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1.
Int J Mol Sci ; 24(2)2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36674703

RESUMO

Synonymous codon usage can be influenced by mutations and/or selection, e.g., for speed of protein translation and correct folding. However, this codon bias can also be affected by a general selection at the amino acid level due to differences in the acceptance of the loss and generation of these codons. To assess the importance of this effect, we constructed a mutation-selection model model, in which we generated almost 90,000 stationary nucleotide distributions produced by mutational processes and applied a selection based on differences in physicochemical properties of amino acids. Under these conditions, we calculated the usage of fourfold degenerated (4FD) codons and compared it with the usage characteristic of the pure mutations. We considered both the standard genetic code (SGC) and alternative genetic codes (AGCs). The analyses showed that a majority of AGCs produced a greater 4FD codon bias than the SGC. The mutations producing more thymine or adenine than guanine and cytosine increased the differences in usage. On the other hand, the mutational pressures generating a lot of cytosine or guanine with a low content of adenine and thymine decreased this bias because the nucleotide content of most 4FD codons stayed in the compositional equilibrium with these pressures. The comparison of the theoretical results with those for real protein coding sequences showed that the influence of selection at the amino acid level on the synonymous codon usage cannot be neglected. The analyses indicate that the effect of amino acid selection cannot be disregarded and that it can interfere with other selection factors influencing codon usage, especially in AT-rich genomes, in which AGCs are usually used.


Assuntos
Aminoácidos , Uso do Códon , Aminoácidos/genética , Timina , Código Genético , Códon/genética , Nucleotídeos/genética , Citosina , Guanina , Adenina , Seleção Genética , Evolução Molecular
2.
Sci Rep ; 13(1): 1215, 2023 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-36681692

RESUMO

The tubulinopathies are an umbrella of rare diseases that result from mutations in tubulin genes and are frequently characterised by severe brain malformations. The characteristics of a given disease reflect the expression pattern of the transcript, the function of a given tubulin gene, and the role microtubules play in a particular cell type. Mouse models have proved to be valuable tools that have provided insight into the molecular and cellular mechanisms that underlie the disease state. In this manuscript we compare two Tuba1a mouse models, both of which express wild-type TUBA1A protein but employ different codon usage. We show that modification of the Tuba1a mRNA sequence results in homozygous lethality and a severe neurodevelopmental phenotype. This is associated with a decrease in the number of post-mitotic neurons, PAX6 positive progenitors, and an increase in the number of apoptotic cells. We attribute this to a decrease in the stability of the modified Tuba1a transcript, and the absence of compensation by the other neurogenic tubulins. Our findings highlight the importance of maintaining the wild-type coding sequence when engineering mouse lines and the impact of synonymous genetic variation.


Assuntos
Tubulina (Proteína) , Camundongos , Animais , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , RNA Mensageiro/genética , Mutação , Fenótipo , Códon/genética
3.
Sci Rep ; 13(1): 1296, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36690654

RESUMO

We evaluated the variation in the frequency of benzimidazole (BZ) resistance-associated alleles at codons 200, 167 and 368 (F200Y, F167Y, V368L) of the ß-tubulin isotype 1 gene during the patent period in isolates of Haemonchus contortus susceptible and resistant to BZ using pyrosequencing. Four lambs 5-6 months old were infected with 5000-6000 infective third-stage larvae (L3) of the susceptible MHco1 and the multi-resistant MHco4 isolates, respectively. Faecal samples were collected 28-times during 20-90 days post-infection (dpi). Coprocultures were subsequently prepared to produce L3 for genotyping. The frequency of the resistant allele (TAC) at codon 200 in MHco1 was lowest at 43 and 76 dpi with at each time point 0% and highest at 36 dpi with 10.85%, with a mean of 6.47% ± 2.39 and a coefficient of variation of 37.01%. The frequency of the TAC at codon 200 in MHco4 was lowest at 76 dpi with 25.6% and highest at 90 dpi with 49.25%, with a mean of 35.7% ± 4.42 and a coefficient of variation of 12.39%. No resistance alleles were detected in MHco1 at either codon 167 or 368. For MHco4 isolate, resistance alleles were detected only on codon 167 with a mean of 8.00% ± 4.83 and a mean coefficient of variation of 60.40%. Our results demonstrate the considerable variation in the frequency of resistant alleles in the susceptible and resistant isolates during the patent period. This variation should be considered when testing for the presence of BZ resistance in populations of gastrointestinal parasites, especially those with a low frequency of TAC.


