RESUMEN

A bicistronic reporter consisting of the promoterless genes aacC1 (conferring gentamycin resistance) and lacZ fused to the catabolic promoter of the phenol degradation genes was used to identify and analyse mutants of Pseudomonas putida with altered carbon catabolite repression (CR) of phenol degradation. Out of approximately 2500 mini-Tn5 mutants analysed so far, 12 mutants that were resistant to gentamycin during growth on succinate were identified. In eight of these mutants mini-Tn5 was inserted into one of the genes of the cyo operon. The cyo operon encodes the cytochrome o ubiquinol oxidase, the terminal oxidase of the cyanide-sensitive branch of the respiratory chain. In these mutants the activity of the PphlA promoter was significantly increased during growth on succinate and reached 15-20% of that found during growth with the non-repressing carbon source pyruvate. During growth on glucose the reduction of CR was less obvious, during growth on lactate CR was unchanged. The possible significance of the cyo operon for the generation of signal(s) for carbon catabolite repression is discussed.

Asunto(s)

Proteínas Bacterianas , Carbono/metabolismo , Complejo IV de Transporte de Electrones/genética , Operón , Fenol/metabolismo , Pseudomonas putida/genética , Carbono/farmacología , División Celular/efectos de los fármacos , División Celular/genética , Clonación Molecular , Elementos Transponibles de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Complejo IV de Transporte de Electrones/metabolismo , Prueba de Complementación Genética , Glucosa/metabolismo , Glucosa/farmacología , Mutagénesis Insercional , Mutación , Fenol/farmacología , Plásmidos/genética , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/metabolismo , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacología , Análisis de Secuencia de ADN , Ácido Succínico/metabolismo , Ácido Succínico/farmacología , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

The activator-encoding gene phlR was identified upstream of the plasmid-encoded operon for phenol degradation in Pseudomonas putida strain H by cassette mutagenesis and DNA sequence analysis. The deduced amino acid sequence of PHLR shows high homology to DmpR of P. putida sp. CF600 and to the chromosomally encoded PhhR of P. putida P35X reported previously. Trans-activation of phenol degradation was observed when phlR was overexpressed in a phlR insertion mutant. Transconjugants of Escherichia coli carrying pPGH11, which contains the complete set of phl genes, are unable to grow on phenol as carbon source. However, two types of mutants were selected for further characterization that were able to metabolize phenol as sole source of carbon and energy. In both types of mutants enhanced expression of phlR is responsible for the Phl+ phenotype. In type I (pPGH13) a deletion of 1 bp made the -35 region and the spacing between the -35 and -10 regions of the phlR promoter more similar to the consensus structure. In type II (pPGH14) a duplication of the phlR 5' region was identified that includes part of the -35 motif and reduces the spacing between the -35 and -10 regions. In addition, due to the duplication of part of phlR, the distance from the phlR promoter to the catabolic phl operon is increased. Different transcriptional start sites have been identified by primer extension analysis in clones harboring pPGH14 or the wild type phlR. Quantitative primer extension analysis revealed that the greatest amount of phlR transcript is expressed from the partial, phlR duplication. Growth on phenol and phenol hydroxylase activity reflect the high level of phlR transcript in E. coli transconjugants. Overexpression of PhlR was also observed when pPGH14 was transferred into P. putida, and results in earlier induction of the phenol degradation operon relative to the wild-type strain.

Asunto(s)

Proteínas Bacterianas , Escherichia coli/genética , Fenoles/metabolismo , Regiones Promotoras Genéticas/genética , Pseudomonas putida/genética , Transactivadores/genética , Activación Transcripcional/genética , Secuencia de Bases , Biodegradación Ambiental , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos/genética , Genes Reguladores/genética , Prueba de Complementación Genética , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mutación , Nitrógeno/metabolismo , Operón/genética , Fenol , Pseudomonas putida/crecimiento & desarrollo , Pseudomonas putida/metabolismo , Transcripción Genética/genética

RESUMEN

Enzymes involved in (methyl)phenol degradation of Pseudomonas putida H are encoded by the catabolic operon (phlA-L) on plasmid pPGH1. Transcription of this operon by the sigma54 (RpoN)-containing RNA polymerase is positively controlled by the gene product of the divergently transcribed phlR in response to the availability of the respective substrate. Additionally, phenol degradation is subject to carbon catabolite repression induced by organic acids (e.g., succinate, lactate, and acetate) or carbohydrates (e.g., glucose and gluconate). Analysis of lacZ fusion to the catabolic promoter and quantified primer extension experiments indicate that carbon catabolite repression also occurs at the transcriptional level of the catabolic operon. In this study, it is furthermore shown that carbon catabolite repression is a negative control. Titration of the postulated negative controlling factor was exclusively observed when extra copies of functional phlR gene were present in the cell. We therefore conclude that PhlR is the target and that carbon catabolite repression of phenol degradation occurs by interfering with the activating function of PhlR.

