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1.
Circ Econ Sustain ; : 1-34, 2022 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-36093032

RESUMEN

The apparel value chain is essential for the livelihood of millions of workers around the globe. However, human rights violations and the lack of a sustained income by apparel workers demonstrate the poor working conditions present in this sector. Circular economy (CE) has been used by incumbent businesses and startups as a framework to achieve sustainability, thus contributing to its economic, environmental and social dimensions. However, there is a lack of knowledge on its social impact. Most of the literature assesses CE's social impacts by focusing only on the number of jobs created. However, the majority of studies agree on the need to analyse further the quality and inclusivity aspects. This paper explores the social impact of the different circular strategies implemented in three countries. It assesses social impacts related to the quality of jobs, workers' sustainable livelihood and gender equality and inclusion. Results corroborate that CE social ambition is low, and that current circular strategies follow the same feminisation and precariousness of working conditions found in the linear apparel value chain. Thus, policymakers and businesses alike need to strengthen their CE social ambition; coordinate policy and strategies with different countries stakeholders of the apparel value chain to minimise trade-offs; and safeguard a just circular transition. This research contributes to the body of literature on CE by introducing a social impact assessment framework for circularity called SIAF-CE⚥. Additionally, it provides evidence on the current CE social impact implemented by startups and incumbents in regional and global contexts. Supplementary Information: The online version contains supplementary material available at 10.1007/s43615-022-00203-8.

2.
J Water Health ; 12(4): 846-57, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25473994

RESUMEN

We investigated the effectiveness of Escherichia coli community fingerprinting for identifying fecal pollution sources impacting a recreational beach. E. coli in water collected from the beach, nearby creek and storm sewer outfall were enumerated using membrane filtration, while E. coli communities were characterized following polymerase chain reaction analysis and denaturing gradient gel electrophoresis (DGGE) fingerprinting. Analysis of E. coli densities to determine the contributions of the creek and storm sewer during dry weather was inconclusive. However, DGGE fingerprinting indicated that the creek E. coli communities had a greater impact on the beach community composition (80-95% similarity), than on storm sewer communities (41-64%). Following rainfall events, E. coli communities in the creek were at least 93% similar to those at the beach, while the similarity of the outfall and beach communities varied from 72 to 96%. Furthermore, E. coli communities at the beach were more similar to creek communities than to storm sewer communities after the first 2 h and 48 h following the onset of rainfall, and of comparable similarity following 24 h of rainfall, suggesting transient contributions from the storm sewer. DGGE analysis of E. coli communities provided evidence that the creek was a consistent source of E. coli to the beach, while the storm sewer was a transient source.


Asunto(s)
Bacterias/aislamiento & purificación , Playas , Heces/microbiología , Lagos/microbiología , Bacterias/genética , Electroforesis en Gel de Gradiente Desnaturalizante , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Ohio
3.
Artículo en Inglés | MEDLINE | ID: mdl-21467139

RESUMEN

Whereas the regulation of a gene is uniquely tailored to respond to specific biological needs, general transcriptional mechanisms are used by diversely regulated genes within and across species. The primary mode of regulation is achieved by modulating specific steps in the transcription cycle of RNA polymerase II (Pol II). Pol II "pausing" has recently been identified as a prevalent rate-limiting and regulated step in the transcription cycle. Many sequence-specific transcription factors (TFs) modulate the duration of the pause by directly or indirectly recruiting positive transcription elongation factor b (P-TEFb) kinase, which promotes escape of Pol II from the pause into productive elongation. These specialized TFs find their target-binding sites by discriminating between DNA sequence elements based on the chromatin context in which these elements reside and can result in productive changes in gene expression or nonfunctional "promiscuous" binding. The binding of a TF can precipitate drastic changes in chromatin architecture that can be both dependent and independent of active Pol II transcription. Here, we highlight heat-shock-mediated gene transcription as a model system in which to study common mechanistic features of gene regulation.


