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1.
J Agric Food Chem ; 67(31): 8626-8631, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31287307

RESUMO

An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X]3C-[X]10-12-C[X]3C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other vicilin allergens (e.g., Car i 2). In many species, however, vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in vicilins might be a result of translocation during evolution.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Prunus dulcis/imunologia , Alérgenos/química , Alérgenos/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/genética , DNA Complementar/genética , Hipersensibilidade Alimentar/imunologia , Humanos , Prunus dulcis/química , Prunus dulcis/genética , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA
2.
Gene ; 712: 143945, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31279712

RESUMO

Reactive oxygen species, generated in all the aerobic organisms, can cause oxidative stress. Excessive ROS may become a source of carcinogen due to DNA damage, lipid peroxidation, cell injury, and cell death. In order to prevent these adverse effects of ROS, antioxidant enzymes have evolved in aerobic organisms. Catalase is a major antioxidant enzyme that breaks down excessive H2O2 and inhibits apoptotic cell death. Here we molecularly characterized catalase from red-lip mullet. The cDNA sequence of LhCAT consists of an ORF of 1545 bp, which encodes a 527 amino acid peptide (~60 kDa). Based on bioinformatics analysis, LhCAT possesses a domain architecture characteristic of catalases, including a catalase proximal active site signature and a catalase proximal heme-ligand signature. It also has heme and NADPH binding sites homologous to previously described catalases. Pairwise alignment with its homologs revealed that LhCAT shares 95.1% identity with Oplegnathus fasciatus catalase and 97.4% similarity with Sparus aurata catalase. An uprooted phylogenetic tree demonstrated that LhCAT resides in a clade with catalases from other teleosts and exhibits a close relationship with Oplegnathus fasciatus catalase. Among twelve tissue types, we observed the highest LhCAT mRNA expression in the liver, followed by blood. Immune challenge by Lactococcus garvieae, or Poly I:C in the blood or spleen resulted in up-regulation at 24 h post injection. We also tested the antioxidant activity of recombinant LhCAT against hydrogen peroxide and found its optimal concentration to be 12.5 µg/mL. Collectively, these data suggested that LhCAT play an important role in antioxidant defense and immune response of red-lip mullet.


Assuntos
Catalase/metabolismo , Proteínas de Peixes/metabolismo , Smegmamorpha , Adjuvantes Imunológicos , Animais , Antioxidantes/metabolismo , Catalase/genética , DNA Complementar/genética , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Heme/química , Peróxido de Hidrogênio/química , Sistema Imunitário , Ligantes , Fígado/enzimologia , Estresse Oxidativo , Filogenia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulação para Cima
3.
Artigo em Inglês | MEDLINE | ID: mdl-31176866

RESUMO

Cysteine oxygenase (CDO) is a mononuclear nonhemoglobin enzyme that catalyzes the production of taurine through the cysteine (Cys) pathway and plays a key role in the biosynthesis of taurine in mammals. However, the function of CDOs in bony fish remains poorly understood. In this study, we cloned CDO genes (CaCDO1 and CaCDO2) from Carassius auratus. The cDNA sequences of both CaCDO1 and CaCDO2 encoded putative proteins with 201 amino acids, which included structural features typical of the CDO protein family. Multiple sequence alignment and phylogenetic analysis showed that CaCDO1 and CaCDO2 shared high sequence identities and similarities with C. carpio homologs. Quantitative real-time polymerase chain reaction (qRT-PCR) results revealed that CaCDO1 and CaCDO2 were both broadly expressed in all selected tissues and developmental stages in C. auratus but had differing mRNA levels. In addition, compared to those of the taurine-free group, the in vivo mRNA expression levels of both CaCDO1 and CaCDO2 significantly decreased with increasing dietary taurine levels from 1.0 to 9.0 g/kg. Furthermore, in vitro taurine treatments showed similar inhibitory effects on the expression of CaCDO1 and CaCDO2 in the intestines of C. auratus. Our results also showed that the mRNA expression of CaCDO2 in the intestines was higher than that of CaCDO1 in response to in vivo and in vitro taurine supplementation. Overall, these data may provide new insights into the regulation of fish CDO expression and provide valuable knowledge for improving dietary formulas in aquaculture.


