The mechanism conferring resistance to paromomycin in Streptomyces rimosus forma paromomycinus, t... more The mechanism conferring resistance to paromomycin in Streptomyces rimosus forma paromomycinus, the producing organism, was studied at the level of both protein synthesis and drug-inactivating enzymes. Ribosomes prepared from this organism grown in either production or nonproduction medium were fully sensitive to paromomycin. A paromomycin acetyltransferase and a paromomycin phosphotransferase, both characteristic of the producer, were highly purified from extracts prepared from two Streptomyces lividans transformants harboring the relevant genes inserted in pIJ702-derived plasmids. In vitro, paromomycin was inactivated by either activity. In vivo, however, S. lividans clones containing the gene for either enzyme inserted in the low-copy-number plasmid pIJ41 were resistant to only low levels of paromomycin. In contrast, an S. lividans transformant containing both genes inserted in the same pIJ41-derived plasmid displayed high levels of resistance to paromomycin. These results indica...
Se analiza, mediante calorimetría diferencial de barrido, muestras de aceite de oliva de diferent... more Se analiza, mediante calorimetría diferencial de barrido, muestras de aceite de oliva de diferentes categorías y mezclas de estos. Los aceites son congelados previamente a ¿100oC, a una velocidad de 5oC/min y calentados desde ésta temperatura hasta los 50oC a la misma velocidad. Los datos del flujo de calor endotérmico producido durante este proceso de fusión, permiten obtener unos perfiles térmicos de los aceites muy característicos y diferenciados entre sí, circunstancia que es utilizada para explorar la viabilidad de esta técnica de análisis térmico, en la caracterización de mezclas del aceite de oliva virgen con los otros dos tipos de aceite: oliva refinado crudo y aceite de oliva de segunda centrifugación inmediata, obteniéndose, igualmente, curvas de fusión diferenciadas respecto del aceite genuino. Los calores de fusión obtenidos para estos aceites han sido de 69,2 ± 0,7 J/g para el aceite de segunda centrifugación inmediata, 73,1 ± 0,9 J/g para el aceite de oliva virgen extr...
A gene (aacC7) encoding an aminocyclitol 3-N-acetyltransferase type VII [AAC(3)-VII] from Strepto... more A gene (aacC7) encoding an aminocyclitol 3-N-acetyltransferase type VII [AAC(3)-VII] from Streptomyces rimosus forma paramomycinus NRRL 2455 was cloned in the Streptomyces plasmid pIJ702 and expressed in Streptomyces lividans 1326. Subcloning experiments located the aacC7 structural gene on a 1.05-kilobase DNA sequence. The direction of transcription of aacC7 was determined by using riboprobes synthesized in vitro from a DNA fragment internal to the gene. A DNA segment encoding the AAC(3)-VII activity and comprising 1,495 base pairs was sequenced. The aacC7 gene was located in an open reading frame of 864 base pairs that encoded a polypeptide of Mr 31,070, consistent with the Mr (32,000) of the AAC(3)-VII enzyme as determined by physicochemical methods. High-resolution S1 nuclease mapping suggested that transcription starts at or near the A residue of the ATG initiator codon. A DNA fragment from the 5' region of aacC7 had promoter activity in the promoter-probe plasmid pIJ486. T...
Pur7 is the product of a gene from the puromycin biosynthetic pur cluster of Streptomyces albonig... more Pur7 is the product of a gene from the puromycin biosynthetic pur cluster of Streptomyces alboniger. It was expressed in Escherichia coli as a recombinant protein fused to a His tag and then was highly purified through a Ni(2+) column. It showed a 3'-amino-3'-dATP pyrophosphohydrolase (nudix) activity which produced 3'-amino-3'-dAMP and pyrophosphate. This is consistent with the presence of a nudix box in its amino acid sequence. As observed with other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for Mg(2+). Among a large variety of other nucleotides tested, only 3'-amino-3'-dTTP was a Pur7 substrate, although at lower reaction rates than 3'-amino-3'-dATP. These findings suggest that Pur7 has a high specificity for the 3' amino group at the ribofuranoside moiety of these two substrates. The K(m) and V(max) values for these dATP and dTTP derivatives were 120 microM and 17 microM/min and 3.45 mM and 12.5 microM/min, respective...
The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2... more The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 alpha-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that alpha-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. alpha-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with en...