Asunto(s)
Drosophila melanogaster/genética , Regulación de la Expresión Génica , Respuesta al Choque Térmico/genética , Modelos Animales , Transcripción Genética , Animales , Secuencia de Bases , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Datos de Secuencia Molecular
4.
Vaccine ; 25(50): 8384-94, 2007 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-17996992

RESUMEN

The effector mechanisms used by CD4+ T cells to control mycobacteria differ between humans and rodent models of TB and should be investigated in additional animal models. In these studies, the bovine model was used to characterize the mycobactericidal CD4+ T cell response induced by vaccination with the attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG). Antigenic stimulation of peripheral blood CD4+ T cells from BCG-vaccinated cattle enhanced expression of perforin and IFNgamma in cells expressing a CD45RA-CD45RO+CD62L+ cell surface phenotype, enhanced transcription of granulysin, IFNgamma, perforin, IL-4, IL-13, and IL-21, and enhanced anti-mycobacterial activity of CD4+ T cells against BCG-infected macrophages.


Asunto(s)
Vacuna BCG/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Memoria Inmunológica , Mycobacterium bovis/inmunología , Tuberculosis Bovina/prevención & control , Animales , Antiinfecciosos/metabolismo , Vacuna BCG/administración & dosificación , Linfocitos T CD4-Positivos/microbiología , Bovinos , Citocinas/metabolismo , Humanos , Interferón gamma/metabolismo , Macrófagos/microbiología , Perforina/metabolismo , Tuberculosis/inmunología , Tuberculosis/prevención & control , Tuberculosis Bovina/inmunología , Vacunación
6.
Mol Cell ; 8(1): 9-19, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11511356

RESUMEN

Activators of RNA polymerase II (Pol II) transcription have been shown to bind several coactivators and basal factors in vitro. Whether such interactions play a primary regulatory role in recruiting these factors to activator-associated chromosomal target sites in living cells remains unclear. Here, we show that upon heat shock the Pol II-free form of Mediator is rapidly recruited to HSF binding sites. Unlike the TAFs and Pol II, the interaction between Mediator and HSF on chromosomal loci is direct and mechanistically separable from the preinitiation complex assembly step. Therefore, the activator-Mediator interaction likely underlies the initiation of signal transfer from enhancer-bound activators to the basal transcription machinery.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Respuesta al Choque Térmico , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Amanitinas/farmacología , Animales , Animales Modificados Genéticamente , Sitios de Unión , Células Cultivadas , Cromosomas , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/fisiología , Genes de Insecto , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Sustancias Macromoleculares , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glándulas Salivales/fisiología , Factores de Tiempo , Factores de Transcripción , Activación Transcripcional
7.
Wiad Lek ; 54(3-4): 171-8, 2001.
Artículo en Polaco | MEDLINE | ID: mdl-11436683

RESUMEN

The clinical picture of infectious mononucleosis and the consequences of EBV infection are due to immune response mechanisms. The level of soluble form of IL-2 receptor (sIL-2R) is thought to be a marker of T-cell activity, especially CD8+. Intercellular adhesion molecule (ICAM-1, CD 54) plays an important role in the process of antigen presentation. The aim of this study was to assess the serum concentration of sIL-2R, ICAM-1 and anti-VCA IgM in patients with infectious mononucleosis. The study group comprised 42 persons: 20 healthy subjects as a control group and 22 individuals with infectious mononucleosis. The highest anti-VCA IgM serum level in all patients was during the 1st day of hospitalization, and decreased in the 8th day of hospitalization. The lowest antibody concentration was observed when the symptoms and sings ceased. The level of sIL-2R was significantly increased and during farther hospitalization we observed lower, but still elevated concentrations. Our study has demonstrated statistically significant elevation of sICAM-1 level during the entire period of hospitalization. This data indicates the importance of antigen presentation process. Although the serum concentration of immune response mediators does not reflect their contents in organism, but is a useful method for in vivo examination.


Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Proteínas de la Cápside , Mononucleosis Infecciosa/diagnóstico , Molécula 1 de Adhesión Intercelular/sangre , Receptores de Interleucina-2/sangre , Adulto , Biomarcadores/análisis , Femenino , Humanos , Mononucleosis Infecciosa/inmunología , Mononucleosis Infecciosa/virología , Masculino
8.
Prehosp Disaster Med ; 16(4): 263-7, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-12090208

RESUMEN

The United Nations Mission in Kosovo (UNMIK) designated that the World Health Organization (WHO) develop health policy to assist in the recovery and rehabilitation of the post-war health system of Kosovo. As a critical part of the pre-policy evaluation, an assessment of current prehospital medical services was performed. This assessment identified a basic healthcare infrastructure upon which additional prehospital capabilities can be built, especially in communications, staffing, equipment, and transport services. To serve Kosovo properly in the future, it is recommended that capacity building must include the parallel development of emergency departments and specialty-trained physicians.


Asunto(s)
Servicios Médicos de Urgencia/organización & administración , Atención a la Salud , Planificación en Desastres , Humanos , Organización Mundial de la Salud , Yugoslavia
9.
Prehosp Disaster Med ; 16(4): 268-74, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-12090209

RESUMEN

Since the return of the refugee population to Kosovo, attempts at development of an emergency medical system in Kosovo have met with varied success, and have been hampered by unforeseen barriers. These barriers have been exacerbated by the lack of detailed health system assessments. A multimodal approach of data collection and analysis was used to identify potential barriers, and determine the appropriate level of intervention for emergency medicine (EM) development in Kosovo. The four step, multi-modal, data collection tool utilized: 1) demographic and health systems data; 2) focus group discussions with health-care workers; 3) individual interviews with key individuals in EM development; and 4) Q-Analysis of the attitudes and opinions of EM leaders. Results indicated that Emergency Medicine in Kosovo is under-developed. This method of combined quantitative and qualitative analysis identified a number of developmental needs in the Kosovar health system. There has been little formal training, the EMS system lacks organization, equipment, and a reliable communication system, and centralized emergency centers, other than the center at Prishtina Hospital, are inadequate. Group discussions and interviews support the desire by Kosovar health-care workers to establish EM, and highlight a number of concerns. A Q-methodology analysis of the attitudes of potential leaders in the field, supported these concerns and identified two attitudinal groups with deeper insights into their opinions on the development of such a system. This study suggests that a multi-modal assessment of health systems can provide important information about the need for emergency health system improvements in Kosovo. This methodology may serve as a model for future, system-wide assessments in post-conflict health system reconstruction.


Asunto(s)
Servicios Médicos de Urgencia/organización & administración , Recolección de Datos , Medicina de Emergencia , Análisis Factorial , Necesidades y Demandas de Servicios de Salud , Humanos , Liderazgo , Guerra , Yugoslavia
10.
Prehosp Disaster Med ; 16(4): 275-80, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-12090210

RESUMEN

The recent crisis in Kosovo led to nearly complete destruction of a healthcare system serving the needs of approximately 2 million people. Even prior to the crisis, the pre-existing healthcare system had inadequate provisions for the delivery of Emergency Medical Services. More than 440 diverse governmental and non-governmental organizations (NGOs) arrived to assist (and often compete) in the rehabilitation of Kosovo's healthcare needs. Each brought with them individual biases and strategies for how this rehabilitation should occur, and each faced numerous unforeseen barriers to the implementation of its programs. The authors used a four-step, multi-modal, needs assessment to gather information on the needs and potential barriers to the implementation of a program to rehabilitate emergency services as discussed in Part II. This paper chronicles the phases of the Emergency Medicine program development and the process of responding to barriers and changing needs. The program's successes and failures are noted, and the actual barriers encountered are reviewed. Overall, the needs assessment tool employed in this program was useful in the implementation of a program to restore and rehabilitate Emergency Services in Kosovo. The authors recommend the use of combined quantitative and qualitative methods for developing priorities for interventions in post-conflict settings following complex emergencies.


Asunto(s)
Servicios Médicos de Urgencia/organización & administración , Desarrollo de Programa , Medicina de Emergencia/educación , Análisis Factorial , Grupos Focales , Necesidades y Demandas de Servicios de Salud , Humanos , Evaluación de Necesidades , Sistemas de Socorro , Guerra , Yugoslavia
11.
Genes Dev ; 14(20): 2635-49, 2000 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-11040217

RESUMEN

Recent studies have demonstrated roles for Spt4, Spt5, and Spt6 in the regulation of transcriptional elongation in both yeast and humans. Here, we show that Drosophila Spt5 and Spt6 colocalize at a large number of transcriptionally active chromosomal sites on polytene chromosomes and are rapidly recruited to endogenous and transgenic heat shock loci upon heat shock. Costaining with antibodies to Spt6 and to either the largest subunit of RNA polymerase II or cyclin T, a subunit of the elongation factor P-TEFb, reveals that all three factors have a similar distribution at sites of active transcription. Crosslinking and immunoprecipitation experiments show that Spt5 is present at uninduced heat shock gene promoters, and that upon heat shock, Spt5 and Spt6 associate with the 5' and 3' ends of heat shock genes. Spt6 is recruited within 2 minutes of a heat shock, similar to heat shock factor (HSF); moreover, this recruitment is dependent on HSF. These findings provide support for the roles of Spt5 in promoter-associated pausing and of Spt5 and Spt6 in transcriptional elongation in vivo.


Asunto(s)
Proteínas Cromosómicas no Histona , Proteínas de Drosophila , Drosophila/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Respuesta al Choque Térmico/genética , Proteínas de Insectos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Elongación Transcripcional , Animales , Cromosomas/metabolismo , Reactivos de Enlaces Cruzados/química , Ciclina T , Ciclinas/inmunología , Ciclinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Proteínas Fúngicas/inmunología , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/inmunología , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/inmunología , Chaperonas de Histonas , Proteínas de Insectos/genética , Proteínas Nucleares/inmunología , Regiones Promotoras Genéticas , ARN Polimerasa II/inmunología , ARN Polimerasa II/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Transcripción Genética
12.
Wiad Lek ; 53(5-6): 355-8, 2000.
Artículo en Polaco | MEDLINE | ID: mdl-10983392

RESUMEN

We presented the case of mumps complicated by left ear's hearing loss in 32-year-old man. Audiological examinations performed after one, two and three months since the date of discharge from hospital showed the maintenance of hearing loss.


Asunto(s)
Pérdida Auditiva Sensorineural/etiología , Paperas/complicaciones , Adulto , Antivirales/uso terapéutico , Pérdida Auditiva Sensorineural/diagnóstico , Humanos , Masculino , Paperas/tratamiento farmacológico , Índice de Severidad de la Enfermedad
13.
Cell ; 101(5): 459-69, 2000 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-10850489

RESUMEN

It has been generally accepted that the TATA binding protein (TBP) is a universal mediator of transcription by RNA polymerase I, II, and III. Here we report that the TBP-related factor TRF1 rather than TBP is responsible for RNA polymerase III transcription in Drosophila. Immunoprecipitation and in vitro transcription assays using immunodepleted extracts supplemented with recombinant proteins reveals that a TRF1:BRF complex is required to reconstitute transcription of tRNA, 5S and U6 RNA genes. In vivo, the majority of TRF1 is complexed with BRF and these two proteins colocalize at many polytene chromosome sites containing RNA pol III genes. These data suggest that in Drosophila, TRF1 rather than TBP forms a complex with BRF that plays a major role in RNA pol III transcription.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Proteínas de Insectos/metabolismo , ARN Polimerasa III/metabolismo , Factor de Transcripción TFIIIB , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN/genética , Drosophila/enzimología , Drosophila/genética , Humanos , Proteínas de Insectos/genética , Ratones , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Proteínas Similares a la Proteína de Unión a TATA-Box , Factores Asociados con la Proteína de Unión a TATA , Proteína de Unión a TATA-Box , Factores de Transcripción/genética
14.
Genes Dev ; 14(7): 792-803, 2000 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-10766736

RESUMEN

P-TEFb, a heterodimer of the kinase Cdk9 and cyclin T, was isolated as a factor that stimulates formation of productive transcription elongation complexes in vitro. Here, we show that P-TEFb is located at >200 distinct sites on Drosophila polytene chromosomes. Upon heat shock, P-TEFb, like the regulatory factor HSF, is rapidly recruited to heat shock loci, and this recruitment is blocked in an HSF mutant. Yet, HSF binding to DNA is not sufficient to recruit P-TEFb in vivo, and HSF and P-TEFb immunostainings within a heat shock locus are not coincident. Insight to the function of P-TEFb is offered by experiments showing that the direct recruitment of a Gal4-binding domain P-TEFb hybrid to an hsp70 promoter in Drosophila cells is sufficient to activate transcription in the absence of heat shock. Analyses of point mutants show this P-TEFb stimulation is dependent on Cdk9 kinase activity and on Cdk9's interaction with cyclin T. These results, coupled with the frequent colocalization of P-TEFb and the hypophosphorylated form of RNA polymerase II (Pol II) found at promoter-pause sites, support a model in which P-TEFb acts to stimulate promoter-paused Pol II to enter into productive elongation.