Asunto(s)

Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Fenoles/metabolismo , Pseudomonas putida/metabolismo , Transactivadores/metabolismo , Secuencia de Bases , ADN Bacteriano , Glucosa/metabolismo , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Operón , Fenoles/farmacología , Piruvatos/metabolismo , Ácido Pirúvico , Succinatos/metabolismo , Ácido Succínico

RESUMEN

The genetic organization of the DNA region encoding the phenol degradation pathway of Pseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via the meta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase and meta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.

Asunto(s)

Dioxigenasas , Fenoles/metabolismo , Pseudomonas putida/genética , Secuencia de Aminoácidos , Secuencia de Bases , Biodegradación Ambiental , Catecol 2,3-Dioxigenasa , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Oxigenasas/genética , Pseudomonas putida/metabolismo

Asunto(s)

Mucopolisacaridosis II , Mucopolisacaridosis II/enzimología , Piel/enzimología , Fibroblastos/enzimología , Humanos , Iduronato Sulfatasa/genética , Cinética , Mucopolisacaridosis II/genética , Fenotipo

RESUMEN

DNA based and biochemical diagnosis of MPS II was performed on 13 unrelated families using Southern blotting. The 35S-sulphate accumulation in cultured fibroblasts was investigated and the iduronate-2-sulphatase (IDS) activity in the serum determined. Sixteen patients and 36 females at risk were screened for structural aberrations and by RFLP analysis using the intragenic probe pc2S15 and probes VK23B, VK21A, and II-10 for the flanking loci DXS297, DXS296, and DXS466. Structural alterations were found in the DNA of two patients. One of them showed a major deletion including the whole coding sequence of the IDS gene. An aberrant Southern fragment occurred in the HindIII/pc2S15 blot of the other patient suggesting a new HindIII restriction site by point mutation in an IDS gene intron. Twenty-nine females were confirmed as carriers, and for five women the heterozygous state could be excluded. Prenatal diagnosis can be offered to 27 women if requested.

Asunto(s)

ADN/análisis , Tamización de Portadores Genéticos/métodos , Iduronato Sulfatasa/genética , Mucopolisacaridosis II/genética , Southern Blotting , Células Cultivadas , Deleción Cromosómica , Femenino , Fibroblastos/metabolismo , Humanos , Iduronato Sulfatasa/sangre , Masculino , Mucopolisacaridosis II/metabolismo , Linaje , Mutación Puntual , Polimorfismo de Longitud del Fragmento de Restricción , Factores de Riesgo

RESUMEN

An immunoquantification protocol based on an enzyme-linked immunosorbent assay was developed to measure the abundance of the microsomal enzyme steroid sulphatase (STS). The two-step sandwich immunoassay is sufficiently sensitive to detect 100-200 pg purified steroid sulphatase in a 50-microliters sample. The steroid sulphatase content in fibroblast, leukocyte and placental extracts correlates with the steroid sulphatase activity in these extracts. No steroid sulphatase protein was found in approximately 350 micrograms plasma proteins from a normal person. In three of four X-linked ichthyosis patients a complete gene deletion was found by Southern hybridization with the full-length STS cDNA as probe. Neither steroid sulphatase protein nor enzymatic activity was found in fibroblast extracts of these three patients. In a fibroblast extract of another X-linked ichthyosis patient, which had a normal Southern blotting pattern, no immunoreactive protein was detected. Residual activity of steroid sulphatase was also not found after prolonged incubation of this fibroblast extract with the natural substrate oestrone sulphate.

Asunto(s)

Arilsulfatasas/metabolismo , Ictiosis Ligada al Cromosoma X/enzimología , Southern Blotting , Western Blotting , Células Cultivadas , Estrona/análogos & derivados , Estrona/metabolismo , Fibroblastos/enzimología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Humanos , Ictiosis Ligada al Cromosoma X/genética , Ictiosis Ligada al Cromosoma X/inmunología , Técnicas para Inmunoenzimas , Leucocitos/enzimología , Placenta/enzimología , Esteril-Sulfatasa

RESUMEN

Phenylalanine hydroxylase (PAH) was purified 105-fold from human liver. The rel mol mass of the subunits in sodium dodecylsulfate-polyacrylamide gel electrophoresis was 54,000 Da and the isoelectric point was estimated to be between pH 5.0 and 5.2. The activity of purified PAH was inhibited by p-Cl-phenylalanine (p-Cl-Phe), 3-J-tyrosine (3-J-Tyr) and 6-F-tryptophane (6-F-Trp) by 73%, 26% and 10%, respectively. The Km value was 0.36 x 10(-3) mol/l for L-Phe and 5.88 x 10(-5) mol/l for the synthetic cofactor dimethyltetrahydrobiopterin (DMPH4). Polyclonal antibodies raised in rabbits against the active human enzyme showed only a slight cross-reaction with purified rat liver PAH. Using the rabbit antibodies an immunoreactive protein with the same mol mass and isoelectric point as purified human liver PAH and PAH from crude liver extract was detected in extracts from kidney, heart, spleen, brain, pancreas, lung, placenta, leucocytes, cultured skin fibroblasts and chorionic villus cells.

