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1.
Medicine (Baltimore) ; 98(31): e16361, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31374005

RESUMO

RATIONALE: Habitual abortion is caused by complex and diverse factors, such as genetic factors, immune factors, endocrine factors, viruses, bacterial infections, and so on. Allogeneic antibodies, generated due to blood-group incompatibilities between a female and her fetus, are sometimes important for habitual abortion. PATIENT CONCERNS: A 26-year-old woman had undergone abortions 3 times in July 2015 (17 weeks pregnant), March 2017 (15 weeks of gestation) and February 2018 (16 weeks pregnant) before she came to the Reproductive Medicine Center of our hospital for prenatal examinations without pregnancy. DIAGNOSES: Unexplained habitual abortion. INTERVENTIONS: A series of serological tests and nucleotide sequence of 1,4-galactosyltransferase (A4GALT) gene were performed. OUTCOMES: The patient was the rare p phenotype in P1P blood system and the patient's habitual abortion was caused by anti-PP1P antibody which was generated naturally in persons with p phenotype. There was a mutation (903C>G, CCC>CCG) in the 3rd exon of A4GALT gene, which is likely a significant contributor to p phenotype. LESSONS: This is the first case of habitual abortion caused by p phenotype due to independent 903C>G homozygous mutation with no similar record reported before, which indicates that it is a new class of mutation that leads to p phenotype.


Assuntos
Aborto Habitual/sangue , Galactosiltransferases/análise , Aborto Habitual/genética , Adulto , China , Feminino , Galactosiltransferases/sangue , Humanos , Fenótipo , Gravidez , Estudos Retrospectivos , Mutação Silenciosa/genética
2.
Nat Commun ; 10(1): 2569, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189880

RESUMO

Synonymous mutations have been viewed as silent mutations, since they only affect the DNA and mRNA, but not the amino acid sequence of the resulting protein. Nonetheless, recent studies suggest their significant impact on splicing, RNA stability, RNA folding, translation or co-translational protein folding. Hence, we compile 659194 synonymous mutations found in human cancer and characterize their properties. We provide the user-friendly, comprehensive resource for synonymous mutations in cancer, SynMICdb ( http://SynMICdb.dkfz.de ), which also contains orthogonal information about gene annotation, recurrence, mutation loads, cancer association, conservation, alternative events, impact on mRNA structure and a SynMICdb score. Notably, synonymous and missense mutations are depleted at the 5'-end of the coding sequence as well as at the ends of internal exons independent of mutational signatures. For patient-derived synonymous mutations in the oncogene KRAS, we indicate that single point mutations can have a relevant impact on expression as well as on mRNA secondary structure.


Assuntos
Bases de Dados de Ácidos Nucleicos , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Mutação Silenciosa/genética , Conjuntos de Dados como Assunto , Humanos , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Dobramento de RNA/genética , Processamento de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética
3.
PLoS Genet ; 15(3): e1007967, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30901340

RESUMO

Mast cell tumours are the most common type of skin cancer in dogs, representing a significant concern in canine health. The molecular pathogenesis is largely unknown, but breed-predisposition for mast cell tumour development suggests the involvement of inherited genetic risk factors in some breeds. In this study, we aimed to identify germline risk factors associated with the development of mast cell tumours in Labrador Retrievers, a breed with an elevated risk of mast cell tumour development. Using a methodological approach that combined a genome-wide association study, targeted next generation sequencing, and TaqMan genotyping, we identified a synonymous variant in the DSCAM gene on canine chromosome 31 that is associated with mast cell tumours in Labrador Retrievers. DSCAM encodes a cell-adhesion molecule. We showed that the variant has no effect on the DSCAM mRNA level but is associated with a significant reduction in the level of the DSCAM protein, suggesting that the variant affects the dynamics of DSCAM mRNA translation. Furthermore, we showed that the variant is also associated with mast cell tumours in Golden Retrievers, a breed that is closely related to Labrador Retrievers and that also has a predilection for mast cell tumour development. The variant is common in both Labradors and Golden Retrievers and consequently is likely to be a significant genetic contributor to the increased susceptibility of both breeds to develop mast cell tumours. The results presented here not only represent an important contribution to the understanding of mast cell tumour development in dogs, as they highlight the role of cell adhesion in mast cell tumour tumourigenesis, but they also emphasise the potential importance of the effects of synonymous variants in complex diseases such as cancer.