In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two bet... more In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two beta-carotene overproducer strains of the red yeast Phaffia rhodozyma. The method used has been pulsed field gel electrophoresis, which has determined 9 DNA chromosomal bands in the yeast genome. The two largest bands are triplets and two other bands, VI and VIII, seem to be doublets. The size of the chromosomal bands varies between 0.35 and 2.5 Mb, suggesting a genome size of 25 Mb. The technique used, complemented with hybridization assays using specific DNA probes, provides direct information about the genomic organization of P. rhodozyma. We have also cloned and located in chromosomal bands different DNA sequences that code for the translation elongation factor 1 alpha (ef-1 alpha), a 7.6 kb BamHI fragment of repetitive DNA (possibly rDNA) and a randomly chosen fragment (named locus R2). Additionally, we have detected a chromosomal length polymorphism between wild-type strains and mutan...
The biologically inactive compound N-acetylpuromycin is the last intermediate of the puromycin an... more The biologically inactive compound N-acetylpuromycin is the last intermediate of the puromycin antibiotic biosynthetic pathway in Streptomyces alboniger. Culture filtrates from either this organism or Streptomyces lividans transformants harboring the puromycin biosynthetic gene cluster cloned in low-copy-number cosmids contained an enzymic activity which hydrolyzes N-acetylpuromycin to produce the active antibiotic. A gene encoding the deacetylase enzyme was located at one end of this cluster, subcloned in a 2.5-kb DNA fragment, and expressed from a high-copy-number plasmid in S. lividans.
Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside ... more Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S. alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S. alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 1H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolas...
The neuroepithelium is a germinal epithelium containing progenitor cells that produce almost all ... more The neuroepithelium is a germinal epithelium containing progenitor cells that produce almost all of the central nervous system cells, including the ependyma. The neuroepithelium and ependyma constitute barriers containing polarized cells covering the embryonic or mature brain ventricles, respectively; therefore, they separate the cerebrospinal fluid that fills cavities from the developing or mature brain parenchyma. As barriers, the neuroepithelium and ependyma play key roles in the central nervous system development processes and physiology. These roles depend on mechanisms related to cell polarity, sensory primary cilia, motile cilia, tight junctions, adherens junctions and gap junctions, machinery for endocytosis and molecule secretion, and water channels. Here, the role of both barriers related to the development of diseases, such as neural tube defects, ciliary dyskinesia, and hydrocephalus, is reviewed.
Page 1. 947 Tras una breve introducción histórica y después de la codificación genérica de los di... more Page 1. 947 Tras una breve introducción histórica y después de la codificación genérica de los diferentes modos de estimu-lación, se describen las técnicas de implantación y se enumeran los requisitos que se consideran ...
The mechanism conferring resistance to paromomycin in Streptomyces rimosus forma paromomycinus, t... more The mechanism conferring resistance to paromomycin in Streptomyces rimosus forma paromomycinus, the producing organism, was studied at the level of both protein synthesis and drug-inactivating enzymes. Ribosomes prepared from this organism grown in either production or nonproduction medium were fully sensitive to paromomycin. A paromomycin acetyltransferase and a paromomycin phosphotransferase, both characteristic of the producer, were highly purified from extracts prepared from two Streptomyces lividans transformants harboring the relevant genes inserted in pIJ702-derived plasmids. In vitro, paromomycin was inactivated by either activity. In vivo, however, S. lividans clones containing the gene for either enzyme inserted in the low-copy-number plasmid pIJ41 were resistant to only low levels of paromomycin. In contrast, an S. lividans transformant containing both genes inserted in the same pIJ41-derived plasmid displayed high levels of resistance to paromomycin. These results indica...
Se analiza, mediante calorimetría diferencial de barrido, muestras de aceite de oliva de diferent... more Se analiza, mediante calorimetría diferencial de barrido, muestras de aceite de oliva de diferentes categorías y mezclas de estos. Los aceites son congelados previamente a ¿100oC, a una velocidad de 5oC/min y calentados desde ésta temperatura hasta los 50oC a la misma velocidad. Los datos del flujo de calor endotérmico producido durante este proceso de fusión, permiten obtener unos perfiles térmicos de los aceites muy característicos y diferenciados entre sí, circunstancia que es utilizada para explorar la viabilidad de esta técnica de análisis térmico, en la caracterización de mezclas del aceite de oliva virgen con los otros dos tipos de aceite: oliva refinado crudo y aceite de oliva de segunda centrifugación inmediata, obteniéndose, igualmente, curvas de fusión diferenciadas respecto del aceite genuino. Los calores de fusión obtenidos para estos aceites han sido de 69,2 ± 0,7 J/g para el aceite de segunda centrifugación inmediata, 73,1 ± 0,9 J/g para el aceite de oliva virgen extr...
