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1.
Tissue Cell ; 80: 101990, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36542947

RESUMEN

Colorectal cancer is the second leading cause of cancer and often has a fatal course. There are many studies in the literature that have described a close functional relationship between the tumor mass and surrounding tissue, or tumor stroma, which is affected by the continuous metabolic exchange that occurs at the interface between tumor and tissues in contact with it. There is much evidence that the presence of adipose tissue in stroma plays a fundamental role in modulating the tumor microenvironment and promote tumor development, growth, and angiogenesis due to its endocrine characteristics. In this analysis, we have studied the alterations of adipose tissue surrounding colorectal tumors with MRI and optical imaging in vivo techniques to monitor tumor progression and also performed histological and molecular analysis. We detected differences in the principal adipose markers expressed by adipocytes residing around the rectal colon and observed that peritumoral adipose tissue is exposed to a mesenchymal transition process that leads to the acquisition of a less differentiated phenotype of adipocyte that represents the main cellular type present in tumor stroma. The mesenchymal transition correlated with the acquisition of more aggressive tumor phenotype and could represent a valid target for tumor therapy.


Asunto(s)
Carcinoma , Neoplasias del Colon , Humanos , Tejido Adiposo/metabolismo , Adipocitos/metabolismo , Neoplasias del Colon/patología , Carcinoma/metabolismo , Carcinoma/patología , Microambiente Tumoral
2.
Int J Biochem Cell Biol ; 95: 17-26, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29242050

RESUMEN

K650M/E substitutions in the Fibroblast growth factor receptor 3 (FGFR3) are associated with Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and Thanatophoric Dysplasia type II (TDII), respectively. Both SADDAN and TDII present with affected endochondral ossification marked by impaired chondrocyte functions and growth plate disorganization. In vitro, K650M/E substitutions confer FGFR3 constitutive kinase activity leading to impaired biosynthesis and accumulation of immature receptors in endoplasmic reticulum (ER)/Golgi. From those compartments, both SADDAN-FGFR3 and TDII-FGFR3 receptors engender uncontrolled signalling, activating PLCγ1, signal transducer and activator of transcription 1, 3 and 5 (STAT1/3/5) and ERK1/2 effectors. Here, we investigated the impact of SADDAN-FGFR3 and TDII-FGFR3 signalling on cytoskeletal organization. We report that SADDAN-FGFR3, but not TDII-FGFR3, affects F-actin organization by inducing tyrosine hyperphosphorylation of paxillin, a key regulator of focal adhesions and actin dynamics. Paxillin phosphorylation was upregulated at tyrosine 118, a functional target of Src and FAK kinases. By using Src-deficient cells and a Src kinase inhibitor, we established a role played by Src activation in paxillin hyperphosphorylation. Moreover, we found that SADDAN-FGFR3 induced FAK phosphorylation at tyrosines 576/577, suggesting its involvement as a Src co-activator in paxillin phosphorylation. Interestingly, paxillin hyperphosphorylation by SADDAN-FGFR3 caused paxillin mislocalization and partial co-localization with the mutant receptor. Finally, the SADDAN-FGFR3 double mutant unable to bind PLCγ1 failed to promote paxillin hyperphosphorylation, pointing to PLCγ1 as an early player in mediating paxillin alterations. Overall, our findings contribute to elucidate the molecular mechanism leading to cell dysfunctions caused by SADDAN-FGFR3 signalling.


Asunto(s)
Acondroplasia/metabolismo , Condrocitos/metabolismo , Citoesqueleto/metabolismo , Mutación , Paxillin/metabolismo , Fosfolipasa C gamma/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Acondroplasia/genética , Acondroplasia/patología , Sustitución de Aminoácidos , Animales , Línea Celular Transformada , Línea Celular Tumoral , Condrocitos/efectos de los fármacos , Condrocitos/patología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/patología , Humanos , Ratones , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Displasia Tanatofórica/genética , Displasia Tanatofórica/metabolismo , Displasia Tanatofórica/patología , Tirosina/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo
3.
J Biol Chem ; 276(8): 6009-15, 2001 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-11087738

RESUMEN

Gerstmann-Sträussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Sträussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.