Asunto(s)

Fenilalanina Hidroxilasa/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoquímica , Punto Isoeléctrico , Hígado/enzimología , Peso Molecular , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/aislamiento & purificación , Embarazo , Distribución Tisular

Asunto(s)

Enzimas/sangre , Errores Innatos del Metabolismo/genética , Diagnóstico Prenatal , Muestra de la Vellosidad Coriónica , Aberraciones Cromosómicas , Femenino , Tamización de Portadores Genéticos , Humanos , Recién Nacido , Masculino , Errores Innatos del Metabolismo/diagnóstico , Mucolipidosis/genética , Embarazo , Esfingolipidosis/genética

RESUMEN

We report about 58 ultrasonographically guided transcervical chorionic villus biopsies from January 1985 to November 1987. Maternal age greater than 35 years (n = 28), followed by trisomy 21 or 18 (n = 10) were the mean indications. Biochemically evaluation of storage diseases (n = 6) and genomically DNA-analysis because of phenylketonuria (n = 2) were combined in each case with cytogenetic diagnosis. In the other cases certain indications were the reasons for biopsy. In 52 of 58 cases we were successful in biopsies and diagnoses. In the other 6 biopsy specimen we didn't found chorionic villi. 3 abortions we observed up to day 3 after operation (n = 3) and after 6 weeks (n = 1). Pathological findings were 1 trisomy 16, 1,47,XYY-karyotype and 1 embryo with phenylketonuria. Another reason for termination of pregnancy was male karyotype in a Morbus Duchenne-risk and 1 risk for Rett-syndrome. Meanwhile 30 healthy babies were born.

Asunto(s)

Muestra de la Vellosidad Coriónica/métodos , Enfermedades Genéticas Congénitas/diagnóstico , Adulto , Aberraciones Cromosómicas/diagnóstico , Trastornos de los Cromosomas , Femenino , Enfermedades Genéticas Congénitas/genética , Humanos , Cariotipificación , Embarazo , Primer Trimestre del Embarazo , Factores de Riesgo , Ultrasonografía

Asunto(s)

Prueba de Complementación Genética , Glicósido Hidrolasas/deficiencia , Iduronidasa/deficiencia , Mucopolisacaridosis/genética , Adulto , Línea Celular , Preescolar , Humanos , Células Híbridas , Iduronidasa/genética , Mucopolisacaridosis/enzimología , Mutación , Fenotipo

RESUMEN

Phenylalanine hydroxylase activity measured in leucocytes and fibroblasts by the fluorometric method is nonspecific and can be released by other aromatic hydroxylases. Investigations with the inhibitors p-Cl-phenylalanine, 3-I-tyrosine and 6-F-tryptophan made evident that these results may be caused by the tryptophan hydroxylase and the tyrosine hydroxylase. Phenylalanine hydroxylase activities in leucocytes could also not be measured by radiochemical investigations with [3-14C] phenylalanine (scanner and liquid scintillation technique).

Asunto(s)

Leucocitos/enzimología , Fenilalanina Hidroxilasa/análisis , Fibroblastos/enzimología , Humanos , Hígado/enzimología , Fenilalanina Hidroxilasa/antagonistas & inhibidores , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/enzimología

RESUMEN

The procedure for the detection of Hunter carriers suggested by Tønnesen et al. (1982) was checked in different mixtures of normal and Hunter cells as well as by examination of five obligate and five potential Hunter carriers. In the presence of fructose 1-phosphate there was a strict correlation between the proportion of mutant cells in the fibroblast culture and sulphate accumulation, both in artificial cell mixtures and in native cell cultures of Hunter carriers. In all obligate heterozygotes studied, sulphate incorporation was increased by a factor of two. The new technique seems to be suitable for carrier diagnosis. Its limitations are discussed.

Asunto(s)

Fructosafosfatos , Tamización de Portadores Genéticos/métodos , Mucopolisacaridosis II/genética , Biopsia , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Masculino , Mutación , Piel/patología , Sulfatos/metabolismo , Radioisótopos de Azufre

Asunto(s)

Pseudomonas aeruginosa/metabolismo , Azufre/metabolismo , Cisteína/genética , Cisteína/metabolismo , Metionina/metabolismo , Mutación , Pseudomonas/crecimiento & desarrollo , Pseudomonas aeruginosa/genética , Selenio/farmacología , Estereoisomerismo , Sulfatos/metabolismo