Assuntos
Cisteína Dioxigenase/genética , Cisteína Dioxigenase/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Carpa Dourada/genética , Carpa Dourada/metabolismo , Taurina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Carpa Dourada/crescimento & desenvolvimento , Isoenzimas/genética , Isoenzimas/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Taurina/farmacologia , Distribuição Tecidual
4.
J Microbiol Biotechnol ; 29(6): 999-1007, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154749

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 and related infection cases have been reported in over 20 countries. Roughly 10,000 human cases have so far been reported in total with fatality rates at up to 40%. The majority of cases have occurred in Saudi Arabia with mostly sporadic outbreaks outside the country except for the one in South Korea in 2015. The Korean MERS-CoV strain was isolated from the second Korean patient and its genome was fully sequenced and deposited. To develop virusspecific protective and therapeutic agents against the Korean isolate and to investigate molecular determinants of virus-host interactions, it is of paramount importance to generate its full-length cDNA. Here we report that two full-length cDNAs from a Korean patientisolated MERS-CoV strain were generated by a combination of conventional cloning techniques and efficient Gibson assembly reactions. The full-length cDNAs were validated by restriction analysis and their sequence was verified by Sanger method. The resulting cDNA was efficiently transcribed in vitro and the T7 promoter-driven expression was robust. The resulting reverse genetic system will add to the published list of MERS-CoV cDNAs and facilitate the development of Korean isolate-specific antiviral measures.


Assuntos
DNA Complementar/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Genética Reversa , Infecções por Coronavirus/virologia , Genes Virais/genética , Engenharia Genética , Genoma Viral/genética , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , RNA Viral/genética , Transcrição Genética
5.
Plant Mol Biol ; 101(1-2): 41-61, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31183604

RESUMO

KEY MESSAGE: Several classes of transcription factors are involved in the activation of defensins. A new type of the transcription factor responsible for the regulation of wheat grain specific defensins was characterised in this work. HD-Zip class IV transcription factors constitute a family of multidomain proteins. A full-length cDNA of HD-Zip IV, designated TaGL7 was isolated from the developing grain of bread wheat, using a specific DNA sequence as bait in the Y1H screen. 3D models of TaGL7 HD complexed with DNA cis-elements rationalised differences that underlined accommodations of binding and non-binding DNA, while the START-like domain model predicted binding of lipidic molecules inside a concave hydrophobic cavity. The 3'-untranslated region of TaGL7 was used as a probe to isolate the genomic clone of TdGL7 from a BAC library prepared from durum wheat. The spatial and temporal activity of the TdGL7 promoter was tested in transgenic wheat, barley and rice. TdGL7 was expressed mostly in ovary at fertilisation and its promoter was active in a liquid endosperm during cellularisation and later in the endosperm transfer cells, aleurone, and starchy endosperm. The pattern of TdGL7 expression resembled that of genes that encode grain-specific lipid transfer proteins, particularly defensins. In addition, GL7 expression was upregulated by mechanical wounding, similarly to defensin genes. Co-bombardment of cultured wheat cells with TdGL7 driven by constitutive promoter and seven grain or root specific defensin promoters fused to GUS gene, revealed activation of four promoters. The data confirmed the previously proposed role of HD-Zip IV transcription factors in the regulation of genes that encode lipid transfer proteins involved in lipid transport and defence. The TdGL7 promoter could be used to engineer cereal grains with enhanced resistance to insects and fungal infections.


Assuntos
Defensinas/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Triticum/genética , DNA Complementar/genética , Grão Comestível/genética , Grão Comestível/metabolismo , Genes Reporter , Hordeum/genética , Hordeum/metabolismo , Especificidade de Órgãos , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Triticum/metabolismo , Técnicas do Sistema de Duplo-Híbrido
6.
Artigo em Inglês | MEDLINE | ID: mdl-31078702