Assuntos
Moléculas de Adesão Celular/genética , Mastocitoma Cutâneo/genética , Mastocitoma Cutâneo/veterinária , Animais , Adesão Celular/genética , Doenças do Cão/genética , Cães , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Células Germinativas , Mutação em Linhagem Germinativa/genética , Mastócitos/metabolismo , Mastócitos/fisiologia , Mastocitoma Cutâneo/metabolismo , Mastocitose Cutânea/genética , Fatores de Risco , Mutação Silenciosa/genética , Neoplasias Cutâneas/genética
4.
Gene ; 694: 111-120, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-30738968

RESUMO

Codon usage bias (CUB) is a universal feature of genomes, and in most species CUB of protein coding genes is positively correlated with expression level and degree of evolutionary conservation. There is mounting experimental evidence that CUB is due in part to selection for translational efficiency and/or accuracy, i.e., translational selection. However, there is a paucity of experimental data on whether and how CUB acts in trans - does the usage of preferred codons in a highly expressed gene affect the translation of other genes by freeing up more ribosomes, thereby increasing their availability to translate all mRNA transcripts in the cell? We investigated this question by creating two extreme versions of the highly expressed Escherichia coli ß-lactamase (bla) gene, one comprised almost entirely of unpreferred codons, and a second comprised almost entirely of preferred codons. We monitored the fitness effects of these synonymous mutations over hundreds of generations in two selective environments that allowed us to disentangle translational effects acting in cis from those acting in trans. In a selective environment for maximizing translational efficiency in trans of a gene (tetA) encoding a tetracycline resistance protein, unpreferred synonymous mutations had a negative impact on long-term fitness, whereas preferred mutations had a positive impact on long-term fitness, providing strong experimental evidence for a pleiotropic effect of translational selection.


Assuntos
Escherichia coli/genética , Mutação Silenciosa/fisiologia , beta-Lactamases/genética , Códon , Proteínas de Escherichia coli/genética , Evolução Molecular , Pleiotropia Genética/genética , Modelos Genéticos , Mutação/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Seleção Genética/genética , Mutação Silenciosa/genética , beta-Lactamases/metabolismo
5.
Int J Biol Macromol ; 116: 23-30, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29738863

RESUMO

CONTEXT: Insulin like growth factor 1 (IGF1) is privotal in the regulation of animal growth and is a single-chain globular protein composed of B, C, A, and D regions, of which the C region is involved in maintaining high affinity binding to the IGF1 receptor (IGF1R). PURPOSE: In this study, significant expression differences between large pigs and miniature pigs were detected and only one synonymous SNP (c.258G>A) in the C region of the coding sequence of IGF1 gene was screened. The aim of this manuscript was to clear the function of the SNP and clarify the mechanism of its influnce. METHODS: The expression vectors contained A allele and G allele were constructed, and the expression assays of the two groups were determined by qRT-PCR and western blotting, then the stability assays of the mRNA and protein were carried out under the inhibitation of actinomycin D and cycloheximide, respectively. At last, the binding affinity of IGF1-G and IGF1-A with IGF1R were indicated by co-immunoprecipitation and double immunofluorescence labeling methods, the conformation difference was detected by differential immunodetection. RESULTS: The IGF1-G expressed higher than IGF1-A in both transcription and translation levels, and the mRNA and protein stabilities of IGF1-G were lower than IGF1-A (P < 0.05). Furthermore, the relative binding affinity of GG-genotype IGF1 with IGF1R was significantly higher than that of the AA-genotype IGF1 (P < 0.05), and there was a difference in the conformation of the IGF1 with two genotypes. CONCLUSION: Our findings indicated the synonymous mutation altered the IGF1 gene expression and confirmed the synonymous mutation affected the IGF1 folding and the interactions with the IGF1R preliminarily.


