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The evolutionary path from protists to multicellular animals remains a mystery. Recent work on the genomes of several unicellular relatives of animals has shaped our understanding of the genetic changes that may have occurred in this transition.1, 2, 3 However, the specific cellular modifications that took place to accommodate these changes remain unclear. To address this, we need to compare metazoan cells with those of their extant relatives, which are choanoflagellates, filastereans, ichthyosporeans, and corallochytreans/pluriformeans. Interestingly, these lineages display a range of developmental patterns potentially homologous to animal ones. Genetic tools have already been established in three of those lineages.4, 5, 6, 7 However, there are no genetic tools available for Corallochytrea. We here report the development of stable transfection in the corallochytrean Corallochytrium limacisporum. Using these tools, we discern previously unknown biological features of C. limacisporum. In particular, we identify two different paths for cell division—binary fission and coenocytic growth—that reveal a non-linear life cycle. Additionally, we found that C. limacisporum is binucleate for most of its life cycle, and that, contrary to what happens in most eukaryotes, nuclear division is decoupled from cellular division. Moreover, its actin cytoskeleton shares characteristics with both fungal and animal cells. The establishment of these tools in C. limacisporum fills an important gap in the unicellular relatives of animals, opening up new avenues of research to elucidate the specific cellular changes that occurred in the evolution of animals., This work was funded by the Gordon and Betty Moore Foundation’s Marine Microbiology Initiative for establishing Emerging Model Systems Grant Number: 4973.01.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways., This collaborative effort was supported by the Gordon and Betty Moore Foundation EMS Program of the Marine Microbiology Initiative (grant nos. GBMF4972 and 4972.01 to F.-Y.B.; GBMF4970 and 4970.01 to M.A. and A.Z.W.; GBMF3788 to A.Z.W.; GBMF 4968 and 4968.01 to H.C.; GBMF4984 to V.H.; GBMF4974 and 4974.01 to C. Brownlee; GBMF4964 to Y. Hirakawa; GBMF4961 to T. Mock; GBMF4958 to P.S.; GBMF4957 to A.T.; GBMF4960 to G.J.S.; GBMF4979 to K.C.; GBMF4982 and 4982.01 to J.L.C.; GBMF4964 to P.J.K.; GBMF4981 to P.v.D.; GBMF5006 to A.E.A.; GBMF4986 to C.M.; GBMF4962 to J.A.F.R.; GBMF4980 and 4980.01 to S.L.; GBMF 4977 and 4977.01 to R.F.W.; GBMF4962.01 to C.H.S.; GBMF4985 to J.M.; GBMF4976 and 4976.01 to C.H.; GBMF4963 and 4963.01 to V.E.; GBMF5007 to C.L.D.; GBMF4983 and 4983.01 to J.L.; GBMF4975 and 4975.01 to A.D.T.; GBMF4973 and 4973.01 to I.R.-T. and GBMF4965 to N.K.), by The Leverhulme Trust (RPG-2017-364) to T. Mock and A. Hopes, and by ERD funds (16_019/0000759) from the Czech Ministry of Education to J.L.

The ciliate Tetrahymena thermophila does not require sterols for growth and synthesizes pentacyclic triterpenoid alcohols, mainly tetrahymanol, as sterol surrogates. However, when sterols are present in the environment, T. thermophila efficiently incorporates and modifies them. These modifications consist of desaturation reactions at positions C5(6), C7(8), and C22(23), and de-ethylation at C24 of 29-carbon sterols (i.e. phytosterols). Three out of four of the enzymes involved in the sterol modification pathway have been previously identified. However, identification of the sterol C22 desaturase remained elusive, as did other basic aspects of this metabolism. To get more insights into this peculiar metabolism, we here perform a whole transcriptome analysis of T. thermophila in response to exogenous cholesterol. We found 356 T. thermophila genes to be differentially expressed after supplementation with cholesterol for 2 h. Among those that were upregulated, we found two genes belonging to the long spacing family of desaturases that we tentatively identified by RNAi analysis as sterol C22 desaturases. Additionally, we determined that the inhibition of tetrahymanol synthesis after supplementation with cholesterol occurs by a transcriptional downregulation of genes involved in squalene synthesis and cyclization. Finally, we identified several uncharacterized genes that are likely involved in sterols transport and signaling., S.R.N. acknowledges a Short‐term Fellowship for Postdoctoral Research Visits from the National Research Council of Argentina (CONICET), which allowed part of this research to be carried out. E.O.P. was supported by a predoctoral FPI grant from MINECO. C.H.S. is funded by the National Science and Engineering Research Council of Canada (Discovery Grant RGPIN/05754‐2015). A.D.U. was funded by a grant from Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT‐PICT 2014‐1311). We thank Michelle M. Leger and Claudio Scazzocchio for critically reading the manuscript and for providing insightful comments that improved the manuscript. S.R.N., C.B.N., A.D.N., and A.D.U. are members of the Carrera del Investigador Científico (CONICET), Argentina.