A gene (aacC7) encoding an aminocyclitol 3-N-acetyltransferase type VII [AAC(3)-VII] from Strepto... more A gene (aacC7) encoding an aminocyclitol 3-N-acetyltransferase type VII [AAC(3)-VII] from Streptomyces rimosus forma paramomycinus NRRL 2455 was cloned in the Streptomyces plasmid pIJ702 and expressed in Streptomyces lividans 1326. Subcloning experiments located the aacC7 structural gene on a 1.05-kilobase DNA sequence. The direction of transcription of aacC7 was determined by using riboprobes synthesized in vitro from a DNA fragment internal to the gene. A DNA segment encoding the AAC(3)-VII activity and comprising 1,495 base pairs was sequenced. The aacC7 gene was located in an open reading frame of 864 base pairs that encoded a polypeptide of Mr 31,070, consistent with the Mr (32,000) of the AAC(3)-VII enzyme as determined by physicochemical methods. High-resolution S1 nuclease mapping suggested that transcription starts at or near the A residue of the ATG initiator codon. A DNA fragment from the 5' region of aacC7 had promoter activity in the promoter-probe plasmid pIJ486. T...
Pur7 is the product of a gene from the puromycin biosynthetic pur cluster of Streptomyces albonig... more Pur7 is the product of a gene from the puromycin biosynthetic pur cluster of Streptomyces alboniger. It was expressed in Escherichia coli as a recombinant protein fused to a His tag and then was highly purified through a Ni(2+) column. It showed a 3'-amino-3'-dATP pyrophosphohydrolase (nudix) activity which produced 3'-amino-3'-dAMP and pyrophosphate. This is consistent with the presence of a nudix box in its amino acid sequence. As observed with other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for Mg(2+). Among a large variety of other nucleotides tested, only 3'-amino-3'-dTTP was a Pur7 substrate, although at lower reaction rates than 3'-amino-3'-dATP. These findings suggest that Pur7 has a high specificity for the 3' amino group at the ribofuranoside moiety of these two substrates. The K(m) and V(max) values for these dATP and dTTP derivatives were 120 microM and 17 microM/min and 3.45 mM and 12.5 microM/min, respective...
The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2... more The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 alpha-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that alpha-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. alpha-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with en...
In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two bet... more In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two beta-carotene overproducer strains of the red yeast Phaffia rhodozyma. The method used has been pulsed field gel electrophoresis, which has determined 9 DNA chromosomal bands in the yeast genome. The two largest bands are triplets and two other bands, VI and VIII, seem to be doublets. The size of the chromosomal bands varies between 0.35 and 2.5 Mb, suggesting a genome size of 25 Mb. The technique used, complemented with hybridization assays using specific DNA probes, provides direct information about the genomic organization of P. rhodozyma. We have also cloned and located in chromosomal bands different DNA sequences that code for the translation elongation factor 1 alpha (ef-1 alpha), a 7.6 kb BamHI fragment of repetitive DNA (possibly rDNA) and a randomly chosen fragment (named locus R2). Additionally, we have detected a chromosomal length polymorphism between wild-type strains and mutan...
The biologically inactive compound N-acetylpuromycin is the last intermediate of the puromycin an... more The biologically inactive compound N-acetylpuromycin is the last intermediate of the puromycin antibiotic biosynthetic pathway in Streptomyces alboniger. Culture filtrates from either this organism or Streptomyces lividans transformants harboring the puromycin biosynthetic gene cluster cloned in low-copy-number cosmids contained an enzymic activity which hydrolyzes N-acetylpuromycin to produce the active antibiotic. A gene encoding the deacetylase enzyme was located at one end of this cluster, subcloned in a 2.5-kb DNA fragment, and expressed from a high-copy-number plasmid in S. lividans.
Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside ... more Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S. alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S. alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 1H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolas...
The neuroepithelium is a germinal epithelium containing progenitor cells that produce almost all ... more The neuroepithelium is a germinal epithelium containing progenitor cells that produce almost all of the central nervous system cells, including the ependyma. The neuroepithelium and ependyma constitute barriers containing polarized cells covering the embryonic or mature brain ventricles, respectively; therefore, they separate the cerebrospinal fluid that fills cavities from the developing or mature brain parenchyma. As barriers, the neuroepithelium and ependyma play key roles in the central nervous system development processes and physiology. These roles depend on mechanisms related to cell polarity, sensory primary cilia, motile cilia, tight junctions, adherens junctions and gap junctions, machinery for endocytosis and molecule secretion, and water channels. Here, the role of both barriers related to the development of diseases, such as neural tube defects, ciliary dyskinesia, and hydrocephalus, is reviewed.
Page 1. 947 Tras una breve introducción histórica y después de la codificación genérica de los di... more Page 1. 947 Tras una breve introducción histórica y después de la codificación genérica de los diferentes modos de estimu-lación, se describen las técnicas de implantación y se enumeran los requisitos que se consideran ...
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