Molecular analysis of nuclear receptors from the anthozoa class (Cnidaria)

FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 1 ACKNOWLEDGES I thank Dr. Agostinho Antunes for give me the opportunity to be part of the Project PTDC/MAR/68106/2006 - “A modelação das vias de sinalização do ácido retinóico por contaminantes ambientais em teleósteos” and in Project PTDC/MAR/115199/2009 -"Estudo da modelação das vias de sinalização dos retinóides e das hormonas da tiróide, por disruptores endócrinos, em invertebrados"; for his support and advices. I am grateful to Barbara Frazão for her helps in the integration in the lab work and for share her knowledge about the protocols implemented; to Anoop Alex for his suggestions and discussions, which helped me to increase the knowledge about molecular techniques and to Cidália Gomes for her PCR recommendations and suggestions for the master thesis. I am grateful to all my lab mates of the molecular biology for the help in the handling of many tools and specially Pedro Leão, Joana Martins and Raquel Branco for their help in the TA cloning; to all my lab mates of the bioinformatics/phylogenetic for teach me about phylogenetic analysis. I thank Dr. Filipe Castro, Dr. Miguel Santos for their helpful suggestions and I acknowledge all team’s of the laboratories of CIIMAR where I was allowed to use their tools: ECOTOX, LETOX, LANUCE and specially the LEGE (Lab Ecotoxicology, Genomics and Evolution) where I developed all my work. I am also grateful to Vincent Laudet, Zdenek Kostrouch and Ann Tarrant, who answered by email to my doubts about their reports. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 2 ABSTRACT Nuclear receptors (NRs) are intracellular transcription factors which are restricted to metazoan organisms. Reduced data has been reported on the diversity of these receptors, especially in early metazoan lineages. Previous studies suggested lineage-specific diversification of several NR genes, such as the diversification of retinoid X receptor (RXR) gene within the Phylum Cnidaria (Reitzel and Tarrant, 2009). Currently, only a few sequences of NRs and/or RXR genes have been reported in Cnidaria, therefore six cnidarian species were analyzed in this study: Actinia equina, Actinia fragacea, Anthothoe sphyrodeta, Anemonia sulcata, Bunodactis verrucosa and Urticina felina. A combination of molecular approaches was used to assess RXR gene in the most basal cnidarian class, the Class Anthozoa. In addition, the amplification of other NRs from the NR2 family previously reported in this phylum was performed. We supported the hypothesis of RXR absence in anthozoans given the non-amplification of DBD fragments of the RXR in the target species. However, in Anemonia sulcata five partial NRs sequences were retrieved, such as the FTZ-F1, TR2/4 and COUPT-TF receptors. In Anthothoe spyrodeta, Bunodactis verrucosa, and Urticina felina, one fragment of TR2/4 was also obtained. Moreover, phylogenetic analysis using representative NRs reinforced the evidence that four A. sulcata sequences found encode also a COUPT-TF receptor, two TR2/4-like receptors and a NR with an inconclusive position within the NR2 family. A conclusion of this work is the need to increase the datasets and knowledge on the NRs existing in the phylum Cnidaria. Key words: nuclear receptor, RXR, Cnidaria, molecular analysis. RESUMO Os recetores nucleares (NRs) são fatores de transcrição intracelulares que são restritos aos metazoários. Poucos dados têm sido documentados acerca da sua diversidade, especialmente nos metazoários menos evoluídos. Artigos anteriores sugerem a diversificação específica de genes de NRs em determinados taxa, tal como a diversificação do gene RXR no filo Cnidária (Reitzel e Tarrant, 2009). Atualmente, poucas sequências de genes de NRs e/ou do gene RXR têm sido publicados em FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 3 metazoários; pelo que neste estudo seis espécies de cnidários foram analisadas: Actinia equina, Actinia fragacea, Anthothoe sphyrodeta, Anemonia sulcata, Bunodactis verrucosa e Urticina felina. Uma combinação de metodologias moleculares foi usada para estudar o gene RXR na classe mais basal dos cnidários, a classe Anthozoa. Para além disso, foi realizada a amplificação de outros NRs da família NR2 anteriormente publicados em cnidários. Nós apoiamos a hipótese que defende a ausência do gene RXR nos antozoários, atendendo à não amplificação do fragmento do DBD do RXR nas espécies-alvo. Mas, na Anemonia sulcata foram obtidas sequências parciais de cinco NRs, nomeadamente dos recetores FTZ-F1, TR2/4 e COUPT-TF. Na espécie Anthothoe sphyrodeta, Bunodactis verrucosa, Urticina felina também foi amplificado um fragmento do TR2/4. A análise filogenética com NRs de taxa representativos reforçou o indício de que quatro sequências descobertas na A. sulcata codificam um recetor COUP-TF, dois recetores TR2/4 e um NR cuja classificação dentro da família NR2 é inconclusiva. Uma conclusão deste trabalho é a necessidade de aumentar os dados e o conhecimento acerca de NRs existentes no filo Cnidária. Palavras-chave: recetor nuclear, RXR, Cnidária, análise molecular. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 4 CONTENTS Acknowledges .................................................................................................... 1 Abstract .............................................................................................................. 2 Resumo .............................................................................................................. 2 Contents ............................................................................................................. 4 List of Abbreviations ........................................................................................... 6 List of Tables ...................................................................................................... 9 List of Figures ..................................................................................................... 9 Introduction....................................................................................................... 11 1. Signalling trough Nuclear Receptors (NRs) ........................................ 11 1.1. Signalling trough Retinoic Acid ........................................................... 13 1.1.1. Retinoic X Receptor (RXR): gene and protein .................................... 13 2. Phylum Cnidaria ................................................................................. 15 2.1. Class Anthozoa .................................................................................. 16 2.2. NRs in Cnidaria .................................................................................. 18 2.2.1. RXR in Cnidaria ................................................................................. 18 3. Objectives .......................................................................................... 19 Material and Methods ....................................................................................... 21 1. Biologic Material ................................................................................. 21 2. Genomic DNA Extraction ................................................................... 21 3. Total RNA Extraction .......................................................................... 22 4. Polymerase Chain Reaction (PCR) .................................................... 23 5. RT (Reverse Transcriptional) PCR ..................................................... 24 6. Rapid Amplification of cDNA Ends (RACE) ........................................ 25 7. TA cloning .......................................................................................... 26 8. Phylogenetic analysis ......................................................................... 26 Results ............................................................................................................. 27 1. Nucleic Acid extraction and comparison ............................................. 27 FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 5 2. FTZ-F1 and TR2/4 gene identification ................................................ 28 3. Partial NR gene isolation in A. sulcata ................................................ 30 4. Three genes amplification of NR2 family in A. sulcata ........................ 33 5. Phylogenetic relationships within NR2 family ..................................... 34 Discussion ........................................................................................................ 37 References ....................................................................................................... 42 Annexes ........................................................................................................... 