RESUMEN
Chronic granulomatous disease (CGD) is an inherited disorder of phagocyte function in which defective superoxide production results in deficient microbicidal activity. CGD patients suffer from recurrent, life-threatening infections, and nearly half develop chronic gastrointestinal (GI) complications (colitis, gastric outlet obstruction, or perirectal abscess) and/or autoimmune/rheumatologic disorders (AIDs). To identify genetic modifiers of disease severity, we studied a cohort of 129 CGD patients, in whom seven candidate genes (myeloperoxidase [MPO], mannose binding lectin [MBL], Fcgamma receptors IIa, IIIa, IIIb, TNF-alpha, and IL-1 receptor antagonist), each containing a physiologically relevant polymorphism predicted to influence the host inflammatory response, were selected for analysis. Genotypes of MPO (P = 0.003) and FcgammaRIIIb (P = 0.007) were strongly associated with an increased risk for GI complications, while an FcgammaRIIa (P = 0.05) genotype was suggestive for an association. Patients with all three associated genotypes had the highest risk for GI complications (P < 0.0001). The risk of AIDs was strongly associated with variant alleles of MBL (P = 0.01) and weakly associated with an FcgammaRIIa genotype (P = 0.04). Patients with variant forms of both MBL and FcgammaRIIa had the highest risk of developing an AID (P = 0.003).
Asunto(s)
Enfermedad Granulomatosa Crónica/genética , Inmunidad/inmunología , Polimorfismo Genético/genética , Enfermedades Autoinmunes/genética , Proteínas Portadoras/genética , Colectinas , Citocinas/genética , Femenino , Genotipo , Enfermedad Granulomatosa Crónica/complicaciones , Enfermedad Granulomatosa Crónica/epidemiología , Humanos , Masculino , Peroxidasa/genética , Reacción en Cadena de la Polimerasa , Receptores de IgG/genética , Estudios Retrospectivos , Factores de RiesgoRESUMEN
The most common, X-linked, form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene located at Xp21.1. The product of this gene is the large subunit of flavocytochrome b558, gp91phox, which forms the catalytic core of the antimicrobial superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene, or more rarely at the intron/exon borders. Alternatively, they are large (often multi-gene) deletions. In addition, four mutations have been found in the promoter region. In contrast, very few intronic mutations have been reported. Here we describe an intronic mutation that causes X-linked CGD. A single nucleotide substitution in the middle of intron V creates a novel 5' splice site and results in multiple abnormal mRNA products.
Asunto(s)
Enfermedad Granulomatosa Crónica/genética , Glicoproteínas de Membrana/genética , Mutación , NADPH Oxidasas/genética , Sitios de Empalme de ARN , Linfocitos B/metabolismo , Exones , Enfermedad Granulomatosa Crónica/metabolismo , Humanos , Lactante , Intrones , Masculino , Glicoproteínas de Membrana/química , NADPH Oxidasa 2 , NADPH Oxidasas/química , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/análisis , ARN Mensajero/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Chronic granulomatous disease is a rare inherited disorder caused by non-existent or severely decreased phagocyte superoxide production that results in a severe defect in host defense and consequent predisposition to microbial infection. The enzyme responsible for superoxide production, NADPH oxidase, involves at least five components. An absence of, or a defect in, any one of four of these proteins (p47(phox), p67(phox), p22(phox) and gp91(phox)) gives rise to the known types of chronic granulomatous disease. The most common form of inheritance is X-linked and is due to mutations in the CYBB gene that encodes gp91(phox), the large subunit of flavocytochrome b, the terminal electron donor of the oxidase. We have recently reported a large number of mutations in this gene revealing a broad range of defects, including large and small deletions, and frameshift, nonsense, missense, splice region and regulatory region mutations. Here we report a patient who has an unusual type of mutation that results in the generation of a 'pseudo-exon' in the gp91(phox) mRNA and an unexpected pattern of splicing.