RESUMO

As one of antimicrobial peptides (AMPs), defensins are involved in invertebrate innate immunity against invading pathogens. In this study, a member of the invertebrate defensins was cloned and characterized from the small abalone Haliotis diversicolor, designated HdDef-2. The HdDef-2 cDNA contained a 201 bp open reading frame encoding 66 amino acids including a signal peptide of 18 amino acids and a mature peptide of 48 amino acids. The mature peptide of HdDef-2 possessed similar features to other AMPs, such as lower molecular mass, net positive charge (+1), and a high hydrophobic residue ratio (45%). In addition, six cysteines in the mature peptide were arranged in the pattern C-X16-C-X3-C-X9-C-X4-C-X1-C and stabilized the α-helix/ß-sheet motif (CSαß) with three disulfide bonds (C1-C4, C2-C5 and C3-C6) in the predicted tertiary structure. Moreover, the similar three-dimensional structure to Anopheles gambiae defensin and a phylogenetic analysis suggest that HdDef-2 may be a new member of the arthropod defensin family. Quantitative real-time PCR analysis revealed that HdDef-2 transcripts were constitutively expressed in the mantle, gill, hepatopancreas, and foot, with the highest level in the hepatopancreas. It was observed that HdDef-2 transcripts were significantly induced in the hepatopancreas after infection by Vibrio harveyi. These results indicate that HdDef-2 may be involved in the immune response against invading pathogenic bacteria, but future work is needed to verify its antimicrobial activity in protein level and elucidate the underlying mechanisms.


Assuntos
Defensinas/genética , Defensinas/imunologia , Gastrópodes/genética , Gastrópodes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Defensinas/química , Gastrópodes/metabolismo , Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade Inata/genética , Modelos Moleculares , Filogenia , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual , Vibrio/patogenicidade
7.
Artigo em Inglês | MEDLINE | ID: mdl-31129291

RESUMO

Mollusk biomineralization is a process controlled by a complex interplay of proteins, ions and external regulators. In spite of several studies, there is a lack of knowledge of who (molecules involved), how (mechanism) and why (evolution and adaptation) mollusk are designed as we know them. In this study, a shell matrix protein, N66, has been purified and characterized biochemically from the shell of Pteria sterna. Two protein bands with carbohydrates associated were separated with a molecular weight of ~60 and 64 kDa. It has carbonic anhydrase activity and it is able to form crystal polymorphs of calcium carbonate in vitro. The mRNA N66 was obtained from the mantle tissue of Pteria sterna and the deduced amino acid sequence contained a carbonic anhydrase (CA) domain and a Asn/Gly-rich domain (aa243-439). The CA domain contained three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu166-Thr525), being thus similar to the human isoform hCAVII. Also, to test whether the posttranslational modifications present on the native N66 affects the CA activity and its crystallization capability in vitro, a recombinant N66 was overexpressed in Escherichia coli and functionally characterized. Our results show that recombinant N66 has higher CA activity and produce larger size crystals in vitro than the native N66 protein, suggesting that intrinsic properties of the native N66, such as glycosylations and/or phosphorylations, might regulate its activity.


Assuntos
Exoesqueleto/metabolismo , Anidrases Carbônicas/isolamento & purificação , Anidrases Carbônicas/metabolismo , Pinctada/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Biomineralização , Anidrases Carbônicas/genética , Cristalização , DNA Complementar/genética , Microscopia Eletrônica de Varredura , Filogenia , Pinctada/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
8.
Plant Physiol Biochem ; 141: 20-29, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31125808

RESUMO

Soil salinity is one of the most abiotic stress factors that severely affects the growth and development of many plants, which can ultimately threaten crop yield. Arbuscular mycorrhiza fungi (AMF) has been proven to be effective in mitigating salinity stress by symbiosis in many crops. Asparagus officinalis are perennial plants grown in saline-alkaline soil, however, limited information on their molecular mechanisms has restricted efficient application of AMF to garden asparagus under salinity stress. In this study, we conducted a transcriptome analysis on the leaves of garden asparagus to identify gene expression under salinity stress. Seedlings were grown in 4 treatments, including non-inoculated AMF using distilled water (NI), inoculated AMF using distilled water (AMF), non-inoculated with salinity stress (NI + S), and inoculated with salinity stress (AMF + S). A total of 6019 novel genes were obtained based on the reference-guided assembly of the garden asparagus transcriptome. Results revealed that 455 differentially expressed genes (DEGs) were identified when comparing NI + S to AMF + S. However, among the up-regulated DEGs, 41 DEGs were down-regulated, while 242 DEGs had no differences in their expression levels when comparing NI to NI + S. These DEGs' expression patterns may be key induced by AMF under salinity stress. Additionally, the GO and KEGG enrichment analyses of 455 DEGs revealed that these genes mainly participate in the improvement of the internal environment in plant cells, nitrogen metabolic-related processes, and possible photoprotection mechanisms. These findings provide insight into enhanced salinity stress adaptation by AMF inoculation, as well as salt-tolerant candidate genes for further functional analyses.


