RESUMEN
PURPOSE: Despite great advances that have been made in the understanding of the molecular complexity of acute myeloid leukemia (AML), very little has been translated into new therapies. Here, we set out to investigate the impact of cytoskeleton regulatory genes on clinical outcomes and their potential as therapeutic targets in AML. METHODS: Gene expression and clinical data were retrieved from The Cancer Genome Atlas (TCGA) AML study and used for survival and functional genomics analyses. For pharmacological tests, AML cells were exposed to ezrin (EZR) inhibitors and submitted to several cellular and molecular assays. RESULTS: High EZR expression was identified as an independent marker of worse outcomes in AML patients from the TCGA cohort (p < 0.05). Functional genomics analyses suggested that EZR contributes to responses to stimuli and signal transduction pathways in leukemia cells. EZR pharmacological inhibition with NSC305787 and NSC668394 reduced viability, proliferation, autonomous clonal growth, and cell cycle progression in AML cells (p < 0.05). NSC305787 had a greater potency and efficiency than NSC668394 in leukemia models. At the molecular level, EZR inhibitors reduced EZR, S6 ribosomal protein and 4EBP1 phosphorylation, and induced PARP1 cleavage in AML cells. NSC305787, but not NSC668394, favored a gene network involving cell cycle arrest and apoptosis in Kasumi 1 AML cells. CONCLUSIONS: From our data we conclude that EZR expression may serve as a prognostic factor in AML. Our preclinical findings indicate that ezrin inhibitors may be employed as a putative novel class of AML targeting drugs.
Asunto(s)
Biomarcadores de Tumor/genética , Proteínas del Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulación Leucémica de la Expresión Génica , Genes Reguladores/genética , Leucemia Mieloide/genética , Enfermedad Aguda , Adamantano/análogos & derivados , Adamantano/farmacología , Adulto , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/metabolismo , Supervivencia sin Enfermedad , Femenino , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/metabolismo , Masculino , Fenoles/farmacología , Pronóstico , Quinolinas/farmacología , Quinolonas/farmacología , Células THP-1 , Células U937RESUMEN
In the N2-fixing symbiont of alfalfa root nodules, Sinorhizobium meliloti 2011, the mmgR gene encodes a 77 nt small untranslated RNA (sRNA) that negatively regulates the accumulation of polyhydroxybutyrate (PHB) when the bacterium is grown under conditions of surplus carbon (C) in relation to nitrogen (N). We previously showed that the expression of mmgR is primarily controlled at the transcriptional level and that it depends on the cellular N status, although the regulatory mechanism and the factors involved were unknown. In this study, we provide experimental data supporting that: (a) mmgR is induced upon N limitation with the maximum expression found at the highest tested C/N molar ratio in the growth medium; (b) a conserved heptamer TTGTGCA located between the -35 and -10 mmgR promoter elements is necessary and sufficient for induction by N limitation; (c) induction of mmgR requires the N-status regulator NtrC; (d) under C limitation, mmgR transcription is repressed by AniA, a global regulator of C flow; (e) the mmgR promoter contains a conserved dyadic motif (TGC[N3]GCA) partially overlapping the heptamer TTGTGCA, which was also found in the promoters of the PHB-related genes phaP1, phaP2, phaZ and phaR (aniA) of S. meliloti and other alpha-proteobacteria. Taken together, these results suggest that the mmgR promoter would integrate signals from the metabolism of C and N through - at least - the global regulators NtrC and AniA, to provide an optimal level of the MmgR sRNA to fine-tune gene expression post-transcriptionally according to varying C and N availability.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN Pequeño no Traducido/genética , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Sitios de Unión , Carbono/metabolismo , Ciclo del Carbono/genética , Secuencia Conservada , Técnicas de Inactivación de Genes , Genes Reguladores/genética , Genes Reguladores/fisiología , Medicago sativa/microbiología , Mutación , Nitrógeno/metabolismo , Fijación del Nitrógeno/genética , Regiones Promotoras Genéticas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Alineación de Secuencia , Sinorhizobium meliloti/crecimiento & desarrollo , SimbiosisRESUMEN
Vibrio cholerae O1 El Tor strains have been responsible for pandemic cholera since 1961. These strains have evolved over time, spreading globally in three separate waves. Wave 3 is caused by altered El Tor (AET) variant strains, which include the strain with the signature ctxB7 allele that was introduced in 2010 into Haiti, where it caused a devastating epidemic. In this study, we used phenotypic analysis to compare an early isolate from the Haiti epidemic to wave 1 El Tor isolates commonly used for research. It is demonstrated that the Haiti isolate has increased production of cholera toxin (CT) and hemolysin, increased motility, and a reduced ability to form biofilms. This strain also outcompetes common wave 1 El Tor isolates for colonization of infant mice, indicating that it has increased virulence. Monitoring of CT production and motility in additional wave 3 isolates revealed that this phenotypic variation likely evolved over time rather than in a single genetic event. Analysis of available whole-genome sequences and phylogenetic analyses suggested that increased virulence arose from positive selection for mutations found in known and putative regulatory genes, including hns and vieA, diguanylate cyclase genes, and genes belonging to the lysR and gntR regulatory families. Overall, the studies presented here revealed that V. cholerae virulence potential can evolve and that the currently prevalent wave 3 AET strains are both phenotypically distinct from and more virulent than many El Tor isolates.