Asunto(s)
Enfermedad Granulomatosa Crónica/genética , Glicoproteínas de Membrana/genética , Mutación , Secuencia de Bases , Secuencia de Consenso , ADN/sangre , ADN/química , Electroforesis en Gel de Agar , Exones , Enfermedad Granulomatosa Crónica/sangre , Humanos , Masculino , Glicoproteínas de Membrana/sangre , Persona de Mediana Edad , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasas/química , NADPH Oxidasas/genética , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/químicaRESUMEN
A gene coding for mature human interferon, IFN-alpha 1, fused to the expression and secretion signals of a staphylokinase gene (sak) derived from Staphylococcus aureus phage 42D, was inserted into the Streptomyces promoter probe vector pIJ487. Streptomyces lividans transformed with the recombinant plasmid (pMG341) secreted biologically active IFN-alpha 1 into the culture medium. Expression of the IFN-alpha 1 gene was at least on the translational level directed by the sak signals since numerous upstream stop codons would have prevented the formation of a fusion protein. Long-term continuous chemostat cultivation under various limitation conditions was used to select clones with an IFN-alpha 1 yield increased about 60-100-fold (1-2 x 10(5) IU/ml). The increase in IFN-alpha 1 formation was accompanied by spontaneous amplification of the adjacent neo gene, but not of the remaining plasmid DNA. Examination of the DNA sequence around the endpoints of the amplified region revealed almost identical stem-loop structures followed by an octanucleotide direct repeat.
Asunto(s)
Clonación Molecular , Amplificación de Genes , Genes , Interleucina-1/genética , Metaloendopeptidasas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/biosíntesis , Streptomyces/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Recombinante/metabolismo , Humanos , Interleucina-1/biosíntesis , Datos de Secuencia Molecular , Fagos de Staphylococcus/genética , Staphylococcus aureus/genéticaAsunto(s)
Eosinófilos , Miocarditis/sangre , Adulto , Eosinófilos/patología , Humanos , Masculino , Miocarditis/patología , Necrosis , Eosinofilia Pulmonar/complicacionesRESUMEN
A nourseothricin-producing Streptomyces noursei strain was continuously cultivated in a chemostat equipped with a stirrer for mechanical fractionation of the mycelium. Different cultivation conditions allowed the selection of six types of differentiation mutants after the culture had reached a population genetically stationary state. The mutants showed an altered control pattern of sporulation as well as altered antibiotic biosynthesis and antibiotic resistance. In addition, the stability of the recombinant plasmid pIJ385 in several differentiation type mutants as host strains was tested. The results suggest that there exists a strong correlation between the cultivation conditions employed and the type of differentiation mutants selected.
Asunto(s)
Antibacterianos/biosíntesis , Genes Bacterianos , Streptomyces/crecimiento & desarrollo , Estreptotricinas/biosíntesis , Medios de Cultivo , Cinética , Mutación , Plásmidos , Streptomyces/efectos de los fármacos , Streptomyces/genética , Streptomyces/metabolismo , Estreptotricinas/farmacologíaRESUMEN
The genetic stability of the capacity of an improved strain of Streptomyces hygroscopicus to produce the macrolide antibiotic turimycin was investigated during long-term continuous culture. Dilution rate, growth-limiting substrate and culture temperature were varied. Certain culture conditions resulted in the stable propagation of the inoculated turimycin-producing population. Other conditions led to segregation of the initial population. Turimycin non-producing phenotypes appeared, and in each case the simultaneous loss of ability to form aerial mycelium was observed. The non-differentiating clones were found to be stable, without any reversion to the parental phenotype, indicating that a loss of genetic information probably took place.
Asunto(s)
Leucomicinas/biosíntesis , Streptomyces/genética , Cloruro de Amonio/farmacología , Técnicas Bacteriológicas , Glucosa/farmacología , Fenotipo , Plásmidos , Temperatura , Factores de TiempoRESUMEN
The aggressivity of cancer chemotherapy is limited by hematologic side effects or makes expensive supportive therapy necessary. This article summarizes known and clinically usable methods of stimulating hematopoiesis to enhance bone marrow recovery after therapy. Longest known is the stimulatory effect of anabolic steroids, which may accelerate the regeneration of granulopoiesis and erythropoiesis. Lithium increases CSA-levels and enhances the regeneration of granulopoiesis and questionably thrombopoiesis according to several publications. The stem cell shift by hypertransfusion promotes granulopoiesis. As malnutrition limits hematopoietic recovery, optimal nutrition after chemotherapy should favor maximal hematopoiesis. Further studies are necessary before the application of the above mentioned methods to stimulate bone marrow - singly or in combination - can be recommended.
