RESUMEN
By analyzing crosses between yeast strains carrying different combinations of mitochondrial (mt) introns, we have shown that the aI5 alpha intron is mobile in vivo. Furthermore, we have observed that the mobility of intron aI5 alpha is affected by both the nuclear and mt genotypes. We have also detected a restriction endonuclease (ENase) activity that cleaves intronless mt genomes close to the aI5 alpha intron insertion site and thus might be involved in intron mobility. This is further supported by the fact that this ENase activity is only detected in a strain containing the aI5 alpha intron. Furthermore, similar to other ENases encoded by mobile mt introns of yeast, the ENase generates a cut with a four-base 3'-OH overhang. Thus, intron aI5 alpha represents a characteristic member of the family of mobile group-I introns.
Asunto(s)
Enzimas de Restricción del ADN/genética , ADN Mitocondrial/genética , Intrones , Saccharomyces cerevisiae/genética , Secuencia de Bases , Southern Blotting , Núcleo Celular/fisiología , Cruzamientos Genéticos , Enzimas de Restricción del ADN/metabolismo , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Genoma Fúngico , Genotipo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Empalme del ARN , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Especificidad por SustratoRESUMEN
Two group I intron-encoded proteins from the yeast mitochondrial genome have already been shown to have a specific DNA endonuclease activity. This activity mediates intron insertion by cleaving the DNA sequence corresponding to the splice junction of an intronless strain. We have discovered in mitochondrial extracts from the yeast strain 777-3A a new DNA endonuclease activity which cleaves the fused exon A3-exon A4 junction sequence of the CO XI gene.
Asunto(s)
Enzimas de Restricción del ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II , Endodesoxirribonucleasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Southern Blotting , Enzimas de Restricción del ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exones , Genes Fúngicos , Intrones , Mitocondrias/enzimología , Datos de Secuencia Molecular , Empalme del ARN , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiaeRESUMEN
The MSS51 gene product has been previously shown to be involved in the splicing of the mitochondrial pre-mRNA of cytochrome oxidase subunit I (COX1). We show here that it is specifically required for the translation of the COX1 mRNA. Furthermore, the paromocyin-resistance mutation (P454R) which affects the 15S mitoribosomal RNA, interferes, directly or indirectly, with the action of the MSS51 gene product. Possible roles of the MSS51 protein on the excision of COX1 introns are discussed.
Asunto(s)
Complejo IV de Transporte de Electrones/genética , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Alelos , Northern Blotting , Farmacorresistencia Microbiana/genética , Complejo IV de Transporte de Electrones/metabolismo , Paromomicina/farmacología , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The pal 4 nuclease (termed I-Sce II) is encoded in the group I al 4 intron of the COX I gene of Saccharomyces cerevisiae. It introduces a specific double-strand break at the junction of the two exons A4-A5 and thus mediates the insertion of the intron into an intronless strain. To define the sequence recognized by pal 4 we introduced 35 single mutations in its target sequence and examined their cleavage properties either in vivo in E. coli (when different forms of the pal 4 proteins were artificially produced) or in vitro with mitochondrial extracts of a mutant yeast strain blocked in the splicing of the al 4 intron. We also detected the pal 4 DNA endonuclease activity in extracts of the wild type strain. The results suggest that 6 to 9 noncontiguous bases in the 17 base-pair region examined are necessary for pal 4 nuclease to bind and cleave its recognition site. We observed that the pal 4 nuclease specificity can be significantly different with the different forms of the protein thus explaining why only some forms are highly toxic in E. coli. This study shows that pal 4 recognition site is a complex phenomenon and this might have evolutionary implications on the transfer properties of the intron.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II , Endodesoxirribonucleasas/genética , Intrones , Saccharomyces cerevisiae/genética , Secuencia de Bases , Southern Blotting , ADN de Hongos/metabolismo , Endodesoxirribonucleasas/metabolismo , Escherichia coli/genética , Exones , Genes Fúngicos , Mitocondrias/enzimología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiaeRESUMEN
We have dissected the mouse H-2Kb gene promoter in order to define the sequences responsible for induction by tumour necrosis factor (TNF-alpha). An enhancer element (-187 to -158) composed of two imperfect direct palindromic repeats has been shown to be necessary and sufficient for TNF-alpha induction of a heterologous promoter. A multimer of either repeat is also responsive, while a single copy is not: this is the situation in the beta 2-microglobulin (beta 2-m) promoter which contains a single palindrome and does not respond to TNF-alpha. We had previously found that the two repeats can bind a factor named KBF1. We show here that in the uninduced state the transcription factor AP2 binds to the interpalindromic region, while in TNF-treated cells an NF kappa B-like activity is induced which displaces both KBF1 and AP2 and binds to the two palindromes. This strongly suggests that induction of an NF kappa B-like activity is responsible for TNF-alpha stimulation of mouse MHC class I genes.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Genes MHC Clase I , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Animales , Secuencia de Bases , Línea Celular , AMP Cíclico/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos H-2/genética , Humanos , Ratones , Datos de Secuencia Molecular , FN-kappa B , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Factor de Transcripción AP-2 , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Using a Y specific probe (pY353/B) taken from a flow sorted mouse Y chromosome library we have identified a family of RNA transcripts encoded by the Y chromosome. These transcripts which are approximately 1.3 Kb in length are present in testis PolyA+ RNA but can not be detected in either male liver, spleen, kidney, brain, heart or lung tissues. Isolation and sequence analysis of a corresponding cDNA shows it to contain a potential coding sequence of 696bp. These data show that the Y chromosome is transcriptionally active in the adult mouse testis.
Asunto(s)
Clonación Molecular , Testículo/citología , Transcripción Genética , Cromosoma Y , Secuencia de Aminoácidos , Animales , Composición de Base , Secuencia de Bases , ADN/genética , ADN/metabolismo , Masculino , RatonesRESUMEN
Major advances in our knowledge of the genetic organization of the mouse X chromosome have been obtained by the use of interspecific crosses involving Mus spretus-derived strains. This system has been used to study sequences detected by three probes 80Y/B, 302Y/B and 371Y/B isolated from a mouse Y-chromosome library which have been shown to recognize both male-female common and male-female differential sequences. These patterns are due to the presence of a family of cross-reacting sequences on the mouse X and Y chromosomes. Detailed genetic analysis of the localization of the X-chromosome-specific sequences using both a somatic cell hybrid panel and an interspecific mouse cross has revealed the presence of at least three discrete clusters of loci (X-Y)A, (X-Y)B and (X-Y)C. Two of these clusters, (X-Y)B and (X-Y)C, lie distally on the mouse X chromosome, the other cluster (X-Y)A being situated close to the centromere. In situ hybridization shows a striking symmetry in the localization of the major sequences on both the X and Y chromosomes detected by these probes, hybridization being preferentially localized to a subcentromeric and subtelomeric region on each chromosome. This striking localization symmetry between the X and Y chromosome sequences is discussed in terms of the extensive pairing of the X-Y chromosomes noted during meiosis.
Asunto(s)
Mapeo Cromosómico , Ratones/genética , Cromosoma X , Cromosoma Y , Animales , Cruzamientos Genéticos , Femenino , Marcadores Genéticos , Variación Genética , Masculino , Hibridación de Ácido Nucleico , Linaje , Polimorfismo GenéticoRESUMEN
To purify mouse Y chromosomes by flow cytometry, a male cell line containing the Robertsonian translocation Rb(9.19)163H has been established by SV40 transformation. Flow karyotypes obtained from these cells exhibit a well-isolated peak of fluorescence corresponding to the single Y chromosome, clearly distinct from that of chromosome 19. From this peak, 650,000 chromosomes were sorted, and two restriction fragment libraries were constructed from the DNA of the sorted chromosomes. The characterization of several Y-specific fragments has shown that the Y DNA was enriched at least 36-fold. Furthermore, given that there are likely homologies between the X and Y chromosomes, we can assume that this calculated value of the purification factor is an underestimation and that the Y DNA was more highly purified by flow sorting.