Assuntos
Anti-Helmínticos , Hemoncose , Haemonchus , Ovinos/genética , Animais , Anti-Helmínticos/farmacologia , Resistência a Medicamentos/genética , Polimorfismo de Nucleotídeo Único , Hemoncose/parasitologia , Frequência do Gene , Benzimidazóis , Carneiro Doméstico/genética , Códon , Tubulina (Proteína)/genética
4.
Sci Rep ; 13(1): 415, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36624167

RESUMO

Mycobacterium tuberculosis (Mtb) is a slow-growing, intracellular pathogen that exhibits a high GC-rich genome. Several factors, including the GC content of the genome, influence the evolution of specific codon usage biases in genomes. As a result, the Mtb genome exhibits strong biases for amino acid usage and codon usage. Codon usage of mRNAs affects several aspects of translation, including accuracy, efficiency, and protein folding. Here we address the effect of codon usage biases in determining the translation efficiency of mRNAs in Mtb. Unlike most commonly studied organisms, Mtb carries a single copy of each tRNA gene. However, we show that the relative levels of tRNAs in the Mtb tRNA pool vary by an order of magnitude. Our results show that the codons decoded by the abundant tRNAs indeed show higher adaptability. Moreover, there is a general positive correlation between genomic codon usage and the tRNA adaptability of codons (TAc). We further estimated the optimality of the codon and mRNAs by considering both the TAc and the tRNA demand. These measures did not show any correlation with mRNA abundance and translation efficiency. There was no correlation between tRNA adaptability and ribosome pausing as well. Taken together, we conclude that the translation machinery, and the tRNA pool of an organism, co-evolve with the codon usage to optimize the translation efficiency of an organism. Thus the deleterious effect of maladapted codons is not pronounced.


Assuntos
Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Biossíntese de Proteínas/genética , Códon/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo
5.
Mikrobiyol Bul ; 57(1): 45-59, 2023 Jan.
Artigo em Turco | MEDLINE | ID: mdl-36636845

RESUMO

Ethambutol (EMB) is one of the first-line drugs used in the standard combination therapy for tuberculosis (TB) caused by Mycobacterium tuberculosis complex (MTC), and resistance to drugs that play a key role in treatment is increasing worldwide. Mutations in the embCAB operon that have been confirmed to be associated with resistance are responsible for EMB resistance. In this study, it was aimed to determine the frequency and patterns of mutations in embA, embB and embC gene regions in clinical MTC isolates found to be phenotypically resistant and susceptible to EMB. A total of 64 MTC isolates, 44 of resistant to EMB and 20 of susceptible to EMB, isoniazid, rifampicin, and streptomycin by conventional phenotypic drug susceptibility test, were included in the study. Following the DNA isolation, embA, embB and embC gene regions associated with EMB resistance were amplified with specific primer sequences. The PCR products were cycle sequenced using the Bigdye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, USA) and electrophoretically separated on the ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA). Mutated gene regions were identified by aligning sequence analysis data in multiple sequence analysis programs. In the study, genomic mutations in the embCAB operon were detected in 68.2% (30/44) of the EMB resistant isolates. Mutations in the embB gene region were detected in 66% (29/44) of the resistant isolates, 76% (22/29) of these mutations were at codon 306 and the most common mutation patterns in this codon were determined as ATG→GTG (M306V; 58.6%; 17/29), ATG→ATA, ATC or ATT (M306I; 17.2%; 5/29). Other mutations in the embB gene region were determined as Y334H (3.4%; 1/29), D354A (6.9%; 2/29), E378A (3.4%; 1/29), G406C (3.4%; 1/29), M423I (3.4%; 1/29) and E521A (3.4%; 1/29). Of the 44 EMB-resistant isolates, mutations were detected in one (2.3%) of the isolate in the embA gene region (L330L) and in two (4.5%) of the isolates in the embC gene region (T270I in one isolate and T270I and E305E in the other isolate). Of the phenotypically EMB susceptible isolates, mutation was detected in only one (5%) of the isolates in the embA gene region (E180G). In our study, it was determined that mutations frequently occur in codon 306 of the embB gene in EMB-resistant MTC isolates and this mutation has a potential role in the development of EMB resistance. However, it was concluded that the absence of mutations does not exclude phenotypic EMB resistance. Our results will shed light on the molecular epidemiology of embCAB operon mutations that cause EMB resistance in our country.