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Cólera/epidemiología , Cólera/microbiología , Vibrio cholerae O1/genética , Vibrio cholerae O1/patogenicidad , Virulencia/genética , Alelos , Animales , Evolución Biológica , Toxina del Cólera/genética , Epidemias , Genes Reguladores/genética , Variación Genética/genética , Haití/epidemiología , Proteínas Hemolisinas/genética , Ratones , Ratones Endogámicos ICR , Fenotipo , FilogeniaRESUMEN
Shifts in pollination syndromes involve coordinated changes in multiple floral traits. This raises the question of how plants can cope with rapid changes in pollinator availability by the slow process of accumulation of mutations in multiple genes. Here we study the transition from bee to hawkmoth pollination in the genus Petunia. Interspecific crosses followed by single locus introgressions were used to recreate putative intermediate evolutionary stages in the evolution of moth pollination. The effect of the loss/gain of petal color was asymmetric: it had no influence on the established pollinator but enhanced visitation by the new pollinator. Therefore, shifts in pollination syndromes may proceed through intermediate stages of reduced specialization and consequently enhanced reproductive assurance. The loss of petal color in moth-pollinated Petunia involves null mutations in a single regulatory gene, An2. Such simple genetic changes may be sufficiently rapid and frequent to ensure survival during pollinator failure.
Asunto(s)
Abejas/fisiología , Evolución Biológica , Flores/genética , Mariposas Nocturnas/fisiología , Petunia/genética , Polinización/fisiología , Análisis de Varianza , Animales , Conducta Animal/fisiología , Color , Cruzamientos Genéticos , Flores/fisiología , Genes Reguladores/genética , Petunia/fisiología , Especificidad de la Especie , UruguayRESUMEN
The genetic characterization of Xanthomonas species remains a challenge. Several DNA-based techniques have been previously employed, including the analysis of the 16S rRNA and 16S-23S rRNA genes in order to differentiate and classify the Xanthomonas species. However, several species could not be distinguished in these studies, due to the high degree of conservation of these molecular markers. In order to obtain more efficient markers, and to better understand the phylogenetic relationships between the Xanthomonas species, two genes commonly found in the different species have been analyzed. The genes rpfB and atpD involved in the regulation of pathogenicity factors and in the synthesis of ATP, respectively, were amplified in Xanthomonas species and further analyzed by PCR-restriction fragment length polymorphism. Dendrograms with the data sets of the rpfB and atpD analyzed separately and combined were constructed. The results obtained revealed that several Xanthomonas species, previously grouped together, could be successfully distinguished using these markers. The results obtained herein provide an alternative method for the distinction of the Xanthomonas species and contribute to a better understanding of the genetic and phylogenetic relationships of Xanthomonas.