Assuntos
Asparagus (Planta)/genética , Micorrizas/fisiologia , Estresse Salino , Transcriptoma , Asparagus (Planta)/microbiologia , DNA Complementar/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genoma de Planta , Metabolômica , Nitrogênio/química , Fotoquímica , Folhas de Planta/genética , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Brotos de Planta/genética , Salinidade , Tolerância ao Sal , Sais/química , Plântula , Análise de Sequência de RNA , Simbiose
9.
Plant Physiol Biochem ; 141: 83-94, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31136934

RESUMO

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases in bananas resulting in significant loss of Cavendish bananas production worldwide. Here we show the agronomic traits and the resistance of 'Guijiao 9' in the field trials from 2012 to 2017. And then we dissect and compare the transcriptome response from these two cultivars (cv. 'Guijiao 9' and cv. Williams) in an attempt to understand the molecular basis that contribute to the enhanced Foc tropical race 4 (Foc-TR4) resistance. 'Guijiao 9' is a Cavendish cultivar with strong resistance to Foc-TR4, which was reflected in a lower disease severity and incidence in glasshouse and field trails, when compared to the susceptible cultivar Williams. Gene expression profiles of 'Guijiao 9' and Williams were captured by performing RNA-Seq analysis on 16 biological samples collected over a six day period post inoculation with Foc-TR4. Transcriptional reprogramming in response to Foc-TR4 was detected in both genotypes but the response was more drastic in 'Guijiao 9' than in Williams. Specific genes involved in plant-pathogen interaction and defense signaling including MAPK, calcium, salicylic acid, jasmonic acid and ethylene pathways were analyzed and compared between 'Guijiao 9' and Williams. Genes associated with defense-related metabolites synthesis such as NB-LRR proteins, calmodulin-binding protein and phenylpropanoids biosynthesis genes were significantly up-regulated in 'Guijiao 9' resistant to Foc-TR4 infection. Taken together, this study highlights the important roles of plant hormone regulation and defense gene activation in mediating resistance in 'Guijiao 9'.


Assuntos
Resistência à Doença/genética , Fusarium/patogenicidade , Regulação da Expressão Gênica de Plantas , Musa/genética , Doenças das Plantas/genética , DNA Complementar/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Biblioteca Gênica , Genes de Plantas , Musa/microbiologia , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Planta/metabolismo , Raízes de Plantas/genética , Ácido Salicílico/metabolismo , Metabolismo Secundário , Especificidade da Espécie , Transcrição Genética , Transcriptoma , Regulação para Cima
10.
BMC Plant Biol ; 19(1): 181, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060493

RESUMO

BACKGROUND: Castor (Ricinus communis L.) is an important seed oil crop. Castor oil is a highly demanded oil for several industrial uses. Current castor bean varieties suffer from low productivity and high risk of insect pests and diseases. High productive and pest/disease resistance varieties are needed. Lignin has been associated to the resistance for pest, disease and lodging. Lignin is produced from several metabolites of the phenylpropanoid pathway. PAL is the key enzyme of the phenylpropanoid pathway. The gene PAL may assist in the improvement of resistance of castor bean. RESULTS: The RcPAL CDs was amplified and its function was examined by transgenic overexpression and antisense expression, lignin histochemical staining, real-time PCR, lignin content measurement and morphological investigation. Its full length was 2145 bp, encoding 714 amino acids. The overexpression of RcPAL (7.2 times) increased significantly the PAL activity, dyeing depth of xylem cells and lignin content (14.44%), resulting in a significantly lower plant height, deeper and thicker blade, more green leaves, shorter internode, thicker stem diameter, and opposite in antisense expression plants (lignin content lowered by 27.1%), demonstrated that the gene RcPAL was a key gene in castor lignin biosynthesis. CONCLUSIONS: The gene RcPAL is a key gene in castor lignin biosynthesis and can be induced to express under mechanical damage stress. When up-regulated, it increased the lignin content significantly and dwarfed the plant height, and opposite when down-regulated. The gene RcPAL may assist in the improvement of resistance and plant type of castor bean.