Assuntos
Etambutol , Mycobacterium tuberculosis , Humanos , Etambutol/farmacologia , Antituberculosos/farmacologia , Farmacorresistência Bacteriana/genética , Mutação , Códon , Testes de Sensibilidade Microbiana
6.
BMC Genom Data ; 24(1): 3, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36624369

RESUMO

BACKGROUND: The Phenomenon of codon usage bias exists in the genomes of prokaryotes and eukaryotes. The codon usage pattern is affected by environmental factors, base mutation, gene flow and gene expression level, among which natural selection and mutation pressure are the main factors. The study of codon preference is an effective method to analyze the source of evolutionary driving forces in organisms. Epimedium species are perennial herbs with ornamental and medicinal value distributed worldwide. The chloroplast genome is self-replicating and maternally inherited which is usually used to study species evolution, gene expression and genetic transformation. RESULTS: The results suggested that chloroplast genomes of Epimedium species preferred to use codons ending with A/U. 17 common high-frequency codons and 2-6 optimal codons were found in the chloroplast genomes of Epimedium species, respectively. According to the ENc-plot, PR2-plot and neutrality-plot, the formation of codon preference in Epimedium was affected by multiple factors, and natural selection was the dominant factor. By comparing the codon usage frequency with 4 common model organisms, it was found that Arabidopsis thaliana, Populus trichocarpa, and Saccharomyces cerevisiae were suitable exogenous expression receptors. CONCLUSION: The evolutionary driving force in the chloroplast genomes of 10 Epimedium species probably comes from mutation pressure. Our results provide an important theoretical basis for evolutionary analysis and transgenic research of chloroplast genes.


Assuntos
Epimedium , Genoma de Cloroplastos , Magnoliopsida , Uso do Códon , Genoma de Cloroplastos/genética , Epimedium/genética , Códon/genética , Eucariotos/genética , Magnoliopsida/genética
7.
BMC Plant Biol ; 23(1): 9, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36604614

