RESUMEN
Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that likely propagate by the retrotranscription of RNA intermediates. Plasmids bearing DNA segments from the left-hand region of a full-length F element fused to the CAT gene were used as templates for transient expression assays in Drosophila Schneider II cultured cells. Protein and RNA analyses led to the identification of two promoters, Fin and Fout, that transcribe in opposite orientations. The Fin promoter drives the synthesis of transcripts that initiate around residue +6 and are directed toward the element. Fin, that probably controls the formation of F transposition RNA intermediates and gene products, is internal to the transcribed region. Sequences important for accumulation of Fin transcripts are included within the +1 to +30 interval; an additional regulatory element may coincide with a heptamer located downstream of this region also found in the 5' end regions of F-like Drosophila retrotransposons. Analysis of the template activity of 3' deletion derivatives indicates that the level of accumulation of Fin RNA is also dependent upon the presence of sequences located within the +175 to +218 interval. The Fout promoter drives transcription in the opposite orientation with respect to Fin. Fout transcripts initiate at nearby sites within the +92 to +102 interval. Sequences downstream of these multiple RNA start sites are not required for the activity of the Fout promoter. Deletions knocking out the Fin promoter do not impair Fout transcription; conversely, initiation at the Fin promoter still takes place in templates that lack the Fout promoter. At a low level, both promoters are active in cultured cells.
Asunto(s)
Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Northern Blotting , Cloranfenicol O-Acetiltransferasa/genética , Clonación Molecular , Datos de Secuencia Molecular , Mutación/genética , Plásmidos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
We have isolated and characterized a heat-inducible gene, hsp82, from the dimorphic pathogenic fungus Histoplasma capsulatum, which is a filamentous mold at 25 degrees C and a unicellular yeast at 37 degrees C. This gene, which has a high degree of homology with other members of the hsp82 gene family, is split into three exons and two introns of 122 and 86 nucleotides, respectively. Contrary to what has been demonstrated in Drosophila melanogaster, Saccharomyces cerevisiae, and other organisms, hsp82 mRNA in H. capsulatum is properly spliced during the severe heat conditions of 37 to 40 degrees C in the temperature-sensitive Downs strain. Splicing accuracy was also observed at 42 degrees C in the temperature-tolerant G222B strain, which showed no evidence of accumulation of primary transcripts. Furthermore, the intron containing the beta-tubulin gene is also properly spliced at the upper temperature range, suggesting that the lack of a block in splicing may be a general phenomenon in this organism.
Asunto(s)
Genes Fúngicos , Proteínas de Choque Térmico/genética , Histoplasma/genética , Intrones , Empalme del ARN , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN de Hongos/genética , Calor , Datos de Secuencia Molecular , Oligonucleótidos , ARN Mensajero/genética , Transcripción Genética , Tubulina (Proteína)/genéticaRESUMEN
We have cloned a nucleotide sequence from Histoplasma capsulatum G222B corresponding to a heat inducible hsp82 gene, and determined its entire sequence and the flanking regions. During the temperature-controlled mycelium-to-yeast phase transition the gene is more actively transcribed at 37 degrees C in the temperature tolerant and mouse-virulent G222B strain, while 34 degrees C is the optimum for transcription in the temperature sensitive and mouse-avirulent Downs strain.