46 FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 6 LIST OF ABBREVIATIONS A - Absorbance AF-2 – Ligand Dependent Transactivation Function Domain 2 As_10 - Anemonia sulcata sequence similar to NvNR10 As_11 - Anemonia sulcata sequence similar to NvNR11 As_14 - Anemonia sulcata sequence similar to NvNR14 As_a – Anemonia sulcata RACE sequence BIC - Bayesian Information Criterion BLAST – Basic Local Alignment Search Tool CCytosine cDNA – complementary DeoxyriboNucleic Acid CDS – Coding Sequence COI - Cytochrome c-oxidase COUP-TF - Chicken Ovalbumin Upstream Promoter Transcription Factor CTAB - CetylTrimethylAmmonium Bromide DBD - DNA Binding Domain ddH2O – ultra pure water DHA - DocoHexaneoic Acid DHR - Diapauses Hormone Receptor EAR2 - V-erbA-Related receptor EcR - Ecdysone Receptor EDTA – DiAminoEthanetetraAcetic Acid ER - Estrogens Receptor ERR - Estrogens Related Receptor EtBr - Ethidium Bromide FTZ-F1 - Fushi tarazu-Factor1 FXR - Farnesoid X Receptor G - discrete Gamma distribution G - Guanine GCNF - Germ Cell Nuclear Factor gDNA – genomic DeoxyriboNucleic Acid GTR - General Time Reversible HNF4 - Hepatocyte Nuclear Factor-4 HREs - Hormone Response Elements JTT - Jones-Taylor-Thornton FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 7 K2 - Kimura 2-parameter LBD - Ligand Binding Domain LEGE - Laboratory of Ecotoxicology, Genomics and Evolution MgCl2 – Magnesium Chloride min – minutes mtDNA – mitochondrial DeoxyriboNucleic Acid NaCl – Sodium Chloride NALC - N-Acetyl-L-Cysteine NCI - Close-Neighbor-Interchange NGFIB - Nerve Growth factor IB NR - Nuclear Receptor NR1B1 – Nuclear Receptor Subfamily 1 Group B Member 1 NR1B2 –Nuclear Receptor Subfamily 1 Group B Member 2 NR1B3 – Nuclear Receptor Subfamily 1 Group B Member 3 NR2B1Nuclear Receptor Subfamily 2 Group B Member 1 NR2B2 – Nuclear Receptor Subfamily 2 Group B Member 2 NR2B3 – Nuclear Receptor Subfamily 2 Group B Member 3 NRR – Nuclear Receptor Resource NvNR – Nuclear receptor of Nematostella vectensis PCR - Polymerase Chain Reaction PPAR - Peroxisome Prolifector-Activated Receptor PUFAs - Polyunsaturated Fatty Acids RA - Retinoic Acid RAR - Retinoic Acid Receptor RAR ɣ - Retinoid X Receptors Gamma Isoform RAR αRetinoid Acid Receptors Alpha Isoform RAR β - Retinoid Acid Receptors Beta Isoform RNAse - Ribonuclease RT – Reverse Transcription RXR - Retinoid X Receptor RXR ɣ - Retinoid X Receptors Gama Isoform RXR α – Retinoid X Receptors Alpha Isoform RXR β - Retinoid X Receptors Beta Isoform s - seconds SF1 - Steroidogenic factor-1 Taq – Thermus aquaticus TLL – Tailless receptor FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 8 TLX - Homologue of the Drosophila tailless gene TR - Testicular receptor TR - Thyroid Hormone Receptor USP - Ultra spiracle VDR - Vitamin D3 Receptor WoRMS - World Register of Marine Species FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 9 LIST OF TABLES Table 1 - Taxonomy of some anthozoan Actinia equina, Actinia fragacea, Actinothoe sphyrodeta, Anemonia sulcata and Urticina felina (WoRMS, www.marinespecies.org). ............................................................................................ 17 Table 2 – Concentration and volume of the components of the Master Mix per PCR reaction. ............................................................................................................. 23 Table 3 - Volume of the components of the 5’/3’ RACE PCR or 5’/3’ Nested RACE per reaction. ..................................................................................................... 25 LIST OF FIGURES Fig. 1 - Modular structure of nuclear receptors (Tarrant et al., 2008).................. 11 Fig. 2 - Phylogenetic tree of NRs (NRR, http://nrresource.org/). ......................... 12 Fig. 3 - RXR cladogram based on LBD ligand-ability (Iwema et al., 2007). ........ 14 Fig. 4 - Consensus of cladograms of classes from Cnidaria, according to 18S rDNA, mtDNA structure and morphological features analyses (Bridge et al., 1995). ... 17 Fig. 5 - 1.5 % Agarose gel electrophoresis; 114: genomic DNA extracted; M - 1 kb plus ladder invitrogen. ............................................................................................ 27 Fig. 6 – 1.5 % Agarose gel electrophoresis; 1 – 16: total RNA extracted; M - 1 kb plus ladder invitrogen. ................................................................................................. 27 Fig. 7 - 3% Agarose gel electrophoresis - PCR products for primers f1 and r1, 1 - Actinia equina, 2 - Actinia fragacea, 3 - Bunodactis verrucosa, 4 - Actinothoe sphyrodeta, 5 - Urticina felina, 6 - Anemonia sulcata, 7 - Negative Control. M - 1 kb plus ladder invitrogen, ................................................................................................. 