RESUMEN
The loggerhead Caretta caretta is the most common sea turtle in the Mediterranean. Currently, sea turtles are considered endangered, mainly due to the impact of human activities. Among traumatic lesions, those involving the skull, if complicated by brain exposure, are often life-threatening. In these cases, death could be the outcome of direct trauma of the cerebral tissue or of secondary meningoencephalitis. This uncontrolled study aims to evaluate the use of a plant-derived dressing (1 Primary Wound Dressing®) in 3 sea turtles with severe lesions of the skull exposing the brain. Following surgical curettage, the treatment protocol involved exclusive use of the plant-derived dressing applied on the wound surface as the primary dressing, daily for the first month and then every other day until the end of treatment. The wound and peri-wound skin were covered with a simple secondary dressing without any active compound (non-woven gauze with petroleum jelly). Data presented herein show an excellent healing process in all 3 cases and no side effects due to contact of the medication with the cerebral tissue.
Asunto(s)
Vendajes , Traumatismos Craneocerebrales/veterinaria , Tortugas , Heridas Penetrantes/veterinaria , Animales , Traumatismos Craneocerebrales/patología , Traumatismos Craneocerebrales/terapia , Cráneo/patología , Heridas Penetrantes/patología , Heridas Penetrantes/terapiaRESUMEN
OBJECTIVE: To observe the efficacy of a plant-derived wound dressing (1 Primary Wound Dressing®), a mixture of hypericum and neem oil, in different types of paediatric burns. METHOD: A retrospective review was conducted over the complete healing course of 9 paediatric patients with a mean age of 8.17±3.35 (1-11 years), presenting mixed, partial or full-thickness burns. The treatment applied by the wound care specialist consisted of daily cleansing of the wound with a saline solution and application of 1 Primary Wound Dressing on the whole wound surface. There was no application of a secondary dressing. The time to heal, wound size, ease of handling, pain and complications were recorded. Procedural and background pain were observed in six of the patients older than 5 years (mean age 9.6±2.39, range 8-11 years). Due to the small number of patients examined during the period studied, it was not possible to perform statistical analyses. RESULTS: The mean wound size was 50.76±48.32cm2 (4.63-132.0cm2). A rapid induction of granulation tissue and re-epithelialisation was observed. Time to complete healing was 16.6±4.69 days (10-22 days). No complications related to wound infection was observed. The 6 patients older than five years reported a strong relief of pain, from an initial value of 7-8 out of 10 to 0 out of 10 within the first week of treatment. This remained at the 0 out of 10 level during the second and third weeks of treatment. CONCLUSION: This retrospective, non-controlled examination suggests that 1 Primary Wound Dressing could be an effective therapy for the treatment of burn wounds, with benefits including pain reduction and simplicity of use. Further evaluations with a larger population are required to document the effectiveness of this plant-derived wound dressing in a controlled fashion. DECLARATION OF INTEREST: There were no external sources of funding for this study. F. Carnevali is a researcher and co-inventor of 1 Primary Wound Dressing®.
Asunto(s)
Quemaduras/tratamiento farmacológico , Glicéridos/uso terapéutico , Fitoterapia/métodos , Extractos Vegetales/uso terapéutico , Terpenos/uso terapéutico , Cicatrización de Heridas , Infección de Heridas/tratamiento farmacológico , Azadirachta/química , Bolivia , Quemaduras/complicaciones , Niño , Preescolar , Femenino , Glicéridos/análisis , Humanos , Hypericum/química , Lactante , Masculino , Apósitos Oclusivos , Dolor/etiología , Dolor/prevención & control , Estudios Retrospectivos , Terpenos/análisis , Infección de Heridas/etiologíaRESUMEN
St. John's wort is a medicinal plant with a long history of use in traditional medicine all over Europe. Traditional preparations and in particular the infused oil from SJW flowers remains one of the most popular and curative topical remedy against ulcerations and burns. The presence of the characteristic polyprenylated acylphloroglucinol derivatives, namely hyperforin and analogs are instead related to the oil's therapeutic activity. Indeed, it is well known that hyperforin has a potent antibacterial activity. In this study we tried to rationalize the production system of the oily preparation in order to obtain the highest concentration and stability of phloroglucinols. Five different samples of SJW oils were evaluated by HPLC-DAD-MS analysis to verify the variability and stability of the constituents according to the following factors: different harvesting time, fresh or dried plant material, use of sunlight or heating systems during extraction. The stability of these oils during 1 year was also tested.