To address biological questions that cannot be answered by current model organisms, we need to develop genetic tools in the specific taxa that can provide the best answers. Such is the case of the origin of animals in which, genetic tools need to be developed among the closest unicellular relatives of animals. To fill this gap, we are developing genetic tools in Corallochytrium limacisporum, a close unicellular relative of animals that also has a fascinating biology. Corallochytrium is a marine free-living walled saprotroph that develops through a choenocyte. Moreover, because of the basal phylogenetic position of Corallochrytria together with Ichthyosporea, this lineage is especially informative to fill the void of information between yeast and metazoans. We have successfully developed transient and a stably transfection protocols by introducing the resistance gene to puromycin, allowing us to select individual transformants. Deep characterization of the established transformed lines has revealed important biological features of this organism such as the plasticity of its genome, the mode of plasmid integration, and some differences between the two known strains (Hawaii and India). Currently we are better characterizing these features and, in parallel, developing genome-editing technologies. Progress and the potential implications of our research will be presented and further discussed.

Genes and genomes can evolve through interchanging genetic material, this leading to reticular evolutionary patterns. However, the importance of reticulate evolution in eukaryotes, and in particular of horizontal gene transfer (HGT), remains controversial. Given that metabolic pathways with taxonomically-patchy distributions can be indicative of HGT events, the eukaryotic nitrate assimilation pathway is an ideal object of investigation, as previous results revealed a patchy distribution and suggested that the nitrate assimilation cluster of dikaryotic fungi (Opisthokonta) could have been originated and transferred from a lineage leading to Oomycota (Stramenopiles). We studied the origin and evolution of this pathway through both multi-scale bioinformatic and experimental approaches. Our taxon-rich genomic screening shows that nitrate assimilation is present in more lineages than previously reported, although being restricted to autotrophs and osmotrophs. The phylogenies indicate a pervasive role of HGT, with three bacterial transfers contributing to the pathway origin, and at least seven well-supported transfers between eukaryotes. In particular, we propose a distinct and more complex HGT path between Opisthokonta and Stramenopiles than the one previously suggested, involving at least two transfers of a nitrate assimilation gene cluster. We also found that gene fusion played an essential role in this evolutionary history, underlying the origin of the canonical eukaryotic nitrate reductase, and of a chimeric nitrate reductase in Ichthyosporea (Opisthokonta). We show that the ichthyosporean pathway, including this novel nitrate reductase, is physiologically active and transcriptionally co-regulated, responding to different nitrogen sources; similarly to distant eukaryotes with independent HGT-acquisitions of the pathway. This indicates that this pattern of transcriptional control evolved convergently in eukaryotes, favoring the proper integration of the pathway in the metabolic landscape. Our results highlight the importance of reticulate evolution in eukaryotes, by showing the crucial contribution of HGT and gene fusion in the evolutionary history of the nitrate assimilation pathway., Author summary One of the most relevant findings in evolution was that lineages, either genes or genomes, can evolve through interchanging genetic material. For example, exon shuffling can lead to genes with complete novel functions, and genomes can acquire novel functionalities by means of horizontal gene transfer (HGT). Whereas HGT is known to be an important driver of metabolic remodelling and ecological adaptations in Bacteria, its importance and prevalence in eukaryotes remains controversial. We show that HGT played a major role in the origin and evolution of the eukaryotic nitrate assimilation pathway, with several bacteria-to-eukaryote and eukaryote-to-eukaryote transfers promoting the acquisition of this ecologically-relevant pathway to autotrophs and to distinct groups of osmotrophs. Moreover, we also show that gene fusion was important in this evolutionary history, underlying the origin of the canonical eukaryotic nitrate reductase, but also of a non-canonical nitrate reductase that we describe in Ichthyosporea, a poorly-characterized eukaryotic group that includes many parasitic species. In conclusion, our results highlight the importance of reticulate evolution in eukaryotes, by showing the contribution of HGT and gene fusion in the evolutionary history of the nitrate assimilation pathway.