Asunto(s)
Proteínas Fúngicas/biosíntesis , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Histoplasma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteínas HSP90 de Choque Térmico , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiaeRESUMEN
Drosophila melanogaster F elements are members of the super-family of LINEs, mobile repeated DNA sequences that lack LTRs and propagate by the reverse transcription of unit-length RNA intermediates. The F 5' end region harbours two promoters (F(in) and F(out)) that transcribe in a convergent manner. Each promoter has been functionally dissected by assaying in D. melanogaster cultured cells templates carrying base substitutions and/or deletions across the +1 to +245 region of the element F12. F(in), that likely controls the synthesis of gene products and transposition intermediates, is internal to the transcribed region. Two elements play a major role in F-sense transcription. The proximal element spans the interval +6 to +14 and includes a major RNA start site. Heterologous DNA featuring a nearly identical purine-pyrimidine sequence can functionally replace the initiator-like module only when properly spaced from downstream F sequences. The distal element is within the interval +18 to +46 and may correspond to a motif (AGACGTTT, +34 to +41) conserved in other Drosophila LINEs. F(out) is a TATA-less promoter that directs transcription predominantly from three nearby sites (a to c). F(out) expression is influenced by multiple elements located upstream of residue -68 relative to site a as +1 within a region (alpha) shown to stimulate the D. melanogaster hsp70 promoter in an orientation and position-independent fashion. Changes within the -43 to +24 interval may suppress or stimulate transcription from sites a and c. Initiation from a site approximately 30 nucleotides upstream of site a is enhanced by alterations of the interval -43 to -5. The expression of the two F promoters, determined by the interaction of the transcriptional machinery with distinct DNA sequences, is influenced by a common element within the alpha region.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular , ADN Recombinante , Proteínas de Choque Térmico/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , ARN sin Sentido , Moldes GenéticosRESUMEN
The basal promoters of three Drosophila long interspersed nuclear elements (LINEs), the I factor and the F and Doc elements, have the same architecture. In each, transcription is directed by an initiator which is faithfully and efficiently recognized only when flanked 3' by a DNA segment approximately 20 bp in length called the B region. The B regions of the three promoters are interchangeable and have a complex structure, comprising three functionally distinct elements: de1, de2 and de3. While de2 is relatively conserved, fitting the consensus RGACGTGY, de1 and de3 vary among the three promoters. At different levels, each downstream element is able to ensure accurate recognition of the initiator. The de2 domain stimulates transcription of the F, I and Doc promoters to the same extent. In contrast, the I de1 domain stimulates transcription much more efficiently than the corresponding domains of the F and Doc elements. The finding that de2 is selectively required in order to detect full activity of enhancer sequences found in the F element suggests that de1 and de2 interact with different proteins. The B regions can be replaced by and synergize with a TATA element, can functionally substitute for downstream promoter sequences in the Drosophila hsp70 gene, and significantly activate the mouse terminal deoxynucleotidyl transferase initiator. Our data suggest that the B regions stimulate transcription by providing sites of interaction for the TFIID complex. Sequences homologous to the del to de3 array are found downstream from the transcription start site(s) both in TATA-less and TATA-containing promoters.
Asunto(s)
Drosophila melanogaster/genética , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Células Cultivadas , Elementos Transponibles de ADN , Ratones , TATA BoxRESUMEN
cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell-cell interactions that play a critical role in the early patterning of the embryo.
Asunto(s)
Factor de Crecimiento Epidérmico , Glicosilfosfatidilinositoles/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Sitios de Unión , Línea Celular Transformada , Membrana Celular/metabolismo , Desarrollo Embrionario y Fetal , Proteínas Ligadas a GPI , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Proteínas de Neoplasias/genética , Fosfatidilinositol Diacilglicerol-Liasa , Conejos , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/metabolismoRESUMEN
Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. Here, we demonstrate that embryonic stem cell (ESC) behaviour relies on a feedback loop that involves the non-essential amino acid L-Proline (L-Pro) in the modulation of the Gcn2-Eif2α-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop generates a highly specific intrinsic shortage of L-Pro that restricts proliferation of tightly packed domed-like ESC colonies and safeguards ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase by halofuginone or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to maintain ESC identity.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Células Madre Embrionarias/metabolismo , Prolina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Retroalimentación Fisiológica , Ratones , Transducción de Señal , Estrés FisiológicoRESUMEN
Stemness was recently depicted as a dynamic condition in normal and tumor cells. We found that the embryonic protein Cripto-1 (CR1) was expressed by normal stem cells at the bottom of colonic crypts and by cancer stem cells (CSCs) in colorectal tumor tissues. CR1-positive populations isolated from patient-derived tumor spheroids exhibited increased clonogenic capacity and expression of stem-cell-related genes. CR1 expression in tumor spheroids was variable over time, being subject to a complex regulation of the intracellular, surface and secreted protein, which was related to changes of the clonogenic capacity at the population level. CR1 silencing induced CSC growth arrest in vitro with a concomitant decrease of Src/Akt signaling, while in vivo it inhibited the growth of CSC-derived tumor xenografts and reduced CSC numbers. Importantly, CR1 silencing in established xenografts through an inducible expression system decreased CSC growth in both primary and metastatic tumors, indicating an essential role of CR1 in the regulation the CSC compartment. These results point to CR1 as a novel and dynamically regulated effector of stem cell functions in colorectal cancer.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Proteínas Ligadas a GPI/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Neoplasias Colorrectales/fisiopatología , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/fisiología , Regulación Neoplásica de la Expresión Génica , Genes src , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/fisiología , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
In the Drosophila I, F and Doc LINEs, basal transcription is ensured by the functional interaction of initiator sequences with intragenic regulatory segments (B regions) which comprise distinct functional modules. Removing the B regions, as changing their composition or location, allowed different activators to stimulate transcription from novel initiators both in Doc and F promoters. The use of distinct initiators plausibly reflects the assembly of transcriptional complexes in which TFIID assumes alternative spatial conformations. The response of I, F and Doc promoters to the same enhancer is significantly influenced by the number, position and type of core elements present.