Asunto(s)
Hypericum/química , Aceites de Plantas/análisis , Plantas Medicinales/química , Química Farmacéutica , Cromatografía Líquida de Alta Presión/métodos , Estabilidad de Medicamentos , Congelación , Espectrometría de Masas/métodos , Extractos Vegetales/química , Aceites de Plantas/química , Temperatura , Factores de TiempoRESUMEN
A 1329-base pair clone isolated from a human placenta cDNA library contains a full-length 837-base pair coding region for a 31.9-kDa protein whose deduced primary structure exhibits high homology to consensus sequences found in other NMN adenylyltransferases. Northern blotting detected a major 3.1-kilobase mRNA transcript as well as a minor 4.1-kilobase transcript in all human tissues examined. In several cancer cell lines, lower levels of mRNA expression were clearly evident. The gene encoding the human enzyme was mapped to chromosome band 1p32-35. High efficiency bacterial expression yielded 1.5 mg of recombinant enzyme/liter of culture medium. The molecular and kinetic properties of recombinant human NMN adenylyltransferase provide new directions for investigating metabolic pathways involving this enzyme.
Asunto(s)
Cromosomas Humanos Par 1/genética , Nicotinamida-Nucleótido Adenililtransferasa/genética , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Catálisis/efectos de los fármacos , Cationes Bivalentes/farmacología , Clonación Molecular , Escherichia coli/genética , Dosificación de Gen , Perfilación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Cinética , Datos de Secuencia Molecular , Nicotinamida-Nucleótido Adenililtransferasa/química , Nicotinamida-Nucleótido Adenililtransferasa/aislamiento & purificación , Mapeo Físico de Cromosoma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Células Tumorales CultivadasRESUMEN
The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.
Asunto(s)
Genes Fúngicos , Nicotinamida-Nucleótido Adenililtransferasa/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia Molecular , Nicotinamida-Nucleótido Adenililtransferasa/aislamiento & purificación , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Screening a maternal Xenopus expression library for activities that synergize with low levels of injected beta-catenin, we have isolated a clone encoding the C-terminal end of x-beta TrCP-2, a highly conserved protein belonging to the F-box/WD40 family of ubiquitin-ligase specificity factors. We show that x-beta TrCP-2 expression reduces dorsal axis formation in Xenopus embryos. A dominant negative mutant lacking the F-box triggers the opposite effect, inducing secondary axes and activating the expression of Wnt responsive genes in ectodermal explants. In light of the existence of beta TrCP transcripts associated with the vegetal cortex, we propose that beta TrCP plays a fundamental role in the establishment of the dorsal determinants during cortical rotation in Xenopus.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Unión al GTP/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores , Proteínas de Xenopus , Xenopus/embriología , Proteínas de Pez Cebra , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Clonación Molecular , Proteínas del Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Biblioteca de Genes , Glucógeno Sintasa Quinasa 3 , Modelos Genéticos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Wnt , beta Catenina , Proteínas con Repetición de beta-TransducinaRESUMEN
METHODS: We analyzed the potential influence that associated risk factors (ARF), such as smoking, alcoholism, overweight, and hypertension, could have on the establishment of chronic chagasic cardiomyopathy (CC). The sample was comprised of 124 individuals, 69 males and 55 females (mean age +/- SD, 41 +/- 9.5 years), who were born in en demic areas of Northern Argentina and migrated further to Rosario City, an area where autochthonous cases of Chagas' disease have never been registered. Assessments included the following: clinical examination to discard previous cardiomyopathies; search for the presence of ARF according to standard criteria; specific serology; frontal chest X-ray, and 12-lead resting electrocardiogram (ECG). Subjects were classified on the basis of their serological status and presence of ARF into four groups: Tc+ARF+ T. cruzi-infected persons with ARF (n = 41); Tc-ARF+ seronegativity in presence of ARF (n = 27); Tc+ARF- individuals showing positive serology that lacked ARF (n = 27), and Tc-ARF- seronegative individuals having no ARF (n = 29). RESULTS: Except for a higher female/male ratio in groups presenting no ARF (p < 0.02), no statistical differences as to age, length of residence in endemicity areas (LR), and ARF distribution were recorded among groups. Forty-one persons presented abnormal ECG tracings, distributed thus: Tc+ARF+, 18/41; Tc-ARF+, 14/27, Tc+ARF-, 14/27, and Tc-ARF, 4/29 (p < 0.01, in relation to the latter group). Subjects from the Tc+ARF+, Tc-ARF+, and Tc+ARF- groups had 4.89-, 6.7-, and 6.7-fold increases, respectively, if having an abnormal ECG when compared with Tc-ARF- individuals. Comparisons on the frequency of abnormal ECG between seropositives carrying ARF or not yielded a non-significant odds ratio, be it estimated as crude, or after adjusting for sex, age, and LR in multivariate analysis. CONCLUSIONS: Presence of ARF was not associated with an increasing risk of cardiac affectation in chronically T. cruzi-infected persons, but resulted in chagasic-compatible ECG abnormalities in those seronegative individuals.
