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1.
BMC Plant Biol ; 19(1): 372, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438864

RESUMO

BACKGROUND: Correct timing of flowering is critical for plants to produce enough viable offspring. In Arabidopsis thaliana (Arabidopsis), flowering time is regulated by an intricate network of molecular signaling pathways. Arabidopsis srr1-1 mutants lacking SENSITIVITY TO RED LIGHT REDUCED 1 (SRR1) expression flower early, particularly under short day (SD) conditions (1). SRR1 ensures that plants do not flower prematurely in such non-inductive conditions by controlling repression of the key florigen FT. Here, we have examined the role of SRR1 in the closely related crop species Brassica napus. RESULTS: Arabidopsis SRR1 has five homologs in Brassica napus. They can be divided into two groups, where the A02 and C02 copies show high similarity to AtSRR1 on the protein level. The other group, including the A03, A10 and C09 copies all carry a larger deletion in the amino acid sequence. Three of the homologs are expressed at detectable levels: A02, C02 and C09. Notably, the gene copies show a differential expression pattern between spring and winter type accessions of B. napus. When the three expressed gene copies were introduced into the srr1-1 background, only A02 and C02 were able to complement the srr1-1 early flowering phenotype, while C09 could not. Transcriptional analysis of known SRR1 targets in Bna.SRR1-transformed lines showed that CYCLING DOF FACTOR 1 (CDF1) expression is key for flowering time control via SRR1. CONCLUSIONS: We observed subfunctionalization of the B. napus SRR1 gene copies, with differential expression between early and late flowering accessions of some Bna.SRR1 copies. This suggests involvement of Bna.SRR1 in regulation of seasonal flowering in B. napus. The C09 gene copy was unable to complement srr1-1 plants, but is highly expressed in B. napus, suggesting specialization of a particular function. Furthermore, the C09 protein carries a deletion which may pinpoint a key region of the SRR1 protein potentially important for its molecular function. This is important evidence of functional domain annotation in the highly conserved but unique SRR1 amino acid sequence.


Assuntos
Brassica napus/genética , Flores/genética , Genes de Plantas , Proteínas de Plantas/genética , Flores/crescimento & desenvolvimento , Dosagem de Genes , Expressão Gênica , Filogenia , Proteínas de Plantas/fisiologia
2.
BMC Plant Biol ; 19(1): 346, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391002

RESUMO

BACKGROUND: The safety assessment and control of stacked transgenic crops is increasingly important due to continuous crop development and is urgently needed in China. The genetic stability of foreign genes and unintended effects are the primary problems encountered in safety assessment. Omics techniques are useful for addressing these problems. The stacked transgenic maize variety 12-5 × IE034, which has insect-resistant and glyphosate-tolerant traits, was developed via a breeding stack using 12-5 and IE034 as parents. Using 12-5 × IE034, its parents (12-5 and IE034), and different maize varieties as materials, we performed proteomic profiling, molecular characterization and a genetic stability analysis. RESULTS: Our results showed that the copy number of foreign genes in 12-5 × IE034 is identical to that of its parents 12-5 and IE034. Foreign genes can be stably inherited over different generations. Proteomic profiling analysis found no newly expressed proteins in 12-5 × IE034, and the differences in protein expression between 12 and 5 × IE034 and its parents were within the range of variation of conventional maize varieties. The expression levels of key enzymes participating in the shikimic acid pathway which is related to glyphosate tolerance of 12-5 × IE034 were not significantly different from those of its parents or five conventional maize varieties, which indicated that without selective pressure by glyphosate, the introduced EPSPS synthase is not has a pronounced impact on the synthesis of aromatic amino acids in maize. CONCLUSIONS: Stacked-trait development via conventional breeding did not have an impact on the genetic stability of T-DNA, and the impact of stacked breeding on the maize proteome was less significant than that of genotypic differences. The results of this study provide a theoretical basis for the development of a safety assessment approach for stacked-trait transgenic crops in China.