RESUMO

BACKGROUND: The Ferula genus encompasses 180-185 species and is one of the largest genera in Apiaceae, with many of Ferula species possessing important medical value. The previous studies provided more information for Ferula, but its infrageneric relationships are still confusing. In addition, its genetic basis of its adaptive evolution remains poorly understood. Plastid genomes with more variable sites have the potential to reconstruct robust phylogeny in plants and investigate the adaptive evolution of plants. Although chloroplast genomes have been reported within the Ferula genus, few studies have been conducted using chloroplast genomes, especially for endemic species in China. RESULTS: Comprehensively comparative analyses of 22 newly sequenced and assembled plastomes indicated that these plastomes had highly conserved genome structure, gene number, codon usage, and repeats type and distribution, but varied in plastomes size, GC content, and the SC/IR boundaries. Thirteen mutation hotspot regions were detected and they would serve as the promising DNA barcodes candidates for species identification in Ferula and related genera. Phylogenomic analyses with high supports and resolutions showed that Talassia transiliensis and Soranthus meyeri were nested in the Ferula genus, and thus they should be transferred into the Ferula genus. Our phylogenies also indicated the monophyly of subgenera Sinoferula and subgenera Narthex in Ferula genus. Twelve genes with significant posterior probabilities for codon sites were identified in the positively selective analysis, and their function may relate to the photosystem II, ATP subunit, and NADH dehydrogenase. Most of them might play an important role to help Ferula species adapt to high-temperatures, strong-light, and drought habitats. CONCLUSION: Plastome data is powerful and efficient to improve the support and resolution of the complicated Ferula phylogeny. Twelve genes with significant posterior probabilities for codon sites were helpful for Ferula to adapt to the harsh environment. Overall, our study supplies a new perspective for comprehending the phylogeny and evolution of Ferula.


Assuntos
Ferula , Genoma de Cloroplastos , Genomas de Plastídeos , Filogenia , Evolução Molecular , Genoma de Cloroplastos/genética , Códon/genética
8.
Sci Adv ; 9(1): eabq5506, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36608134

RESUMO

Abnormal temperature caused by global climate change threatens the rice production. Defense signaling network for chilling has been uncovered in plants. However, less is known about repairing DNA damage produced from overwhelmed defense and its evolution during domestication. Here, we genetically identified a major QTL, COLD11, using the data-merging genome-wide association study based on an algorithm combining polarized data from two subspecies, indica and japonica, into one system. Rice loss-of-function mutations of COLD11 caused reduced chilling tolerance. Genome evolution analysis of representative rice germplasms suggested that numbers of GCG sequence repeats in the first exon of COLD11 were subjected to strong domestication selection during the northern expansion of rice planting. The repeat numbers affected the biochemical activity of DNA repair protein COLD11/RAD51A1 in renovating DNA damage under chilling stress. Our findings highlight a potential way to finely manipulate key genes in rice genome and effectively improve chilling tolerance through molecular designing.


Assuntos
Oryza , Oryza/genética , Oryza/metabolismo , Estudo de Associação Genômica Ampla , Códon/metabolismo , Temperatura Baixa
9.
Philos Trans R Soc Lond B Biol Sci ; 378(1871): 20220029, 2023 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-36633285

RESUMO

By linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. We recently found that a naturally occurring tRNASer variant decodes phenylalanine codons with serine and inhibits protein synthesis. Here, we hypothesized that human tRNA variants that misread glycine (Gly) codons with alanine (Ala) will also disrupt protein homeostasis. The A3G mutation occurs naturally in tRNAGly variants (tRNAGlyCCC, tRNAGlyGCC) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNAAlaAGC G35C (tRNAAlaACC) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNAGly and tRNAAla variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNAGlyGCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNAGlyGCC A3G and tRNAAlaAGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.


Assuntos
Alanina-tRNA Ligase , Biossíntese de Proteínas , Humanos , Alanina/genética , Alanina/química , Alanina/metabolismo , Alanina-tRNA Ligase/química , Alanina-tRNA Ligase/genética , Alanina-tRNA Ligase/metabolismo , Códon/genética , Glicina/genética , Glicina/metabolismo , Proteostase , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Alanina/química , RNA de Transferência de Alanina/genética , RNA de Transferência de Alanina/metabolismo , RNA de Transferência de Glicina/metabolismo
10.
Sci Rep ; 13(1): 896, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36650197