Asunto(s)
Ratones/genética , Cromosoma Y , Animales , Bacteriófago lambda , Fraccionamiento Celular/métodos , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Citometría de Flujo , Vectores Genéticos , Masculino , Hibridación de Ácido Nucleico , Translocación GenéticaRESUMEN
Restriction fragment length polymorphisms (RFLPs) of maternally inherited mitochondrial DNA have revealed that genetic exchanges can occur between populations, subspecies or even species. From the point of view of population genetics the mammalian Y chromosome represents a genomic analogue of the mitochondrion; it is inherited only paternally and remains perpetually monosomic, showing little meiotic recombination with other chromosomes. Using a Y-specific genomic DNA probe obtained from a flow-sorted mouse Y-chromosome library, we have examined the RFLPs in 10 newly established mouse lines of the European semispecies Mus musculus domesticus and Mus musculus musculus, and identified two variant forms of the Y chromosome, each of which is characteristic of one of the semispecies. As reported here, probing the DNA from nine established inbred laboratory strains reveals that the strain SJL carries a M.m. domesticus type Y whereas, surprisingly, the Y chromosomes of the other eight strains are of M.m. musculus origin. Hence, these strains cannot be regarded as archetypes of M.m. domesticus as suggested by protein polymorphisms and mitochrondrial DNA sequence data, but rather as genetic hybrids between the two semispecies.
Asunto(s)
Ratones/genética , Cromosoma Y , Animales , ADN/genética , Enzimas de Restricción del ADN , ADN Mitocondrial/genética , Masculino , Ratones Endogámicos/genética , Hibridación de Ácido Nucleico , Polimorfismo Genético , Especificidad de la EspecieRESUMEN
Human interferons-alpha, -beta and -gamma enhance HLA-DR mRNAs in all the human lymphoblastoid and melanoma cell lines studied. The increase concerns both alpha and beta chain mRNAs. Moreover, we show that immune interferon-gamma preferentially enhances class II MHC mRNA. This effect of IFN-gamma on the synthesis of alpha and beta HLA-DR chains has been also analysed by immunoprecipitation. It is abolished by a monoclonal antibody directed against human IFN-gamma. The effect of interferon on the cell surface level of HLA-DR molecules does not always correspond to the enhancement of HLA-DR mRNA. Our experiments suggest that this discrepancy between the enhancement of HLA-DR mRNA and cell surface antigen might be due to a constitutively high level of the corresponding antigens on several of the human cells studied.
Asunto(s)
Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Interferones/farmacología , ARN Mensajero/genética , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos HLA-A/metabolismo , Antígenos HLA-B/metabolismo , Antígenos HLA-C/metabolismo , Humanos , Interferón-alfa/farmacología , Interferón beta/farmacología , Interferón gamma/farmacología , Melanoma/inmunologíaRESUMEN
Sodium periodate treatment of Mouse spleen cells activated suppressor cells which act on the in vitro antibody response towards sheep erythrocytes or trinitrophenyl-polyacrylamide beads. Present study shows that suppression can be mediated by soluble factor (s) released in the cultures of periodate treated cells. Production of such suppressor factor (s) was associated to the presence of free aldehyde groups generated by periodate on the lymphocyte membrane. Control or cells successively treated by periodate and then reduced by borohydride were unable to produce suppressor factor (s). Suppressor factor (s) were stable at 56 degrees C (30 mn) and inactivated at 80 degrees C (10 mn).
