Anti-RPL10A monoclonal antibody, clone 4H3

Symbiotic associations between green algae (Chlorophyta) and fungi give rise to morphologically and eco-physiologically distinct entities, or so-called, lichens. In one of the most peculiar of these associations, the partners are species of the macroscopic genus Prasiola (Trebouxiophyceae) and the ascomycete Mastodia tessellata (Verrucariaceae). This is the only known case of a lichen symbiosis involving a foliose green alga. Despite intense research targeted at understanding the biology of this particular association, little is known about the genetic variability of its symbionts. This study focuses on the photobiont partner of this lichen and was designed to explore and compare its genetic diversity along a latitudinal axis from Alaska to Antarctica. Molecular sequence data were generated for three loci: two nuclear markers (nrITS, RPL10A) and one plastid-encoded marker (tufA). The usefulness of the Prasiola nrITS and RPL10A data was examined at the species and intraspecific levels. We used the population assignment tests implemented in BAPS and STRUCTURE and two algorithmic species delimitation procedures (ABGD, GMYC) to generate species boundary discovery hypotheses, which were subsequently tested using Bayes factors. Population genetic differentiation and structure were also assessed through fixation indices, polymorphism statistics and haplotype networks. Based on the results of the species validation method, we propose that at least two species of Prasiola associate with the lichen-forming fungus Mastodia tessellata. Of these, P. borealis is broadly distributed in Alaska, Tierra del Fuego and the Antarctic Peninsula, whereas the second, undescribed, species is restricted to the Antarctic Peninsula. We detected significant phylogeographic substructure in P. borealis, including greater haplotype diversity in the Tierra del Fuego populations. Our findings provide new data that will be useful to unravel the cryptic diversity and phylogeographic patterns of the green alga partners of lichens. (C) 2016 Elsevier Inc. All rights reserved.

In many organisms, tissue complexity and cellular diversity create a barrier that can hinder our understanding of gene expression programs. To address this problem, methods have been developed that allow for easy isolation of translated mRNAs from genetically defined cell populations. A prominent example is the Translating Ribosome Affinity Purification method also called TRAP. Here, ribosome associated mRNAs are isolated via purification of the ribosomal protein RPL10A/uL1, which is expressed under the control of a tissue specific promoter. Originally developed to investigate gene expression in mouse neurons, it has by now been adopted to many different organisms and tissues. Interestingly, TRAP has never been used successfully to analyze mRNA translation in germ cells. Employing a combination of genetic and biochemical approaches, I assessed several ribosomal proteins for their suitability for TRAP using the Caenorhabditis elegans germline as a target tissue. Surprisingly, I found that RPL10A/uL1 is not the ideal ribosomal component to perform such an analysis in germ cells. Instead other proteins such as RPL4/uL4 or RPL9/eL6 are much better suited for this task. Tagged variants of these proteins are well expressed in germ cells, integrated into translating ribosomes and do not influence germ cell functions. Furthermore, germ cell-specific mRNAs are much more efficiently co-purified with RPL4/uL4 and RPL9/uL6 compared to RPL10A/uL1. This study provides a solid basis upon which future germ cell TRAP experiments can be built, and it highlights the need for rigorous testing when adopting such methods to a new biological system.