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Biomedical subjects

M Espinosa

Publications and source records attributed to M Espinosa.

At least 73 records · Page 4Linked to original sources
PubMed1995

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein.

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.

Amino Acid Sequence↗
PubMed1994

Correlation between DNA bending and transcriptional activation at a plasmid promoter.

Plasmid pLS1 replicates in both Gram-positive and Gram-negative bacteria. One of the elements controlling plasmid replication is an antisense RNA (ctRNA II), transcribed from promoter PctII, which should be expressed in all the hosts where the plasmid replicates. We show here that expression from PctII and the presence of curved DNA regions located upstream of this promoter are related. DNA fragments containing these upstream regions exhibit anomalous electrophoretic mobility, and their presence increases in vivo and in vitro transcription, apparently with an independence of any activator protein. RNA polymerase is able to bend a DNA fragment containing PctII, suggesting that an increase in the RNA polymerase-DNA contacts is an important step in transcription initiation. Furthermore, the upstream activating region could be substituted by targets of unrelated DNA-bending proteins. This finding supports the role of curved DNA as a transcriptional modulator.

Base Sequence↗
PubMed1994

Chemical synthesis of a fully active transcriptional repressor protein.

Plasmid pLS1-encoded 45-amino acid transcriptional repressor CopG (formerly RepA) has been chemically synthesized. A one-step purification of the synthetic protein has been developed, which yields high levels of pure protein with low or no contamination of truncated products. We have compared some properties of the chemical CopG protein with those of the biologically purified CopG. The two proteins were indistinguishable in (i) their ability to generate specific protein-DNA complexes, (ii) their capacity to protect a restriction site included within the CopG DNA target, and (iii) in their in vitro capacity to specifically repress synthesis of copG mRNA.

Amino Acid Sequence↗
PubMed1994

Fractures of the C-2 vertebral body.

Vertical C-2 body fractures are presented in 15 patients with clinical and imaging correlations that suggest the existence of a variety of mechanisms of injury. In these patients, clinical and imaging correlations were derived by: 1) defining the point of impact by clinical examination; 2) defining the point of impact by soft-tissue changes on cranial magnetic resonance (MR) imaging or computerized tomography (CT); 3) obtaining an accurate history of the mechanism of injury; and 4) spine imaging (x-ray studies, CT, and MR imaging) of the C-2 body fracture and surrounding bone and soft tissue. The cases presented involve the region located between the dens and the pars interarticularis of the axis. Although these fractures are rarely reported, they are not uncommon. An elucidation of their pathological anatomy helps to further the understanding of the mechanistic etiology of upper cervical spine trauma. A spectrum of mechanisms of injury causing upper cervical spine fractures was observed. The type of injury incurred is determined predominantly by the force vector applied during impact and the intrinsic strength and anatomy of C-2 and its surrounding spinal elements. From this clinical experience, two types of vertical C-2 body fractures are defined and presented: coronally oriented (Type 1) and sagittally oriented (Type 2). A third type of C-2 body fracture, the horizontal rostral C-2 fracture (Type 3), is added for completeness; this Type 3 fracture is the previously described Type III odontoid process fracture described by Anderson and D'Alonzo.

Adult↗
PubMed1993

Characterization of the Streptococcus pneumoniae maltosaccharide regulator MalR, a member of the LacI-GalR family of repressors displaying distinctive genetic features.

The gene encoding a transcriptional repressor of the maltosaccharide utilization operons of the Gram-positive bacterium Streptococcus pneumoniae (malR) has been cloned and sequenced. The genetic structure of the locus reveals the presence of an upstream gene necessary for growth on maltotetraose medium (malA). The phenotype of malR- and malA- mutants obtained by interruption of the coding regions suggests that both genes could belong to the same transcription unit. Two copies of a DNA motif consisting of three conserved regions of 59, 42, and 49 nucleotides were found located upstream and downstream of the malA-malR putative operon. These DNA structures are almost identical to the reported Box sequences associated with several genes of S. pneumoniae. The protein encoded by malR was visualized and partially purified after selective expression in Escherichia coli, whereas the product of malA was identified in vitro. The amino acid sequence of MalR displays similarities with the Lac and Gal family of repressors. The highest similarities were found when comparing MalR with the E. coli MalI repressor, which is related with an indirect induction pathway of the maltose regulon. The significance of these similarities is discussed in terms of the possible evolutionary pathways followed by structural and regulatory genes of sugar utilization systems in bacteria.