Asunto(s)
Drosophila melanogaster/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Línea Celular , Mapeo Cromosómico , Ciclina T , Quinasa 9 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Dimerización , Proteínas de Drosophila , Regulación de la Expresión Génica , Calor , Fosforilación , Factor B de Elongación Transcripcional Positiva , Proteínas Recombinantes/metabolismo , Glándulas Salivales/fisiología , Transcripción Genética , Transfección
15.
J Affect Disord ; 57(1-3): 63-71, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-10708817

RESUMEN

BACKGROUND: There is a dearth of placebo-controlled studies of cognitive behavior therapy (CBT) of depression and the largest such study, by Elkin et al. (Arch. Gen. Psychiatry 46 (1989) 971-982), failed to find a significant difference between CBT and a clinical management plus placebo condition. METHODS: The outcomes of two consecutive cohorts of out-patients with major depressive disorder, treated with either CBT (n=90) or a nonspecific control condition (support-counseling-placebo; SCP: n=100), were compared. Although the principal comparisons between the CBT and SCP conditions were delimited to the first 4 weeks of treatment, a secondary set of analyses addressed the subset of 16 patients who received 12 additional weeks of supportive therapy. RESULTS: A consistent pattern of statistically and clinically significant differences favoring CBT over SCP was found in both weeks 4 and 16. LIMITATIONS: Interpretation of these findings are subject to several potential confounds, including the non-randomized nature of the groups and the greater amount of therapeutic contact during the first 4 weeks of CBT. CONCLUSIONS: While these results do not lessen the need for additional prospective studies, our findings do suggest that CBT has therapeutic effects beyond those attributable to placebo-expectancy and other nonspecific factors.


Asunto(s)
Terapia Cognitivo-Conductual/métodos , Trastorno Depresivo Mayor/terapia , Adulto , Ensayos Clínicos como Asunto , Estudios de Cohortes , Consejo , Trastorno Depresivo Mayor/diagnóstico , Femenino , Humanos , Masculino , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Resultado del Tratamiento
16.
Mol Cell Biol ; 20(1): 181-6, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10594020

RESUMEN

B52, an essential SR protein of Drosophila melanogaster, stimulates pre-mRNA splicing in splicing-deficient mammalian S100 extracts. Surprisingly, mutant larvae depleted of B52 were found to be capable of splicing at least several pre-mRNAs tested (H. Z. Ring and J. T. Lis, Mol. Cell. Biol. 14:7499-7506, 1994). In a homologous in vitro system, we demonstrated that B52 complements a Drosophila S100 extract to allow splicing of a Drosophila fushi tarazu (ftz) mini-pre-mRNA. Moreover, Kc cell nuclear extracts that were immunodepleted of B52 lost their ability to splice this ftz pre-mRNA. In contrast, splicing of this same ftz pre-mRNA occurred in whole larvae homozygous for the B52 deletion. Other SR protein family members isolated from these larvae could substitute for B52 splicing activity in vitro. We also observed that SR proteins are expressed variably in different larval tissues. B52 is the predominant SR protein in specific tissues, including the brain. Tissues in which B52 is normally the major SR protein, such as larval brain tissue, failed to produce ftz mRNA in the B52 deletion line. These observations support a model in which the lethality of the B52 deletion strain is a consequence of splicing defects in tissues in which B52 is normally the major SR protein.


Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Precursores del ARN/genética , Empalme del ARN , Animales , Regulación de la Expresión Génica , Mutación , Especificidad de Órganos , Factores de Empalme de ARN , Proteínas de Unión al ARN/genética
17.
Genes Dev ; 13(22): 2934-9, 1999 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-10580000

RESUMEN

Activated transcription by RNA polymerase II (Pol II) requires coactivators, one of which is the SRB/mediator. Whereas Srb4, an essential subunit of the SRB/mediator, is broadly required for Pol II transcription in yeast, we have shown that it is dispensable for the transcriptional activation of some genes. Here, we show that transcriptional activation by different natural activators, and by artificial recruitment of various transcription factors, have very different degrees of Srb4 independence. These data, and the analysis of an rgr1 mutant, point to an Rgr1 subcomplex of the SRB/mediator as the mechanistic route of activation by Srb4-independent activators in vivo.


Asunto(s)
Proteínas Fúngicas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transactivadores/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional/fisiología , Proteínas Portadoras , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/genética , Proteínas de Choque Térmico/fisiología , Sustancias Macromoleculares , Complejo Mediador , Metalotioneína/biosíntesis , Metalotioneína/genética , Estructura Terciaria de Proteína , ARN Polimerasa II/metabolismo , Proteínas Represoras/fisiología , Factores de Transcripción/genética
18.
Proc Natl Acad Sci U S A ; 96(18): 10033-8, 1999 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-10468557

RESUMEN

RNA aptamers selected against proteins can be used to modulate specific protein function. Expression of such reagents in cells and whole organisms could provide a means of dissecting and controlling molecular mechanisms in vivo. We demonstrate that Drosophila B52 protein can be specifically inhibited in vitro and in vivo by a multivalent RNA aptamer. This inhibitory aptamer RNA binds B52 avidly and inhibits B52-stimulated pre-mRNA splicing. It can be expressed in cultured cells and whole animals in a stable form that accumulates up to 10% of total mRNA. It binds B52 in vivo and suppresses all phenotypes caused by B52 overexpression. The strategies presented here should prove general in design and expression of functional and therapeutic RNAs.


Asunto(s)
Proteínas de Drosophila , Drosophila/genética , Proteínas Nucleares/antagonistas & inhibidores , Oligorribonucleótidos/farmacología , Fosfoproteínas/antagonistas & inhibidores , Empalme del ARN , ARN Mensajero/genética , ARN/química , ARN/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Células Cultivadas , Cruzamientos Genéticos , Drosophila/metabolismo , Femenino , Genotipo , Masculino , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fenotipo , Factores de Empalme de ARN , Proteínas de Unión al ARN/antagonistas & inhibidores , Transcripción Genética
19.
Mol Cell Biol ; 19(8): 5652-8, 1999 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10409754

RESUMEN

The Rad25 protein in yeast is a DNA helicase and a subunit of the general transcription factor TFIIH. While in vitro studies have led to the hypothesis that TFIIH helicase activity plays a role in promoter melting, in vivo tests are lacking. Using potassium permanganate, which preferentially modifies single-stranded DNA, we show that a temperature-sensitive rad25(ts) mutant severely reduces the normally extensive promoter melting observed in vivo on the highly expressed genes TDH2 and PDC1 and on the induced heat shock gene HSP82. Loss of promoter melting can be observed in as little as 30 s after a shift to the nonpermissive temperature and is accompanied by a dramatic reduction in transcription. These effects on the promoter are specific, since the mutation does not affect TATA box occupancy or, in the case of HSP82, recruitment of TATA-binding protein to the TATA element or that of heat shock factor to heat shock elements. Additionally, using the technique of formaldehyde cross-linking coupled with restriction endonuclease cleavage and ligation-mediated PCR, we were able to map the polymerase density on the promoter of HSP82. This high-resolution mapping allowed us to determine that the polymerase II (Pol II) density on the promoter is also dramatically reduced after inactivation of TFIIH. These data provide strong support for the hypothesis that TFIIH functions with Pol II in the transcriptionally required step of promoter melting and show, surprisingly, that the extent of TFIIH-dependent promoter melting observed in vivo is several times larger than that seen in vitro.


Asunto(s)
ADN de Hongos/química , Proteínas Fúngicas/fisiología , Genes Fúngicos , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción TFII , Factores de Transcripción/fisiología , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Helicasas/fisiología , ADN de Hongos/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas HSP90 de Choque Térmico , Proteínas de Choque Térmico/genética , Desnaturalización de Ácido Nucleico , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIIH
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