Antonio Moreno Jiménez

Cloning and expression of a puromycin N-acetyl transferase gene from Streptomyces alboniger in Streptomyces lividans and Escherichia coli.

A gene (pac) encoding a puromycin N-acetyl transferase (PAC) of Streptomyces alboniger ATCC12461 was cloned in the Streptomyces plasmid pIJ702 and expressed in S. lividans 1326. Several clones resistant to puromycin were isolated and shown to carry pIJ702 with different inserts of S. alboniger DNA. They were classified as of low and high activity according to the levels of enzymatic activity expressed by them. The different levels of expression were related to the two possible orientations of the S. alboniger DNA inserts in the pIJ702 vector. Six of the recombinant plasmids contain a common 1.6-kb DNA sequence which, by subcloning experiments, was shown to carry a pac gene encoding PAC activity. The pac gene was subcloned next to the lac promoter of Escherichia coli plasmid pUC19. Only one of the two possible orientations of insertion expressed PAC activity, suggesting that it was dependent on the lac promoter. Accordingly, isopropylthio-beta-D-galactoside (IPTG) was able to stimulate the expression of the enzyme activity. These results allowed the direction of transcription of the pac gene to be determined.

Bruceantin, a novel inhibitor of peptide bond formation.

Abstract The effects of bruceantin on a number of steps of the protein synthesis process have been studied using resolved model systems from both yeast and reticulocytes. Bruceantin is a potent inhibitor of polyphenylalanine synthesis as directed by poly(U). However, inhibition is less pronounced on protein synthesis as directed by endogenous mRNA and the compound inhibits the poly(U) system only poorly if added after polyphenylalanine synthesis has been initiated. Peptide bond formation as assayed in both the fragment reaction and in the puromycin reaction with a preformed initiation complex containing ribosomes and [ 35 S]Met-tRNA F is totally blocked by bruceantin. Neither the enzymic binding of Phe-tRNA to reticulocyte ribosomes nor the formation of the 35 S-labeled tRNA · ribosome initiation complex is inhibited by bruceantin. The binding of [ 14 C]trichodermin to yeast ribosomes is strongly inhibited by bruceantin. A Klotz plot shows that both these drugs bind to ribosomes in mutually exclusive fashion and it can be calculated that bruceantin binds to the peptidyltransferase center with K d = 0.34 μ M. This high affinity is considerably lower for polyribosomes ( K d = 557 μ M), which may explain the earlier finding that bruceantin only stabilizes polyribosomes at high drug concentrations.

Cycloheximide resistance as a yeast cloning marker

SummaryIn CYH2/cyh2 heterozygous diploids of the yeast Saccharomyces cerevisiae resistance is dominant over sensitivity at low (0.5–5 μg/ml) cycloheximide (cyh) concentrations. The cyh-resistant haploid strain MMY1 confers relatively high (10 μg/ml) cyh-resistance to heterozygous diploids constructed by mating this strain with cyh-sensitive haploid strains. We present here a genetic and biochemical study of strain MMY1. Analysis of tetrads obtained from a MMY1 heterozygous diploid showed that two unlinked nuclear mutations, determining high-and low-cycloheximide resistance, were present in MMY1. From a genomic library of this strain, constructed in vector YCp50, two plasmids (pRC1 and pRC13) have been isolated which, respectively, confer high-and low-resistance phenotypes to cyh-sensitive S. cerevisiae strains. The restriction maps of pRC1 and pRC13 are totally unrelated. This finding suggests that the genes harboring the two mutations encoding cyh-resistance from MMY1 were cloned in plasmids pRC1 and pRC13, respectively. Pulse field gel electrophoresis showed that the DNA insert of pRC1 maps at either chromosome VII or XV, whereas that from pRC13 maps at chromosome XI. This latter gene appears to define a previously unreported locus and has been named cyh5. By restriction and nucleotide sequencing analysis, the cyh gene present in pRC1 has been shown to correspond to cyh2, which maps at chromosome VII. These results suggest that the dominant cyh-resistance phenotype conferred by MMY1 in heterozygous diploids is promoted by the presence of both cyh2 and cyh5. A 2.3 kb NcoI fragment, containing cyh2 from pRC1, has been inserted in vectors YCp50, YEp13 and YIp5. By selecting at discriminating cyh concentrations, the resulting constructs efficiently transform a variety of haploid and diploid yeasts of both laboratory and industrial origin.

Acetylation of puromycin by Streptomyces, alboniger the producing organism

Streptomyces alboniger produces the antibiotic puromycin and expresses an enzymic activity which acetylates the drug using acetyl CoA. The N-acetyl-puromycin formed is biologically inactive against protein synthesis in Bacillus subtilis (as assayed in vivo).