Asunto(s)
Enfermedades Cardiovasculares/etiología , Enfermedad de Chagas/fisiopatología , Corazón/fisiopatología , Adulto , Alcoholismo/complicaciones , Animales , Enfermedad de Chagas/complicaciones , Electrocardiografía , Femenino , Humanos , Hipertensión/complicaciones , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Factores de Riesgo , Fumar , Trypanosoma cruziRESUMEN
During embryogenesis, inductive interactions underlie the development of much of the body plan. In Xenopus laevis, factors secreted from the vegetal pole induce mesoderm in the adjacent marginal zone; members of both the transforming growth factor-beta (TGF-beta) and fibroblast growth factor (FGF) ligand families seem to have critical roles in this process. Here we report the identification and characterization of laloo, a novel participant in the signal transduction cascade linking extracellular, mesoderm-inducing signals to the nucleus, where alteration of cell fate is driven by changes in gene expression. Overexpression of laloo, a member of the Src-related gene family, in Xenopus embryos gives rise to ectopic posterior structures that frequently contain axial tissue. Laloo induces mesoderm in Xenopus ectodermal explants; this induction is blocked by reagents that disrupt the FGF signalling pathway. Conversely, expression of a dominant-inhibitory Laloo mutant blocks mesoderm induction by FGF and causes severe posterior truncations in vivo. This work provides the first evidence that a Src-related kinase is involved in vertebrate mesoderm induction.
Asunto(s)
Inducción Embrionaria , Factores de Crecimiento de Fibroblastos/fisiología , Mesodermo/fisiología , Proteínas de Xenopus , Familia-src Quinasas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Técnicas de Cultivo , ADN Complementario , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Transducción de Señal , Xenopus , Proteínas ras/fisiología , Familia-src Quinasas/aislamiento & purificaciónRESUMEN
The cDNAs coding for the alpha and beta components of XrpFI, an activator of ribosomal protein genes transcription, were isolated. RNA analysis showed that transcripts of the beta genes are present at constant level throughout Xenopus development unlike the beta proteins which are undetectable in the early stages of embryogenesis when transcription is silent. On the contrary, alpha transcripts and proteins were found in all the embryo stages examined. The role of the XrpFI alpha and beta subunits in rp-gene transcription has been studied by mRNA microinjection into X.laevis oocytes. The presence of the beta subunit appears to be critical for the proper rp-gene promoter function.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Expresión Génica , Proteínas Nucleares/biosíntesis , Oocitos/metabolismo , Proteínas de Xenopus , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/biosíntesis , Clonación Molecular , Sondas de ADN , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/fisiología , Femenino , Técnicas In Vitro , Sustancias Macromoleculares , Ratones , Microinyecciones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Transcripción Genética , Xenopus laevis , beta-Galactosidasa/biosíntesisRESUMEN
The cDNA coding for the Xenopus laevis homolog of the transcriptional activator/repressor protein delta/YY1 was isolated from a lambda gt11 oocyte cDNA library. The deduced aminoacid sequence shows that the four zinc fingers of the DNA binding domain are 99% conserved when compared to the mouse (delta) and 95% to the human (YY1) proteins, while differences are found in the N-terminal region. In particular, the long run of consecutive glycines and histidines of delta and YY1 is missing. The protein, named FIII/YY1, was overexpressed into Xenopus oocytes from the cDNA under direction of the L14 rp-promoter and found to share antigenic and DNA-binding properties with the oocyte endogenous protein binding to the first exon of the X.laevis ribosomal protein genes (rp-genes) L1 and L14.