Asunto(s)
Drosophila melanogaster/genética , Regiones Promotoras Genéticas , Activación Transcripcional , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genes Reporteros , Datos de Secuencia Molecular , Mutación/genética , Análisis de Secuencia de ADN , Factor de Transcripción TFIID , Factores de Transcripción TFII/metabolismo , TransfecciónAsunto(s)
Factor de Crecimiento Epidérmico , Glicoproteínas de Membrana , Proteínas de Neoplasias/genética , Neoplasias/patología , Secuencia de Aminoácidos , Animales , Mama/metabolismo , Neoplasias de la Mama/química , ADN Complementario/química , Desarrollo Embrionario y Fetal , Femenino , Proteínas Ligadas a GPI , Neoplasias Gastrointestinales/química , Humanos , Péptidos y Proteínas de Señalización Intercelular , Lactancia , Masculino , Datos de Secuencia Molecular , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Neoplasias/química , Seudogenes , Proteínas Recombinantes/química , Alineación de Secuencia , Bazo/metabolismo , Testículo/metabolismoRESUMEN
Mobile elements known as LINEs are members of a superfamily of repeated DNA conserved from protozoa to man. These sequences propagate by the retrotranscription of RNA intermediates and differ in many respects from retroviruses. Whereas most eukaryotic genomes host a single LINE family, several families of LINE-like sequences or type II retrotransposons coexist in the fruit fly Drosophila melanogaster. Properties and features of these elements are discussed in this work.
Asunto(s)
Drosophila melanogaster/genética , Retroelementos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genéticaRESUMEN
Long interspersed nuclear elements (LINEs) are mobile DNA elements which propagate by reverse transcription of RNA intermediates. LINEs lack long terminal repeats, and their expression is controlled by promoters located inside to the transcribed region of unit-length DNA copies. Doc elements constitute one of the seven families of LINEs found in Drosophila melanogaster. Plasmids in which the chloramphenicol acetyltransferase (CAT) gene is preceded by DNA segments from different Doc family members were used as templates for transient expression assays in Drosophila S2 cells. Transcription is initiated at the 5' end of Doc elements within hexamers fitting the consensus (C/G)AYTCG and is regulated by a DNA region which is located approximately 20 base pairs (bp) downstream from the RNA start site(s). The region includes a sequence (RGACGTGY motif, or DE2) which stimulates transcription in other Drosophila LINEs, and two adjacent elements, DE1 and DE3. Moving the downstream region either 4 bp away from, or 5 bp closer to the RNA start site region inhibited transcription. Sequences located approximately 200 bp downstream from the Doc 5' end repressed CAT expression in an orientation- and position-dependent manner. The inhibition reflects impaired translation of the CAT gene possibly consequent to the interaction of specific Doc RNA sequences with a cellular component.
