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1.
Methods Mol Biol ; 2626: 323-333, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36715913

RESUMO

The production of eggs in the Drosophila ovary requires complex interactions between multiple cell types that coexist within the same solid tissue. This cellular heterogeneity makes the ovary a rich subject of study, but also makes it challenging to identify transcriptional differences between individual cell types using methods such as bulk RNA sequencing. The development of single-cell RNA sequencing (scRNA-seq) techniques addresses this limitation by providing an avenue to profile genetic and functional heterogeneity at a cellular resolution. Here, we describe the isolation and preparation of the Drosophila ovary for scRNA-seq. This protocol emphasizes a short preparation time, high cell viability, prevention of RNA-degradation, and reduction of technical variation to achieve highly reproducible single-cell profiles.


Assuntos
Drosophila , Análise de Célula Única , Animais , Feminino , Drosophila/genética , Análise de Célula Única/métodos , Análise de Sequência de RNA/métodos , Ovário/metabolismo , Sequência de Bases , RNA/genética , RNA/metabolismo , Perfilação da Expressão Gênica/métodos
2.
Arch Virol ; 168(2): 50, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36609709

RESUMO

The whole genome sequence of mulberry crinivirus (MuCV), a novel member of the genus Crinivirus (family Closteroviridae) identified in mulberry (Morus alba L), was determined. The virus possesses a bipartite genome. RNA1 contains 8571 nucleotides (nt) with four open reading frames (ORFs). ORF1a encodes a putative polyprotein with papain-like protease, methyltransferase, and RNA helicase domains. ORF1b putatively encodes an RNA-dependent RNA polymerase (RdRp), which is probably expressed via a + 1 ribosomal frameshift. RNA2 consists of 8082 nt, containing eight ORFs that are similar in size and position to orthologous genes of other criniviruses. Phylogenetic analysis based on RdRp amino acid sequences of criniviruses placed MuCV in group 1.


Assuntos
Crinivirus , Morus , Crinivirus/genética , Sequência de Bases , Filogenia , Genoma Viral , Nucleotídeos , Fases de Leitura Aberta , RNA Polimerase Dependente de RNA/genética , RNA Viral/genética
3.
Arch Virol ; 168(2): 51, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36609930

RESUMO

Bovine respiratory syncytial virus (BRSV) strains that were detected in Kagoshima prefecture and isolated in Hokkaido between 2017 and 2019, together with a BRSV vaccine strain, were subjected to full-genome sequencing. The BRSV strains identified in Japan were found to be genetically close to each other but distant from the vaccine strains. The deduced amino acids at positions 206 and 208 of the glycoprotein (G protein), which form one of the major epitopes of the recent Japanese BRSV strains, were different from those of the vaccine strains. Therefore, the recent Japanese BRSV strains might be antigenically different from the BRSV vaccine strains.


Assuntos
Doenças dos Bovinos , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Bovino , Animais , Bovinos , Vírus Sincicial Respiratório Bovino/genética , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/veterinária , Infecções por Vírus Respiratório Sincicial/genética , Japão , Sequência de Bases , Anticorpos Antivirais
4.
Vector Borne Zoonotic Dis ; 23(1): 35-43, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36595376

RESUMO

Background: In August 2013, a virus strain (DH13M98) was isolated from Culex tritaeniorhynchus Giles collected in Mangshi, the southwestern border area of Yunnan Province, China. The virus replicated and caused cytopathic effects (CPE) in Aedes albopictus (C6/36) cells, but not in baby hamster Syrian kidney (BHK-21) cells. Materials and Methods: Agarose gel electrophoresis (AGE) analysis revealed that the DH13M98 virus was a 10-segment double-stranded RNA (dsRNA) virus, with a "1-1-1-2-1-1-2-1" pattern. The full genome of the DH13M98 virus was sequenced by full-length amplification of complementary DNAs (FLAC). Results: Phylogenetic analysis of the viral RNA-dependent RNA polymerase (Pol), major subcore-shell (T2), and major core-surface (T13) protein showed that DH13M98 clustered with Umatilla virus (UMAV), and the amino acid (aa) sequences of DH13M98 shared more than 89.5% (Pol), 95% (T2), and 91.1% (T13) identity with UMAV. However, the aa identity of outer capsid protein one (OC1) of DH13M98 with other UMAV was 57.1-79.2%, suggesting that DH13M98 was UMAV, but distinct from other strains of UMAV from the United States, Japan, and Germany at OC1, and it may be a high variant strain of UMAV, even a new serotype. Conclusion: This is the first isolation of UMAV in China, which enriches the resources of virus species in China and provides new insights into the genetic diversity and geographical distribution of the virus.