Asunto(s)
Secuencia Conservada , Proteínas de Unión al ADN/metabolismo , Exones , Proteínas Represoras/metabolismo , Proteínas Ribosómicas/genética , Factores de Transcripción/metabolismo , Xenopus laevis/genética , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Cloranfenicol O-Acetiltransferasa/biosíntesis , Clonación Molecular , Proteínas de Unión al ADN/biosíntesis , Factores de Unión al ADN Específico de las Células Eritroides , Femenino , Expresión Génica , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Oocitos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Factores de Transcripción/biosíntesis , Proteínas de Xenopus , Factor de Transcripción YY1RESUMEN
XrpFI, first identified in the extract of Xenopus laevis oocyte nuclei, binds to a proximal sequence of the L14 ribosomal protein gene promoter. Its target sequence, 5'-TAACCGGAAGTTTGT-3', is required to fully activate the promoter, and the two G's of the central motif are essential for factor binding and transcriptional activation; our data also suggest that XrpFI may play a role in cap site positioning. The binding site of XrpFI is homologous to the sequence recognized by the family of ets genes. Antibodies specific for Ets-1 and Ets-2 proteins did not react with XrpFI, but those raised against the rat alpha and beta GA-binding proteins both supershifted the retarded bands formed by XrpFI. The Xenopus polypeptides related to GA-binding protein alpha interact with DNA both as monomers and as heterodimers associated with beta-related proteins. Oocyte nuclei contain multiple forms of alpha- and beta-related proteins: the alpha-like proteins remain throughout development, while the pattern of the beta species changes in the embryonic stages examined. beta-like proteins are undetectable in the cleavage period up to the neurula stage, but at later stages, when ribosomal protein genes are actively transcribed, two beta-related polypeptides reappear.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/biosíntesis , Animales , Secuencia de Bases , ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas c-ets , Proteínas Ribosómicas/genética , Factores de Transcripción/química , Factores de Transcripción/inmunología , Transcripción Genética , Xenopus laevisRESUMEN
The identification in HeLa nuclei of a novel DNA-binding protein, designated HrpF, is presented. This factor recognizes and binds a sequence of the Xenopus laevis L14 ribosomal protein (r-p) gene promoter bound by the Xenopus r-p transcription factor I (XrpFI). We show here that XrpFI and HrpF share a conserved DNA-binding domain. We also present evidences suggesting that the two factors perform similar functions in the cell. We discuss the hypothesis that closely related factors might be involved in the control of rp-gene transcription in vertebrates.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Ribosómicas/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/metabolismo , Transcripción Genética , Xenopus laevis/genéticaRESUMEN
The purpose of this experiment was to determine if pituitary stores of LH could be replenished by administration of GnRH when circulating concentrations of both progesterone and estradiol-17 beta (estradiol) were present at levels observed during late gestation. Ten ovariectomized (OVX) ewes were administered estradiol and progesterone via Silastic implants for 69 days. One group of 5 steroid-treated OVX ewes was given GnRH for an additional 42 days (250 ng once every 4 h). Steroid treatment alone reduced (p less than 0.01) the amount of LH in the anterior pituitary gland by 77%. Pulsatile administration of GnRH to steroid-treated ewes resulted in a further decrease (p less than 0.01) in pituitary content of LH. Compared to the OVX ewes, concentrations of mRNAs for alpha- and LH beta-subunits were depressed (p less than 0.01) in all steroid-treated ewes, whether or not they received GnRH. The ability of the dosage of GnRH used to induce release of LH was examined by collecting blood samples for analysis of LH at 15 days and 42 days after GnRH treatment was initiated. Two of 5 and 3 of 5 steroid-treated ewes that received pulses of GnRH responded with increased serum concentrations of LH after GnRH administration during the first and second bleedings, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Estradiol/fisiología , Hipófisis/efectos de los fármacos , Ovinos/fisiología , Animales , Estradiol/sangre , Femenino , Hormona Liberadora de Gonadotropina/farmacología , Hormona Luteinizante/análisis , Hormona Luteinizante/sangre , Ovariectomía , Hipófisis/química , Hipófisis/metabolismo , Progesterona/sangre , Progesterona/fisiologíaRESUMEN