Assuntos
Expressão Gênica/genética , Fator de Crescimento Insulin-Like I/genética , Mutação Silenciosa/genética , Células 3T3 , Alelos , Animais , Linhagem Celular , Genótipo , Camundongos , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Receptores de Somatomedina/genética , Suínos , Transcrição Genética/genética
6.
J Basic Microbiol ; 58(5): 414-424, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29534300

RESUMO

Lyme disease, caused by Borrelia burgdorferi, is a focally endemic tick-transmitted zoonotic infection. In this study, the major factors underlying synonymous codon-related amino acid usage in the B. burgdorferi genome and bias in synonymous codon usage of the translation initiation region of coding sequences were analyzed. Additionally, adaptation of B. burgdorferi to several of its hosts was analyzed in the context of synonymous codon usage. Principal component analysis (PCA) revealed that nucleotide content at the third synonymous position of a codon influenced the synonymous codon usage pattern, but the strand-specific factor did not influence the synonymous codon usage pattern of B. burgdorferi. In terms of the low GC content of B. burgdorferi coding sequences, the effective number of codons (ENC) showed a significant correlation with GC3 content (at the synonymous position). For the amino acid usage pattern for B. burgdorferi, PCA showed that the strand-specific factor did not contribute to this pattern, while the properties (aromaticity and hydrophobicity) of the amino acids themselves showed strong correlations with this pattern. Under-represented codons, which were frequently selected in the translation initiation region, possibly play roles in regulating gene expression in B. burgdorferi. In terms of co-evolution and synonymous codon usage patterns, adaptation of B. burgdorferi to different intermediate hosts was apparent to different degrees, and the degree of adaptation of this spirochete to wild animals was stronger than that of humans or mice.


Assuntos
Adaptação Fisiológica/genética , Aminoácidos/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/fisiologia , Códon/genética , Mutação Silenciosa/genética , Animais , Composição de Bases , Borrelia burgdorferi/patogenicidade , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Interações Hospedeiro-Patógeno , Humanos , Doença de Lyme/microbiologia , Fases de Leitura Aberta , Análise de Componente Principal , Análise de Sequência
7.
BMC Oral Health ; 18(1): 39, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29530000

RESUMO

BACKGROUND: The VicRK two-component signalling system regulates virulence and cariogenicity in Streptococcus mutans (S. mutans). The purpose of this study was to explore the genetic polymorphisms of the vicR and vicK genes, which are associated with dental caries in children with S. mutans. METHODS: In this study, 121 (from each group) clinical S. mutans strains were isolated from caries-free children and children with high-severity caries to sequence the vicR and vicK genes. Genomic DNA was extracted from S. mutans strains and amplified using PCR. The PCR products were purified and sequenced. A chi-squared test and ABI Variant Reporter software were used to analyse the sequencing results. RESULTS: The 242 clinically isolated S. mutans strains contained the full-length vicR and vicK genes. No nucleotide sequence insertions or deletions were observed in the two genes. Four silent point mutations were identified in the vicR genes, and no missense mutations could be detected. Forty-one mutations were identified in the vicK genes. In addition to 32 silent mutations, 9 missense mutations at the 173, 337, 470, 1051, 1132, 1258, 1260, 1277, and 1348 bp positions were found. The distribution frequencies of the missense mutations were not significantly different between the groups, except for the C470T mutation. The frequency of the C470T missense mutation was higher in the high-severity caries group than in the caries-free group. CONCLUSIONS: vicR sequences are highly conserved in S. mutans clinical isolates. The locus 470 missense mutation of the vicK gene may be related to caries in children with S. mutans.


Assuntos
Proteínas de Bactérias/genética , Cárie Dentária/microbiologia , Genes Bacterianos/genética , Polimorfismo Genético/genética , Streptococcus mutans/genética , Pré-Escolar , Cárie Dentária/etiologia , Humanos , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Mutação Silenciosa/genética , Streptococcus mutans/patogenicidade
8.
RNA Biol ; 15(1): 62-69, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-28873329

RESUMO

As a genetic disease, cancer is caused by the activation of oncogenes and the inhibition of tumor suppressor genes via genetic and epigenetic mechanisms. Given the important role of energy metabolism in tumors, we analyzed the cancer-derived mutations occurring in the DNA of the mitochondrion. Mutations in the mitochondrial DNA (mtDNA) compared to nuclear DNA are 62% decreased relative to the coding length per chromosome. We find that the majority of these mutations affects highly conserved nucleotides - significantly exceeding the conservation of the mtDNA - and are devoid of single nucleotide polymorphisms (SNPs). Surprisingly, the leading resources for tumor genetics information universally use the standard genetic code for translation of nucleotide into amino acid sequences in their online resources. However, the nuclear and mitochondrial genetic codes differ for four codons and the usage of incomplete STOP codons. Hence, we analyze and curate the consequences for all mutations in the mtDNA and comprehensively reclassify missense, nonsense and synonymous mutations accordingly. In total, 10% of the mutations are incorrectly translated leading to significant changes in the distribution of mutation types with tripling of nonsense and 69% loss of nonstop extension mutations. Lastly, we provide a curated dataset of coding and non-coding mitochondrial mutations in cancer merged, standardized, duplicate-free and aggregated from two databases as a resource including orthogonal data on their high conservation and SNPs. This study generally highlights the need to universally regard the important differences between the standard and mitochondrial genetic code in life science research.