Assuntos
Culex , Orbivirus , Cricetinae , Animais , China , Filogenia , Orbivirus/genética , Sequência de Bases
5.
Biotechniques ; 74(1): 5-7, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36625391

RESUMO

Standfirst: In the last decade, ancient DNA research has provided invaluable insights into the lives of ancient populations, adding detail and enriching the story of human evolution and development. [Formula: see text].


Assuntos
DNA Antigo , Humanos , Análise de Sequência de DNA , Sequência de Bases
6.
Trends Genet ; 39(2): 109-124, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36604282

RESUMO

In addition to the canonical right-handed double helix, other DNA structures, termed 'non-B DNA', can form in the genomes across the tree of life. Non-B DNA regulates multiple cellular processes, including replication and transcription, yet its presence is associated with elevated mutagenicity and genome instability. These discordant cellular roles fuel the enormous potential of non-B DNA to drive genomic and phenotypic evolution. Here we discuss recent studies establishing non-B DNA structures as novel functional elements subject to natural selection, affecting evolution of transposable elements (TEs), and specifying centromeres. By highlighting the contributions of non-B DNA to repeated evolution and adaptation to changing environments, we conclude that evolutionary analyses should include a perspective of not only DNA sequence, but also its structure.


Assuntos
Elementos de DNA Transponíveis , Genômica , Humanos , Elementos de DNA Transponíveis/genética , Sequência de Bases , Instabilidade Genômica/genética , Evolução Molecular
7.
Chromosome Res ; 31(1): 3, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36692656

RESUMO

Like other cecidomyiid Diptera, Hessian fly has stable S chromosomes and dispensable E chromosomes that are retained only in the germ line. Amplified fragment length polymorphisms (AFLP), suppressive subtractive hybridization (SSH), fluorescent in-situ hybridization (FISH), and sequencing were used to investigate similarities and differences between S and E chromosomes. More than 99.9% of AFLP bands were identical between separated ovary and somatic tissue, but one band was unique to ovary and resembled Worf, a non-LTR retrotransposon. Arrayed clones, derived by SSH of somatic from ovarian DNA, showed no clones that were unique to ovary. FISH with BAC clones revealed a diagnostic banding pattern of BAC positions on both autosomes and both sex chromosomes, and each E chromosome shared a pattern with one of the S chromosomes. Sequencing analysis showed that E chromosomes are nearly identical to S chromosomes, since no sequence could be confirmed to belong only to E chromosomes. There were a few questionably E-specific sequences that are candidates for further investigation. Thus, the E chromosomes appear to be derived from S chromosomes by the acquisition or conversion of sequences that produce the negatively heteropycnotic region around the centromere.


Assuntos
Dípteros , Animais , Feminino , Dípteros/genética , Sequência de Bases , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Cromossomos Sexuais/genética , DNA/genética
8.
Planta ; 257(2): 45, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36695892