Asunto(s)
Enfermedades de la Médula Ósea/terapia , Médula Ósea/efectos de los fármacos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Anabolizantes/uso terapéutico , Animales , Transfusión Sanguínea , Busulfano/uso terapéutico , Corynebacterium , Ciclofosfamida/farmacología , Tolerancia a Medicamentos , Endotoxinas/uso terapéutico , Granulocitos , Hematopoyesis/efectos de los fármacos , Humanos , Litio/uso terapéutico , Ratones , Nutrición Parenteral TotalRESUMEN
A Bacillus amyloliquefaciens strain high-producing for beta-1.3-1.4-glucanase has gradually lost the ability to produce this enzyme during long-time continuous cultivation, independent of the culture conditions. Mutant strains isolated after long-term cultivation exhibited changed behaviour concerning extracellular enzyme formation and sporulation. By agarose gel electrophoresis of alkaline DNA extracts isolated form original and mutant strains we demonstrate that the observed pleiotropic phenomena are not caused by the loss of a complete plasmid present in the original strain. From extracts of both the original and mutant strains plasmid DNAs with approximately the same molecular weight of about 35 Mdal were isolated.
Asunto(s)
Bacillus/enzimología , Glicósido Hidrolasas/biosíntesis , Bacillus/genética , Bacillus/crecimiento & desarrollo , Glicósido Hidrolasas/genética , Mutación , Plásmidos , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
A new method has been developed for the selection of antibiotic-resistant clones after transformation of Streptomyces protoplasts with plasmid DNA. This method is based on establishing a spatial concentration gradient for the antibiotic, the resistance to which is encoded by the transforming plasmid. By this method, the resistance development of regenerating protoplasts can be followed. The results suggest that antibiotic resistance is inducible. In addition, we were able to show that resident plasmids incompatible with the incoming ones are eliminated when this direct selection principle is used. Moreover, this method, which may facilitate the application of gene technology in Streptomyces, works even though the transformation procedure gives variable results.
Asunto(s)
ADN Bacteriano/genética , Plásmidos , Protoplastos , Streptomyces/genética , Transformación Bacteriana , ADN Circular/genética , Farmacorresistencia Microbiana , Kanamicina Quinasa , Neomicina/farmacología , Fosfotransferasas/genética , Streptomyces/efectos de los fármacos , Streptomyces/crecimiento & desarrolloRESUMEN
The nourseothricin producer Streptomyces noursei is resistant to its own antibiotic in submerged as well as in surface culture. The strain shows no cross-resistance to miscoding inducing aminoglycoside antibiotics. Cell free extracts of Streptomyces noursei inactivate nourseothricin by enzymatic acetylation. The pattern of cross-resistance of Streptomyces noursei correlates well with the substrate specificity of the nourseothricin acetyltransferase. Furthermore, the acetyltransferase activity parallels the resistance level in nourseothricin-producing strains and nonproducing mutants. The results suggest that the nourseothricin acetyltransferase is important in the self-defence strategy of the nourseothricin-producing strain.
Asunto(s)
Acetiltransferasas/metabolismo , Antibacterianos/farmacología , Streptomyces/efectos de los fármacos , Estreptotricinas/farmacología , Farmacorresistencia Microbiana , Streptomyces/enzimología , Estreptotricinas/metabolismo , Especificidad por SustratoRESUMEN
The slightly modified procedure for the transformation of protoplasts of S. coelicolor A3 (2) with SCP2 plasmid DNA and polyethylenglycol (PEG) (Bibb et al., 1978) was extended to infection of protoplasts of S. lividans 66 with actinophage SH10 DNA (Klaus et al., 1979). Maximal yield of transfected protoplasts was obtained at 20% PEG, 3 MM sodium-citrate and 150 mM NaCl final concentrations. The efficiency of transfection was determined to be about 2 x 10(-8) to 2 x 10(-7). The average value of competent protoplasts was about 1-2 x 10(-4) of regenerating protoplasts. In comparison with outgrowing spores infected with phage particles the average burst size of transfected protoplasts was reduced from 100 to 10 pfu/infected cell, the latent period prolonged from 45 min to 120 min and the rise period was not affected.
