population. NGS data and corresponding clinicopathological features (sex, age, tumor size, extrathyroidal invasion, metastasis, multifocality, and TNM stage) were collected and analyzed retrospectively from 2844 individual thyroid tumor samples from July 2021 to August 2022. Among the cohort, 2337 (82%) cases possess genetic alterations, including BRAF (71%), RAS (4%), RET/PTC (4%), TERT (3%), RET (2.2%), and TP53 (1.4%). Diagnostic sensitivity before surgery can be significantly increased from 0.76 to 0.91 when cytology is supplemented by NGS. Our results show that BRAF-positive papillary thyroid cancer (PTC) patients tend to have older age, smaller tumor size, less vascular invasion, more frequent tumor multifocality, and a significantly higher cervical lymph node metastatic rate. Mutation at RET gene codons 918 and 634 is strongly correlated with medullary thyroid cancer. However, it did not display more invasive clinical characteristics. TERT-positive patients are more likely to have older age, and have larger tumor size, more tumor invasiveness, and more advanced TNM stage, indicating a poor prognosis. Patients with TERT, RET/PTC1, and CHEK2 mutations are more susceptible to lateral lymph node metastasis. In conclusion, NGS can be a useful tool that provides practical gene evidence in the process of diagnosis and treatment in thyroid tumors.

Characterization of Ucp1-iCre knockin mice reveals the recombination activity in male germ cells.

Ucp1 promoter-driven Cre transgenic mice are useful in the manipulation of gene expression specifically in thermogenic adipose tissues. However, the wildly used Ucp1-Cre line was generated by random insertion into the genome and showed ectopic activity in some tissues beyond adipose tissues. Here, we characterized a knockin mouse line Ucp1-iCre generated by targeting IRES-Cre cassette immediately downstream the stop codon of the Ucp1 gene. The Cre insertion had little to no effect on uncoupling protein 1 (UCP1) levels in brown adipose tissue. Ucp1-iCre mice of both genders exhibited normal thermogenesis and cold tolerance. When crossed with Rosa-tdTomato reporter mice, Ucp1-iCre mice showed robust Cre activity in thermogenic adipose tissues. In addition, limited Cre activity was sparsely present in the ventromedial hypothalamus (VMH), choroid plexus, kidney, adrenal glands, ovary, and testis in Ucp1-iCre mice, albeit to a much lesser extent and with reduced intensity compared with the conventional Ucp1-Cre line. Single-cell transcriptome analysis revealed Ucp1 mRNA expression in male spermatocytes. Moreover, male Ucp1-iCre mice displayed a high frequency of Cre-mediated recombination in the germline, whereas no such effect was observed in female Ucp1-iCre mice. These findings suggest that Ucp1-iCre mice offer promising utility in the context of conditional gene manipulation in thermogenic adipose tissues, while also highlighting the need for caution in mouse mating and genotyping procedures.NEW & NOTEWORTHY Ucp1 promoter-driven Cre transgenic mice are useful in the manipulation of gene expression specifically in thermogenic adipose tissues. The widely used Ucp1-Cre mouse line (Ucp1-CreEvdr), which was generated using the bacterial artificial chromosome (BAC) strategy, exhibits major brown and white fat transcriptomic dysregulation and ectopic activity beyond adipose tissues. Here, we comprehensively validate Ucp1-iCre knockin mice, which serve as another optional model besides Ucp1-CreEvdr mice for specific genetic manipulation in thermogenic tissue.