RESUMO

MAIN CONCLUSION: This study reported seven new plastomes from Impatiens and observed three highly variable regions for phylogeny and DNA barcoding, which resolved the relationships among sections of subgenus Impatiens. Impatiens L. (Balsaminaceae, Ericales) is one of the largest and most diverse genera of angiosperms, widely known for its taxonomic difficulty. In this study, we reevaluated the infrageneric relationships within the genus Impatiens, using complete plastome sequence data. Seven complete plastomes of Impatiens (representing 6 species) were newly sequenced and characterized along with 20 previously published plastomes of other Impatiens species, plus 2 plastomes of outgroups (Hydrocera triflora, Balsaminaceae; Marcgravia coriacea, Marcgraviaceae). The total size of these 29 plastomes ranged from 151,538 bp to 152,917 bp, except 2 samples of Impatiens morsei, which exhibited a shorter length and lost some genes encoding NADH dehydrogenase subunits. Moreover, the number of simple sequence repeats (SSRs) ranged from 51 to 113, and the number of long repeats from 17 to 26. In addition, three highly variable regions were identified (trnG-GCC (The previous one), ndhF-rpl32-trnL-UGA-ccsA, and ycf1). Our phylogenomic analysis based on 80 plastome-derived protein-coding genes strongly supported the monophyly of Impatiens and its two subgenera (Clavicarpa and Impatiens), and fully resolved relationships among the six (out of seven) sampled sections of subgenus Impatiens. Overall, the plastome DNA markers and phylogenetic results reported in this study will facilitate future identification, taxonomic and DNA barcoding studies in Impatiens as well as evolutionary studies in Balsaminaceae.


Assuntos
Balsaminaceae , Impatiens , Balsaminaceae/genética , Impatiens/genética , Filogenia , Sequência de Bases , Evolução Molecular
9.
Sci Adv ; 9(2): eadd3783, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36638170

RESUMO

Microcystis, a common harmful algal bloom (HAB) taxon, threatens water supplies and human health, yet species delimitation is contentious in this taxon, leading to challenges in research and management of this threat. Historical and common morphology-based classifications recognize multiple morphospecies, most with variable and diverse ecologies, while DNA sequence-based classifications indicate a single species with multiple ecotypes. To better delimit Microcystis species, we conducted a pangenome analysis of 122 genomes. Core- and non-core gene phylogenetic analyses placed 113 genomes into 23 monophyletic clusters containing at least two genomes. Overall, genome-related indices revealed that Microcystis contains at least 16 putative genospecies. Fifteen genospecies included at least one Microcystis aeruginosa morphospecies, and 10 genospecies included two or more morphospecies. This classification system will enable consistent taxonomic identification of Microcystis and thereby aid in resolving some of the complexities and controversies that have long characterized eco-evolutionary research and management of this important HAB taxon.


Assuntos
Microcystis , Humanos , Microcystis/genética , Filogenia , Sequência de Bases , Ecologia
10.
Methods Mol Biol ; 2630: 13-24, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36689173

RESUMO

Micro-RNAs (mi-RNAs) are regulatory elements that play a vital role in the growth, development, and metabolic regulation of plants. In current research, the isolation of miRNAs is a tedious and difficult task using in vitro methods. However, recent exploration into the remarkably highly conserved nature of nucleotide sequences of miRNAs assists in the identification of miRNAs in plant species through homologous approaches. Here, we describe the in silico-based method for identification of miRNAs from the EST database which is emerging as a faster and more reliable approach along with the development of miRNA-SSR markers. This approach has the potential to accelerate research into the regulation of gene expression in various plant species such as tea, potato, tomato, tobacco, and orphan crops like cluster bean.


Assuntos
MicroRNAs , MicroRNAs/genética , Sequência de Bases , Plantas/genética , Tabaco/genética , Regulação da Expressão Gênica de Plantas , RNA de Plantas/genética
11.
Methods Mol Biol ; 2630: 179-213, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36689184

RESUMO

The current versions of the microRNA databases MiRgeneDB, miRBase, and PmiREN contain annotations for a total of 358 different species. Public repositories, however, host small RNA sequencing data for over 800 species. This discrepancy implies that microRNA research is also very active in species that neither have an available high-quality genome assembly nor annotations for microRNAs or other types of noncoding genes. These cases are particularly challenging to analyze because reference sequences need to be collected from different sources and processed and formatted appropriately so that the dedicated small RNA analysis tools can make use of them. In this protocol we describe how small RNA sequencing data can be easily analyzed by means of a dockerized version of the well-established sRNAtoolbox/sRNAbench small RNA tools. We outline the analysis of two publicly available datasets to demonstrate basic aspects like the preparation of the local database, expression profiling, or differential expression analysis as well as more advanced features such as quantification of exogenous RNA content and data analysis in non-model species.