Assuntos
Núcleo Celular/genética , DNA Mitocondrial/genética , Mitocôndrias/genética , Neoplasias/genética , Códon sem Sentido/genética , Códon de Terminação/genética , Bases de Dados Genéticas , Código Genético , Humanos , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único/genética , Biossíntese de Proteínas , Mutação Silenciosa/genética
9.
J Clin Lab Anal ; 32(4): e22330, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28926139

RESUMO

BACKGROUND: D antigen is one of the most clinically significant blood group antigens. Variation of the RHD gene can cause weak D or partial D phenotypes. While most variations are missense substitutions with amino acid changes, those without are called "silent" or "synonymous" substitutions. Synonymous substitutions often have little effect on the protein, not altering the phenotype. However, effect on splicing can affect end-product protein. We report a new synonymous variation, RHD 1056C>G, that resulted in weak D phenotype, and predicted its effect with various in silico methods. METHODS: Serologic testing of the D antigen with full sequencing of the RHD gene was done. Human Splice Finder was used to predict the effect of this variation, and validation of this method was done with all known RHD variations reported in the literature. RESULTS: RHD 1056C>G was predicted to cause the formation of an exonic splicing silencer (ESS) site. The creation of new ESS site potentially inhibits the splicing event, resulting alteration of splicing. This is similar to remodeling of splice acceptor or donor site, as this kind of deep exonic variation could affect the D antigen's quality or quantity. This is in concordance with serologic results, which showed only delayed weak agglutination to anti-D reagents. CONCLUSIONS: The analytic methods we applied showed good correlation with the actual phenotype, along with concordant results when analyzing other known variants reported in the literature. We conclude that RHD 1056C>G results in serologic weak D phenotype.


Assuntos
Biologia Computacional/métodos , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Sistema do Grupo Sanguíneo Rh-Hr/genética , Mutação Silenciosa/genética , Genótipo , Humanos , Masculino
10.
Mol Biol Evol ; 34(11): 2944-2958, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28961875

RESUMO

Codon usage bias (CUB) refers to the observation that synonymous codons are not used equally frequently in a genome. CUB is stronger in more highly expressed genes, a phenomenon commonly explained by stronger natural selection on translational accuracy and/or efficiency among these genes. Nevertheless, this phenomenon could also occur if CUB regulates gene expression at the mRNA level, a hypothesis that has not been tested until recently. Here, we attempt to quantify the impact of synonymous mutations on mRNA level in yeast using 3,556 synonymous variants of a heterologous gene encoding green fluorescent protein (GFP) and 523 synonymous variants of an endogenous gene TDH3. We found that mRNA level was positively correlated with CUB among these synonymous variants, demonstrating a direct role of CUB in regulating transcript concentration, likely via regulating mRNA degradation rate, as our additional experiments suggested. More importantly, we quantified the effects of individual synonymous mutations on mRNA level and found them dependent on 1) CUB and 2) mRNA secondary structure, both in proximal sequence contexts. Our study reveals the pleiotropic effects of synonymous codon usage and provides an additional explanation for the well-known correlation between CUB and gene expression level.


Assuntos
Códon/genética , RNA Mensageiro/genética , Mutação Silenciosa/genética , Evolução Molecular , Regulação da Expressão Gênica/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Proteínas de Fluorescência Verde/genética , Modelos Genéticos , Mutação , Biossíntese de Proteínas/genética , Estabilidade de RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Seleção Genética/genética
11.
BMC Oral Health ; 17(1): 114, 2017 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-28705197