Two genes in Streptomyces alboniger puromycin biosynthesis pathway are closely linked

Several recombinant plasmids, derived from the Streptomyces vector pIJ702 and carrying different stretches of Streptomyces alboniger DNA encoding the gene (pac) for puromycin N-acetyl transferase [Vara et al., Gene 33 (1985) 197-206] were found to also include the gene (dmpM) for the O-demethylpuromycin O-methyl transferase enzyme. Both genes are present on the same 2.4-kb DNA fragment. Coupled transcription-translation experiments suggested that the dmpM gene product is a 44-kDa polypeptide and that both dmpM and pac might belong to different transcriptional units. The level of expression of the dmpM gene was dependent upon the orientation of insertion in the vector.

The mechanism of resistance to puromycin and to the puromycin-precursor O-demethyl-puromycin in Streptomyces alboniger

Ribosomes from Streptomyces alboniger are sensitive in vitro to puromycin and, to a lesser extent, to the puromycin-precursor O-demethyl-puromycin. The puromycin-inactivating enzyme (puromycin N-acetyltransferase) from S. alboniger also N-acetylates O-demethyl-puromycin. This finding indicates that in certain antibiotic-producing organisms the antibiotic-inactivating enzymes may play a role in self-defence against toxic precursor molecules.

ENTENDIMIENTO Y NATURALEZA DE LA CIENTIFICIDAD GEOTECNOLÓGICA: UNA APROXIMACIÓN DESDE EL PRAGMATISMO EPISTEMOLÓGICO

Understanding and nature of geo-technological science: an epistemological pragmatism based approach Geographical information technologies (GIT) are producing a deep impact in Geography and other disciplines. According to the thesis of some philosophers of science and geographers, in this paper it is proposed and argued that the observed changes in the research encompass the emersion of a new and distinct scientific praxis. Based on the “research traditions” concept made in the pragmatist epistemology and on the recent contributions of philosophers of technology, the main elements and characteristics of this GIT based emerging science nature are exposed. In this way, it is intended to uncover that GIT have a much more essential and deeper role on research than a simple tool.

The promoter element GTACAAG of the SGA and STA2 genes is a possible target site for repression by the STA10 gene product from Saccharomyces cerevisiae

The SGA and STA2 genes that, respectively, encode the intra- and extracellular glucoamylases of Saccharomyces cerevisiae are coregulated negatively, at the level of transcription, by the STA10 gene. This finding was re-examined by determining the effects of STA10 on the expression of gene constructs containing different fragments from the SGA and STA2 promoter regions fused to the lacZ gene. Repression was observed only for promoter fragments carrying the sequence GTACAAG indicating that this element is responsible for the coregulation of SGA and STA2 by STA10.

A Visual Language for Modelling and Simulation of Networks of Evolutionary Processors

The goal of this work is to provide the jNEP user with a visual tool to graphically design the NEPs under consideration. jNEP is a Java simulator for NEPs previously developed by some of the authors of this paper. We have designed a domain specific visual language for NEPs by means of AToM3.We have also taken advantage of the AToM3’s graph grammar modules to automate some mechanical and time-consuming designing tasks, such as properly placing filters close to their processors, and defining some kinds of standard graph topologies. AToM3 is a python tool previously developed by some of the authors of this paper.

EL CONTRASTE INTRAURBANO DE LA CONTAMINACIÓN DEL AIRE POR NO2 Y O3: ESTUDIO EN GRANDES CIUDADES ESPAÑOLAS CON DATOS OBSERVADOS E INTERPOLADOS CON SIG

Dos de los contaminantes mas significativos de la calidad del aire urbano son el dioxido de nitrogeno y el ozono troposferico, ya que ocasionan problemas de incumplimiento de normativa y de salud en los ciudadanos. El hecho de que uno de los precursores del ozono sean los oxidos de nitrogeno anticipa una relacion espacial negativa entre ambos contaminantes. El objetivo principal de este trabajo estriba en comparar el patron espacial de ellos en las ciudades de Madrid, Barcelona y Sevilla, a fin de mostrar y medir dicha relacion. A tal fin, se utilizan datos observados en las estaciones y estimados para toda la ciudad con tecnicas de interpolacion espacial. Mediante diversas herramientas cuantitativas, graficas y de geoprocesamiento aplicadas con SIG y software estadistico se desvela y comprueba la intensidad y el sentido de la relacion hipotetica, asi como tambien los problemas de la escasez de datos. Con ello se aporta luz adicional sobre los sindromes de contaminacion que en la compleja atmosfera urbana afloran.