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Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana/métodos , Dermatoglifia del ADN/métodos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Xanthomonas/clasificación , Análisis por Conglomerados , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Electroforesis en Gel de Agar , Genes Reguladores/genética , Genotipo , Filogenia , Factores de Transcripción/genética , Xanthomonas/genéticaRESUMEN
Sonic Hedgehog (SHH) plays a fundamental role in numerous developmental processes including morphogenesis of limbs, nervous system, and teeth. Using a Bayesian alignment algorithm for phylogenetic footprinting we analyzed approximately 28 kb of noncoding DNA in the SHH locus of human and mouse. This showed that the length of conserved noncoding sequences (4196 nt) shared by these species was approximately 3 times larger than the SHH coding sequence (1386 nt). Most segments were located in introns (53%) or within 2-kb regions upstream (16%) or downstream (20%) of the first and last SHH codon. Even though regions more than 2 kb upstream or downstream of the first and last SHH codon represented 57% (16 kb) of the sequence compared, they accounted for only 11% (494 nt) of the total length of conserved noncoding segments. One region of 650 nt downstream of SHH was identified as a putative scaffold/matrix attachment region (SMAR). Human-mouse analysis was complemented by sequencing in apes, monkeys, rodents, and bats, thus further confirming the evolutionary conservation of some segments. Gel-shift assays indicated that conserved segments are targeted by nuclear proteins and showed differences between two cell types that expressed different levels of SHH, namely human endothelial cells and breast cancer cells. The relevance of these findings with respect to regulation of SHH expression during normal and pathologic development is discussed.
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ADN Intergénico/genética , Genes Reguladores/genética , Mamíferos/genética , Filogenia , Transactivadores/genética , Animales , Secuencia de Bases , Teorema de Bayes , Sitios de Unión , Secuencia Conservada/genética , Huella de ADN , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Proteínas Hedgehog , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.
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Núcleo Celular/metabolismo , Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , Uniones Estrechas/metabolismo , Actinas/metabolismo , Transporte Activo de Núcleo Celular/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos/genética , Animales , Línea Celular , Núcleo Celular/ultraestructura , Perros , Células Epiteliales/ultraestructura , Ácidos Grasos Insaturados/farmacología , Genes Reguladores/genética , Lamina Tipo B/metabolismo , Datos de Secuencia Molecular , Matriz Nuclear/metabolismo , Matriz Nuclear/ultraestructura , Ovalbúmina/metabolismo , Regiones Promotoras Genéticas/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Uniones Estrechas/ultraestructura , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Repeticiones de Trinucleótidos/genética , Proteína de la Zonula Occludens-2RESUMEN
Bacillus subtilis aprE gene codes for the extracellular protease subtilisin. Its expression is controlled by AbrB, DegU, Hpr, SinI, SinR and Spo0A transition state protein regulators. To determine in vivo the protein-protein interactions among these regulators, we used the LexA-based bacterial genetic two-hybrid system. Our results show homo-dimerization to all the analyzed proteins and hetero-dimerization between SinR-SinI and SinR-Hpr.
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Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas de Transporte de Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , Proteínas Bacterianas/genética , Genes Reguladores/genética , Proteínas de Transporte de Membrana/genética , Serina Endopeptidasas/genéticaRESUMEN
A second regulatory gene (jadR(1)) is located immediately upstream of the putative repressor gene (jadR(2)) in the jad cluster for biosynthesis of the antibiotic jadomycin B in Streptomyces venezuelae ISP5230. It encodes a 234-amino acid polypeptide with a sequence resembling those of response regulator proteins in two-component control systems. Features in the conserved C-terminal domain of JadR(1) place the protein in the OmpR-PhoB subfamily of response regulators. In mutants where jadR(1) was deleted or disrupted, jadomycin B was not produced, implying that the gene has an essential role in biosynthesis of the antibiotic. Cloning jadR(1) from S. venezuelae in pJV73A, and introducing additional copies of the gene into the wild-type parent by plasmid transformation gave unstable strains with pJV73A integrated into the chromosome. The transformants initially showed increased production of jadomycin B but gave lower titers as excess copies of jadR(1) were lost; mature cultures stabilized with a wild-type level of antibiotic production. The mutant from which jadR(1) had been deleted could not be transformed with pJV73A. Altering the composition of jadR genes in the chromosome by integration of vectors carrying intact and disrupted copies of jadR(1) and jadR(2) provided evidence that the two genes form a regulatory pair different in function from previously reported two-component systems controlling antibiotic biosynthesis in streptomycetes.