Asunto(s)
Formación de Anticuerpos/efectos de los fármacos , Inmunosupresores/inmunología , Linfocitos/inmunología , Ácido Peryódico/farmacología , Animales , Terapia de Inmunosupresión , Cinética , Activación de Linfocitos , Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos DBA , Compuestos Orgánicos , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
Mild hydrolysis of Haemophilus influenzae type a lipopolysaccharide by ion exchangers in the presence of chloroform, to remove the lipid moiety, yielded a nontoxic and immunogenic polysaccharide fraction. This polysaccharide selectively triggered murine B lymphocytes in vitro: (i) it induced enhancement of thymidine incorporation and stimulated antibody secretion in cultures of normal and nude mouse spleen cells; (ii) it did not stimulate splenic T lymphocytes; (iii) the activation of B lymphocytes was not absolutely dependent on the presence of macrophages. Sepharose 4B gel filtration showed that this polysaccharide consisted at least of two fractions: PS I (molecular weight [MW] 10(6)) and PS II (MW 10(4)). Only PS I was found to act as a polyclonal B cell activator. EDTA treatment dissociated the polysaccharide into PS III (MW 10(6)) and PS IV (MW 10(4)), which was not reassembled after the addition of 0.02 M CaCl2. Both fractions PS III and PS IV were unable to stimulate B lymphocytes. The immunological active fraction of H. influenzae polysaccharide is PS I. This fraction consists of a high-molecular-weight group (10(6)) and an association of 10(4)-MW aggregated units.
Asunto(s)
Endotoxinas/análisis , Haemophilus influenzae/inmunología , Activación de Linfocitos , Polisacáridos Bacterianos/inmunología , Linfocitos B/inmunología , Mitógenos/farmacología , Polisacáridos Bacterianos/análisis , Polisacáridos Bacterianos/farmacología , Linfocitos T/inmunologíaRESUMEN
A polysaccharide fraction (PS) was separated by mild hydrolysis from Haemophilus influenzae lipopolysaccharide. This preparation contained glycosyl-galactosyl, rhamnosyl, glucosaminyl and mannosyl residues (molar ratio: 4-1-1-2-2). It was nontoxic and immunogenic and consisted of at least one stable molecular group (fraction A; MW approximately equal to 10(6)) and an association of aggregated units (fraction B;MW approximately equal to 10(4)). This study evaluated the capacity of phagocytosis and quantitative nitroblue-tetrazolium reduction of mouse macrophages in presence of these polysaccharide fractions. After a 24-h incubation period, PS and fraction A, at 1 mg/ml, increased both phagocytosis and reduction potential of mouse peritoneal macrophages by 100%. In contrast, 1-h incubation with PS or fraction A induced a decrease of 50% in phagocytosis but no modification of NBT reduction. An identical incubation with various sugars showed that only mannosyl polymers could significantly decrease this phagocytic process. As in the case of toxic lipopolysaccharides, macrophages responded to a nontoxic preparation obtained from an endotoxin. We confirmed the role of mannosyl residues in recognition of macrophage binding receptors. Moreover, we suggest that this mannose binding ability was dependent on dose, aggregation state and molecular weight of the preparation.U
Asunto(s)
Haemophilus influenzae/fisiología , Lectinas Tipo C , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Lectinas de Unión a Manosa , Receptores de Superficie Celular/metabolismo , Animales , Técnicas In Vitro , Activación de Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Receptor de Manosa , Ratones , Nitroazul de Tetrazolio/metabolismo , Fagocitosis/efectos de los fármacosRESUMEN
A scale of response, called phagocytic index (PI), is established to study the phagocytic function by measure of the Nitrobluetetrazoluim (NBT) reduction. 0 % and 100 % opsonic activity must be evaluated before the determination of PI which is calculated as follows: (Formula: see text) OD 0% vary with leukocyte species and the NBT batches, OD 100 % is depended on bacteria strains and nature of cells. Therefore PI must be performed in a system strictly characterized for bacteria, cells, and batch of NBT used. PI provides a better evaluation of bacterial phagocytosis as shown with antisera from rabbits immunized with Haemophilus influenzae.