RESUMO

Chloroplasts have evolved from photosynthetic cyanobacteria-like progenitors through endosymbiosis. The chloroplasts of present-day land plants have their own transcription and translation systems that show several similarities with prokaryotic organisms. A remarkable feature of the chloroplast translation system is the use of non-AUG start codons in the protein synthesis of certain genes that are evolutionarily conserved from Algae to angiosperms. However, the biological significance of such use of non-AUG codons is not fully understood. The present study was undertaken to unravel the significance of non-AUG start codons in vivo using the chloroplast genetic engineering approach. For this purpose, stable transplastomic tobacco plants expressing a reporter gene i.e. uidA (GUS) under four different start codons (AUG/UUG/GUG/CUG) were generated and ß-glucuronidase (GUS) expression was compared. To investigate further the role of promoter sequences proximal to the start codon, uidA was expressed under two different chloroplast gene promoters psbA and psbC that use AUG and a non-AUG (GUG) start codons, respectively, and also showed significant differences in the DNA sequence surrounding the start codon. Further, to delineate the role of RNA editing that creates AUG start codon by editing non-AUG codons, if any, which is another important feature of the chloroplast transcription and translation system, transcripts were sequenced. In addition, a proteomic approach was used to identify the translation initiation site(s) of GUS and the N-terminal amino acid encoded when expressed under different non-AUG start codons. The results showed that chloroplasts use non-AUG start codons in combination with the translation initiation site as an additional layer of gene regulation to over-express proteins that are required at high levels due to their high rates of turnover.


Assuntos
Biossíntese de Proteínas , Proteômica , Códon de Iniciação/genética , Biossíntese de Proteínas/genética , Códon/genética , Cloroplastos/genética , Iniciação Traducional da Cadeia Peptídica/genética
11.
Sci Adv ; 9(1): eade9120, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36608131

RESUMO

Utilization of specific codons varies between organisms. Cancer represents a model for understanding DNA sequence evolution and could reveal causal factors underlying codon evolution. We found that across human cancer, arginine codons are frequently mutated to other codons. Moreover, arginine limitation-a feature of tumor microenvironments-is sufficient to induce arginine codon-switching mutations in human colon cancer cells. Such DNA codon switching events encode mutant proteins with arginine residue substitutions. Mechanistically, arginine limitation caused rapid reduction of arginine transfer RNAs and the stalling of ribosomes over arginine codons. Such selective pressure against arginine codon translation induced an adaptive proteomic shift toward low-arginine codon-containing genes, including specific amino acid transporters, and caused mutational evolution away from arginine codons-reducing translational bottlenecks that occurred during arginine starvation. Thus, environmental availability of a specific amino acid can influence DNA sequence evolution away from its cognate codons and generate altered proteins.


Assuntos
Arginina , Neoplasias Colorretais , Humanos , Sequência de Bases , Arginina/genética , Arginina/metabolismo , Biossíntese de Proteínas , Proteômica , Escherichia coli/metabolismo , Códon/metabolismo , Neoplasias Colorretais/genética , Microambiente Tumoral
12.
Int J Mol Sci ; 24(2)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36674611

RESUMO

Plants experience different stresses, i.e., abiotic, or biotic, and to combat them, plants re-program the expression of growth-, metabolism-, and resistance-related genes. These genes differ in their synonymous codon usage frequency and show codon usage bias. Here, we investigated the correlation among codon usage bias, gene expression, and underlying mechanisms in rice under abiotic and biotic stress conditions. The results indicated that genes with higher expression (up- or downregulated) levels had high GC content (≥60%), a low effective number of codon usage (≤40), and exhibited strong biases towards the codons with C/G at the third nucleotide position, irrespective of stress received. TTC, ATC, and CTC were the most preferred codons, while TAC, CAC, AAC, GAC, and TGC were moderately preferred under any stress (abiotic or biotic) condition. Additionally, downregulated genes are under mutational pressure (R2 ≥ 0.5) while upregulated genes are under natural selection pressure (R2 ≤ 0.5). Based on these results, we also identified the possible target codons that can be used to design an optimized set of genes with specific codons to develop climate-resilient varieties. Conclusively, under stress, rice has a bias towards codon usage which is correlated with GC content, gene expression level, and gene length.