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Genes Reguladores/genética , Isoquinolinas/metabolismo , Streptomyces/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano/química , ADN Bacteriano/genética , Dosificación de Gen , Genes Reguladores/fisiología , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Streptomyces/metabolismoRESUMEN
The B-Peru allele of the maize b regulatory gene is unusual relative to most b alleles in that it is expressed in the aleurone layer of the seed. It is also expressed in a subset of plant vegetative tissues. Transgenic maize plants containing the B-Peru gene with the first 710 bases of upstream sequence conferred the same levels of aleurone expression as nontransgenic B-Peru plants, but no pigment was made in vegetative tissues. Transient transformation assays in aleurone tissue localized the aleurone-specific promoter to the first 176 bases of the B-Peru upstream region and identified two critically important regions within this fragment. Mutation of either region alone reduced expression greater than fivefold. Surprisingly, the double mutation actually increased expression to twice the native promoter level. Our results suggest that these two critical sequences, which lie close together in the promoter, may form a negative regulatory element. Several lines of evidence suggest that the B-Peru promoter arose through the translocation of an existing aleurone-specific promoter to the b locus. Immediately upstream of the aleurone-specific promoter elements and in the opposite orientation to the b coding sequence is a pseudogene sequence with strong similarity to a known class of proteins. Our findings that novel aleurone-specific promoter sequences of the B-Peru transcription factor are found adjacent to part of another gene in a small insertion are quite unexpected and have interesting evolutionary implications.
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Genes de Plantas , Genes Reguladores/genética , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Translocación Genética , Zea mays/genética , Alelos , Secuencia de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Exones , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Mutagénesis Insercional , Especificidad de Órganos/genética , Proteínas de Plantas/biosíntesis , Plantas Modificadas Genéticamente , Seudogenes , Semillas/genética , Factores de Transcripción/biosíntesis , Zea mays/embriologíaRESUMEN
In the last decade, two types of genes participating in the etiology of hypertension have been identified. The primary genes or blood pressure regulators are those that codify enzymes (renin, kallikrein, kininase, aminopeptidase), hormones (angiotensins, vasopressin, aldosterone, prostaglandins, and atrial natriuretic peptide) and substrates (angiotensinogen and kininogen). They cause arteriolar vasodilation or vasoconstriction or sodium retention in the extravascular space. Allelic polymorphisms associated to essential hypertension have been described. The secondary genes are those that produce hereditary diseases of low prevalence, associated to hypertension in 20 to 80% of patients (polycystic kidney disease, pheochromocytoma, adrenal hyperplasia, hereditary nephritis). Forty genes located in all chromosomes, that are dominantly, recessively or X-linked transmitted, have thus far been identified. Chromosomal maps with all genic loci are presented.
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Genoma Humano , Hipertensión/genética , Mapeo Cromosómico , Genes/genética , Genes Reguladores/genética , HumanosRESUMEN
OBJECTIVE: To compare differences in epithelial chloride conductance according to class of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. METHODS: We evaluated the relationship between the functional classes of CFTR mutations and chloride conductance using the first diagnostic sweat chloride concentration in a large cystic fibrosis (CF) population. RESULTS: There was no difference in sweat chloride value value between classes of CFTR mutations that produce no protein (class I), fail to reach the apical membrane because of defective processing (class II), or produce protein that fails to respond to cyclic adenosine monophosphate (class III). Those mutations that produce a cyclic adenosine monophosphate-responsive channel with reduced conductance (class IV) were associated with a significantly lower, intermediate sweat chloride value. However, patients with the mutations that cause reduced synthesis or partially defective processing of normal CFTR (class V) had sweat chloride concentrations similar to those in classes I to III. CONCLUSION: Studies of differences in chloride conductance between functional classes of CFTR mutations provide insight into phenotypic expression of the disease.