The upstream region of the Xenopus laevis L14 ribosomal protein gene was deleted starting from the 5' extremity in order to define the promoter length necessary to express a linked reporter CAT gene. The functional analysis indicated that a sequence located between -63 and -49 from the capsite is important for an efficient promoter activity. Band shift and ExoIII protection assays evidenced the binding to this region of a factor, called XrpFI, present in the crude nuclear extract from X.laevis oocytes. Methylation interference analysis localized the contacts in the G residues belonging to a short box, 5' CTTCC 3', positioned between -53 and -49 from the capsite. An additional factor, XrpFII, makes contacts with the sequence 5'GCCTGTTCGCC 3' located between -27 and -17 from the capsite. The deletion mutant still containing this sequence is poorly transcribed, but resumes activity when a short fragment containing the binding site for factor XrpFI is cloned in an upstream position.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genes , Regiones Promotoras Genéticas , Proteínas Ribosómicas/genética , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Deleción Cromosómica , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/síntesis química , Oocitos/enzimología , Plásmidos , Transcripción Genética , Xenopus laevisRESUMEN
We describe an improvement on the procedure of Scalenghe et al. (F. Scalenghe, M. Buscaglia, C. Steinheil, and M. Crippa (1978) Chromosoma 66, 299-308) for the large scale isolation of nuclei from Xenopus laevis oocytes. The nuclear extract obtained was tested for its ability to transcribe a cloned Xenopus 5 S RNA gene and for the presence of nuclear factors interacting with a X. laevis ribosomal protein gene promoter. Efficiency of accurate transcription and of factor binding is comparable with that of an extract prepared from manually isolated nuclei.
Asunto(s)
Fraccionamiento Celular/métodos , Núcleo Celular/metabolismo , Oocitos/ultraestructura , Animales , Sondas de ADN , Proteínas de Unión al ADN/análisis , Femenino , Técnicas In Vitro , Oocitos/metabolismo , Plásmidos , Regiones Promotoras Genéticas , ARN Ribosómico 5S/genética , Transcripción Genética , Xenopus laevisRESUMEN
Activation of in vitro transcription of otherwise inert DNA sequences by purified yeast RNA polymerase II has been observed following the introduction in closed DNA domains of fragments of various origin. This enhancer-like effect on the in vitro transcriptional capacity is only detected in negatively supercoiled DNA domains and is characterized for each chimaeric plasmid by the superhelical density (- sigma) at which a sharp transition toward activation takes place. We have analyzed the topological state (as defined by localization and evaluation of the relative occurrence of secondary structures sensitive to S1 endonuclease) of the activated closed domains as a function of the conditions that determine the transcriptional enhancer effect, i.e. superhelical density, size, and nature of the components of the domains. We observe that variations in transcriptional capacity coincide with a defined pattern of secondary structures. These observations support a cause-effect relation between topology and regulation of transcription.
Asunto(s)
ADN Superhelicoidal/genética , ARN Polimerasa II/metabolismo , Transcripción Genética , Secuencia de Bases , Quimera , Enzimas de Restricción del ADN , ADN Recombinante/análisis , Escherichia coli/enzimología , Vectores Genéticos , Plásmidos , Saccharomyces cerevisiae/enzimologíaRESUMEN
We present an analysis of the influence of DNA superhelicity on the topology of chimeric plasmids. A correlation is found between topological variations and in vivo/in vitro functions. Data refer to the topological transitions observed in function of variations of the superhelical density in the three closed DNA domains that represent the three extreme examples, from the point of view of the topological organization, among many chimeric systems analyzed. The plasmids studied are ADR2-BS-pBR322 (the vector pBR322 + the yeast gene ADR2), ADR3-5c-yRp7 (containing yeast TRP1, a constitutive mutant of yeast ADR2, a fragment of 5 kilobases of unanalyzed chromosomal DNA and the vector pBR322), and p31 (pBR322 + a yeast DNA fragment encompassing the right moiety of the Ty1 element and its in vivo promoter). In the model systems analyzed, the topological transitions observed in the eukaryotic sequences (relevant in one case for activation of selective in vitro transcription (Ty), in another (ADR2) for the in vivo expression) take place in the range of superhelical densities predicted on theoretical ground (Vologodskii, A. V., Lukashin, A. V. Anshelevich, V. V. & Frank-Kamenetskii, M. D. (1979) Nucleic Acids Res. 6, 967-982).