Assuntos
Vias Biossintéticas/genética , Genes de Plantas , Lignina/biossíntese , Fenilalanina Amônia-Liase/genética , Ricinus/genética , Ricinus/metabolismo , Cinamatos/farmacologia , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Fenilalanina Amônia-Liase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estresse Mecânico , Transformação Genética
11.
Biol Pharm Bull ; 42(5): 764-769, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061318

RESUMO

Werner helicase-interacting protein 1 (WRNIP1) was originally identified as a protein that interacts with WRN, the product of the gene responsible for Werner syndrome. Our previous studies suggested that WRNIP1 is implicated in translesion synthesis (TLS), a process in which specialized TLS polymerases replace replicative DNA polymerase and take over DNA synthesis on damaged templates. We proposed that a novel error-free pathway involving DNA polymerase δ and primase-polymerase (PrimPol) functions to synthesize DNA on UV-damaged DNA templates in the absence of WRNIP1 and the TLS polymerase Polη. Hence, in the current study, we analyzed the relationship between WRNIP1 and PrimPol. We found that WRNIP1 and PrimPol form a complex in cells. PrimPol protein expression was reduced in cells overexpressing WRNIP1, but was increased in WRNIP1-depleted cells. The WRNIP1-mediated reduction in the amount of PrimPol was suppressed by treatment of the cells with proteasome inhibitors, suggesting that WRNIP1 is involved in the degradation of PrimPol via the proteasome.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , DNA Primase/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Células HEK293 , Humanos , Enzimas Multifuncionais/genética , Plasmídeos , Inibidores de Proteassoma/farmacologia , RNA Mensageiro/metabolismo , Transfecção
12.
Nat Commun ; 10(1): 1634, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967552

RESUMO

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.


Assuntos
DNA Complementar/genética , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Subunidade gama Comum de Receptores de Interleucina/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Animais , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular , Códon de Iniciação/genética , Éxons/genética , Sangue Fetal/citologia , Vetores Genéticos/genética , Voluntários Saudáveis , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Mutação , Parvovirinae/genética , Cultura Primária de Células , Fatores de Tempo , Transdução Genética/métodos , Quimeras de Transplante/genética , Transplante Heterólogo/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
13.
Arch Insect Biochem Physiol ; 101(2): e21553, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31004387

RESUMO

In this study, we identified and characterized a phosphoserine aminotransferase (bmPSAT) from Bombyx mori (B. mori) that is responsible for l-serine biosynthesis. A complementary DNA that encodes bmPSAT was cloned by reverse transcriptase polymerase reaction and sequenced. The presumed amino acid sequence revealed 47-87% identity with known PSATs from insects, humans, plants, and bacteria. Through phylogenetic analysis, we found that bmPSAT is evolutionary related to insect PSATs. Recombinant bmPSAT was produced in Escherichia coli by using a cold-shock promotor and purified to homogeneity. This enzyme utilizes phosphohydroxypyruvate and glutamate for transamination. bmPSAT messenger RNA (mRNA) was expressed at higher levels in several tissues of standard strain silkworm including the silk gland, whereas a sericin-deficient silkworm strain exhibited a diminished expression of bmPSAT mRNA in the silk gland. These findings indicate that bmPSAT may play an important role in synthesizing and supplying l-serine in the larva of B. mori.


Assuntos
Bombyx/enzimologia , Serina/biossíntese , Transaminases/química , Animais , Bombyx/genética , Bombyx/metabolismo , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/biossíntese , Proteínas de Insetos/metabolismo , Larva/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Transaminases/genética , Transaminases/metabolismo
14.
J Therm Biol ; 81: 59-65, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30975424

RESUMO

Heat shock proteins (HSPs) play important roles in the adaption of Pomacea canaliculata to unsuitable environments. In the present study, a cDNA encoding HSP40 in P. canaliculata (PocaHSP40) was cloned and characterized. The PocaHSP40 cDNA was 1466 bp, containing an ORF of 954 bp encoding 317 amino acids. Bioinformatics analysis showed that PocaHSP40 belonged to type II HSP40s and had four predicted phosphorylation sites. Phylogenetic analysis proved the conservation of HSP40s in mollusks. PocaHSP40 was widely expressed in the gill, digestive gland, kidney, and foot muscle of P. canaliculata. Challenged by different temperatures, the expression of PocaHSP40 was up-regulated under low temperatures but not high temperatures, which was contrary to the expression change of PocaHSP70 under low and high temperatures. These results implied that P. canaliculata evolved different strategies for survival under low temperature and high temperature through the regulation of HSPs.