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Cloruros/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Genes Reguladores/genética , Mutación , Sudor/química , Niño , Preescolar , Fibrosis Quística/clasificación , Regulador de Conductancia de Transmembrana de Fibrosis Quística/clasificación , Genotipo , Heterocigoto , Homocigoto , Humanos , Fenotipo , Estudios RetrospectivosRESUMEN
When grown on low-Pi medium, the chaA1 pabaA1 palB7 mutant of Aspergillus nidulans excretes an acid phosphatase with steady-state kinetic properties, temperature sensitivity and electrophoretic mobility different from those of the enzyme excreted by the pabaA1 strain. The enzyme excreted by the pabaA1 strain at pH 6.5 showed PNP-P activity with negative cooperativity (K0.5 = 0.87 +/- 0.06 mM, n = 0.68 +/- 0.03) whereas the enzyme excreted by the chaA1 pabaA1 palB7 mutant showed Michaelian kinetics (Km = 0.46 +/- 0.03 mM, n = 1.00 +/- 0.02). The apparent half-lives at 60 degrees C, pH 5.5, of acid phosphatase excreted by the pabaA1 and chaA1 pabaA1 palB7 strains were 58.6 +/- 4.9 min and 21.5 +/- 1.8 min, respectively. Furthermore, the electrophoretic mobility of acid phosphatases excreted by the palA1, palB7, palC4, palE11 and palF15 mutants of A. nidulans was altered and differed from the electrophoretic mobility of the enzyme excreted by the wild-type strain. Also, the palB7 mutation altered the electrophoretic pattern of acid phosphatases synthesized on high-Pi medium. These results are compatible with the post-translational modifications in the Pi-repressible phosphatases rather than with the action of gene palB in controlling the transcription of structural genes of these enzymes.
Asunto(s)
Fosfatasa Ácida/metabolismo , Aspergillus nidulans/genética , Regulación Fúngica de la Expresión Génica/genética , Genes Reguladores/genética , Procesamiento Proteico-Postraduccional/genética , Transcripción Genética/genética , Fosfatasa Ácida/genética , Aspergillus nidulans/enzimología , Represión Enzimática , FosfatosRESUMEN
When grown on low-Pi medium, the chaA1 pabaA1 palB7 mutant of Aspergillus nidulans excretes an acid phosphatase with steady-state kinetic properties, temperature sensitivity and electrophoretic mobility different from those of the enzyme excreted by the pabaA1 strain. The enzyme excreted by the pabaA1 strain at pH 6.5 showed PNP-P activity with negative cooperativity (K0.5 = 0.87 + or - 0.06 mM, n = 0.68 + or - 0.03) whereas the enzyme excreted by the chaA1 pabaA1 palB7 mutant showed Michaelian kinetics (Km = 0.46 + or - 0.03 mM, n = 1.00 + or - 0.02). The apparent half-lives at 60§C, pH 5.5, of acid phosphatase excreted by the pabaA1 and chaA1 pabaA1 palB7 strains were 58.6 + or - 4.9 min and 21.5 + or - 1.8 min, respectively. Furthermore, the electrophoretic mobility of acid phosphatases excreted by the palA1, palB7, palC4, palE11 and palF15 mutants of A. nidulans was altered and differed from the electrophoretic mobility of the enzyme excreted by the wild-type strain. Also, the palB7 mutation altered the electrophoretic pattern of acid phosphatases synthesized on high-Pi medium. These results are compatible with the post-translational modifications in the Pi-repressible phosphatases rather than with the action of gene palB in controlling the transcription of structural genes of these enzymes
Asunto(s)
Aspergillus nidulans/genética , Fosfatasa Ácida/farmacocinética , Genes Reguladores/genética , Regulación Fúngica de la Expresión Génica/genética , Transcripción Genética/genética , Aspergillus nidulans/enzimología , Cromatografía DEAE-Celulosa , Fosfatasa Ácida/aislamiento & purificaciónRESUMEN
An open reading frame, rbcR, was identified 226 bp upstream of rbcAB, i.e., the ribulose 1,5-bisphosphate carboxylase genes expressed in the phototrophic purple bacterium Chromatium vinosum. Several features reveal that rbcR encodes a member of the LysR family of transcriptional regulators, in which an anomalous content of lysine and arginine residues (Lys/Arg anomaly) was found. The expression of rbcR in Escherichia coli as a protein fused to the N-terminal region of beta-galactosidase led to reduced expression of rbcAB. Thus, rbcR is likely to encode a trans-acting transcriptional regulator of rbcAB expression in C. vinosum.