Assuntos
MicroRNAs , Software , MicroRNAs/genética , Análise de Sequência de RNA , Bases de Dados de Ácidos Nucleicos , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala/métodos
12.
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
13.
Genome Biol Evol ; 15(1)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36617272

RESUMO

Improved sequencing technologies have profoundly altered global views of fungal diversity and evolution. High-throughput sequencing methods are critical for studying fungi due to the cryptic, symbiotic nature of many species, particularly those that are difficult to culture. However, the low coverage genome sequencing (LCGS) approach to phylogenomic inference has not been widely applied to fungi. Here we analyzed 171 Kickxellomycotina fungi using LCGS methods to obtain hundreds of marker genes for robust phylogenomic reconstruction. Additionally, we mined our LCGS data for a set of nine rDNA and protein coding genes to enable analyses across species for which no LCGS data were obtained. The main goals of this study were to: 1) evaluate the quality and utility of LCGS data for both phylogenetic reconstruction and functional annotation, 2) test relationships among clades of Kickxellomycotina, and 3) perform comparative functional analyses between clades to gain insight into putative trophic modes. In opposition to previous studies, our nine-gene analyses support two clades of arthropod gut dwelling species and suggest a possible single evolutionary event leading to this symbiotic lifestyle. Furthermore, we resolve the mycoparasitic Dimargaritales as the earliest diverging clade in the subphylum and find four major clades of Coemansia species. Finally, functional analyses illustrate clear variation in predicted carbohydrate active enzymes and secondary metabolites (SM) based on ecology, that is biotroph versus saprotroph. Saprotrophic Kickxellales broadly lack many known pectinase families compared with saprotrophic Mucoromycota and are depauperate for SM but have similar numbers of predicted chitinases as mycoparasitic.


Assuntos
Artrópodes , Fungos , Humanos , Animais , Filogenia , Fungos/genética , Artrópodes/genética , Sequência de Bases , Genoma
14.
Emerg Infect Dis ; 29(2): 448-450, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36648111

RESUMO

Monkeypox virus (MPXV) has gained interest because of a multicountry outbreak of mpox (formerly monkeypox) cases with no epidemiologic link to MPXV-endemic regions. We sequenced the complete genome of MPXV isolated from a patient in northern Mexico. Phylogenetic analysis grouped the virus with isolates from Germany.


Assuntos
Vírus da Varíola dos Macacos , Varíola dos Macacos , Humanos , Vírus da Varíola dos Macacos/genética , Filogenia , México/epidemiologia , Varíola dos Macacos/diagnóstico , Varíola dos Macacos/epidemiologia , Sequência de Bases
15.
Brief Bioinform ; 24(1)2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36611257

RESUMO

Trans-splicing of a spliced leader (SL) to the 5' ends of mRNAs is used to produce mature mRNAs in several phyla of great importance to human health and the marine ecosystem. One of the consequences of the addition of SL sequences is the change or disruption of the open reading frames (ORFs) in the recipient transcripts. Given that most SL sequences have one or more of the trinucleotide NUG, including AUG in flatworms, trans-splicing of SL sequences can potentially supply a start codon to create new ORFs, which we refer to as slORFs, in the recipient mRNAs. Due to the lack of a tool to precisely detect them, slORFs were usually neglected in previous studies. In this work, we present the tool slORFfinder, which automatically links the SL sequences to the recipient mRNAs at the trans-splicing sites identified from SL-containing reads of RNA-Seq and predicts slORFs according to the distribution of ribosome-protected footprints (RPFs) on the trans-spliced transcripts. By applying this tool to the analyses of nematodes, ascidians and euglena, whose RPFs are publicly available, we find wide existence of slORFs in these taxa. Furthermore, we find that slORFs are generally translated at higher levels than the annotated ORFs in the genomes, suggesting they might have important functions. Overall, this study provides a tool, slORFfinder (https://github.com/songbo446/slORFfinder), to identify slORFs, which can enhance our understanding of ORFs in taxa with SL machinery.