Asunto(s)
Amiloide/genética , Enfermedad de Gerstmann-Straussler-Scheinker/etiología , Fragmentos de Péptidos/aislamiento & purificación , Priones/patogenicidad , Precursores de Proteínas/genética , Adulto , Alelos , Corteza Cerebral/patología , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Heterocigoto , Humanos , Masculino , Metionina/genética , Proteínas Priónicas , Priones/aislamiento & purificación , Análisis de Secuencia de Proteína , Síndrome , Valina/genética
5.
Am J Pathol ; 153(5): 1561-72, 1998 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9811348

RESUMEN

We have investigated the proteolytic cleavage of the cellular (PrPC) and pathological (PrPSc) isoforms of the human prion protein (PrP) in normal and prion-affected brains and in tonsils and platelets from neurologically intact individuals. The various PrP species were resolved after deglycosylation according to their electrophoretic mobility, immunoreactivity, Sarkosyl solubility, and, as a novel approach, resistance to endogenous proteases. First, our data show that PrPC proteolysis in brain originates amino-truncated peptides of 21 to 22 and 18 (C1) kd that are similar in different regions and are not modified by the PrP codon 129 genotype, a polymorphism that affects the expression of prion disorders. Second, this proteolytic cleavage of PrPC in brain is blocked by inhibitors of metalloproteases. Third, differences in PrPC proteolysis, and probably in Asn glycosylation and glycosylphosphatidylinositol anchor composition, exist between neural and non-neural tissues. Fourth, protease-resistant PrPSc cores in sporadic Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker F198S disease brains all have an intact C1 cleavage site (Met111-His112), which precludes disruption of a domain associated with toxicity and fibrillogenesis. Fifth, the profile of endogenous proteolytic PrPSc peptides is characteristic of each disorder studied, thus permitting the molecular classification of these prion diseases without the use of proteinase K and even a recognition of PrPSc heterogeneity within type 2 CJD patients having different codon 129 genotype and neuropathological phenotype. This does not exclude the role of additional factors in phenotypic expression; in particular, differences in glycosylation that may be especially relevant in the new variant CJD. Proteolytic processing of PrP may play an important role in the neurotropism and phenotypic expression of prion diseases, but it does not appear to participate in disease susceptibility.


Asunto(s)
Enfermedad de Alzheimer/patología , Síndrome de Creutzfeldt-Jakob/patología , Enfermedad de Gerstmann-Straussler-Scheinker/patología , Neuronas/química , Proteínas PrPC/análisis , Proteínas PrPSc/análisis , Anciano , Enfermedad de Alzheimer/genética , Plaquetas/química , Encéfalo/patología , Química Encefálica , Codón , Síndrome de Creutzfeldt-Jakob/genética , Genotipo , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Glicosilación , Humanos , Metaloendopeptidasas/metabolismo , Persona de Mediana Edad , Tonsila Palatina/química , Fragmentos de Péptidos/análisis , Mapeo Peptídico
6.
J Neuropathol Exp Neurol ; 57(10): 979-88, 1998 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9786248

RESUMEN

Gerstmann-Sträussler-Scheinker disease (GSS), a cerebello-pyramidal syndrome associated with dementia and caused by mutations in the prion protein gene (PRNP), is phenotypically heterogeneous. The molecular mechanisms responsible for such heterogeneity are unknown. Since we hypothesize that prion protein (PrP) heterogeneity may be associated with clinico-pathologic heterogeneity, the aim of this study was to analyze PrP in several GSS variants. Among the pathologic phenotypes of GSS, we recognize those without and with marked spongiform degeneration. In the latter (i.e. a subset of GSS P102L patients) we observed 3 major proteinase-K resistant PrP (PrPres) isoforms of ca. 21-30 kDa, similar to those seen in Creutzfeldt-Jakob disease. In contrast, the 21-30 kDa isoforms were not prominent in GSS variants without spongiform changes, including GSS A117V, GSS D202N, GSS Q212P, GSS Q217R, and 2 cases of GSS P102L. This suggests that spongiform changes in GSS are related to the presence of high levels of these distinct 21-30 kDa isoforms. Variable amounts of smaller, distinct PrPres isoforms of ca. 7-15 kDa were seen in all GSS variants. This suggests that GSS is characterized by the presence PrP isoforms that can be partially cleaved to low molecular weight PrPres peptides.