Asunto(s)
Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa/biosíntesis , Clonación Molecular , Secuencia de Consenso , Drosophila melanogaster/genética , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , TransfecciónRESUMEN
ERV9 is a low repeated family of human endogenous retroviral elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. In this report we have analyzed the minimal promoter region located within the ERV9 LTR. Using the transient CAT expression assay we have identified the minimal promoter region, which includes sequences spanning from -70 to +6 relative to the major transcription start site. Deletion analysis, primer extension mapping of the transcription start sites and DNA-protein interactions assays have allowed us to define two important regions within the ERV9 minimal promoter. One region located between -70 to -39 acts as a transcriptional activating sequence and contains an Sp 1 binding site. The second region from -7 to +6, which resembles an initiator element (Inr), was necessary for the correct transcription start site utilization, and binds to a regulatory protein. Cross-competition experiments using various Inr elements have indicated that the protein that binds to the ERV9 Inr element can be competed by the HIV-1 and TdT Inr sequences.
Asunto(s)
Regulación Viral de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Provirus/genética , Retroviridae/genética , Transcripción Genética/genética , Secuencia de Bases , Sitios de Unión/genética , Células Madre de Carcinoma Embrionario , Humanos , Datos de Secuencia Molecular , Células Madre Neoplásicas , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
Cripto is the founding member of the family of EGF-CFC genes, a class of extracellular factors essential for early vertebrate development. In this study we show that injection of Cripto recombinant protein in mid to late zebrafish Maternal-Zygotic one-eyed pinhead (MZoep) blastulae was able to fully rescue the mutant phenotype, thus providing the first direct evidence that Cripto activity can be added extracellularly to recover oep-encoded function in zebrafish early embryos. Moreover, 15 point mutations and two deletion mutants were generated to assess in vivo their functional relevance by comparing the ability of cripto wild-type and mutant RNAs to rescue the zebrafish MZoep mutant. From this study we concluded that the EGF-CFC domain is sufficient for Cripto biological activity and identified ten point mutations with a functional defective phenotype, two of which, located in the EGF-like domain, correspond to loss-of-function mutations. Finally, we have developed a three-dimensional structural model of Cripto protein and used it as a guide to predict amino acid residues potentially implicated in protein-protein interaction.
Asunto(s)
Inducción Embrionaria , Proteínas de Neoplasias/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Simulación por Computador , Cisteína , Factor de Crecimiento Epidérmico , Prueba de Complementación Genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/administración & dosificación , Proteínas de Neoplasias/genética , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/administración & dosificación , Proteínas de Pez Cebra/genéticaRESUMEN
Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that probably propagate by the retrotranscription of RNA intermediates. Polyadenylated transcripts corresponding in size to full-length (4.7 kb) family members were detected in the Drosophila melanogaster Canton-S strain from 2nd larval instar to the adult stage. RNA accumulation reached a maximum in pupae. In the adult, F elements are transcribed in both sexes. F expression is directed in vivo by the intragenic promoter (Fin) located at the 5' end of F. Whole-mount hybridizations were carried out to define the site of synthesis of full-length transcripts found in the ovary. Selective RNA accumulation was not detected in the cytoplasm of any specific cell type. Stained nuclear dots were observed in nurse cells from stage 2-3 to the end of oogenesis. RNase treatment of egg chambers prior to the addition of the probe led to disappearance of the nuclear dots and appearance of a cytoplasmic hybridization signal suggesting leakage of nuclear transcripts. Transgenic lines harbouring the chloramphenicol acetyltransferase (CAT) gene under the control of the Fin promoter were obtained. In independent lines, CAT enzyme levels mirror the ontogenetic profile of F expression drawn from Northern RNA blotting data. An antisense promoter (Fout) that is located downstream from the Fin promoter and transcribe too bords the 5' end of F seems to be constitutively expressed in the fly.
Asunto(s)
Drosophila melanogaster/genética , Regulación de la Expresión Génica , Retroelementos , Animales , Secuencia de Bases , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero , Femenino , Larva , Masculino , Datos de Secuencia Molecular , Ovario/química , Pupa , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Retroelementos/genéticaRESUMEN
Nodal proteins have crucial roles in mesendoderm formation and left-right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling.