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Biomedical subjects

F Rojo

Publications and source records attributed to F Rojo.

At least 19 recordsLinked to original sources
PubMed2000

Health services accessibility among Spanish elderly.

The paper aims to identify the variables that best explain the use of health services by people aged 65 and over in Spain. The data comes from the 1993 Spanish National Health Survey (ENSE 93). The conceptual framework is the model proposed by Andersen, who suggests that utilisation is a function of predisposition to use the services, the ability to use them and of need. A bivariate and multivariate analysis (SPSS-X Discriminant Procedure) is conducted to define the predictors that best discriminate users and non-users. The use of each health service is explained by a different set of variables. The need variables play a more important role in predicting the use of non-discretionary services that are more closely related to healing processes (medical consultations, emergencies and hospitalisation). The predisposing and enabling variables are more relevant in explaining the use of dental services, indicating a certain degree of inequity of these discretionary services.

Aged↗
PubMed1999

The switch from early to late transcription in phage GA-1: characterization of the regulatory protein p4G.

The transcription program of the Bacillus phage GA-1, a distant relative of phage Phi29, has been studied. Transcription of the GA-1 genome occurred in two stages, early and late. Early genes were expressed from two promoters equivalent to the Phi29 A2b and A2c promoters, whereas late transcription started at a site equivalent to the Phi29 late A3 promoter. The activity of the GA-1 early A2b and A2c promoters diminished 10 minutes after infection, a time at which expression of the late promoter increased significantly. The switch from early to late transcription required protein synthesis, suggesting the need for viral protein(s). An open reading frame was found in the GA-1 genome coding for a protein showing a 53 % similarity to Phi29 regulatory protein p4, and was named p4G. In Phi29, protein p4 represses the early A2b and A2c promoters and activates the late A3 promoter by recruiting RNA polymerase to it. A binding site for protein p4Gwas localized upstream from the GA-1 late A3 promoter, overlapping with the early A2b promoter. In vitro, protein p4Gprevented the binding of RNA polymerase to the GA-1 early A2b promoter but, unlike in Phi29, had no effect on the expression of the late A3 promoter: RNA polymerase could efficiently bind and initiate transcription from the A3 promoter in the absence of protein p4G. Therefore, activation of late transcription occurs differently in GA-1 and Phi29. We propose that protein p4Gis an anti-repressor which inhibits the binding to the late promoter of an unknown repressor factor present in the host strain.

Amino Acid Sequence↗
PubMed1999

The prokaryotic beta-recombinase catalyzes site-specific recombination in mammalian cells.

The development of new strategies for the in vivo modification of eukaryotic genomes has become an important objective of current research. Site-specific recombination has proven useful, as it allows controlled manipulation of murine, plant, and yeast genomes. Here we provide the first evidence that the prokaryotic site-specific recombinase (beta-recombinase), which catalyzes only intramolecular recombination, is active in eukaryotic environments. beta-Recombinase, encoded by the beta gene of the Gram-positive broad host range plasmid pSM19035, has been functionally expressed in eukaryotic cell lines, demonstrating high avidity for the nuclear compartment and forming a clear speckled pattern when assayed by indirect immunofluorescence. In simian COS-1 cells, transient beta-recombinase expression promoted deletion of a DNA fragment lying between two directly oriented specific recognition/crossing over sequences (six sites) located as an extrachromosomal DNA substrate. The same result was obtained in a recombination-dependent lacZ activation system tested in a cell line that stably expresses the beta-recombinase protein. In stable NIH/3T3 clones bearing different number of copies of the target sequences integrated at distinct chromosomal locations, transient beta-recombinase expression also promoted deletion of the intervening DNA, independently of the insertion position of the target sequences. The utility of this new recombination tool for the manipulation of eukaryotic genomes, used either alone or in combination with the other recombination systems currently in use, is discussed.

Animals↗
PubMed1999

Role of the alternative sigma factor sigmaS in expression of the AlkS regulator of the Pseudomonas oleovorans alkane degradation pathway.