Asunto(s)
Enfermedad de Gerstmann-Straussler-Scheinker/genética , Enfermedad de Gerstmann-Straussler-Scheinker/metabolismo , Priones/genética , Priones/metabolismo , Adulto , Anciano , Western Blotting , Encéfalo/patología , Química Encefálica , ADN/análisis , ADN/genética , Femenino , Enfermedad de Gerstmann-Straussler-Scheinker/patología , Humanos , Masculino , Persona de Mediana Edad , Peso Molecular , Fenotipo
7.
J Biol Chem ; 273(33): 20779-84, 1998 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-9694822

RESUMEN

The STAT5 activation has important roles in cell differentiation, cell cycle control, and development. However, the potential implications of STAT5 in the control of apoptosis remain unexplored. To evaluate any possible link between the erythropoietin receptor (EpoR) JAK2/STAT5 transduction pathway and apoptosis, we have investigated apoptosis-resistant cells (ApoR) that arose from positive selection of the erythroid-committed Ba/F3EpoR cells triggered to apoptosis by ectopic expression of the HOX-B8 homeotic gene. We show that JAK2 is normally activated by Epo in both Ba/F3EpoR and ApoR cells. In contrast, both STAT5a and STAT5b isoforms are uniquely activated in a C-truncated form (86 kDa) only in ApoR cells. Analysis of ApoR and Ba/F3EpoR subclones confirmed that the switch to the truncated STAT5 isoform coincides with apoptosis survival and that ApoR do not derive from preexisting cells with a shortened STAT5. In addition, ApoR cells die in the absence of Epo. This indicates that resistance to apoptosis is not because of a general defect in the apoptotic pathway of ApoR cells. Furthermore, we show that the 86-kDa STAT5 protein presents a dominant-negative (DN) character. We hypothesize that the switch to a DN STAT5 may be part of a mechanism that allows ApoR cells to be selectively advantaged during apoptosis. In conclusion, we provide evidence for a functional correlation between a naturally occurring DN STAT5 and a biological response.


Asunto(s)
Apoptosis , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/genética , Proteínas de la Leche , Transactivadores/metabolismo , Animales , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/genética , Eritropoyetina/farmacología , Genes Dominantes , Ratones , Fosforilación , Factor de Transcripción STAT5 , Transducción de Señal , Células Madre/metabolismo , Transactivadores/genética
8.
Gene ; 197(1-2): 73-81, 1997 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-9332351

RESUMEN

Human CDP/cut and its murine counterpart, cux1/CDP are homeodomain repressor proteins in the family of Drosophila Cut. Northern blot analysis reveals complex alternative splicing, including forms too small to encode the full 1505 amino acid protein. We have characterized a CDP/cut alternatively spliced cDNA (CASP) of 3.4 kb. Human CASP, a predicted 678 amino acid polypeptide, shares 400 amino acids with CDP, but has an alternate N terminal exon of 20 aa, and the C-terminal 258 amino acids diverge from CDP/cut entirely. As the unique C-terminus of CASP lacks the three 'cut-repeats' and homeodomain of CDP/cut, we predict it does not bind DNA. Murine CASP, 96% similar to human, shares these features. Database searches identify homologs in chicken (86% identical to human CASP) and yeast (29% identical to human). Murine CASP mRNA is ubiquitous in mouse tissues and in tissue-culture cell lines. We generated a specific antiserum against the unique C-terminus of CASP, and used this reagent to demonstrate that CASP protein is expressed as an approx. 80 kDa protein in human and murine cells. Co-translation of in vitro-translated CDP and CASP mRNA, followed by immunoprecipitation with specific anti-CASP IgG, shows that CASP polypeptide can from a complex with CDP. Studies of the intron/exon structure of the murine cux/CDP/mCASP locus (>> 100 kb) reveal that the unique 3' exons of CASP are interposed between cut-repeats 2 and 3 of the cux gene. We speculate that a primordial CASP-like gene captured a cut-repeat-homeobox gene to give rise to the eukaryotic Cut/CDP family of proteins.


Asunto(s)
Empalme Alternativo/fisiología , ADN Complementario/genética , Proteínas de Homeodominio/genética , Proteínas Nucleares/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Extractos Celulares , Línea Celular , Núcleo Celular , Clonación Molecular , Exones/genética , Genes Homeobox/genética , Humanos , Intrones/genética , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción
9.
Science ; 276(5315): 1119-22, 1997 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-9148807

RESUMEN

Prion diseases are transmissible neurodegenerative conditions characterized by the accumulation of protease-resistant forms of the prion protein (PrP), termed PrPres, in the brain. Insoluble PrPres tends to aggregate into amyloid fibrils. The anthracycline 4'-iodo-4'-deoxy-doxorubicin (IDX) binds to amyloid fibrils and induces amyloid resorption in patients with systemic amyloidosis. To test IDX in an experimental model of prion disease, Syrian hamsters were inoculated intracerebrally either with scrapie-infected brain homogenate or with infected homogenate coincubated with IDX. In IDX-treated hamsters, clinical signs of disease were delayed and survival time was prolonged. Neuropathological examination showed a parallel delay in the appearance of brain changes and in the accumulation of PrPres and PrP amyloid.