Assuntos
RNA Líder para Processamento , Trans-Splicing , Humanos , RNA Líder para Processamento/genética , RNA Líder para Processamento/metabolismo , Fases de Leitura Aberta , Ecossistema , Sequência de Bases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Splicing de RNA
16.
Brief Bioinform ; 24(1)2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36642413

RESUMO

The coronavirus disease of 2019 pandemic has catalyzed the rapid development of mRNA vaccines, whereas, how to optimize the mRNA sequence of exogenous gene such as severe acute respiratory syndrome coronavirus 2 spike to fit human cells remains a critical challenge. A new algorithm, iDRO (integrated deep-learning-based mRNA optimization), is developed to optimize multiple components of mRNA sequences based on given amino acid sequences of target protein. Considering the biological constraints, we divided iDRO into two steps: open reading frame (ORF) optimization and 5' untranslated region (UTR) and 3'UTR generation. In ORF optimization, BiLSTM-CRF (bidirectional long-short-term memory with conditional random field) is employed to determine the codon for each amino acid. In UTR generation, RNA-Bart (bidirectional auto-regressive transformer) is proposed to output the corresponding UTR. The results show that the optimized sequences of exogenous genes acquired the pattern of human endogenous gene sequence. In experimental validation, the mRNA sequence optimized by our method, compared with conventional method, shows higher protein expression. To the best of our knowledge, this is the first study by introducing deep-learning methods to integrated mRNA sequence optimization, and these results may contribute to the development of mRNA therapeutics.


Assuntos
COVID-19 , Aprendizado Profundo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , COVID-19/genética , Sequência de Bases , Sequência de Aminoácidos
17.
Biomolecules ; 13(1)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36671542

RESUMO

In a previous work, the common gonadotrophic hormone α-subunit (ag-GTHα), the ag-FSH ß- and ag-LH ß-subunit cDNAs, were isolated and characterized by our research group from A. gigas pituitaries, while a preliminary synthesis of ag-FSH was also carried out in human embryonic kidney 293 (HEK293) cells. In the present work, the cDNA sequence encoding the ag-growth hormone (ag-GH) has also been isolated from the same giant Arapaimidae Amazonian fish. The ag-GH consists of 208 amino acids with a putative 23 amino acid signal peptide and a 185 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the Elopiformes (82.0%), followed by Anguilliformes (79.7%) and Acipenseriformes (74.5%). The identity with the corresponding human GH (hGH) amino acid sequence is remarkable (44.8%), and the two disulfide bonds present in both sequences were perfectly conserved. Three-dimensional (3D) models of ag-GH, in comparison with hGH, were generated using the threading modeling method followed by molecular dynamics. Our simulations suggest that the two proteins have similar structural properties without major conformational changes under the simulated conditions, even though they are separated from each other by a >100 Myr evolutionary period (1 Myr = 1 million years). The sequence found will be used for the biotechnological synthesis of ag-GH while the ag-GH cDNA obtained will be utilized for preliminary Gene Therapy studies.


Assuntos
Hormônio do Crescimento , Hormônio do Crescimento Humano , Animais , Humanos , Hormônio do Crescimento/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Células HEK293 , Sequência de Bases , Clonagem Molecular , Peixes/genética , Peixes/metabolismo , Hormônio do Crescimento Humano/genética
18.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675305