Assuntos
Variação Genética , Melhoramento Vegetal , Plantas Geneticamente Modificadas , Zea mays/genética , China , Inocuidade dos Alimentos , Dosagem de Genes , Instabilidade Genômica , Análise de Perigos e Pontos Críticos de Controle , Proteômica
3.
Nat Commun ; 10(1): 3101, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308377

RESUMO

The identification of cancer-promoting genetic alterations is challenging particularly in highly unstable and heterogeneous cancers, such as esophageal adenocarcinoma (EAC). Here we describe a machine learning algorithm to identify cancer genes in individual patients considering all types of damaging alterations simultaneously. Analysing 261 EACs from the OCCAMS Consortium, we discover helper genes that, alongside well-known drivers, promote cancer. We confirm the robustness of our approach in 107 additional EACs. Unlike recurrent alterations of known drivers, these cancer helper genes are rare or patient-specific. However, they converge towards perturbations of well-known cancer processes. Recurrence of the same process perturbations, rather than individual genes, divides EACs into six clusters differing in their molecular and clinical features. Experimentally mimicking the alterations of predicted helper genes in cancer and pre-cancer cells validates their contribution to disease progression, while reverting their alterations reveals EAC acquired dependencies that can be exploited in therapy.


Assuntos
Adenocarcinoma/genética , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias Esofágicas/genética , Perfilação da Expressão Gênica/métodos , Medicina de Precisão/métodos , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Progressão da Doença , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Instabilidade Genômica , Humanos , Aprendizado de Máquina , Modelos Genéticos , Família Multigênica/efeitos dos fármacos , Taxa de Mutação , Polimorfismo de Nucleotídeo Único
4.
Arch Virol ; 164(9): 2285-2295, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31250104

RESUMO

Examination of lumpy skin disease virus (LSDV) isolates from different geographic regions and times revealed that assays developed in our laboratory for differentiating between virulent Israeli viruses and Neethling vaccine virus (NVV) are generally useful in most, if not all, endemic areas in which NVV-based vaccines are used. Recently it was revealed that the LSDV126 gene of field isolates contains a duplicated region of 27 bp (9 aa), while the vaccine viruses have only one copy. Phylogenetic analysis of a 532-bp segment carrying the LSDV126 gene and whole virus genome sequences revealed that LSDV isolates formed two groups: virulent and vaccine viruses. In this analysis, all of the capripox viruses that lack the ability to efficiently infect cattle were found to carry only one copy of the 27-bp fragment, suggesting that the LSDV126 gene plays an important role in the ability of capripox viruses to infect cattle. In silico analysis of potential antigenic sites in LSDV126 revealed that LSDV126 variants with only one copy of the repeat lack a potentially important antigenic epitope, supporting its possible significance in cattle infection. This study provides new information about the nature of the LSDV126 gene and its possible role in the life cycle of LSDV.


Assuntos
Doença Nodular Cutânea/virologia , Vírus da Doença Nodular Cutânea/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Mapeamento de Epitopos , Dosagem de Genes , Doença Nodular Cutânea/diagnóstico , Vírus da Doença Nodular Cutânea/química , Vírus da Doença Nodular Cutânea/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética
5.
Prep Biochem Biotechnol ; 49(8): 822-829, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156045

RESUMO

Therapeutic monoclonal antibodies (mAbs) have become the dominant products in biopharmaceutical industry. Mammalian cell expression systems including Chinese hamster ovary (CHO) cells are the most commonly used hosts for the production of complex recombinant proteins. However, development of stable, high producing CHO cell lines suffers from the low expression level and instability of the transgene. The increasing efforts in the development of novel therapeutic antibodies and the advent of biosimilars have revealed the necessity for the development of improved platforms for rapid production of products for initial characterization and testing. In line with this premise, vector design and engineering has been applied to improve the expression level and stability of the transgene. This study reports the application of an improved lentiviral vector system containing the human interferon-ß scaffold attachment region (IFN-SAR) for the development of antibody producing stable CHO cells. mAb expressing clones producing 1100 µg/L of IgG1 monoclonal antibody were isolated without extensive screening of a large number of clones. Our results here indicate the positive effects of IFN-SAR on stable mAb expression using lentiviral based expression vectors. We also observed that although IFN-SAR can improve light chain (LC) and heavy chain (HC) gene copy numbers in stable cell pools, mAb expression in single cell clones was not affected by the transgene copy number.