Asunto(s)
Doxorrubicina/análogos & derivados , Priones/metabolismo , Scrapie/tratamiento farmacológico , Amiloide/metabolismo , Animales , Conducta Animal , Encéfalo/metabolismo , Encéfalo/patología , Síndrome de Creutzfeldt-Jakob/metabolismo , Cricetinae , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Femenino , Humanos , Mesocricetus , ARN Mensajero/genética , ARN Mensajero/metabolismo , Scrapie/metabolismo , Scrapie/patología , Tubulina (Proteína)/análisis
10.
Biochem J ; 320 ( Pt 2): 563-70, 1996 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-8973567

RESUMEN

Prion-related encephalopathies are characterized by the intracerebral accumulation of an abnormal isoform of the cellular prion protein (PrPC) named scrapie prion protein (PrPSc). The pathological forms of this protein and its cellular precursor are not only expressed in the brain but also, at lower concentrations, in peripheral tissues. We recently showed that a synthetic peptide corresponding to residues 106-126 [PrP-(106-126)] of the human PrP is toxic to neurons and trophic to astrocytes in vitro. Our experiments were aimed at verifying whether PrP-(106-126) and other peptides corresponding to fragments of the amyloid protein purified from brains of patients with Gerstmann-Sträussler-Scheinker disease-namely PrP-(89-106), PrP-(106-114), PrP-(127-147)-were capable of stimulating circulating leucocytes. Native PrP expression in human lymphocytes, monocytes and neutrophils was first confirmed using PCR amplification of total RNA, after reverse transcription, and immunoblot analysis of cell extracts with anti-PrP antibodies. PrP-(106-126), but not the other peptides, increased membrane microviscosity, intracellular Ca2+ concentration and cell migration in circulating leucocytes, and O2-. production in monocytes and neutrophils. Membrane microviscosity was determined by the fluorescence polarization technique, using diphenylhexatriene as a probe, 300 s after the addition of PrP-(106-126) to the cell suspension in the concentration range 5-50 microM. The increase in intracellular Ca2+ elicited by PrP-(106-126) was dose-dependent in the range 5-500 microM. PrP-(106-126) stimulated O2-. production in monocytes and neutrophils in a dose- (10-300 microM) and time-(5-30 min) dependent manner in the presence of 10 microM dihydrocytochalasin B. Both the increase in Ca2+ concentration and the O2-. production were partially sensitive to pertussis toxin. PrP-(106-126) stimulated leucocyte migration in a dose-dependent (30-300 microM) manner and, at the highest concentration used, this migration was comparable with that elicited by 2.5 nM interleukin 8 or 10 nM fMet-Leu-Phe peptide.


Asunto(s)
Actinas/biosíntesis , Quimiotaxis de Leucocito/efectos de los fármacos , Leucocitos/fisiología , Fragmentos de Péptidos/farmacología , Priones/biosíntesis , Priones/farmacología , Transcripción Genética/efectos de los fármacos , Secuencia de Aminoácidos , Calcio/sangre , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Cartilla de ADN , Humanos , Cinética , Leucocitos/efectos de los fármacos , Linfocitos/fisiología , Datos de Secuencia Molecular , Monocitos/fisiología , Neutrófilos/fisiología , Reacción en Cadena de la Polimerasa , Priones/sangre , Superóxidos/sangre
11.
J Biol Chem ; 270(21): 12745-50, 1995 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-7759529