RESUMO

BACKGROUND: To explore and analyse the association between biofilm and the genetic polymorphisms of scrA gene of EnzymeIIscr found in clinical isolates of Streptococcus mutans (S. mutans) from severe early childhood caries (S-ECC) in 3 years old children. METHODS: Clinical strains of S. mutans were conserved from a previous study. Thirty strains of S. mutans from the S-ECC group and 30 strains of S. mutans from the caries free (CF) group were selected. Biomass and viability of biofilm formed by the strains were evaluated by crystal violet and alamar blue assay. Genomic DNA was extracted from the S. mutans isolates. PCR was conducted to amplify scrA gene. After purified and sequenced the PCR products, BioEdit sofeware was used to analyse the sequence results. A chi-square test was used to compare the results. RESULTS: Compared to the CF group, the biomass of S-ECC group was higher (P = 0.0424). However, the viability of the two groups showed no significant difference. All 60 clinically isolated S. mutans strains had a 1995 base pair (bp) scrA gene. Forty-nine point mutations were identified in scrA from the 60 clinical isolates. There were 17 missense point mutations at the 10, 65, 103, 284, 289, 925, 1444, 1487, 1494, 1508, 1553, 1576, 1786, 1822, 1863, 1886, and 1925 bp positions. The other 32 mutations were silent point mutations. No positions were found at active sites of ScrA. The statistic analyse showed no significant missense mutation rates between the two groups. CONCLUSIONS: There was no association between biofilm and genetic polymorphisms of scrA from S. mutans with S-ECC in 3 years old children.


Assuntos
Biofilmes , Cárie Dentária/microbiologia , Genes Bacterianos/genética , Streptococcus mutans/genética , Biofilmes/crescimento & desenvolvimento , Estudos de Casos e Controles , Pré-Escolar , DNA Bacteriano/genética , Humanos , Mutação de Sentido Incorreto/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Mutação Silenciosa/genética , Streptococcus mutans/isolamento & purificação
12.
Hum Mutat ; 38(10): 1336-1347, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28649752

RESUMO

Synonymous single-nucleotide variants (SNVs), although they do not alter the encoded protein sequences, have been implicated in many genetic diseases. Experimental studies indicate that synonymous SNVs can lead to changes in the secondary and tertiary structures of DNA and RNA, thereby affecting translational efficiency, cotranslational protein folding as well as the binding of DNA-/RNA-binding proteins. However, the importance of these various features in disease phenotypes is not clearly understood. Here, we have built a support vector machine (SVM) model (termed DDIG-SN) as a means to discriminate disease-causing synonymous variants. The model was trained and evaluated on nearly 900 disease-causing variants. The method achieves robust performance with the area under the receiver operating characteristic curve of 0.84 and 0.85 for protein-stratified 10-fold cross-validation and independent testing, respectively. We were able to show that the disease-causing effects in the immediate proximity to exon-intron junctions (1-3 bp) are driven by the loss of splicing motif strength, whereas the gain of splicing motif strength is the primary cause in regions further away from the splice site (4-69 bp). The method is available as a part of the DDIG server at http://sparks-lab.org/ddig.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas/genética , Mutação Silenciosa/genética , DNA/química , Proteínas de Ligação a DNA/química , Predisposição Genética para Doença , Humanos , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único/genética , Dobramento de Proteína , Proteínas/química , RNA/química , RNA/genética
13.
Mol Biol Evol ; 34(7): 1613-1628, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28369510

RESUMO

TYRO3, AXL, and MERTK (TAM) receptors are a family of receptor tyrosine kinases that maintain homeostasis through the clearance of apoptotic cells, and when defective, contribute to chronic inflammatory and autoimmune diseases such as atherosclerosis, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and Crohn's disease. In addition, certain enveloped viruses utilize TAM receptors for immune evasion and entry into host cells, with several viruses preferentially hijacking MERTK for these purposes. Despite the biological importance of TAM receptors, little is understood of their recent evolution and its impact on their function. Using evolutionary analysis of primate TAM receptor sequences, we identified strong, recent positive selection in MERTK's signal peptide and transmembrane domain that was absent from TYRO3 and AXL. Reconstruction of hominid and primate ancestral MERTK sequences revealed three nonsynonymous single nucleotide polymorphisms in the human MERTK signal peptide, with a G14C mutation resulting in a predicted non-B DNA cruciform motif, producing a significant decrease in MERTK expression with no significant effect on MERTK trafficking or half-life. Reconstruction of MERTK's transmembrane domain identified three amino acid substitutions and four amino acid insertions in humans, which led to significantly higher levels of self-clustering through the creation of a new interaction motif. This clustering counteracted the effect of the signal peptide mutations through enhancing MERTK avidity, whereas the lower MERTK expression led to reduced binding of Ebola virus-like particles. The decreased MERTK expression counterbalanced by increased avidity is consistent with antagonistic coevolution to evade viral hijacking of MERTK.


Assuntos
Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Apoptose/genética , Sequência de Bases/genética , Movimento Celular , Evolução Molecular , Homeostase , Humanos , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Primatas/genética , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Mutação Silenciosa/genética , c-Mer Tirosina Quinase
14.
Biol Pharm Bull ; 40(7): 1086-1091, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28420819

RESUMO

Middle East respiratory syndrome coronavirus (MERS CoV) is a recently evolved fatal respiratory disease that poses a concern for a global epidemic. MERS CoV encodes 2 proteases, 3C-like protease (3CLpro) and papain-like protease (PLpro). These proteases share in processing MERS CoV polyproteins at different sites to yield 16 nonstructural proteins. In this work, we provide evidence that MERS CoV 3CLpro and PLpro are subject to different genetic and evolutionary influences that shape the protein sequence, codon usage pattern, and codon usage bias. Compositional bias is present in both proteins due to a preference for AT nucleotides. Thymidine (T) was highly preferred at the third position of codons, preferred and overrepresented codons in PLpro, but was replaced by guanosine (G) in 3CLpro. Compositional constraints were important in PLpro but not in 3CLpro. Directed mutation pressure seems to have a strong influence on 3CLpro codon usage, which is more than 30-fold higher than that in PLpro. Translational selection was evident with PLpro but not with 3CLpro. Both proteins are less immunogenic by showing low CpG frequencies. Correspondence analysis reveals the presence of 3 genetic clusters based on codon usage in PLpro and 3CLpro. Every protein had one common cluster and 2 different clusters. As revealed by correspondence analysis, the number of influences on codon usage are restricted in MERS CoV 3CLpro. In contrast, PLpro is controlled by a broader range of compositional, mutational, and other influences. This may be due to the multifunctional protease, deubiquitination, and innate immunity suppressing profiles of PLpro.


Assuntos
Biologia Computacional/métodos , Cisteína Endopeptidases/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Papaína/genética , Mutação Silenciosa/genética , Proteínas Virais/genética , Cisteína Endopeptidases/química , Evolução Molecular , Genoma Viral , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Papaína/química , Processamento de Proteína Pós-Traducional , Proteínas Virais/química , Replicação Viral/genética
15.
Trends Genet ; 33(4): 283-297, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28292534

RESUMO

The genetic code, which defines the amino acid sequence of a protein, also contains information that influences the rate and efficiency of translation. Neither the mechanisms nor functions of codon-mediated regulation were well understood. The prevailing model was that the slow translation of codons decoded by rare tRNAs reduces efficiency. Recent genome-wide analyses have clarified several issues. Specific codons and codon combinations modulate ribosome speed and facilitate protein folding. However, tRNA availability is not the sole determinant of rate; rather, interactions between adjacent codons and wobble base pairing are key. One mechanism linking translation efficiency and codon use is that slower decoding is coupled to reduced mRNA stability. Changes in tRNA supply mediate biological regulationfor instance,, changes in tRNA amounts facilitate cancer metastasis.


Assuntos
Códon/genética , Código Genético , Biossíntese de Proteínas , Mutação Silenciosa/genética , Sequência de Aminoácidos/genética , Pareamento de Bases , Regulação da Expressão Gênica , Estabilidade de RNA/genética , RNA de Transferência/genética , Ribossomos/genética
16.
Mol Biol Evol ; 34(6): 1417-1428, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28333215

RESUMO

A central question in evolutionary biology is why some species have more genetic diversity than others and a no less important question is why selection efficacy varies among species. Although these questions have started to be tackled in animals, they have not been addressed to the same extent in plants. Here, we estimated nucleotide diversity at synonymous, πS, and nonsynonymous sites, πN, and a measure of the efficacy of selection, the ratio πN/πS, in 34 animal and 28 plant species using full genome data. We then evaluated the relationship of nucleotide diversity and selection efficacy with effective population size, the distribution of fitness effect and life history traits. In animals, our data confirm that longevity and propagule size are the variables that best explain the variation in πS among species. In plants longevity also plays a major role as well as mating system. As predicted by the nearly neutral theory of molecular evolution, the log of πN/πS decreased linearly with the log of πS but the slope was weaker in plants than in animals. This appears to be due to a higher mutation rate in long lived plants, and the difference disappears when πS is rescaled by the mutation rate. Differences in the distribution of fitness effect of new mutations also contributed to variation in πN/πS among species.