The AlkS protein activates transcription from the PalkB promoter, allowing the expression of a number of genes required for the assimilation of alkanes in Pseudomonas oleovorans. We have identified the promoter from which the alkS gene is transcribed, PalkS, and analyzed its expression under different conditions and genetic backgrounds. Transcription from PalkS was very low during the exponential phase of growth and increased considerably when cells reached the stationary phase. The PalkS -10 region was similar to the consensus described for promoters recognized by Escherichia coli RNA polymerase bound to the alternative sigma factor sigmaS, which directs the expression of many stationary-phase genes. Reporter strains containing PalkS-lacZ transcriptional fusions showed that PalkS promoter is very weakly expressed in a Pseudomonas putida strain bearing an inactivated allele of the gene coding for sigmaS, rpoS. When PalkS was transferred to E. coli, transcription started at the same site and expression was higher in stationary phase only if sigmaS-RNA polymerase was present. The low levels of AlkS protein generated in the absence of sigmaS were enough to support a partial induction of the PalkB promoter. The -10 and -35 regions of PalkS promoter also show some similarity to the consensus recognized by sigmaD-RNA polymerase, the primary form of RNA polymerase. We propose that in exponential phase PalkS is probably recognized both by sigmaD-RNA polymerase (inefficiently) and by sigmaS-RNA polymerase (present at low levels), leading to low-level expression of the alkS gene. sigmaS-RNA polymerase would be responsible for the high level of activity of PalkS observed in stationary phase.

Alkanes↗
PubMed1998

Binding of phage phi29 protein p4 to the early A2c promoter: recruitment of a repressor by the RNA polymerase.

Regulatory protein p4 from Bacillus subtilis phage Phi29 represses the early A2c promoter by binding upstream from RNA polymerase and interacting with the C-terminal domain of the RNA polymerase alpha subunit. This interaction stabilizes the RNA polymerase at the promoter in such a way that promoter clearance is prevented. Here, the binding of protein p4 to the A2c promoter has been studied. In the absence of RNA polymerase, protein p4 was found to bind with low affinity to a site centered at position -39 relative to the transcription start site. When RNA polymerase was present, protein p4 was displaced from this site and bound instead to a different target centered at position -71. Stable binding to this site requires the interaction of protein p4 with the C-terminal domain of the RNA polymerase alpha-subunit. Both sites contain sequences resembling the well-characterized p4 binding site present at the late A3 promoter, to which p4 binds with high affinity. A mutational analysis revealed that the site at -71 is critical for a stable interaction between protein p4 and RNA polymerase, and for efficient repression, whereas mutation of the site at -39 had only a small effect on repression efficiency. Therefore, RNA polymerase plays an active role in the repression mechanism by stabilizing the repressor at the promoter, generating a nucleoprotein complex that is too stable to allow promoter clearance.

Bacillus subtilis↗
PubMed1998

Selection and development of a Spanish precocious strain of Eimeria necatrix.

A precocious line of Eimeria necatrix (PEN E-281/20) with an abbreviated life cycle was derived from a Spanish field strain (E-281) by repeated passages of the first shed oocysts recovered from the caecal contents of previously infected chickens. After 20 passages, the 'useful' prepatent period (time from infection to obtaining sufficient oocysts to repassage) of the parasite was reduced by 30 h (from 148 to 118 h). The earliest oocysts found in the caecal content were 114 h postinfection (hpi), on the 19th passage. The pathogenicity of the parasite was reduced in comparison with the parent strain, its immunogenicity against homologous and heterologous strains was maintained and its reproductive capacity was similar to or higher than that of the parent strain. Compared with the parent strain, the second generation of schizonts was reduced in size (reduced pathogenicity), third generation schizonts were bigger and with more merozoites (maintenance of the reproductive index) and the life cycle progressed faster from the second generation of schizonts (reduction of prepatent period). Complete second schizogony, from trophozoites to mature schizonts was observed frequently in the caeca of birds infected with both parent and precocious lines.

Animals↗
PubMed1998

beta Recombinase catalyzes inversion and resolution between two inversely oriented six sites on a supercoiled DNA substrate and only inversion on relaxed or linear substrates.

