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Biomedical subjects

Ignacio G Bravo

Publications and source records attributed to Ignacio G Bravo.

11 recordsLinked to original sources
PubMed2025

Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF.

Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the "main" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus-like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.

Humans↗
PubMed2006

Phylogeny and evolution of papillomaviruses based on the E1 and E2 proteins.

Papillomaviridae are a family of small double-stranded DNA viruses that infect stratified squamous epithelia in vertebrates. Members of this family are causative agents of malignant tumours, such as cervical cancer while others are associated with benign proliferative lesions. So far, Papillomaviruses (PVs) are classified according to the sequence identity in the capsid gene L1. However, evidence has accumulated indicating a discontinuity in the evolutionary history of the L1 and L2 genes of many PVs, giving rise to differences in the phylogenetic reconstructions of the early and of the late genes. Neither the oncogenes E5, E6 and E7 nor the upstream regulatory region are suitable for phylogenetic inference due to the poor conservation along the Papillomaviridae family. We have analysed here the evolutionary relationships of the PVs with respect to the E1 and E2 proteins, and the results provide both phylogeny and biologic behaviour of the viruses. The hierarchical taxonomic relationships can be structured as an alternative classification system in which mucosal high-risk viruses, mucosal low-risk viruses and viruses associated with cutaneous lesions are grouped separately and do not appear intermingled. Some important trends are also observed: first, evolution of the PVs has not been homogeneous, even in viruses that infect the same host, and second mucosal human PVs have evolved faster than their cutaneous counterparts. The evolutionary analysis based on the E1 and E2 proteins will allow us to better understand the generation of the diversity of the PVs and the development of malignancy associated with these viruses.

Animals↗
PubMed2006

Different papillomaviruses have different repertoires of transcription factor binding sites: convergence and divergence in the upstream regulatory region.

BACKGROUND: Papillomaviruses (PVs) infect stratified squamous epithelia in warm-blooded vertebrates and have undergone a complex evolutionary process. The control of the expression of the early ORFs in PVs depends on the binding of cellular and viral transcription factors to the upstream regulatory region (URR) of the virus. It is believed that there is a core of transcription factor binding sites (TFBS) common to all PVs, with additional individual differences, although most of the available information focuses only on a handful of viruses. RESULTS: We have studied the URR of sixty-one PVs, covering twenty different hosts. We have predicted the TFBS present in the URR and analysed these results by principal component analysis and genetic algorithms. The number and nature of TFBS in the URR might be much broader than thus far described, and different PVs have different repertoires of TFBS. CONCLUSION: There are common fingerprints in the URR in PVs that infect primates, although the ancestors of these viruses diverged a long time ago. Additionally, there are obvious differences between the URR of alpha and beta PVs, despite these PVs infect similar histological cell types in the same host, i.e. human. A thorough analysis of the TFBS in the URR might provide crucial information about the differential biology of cancer-associated PVs.

Binding Sites↗
PubMed2006

Feline foamy virus-mediated marker gene transfer: identification of essential genetic elements and influence of truncated and chimeric proteins.

Retroviral vectors derived from foamy or spumaretroviruses are considered promising tools for targeted gene delivery and vaccination purposes. In order to fully exploit this potential, we identified essential cis-acting sequences on the feline foamy virus (FFV) genome by constructing and analyzing a series of FFV-based replication-deficient vector genomes. Cis-acting sequences essentially required for marker gene transfer were found to be localized at two sites on the FFV genome: (i) in the 5'-untranslated region and close to the gag ATG and (ii) in the central part of the pol gene. The presence of two cis-acting sequences and their relative location on the FFV genome are similar but not identical to the functionally corresponding elements described for simian and primate foamy viruses.

5' Untranslated Regions↗
PubMed2005

Papillomaviruses: different genes have different histories.

Papillomaviruses (PVs) infect stratified squamous epithelia in vertebrates. Some PVs are associated with different types of cancer and with certain benign lesions. It has been assumed that PVs coevolved with their hosts. However, recently it has been shown that different regions of the genome have different evolutionary histories. The PV genome has a modular nature and appeared after the addition of pre-existent blocks. This order of appearance in the PV genome is evident today in the different evolutionary rates of the different genes, with new genes--E5, E6 and E7--diverging faster than old genes--E1, E2, L2 and L1. Here, we propose an evolutionary framework aiming to integrate genome evolution, PV biology and epidemiology of PV infections.

Evolution, Molecular↗
PubMed2004

Multiple independent kinase cascades are targeted by hyperosmotic stress but only one activates stress kinase p38.