RESUMEN

CCAAT displacement protein (CDP)/cut is implicated in several systems as a transcriptional repressor of developmentally regulated genes. In myeloid leukemia cells, CDP/cut binding activity as assayed on the promoter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves as a repressor of gp91-phox gene expression. First, we used transient transfection assays in 3T3 cells to demonstrate that the CDP/cut binding site from the gp91-phox promoter acts as a negative regulatory element in artificial promoter constructs. Second, we isolated a stable transformant of HL-60 myeloid cells constitutively expressing transfected CDP/cut cDNA. Stable transformants carrying expression vector alone or expressing CDP/cut mRNA were induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retinoic acid/dimethylformamide. Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosolic components, p47 and p67. In the stable transformant expressing transfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whereas morphologic differentiation and induction of mRNA for myeloid oxidase components p47 and p67 were unaffected. These data provide persuasive evidence that CDP/cut acts to repress the gp91-phox gene.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide/genética , NADPH Oxidasas , Proteínas Nucleares/genética , Proteínas Represoras/genética , Células 3T3 , Animales , Sitios de Unión , Diferenciación Celular , ADN Recombinante , Dimetilsulfóxido/farmacología , Dimetilformamida/farmacología , Globinas/genética , Proteínas de Homeodominio , Humanos , Macrófagos/fisiología , Glicoproteínas de Membrana/genética , Ratones , NADPH Oxidasa 2 , Neutrófilos/fisiología , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción , Transformación Genética , Tretinoina/farmacología , Células Tumorales Cultivadas
13.
Biochim Biophys Acta ; 1171(2): 147-52, 1992 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-1339297

RESUMEN

In the 5'-flanking region of the bovine pancreatic ribonuclease gene a sequence has been identified which specifically binds one or more factors present in nuclear protein extracts prepared from bovine pancreas. The binding site, as delineated by footprinting analysis, is located in a region extending from positions -113 to -146 relative to the transcription initiation site of the ribonuclease gene. This region contains consensus sequences for known control transcriptional elements. The observed pattern of protein-DNA interactions is likely to be pancreas-specific as it could not be detected with nuclear extracts prepared from HeLa or bovine aorta endothelium cells.


Asunto(s)
ADN/metabolismo , Genes Reguladores , Proteínas Nucleares/metabolismo , Ribonucleasa Pancreática/genética , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Núcleo Celular/fisiología , ADN/genética , ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I , Factor C1 de la Célula Huésped , Datos de Secuencia Molecular , Factor 1 de Transcripción de Unión a Octámeros , Oligodesoxirribonucleótidos , Páncreas/enzimología , Plásmidos , Mapeo Restrictivo , Factores de Transcripción/metabolismo
14.
Nat Genet ; 1(1): 50-5, 1992 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-1301999

RESUMEN

Human CCAAT displacement protein (CDP), a putative repressor of developmentally regulated gene expression, was purified from HeLa cells by DNA binding-site affinity chromatography. cDNA encoding CDP was obtained by immunoscreening a lambda gt11 library with antibody raised against purified protein. The deduced primary amino acid sequence of CDP reveals remarkable homology to Drosophila cut with respect to the presence of a unique homeodomain and "cut repeats". As cut participates in determination of cell fate in several tissues in Drosophila, the similarity predicts a broad role for CDP in mammalian development.


Asunto(s)
Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Drosophila/genética , Proteínas de Drosophila , Expresión Génica , Células HeLa , Proteínas de Homeodominio , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factores de Transcripción
15.
Res Microbiol ; 141(6): 659-70, 1990.
Artículo en Inglés | MEDLINE | ID: mdl-2284501

RESUMEN

A simple procedure for genetic transformation of Streptococcus sanguis Challis was developed and standardized. During the exponential phase of growth, cells became competent while growing as diplococci in broth containing 10% foetal calf serum. High levels of competence were maintained by the cultures for 60 min. Competent cells could be stored frozen without loss of competence for at least three years. Using total chromosomal DNA as donor, the dose-response curve for transformation of a point mutation (streptomycin resistance) showed one-hit kinetics, as the DNA concentration varied from 0.000001 to 10 micrograms/ml. At 10 micrograms/ml, more than 2.2% of the colony-forming units were transformed to streptomycin resistance, while transforming activity remained detectable with 1 pg of DNA/ml. Optimal time of exposure of competent cells to transforming DNA was 30 min. The transformation reaction was inhibited at 0 and 4 degrees C, whereas it occurred efficiently both at 25 and 37 degrees C.


Asunto(s)
Streptococcus sanguis/genética , Transformación Genética/fisiología , ADN Bacteriano/genética , Técnicas In Vitro , Cinética , Streptococcus sanguis/crecimiento & desarrollo , Temperatura , Factores de Tiempo
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