Assuntos
Anticorpos Monoclonais/genética , Clonagem Molecular/métodos , Vetores Genéticos/genética , Lentivirus/genética , Animais , Células CHO , Linhagem Celular , Cricetulus , Dosagem de Genes , Humanos , Proteínas Recombinantes/genética , Transdução Genética
6.
World J Microbiol Biotechnol ; 35(6): 79, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134410

RESUMO

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Pichia/genética , Plasmídeos/genética , Transformação Genética , Replicação do DNA , Escherichia coli/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos , Instabilidade Genômica , Microbiologia Industrial , Kluyveromyces/genética , Regiões Promotoras Genéticas , RNA Guia , Biologia Sintética
7.
World J Microbiol Biotechnol ; 35(6): 84, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134444

RESUMO

Pectin is a type of complex hydrophilic polysaccharide widely distributed in plant resources. Thermal stable pectinase has its advantage in bioapplication in the fields of food processing, brewing, and papermaking, etc. In this study, we enzymatically characterized a putative endo-polygalacturonase TcPG from a Talaromyces cellulolyticus, realized its high-level expression in Pichia pastoris by in vitro constructing of a series of multi-copy expression cassettes and real time quantitative PCR screening. The secretive expression level of TcPG was nonlinear correlated to the gene dosage. Recombinants with five-copy TcPG gene in the host genome showed the highest expression. After cultivation in a bioreactor for about 96 h, the enzyme activity reached 7124.8 U/mL culture. TcPG has its optimal temperature of 70 °C. Under the optimized parameters, the pectin could be efficiently hydrolyzed into oligosaccharides.


Assuntos
Dosagem de Genes , Pectinas/metabolismo , Pichia/genética , Poligalacturonase/biossíntese , Poligalacturonase/genética , Talaromyces/enzimologia , Talaromyces/genética , Reatores Biológicos , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Hidrólise , Pichia/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/genética , Temperatura Ambiente , Fatores de Tempo
8.
Life Sci ; 228: 128-134, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31054270

RESUMO

AIMS: Forkhead box (FOX) proteins constitute a huge family of transcriptional regulators, which are involved in a wide range of cancers. FOXK1 is a little studied member of FOXK subfamily. This study aimed to investigate the potential prognostic value of FOXK1 in human hepatocellular carcinoma (HCC) and explore potential underlying mechanisms. MAIN METHODS: We performed bioinformatic analyses to evaluate the prognostic value of FOXK1 expression in human HCC and to reveal the underlying mechanism by which FOXK1 regulates HCC. RT-PCR, FACS analysis and sphere formation assay were carried out to investigate the role of FOXK1 in regulating liver cancer stem cells. KEY FINDINGS: Our results demonstrated that FOXK1 was overexpressed in human HCC and positively correlated with cancer progression. DNA hypomethylation and gene copy number variation contributed to the overexpression of FOXK1. Importantly, high FOXK1 expression was associated with both low overall survival probability (OS) and low relapse free survival probability (RFS) of HCC patients. Intriguingly, we found that high FOXK1 expression was correlated with activation of stem cell-regulating pathways in human HCC. Knockdown of FOXK1 resulted in downregulation of the cancer stem cell marker EpCAM and ALDH1 and decreased sphere-forming ability of hepatocellular carcinoma cells. SIGNIFICANCE: Overall, our study identified FOXK1 as a new biomarker for prognosis of HCC patients and revealed its role in regulating stemness of hepatocellular carcinoma cells.


Assuntos
Carcinoma Hepatocelular/genética , Fatores de Transcrição Forkhead/genética , Neoplasias Hepáticas/genética , Regulação para Cima , Adulto , Idoso , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Metilação de DNA , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida
9.
Iran J Allergy Asthma Immunol ; 18(2): 143-152, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31066250

RESUMO

Assessment of the number of T-cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) copies has been recently described as biomarkers of newly formed T and B cells respectively. In this study, we aimed to explore the effects of demographic variables including age, gender, weight, height and ethnicity on these two episomal DNA molecules. Second, for the first time in our country, we determined the reference values of TREC and KREC copy numbers in different age groups of Iranian healthy individuals as a threshold for identifying T cell and B cell lymphopenia. The TREC and KREC copy numbers were evaluated in 251 dried blood spot (DBS) samples from healthy volunteers (age range: 0-60 years). Six primary immunodeficiency (PID) patients were included as disease controls. TREC and KREC copies were markedly reduced with increasing age. Although the levels of TREC and KREC were higher in females than males, this difference did not reach statistical significance. These findings suggest that demographic variables including age should be considered for interpretation results of the TREC/KREC assay.


