RESUMEN

Osimertinib, an orally administered third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is widely approved for the first-line and second-line treatment of advanced non-small-cell lung cancer (NSCLC) with EGFR mutations. However, the rapid development of osimertinib resistance renders the unsustainable treatment benefit. Patients with EGFR -mutated NSCLC who develop osimertinib resistance, especially those acquiring relatively rare and 'off-target' resistance mutations, still lack effective therapeutic options for postosimertinib therapy. Herein, we reported a 73-year-old woman diagnosed with T1N3M1 lung adenocarcinoma harboring EGFR L858R mutation, who acquired two GNAS mutations (R201C and R201H) and lost the EGFR L858R mutation after progression on icotinib and osimertinib. The patient was subsequently treated with trametinib and there was no obvious tumor increase. Our study revealed that GNAS R201 can confer the osimertinib resistance in EGFR -positive NSCLC, and present the first report of the prevalence of GNAS R201C and R201H mutants in NSCLC which response to trametinib treatment. Our case suggests that trametinib could be a treatment option in NSCLC patients harboring GNAS -activating mutations.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Acrilamidas , Anciano , Compuestos de Anilina/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Cromograninas/genética , Cromograninas/uso terapéutico , Receptores ErbB/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/uso terapéutico , Humanos , Indoles , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridonas , Pirimidinas , Pirimidinonas

RESUMEN

OBJECTIVES: Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (MTB), has similar clinical, radiological, and histopathological characteristics to sarcoidosis (SA). Accurately distinguishing SA from TB remains a clinical challenge. METHODS: A total of 44 TB patients and 47 SA patients who were clinically diagnosed using chest radiography, pathological examination, routine smear microscopy, and microbial culture were enrolled in this study. The MTB genome was captured and sequenced directly from tissue specimens obtained upon operation or biopsy, and the feasibility of next-generation sequencing (NGS) for the MTB genome in the differential diagnosis of TB from SA was evaluated. RESULTS: Using a depth >10× and coverage >15% of the sequencing data, TB patients were identified via the NGS approach directly using operation or biopsy specimens without clinical pretreatment. The sensitivity, specificity, and concordance of the NGS method were 81.8% (36/44), 95.7% (45/47), and 89.0% (81/91), respectively (kappa = 0.78, 95% confidence interval 0.65-0.91; P<0.001). CONCLUSIONS: This study established an improved NGS strategy for rapidly distinguishing patients with TB from those with SA and has potential clinical benefits.

Asunto(s)

Mycobacterium tuberculosis , Sarcoidosis , Tuberculosis , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mycobacterium tuberculosis/genética , Sarcoidosis/diagnóstico , Sensibilidad y Especificidad , Tuberculosis/diagnóstico

RESUMEN

BACKGROUND: Genetic skeletal disorders (GSDs) are clinically and genetically heterogeneous with more than 350 genes accounting for the diversity of disease phenotypes. Prenatal diagnosis of these disorders has been challenging because of the limited but variable prenatal phenotypes, highlighting the need of a novel genetic approach. Short-rib polydactyly syndrome (SRPS) Type III is an autosomal recessive GSD characterized by extreme narrowness of the thorax, severely shortened tubular bones, polydactyly and multiple malformations. METHODS: Cytogenetic and molecular analyses using GTG-banding, single nucleotide polymorphism array and a novel GSDs targeted gene panel sequencing were performed in a 24 weeks fetus with increased biparietal diameter (BPD), short limbs, narrow thorax and polyhydramnios. RESULTS: No chromosomal abnormalities and pathogenic copy number variations (CNVs) were detected in the fetus. Two novel compound heterozygous mutations c.2992C > T and c.12836G > C in the DYNC2H1 gene were identified by targeted genes panel sequencing. A literature review was performed to delineate the prenatal phenotype of SRPS Type III. CONCLUSION: This is the first report of prenatal diagnosis of DYNC2H1 mutations causing SRPS Type III in a fetus with increased BPD associated with polyhydramnios in China. Our findings expand the mutation spectrum of DYNC2H1 in this rare disease and demonstrate that targeted gene panel capture followed by next-generation sequencing (NGS) is an efficient and cost-effective method to perform a molecular prenatal diagnosis of a rare genetic skeletal disorder.