Codon usage and expression-based features significantly improve prediction of CRISPR efficiency

CRISPR is a precise and effective genome editing technology; but despite several advancements during the last decade, our ability to computationally design gRNAs remains limited. Most predictive models have relatively low predictive power and utilize only the sequence of the target site as input. Here we suggest a new category of features, which incorporate the target site genomic position and the presence of genes close to it. We calculate four features based on gene expression and codon usage bias indices. We show, on CRISPR datasets taken from 3 different cell types, that such features perform comparably with 425 state-of-the-art predictive features, ranking in the top 2–12% of features. We trained new predictive models, showing that adding expression features to them significantly improves their r2 by up to 0.04 (relative increase of 39%), achieving average correlations of up to 0.38 on their validation sets; and that these features are deemed important by different feature importance metrics. We believe that incorporating the target site’s position, in addition to its sequence, in features such as we have generated here will improve our ability to predict, design and understand CRISPR experiments going forward.

Analysis of Codon Usage Bias in the Plastid Genome of Diplandrorchis sinica (Orchidaceae).

In order to understand the bias and main affecting factors of codon usage in the plastid genome of Diplandrorchis sinica, which is a rare and endangered plant species in the Orchidaceae family, the complete plastid genome sequence of D. sinica was downloaded from the GenBank database and 20 protein-coding sequences that met the analysis requirements were finally selected. The GC content, length of the amino acid (Laa), relative synonymous codon usage (RSCU), and effective number of codon (ENC) of each gene and codon were calculated using the CodonW and EMBOSS online programs. Neutral plot analysis, ENC-plot analysis, PR2-plot analysis, and correspondence analysis were performed using Origin Pro 2024 software, and correlation analysis between various indicators was performed using SPSS 23.0 software. The results showed that the third base of the codon in the plastid genome of D. sinica was rich in A and T, with a GC3 content of 27%, which was lower than that of GC1 (45%) and GC2 (39%). The ENC value ranged from 35 to 57, with an average of 47. The codon usage bias was relatively low, and there was a significant positive correlation between ENC and GC3. There were a total of 32 codons with RSCU values greater than 1, of which 30 ended with either A or U. There were a total of nine optimal codons identified, namely, UCU, UCC, UCA, GCA, UUG, AUA, CGU, CGA, and GGU. This study indicated that the dominant factor affecting codon usage bias in the plastid genome of D. sinica was natural selection pressure, while the impact of base mutations was limited. The codon usage patterns were not closely related to gene types, and the distribution of photosynthetic system genes and ribosomal protein-coding gene loci was relatively scattered, indicating significant differences in the usage patterns of these gene codons. In addition, the codon usage patterns may not be related to whether the plant is a photosynthetic autotrophic or heterotrophic nutritional type. The results of this study could provide scientific references for the genomic evolution and phylogenetic research of plant species in the family Orchidaceae.

Genomic factors shaping codon usage across the Saccharomycotina subphylum.

Codon usage bias, or the unequal use of synonymous codons, is observed across genes, genomes, and between species. It has been implicated in many cellular functions, such as translation dynamics and transcript stability, but can also be shaped by neutral forces. We characterized codon usage across 1,154 strains from 1,051 species from the fungal subphylum Saccharomycotina to gain insight into the biases, molecular mechanisms, evolution, and genomic features contributing to codon usage patterns. We found a general preference for A/T-ending codons and correlations between codon usage bias, GC content, and tRNA-ome size. Codon usage bias is distinct between the 12 orders to such a degree that yeasts can be classified with an accuracy >90% using a machine learning algorithm. We also characterized the degree to which codon usage bias is impacted by translational selection. We found it was influenced by a combination of features, including the number of coding sequences, BUSCO count, and genome length. Our analysis also revealed an extreme bias in codon usage in the Saccharomycodales associated with a lack of predicted arginine tRNAs that decode CGN codons, leaving only the AGN codons to encode arginine. Analysis of Saccharomycodales gene expression, tRNA sequences, and codon evolution suggests that avoidance of the CGN codons is associated with a decline in arginine tRNA function. Consistent with previous findings, codon usage bias within the Saccharomycotina is shaped by genomic features and GC bias. However, we find cases of extreme codon usage preference and avoidance along yeast lineages, suggesting additional forces may be shaping the evolution of specific codons.