Amino Acid Sequence↗
PubMed1993

Protein-induced bending as a transcriptional switch.

The question of whether protein-induced DNA bending can act as a switch factor when placed upstream of an array of promoters located in tandem was investigated in vivo. The catabolite activating protein binding site of the fur operon was replaced by the binding site of the RepA repressor protein, which is able to bend DNA immediately after binding. Appropriately phased induced bending could act as a transcriptional switch factor in vivo.

Bacterial Proteins↗
PubMed1993

Structure of the maltodextrin-uptake locus of Streptococcus pneumoniae. Correlation to the Escherichia coli maltose regulon.

The mechanism of induction of the maltose/maltodextrin regulon of the Gram-positive bacterium Streptococcus pneumoniae seems to be different to the positively controlled maltose regulons of the enteric bacteria Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. In this work, we report on the structure of the S. pneumoniae genes involved in maltodextrin uptake malX, malC and malD. Comparisons of the amino acid sequences encoded by these genes indicate that they are homologous to the E. coli MalE periplasmic maltose binding protein and the two maltose permeases MalG and MalF. The analysis of transcription start points indicates that malXCD could be transcribed from a single consensus promoter sequence. Northern analysis of the mRNA molecules pertaining to this region reveals that the transcript encompassing all these three genes is apparently cleaved at a large putative mRNA secondary structure, yielding two mRNA molecules. The smaller of these molecules would include only the malX gene while a larger fragment spans through malC and malD. The processing of mRNA has not been reported in the Gram-negative maltose regulons, and may suggest either a less evolved or a divergent system for the control of gene expression of this regulon in S. pneumoniae.

ATP-Binding Cassette Transporters↗
PubMed1993

Replication of the promiscuous plasmid pLS1: a region encompassing the minus origin of replication is associated with stable plasmid inheritance.

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RSB.

Bacillus subtilis↗
PubMed1993

In vivo definition of the functional origin of replication (ori(+)) of the promiscuous plasmid pLS1.

We have defined the minimal origin of replication of the plasmid pLS1 leading strand, as comprised within a 247 bp region, by in vivo deletion analyses. Cloning of pLS1 DNA regions containing its oriV(+) into a compatible replicon resulted in weak incompatibility towards pLS1, but only when the cloned fragment included the entire pLS1 oriV(+). Plasmids lacking a functional repB gene (which encodes the pLS1 initiator of replication RepB protein) could be established in Streptococcus pneumoniae only when RepB was supplied in trans. We conclude that all the pLS1-encoded gene products involved in its replication and control are efficient trans-complementing plasmid elements.

Bacterial Proteins↗
PubMed1993

Molecular cloning of a chromosomal DNA region encompassing the dihydrofolate reductase gene of Streptococcus pneumoniae.

Natural competence of Streptococcus pneumoniae was used to locate and enrich DNA restriction fragments, biologically active for transformation of thymidine-deficient to thymidine-proficient cells. Mutations in the dihydrofolate reductase gene are accompanied by resistance to the drug trimethoprim (Tp). A 6.5-kb region of the pneumococcal chromosome encompassing the dihydrofolate reductase gene has been cloned in plasmid pLS1. Escherichia coli mutants, resistant to Tp, became fully sensitive to the drug when they harbored the recombinant plasmid. The pneumococcal dfrA mutation has been mapped within a 500-bp DNA region.

Chromosome Mapping↗
PubMed1993

Rolling circle-replicating plasmids from gram-positive and gram-negative bacteria: a wall falls.

Rolling circle-replicating plasmids constitute a group of small, promiscuous multicopy replicons spread among eubacteria. Until recently, rolling circle replication seemed to be limited to small plasmids from Gram-positive hosts and to single-stranded bacteriophages from Gram-negative bacteria. However, characterization of two small plasmids from Gram-negative hosts has shown that this replication mechanism is general among eubacteria. This review focuses on a family of highly related promiscuous plasmids that replicate by the rolling circle mechanism, and that have been isolated from various Gram-positive bacteria and from the Gram-negative bacterium Helicobacter. They all share homologies at the leading-strand origins and at the initiator of replication proteins. The plasmids of this family have directly repeated sequences at their plus origin of replication, which is located 5' from the start point of the mRNA for the initiation of replication protein. Replication is controlled by an antisense RNA and by a transcriptional repressor protein. The features and regulatory circuits of replication of this plasmid family seem to be unique among rolling circle-replicating plasmids. Members of this family replicate autonomously in Gram-positive and -negative hosts.

Amino Acid Sequence↗
PubMed1993

Effect of clomiphene citrate on hormonal profile in male hemodialysis and kidney transplant patients.

The aim of this study was to evaluate the role of clomiphene citrate (CC) therapy in the hypothalamus-pituitary-gonadal axis of male uremic subjects. Thirty-four patients on hemodialysis (HD) and 8 successful kidney transplant subjects (RT) were evaluated. Nine healthy males were used as controls (C). At baseline, zinc, testosterone (TEST), prolactin (PRL), FSH, LH and estradiol plasma concentrations were measured. All subjects were treated with CC (100 mg/day) for a week. The aforementioned parameters were determined again on the seventh day of CC therapy, and 3 days after drug withdrawal. Following CC, there was a rise in FSH, LH and TEST levels in all subjects (p < 0.05); it is interesting to stress that TEST became normal in HD. In addition, we observed a decrease of PRL after CC only in HD patients (p < 0.01). In summary, CC was able to partially correct most of the hormonal disturbances of the gonadal axis in uremic patients.

Adult↗
PubMed1993

Gene deletion suggests a role for Trypanosoma cruzi surface glycoprotein GP72 in the insect and mammalian stages of the life cycle.

We have explored the biological function of a surface glycoprotein (GP72) of Trypanosoma cruzi by studying a null mutant parasite, generated by targeted gene deletion. GP72 deletion affected parasite morphology in several stages of the life cycle. Insect midgut (epimastigote) forms had a detached flagellum (apomastigote) in the null mutant. The abnormal flagellar phenotype persisted during development of the infective (metacyclic) forms but there was no impairment in the acquisition of complement resistance, sialidase expression or cell infectivity. The GP72 null mutant could efficiently infect and proliferate in mouse macrophages and non-phagocytic L6E9 cells. The mammalian stages of the life cycle also showed major morphological abnormalities. During early subcultures in L6E9 cells, few extracellular fully flagellated forms, expressing markers characteristic of trypomastigotes, were seen. The extracellular population consisted almost exclusively of rounded forms with short flagella (micromastigote), which expressed an amastigote-specific surface marker and no sialidase. The propagation of the parasite was not affected, despite the apparent lack of the trypomastigote forms, which are thought to be primarily responsible for cell invasion. After some subcultures, the extracellular population changed to about equal numbers of micromastigotes and a range of flagellated forms that still did not include true trypomastigotes. Instead, the kinetoplast remained close to the nucleus and the flagellum emerged from the middle of the cell (mesomastigote). Half of the flagellum adhered to the cell body and the remainder was free at the anterior end. In Triatoma infestans, the survival of the mutant was dramatically reduced, suggesting that either GP72 itself, or the altered properties of the flagellum, were critical for establishment in the insect vector.

Animals↗
PubMed1992

A genetic system to study the in vivo role of transcriptional regulators in Escherichia coli.

A genetic system for studying in vivo the interactions between a transcriptional regulatory protein and its target DNA has been developed for Escherichia coli. It is composed of two compatible plasmids: one high-copy-number promoter-probe vector, and one low-copy-number vector in which the gene encoding the desired protein is cloned under the control of an inducible promoter. The system was successfully tested for its specificity and for dosage analysis by using a combination of the plasmid pLS1-encoded RepA repressor and its target DNA.

Bacterial Proteins↗
PubMed1992

The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNA II and the repressor protein, RepA.

The promiscuous plasmid pLS1 encodes two transacting elements that regulate its copy number: protein RepA and antisense RNA II. In vitro transcription showed that RNAs for both repressors are synthesized from two promoters, PAB and PII. From PAB, genes encoding RepA (transcriptional repressor) and RepB (initiator of replication) are cotranscribed, the target of RepA being located within PAB. Mutants in repA or in PAB are still sensitive to RepA. However, cloning of the repA gene in a compatible replicon did not result in incompatibility towards pLS1. From PII, the 50-nucleotide RNA II is synthesized. The main incompatibility determinant towards pLS1 corresponds to the coding sequence for RNA II. The RNA II target could be reduced to 21 nucleotides, including the RepB initiation of translation signals. We propose that plasmids of the pLS1 family (pE194, pADB201, and pLB4) share functional and structural characteristics for the regulation of their copy numbers.

Bacterial Proteins↗