Asunto(s)

Dineínas Citoplasmáticas/genética , Feto , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Polihidramnios , Diagnóstico Prenatal , Síndrome de Costilla Pequeña y Polidactilia , Femenino , Humanos , Polihidramnios/diagnóstico , Polihidramnios/genética , Embarazo , Síndrome de Costilla Pequeña y Polidactilia/diagnóstico , Síndrome de Costilla Pequeña y Polidactilia/genética

RESUMEN

UNLABELLED: Phenylketonuria (PKU) is caused by variants in the phenylalanine hydroxylase (PAH) gene. We systematically investigated all 13 exons of the PAH gene and their flanking introns in 31 unrelated patients and their parents using next-generation sequencing (NGS). A total of 33 different variants were identified in 58 of 62 mutant PAH alleles. The prevalent variants with a relative frequency of 5 % or more were c.721C > T, c.1068C > A, c.611A > G, c.1197A > T, c.728G > A, c.331C > T, and c.442-1G > A. One novel variant was identified in this study-c.699C > G. We studied genotype-phenotype correlations using the Guldberg arbitrary value (AV) system, which revealed a consistency rate of 38 % (8/21) among the 21 predicted phenotypes. The genotype-based prediction of BH4 responsiveness was also evaluated, and 14 patients (45.2 %) were predicted to be BH4 responsive. CONCLUSION: This study presents the spectrum of PAH variants in Jiangsu province. The information obtained from the genotype-based prediction of BH4 responsiveness might be used for the rational selection of candidates for BH4 testing. WHAT IS KNOWN: • Phenylketonuria (PKU) is caused by variants in the phenylalanine hydroxylase (PAH) gene. • The spectrum of PAH variants in different Chinese populations has been reported. What is new: • This is the first report on the spectrum of PAH variants in Jiangsu province. • This study identified one novel PAH variant-c.699C>G-and and tries to show a genotype-phenotype relationship also regarding BH4-responsiveness.

Asunto(s)

ADN/genética , Estudios de Asociación Genética/métodos , Mutación , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/genética , Alelos , China/epidemiología , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Recién Nacido , Masculino , Fenotipo , Fenilalanina Hidroxilasa/metabolismo , Fenilcetonurias/enzimología , Fenilcetonurias/epidemiología , Prevalencia , Estudios Retrospectivos

RESUMEN

Elevated plasma phosphate levels are signifcantly associated with progression of chronic kidney disease (CKD). Interstitial fibrosis is an important factor in the progression of CKD. In this study we investigate the role of inorganic phosphate in stimulating fibronectin (FN) synthesis in a kidney fibroblast cell line (NRK-49F). We find that phosphate increases FN abundance and message in a dose-dependent fashion and that both ERK1/2 and AKT are important signaling pathways that mediate phosphate-dependent FN expression in NRK-49F cells. Moreover phosphate srimulates the expression of the transcription factors osterix and NFATc1, which form complexes and mediate FN synthesis. Another transcription factor involved in phosphate-dependent FN synthesis is the AP1 family member c-Fos. In summary we show that even mildly elevated serum phosphate levels can induce synthesis of the interstitial matrix protein fibronectin through activation of ERK1/2 and AKT signaling pathways in kidney fibroblasts and that the synthesis of fibronectin is mediated by a transcriptional complex consisting of NFATc1, osterix and c-Fos.

Asunto(s)

Fibroblastos/metabolismo , Fibronectinas/metabolismo , Expresión Génica , Riñón/citología , Fosfatos/fisiología , Animales , Línea Celular , Fibronectinas/genética , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos , Factores de Transcripción NFATC/metabolismo , Fosfatos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Factores de Transcripción/metabolismo

RESUMEN

BACKGROUND: Hyperosmotic stress causes cell death through activation of apoptotic pathways if the protective osmolyte response is impaired. In this study we attempt to elucidate the molecular mechanisms of hypertonicity-induced apoptosis and the effect of major organic osmolytes upon those. METHODS: Hypertonicity-induced changes in Bcl2-family protein abundance and the presence of cytochrome c and apoptosis inducing factor (AIF) in the cytoplasm, were measured using western blot and immunofluorescence labeling. To determine dissipation of mitochondrial membrane potential (Delta Psi) though the permeability transition pore (PTP), the lipophilic cationic carbocyanine fluorescence probe JC-1 and TMRM fluorescence probes were used. RESULTS: Hypertonic culture conditions increase the abundance of proapoptotic Bax and the concentration of cytochrome c and apoptosis inducing factor (AIF) in the cytoplasm. These changes are associated with a dissipation of Delta Psi and increased permeability of the PTP. We further show that organic osmolytes stabilize the Delta Psi and decrease the concentration of cytochrome c and AIF in the cytoplasm. CONCLUSION: Our study shows that organic osmolytes prevent hypertonicity-induced apoptosis by preventing dissipation of Delta Psi through stabilization of the PTP. These findings further support the important role of organic osmolytes in preventing hypertonicity-mediated cell death in medullary kidney cells.

Asunto(s)

Apoptosis , Médula Renal/citología , Membranas Mitocondriales/metabolismo , Animales , Factor Inductor de la Apoptosis/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Citoplasma/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Concentración Osmolar , Permeabilidad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

mAtNOS1 is a novel gene recently reported in mammalian genome with functions that are not fully understood. The present study shows that in human mammary adenocarcinoma MCF-7 cells, mAtNOS1 expression increases mitochondrial nitric oxide and calcium. Our study further shows that overexpression of mAtNOS1 induces apoptosis in MCF-7 cells by increasing mitochondrial protein tyrosine nitration and cytochrome c release. The present study suggests a novel function for mAtNOS1 in regulating mitochondrial nitric oxide and calcium and inducing apoptosis of MCF-7 cells.

Asunto(s)

Adenocarcinoma/patología , Apoptosis , Proteínas de Arabidopsis/biosíntesis , Neoplasias de la Mama/patología , Óxido Nítrico Sintasa/biosíntesis , Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Femenino , Humanos , Inmunoquímica , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Transfección , Tirosina/metabolismo

RESUMEN

mAtNOS1 is a novel gene recently reported in mammalian cells with functions that are not fully understood. The present study generated human neuroblastoma SHSY cells over- and underexpressing mAtNOS1 and shows that mAtNOS1 is involved in regulating mitochondrial nitric oxide, mitochondrial transmembrane potential, protein tyrosine nitration, cytochrome c release, and apoptosis of those cells.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas de Unión al GTP/metabolismo , Mitocondrias/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Óxido Nítrico/metabolismo , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , GTP Fosfohidrolasas , Proteínas de Unión al GTP/genética , Humanos , Potenciales de la Membrana/genética , Ratones , Mitocondrias/genética , Membranas Mitocondriales/metabolismo , Mutación , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Homología de Secuencia de Aminoácido , Timo/embriología , Timo/metabolismo

RESUMEN

A cDNA clone (designated as SsPR10, GenBank Accession Number AY660753 ) encoding a PR10 protein from yellow-fruit nightshade (Solanum surattense) was isolated and characterized. SsPR10 encoded a 160-amino-acid polypeptide with a predicted molecular mass of 17.58 kDa and pI of 5.29. Sequence alignments showed that SsPR10 had high identity (68.1%) with CaPR10, but had only about 31.7% identity with JIOsPR10 at the amino acid level. Genomic DNA gel blot analysis indicated that SsPR10 belonged to a multigene family. The constitutively expressed SsPR10 was detected to be the highest in roots of the sterile seedlings cultured in jars, while SsPR10 expression was the highest in old yellow leaves from the seedlings incubated with sap containing TMV. SsPR10 always expressed at slightly higher level in senescent leaves than in tender ones under both conditions. Further expression analysis revealed that the signaling components of defense/stress pathways (MeJA, SA, ABA, GA3, H2O2 and Cu2+) up-regulated significantly the SsPR10 mRNA levels over the control. However, darkness failed to induce SsPR10 expression and its expression was also inhibited by cold treatment. The SsPR10 was successfully expressed in Eschericha coli and the expressed protein was purified to near homogeneity. The dialytically renatured SsPR10 protein without phosphorylation exhibited ribonucleolytic activity against S. surattense leaf total RNA preparations and could inhibit hyphal growth of Pyricularia oryzae. Our findings suggest that the novel stress- and pathogen-inducible SsPR10 with ribonucleolytic and antimicrobial activity participates not only in the defense/stress response pathways but also in plants' growth, development and senescence.

Asunto(s)

Antiinfecciosos/metabolismo , Proteínas de Plantas/metabolismo , Ribonucleasas/metabolismo , Solanum/metabolismo , Secuencia de Aminoácidos , Antiinfecciosos/farmacología , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Escherichia coli/genética , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/farmacología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Plantones/genética , Plantones/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Solanum/genética , Solanum/virología , Regulación hacia Arriba

RESUMEN

Recombinant Arisaema heterophyllum agglutinin (AHA) was expressed in Escherichia coli as N-terminal His-tagged fusions. After induction with isopropylthio-beta-D-galactoside, the recombinant AHA was purified by metal-affinity chromatography. The purified AHA protein was incorporated into artificial diet at 0.1% (w/v) concentration in insect bioassay trial and the result showed that artificial diet containing AHA could significantly inhibit the growth of the third-instar nymphs of peach potato aphid (Myzus persicae). This study suggested that AHA could be an effective candidate for the control of peach potato aphid, one of the most serious sap-sucking insect pests causing significant yield loss of crops.

Asunto(s)

Aglutininas/biosíntesis , Arisaema/química , Escherichia coli/genética , Proteínas Recombinantes de Fusión/biosíntesis , Aglutininas/aislamiento & purificación , Aglutininas/farmacología , Animales , Áfidos/efectos de los fármacos , Productos Agrícolas/parasitología , Escherichia coli/metabolismo , Vectores Genéticos , Ninfa/efectos de los fármacos , Pruebas de Toxicidad Crónica

RESUMEN

From the middle of the 20th century, Chinese scientists have been actively involved in biotechnology. However, biotechnology education in China is a relatively recent phenomenon. This subject has not been addressed at the undergraduate level in a serious way until recently. In the last decade, biotechnology education developed rapidly and reached a new level in Chinese universities. The Chinese scientific establishment is very much aware of the importance of biotechnology and has identified this subject as one of the priority areas. Some universities are taking positive steps toward enhancing biotechnology education. This article focuses on the emergence, as well as the problems and prospects, of biotechnology education in China.

RESUMEN

Using RNA extracted from Zantedeschia aethiopica young leaves and primers designed according to the conservative regions of Araceae lectins, the full-length cDNA of Z. aethiopica agglutinin (ZAA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zaa was 871 bp and contained a 417 bp open reading frame (ORF) encoding a lectin precursor of 138 amino acids. Through comparative analysis of zaa gene and its deduced amino acid sequence with those of other Araceae species, it was found that zaa encoded a precursor lectin with signal peptide. Secondary and three-dimensional structure analyses showed that ZAA had many common characters of mannose-binding lectin superfamily and ZAA was a mannose-binding lectin with three mannose-binding sites. Southern blot analysis of the genomic DNA revealed that zaa belonged to a multi-copy gene family.