RESUMO

Current molecular classification approaches for endometrial cancer (EC) often employ multiple testing platforms. Some subtypes still lack univocal prognostic significance, highlighting the need for risk sub-stratification. The tumor immune microenvironment (TIME) is associated with tumor progression and prognosis. We sought to investigate the feasibility of classifying EC via DNA sequencing and interrogate immunologic signatures and prognostic markers across and within subtypes, respectively. Formalin-fixed paraffin-embedding (FFPE) samples from 50 EC patients underwent targeted DNA and RNA sequencing, and multiplex immunofluorescence assay for TIME. DNA sequencing classified 10%, 20%, 52%, and 18% of patients into the subtype of POLE-mutant, microsatellite instability-high (MSI-H), TP53-wt, and TP53-mutant. POLE-mutant tumors expressed the highest T-effector and IFN-γ signature and the lowest innate anti-PD-1 resistance signature among subtypes. TP53-wt revealed a converse enrichment trend for these immunologic signatures. Survival analyses using the Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA-UCEC) dataset identified associations of CCR5 (hazard ratio (HR) = 0.71, p = 0.035), TNFRSF14 (HR = 0.58, p = 0.028), and IL-10 (HR = 2.5, p = 0.012) with overall survival within MSI-H, TP53-mutant, and TP53-wt subtype, respectively. A TIME comparison between the sub-stratified subgroups of our cohort revealed upregulated tumor infiltration of immune cells in the low-risk subgroups. Our study demonstrates that targeted DNA sequencing is an effective one-stop strategy to classify EC. Immunomodulatory genes may serve as prognostic markers within subtypes.


Assuntos
Neoplasias do Endométrio , Feminino , Humanos , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Instabilidade de Microssatélites , Biomarcadores , Sequência de Bases , Modelos de Riscos Proporcionais , Biomarcadores Tumorais/genética , Microambiente Tumoral/genética
19.
Molecules ; 28(2)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36677740

RESUMO

During the last decade, the evidence for the biological relevance of i-motif DNA (i-DNA) has been accumulated. However, relatively few molecules were reported to interact with i-DNA, and a controversy concerning their binding mode, affinity, and selectivity persists in the literature. In this context, the cholestane derivative IMC-48 has been reported to modulate bcl-2 gene expression by stabilizing an i-motif structure in its promoter. In the present contribution, we report on a novel, more straightforward, synthesis of IMC-48 requiring fewer steps compared to the previous approach. Furthermore, the interaction of IMC-48 with four different i-motif DNA sequences was thoroughly investigated by bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy. Surprisingly, our results show that IMC-48 is a very weak ligand of i-DNA as no quantifiable interaction or significant stabilization of i-motif structures could be observed, stimulating a quest for an alternative mechanism of its biological activity.


Assuntos
Colestanos , DNA , Sequência de Bases , DNA/genética , DNA/química , Piperidinas/química , Colestanos/química , Dicroísmo Circular , Ligantes
20.
Nucleic Acids Res ; 51(2): 595-609, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36629271

RESUMO

Protein translation is tightly and precisely controlled by multiple mechanisms including upstream open reading frames (uORFs), but the origins of uORFs and their role in maize are largely unexplored. In this study, an active transposition event was identified during the propagation of maize inbred line B73. The transposon, which was named BTA for 'B73 active transposable element hAT', creates a novel dosage-dependent hypomorphic allele of the hexose transporter gene ZmSWEET4c through insertion within the coding sequence in the first exon, and results in reduced kernel size. The BTA insertion does not affect transcript abundance but reduces protein abundance of ZmSWEET4c, probably through the introduction of a uORF. Furthermore, the introduction of BTA sequence in the exon of other genes can regulate translation efficiency without affecting their mRNA levels. A transposon capture assay revealed 79 novel insertions for BTA and BTA-like elements. These insertion sites have typical euchromatin features, including low levels of DNA methylation and high levels of H3K27ac. A putative autonomous element that mobilizes BTA and BTA-like elements was identified. Together, our results suggest a transposon-based origin of uORFs and document a new role for transposable elements to influence protein abundance and phenotypic diversity by affecting the translation rate.


Assuntos
Biossíntese de Proteínas , Alelos , Sequência de Bases , RNA Mensageiro/genética , Fases de Leitura Aberta/genética
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