Asunto(s)
ADN Superhelicoidal/metabolismo , Plásmidos , Alcohol Deshidrogenasa , Oxidorreductasas de Alcohol/genética , ADN Recombinante/metabolismo , Endonucleasas , Genes , Genes Fúngicos , Genes Reguladores , Cinética , Mutación , Hibridación de Ácido Nucleico , Saccharomyces cerevisiae/enzimología , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Transcripción GenéticaRESUMEN
The effect of the insertion of foreign genes or gene systems in closed DNA domains has been investigated in vitro in purified systems. We observe that in chimaeric plasmids two apparently independent classes of modifications, (1) functional and (2) topological, do take place in defined instances. (1) Among the screened yeast gene systems, examples have been found of DNA sequences that upon insertion cause activation of in vitro transcription of distant genes. (2) Foreign DNA sequences may lead to new topological features of the harbouring plasmids; it is shown that more than one S1-sensitive secondary structure may be contemporaneously present on the same chimaeric plasmid. DNA superhelicity is a prerequisite of these modifications. The two classes of effects (1) functional and (2) topological are not a priori directly related one to the other but appear to be two independent consequences of the same cause: the insertion of foreign DNA sequences into closed DNA domains. These observations suggest a regulatory model of gene expression based on alternative topologies of closed DNA domains.
Asunto(s)
Quimera , Elementos Transponibles de ADN , Plásmidos , Enzimas de Restricción del ADN , ADN-Topoisomerasas de Tipo I , ADN Recombinante , ADN Superhelicoidal , Electroforesis en Gel de Agar , Endonucleasas , ARN Polimerasa II , Saccharomyces cerevisiae/enzimología , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Moldes Genéticos , Transcripción GenéticaRESUMEN
Clones of the yeast Tyl element and 2 microns plasmid have been selectively transcribed in vitro by partially or completely purified yeast RNA polymerase II. Electrophoretic analysis of whole and restricted ternary transcription complexes allows the localization of the in vitro actively transcribed regions of the analyzed genes. The DNA regions that actively promote in vitro transcription correspond to the nucleotide sequence that in the Tyl element encompasses the in vivo transcription initiation sites and that in the 2 micrometer plasmid encompasses the starting codons of two oppositely oriented potential protein coding frames. The transcription assay that we describe herein may be applied to analyze rapidly the in vitro transcription of clones genes, to localize transcription initiation sites on supercoiled templates and to evaluate the differential in vitro promoter strength in RNA polymerase II served genes. Data obtained with RNA polymerase II at two different stages of purification are presented in parallel. Studies with a completely purified enzyme should certainly be preferred although the use of a partially purified RNA polymerase II may be convenient and may reveal factors which affect specificity.
Asunto(s)
Clonación Molecular , ADN Recombinante , ARN Polimerasas Dirigidas por ADN/metabolismo , Operón , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Transcripción Genética , Secuencia de Bases , Enzimas de Restricción del ADN , Plásmidos , Saccharomyces cerevisiae/genéticaRESUMEN
The in vitro transcription properties of purified yeast RNA polymerase II have been analyzed on prokaryotic plasmids (pBR322 and pBR313) and chimaeric plasmids bearing yeast 2 micron sequences (BTYP 1, BTYH 2 and BTYH 3). Conditions for selective transcription of the 2 micron DNA sequences in chimaeric plasmids have been determined. pBR322 and pBR313 are not transcribed by the purified RNA polymerase II when not bearing eukaryotic inserts. We show that the agarose gel electrophoretic analysis of ternary transcription complexes allows the localization of nascent RNA chains. The RNA produced has been visualized by electron microscopy (nascent RNA hybridization loops) and by gel electrophoretic analysis. All the observed properties are shared by RNA polymerase II purified by a conventional method (1) and by a rapid alternative procedure described herein. The peculiar properties of a partially purified form of RNA polymerase II are reported.