Asunto(s)

Lectina de Unión a Manosa/química , Zantedeschia/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Southern Blotting , Clonación Molecular , Cartilla de ADN/química , ADN Complementario/metabolismo , Lectinas/química , Lectinas/metabolismo , Manosa/química , Lectina de Unión a Manosa/genética , Modelos Moleculares , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Plantas , Conformación Proteica , Señales de Clasificación de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

Using RNA extracted from Zantedeschia aethiopica young leaves and primers designed according to the conservative regions of Araceae lectins, the full-length cDNA of Z. aethiopica agglutinin (ZAA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zaa was 871 bp and contained a 417 bp open reading frame (ORF) encoding a lectin precursor of 138 amino acids. Through comparative analysis of zaa gene and its deduced amino acid sequence with those of other Araceae species, it was found that zaa encoded a precursor lectin with signal peptide. Secondary and three-dimensional structure analyses showed that ZAA had many common characters of mannose-binding lectin superfamily and ZAA was a mannose-binding lectin with three mannose-binding sites. Southern blot analysis of the genomic DNA revealed that zaa belonged to a multi-copy gene family

Asunto(s)

Lectina de Unión a Manosa , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/fisiología , Proteínas de Plantas/fisiología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Genes de Plantas/genética , Genes de Plantas/fisiología

RESUMEN

Using RNA extracted from Zantedeschia aethiopica young leaves and primers designed according to the conservative regions of Araceae lectins, the full-length cDNA of Z. aethiopica agglutinin (ZAA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zaa was 871 bp and contained a 417 bp open reading frame (ORF) encoding a lectin precursor of 138 amino acids. Through comparative analysis of zaa gene and its deduced amino acid sequence with those of other Araceae species, it was found that zaa encoded a precursor lectin with signal peptide. Secondary and three-dimensional structure analyses showed that ZAA had many common characters of mannose-binding lectin superfamily and ZAA was a mannose-binding lectin with three mannose-binding sites. Southern blot analysis of the genomic DNA revealed that zaa belonged to a multi-copy gene family

Asunto(s)

Lectina de Unión a Manosa/fisiología , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa , Proteínas de Plantas/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/química , Genes de Plantas/fisiología , Genes de Plantas/genética , Plantas Modificadas Genéticamente/fisiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/química , Regulación de la Expresión Génica de las Plantas/fisiología , Regulación de la Expresión Génica de las Plantas/genética

RESUMEN

A new dehydrin ERD10 gene was cloned and characterized from Brassica napus (designated as Bndhn ERD10). The full-length cDNA of Bndhn ERD10 was 1114 bp and contained an open reading frame of 816 bp encoding a protein of 271 amino acid residues. The deduced Bndhn ERD10 protein contained an 8-serine residue domain and two conserved repeats of the characterized lysine-rich-K-segment (KIKEKLPG). Analysis of full-length cDNA and genomic DNA indicated that there were no introns in Bndhn ERD10 gene. The promoter of Bndhn ERD10 was further obtained by genomic walking technology, and analysis of the promoter indicated that the regulation of Bndhn ERD10 was ABA-dependent. Semi-quantitative RT-PCR of different tissues in unstressed B. napus plants indicated that the transcript of Bndhn ERD10 was more abundant in leaf than in stem and root. The expression profiles of Bndhn ERD10 in B. napus seedlings under various stress conditions including cold, salt and ABA were also investigated. Upon cold, salt and ABA stresses, increased transcript accumulations of the Bndhn ERD10 mRNAs were detected in young leaves 8 h after treatment.

Asunto(s)

Brassica napus/genética , Genes de Plantas , Proteínas de Plantas/genética , Ácido Abscísico/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Brassica napus/efectos de los fármacos , Clonación Molecular , Frío , ADN Complementario/genética , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Datos de Secuencia Molecular , Proteínas de Plantas/química , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Cloruro de Sodio

RESUMEN

Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed that zoa expressed in all the tested tissues of Z. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed in Escherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae.

Asunto(s)

Expresión Génica , Lectina de Unión a Manosa/genética , Filogenia , Rizoma/química , Zingiber officinale/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Análisis por Conglomerados , Biología Computacional , Cartilla de ADN , ADN Complementario/genética , Hemaglutinación , Lectina de Unión a Manosa/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN

RESUMEN

Using RNA extracted from Dendrobium officinale young leaves and primers designed according to the conservative regions of orchidaceae lectins, the full-length cDNA of Dendrobium officinale agglutinin2 (DOA2) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of doa2 was 777 bp and contained a 513 bp open reading frame (ORF) encoding a lectin precursor of 170 amino acids. Through comparative analysis of doa2 gene and its deduced amino acid sequence with those of other orchidaceae species and Amaryllidaceae species, it was found that DOA2 had many common characters of mannose-binding lectin superfamily including three mannose-binding sites. Semi-Quantitative RT-PCR analysis revealed that doa2 mRNA expression was detected in all tested tissues including root, stem and leaf, however, the expression was higher in stem, and lower in leaf. As the doa2 mRNA was detected in all the tested plant tissues, the doa2 was considered to be a constitutively expressed gene. The recombinant protein was expressed in E. coli and purified. Anti-fungal assay showed that DOA2 has anti-fungal activity towards Gibberella zeae. To our knowledge, this is the first report on cDNA cloning of mannose binding lectin from Dendrobium officinale.

Asunto(s)

Aglutininas/genética , Dendrobium/genética , Lectina de Unión a Manosa/genética , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , China , Clonación Molecular , Cartilla de ADN , ADN Complementario/genética , Lectina de Unión a Manosa/metabolismo , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad de la Especie

RESUMEN

Using RNA extracted from Zantedeschia aethiopica young leaves and primers designed according to the conservative regions of Araceae lectins, the full-length cDNA of Z. aethiopica agglutinin (ZAA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zaa was 871 bp and contained a 417 bp open reading frame (ORF) encoding a lectin precursor of 138 amino acids. Through comparative analysis of zaa gene and its deduced amino acid sequence with those of other Araceae species, it was found that zaa encoded a precursor lectin with signal peptide. Secondary and three-dimensional structure analyses showed that ZAA had many common characters of mannose-binding lectin superfamily and ZAA was a mannose-binding lectin with three mannose-binding sites. Southern blot analysis of the genomic DNA revealed that zaa belonged to a multi-copy gene family.

RESUMEN

A new CBF gene was cloned from Capsella bursa-pastoris(shepherd's purse) by rapid amplification of cDNA ends (RACE). The full-length cDNA of C. bursa-pastoris CBF gene (designated as Cbcbf) was 1034 bp long and contained a 657 bp open reading frame (ORF) encoding a putative DRE/CRT (LTRE)-binding protein of 219 amino acids. The predicted CbCBF protein was found to have a potential nuclear localization signal (NLS) in its N-terminal region followed by an AP2 DNA-binding motif and an acidic C-terminal half that might act as an activator domain. Bioinformatic analysis revealed that Cbcbf strongly resembled other CBF genes from Arabidopsis thaliana (cbf1, cbf2, cbf3) and Brassica napus (Bncbf5, Bncbf 7, Bncbf16 and Bncbf17). Subsequent cold acclimation assay showed that Cbcbf was relevant to cold acclimation. Our study implies that Cbcbf might have similar functions possessed by other CBF genes such as inducing the expression of some cold-regulated genes and increasing plants' freezing tolerance.

Asunto(s)

Adaptación Biológica/genética , Capsella/genética , Frío , Proteínas de Unión al ADN/genética , Transactivadores/genética , Secuencia de Aminoácidos , Secuencia de Bases , Capsella/metabolismo , Biología Computacional , Cartilla de ADN , ADN Complementario/genética , Modelos Moleculares , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , Sistemas de Lectura Abierta/genética , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia

RESUMEN

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5' flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3' flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5' flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3'-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49-54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.

Asunto(s)

Lectinas/genética , Proteínas de Plantas/genética , Zantedeschia/química , Zantedeschia/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Lectinas/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas de Plantas/química , Alineación de Secuencia