Assuntos
Fatores Etários , Linfócitos B/fisiologia , Linfopenia/diagnóstico , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/fisiologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Dosagem de Genes , Humanos , Lactente , Recém-Nascido , Irã (Geográfico) , Linfopenia/imunologia , Masculino , Pessoa de Meia-Idade , Padrões de Referência , Adulto Jovem
10.
Bioelectrochemistry ; 129: 18-25, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31075535

RESUMO

Periplasmic c-type cytochromes are essential for the electron transport between the cytoplasmic membrane bound menquinol oxidase CymA and the terminal ferric iron reductase MtrABC in the outer membrane of Shewanella oneidensis cells. Either STC or FccA are necessary for periplasmic electron transfer. We followed the hypothesis that the elimination of potential competing reactions in the periplasm and the simultaneous overexpression of STC (cctA) could lead to an accelerated electron transfer to the cell surface. The genes nrfA, ccpA, napB and napA were replaced by cctA. This led to a 1.7-fold increased ferric iron reduction rate and a 23% higher current generation in a bioelectrochemical system. Moreover, the quadruple mutant had a higher periplasmic flavin content. Further deletion of fccA and its replacement by cctA resulted in a strain with ferric iron reduction rates similar to the wild type and a lower concentration of periplasmic flavin compared to the quadruple mutant. A transcriptomic analysis revealed that the quadruple mutant had a 3.7-fold higher cctA expression which could not be further increased by the replacement of fccA. This work indicates that a synthetic adaptation of Shewanella towards extracellular respiration holds potential for increased respiratory rates and consequently higher current densities.


Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Proteínas Periplásmicas/metabolismo , Shewanella/metabolismo , Proteínas de Bactérias/genética , Grupo dos Citocromos c/genética , Transporte de Elétrons , Desenho de Equipamento , Fumaratos/metabolismo , Deleção de Genes , Dosagem de Genes , Engenharia Genética/métodos , Lactatos/metabolismo , Proteínas Periplásmicas/genética , Shewanella/genética , Transcriptoma , Regulação para Cima
11.
Scand J Immunol ; 90(2): e12776, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31069824

RESUMO

The number of the X chromosome-linked genes has been previously suggested to influence immune responses and the development of autoimmune diseases. In the present study, we aimed at evaluating the level of expression of CD40L (an X-linked gene involved in adaptive immunity) and TLR7 (an X-linked gene involved in innate immunity) in a variety of different karyotypes. Those included males, females and patients with X chromosome aneuploidy. Healthy females (46, XX; n = 10) and healthy males (46, XY; n = 10) were compared to females with Turner syndrome (TS) (45, X; n = 11) and males with Klinefelter syndrome (KS) (47, XXY; n = 5). Stimulation of peripheral blood mononuclear cells (PBMCs) with PMA and ionomycin resulted in higher percentage of CD3 + CD40L+ T cells (P < 0.001) and higher level expression of CD40L in T cell (P < 0.001) in female and KS patients compared with male and TS patients. TLR7-mediated IFN-alpha production by HLADR + CD3- CD19- cells was significantly upregulated in healthy women compared with healthy males, TS and KS patients (P < 0.001). TLR7 agonist-stimulated PBMCs from healthy females and KS patients expressed significantly higher levels of TLR7 mRNA than those from male and TS patients (P < 0.05). The increased expression of the X-linked genes TLR7 and CD40L in healthy females and KS patients suggests that the presence of two X chromosomes plays a major role in enhancing both innate and adaptive immune responses. These results may contribute to the explanation of sex-based differences in immune biology and the sex bias in predisposition to autoimmune diseases.


