The birth of new exons: mechanisms and evolutionary consequences

A significant amount of literature was dedicated to hypotheses concerning the origin of ancient introns and exons, but accumulating evidence indicates that new exons are also constantly being added to evolving genomes. Several mechanisms contribute to the creation of novel exons in metazoan genomes, including whole gene and single exon duplications, but perhaps the most intriguing are events of exonization, where intronic sequences become exons de novo. Exonizations of intronic sequences, particularly those originating from repetitive elements, are now widely documented in many genomes including human, mouse, dog, and fish. Such de novo appearance of exons is very frequently associated with alternative splicing, with the new exon-containing variant typically being the rare one. This allows the new variant to be evolutionarily tested without compromising the original one, and provides an evolutionary strategy for generation of novel functions with minimum damage to the existing functional repertoire. This review discusses the molecular mechanisms leading to exonization, its extent in vertebrate genomes, and its evolutionary implications.     

A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction

Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases.     

Cxcl12 evolution--subfunctionalization of a ligand through altered interaction with the chemokine receptor

The active migration of primordial germ cells (PGCs) from their site of specification towards their target is a valuable model for investigating directed cell migration within the complex environment of the developing embryo. In several vertebrates, PGC migration is guided by Cxcl12, a member of the chemokine superfamily. Interestingly, two distinct Cxcl12 paralogs are expressed in zebrafish embryos and contribute to the chemotattractive landscape. Although this offers versatility in the use of chemokine signals, it also requires a mechanism through which migrating cells prioritize the relevant cues that they encounter. Here, we show that PGCs respond preferentially to one of the paralogs and define the molecular basis for this biased behavior. We find that a single amino acid exchange switches the relative affinity of the Cxcl12 ligands for one of the duplicated Cxcr4 receptors, thereby determining the functional specialization of each chemokine that elicits a distinct function in a distinct process. This scenario represents an example of protein subfunctionalization--the specialization of two gene copies to perform complementary functions following gene duplication--which in this case is based on receptor-ligand interaction. Such specialization increases the complexity and flexibility of chemokine signaling in controlling concurrent developmental processes.     

Power-law input-output transfer functions explain the contrast-response and tuning properties of neurons in visual cortex

We develop a unified model accounting simultaneously for the contrast invariance of the width of the orientation tuning curves (OT) and for the sigmoidal shape of the contrast response function (CRF) of neurons in the primary visual cortex (V1). We determine analytically the conditions for the structure of the afferent LGN and recurrent V1 inputs that lead to these properties for a hypercolumn composed of rate based neurons with a power-law transfer function. We investigate what are the relative contributions of single neuron and network properties in shaping the OT and the CRF. We test these results with numerical simulations of a network of conductance-based model (CBM) neurons and we demonstrate that they are valid and more robust here than in the rate model. The results indicate that because of the acceleration in the transfer function, described here by a power-law, the orientation tuning curves of V1 neurons are more tuned, and their CRF is steeper than those of their inputs. Last, we show that it is possible to account for the diversity in the measured CRFs by introducing heterogeneities either in single neuron properties or in the input to the neurons. We show how correlations among the parameters that characterize the CRF depend on these sources of heterogeneities. Comparison with experimental data suggests that both sources contribute nearly equally to the diversity of CRF shapes observed in V1 neurons.     

Whole-genome immunoinformatic analysis of F. tularensis: predicted CTL epitopes clustered in hotspots are prone to elicit a T-cell response

The cellular arm of the immune response plays a central role in the defense against intracellular pathogens, such as F. tularensis. To date, whole genome immunoinformatic analyses were limited either to relatively small genomes (e.g. viral) or to preselected subsets of proteins in complex pathogens. Here we present, for the first time, an unbiased bacterial global immunoinformatic screen of the 1740 proteins of F. tularensis subs. holarctica (LVS), aiming at identification of immunogenic peptides eliciting a CTL response. The very large number of predicted MHC class I binders (about 100,000, IC(50) of 1000 nM or less) required the design of a strategy for further down selection of CTL candidates. The approach developed focused on mapping clusters rich in overlapping predicted epitopes, and ranking these "hotspot" regions according to the density of putative binding epitopes. Limited by the experimental load, we selected to screen a library of 1240 putative MHC binders derived from 104 top-ranking highly dense clusters. Peptides were tested for their ability to stimulate IFNγ secretion from splenocytes isolated from LVS vaccinated C57BL/6 mice. The majority of the clusters contained one or more CTL responder peptides and altogether 127 novel epitopes were identified, of which 82 are non-redundant. Accordingly, the level of success in identification of positive CTL responders was 17-25 fold higher than that found for a randomly selected library of 500 predicted MHC binders (IC(50) of 500 nM or less). Most proteins (ca. 2/3) harboring the highly dense hotspots are membrane-associated. The approach for enrichment of true positive CTL epitopes described in this study, which allowed for over 50% increase in the dataset of known T-cell epitopes of F. tularensis, could be applied in immunoinformatic analyses of many other complex pathogen genomes.