The beta recombinase, in the presence of a chromatin-associated protein such as Hbsu, catalyzes DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented six sites. Hbsu stabilizes the formation of the recombination complex (Alonso, J. C., Weise, F., and Rojo, F. (1995) J. Biol. Chem. 270, 2938-2945). In this study we show that resolution by beta recombinase strictly requires supercoiled DNA, but inversion does not. On a substrate with two inversely oriented six sites, beta recombinase catalyzed both resolution and inversion if the DNA was supercoiled but only inversion if the substrate was relaxed or linear. Hbsu was critical for the formation of synaptic complexes; its concentration relative to that of the supercoiled DNA substrate determined whether resolution or inversion products were preferentially formed. The results suggest that the beta recombinase forms unproductive short-lived synaptic complexes between two juxtaposed inversely oriented six sites; the presence of 3 to 13 Hbsu dimers per supercoiled DNA molecule would stabilize a synaptic complex with a relative geometry of the six sites allowing beta recombinase preferentially to achieve resolution. Supercoiling probably helps to overcome an energetic barrier, since resolution does not occur in relaxed DNA. The presence of >30 Hbsu dimers per DNA molecule probably favors the formation of a recombination complex with a different geometry since the reaction is directed preferentially toward DNA inversion.

Catalysis↗
PubMed1998

Substitution of the C-terminal domain of the Escherichia coli RNA polymerase alpha subunit by that from Bacillus subtilis makes the enzyme responsive to a Bacillus subtilis transcriptional activator.

Regulatory protein p4 of Bacillus subtilis phage phi 29 activates transcription from the viral late A3 promoter by interacting with the C-terminal domain (CTD) of the B. subtilis RNA polymerase alpha subunit, thereby stabilizing the holoenzyme at the promoter. Protein p4 does not interact with the Escherichia coli RNA polymerase and cannot activate transcription with this enzyme. We have constructed a chimerical alpha subunit containing the N-terminal domain of the E. coli alpha subunit and the CTD of the B. subtilis alpha subunit. Reconstitution of RNA polymerases containing this chimerical alpha subunit, the E. coli beta and beta' subunits, and the vegetative sigma factor from either E. coli (sigma 70) or B. subtilis (sigma A), generated hybrid enzymes that were responsive to protein p4 and efficiently supported activation at the A3 promoter. Protein p4 activated transcription with the chimerical enzymes through the same activation surface used with B. subtilis RNA polymerase. Therefore, the B. subtilis alpha-CTD allowed activation by p4 even when the rest of the RNA polymerase subunits belonged to E. coli, a distantly related bacterium. These results strongly suggest that protein p4 works essentially by serving as an anchor that stabilizes RNA polymerase at the promoter.

Amino Acid Sequence↗
PubMed1998

Transcription activation and repression by interaction of a regulator with the alpha subunit of RNA polymerase: the model of phage phi 29 protein p4.

Regulatory protein p4, encoded by Bacillus subtilis phage phi 29, has proved to be a very useful model to analyze the molecular mechanisms of transcription regulation. Protein p4 modulates the transcription of phage phi 29 genome by activating the late A3 promoter (PA3) and simultaneously repressing the two main early promoters, A2b and A2c (or PA2b and PA2c). This review describes in detail the regulatory mechanism leading to activation or repression, and discusses them in the context of the recent findings on the role of the RNA polymerase alpha subunit in transcription regulation. Activation of PA3 implies the p4-mediated stabilization of RNA polymerase at the promoter as a closed complex. Repression of the early A2b promoter occurs by binding of protein p4 to a site that partially overlaps the -35 consensus region of the promoter, therefore preventing the binding of RNA polymerase to the promoter. Repression of the A2c promoter, located 96 bp downstream from PA2b, occurs by a different mechanism that implies the simultaneous binding of protein p4 and RNA polymerase to the promoter in such a way that promoter clearance is inhibited. Interestingly, activation of PA3 and repression of PA2c require an interaction between protein p4 and RNA polymerase, and in both cases this interaction occurs between the same surface of protein p4 and the C-terminal domain of the alpha subunit of RNA polymerase, which provides new insights into how a protein can activate or repress transcription by subtle variations in the protein-DNA complexes formed at promoters.

Bacillus Phages↗
PubMed1998

Transcriptional activation of the Bacillus subtilis spoIIG promoter by the response regulator Spo0A is independent of the C-terminal domain of the RNA polymerase alpha subunit.

In vitro transcription from the spoIIG promoter by Bacillus subtilis RNA polymerase reconstituted with wild-type alpha subunits and with C-terminal deletion mutants of the alpha subunit was equally stimulated by the response regulator Spo0A. Some differences in the structure of open complexes formed by RNA polymerase containing alpha subunit mutants were noted, although the wild-type and mutant polymerases appeared to use the same initiation mechanism.

Bacillus subtilis↗
PubMed1998

Carbon-source-dependent expression of the PalkB promoter from the Pseudomonas oleovorans alkane degradation pathway.

Pseudomonas oleovorans GPo1 can metabolize medium-chain-length alkanes by means of an enzymatic system whose induction is regulated by the AlkS protein. In the presence of alkanes, AlkS activates the expression of promoter PalkB, from which most of the genes of the pathway are transcribed. In addition, expression of the first enzyme of the pathway, alkane hydroxylase, is known to be influenced by the carbon source present in the growth medium, indicating the existence of an additional overimposed level of regulation associating expression of the alk genes with the metabolic status of the cell. Reporter strains bearing PalkB-lacZ transcriptional fusions were constructed to analyze the influence of the carbon source on induction of the PalkB promoter by a nonmetabolizable inducer. Expression was most efficient when cells grew at the expense of citrate, decreasing significantly when the carbon source was lactate or succinate. When cells were grown in Luria-Bertani rich medium, PalkB was strongly down-regulated. This effect was partially relieved when multiple copies of the gene coding for the AlkS activator were present and was not observed when the promoter was moved to Escherichia coli, a heterologous genetic background. Possible mechanisms responsible for PalkB regulation are discussed.

Alkanes↗
PubMed1997

Mutational analysis of a site-specific recombinase: characterization of the catalytic and dimerization domains of the beta recombinase of pSM19035.

The beta recombinase encoded by the streptococcal plasmid pSM19035, which shows 28 to 34% identity with DNA resolvases and DNA invertases, can catalyze formation of deletions or inversions between properly oriented target sites. We have constructed a number of site-directed mutations at residues that are conserved between the beta protein and other DNA recombinases of the resolvase/invertase family. The analysis of the recombination and DNA-binding ability of each mutant protein shows that the mutations affect the catalytic activity and, in two cases, the dimerization of the protein. The results suggest that the beta protein probably mediates recombination by a catalytic mechanism similar to that proposed for the resolvase/invertase family. Since the beta recombinase differs from DNA resolvases and DNA invertases in its lack of bias towards either of these reactions, the results presented support the hypothesis that its unique properties might depend on details of the architecture or assembly of the recombination complex. In addition, two beta protein mutants that can no longer form dimers in solution have provided new insights into the way the protein binds to DNA.

Amino Acid Sequence↗
PubMed1997

Transcription activation or repression by phage psi 29 protein p4 depends on the strength of the RNA polymerase-promoter interactions.

Phage psi 29 protein p4 activates the late A3 promoter and represses the early A2c promoter, in both cases by binding upstream from RNA polymerase (RNAP) and interacting with the C-terminal domain of the RNAP alpha subunit. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. We show that the position of protein p4 relative to that of RNAP, which is different at each promoter, does not dictate the outcome of the interaction. Rather, in the absence of a-35 consensus box for sigma A-RNAP activation was observed, while in its presence repression occurred. The results support the view that stabilization of RNAP at the promoter over a threshold level leads to repression.

Bacillus Phages↗
PubMed1997

The Bacillus subtilis chromatin-associated protein Hbsu is involved in DNA repair and recombination.

The Bacillus subtilis hbs gene encodes an essential chromatin-associated protein termed Hbsu. Hbsu, the counterpart of the Escherichia coli HU protein, binds DNA in a non-specific way but has a clear preference for bent, kinked or altered DNA sequences. To investigate the role of Hbsu in DNA repair and DNA recombination we have constructed a series of site-directed mutants in the hbs gene and used these mutant genes to substitute the wild-type chromosomal hbs gene. The hbs47 mutation, which codes for a mutant protein in which residue Phe-47 has been replaced by Trp, does not cause any discernible phenotype. Additional substitution of residue Arg-55 by Ala (hbs4755 mutation) rendered cells deficient in DNA repair, homologous recombination and beta protein-mediated site-specific recombination. We have also tested the effect on DNA repair of the hbs4755 mutation in combination with mutations in different functions of homologous DNA recombination (recA, recF, recG, recH and addAB). The hbs4755 mutation did not modify the sensitivity of recH and addAB cells to the DNA-damaging agents methylmethane sulphonate (MMS) or 4-nitroquinoline-1-oxide (4NQO), and it only marginally affected recF and recG cells. The hbs4755 mutation blocked intermolecular recombination in recH cells and markedly reduced it (20- to 50-fold) in recF and recG cells, but had no effect on addAB cells. Taken together, these data indicate that the Hbsu protein is required for DNA repair and for homologous DNA recombination.

Amino Acid Sequence↗
PubMed1996

Protein p4 represses phage phi 29 A2c promoter by interacting with the alpha subunit of Bacillus subtilis RNA polymerase.

Regulatory protein p4 from Bacillus subtilis phage phi 29 represses the strong viral A2c promoter (PA2c) by preventing promoter clearance; it allows RNA polymerase to bind to the promoter and form an initiated complex, but the elongation step is not reached. Protein p4 binds at PA2c immediately upstream from RNA polymerase; repression involves a contact between both proteins that holds the RNA polymerase at the promoter. This contact is held mainly through p4 residue Arg120, which is also required for activation of the phi 29 late A3 promoter. We have investigated which region of RNA polymerase contacts protein p4 at PA2c. Promoter repression was impaired when a reconstituted RNA polymerase lacking the 15 C-terminal residues of the alpha subunit C-terminal domain was used; this polymerase was otherwise competent for transcription. Binding cooperativity assays indicated that protein p4 cannot interact with this mutant RNA polymerase at PA2c. Protein p4 could form a complex at PA2c with purified wild-type alpha subunit, but not with a deletion mutant lacking the 15 C-terminal residues. Our results indicate that protein p4 represses PA2c by interacting with the C-terminal domain of the alpha subunit of RNA polymerase. Therefore, this domain of the alpha subunit can receive regulatory signals not only from transcriptional activators, but from repressors also.

Bacillus Phages↗
PubMed1996

Site-specific recombination in gram-positive theta-replicating plasmids.

This review summarises current information on the site-specific recombinases encoded by the plasmids of the Gram-positive bacteria that have low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of the recombination systems encoded by the theta-replicating plasmids and compares them with the site-specific recombinases encoded by transposons or plasmids originally isolated from Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1996

Site-specific recombination by the beta protein from the streptococcal plasmid pSM19035: minimal recombination sequences and crossing over site.

The beta recombinase from the broad host range Grampositive plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented recombination sites in the presence of a chromatin-associated protein (Hbsu). The recombination site had been localized to a 447 bp DNA segment from pSM19035. This segment includes a 90 bp region that contains two adjacent binding sites (I and II) for beta protein dimers. Using in vitro recombination assays, we show that this 90 bp region is necessary and sufficient for beta protein-mediated recombination; this defines the six site as the region required for beta protein binding. The point of crossing over has been localized to the center of site I. Hbsu has a strong binding affinity for an unknown site located within the 447 bp segment containing the six site. We discuss the possibility that Hbsu recognizes an altered DNA structure, rather than a specific sequence, generated in the synaptic complex.

Bacterial Proteins↗