Author(s): Faktorova, Drahomira; Nisbet, R Ellen R; Fernandez Robledo, Jose A; Casacuberta, Elena; Sudek, Lisa; Allen, Andrew E; Ares, Manuel; Areste, Cristina; Balestreri, Cecilia; Barbrook, Adrian C; Beardslee, Patrick; Bender, Sara; Booth, David S; Bouget, Francois-Yves; Bowler, Chris; Breglia, Susana A; Brownlee, Colin; Burger, Gertraud; Cerutti, Heriberto; Cesaroni, Rachele; Chiurillo, Miguel A; Clemente, Thomas; Coles, Duncan B; Collier, Jackie L; Cooney, Elizabeth C; Coyne, Kathryn; Docampo, Roberto; Dupont, Christopher L; Edgcomb, Virginia; Einarsson, Elin; Elustondo, Pia A; Federici, Fernan; Freire-Beneitez, Veronica; Freyria, Nastasia J; Fukuda, Kodai; Garcia, Paulo A; Girguis, Peter R; Gomaa, Fatma; Gornik, Sebastian G; Guo, Jian; Hampl, Vladimir; Hanawa, Yutaka; Haro-Contreras, Esteban R; Hehenberger, Elisabeth; Highfield, Andrea; Hirakawa, Yoshihisa; Hopes, Amanda; Howe, Christopher J; Hu, Ian; Ibanez, Jorge; Irwin, Nicholas AT; Ishii, Yuu; Janowicz, Natalia Ewa; Jones, Adam C; Kachale, Ambar; Fujimura-Kamada, Konomi; Kaur, Binnypreet; Kaye, Jonathan Z; Kazana, Eleanna; Keeling, Patrick J; King, Nicole; Klobutcher, Lawrence A; Lander, Noelia; Lassadi, Imen; Li, Zhuhong; Lin, Senjie; Lozano, Jean-Claude; Luan, Fulei; Maruyama, Shinichiro; Matute, Tamara; Miceli, Cristina; Minagawa, Jun; Moosburner, Mark; Najle, Sebastian R; Nanjappa, Deepak; Nimmo, Isabel C; Noble, Luke; Novak Vanclova, Anna MG; Nowacki, Mariusz; Nunez, Isaac; Pain, Arnab; Piersanti, Angela; Pucciarelli, Sandra; Pyrih, Jan; Rest, Joshua S | Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.

This is a protocol to transiently transfect Capsaspora owczarzaki, a unicellular eukaryote

