Biomedical subjects

F Macián

Publications and source records attributed to F Macián.

5 recordsLinked to original sources

PubMed1995

Transcription termination in the Escherichia coli dnaA gene is not mediated by the internal DnaA box.

DnaA protein is a DNA-binding protein which recognizes a 9-bp consensus sequence called the DnaA box. By binding to DnaA boxes, DnaA protein regulates initiation of chromosomal replication and transcription of several genes. The dnaA gene contains two DnaA boxes, one located in the regulatory region and one within the structural gene. In this paper, we explore the role of the internal DnaA box in dnaA expression because it has been proposed that the DnaA box-DnaA protein complex can block transcribing RNA polymerase. Firstly, we analyzed the degree of derepression of the dnaA gene, measured as beta-galactosidase activity of a dnaA-lacZ fusion inserted onto the bacterial chromosome, produced by an extra copy number of the dnaA DnaA boxes carried by multicopy plasmids. Secondly, we analyzed repression produced by elevated levels of DnaA protein on single-copy dnaA-lacZ fusions containing or not containing the internal DnaA box. Our results indicate that the internal DnaA box does not play a regulatory role in dnaA expression.

Bacterial Proteins↗

PubMed1994

An improved vector system for constructing transcriptional lacZ fusions: analysis of regulation of the dnaA, dnaN, recF and gyrB genes of Escherichia coli.

We describe a new vector system for the in vitro construction of transcriptional fusions to the lacZ gene, which is expressed from the translational start signals of galK. The galK ribosome-binding site (RBS) and its natural preceding region ensure a constant efficiency for lacZ translation and, thus, the beta-galactosidase (beta Gal) production of a given fusion is directly proportional to the in vivo transcriptional activity of the inserted DNA fragment. Single-copy lambda prophage versions of multicopy constructs can be made by in vivo recombination. We use this system to compare the transcriptional activities of the promoters present in the dnaA-dnaN-recF-gyrB cluster. The order of strength of these promoters is gyrB > dnaA > recF > dnaN. It is assumed that gyrB belongs to the dnaA-dnaN-recF operon, because the short recF-gyrB intercistronic region does not contain a terminator. By using this new vector system, we have detected strong termination signals within recF that are functional even when recF is translated at its normal rate. The low level of transcription coming to the end of recF, and the highest activity of the gyrB promoter, as well as results obtained with several gyrB::lacZ translational fusions, support the conclusion that gyrB is predominantly expressed from its own promoter under standard growth conditions. Finally, we have found that transcription from the dnaA promoters is constant at different growth rates. This supports the idea that autoregulation of the dnaA gene is responsible for the coupling of the DnaA protein synthesis to cell mass increase, and accumulation of DnaA protein governs the initiation of chromosome replication.

Base Sequence↗

PubMed1991

Tandem transcription termination sites in the dnaN gene of Escherichia coli.

The dnaN gene of Escherichia coli encodes the beta-subunit of DNA polymerase III and maps between the dnaA and recF genes. We demonstrated previously that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. However, the recF gene has its own promoter region located in the middle of the dnaN structural gene. In this report, we use S1 mapping of mRNAs, transcriptional and translational fusions to the galK and lacZ genes, and in vitro mutagenesis to identify and characterize three tandem transcription termination sites responsible for transcriptional polarity in the dnaN-recF operon. These sites are located in the dnaN gene, downstream from the recF promoter region. Cumulatively, they terminate about 80% of the untranslated transcripts started at the recF promoters. As expected, they do not reduce transcription coming from the dnaN promoters unless dnaN translation was prematurely disrupted by the presence of a nonsense codon. The particular arrangement of regulatory elements (promoters and terminators) in the dnaN-recF region provides an exceptional in vivo system to confirm the latent termination site model of transcriptional polarity. In addition, our results contribute to the understanding of the complex regulation of the dnaA, dnaN, and recF genes. We propose that these three genes constitute an operon and that the terminators described in this work could be used to reduce expression of the distal genes of the operon under circumstances in which the dnaN translation happens to be slowed down.

Base Sequence↗

PubMedDate not supplied

Positive and negative regulatory elements in the dnaA-dnaN-recF operon of Escherichia coli.

The recF gene of E coli lies within a cluster of genes which play essential roles in DNA replication; the gene order is dnaA dnaN recF gyrB. Each of these genes has its own promoters which, with the exception of dnaA promoters, reside entirely within the translated region of the respective preceding gene. In this report, we analyze the effect of the dnaA and dnaN promoters on recF expression by translational fusions between recF and the lacZ reporter gene. Our results indicate that recF is a distal gene of the dnaA operon, and support the previous proposal that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. They also suggest that dnaA, dnaN and recF are predominantly expressed from the same mRNA although transcriptional and/or post-transcriptional mechanisms should be specifically involved in lowering expression of the recF gene. Recently, we have localized 3 tandem transcription termination sites in the second half of the dnaN gene, downstream from the recF promoters. Neither of them shows the typical features of simple terminators and apparently they do not work in a minimal system of in vitro transcription. In this report, we present evidence that only one of them is dependent on the Rho protein. Although the operon structure allows coordinate expression of dnaA, dnaN and recF, the presence of internal promoters (the dnaN and recF promoters), which appear to be inducible by DNA damage, and intracistronic terminators, whose activity is inversely proportional to the efficiency of translation, permits expression of individual genes to be independently regulated in response to altered growth conditions.

Bacterial Proteins↗

PubMedDate not supplied

Nerve conduction velocity decrease and synaptic transmission alterations in caffeine-treated rats.

The action of caffeine on peripheral neuromuscular function was studied by means of in vivo determinations of electrophysiological parameters, i.e., amplitude of extracellularly recorded muscle action potentials and nerve conduction velocity in the dorsal skeletal muscle and caudal nerve of the rat tail, respectively. Repeated exposure of the rats was carried out by adding caffeine to the drinking water for 10 days. Here we report the novel finding that motor nerve conduction velocity showed a significant decrease in caffeine-treated animals, whereas no change was observed in the amplitude of indirectly evoked extracellular muscle action potentials. The physiological recovery of the amplitude of the compound muscle action potential observed in nonintoxicated rats after high-frequency stimulation (10 Hz) was not observed in intoxicated animals and is also discussed.

Action Potentials↗