Asunto(s)
Bacteriófagos/genética , Vectores Genéticos , Streptomyces/genética , Transfección , ADN Viral/genética , Protoplastos/metabolismo , Factores de TiempoRESUMEN
Streptomyces hygroscopicus JA 6599 is the producer of the macrolide antibiotic turimycin. Mapping analysis by conventional matings and protoplast fusion techniques were carried out. The sequence of auxotrophic markers determined by using the method of minimizing the frequency of quadruple crossover recombinants, could be shown to be in accordance with the related marker sequence of Streptomyces coelicolor after both conjugation and protoplast fusion. However, the tur locus could localized between chromosomal markers only assuming quadruple crossover. Moreover, after conventional crosses the tur marker has to be localized at quite another site than after protoplast fusion. Regarding also our results on the evidence of extrachromosomal DNA in strains of S. hygroscopicus, the following hypothesis is proposed: the structural genes for turimycin biosynthesis are localized on the bacterial genome, but plasmid-borne genes might be involved in the control of the antibiotic production in a yet unknown way, possibly by inducing chromosomal rearrangements.
Asunto(s)
Leucomicinas/genética , Plásmidos , Streptomyces/genética , ADN Bacteriano , Herencia Extracromosómica , Regulación de la Expresión Génica , Peso MolecularRESUMEN
The streptococcal erythromycin resistance plasmid pSM9 was used to study the problem of how the transforming activity of mixtures of two unique linear products of restriction enzyme digestion depends on the distance between the cleavage sites. In transformation of the Challis strain of S. sanguis, the transforming activity of mixed digests increased with increasing relative distances (x) between the restriction sites, where 0 less than or equal to X less than or equal to 0.5. To explain the experimental results, a mathematical model was proposed according to which the overall probability (p) of transformation resulting in a functional replicon is the product of the partial probabilities of initial single-strand pairing, circularization, and stability of the paired intermediate, all of which were assumed to depend on x. A linear relationship found between transformation frequency and p was taken to support the model. Transformation of Challis by mixtures of two linearized plasmid molecules with regions of internal nonhomology resulting in paired intermediates with insertion or substitution loops allowed either donor molecule to contribute to the transformation yield.
Asunto(s)
Plásmidos , Streptococcus/genética , Transformación Bacteriana , Enzimas de Restricción del ADN , Farmacorresistencia Microbiana , Eritromicina/farmacología , Streptococcus/efectos de los fármacosRESUMEN
A stable temperature sensitive mutant of Streptomyces hygroscopicus JA6599 defective in both DNA and RNA syntheses is described. The mutant ts35 is characterized by an immediate stop of DNA synthesis and continued protein synthesis after transfer to restrictive temperature. The reinitiation of DNA synthesis begins immediately after a return to the permissive temperature. This kinetics of macromolecular synthesis at restrictive temperature appears to share similarities with a defect in the DNA elongation process, as described for Escherichia coli (Carl 1970, Hanna and Carl 1975). The simultaneous stop of both DNA and RNA syntheses may be caused by an additional mutational event affecting also the RNA synthesis. The data were discussed with respect to similar results in E. coli.
Asunto(s)
ADN Bacteriano/biosíntesis , ARN Bacteriano/biosíntesis , Streptomyces/genética , Calor , Cinética , Mutación , Streptomyces/crecimiento & desarrolloRESUMEN
The streptomycin-producing Streptomyces griseus HP spontaneously segregated non-reverting derivatives with altered phenotypes. Clones characterized by increased spore formation and decreased streptomycin production were found. Two other types of derivatives were defective in aerial mycelium and streptomycin formation as well, but differed in the capacity to synthesize a yellow pigment. These derivatives were examined with respect to further properties. The stability of S. griseus HP was investigated in relation to conditions of continuous culture. Both at 26 and 30 degrees C, under glycerol and NH4Cl limitation a rapid segregation and enrichment of streptomycin-non-producing derivatives occurred. At 34 degrees C and glycerol limitation segregation began only after about 35 generations of continuous culture. In NH4Cl-limited chemostats the original strain was stable during 80 generations. In the course of the continuous culture experiments it was shown that the onset of genetic segregation within mycelia can be detected before it becomes obvious in colonies grown from the mycelia. This was achieved by fractionation of the mycelia by protoplast formation and subsequent plating on regeneration medium allowing colony growth and differentiation.
Asunto(s)
Genes Bacterianos , Streptomyces griseus/genética , Estreptomicina/biosíntesis , Cloruro de Amonio/metabolismo , Glicerol/metabolismo , Fenotipo , Plásmidos , Esporas Bacterianas/fisiología , Streptomyces griseus/citología , Streptomyces griseus/fisiología , TemperaturaRESUMEN
The maintenance and genetic stability of the vector plasmids pBR322 and pBR325 in two genetically different Escherichia coli hosts were studied during chemostat cultivation with glucose and ammonium chloride limitation and at two different dilution rates. The plasmid pBR322 was stably maintained under all growth conditions tested. However pBR325 segregated from both hosts preferentially during glucose limitation and at low dilution rate. In addition to this general segregation process a separate loss of tetracycline resistance was observed. The remaining plasmid conferred resistance to ampicillin and chloramphenicol only, without any remarkable alteration of its molecular weight. Cultivation conditions in the chemostat were found that allowed the stable genetic inheritance of both plasmids in the hosts studied.
Asunto(s)
Medios de Cultivo/farmacología , Escherichia coli/genética , Vectores Genéticos/efectos de los fármacos , Plásmidos/efectos de los fármacos , Cloruro de Amonio/farmacología , Ampicilina/farmacología , Cloranfenicol/farmacología , Glucosa/farmacología , Resistencia a las Penicilinas , Fenotipo , Tetraciclina/farmacologíaRESUMEN
The most common form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene that is carried on the X-chromosome and give rise to the X-linked form of the disease. The product of this gene is the large subunit of flavocytochrome b558, gp91phox, the catalytic core of the superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene or, less frequently, at the intron/exon borders. In addition, there are large, often multi-gene, deletions. Four mutations have also been found in the promoter regions. In contrast, very few intronic mutations have been reported. Here we describe an unusual intronic mutation that causes CGD. The mutation is the insertion of 12 bp in intron XI, accompanied by the deletion of exon 12. Remarkably, the grandmother of this patient is chimeric, carrying a normal allele, the patient's allele, and an allele with a 4-nucleotide insertion at a site adjacent to the patient's insertion, in combination with a 1.5-kb deletion within intron XI. The patient's mother carries a normal allele and the patient's allele. We propose that an initial mutational event during the grandmother's embryogenesis has undergone unsuccessful DNA repair and has resulted in two aberrant alleles, one of which has been inherited by the patient and his mother. Remarkably, in the only two kindreds that have been examined in detail where deletions originating within introns have led to CGD, both families have contained members with triple somatic mosaicism.
Asunto(s)
Enfermedad Granulomatosa Crónica/genética , Glicoproteínas de Membrana/genética , Mosaicismo/genética , NADPH Oxidasas , Preescolar , Análisis Mutacional de ADN , Cartilla de ADN/química , Exones/genética , Femenino , Humanos , Intrones/genética , Masculino , Glicoproteínas de Membrana/deficiencia , Datos de Secuencia Molecular , NADPH Oxidasa 2 , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/análisis , ARN Mensajero/metabolismoRESUMEN
C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. A murine genomic library was screened, and five clones were selected that covered the remaining four exons in the 5'-region of the C4BP gene. Together with previous work (Barnum, S. R., Kristensen, T., Chaplin, D. D., Seldin, M. F., and Tack, B. F. (1989) Biochemistry 28, 8312-8317), the entire C4BP gene has now been shown to be 23 kilobases (kb) long and comprised of 10 exons ranging in size from 86 to 442 base pairs (bp). Primer extension analysis revealed the major transcription start site to be 46 bp upstream of the published cDNA start site. Northern blot analysis of RNA isolated from several mouse tissues demonstrated that the C4BP gene is expressed in a liver-specific manner. Several regions homologous to known response elements were identified upstream of the C4BP gene including a strong hepatocyte nuclear factor 1 binding site and four putative glucocorticoid response elements. Furthermore, dexamethasone increased C4BP mRNA and protein levels in the mouse liver cell line, NMuLi. The stimulation of C4BP gene expression was rapid and independent of protein synthesis. These results suggest dexamethasone induction of the C4BP gene is a primary response and therefore a transcriptional effect. Inhibition of the dexamethasone effect on C4BP by actinomycin D supports this theory. These studies also provide evidence that, for optimal induction of the C4BP gene, the glucocorticoid receptor complex may cooperatively interact with accessory transcription factors. It is likely that stimulation of C4BP gene expression by dexamethasone may allude to a mechanism by which glucocorticoids exert their anti-inflammatory effects.