Assuntos
Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Caramujos/genética , Temperatura Ambiente , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , Evolução Molecular , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP70/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
15.
Biosens Bioelectron ; 133: 160-168, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30933710

RESUMO

An efficient and new electrochemical biosensor for detection of DNA damage, induced by the interaction of the hybrid anti-cancer compound (7ESTAC01) with DNA, was studied by differential pulse voltammetry (DPV). The biosensor consists of a Stem-Loop DNA (SL-DNA) probe covalently attached to the gold electrode (GE) surface that hybridizes to a complementary DNA strand (cDNA) to form a double-stranded DNA (dsDNA). The interaction and DNA damage induced by 7ESTAC01 was electrochemically studied based on the oxidation signals of the electroactive nucleic acids on the surface of the GE by DPV. As a result, the SL-DNA/GE and dsDNA/GE were tested with the reduced 7ESTAC01, showing the voltammetric signal of guanine and adenine, increase in the presence of 7ESTAC01. Under optimum conditions, the dsDNA/GE biosensor exhibited excellent DPV response in the presence of 7ESTAC01. The bonding interaction between 7ESTAC01 and calf thymus DNA (ctDNA) was confirmed by UV-Vis absorption spectroscopy, dynamic simulations (performed to investigate the DNA structure under physiological conditions), and molecular docking. Theoretical results showed the presence of hydrogen bonding and intercalation in the minor groove of DNA, involving hydrophobic interactions.


Assuntos
Antineoplásicos/química , Técnicas Biossensoriais , DNA/isolamento & purificação , Técnicas Eletroquímicas , Antineoplásicos/farmacologia , DNA/química , DNA/genética , Dano ao DNA/efeitos dos fármacos , DNA Complementar/química , DNA Complementar/genética , Ouro/química , Humanos , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Sequências Repetidas Invertidas/genética , Simulação de Acoplamento Molecular , Oxirredução/efeitos dos fármacos , Raios Ultravioleta
16.
Biomed Res Int ; 2019: 1321287, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016183

RESUMO

SERPINA1 is a member of serine protease inhibitors and is increasingly considered to be a regulator of innate immunity in human and animals. However, the expression and function of SERPINA1 gene in immune defense against viral infection remain unknown in ducks. The full-length du SERPINA1 cDNA sequence was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). It contained 1457 nucleotide, including 47-bp 5' UTR, 135-bp 3' UTR, and 1275-bp open reading frame (ORF), and encodes a 424-amino acid protein. Then, the tissue expression profile of du SERPINA1 gene was determined. Real-time quantitative polymerase chain reaction (real-time qPCR) analysis revealed that du SERPINA1 mRNA is ubiquitous in various tissues, but higher expression levels were observed in lung and liver tissues. In addition, the expression pattern was investigated when the ducklings were challenged with duck hepatitis virus 1(DHV-1) and polyriboinosinic polyribocytidylic acid (poly I:C). After DHV-1 injection or poly I:C treatment, du SERPINA1 mRNA was up-regulated in the liver and kidney tissues. However, the peak time in two tissues was not consistent. In kidney, the expression lever of SERPINA1 increased immediately after the treatment while in liver tissue it kept steady until 12 h post-infection. Our results indicate that SERPINA1 has an active role in the antiviral response, and thus improve our understanding of the role of this protein.