Assuntos
Variação Genética/genética , Seleção Genética/genética , Animais , Evolução Biológica , Evolução Molecular , Aptidão Genética/genética , Genoma , Genoma de Planta , Modelos Genéticos , Mutação , Filogenia , Polimorfismo Genético/genética , Densidade Demográfica , Mutação Silenciosa/genética
17.
Jpn J Clin Oncol ; 47(6): 576-580, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334867

RESUMO

The proband was a 67-year-old man with transverse and sigmoid colon cancer. Microsatellite instability analysis revealed a high frequency of microsatellite instability, and immunohistochemical staining showed the absence of both MLH1 and PMS2 proteins in the sigmoid colon cancer tissue specimens from the patient. DNA sequencing revealed a nucleotide substitution c.543C>T in MLH1, but this variant did not substitute an amino acid. The MLH1 c.543C>T variant was located 3 bases upstream from the end of exon 6 and created a new splice donor site 4 bases upstream from the end of exon 6. Consequently, the last 4 bases of exon 6 were deleted and frameshift occurred. Thus, the MLH1 c.543C>T silent mutation is considered 'pathogenic'.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteína 1 Homóloga a MutL/genética , Mutação Silenciosa/genética , Idoso , Processamento Alternativo/genética , Sequência de Bases , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de RNA
19.
Biotechnol Bioeng ; 114(5): 1054-1064, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27943233

RESUMO

Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon-optimized (i.e., rare codon-free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha-helix, beta-strand, or linker in the first half of rare codon-free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta-strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi-SRC-containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP-folding reporter when it was fused to the 3' end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six-fold enhancement in soluble protein expression level as compared to the wild-type and rare codon-free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co-translational folding, resulting in enhanced functional protein expression. Biotechnol. Bioeng. 2017;114: 1054-1064. © 2016 Wiley Periodicals, Inc.


Assuntos
Códon/genética , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosiltransferases/genética , Mutação Silenciosa/genética , Clonagem Molecular , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Modelos Moleculares , Biossíntese de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermoanaerobacterium/enzimologia , Thermoanaerobacterium/genética
20.
J Bone Miner Res ; 32(1): 99-105, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27468155

RESUMO

Autosomal recessive osteopetroses (AROs) are rare, genetically heterogeneous skeletal diseases with increased bone density that are often lethal if left untreated. A precise molecular classification is relevant for the patient's management, because in some subgroups hematopoietic stem cell transplantation (HSCT), which is the only curative therapy, is contraindicated. In two unrelated ARO patients, the molecular analysis revealed the presence of a synonymous variant in known ARO genes, namely in the TCIRG1 gene in one patient and in the CLCN7 in the other patient, predicted to impact on the splicing process. In the latter case, sequencing of the transcript confirmed the splicing defect, whereas in the former, for whom an RNA sample was not available, the defect was reconstructed in vitro by the minigene technology. These results strongly suggest that these synonymous changes were responsible for the disease in our patients. Our findings are novel with respect to ARO and add to the few reports in literature dealing with different diseases, underlining the importance of cDNA analysis for the correct assessment of exonic changes, even when exome sequencing is performed. In particular, we highlight the possibility that at least in some cases ARO is due to synonymous changes, erroneously considered clinically silent, in the genes already described in literature, and suggest carefully reevaluating the sequencing results of these genes when mutations are not found at a first analysis. In addition, with respect to the CLCN7 gene, we suggest that synonymous variants might also contribute to the large spectrum of severity typical of CLCN7-dependent osteopetrosis through more subtle, but not negligible, effects on protein availability and functionality. © 2016 American Society for Bone and Mineral Research.


Assuntos
Osteopetrose/diagnóstico , Osteopetrose/genética , Mutação Silenciosa/genética , Sequência de Aminoácidos , Sequência de Bases , Canais de Cloreto/genética , Evolução Fatal , Feminino , Humanos , Lactente , Masculino , Osteopetrose/diagnóstico por imagem , Gravidez , Alinhamento de Sequência , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genética
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