How animals emerged from their unicellular ancestor remains a major evolutionary question. New genome data from the closest unicellular relatives of animals have provided important insights into the evolution of animal multicellularity. We know that the unicellular ancestor of animals had an unexpectedly complex genetic repertoire, including many genes that are key to animal development and multicellularity. Thus, assessing the function of these genes among unicellular relatives of animals is key to understanding how they were co-opted at the onset of the Metazoa. However, such analyses have been hampered by the lack of genetic tools. Progress has been made in choanoflagellates and teretosporeans, two of the three lineages closely related to animals, whereas no tools are yet available for functional analysis in the third lineage: the filastereans. Importantly, filastereans have a striking repertoire of genes involved in transcriptional regulation and other developmental processes. Here, we describe a reliable transfection method for the filasterean Capsaspora owczarzaki. We also provide a set of constructs for visualising subcellular structures in live cells. These tools convert Capsaspora into a unique experimentally tractable organism to use to investigate the origin and evolution of animal multicellularity. Fil: Parra Acero, Helena. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España Fil: Ros Rocher, Núria. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España Fil: Perez Posada, Alberto. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España Fil: Kożyczkowska, Aleksandra. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España Fil: Sánchez Pons, Núria. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España Fil: Nakata, Azusa. Prefectural University of Hiroshima; Japón Fil: Suga, Hiroshi. Prefectural University of Hiroshima; Japón Fil: Najle, Sebastián Rodrigo. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Ruiz Trillo, Iñaki. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales. Departamento de Biodiversidad y Biología Evolutiva; España. Institució Catalana de Recerca i Estudis Avancats; España. Universidad de Barcelona; España

How animals emerged from their unicellular ancestor remains a major evolutionary question. New genome data from the closest unicellular relatives of animals have provided important insights into the evolution of animal multicellularity. We know that the unicellular ancestor of animals had an unexpectedly complex genetic repertoire, including many genes that are key to animal development and multicellularity. Thus, assessing the function of these genes among unicellular relatives of animals is key to understanding how they were co-opted at the onset of the Metazoa. However, such analyses have been hampered by the lack of genetic tools. Progress has been made in choanoflagellates and teretosporeans, two of the three lineages closely related to animals, whereas no tools are yet available for functional analysis in the third lineage: the filastereans. Importantly, filastereans have a striking repertoire of genes involved in transcriptional regulation and other developmental processes. Here, we describe a reliable transfection method for the filasterean

Sterols are essential for several physiological processes in most eukaryotes. Sterols regulate membrane homeostasis and participate in different signalling pathways not only as precursors of steroid hormones and vitamins, but also through its role in the formation of lipid rafts. Two major types of sterols, cholesterol and ergosterol, have been described so far in the opisthokonts, the clade that comprise animals, fungi and their unicellular relatives. Cholesterol predominates in derived bilaterians, whereas ergosterol is what generally defines fungi. We here characterize, by a combination of bioinformatic and biochemical analyses, the sterol metabolism in the filasterean Capsaspora owczarzaki, a close unicellular relative of animals that is becoming a model organism. We found that C. owczarzaki sterol metabolism combines enzymatic activities that are usually considered either characteristic of fungi or exclusive to metazoans. Moreover, we observe a differential transcriptional regulation of this metabolism across its life cycle. Thus, C. owczarzaki alternates between synthesizing 7-dehydrocholesterol de novo, which happens at the cystic stage, and the partial conversion—via a novel pathway—of incorporated cholesterol into ergosterol, the characteristic fungal sterol, in the filopodial and aggregative stages., This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT-PICT 2012-1301 and ANPCYT-PICT 2014-1311) to A.D.U; a European Research Council Consolidator Grant (ERC-2012-Co -616960) grant and a grant (BFU2014-57779-P) from Ministerio de Economía y Competitividad (MINECO) to I.R.-T., the latter co-funded by the European Regional Development Fund (fondos FEDER). S.R.N. and A.D.U. are members of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. We also acknowledge financial support from Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya (Project 2014 SGR 619).

Background The DesK-DesR two-component system regulates the order of membrane lipids in the bacterium Bacillus subtilis by controlling the expression of the des gene coding for the delta 5-acyl-lipid desaturase. To activate des transcription, the membrane-bound histidine kinase DesK phosphorylates the response regulator DesR. This covalent modification of the regulatory domain of dimeric DesR promotes, in a cooperative fashion, the hierarchical occupation of two adjacent, non-identical, DesR-P binding sites, so that there is a shift in the equilibrium toward the tetrameric active form of the response regulator. However, the mechanism of regulation of DesR activity by phosphorylation and oligomerization is not well understood. Methods We employed deletion analysis and reporter fusions to study the role of the N-terminal domain on DesR activity. In addition, electromobility shift assays were used to analyze the binding capacity of the transcription factor to deletion mutants of the des promoter. Results We show that DesR lacking the N-terminal domain is still able to bind to the des promoter. We also demonstrate that if the RA site is moved closer to the − 35 region of P des , the adjacent site RB is dispensable for activation. General significance Our results indicate that the unphosphorylated regulatory domain of DesR obstructs the access of the recognition helix of DesR to its DNA target. In addition, we present evidence showing that RB is physiologically relevant to control the activation of the des gene when the levels of DesR-P reach a critical threshold.