Assuntos
Patos/genética , Expressão Gênica/genética , alfa 1-Antitripsina/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular/métodos , DNA Complementar/genética , Vírus da Hepatite do Pato/genética , Rim/fisiologia , Fígado/fisiologia , Fases de Leitura Aberta/genética , Filogenia , RNA Mensageiro/genética , Alinhamento de Sequência , Análise de Sequência de DNA/métodos , Regulação para Cima/genética
17.
Zhongguo Zhong Yao Za Zhi ; 44(5): 935-941, 2019 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-30989852

RESUMO

1-deoxy-D-xylulose-5-phosphate synthase2(DXS2) is the first key enzyme of the MEP pathway,which plays an important role in terpene biosynthesis of plants. According to the data of Swertia mussotii transcriptome, DXS2 gene(Gen Bank number MH535905) was cloned and named as Sm DXS2. The bioinformatics results showed that Sm DXS2 has no intron,with a 2 145 bp open reading frame encoding a polypeptide of 714 amino acids. They are belonging to 20 kinds of amino acids,and the most abundant amino acids include Ala,Gly and Trp. The predicted protein molecular weight was 76. 91 k Da and its theoretical isoelectric point(p I) was6. 5,which belonging to a hydrophilic protein. α-Helix and loop were the major motifs of predicted secondary structure of DXS2. The three function domains are TPP_superfamily,Transket_pyr_ superfamily and Transketolase_C superfamily,respectively. The Sm DXS2 protein shared high identity with other DXS2 proteins of plants. Phylogenetic analysis showed that Sm DXS2 protein is grouped with the gentian DXS2 protein. The recombinant protein of Sm DXS2 gene in Escherichia coli was approximately 92. 00 k Da(containing sumo-His tag protein 13 k Da),which was consistent with the anticipated size.This work will provide a foundation for further functional research of Sm DXS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.


Assuntos
Proteínas de Plantas/genética , Swertia/genética , Transferases/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , Genes de Plantas , Iridoides , Filogenia , Swertia/enzimologia , Transcriptoma
18.
Adv Exp Med Biol ; 1129: 1-17, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30968357

RESUMO

This review describes the features of molecular biology techniques for single-cell RNA sequencing (scRNA-seq), including methods developed in our laboratory. Existing scRNA-seq methods require the conversion of first-strand cDNA to amplifiable cDNA followed by whole-transcript amplification. There are three primary strategies for this conversion: poly-A tagging, template switching, and RNase H-DNA polymerase I-mediated second-strand cDNA synthesis for in vitro transcription. We discuss the merits and limitations of these strategies and describe our Reverse Transcription with Random Displacement Amplification technology that allows for direct first-strand cDNA amplification from RNA without the need for conversion to an amplifiable cDNA. We believe that this review provides all users of single-cell transcriptome technologies with an understanding of the relationship between the quantitative performance of various methods and their molecular features.


Assuntos
DNA Complementar/genética , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma
19.
Methods Mol Biol ; 1979: 25-44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028630

RESUMO

In the last few years single-cell RNA sequencing (scRNA-seq) has enabled the investigation of cellular heterogeneity at the transcriptional level, the characterization of rare cell types as well as the detailed analysis of the stochastic nature of gene expression. A large number of methods have been developed, varying in their throughput, sensitivity, and scalability. A major distinction is whether they profile only 5'- or 3'-terminal part of the transcripts or allow for the characterization of the entire length of the transcripts. Among the latter, Smart-seq2 is still considered the "gold standard" due to its sensitivity, precision, lower cost, scalability and for being easy to set up on automated platforms. In this chapter I describe how to efficiently generate sequencing-ready libraries, highlight common issues and pitfalls, and offer solutions for generating high-quality data.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Análise de Célula Única/métodos , Animais , DNA Complementar/genética , DNA Complementar/metabolismo , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Humanos , Transcrição Reversa , Transcriptoma , Transposases/metabolismo
20.
Methods Mol Biol ; 1979: 45-56, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028631

RESUMO

Single-cell RNA sequencing has revolutionized the way we look at cell populations. Of the methods available, CEL-Seq was the first to use linear RNA amplification. With early barcoding and 3' sequencing, it is sensitive, cost-effective and easy to perform. Here we describe a protocol for performing CEL-Seq2 on sorted cells, which can be performed without any special equipment.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Sequência de Bases , DNA Complementar/genética , Citometria de Fluxo , Perfilação da Expressão Gênica/economia , Biblioteca Gênica , Humanos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de RNA/economia , Análise de Célula Única/economia
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