Assuntos
Imunidade Adaptativa/genética , Ligante de CD40/biossíntese , Ligante de CD40/genética , Cromossomos Humanos X/genética , Dosagem de Genes/genética , Imunidade Inata/genética , Receptor 7 Toll-Like/biossíntese , Receptor 7 Toll-Like/genética , Imunidade Adaptativa/imunologia , Antígenos CD19/biossíntese , Complexo CD3/biossíntese , Células Cultivadas , Variações do Número de Cópias de DNA/genética , Feminino , Humanos , Imunidade Inata/imunologia , Interferon-alfa/biossíntese , Ionomicina/farmacologia , Síndrome de Klinefelter/genética , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Ácidos Polimetacrílicos/farmacologia , RNA Mensageiro/biossíntese , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Síndrome de Turner/genética
12.
J Clin Pathol ; 72(9): 642-646, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31123138

RESUMO

AIMS: To elucidate the clinicopathological and molecular features of intravascular NK/T-cell lymphoma (IVNKTCL). METHODS: Two cases of IVNKTCL were retrieved from a single-centre cohort composed of 25 intravascular lymphomas. Whole-exome and RNA sequencing and immunohistochemistry were performed. RESULTS: We identified somatic mutations in the following epigenetic regulators: four histone genes (HIST1H2AN, HIST1H2BE, HIST1H2BN and H3F3A), histone deacetylase (HDAC5), two helicases (WRN and DDX3X), two methylation-related enzymes (TET2 and DNMT1) and the SNI/SWF pathway (ARID1A). Copy number analysis identified driver gene alterations comprising the loss of ARID1B, HACE1 and SMAD4, and the gain of SOX2 and histone clusters. RNA sequencing analysis did not indicate the presence of any fusion gene. Both cases were positive for Epstein-Barr virus (EBV) and showed strong expression of programmed death-ligand 1 (PD-L1). CONCLUSIONS: This study raises the possibility that, at least for some patients, IVNKTCL may be considered an epigenetic disease with EBV infection-associated aetiopathogenesis.


Assuntos
Biomarcadores Tumorais/genética , Variações do Número de Cópias de DNA , Dosagem de Genes , Linfoma Extranodal de Células T-NK/genética , Linfoma Extranodal de Células T-NK/patologia , Mutação , Idoso , Antígeno B7-H1/análise , Biomarcadores Tumorais/análise , Epigênese Genética , Predisposição Genética para Doença , Herpesvirus Humano 4/isolamento & purificação , Humanos , Imuno-Histoquímica , Linfoma Extranodal de Células T-NK/química , Linfoma Extranodal de Células T-NK/virologia , Masculino , Fenótipo , Estudos Retrospectivos , Análise de Sequência de RNA , Sequenciamento Completo do Exoma , Adulto Jovem
13.
Plant Cell Rep ; 38(9): 1081-1097, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31134349

RESUMO

KEY MESSAGE: Duplicate POT1 genes must rapidly diverge or be inactivated. Protection of telomeres 1 (POT1) encodes a conserved telomere binding protein implicated in both chromosome end protection and telomere length maintenance. Most organisms harbor a single POT1 gene, but in the few lineages where the POT1 family has expanded, the duplicate genes have diversified. Arabidopsis thaliana bears three POT1-like loci, POT1a, POT1b and POT1c. POT1a retains the ancestral function of telomerase regulation, while POT1b is implicated in chromosome end protection. Here we examine the function and evolution of the third POT1 paralog, POT1c. POT1c is a new gene, unique to A. thaliana, and was derived from a duplication event involving the POT1a locus and a neighboring gene encoding ribosomal protein S17. The duplicate S17 locus (dS17) is highly conserved across A. thaliana accessions, while POT1c is highly divergent, harboring multiple deletions within the gene body and two transposable elements within the promoter. The POT1c locus is transcribed at very low to non-detectable levels under standard growth conditions. In addition, no discernable molecular or developmental defects are associated with plants bearing a CRISPR mutation in the POT1c locus. However, forced expression of POT1c leads to decreased telomerase enzyme activity and shortened telomeres. Evolutionary reconstruction indicates that transposons invaded the POT1c promoter soon after the locus was formed, permanently silencing the gene. Altogether, these findings argue that POT1 dosage is critically important for viability and duplicate gene copies are retained only upon functional divergence.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Dosagem de Genes , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/genética , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Elementos de DNA Transponíveis/genética , Evolução Molecular , Duplicação Gênica , Mutação , Regiões Promotoras Genéticas/genética , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética
14.
Nat Commun ; 10(1): 1519, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944321

RESUMO

Hyperdiploidy, i.e. gain of whole chromosomes, is one of the most common genetic features of childhood acute lymphoblastic leukemia (ALL), but its pathogenetic impact is poorly understood. Here, we report a proteogenomic analysis on matched datasets from genomic profiling, RNA-sequencing, and mass spectrometry-based analysis of >8,000 genes and proteins as well as Hi-C of primary patient samples from hyperdiploid and ETV6/RUNX1-positive pediatric ALL. We show that CTCF and cohesin, which are master regulators of chromatin architecture, display low expression in hyperdiploid ALL. In line with this, a general genome-wide dysregulation of gene expression in relation to topologically associating domain (TAD) borders were seen in the hyperdiploid group. Furthermore, Hi-C of a limited number of hyperdiploid childhood ALL cases revealed that 2/4 cases displayed a clear loss of TAD boundary strength and 3/4 showed reduced insulation at TAD borders, with putative leukemogenic effects.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transcrição Genética , Adolescente , Aneuploidia , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/genética , Criança , Pré-Escolar , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Aberrações Cromossômicas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Lactente , Recém-Nascido , Masculino , Proteogenômica/métodos , Proteoma/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Análise de Sequência de RNA
15.
J Cancer Res Clin Oncol ; 145(7): 1845-1856, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31025094

RESUMO

PURPOSE: The protein tyrosine phosphatase PTPN2 dephosphorylates several tyrosine kinases in cancer-related signalling pathways and is thought to be a tumour suppressor. As PTPN2 is not frequently studied in breast cancer, we aimed to explore the role of PTPN2 and the effects of its loss in breast cancer. METHODS: Protein expression and gene copy number of PTPN2 were analysed in a cohort of pre-menopausal breast cancer patients with immunohistochemistry and droplet digital PCR, respectively. PTPN2 was knocked down in three cell lines, representing different breast cancer subtypes, with siRNA transfection. Several proteins related to PTPN2 were analysed with Western blot. RESULTS: Low PTPN2 protein expression was found in 50.2% of the tumours (110/219), gene copy loss in 15.4% (33/214). Low protein expression was associated with a higher relapse rate in patients with Luminal A and HER2-positive tumours, but not triple-negative tumours. In vitro studies further suggested a subtype-specific role of PTPN2. Knockdown of PTPN2 had no effect on the triple-negative cell line, whilst knockdown in MCF7 inhibited phosphorylation of Met and promoted that of Akt. Knockdown in SKBR3 led to increased Met phosphorylation and decreased Erk phosphorylation as well as EGF-mediated STAT3 activation. CONCLUSION: We confirm previous studies showing that the PTPN2 protein is lost in half of the breast cancer cases and gene deletion occurs in 15-18% of the cases. Furthermore, the results suggest that the role of PTPN2 is subtype-related and should be further investigated to assess how this could affect breast cancer prognosis and treatment response.


Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Comunicação Celular , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Dosagem de Genes , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Células MCF-7 , Prognóstico , Proteína Tirosina Fosfatase não Receptora Tipo 2/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 2/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Receptor ErbB-2/biossíntese , Neoplasias de Mama Triplo Negativas/classificação , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
16.
Genome Biol ; 20(1): 64, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30935422

RESUMO

BACKGROUND: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. RESULTS: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. CONCLUSIONS: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus's strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes.


Assuntos
Evolução Molecular , Genoma de Inseto , Hemípteros/genética , Sequência de Aminoácidos , Animais , Dedos de Zinco CYS2-HIS2 , Comportamento Alimentar , Dosagem de Genes , Perfilação da Expressão Gênica , Transferência Genética Horizontal , Genes Homeobox , Hemípteros/crescimento & desenvolvimento , Hemípteros/metabolismo , Pigmentação/genética , Olfato , Fatores de Transcrição/genética
17.
Int J Mol Sci ; 20(8)2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995757

RESUMO

The amplification of estrogen receptor alpha (ERα) encoded by the ESR1 gene has been described as having a prognostic role in breast cancer patients. However, increased dosage of the ESR1 gene (tested by real-time PCR) is also observed in ER-negative breast cancers, which might suggest the expression of alternative isoforms of ERα (other than classical ERα of 66 kDa). In the current work, we have investigated the ESR1 gene dosage in 402 primary breast cancer patients as well as the expression of ERα isoforms-ERα66 and ERα36-on mRNA and protein levels. The obtained results were correlated with clinicopathological data of the patients. Results showed that increased ESR1 gene dosage is not related to ESR1 gene amplification measured by fluorescent in situ hybridization (FISH), but it correlates with the decreased expression of ERα66 isoform (p = 0.01). Interestingly, the short ER isoform ERα36 was expressed in samples with increased ESR1 gene dosage, suggesting that genomic aberration might influence the expression of that particular isoform. Similarly to ESR1 increased gene dosage, high ERα36 expression was linked with the decreased disease-free survival of the patients (p = 0.05), which was independent of the status of the classical ERα66 level in breast tumors.


Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Mama/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Prognóstico , Isoformas de Proteínas/genética , Regulação para Cima
18.
Biomed Res Int ; 2019: 5921725, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30941367

RESUMO

Ephedrae Herba and Ephedrae Radix et Rhizoma (Mahuang) have been used as Chinese herbal medicines. Ephedra plants mainly live in deserts and have good governance of desertification. Despite their important medicinal and environmental protection value, dietary supplements containing ephedrine from Ephedra species may threaten the health of people. Morphological resemblance amongst species causes difficulty in identifying the original species of Ephedra herbs. Chloroplast (CP) genome shows good prospects in identification and phylogenetic analysis. This study introduced the structures of the CP genomes of three Ephedra species and analysed their phylogenetic relationships. Three complete CP genomes of Ephedra showed four-part annular structures, namely, two single-copy regions and two inverted repeat regions. The entire CP genomes of three Ephedra species in terms of size were 109,550 bp (E. sinica), 109,667 bp (E. intermedia), and 109,558 bp (E. equisetina). Each CP genome of the three Ephedra species encoded 118 genes, including 73 protein-coding genes, 37 tRNA genes and 8 ribosomal RNA genes. Eleven high-variation regions were screened through mVISTA to be potential specific DNA barcodes for identifying Ephedra species. Maximum likelihood and maximum parsimony trees showed that CP genomes could be used to identify Ephedra species. The Ephedra species had a close phylogenetic relationship with Gnetum species and Welwitschia mirabilis. This research provided valuable information for the identification and phylogenetic analysis of gymnosperms and drug safety of Ephedra.


Assuntos
Ephedra/classificação , Ephedra/genética , Efedrina/metabolismo , Genoma de Cloroplastos , Filogenia , Mapeamento Cromossômico , Códon/genética , Dosagem de Genes , Sequências Repetidas Invertidas/genética , Funções Verossimilhança , Repetições de Microssatélites/genética , Sequências Repetitivas de Ácido Nucleico/genética , Especificidade da Espécie
19.
BMC Cancer ; 19(1): 352, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975103

RESUMO

BACKGROUND: High risk human papillomaviruses (HPV) plays important roles in the development of cervical cancer, a number of other anogenital cancer and they are increasingly found in oropharyngeal squamous cell carcinoma (OPSCC), however there has not been comprehensive analysis about the role how these viruses play in the development of OPSCC. METHODS: To characterize the physical status of HPV within OPSCC and to determine the effect this has throughout the host genome, we have performed 30-40X whole genome sequencing (WGS) on the BGI sequencing platform on 34 OPSCCs: 28 of which were HPV positive. We then examined the sequencing data to characterize the HPV copy number and HPV physical status to determine what effect they have on both HPV and human genome structural changes. RESULTS: WGS determined the HPV copy number across the viral genome. HPV copy number ranged from 1 copy to as high as 150 copies in each individual OPSCC. Independent of HPV copy number, most tumors had either a small or a very large deletion in the viral genome. We discovered that these deletions were the result of either HPV integration into the human genome or HPV-HPV sequence junctions. WGS revealed that ~ 70% of these tumors had HPV integrations within the human genome and HPV integration occurred independent of HPV copy number. Individual HPV integrations were found to be highly disruptive resulting in structural variations and copy number changes at or around the integration sites. CONCLUSIONS: WGS reveals that there is a great complexity in both HPV sequences present and the HPV integrations events in HPV positive OPSCCs tumors. Thus HPV may be playing different roles in the development of different OPSCCs and this further challenge the HPV-driven carcinogenesis model first proposed for cervical cancer.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Neoplasias Orofaríngeas/genética , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Adulto , Idoso , Carcinogênese/genética , Carcinoma de Células Escamosas/patologia , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Dosagem de Genes , Genoma Humano/genética , Genoma Viral/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Orofaríngeas/patologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/patologia , Integração Viral/genética , Sequenciamento Completo do Genoma
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