The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila , DES5A , has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.

The ciliate Tetrahymena thermophila incorporates sterols from its environment that desaturates at positions C5(6), C7(8), and C22(23). Phytosterols are additionally modified by removal of the ethyl group at carbon 24 (C24). The enzymes involved are oxygen-, NAD(P)H-, and cytochrome b5 dependent, reason why they were classified as members of the hydroxylases/desaturases superfamily. The ciliate’s genome revealed the presence of seven putative sterol desaturases belonging to this family, two of which we have previously characterized as the C24-de-ethylase and C5(6)-desaturase. A Rieske oxygenase was also identified; this type of enzyme, with sterol C7(8)-desaturase activity, was observed only in animals, called Neverland in insects and DAF-36 in nematodes. They perform the conversion of cholesterol into 7-dehydrocholesterol, first step in the synthesis of the essential hormones ecdysteroids and dafachronic acids. By adapting an RNA interference-by-feeding protocol, we easily screened six of the eight genes described earlier, allowing the characterization of the Rieske-like oxygenase as the ciliate’s C7(8)-desaturase (Des7p). This characterization was confirmed by obtaining the corresponding knockout mutant, making Des7p the first nonanimal Rieske-sterol desaturase described. To our knowledge, this is the first time that the feeding-RNAi technique was successfully applied in T. thermophila, enabling to consider such methodology for future reverse genetics high-throughput screenings in this ciliate. Bioinformatics analyses revealed the presence of Des7p orthologs in other Oligohymenophorean ciliates and in nonanimal Opisthokonts, like the protists Salpingoeca rosetta and Capsaspora owczarzaki. A horizontal gene transfer event from a unicellular Opisthokont to an ancient phagotrophic Oligohymenophorean could explain the acquisition of the Rieske oxygenase by Tetrahymena. Fil: Najle, Sebastián Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Nusblat, Alejandro David. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Nudel, Berta Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Uttaro, Antonio Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

The gene TTHERM_00438800 (DES24) from the ciliate Tetrahymena thermophila encodes a protein with three conserved histidine clusters, typical of the fatty acid hydroxylase superfamily. Despite its high similarity to sterol desaturase-like enzymes, the phylogenetic analysis groups Des24p in a separate cluster more related to bacterial than to eukaryotic proteins, suggesting a possible horizontal gene transfer event. A somatic knockout of DES24 revealed that the gene encodes a protein, Des24p, which is involved in the dealkylation of phytosterols. Knocked-out mutants were unable to eliminate the C-24 ethyl group from C29 sterols, whereas the ability to introduce other modifications, such as desaturations at positions C-5(6), C-7(8), and C-22(23), were not altered. Although C-24 dealkylations have been described in other organisms, such as insects, neither the enzymes nor the corresponding genes have been identified to date. Therefore, this is the first identification of a gene involved in sterol dealkylation. Moreover, the knockout mutant and wild-type strain differed signifi- cantly in growth and morphology only when cultivated with C29 sterols; under this culture condition, a change from the typical pear-like shape to a round shape and an alteration in the regulation of tetrahymanol biosynthesis were observed. Sterol analysis upon culture with various substrates and inhibitors indicate that the removal of the C-24 ethyl group in Tetrahymena may proceed by a mechanism different from the one currently known. Fil: Tomazic, Mariela Luján. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Najle, Sebastián Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Nusblat, Alejandro David. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Uttaro, Antonio Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Nudel, Berta Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina