New Insights into the Evolution of Metazoan Tyrosinase Gene Family

Tyrosinases, widely distributed among animals, plants and fungi, are involved in the biosynthesis of melanin, a pigment that has been exploited, in the course of evolution, to serve different functions. We conducted a deep evolutionary analysis of tyrosinase family amongst metazoa, thanks to the availability of new sequenced genomes, assessing that tyrosinases (tyr) represent a distinctive feature of all the organisms included in our study and, interestingly, they show an independent expansion in most of the analyzed phyla. Tyrosinase-related proteins (tyrp), which derive from tyr but show distinct key residues in the catalytic domain, constitute an invention of chordate lineage. In addition we here reported a detailed study of the expression territories of the ascidian Ciona intestinalis tyr and tyrps. Furthermore, we put efforts in the identification of the regulatory sequences responsible for their expression in pigment cell lineage. Collectively, the results reported here enlarge our knowledge about the tyrosinase gene family as valuable resource for understanding the genetic components involved in pigment cells evolution and development

Natural Variation of Model Mutant Phenotypes in Ciona intestinalis

BACKGROUND: The study of ascidians (Chordata, Tunicata) has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. CONCLUSIONS/SIGNIFICANCE: Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity

Expression of caspase 3 in ovarian follicle cells of the lizard Podarcis sicula.

In this study, our aim was to determine whether caspase 3 plays a role, during previtellogenesis, in the ovarian follicular epithelium of the lizard Podarcis sicula. We investigated the presence and localization of proform and active caspase 3 by enzyme assay, Western blotting and immunocytochemistry. In parallel, a fragment of caspase 3 was cloned for the first time in this species, sequenced and used for in situ hybridization to localize messengers and analysed by a phylogenetic survey to shed light on its homology with reptilian caspases. Results demonstrated that: (1) the follicle cells expressed a caspase of the 3/7 group and the mRNA for caspase 3 was transcribed in the stem phase and was completely translated during cell differentiation; (2) the proform protein was stored during the differentiated (nurse) stage and activated at the end of previtellogenesis provoking the degeneration of cells; (3) the predicted protein sequence, although partial, had a strong similarity with the known reptilian caspases 3. The epithelial cells of the ovarian follicle, therefore, do not employ caspase 3 during the nurse stage but, instead, prepare for apoptosis long before the process actually begins. The relevance of this strategy is discussed

Neurons and genes involved in chemical sensitivity in nematodes

Organelles and neurons of nematodes involved in sensing chemical signals present in the environment are described. Laser ablation of neurons has helped assign them a specific function. Genetic mutational analysis has led to the identification of genes controlling the behaviour of the worms and/or some cellular properties of the chemosensory neurons. Some conclusions on the general organization and functioning of chemoreception in nematodes can be drawn

Cuticlin genes of nematodes

Two genes coding for cuticlin components of Caenorhabditis elegans have been cloned and their structure is described. Recombinant proteins have been produced in E. coli and antibodies raised against them. Nucleic acid and specific antibodies are being used to isolate the homologues from the parasitic species Ascaris lumbricoides and Brugia pahangi

A Rapid and Cheap Methodology for CRISPR/Cas9 Zebrafish Mutant Screening

The introduction of new genome editing tools such as ZFNs, TALENs and, more recently, the CRISPR/Cas9 system, has greatly expanded the ability to knock-out genes in different animal models, including zebrafish. However, time and costs required for the screening of a huge number of animals, aimed to identify first founder fishes (F0), and then carriers (F1) are still a bottleneck. Currently, high-resolution melting (HRM) analysis is the most efficient technology for large-scale InDels detection, but the very expensive equipment demanded for its application may represent a limitation for research laboratories. Here, we propose a rapid and cheap method for high-throughput genotyping that displays efficiency rate similar to the HRM. In fact, using a common ViiA™7 real-time PCR system and optimizing the parameters of the melting analysis, we demonstrated that it is possible to discriminate between the mutant and the wild type melting curves. Due to its simplicity, rapidity and cheapness, our method can be used as a preliminary one-step approach for massive screening, in order to restrict the scope at a limited number of embryos and to focus merely on them for the next sequencing step, necessary for the exact sequence identification of the induced mutation. Moreover, thanks to its versatility, this simple approach can be readily adapted to the detection of any kind of genome editing approach directed to genes or regulatory regions and can be applied to many other animal models