In this report, we analyse the effects of osmotic shock on signal transduction in CHO cells. We demonstrate that at least three different kinase cascades are switched on upon osmotic shock, namely PKA, AMPK, and MLTK. Whereas PKA from cells treated with forskolin activated stress kinase p38, PKA from cells treated with sorbitol did not activate p38, although the enzyme is activated in both cases as analysed in vitro using a specific peptide target. Further, osmolar shock activated AMPK but treatment of the cells with the AMPK activator 5-amino-4-imidazolecarboxamide (AICAr) did not result in p38 activation, strongly suggesting that AMPK is not involved in stress kinase activation. Transfection of CHO cells with dominant negative recombinants of MLTKalpha resulted in inhibition of sorbitol-mediated p38 activation, indicating that the mixed-lineage kinase is involved in the activation of p38 by sorbitol. Finally, in CHO cells overexpressing wild-type MLTKalpha, no activation of AMPK of PKA could be demonstrated, indicating that the activated kinase cascades are not involved in a cross-talk process.

AMP-Activated Protein Kinases↗
PubMed2004

Application of a normalised plot to the study of ter ter enzyme systems.

The kinetic characterisation of multisubstrate systems is not a trivial task. Common approaches simplify the experimental procedures by sequentially fixing saturating concentrations of different substrates/products, thereby attempting to isolate the influence of the varying molecule. Even after such tedious work, only apparent Km values can be determined, preventing serious comparison among differential substrate behaviours. Moreover, the choice among rival kinetic models is not statistically guaranteed; instead, classical tools such as re-plots continue to be used. Here, we report the application of a normalisation of kinetic data, formerly applied to simpler systems, to the description of ter ter systems. This data treatment is able to provide true Km values and a reliable description of the system, at the same time reducing the experimental work and statistically supporting the choice of kinetic schemes.

Computer Simulation↗
PubMed2004

Mucosal human papillomaviruses encode four different E5 proteins whose chemistry and phylogeny correlate with malignant or benign growth.

We performed a phylogenetic study of the E2-L2 region of human mucosal papillomaviruses (PVs) and of the proteins therein encoded. Hitherto, proteins codified in this region were known as E5 proteins. We show that many of these proteins could be spurious translations, according to phylogenetic and chemical coherence criteria between similar protein sequences. We show that there are four separate families of E5 proteins, with different characteristics of phylogeny, chemistry, and rate of evolution. For the sake of clarity, we propose a change in the present nomenclature. E5alpha is present in groups A5, A6, A7, A9, and A11, PVs highly associated with malignant carcinomas of the cervix and penis. E5beta is present in groups A2, A3, A4, and A12, i.e., viruses associated with certain warts. E5gamma is present in group A10, and E5delta is encoded in groups A1, A8, and A10, which are associated with benign transformations. The phylogenetic relationships between mucosal human PVs are the same when considering the oncoproteins E6 and E7 and the E5 proteins and differ from the phylogeny estimated for the structural proteins L1 and L2. Besides, the protein divergence rate is higher in early proteins than in late proteins, increasing in the order L1 < L2 < E6 approximately E7 < E5. Moreover, the same proteins have diverged more rapidly in viruses associated with malignant transformations than in viruses associated with benign transformations. The E5 proteins display, therefore, evolutionary characteristics similar to those of the E6 and E7 oncoproteins. This could reflect a differential involvement of the E5 types in the transformation processes.

Amino Acid Sequence↗
PubMed2002

Application of a normalised plot to the study of uni-uni enzyme-inhibitor systems.

Normalisation of kinetic data is a useful tool in the study of complex enzyme systems. In the present paper, we have applied the premises of the normalised plot to the description of uni-uni enzyme inhibition. Guidelines to the design of the experiments and to data managing using the freeware program SIMFIT (http:\\www.simfit.man.ac.uk) are offered. The treatment has a lessened demand in experimental data while ensuring biological consistence of the results. Moreover, the results are obtained without resorting to secondary plots, and the election between rival mechanisms is statistically granted. Hyperbolic mixed-type inhibition is studied as a general model for enzyme-inhibitor/activator interaction, and equations describing classical cases of linear inhibition are also considered.

Enzyme Inhibitors↗
PubMed2002

Transport of N-acetyl-D-mannosamine and N-acetyl-D-glucosamine in Escherichia coli K1: effect on capsular polysialic acid production.

N-Acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc) are the essential precursors of N-acetylneuraminic acid (NeuAc), the specific monomer of polysialic acid (PA), a bacterial pathogenic determinant. Escherichia coli K1 uses both amino sugars as carbon sources and uptake takes place through the mannose phosphotransferase system transporter, a phosphoenolpyruvate-dependent phosphotransferase system that shows a broad range of specificity. Glucose, mannose, fructose, and glucosamine strongly inhibited the transport of these amino-acetylated sugars and GlcNAc and ManNAc strongly affected ManNAc and GlcNAc uptake, respectively. The ManNAc and the GlcNAc phosphorylation that occurs during uptake affected NeuAc synthesis in vitro. These findings account for the low in vivo PA production observed when E. coli K1 uses ManNAc or GlcNAc as a carbon source for growth.

Acetylglucosamine↗