Assuntos
Uso do Códon , Oryza , Oryza/genética , Códon/genética , Mutação , Aclimatação
13.
ACS Chem Biol ; 18(1): 81-90, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36607609

RESUMO

Advances in genetic code reprogramming have allowed the site-specific incorporation of noncanonical functionalities into polypeptides and proteins, providing access to wide swaths of chemical space via in vitro translation techniques like mRNA display. Prior efforts have established that the translation machinery can tolerate amino acids with modifications to both the peptide backbone and side chains, greatly broadening the chemical space that can be interrogated in ligand discovery efforts. However, existing methods for confirming the translation yield of new amino acid building blocks for these technologies necessitate multistep workups and, more importantly, are not relevant for measuring translation within the context of a combinatorial library consisting of multiple noncanonical amino acids. In this study, we developed a luminescence-based assay to rapidly assess the relative translation yield of any noncanonical amino acid in real time. Among the 59 amino acids tested here, we found that many translate with high efficiency, but translational yield is not necessarily correlated to whether the amino acid is proteinogenic or has high tRNA acylation efficiency. Interestingly, we found that single-template translation data can inform the library-scale translation yield and that shorter peptide libraries are more tolerant of lower-efficiency amino acid monomers. Together our data show that the luminescence-based assay described herein is an essential tool in evaluating new building blocks and codon table designs within mRNA display toward the goal of developing druglike peptide-based libraries for drug discovery campaigns.


Assuntos
Aminoácidos , Biblioteca de Peptídeos , Aminoácidos/química , Proteínas/metabolismo , Peptídeos/química , Códon
14.
Nat Chem ; 15(1): 61-69, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36550233

RESUMO

The direct genetically encoded cell-based synthesis of non-natural peptide and depsipeptide macrocycles is an outstanding challenge. Here we programme the encoded synthesis of 25 diverse non-natural macrocyclic peptides, each containing two non-canonical amino acids, in Syn61Δ3-derived cells; these cells contain a synthetic Escherichia coli genome in which the annotated occurrences of two sense codons and a stop codon, and the cognate transfer RNAs (tRNAs) and release factor that normally decode these codons, have been removed. We further demonstrate that pyrrolysyl-tRNA synthetase/tRNA pairs from distinct classes can be engineered to direct the co-translational incorporation of diverse alpha hydroxy acids, with both aliphatic and aromatic side chains. We define 49 engineered mutually orthogonal pairs that recognize distinct non-canonical amino acids or alpha hydroxy acids and decode distinct codons. Finally, we combine our advances to programme Syn61Δ3-derived cells for the encoded synthesis of 12 diverse non-natural depsipeptide macrocycles, which contain two non-canonical side chains and either one or two ester bonds.


Assuntos
Aminoacil-tRNA Sintetases , Depsipeptídeos , Códon , Aminoácidos/metabolismo , RNA de Transferência/genética , Aminoacil-tRNA Sintetases/química , Hidroxiácidos
15.
Vet Microbiol ; 277: 109625, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36563582

RESUMO

In ovo vaccination is an attractive immunization approach for the poultry industry. However, commonly used Newcastle disease virus (NDV) vaccines cannot be administered in ovo because of the reduced hatchability and embryo mortality. The codon pair deoptimization (CPD) approach has been used to efficiently and rapidly attenuate viruses by targeting the virulence genes. In this study, we aimed to attenuate the NDV LaSota (LS) vaccine strain for in ovo vaccination by CPD of the fusion (F) or/and hemagglutinin-neuraminidase (HN) genes with approximately 44 % suboptimal codon substitutions. Three NDV LS recombinants expressing codon deoptimized F (rLS/F-d), HN (rLS/HN-d), or both genes (rLS/F+HN-d) were generated using reverse genetics technology. Biological assays showed that the CPD viruses retained similar hemagglutination activity and growth ability to the parental rLS virus. The CPD of the HN gene slightly attenuated the rLS/HN-d and rLS/F+HN-d viruses, whereas the CPD of the F gene marginally increased the rLS/F-d virus pathogenicity compared to rLS. Nevertheless, all three CPD rLS viruses were still lethal to 10-day-old specific-pathogen-free (SPF) chicken embryos. In ovo inoculation of 18-day-old SPF chicken embryos with the CPD viruses severely reduced chicken's hatch and survival rates. These results suggested that the CPD of the surface glycoprotein genes of the LS strain at the current level of suboptimal codon substitutions could not sufficiently attenuate the virus for use as an in ovo vaccine, and codon deoptimizing a greater proportion of the F and HN genes or additional gene(s) may be required for sufficient attenuation of the LS strain.


Assuntos
Doença de Newcastle , Vacinas Virais , Embrião de Galinha , Animais , Vírus da Doença de Newcastle , Doença de Newcastle/prevenção & controle , Galinhas , Vacinação/veterinária , Vacinação/métodos , Proteína HN/genética , Códon
16.
Nucleic Acids Res ; 51(1): 271-289, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36546769

RESUMO

During initiation, the ribosome is tasked to efficiently recognize open reading frames (ORFs) for accurate and fast translation of mRNAs. A critical step is start codon recognition, which is modulated by initiation factors, mRNA structure, a Shine Dalgarno (SD) sequence and the start codon itself. Within the Escherichia coli genome, we identified more than 50 annotated initiation sites harboring AUGUG or GUGUG sequence motifs that provide two canonical start codons, AUG and GUG, in immediate proximity. As these sites may challenge start codon recognition, we studied if and how the ribosome is accurately guided to the designated ORF, with a special focus on the SD sequence as well as adenine at the fourth coding sequence position (A4). By in vitro and in vivo experiments, we characterized key requirements for unambiguous start codon recognition, but also discovered initiation sites that lead to the translation of both overlapping reading frames. Our findings corroborate the existence of an ambiguous translation initiation mechanism, implicating a multitude of so far unrecognized ORFs and translation products in bacteria.


Assuntos
Escherichia coli , Biossíntese de Proteínas , Códon de Iniciação , Escherichia coli/genética , Escherichia coli/metabolismo , Códon , RNA Mensageiro/metabolismo , Fases de Leitura Aberta
17.
Gene ; 855: 147121, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36535463

RESUMO

Scrapie is a fatal prion protein disease stiffly associated with single nucleotide polymorphism (SNPs) of the prion protein gene (PRNP). The prevalence of this deadly disease has been reported in small ruminants, including goats. The Nigerian goats are hardy, trypano-tolerant, and contribute to the protein intake of the increasing population. Although scrapie has been reported in Nigerian goats, there is no study on the polymorphism of the PRNP gene. Herein, we evaluated the genetic and allele distributions of PRNP polymorphism in 132 Nigerian goats and compared them with publicly available studies on scrapie-affected goats. We utilized Polyphen-2, PROVEAN and AMYCO programs to examine structural variations produced by the non-synonymous SNPs. Our study revealed 29 SNPs in Nigerian goats, of which 14 were non-synonymous, and 23 were novel. There were significant differences (P < 0.001) in the allele frequencies of PRNP codons 139, 146, 154 and 193 in Nigerian goats compared with scrapie-affected goats, except for Northern Italian goats at codon 154. Based on the prediction by Polyphen-2, R139S and N146S were 'benign', R154H was 'probably damaging', and T193I was 'possibly damaging'. In contrast, PROVEAN predicted 'neutral' for all non-synonymous SNPs, while AMYCO showed a similar amyloid propensity of PRNP for resistant haplotype and two haplotypes of Nigerian goats. Our study is the first to investigate the polymorphism of scrapie-related genes in Nigerian goats.


Assuntos
Doenças das Cabras , Príons , Scrapie , Animais , Ovinos/genética , Príons/genética , Proteínas Priônicas/genética , Scrapie/genética , Scrapie/epidemiologia , Cabras/genética , Doenças das Cabras/genética , Polimorfismo de Nucleotídeo Único , Códon
18.
Zhongguo Zhong Yao Za Zhi ; 47(22): 6058-6065, 2022 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-36471930

RESUMO

Artemisia indica is an important medicinal plant in the Asteraceae family, but its molecular genetic information has been rarely reported. In this study, the chloroplast genome of A. indica was sequenced, assembled, and annotated by the high-throughput sequencing technology, and its sequence characteristics, repeat sequences, codon usage bias, and phylogeny were analyzed. The results showed that the length of the chloroplast genome for A. indica was 151 161 bp, which was a typical circular four-segment structure, including two inverted repeat regions(IRs), a large single-copy(LSC) region, and a small single-copy(SSC) region, with a GC content of 37.47%. A total of 132 genes were annotated, and 114 were obtained after de-duplication, including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Fifty long repeat sequences and 191 SSRs were detected in the chloroplast genome of A. indica, and SSRs were mainly single nucleotides. Codon usage bias analysis showed that leucine was the most frequently used amino acid(10.77%) in the chloroplast genome, and there were 30 codons with relative synonymous codon usage(RSCU)>1 and all ended with A/U. The phylogenetic tree constructed based on the chloroplast genomes of the 19 species from the Asteraceae family showed that A. indica and A. argyi were closest in the genetic relationship, and Artemisia species clustered into separate evolutionary branches. The results of this study are expected to provide a theoretical basis for the genetic diversity and resource conservation of Artemisia medicinal plants.


Assuntos
Artemisia , Genoma de Cloroplastos , Plantas Medicinais , Filogenia , Artemisia/genética , Códon/genética , Composição de Bases , Plantas Medicinais/genética
19.
Nat Struct Mol Biol ; 29(12): 1277-1290, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36482253

RESUMO

Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis and products of mRNA translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin-binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome biogenesis, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing activity of eIF4EBP1, a direct inhibitor of ribosome biogenesis, is concurrent with ribosome downregulation and affects neurogenesis of the Satb2 lineage. Thus, the molecular logic of brain development includes the refinement of transcriptional programs by translation. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource at https://shiny.mdc-berlin.de/cortexomics .


Assuntos
Biossíntese de Proteínas , Ribossomos , Camundongos , Animais , Ribossomos/genética , Ribossomos/metabolismo , Proteínas Ribossômicas/metabolismo , Códon , Encéfalo/metabolismo
20.
Nat Cancer ; 3(12): 1484-1497, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36510010

RESUMO

The human genome contains 61 codons encoding 20 amino acids. Synonymous codons representing a given amino acid are decoded by a set of transfer RNAs (tRNAs) called isoacceptors. We report the surprising observation that two isoacceptor tRNAs that decode synonymous codons become modulated in opposing directions during breast cancer progression. Specifically, tRNAIleUAU became upregulated, whereas tRNAIleGAU became repressed as breast cancer cells attained enhanced metastatic capacity. Functionally, tRNAIleUAU promoted and tRNAIleGAU suppressed metastatic colonization in mouse xenograft models. These tRNAs mediated opposing effects on codon-dependent translation of growth-promoting genes, consistent with genomic enrichment or depletion of their cognate codons in mitotic genes. Our findings uncover a specific isoacceptor tRNA pair that act in opposition, divergently impacting growth-regulating genes and a disease phenotype. Degeneracy of the genetic code can thus be biologically exploited by human cancer cells via tRNA isoacceptor shifts that causally facilitate the transition toward a growth-promoting state.


Assuntos
Neoplasias da Mama , Animais , Camundongos , Humanos , Feminino , Neoplasias da Mama/genética , RNA de Transferência de Isoleucina , Códon/genética , RNA de Transferência/genética , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoácidos/genética , Proliferação de Células/genética
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