28 Fig. 10 - 1.3 % Agarose gel electrophoresis - PCR products for primers f4 and r4, 1 - Actinia equina, 2 - Actinia fragacea, 3 Anemonia sulcata, 4 - Bunodactis verrucosa, 5 - Urticina felina, 6 – Negative Control, M - 1kb plus ladder invitrogen. ..................... 30 Fig. 11 - Fig. 11 – 1.5 % Agarose gel electrophoresis; 16: PCR products for primers LCO1490 and HC02198 using cDNA as the template; M - 1 kb plus ladder Invitrogen. ................................................................................................................... 30 Fig. 12 - 1.5 % Agarose gel electrophoresis; RACE-PCR products. A - Biotaq TM DNA Taq polymerase (Bioline) master mix test with the 5’ RACE-cDNA of Control Mouse Heart Total RNA of Clontech, 1 – amplification with Control 5'RACE TFR Primer FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 16 endpoints have been established. Thus, it is urgent to develop more studies about the metazoan bioregulation; new released genomic sequences and better molecular tools will improve the evaluation of the regulation and/or disruption of cnidarian signalling (Tarrant, 2007). 2.1. Class Anthozoa Bridge et al. (1992) suggested a basal position for class Anthozoa within the phylum Cnidaria, given the presence of a structural alteration in the mitochondrial DNA (mtDNA) of the Hydrozoa, Scyphozoa and Cubozoa classes, which is not evidenced in the class Anthozoa. This fact indicates that these three classes form a monophyletic group relative to the class Anthozoa. A more comprehensive study of the evolutionary relationships among these groups [trough an analysis based on 18S ribosomal DNA (rDNA), mitochondrial 16S rDNA, mitochondrial genome structure and morphological characters] also supports that Anthozoa is the earliest cnidarians class (Fig. 4). The class Anthozoa is among the most slowly evolving metazoan lineages; because rates of mitochondrial evolution are slower than those of other marine invertebrates, nonetheless, the rate of evolution for nuclear genes has not been wellstudied (Daly et al., 2008). It is supported the split of Anthozoa into the clades Hexacorallia and Octocorallia (Daly et al, 2010). Within the subclass Hexacorallia, the order Actiniaria (sea anemones) is the most diverse and successful. Curiously, studies suggested that an unusually slow molecular evolution is shown within this group and that Actiniaria is monophyletic with respect to other extant hexacorallians (Daly et al., 2008). The order Actiniaria comes out as an interesting group to study genes, such as nuclear genes, and their rate of evolution. Therefore, the species studied (Table 1) are sea anemones belonging to this order and phylogenetic closer to Anemonia sulcata, which has been the first anthozoan reported with the RXR gene (Escriva et al., 1997). Within the studied species there are some morphological identifying features: Actinia equina is an anemone with tentacles arranged radially in six circles around the opening of gastrovascular cavity. Bright blue spots, called acrorhagi, are below the tentacles on the outer margin of the column, which distinguish A. equina and A. fragacea. These species are likely to be confused, however, Actinia fragacea have larger size, have spots all over the column and have less variable coloration (Stachowitsch, 1992). Actinothoe sphyrodeta has up to 100 white tentacles irregularly arranged around a yellow/orange disc. The translucent dirty-white column has faint longitudinal greenish stripes and a diameter base up to 2 cm (Picton and Morrow, FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 17 2010). Anemonia sulcata is a sea anemone with long, sinuous tentacles, which are rarely retracted. The column is smooth, with a row of inconspicuous warts and its colour is brownish or greyish. Bunodactis verrucosa is an anemone with a brownish base and a column surrounded by rows of many white, greyish, long and/or small warts. They have four rows of circles of tentacles around the only opening. Urticina felina may occur in almost any colour and may have short tentacles are arranged in multiples of 10. They have small size; its diameter of base is around to 120mm (Picton and Morrow, 2010). Fig. 4 - Consensus of cladograms of classes from Cnidaria, according to 18S rDNA, mtDNA structure and morphological features analyses (Bridge et al., 1995). Class Anthozoa Subclass Order Suborder Family Genus Specie Hexacorallia Actiniaria Nynantheae Actiniidae Actinia Actinia equina Hexacorallia Actiniaria Nynantheae Actiniidae Actinia Actinia fragacea Hexacorallia Actiniaria Nynantheae Sagartiidae Actinothoe Actinothoe sphyrodeta Hexacorallia Actiniaria Nynantheae Actiniidae Anemonia Anemonia sulcata Hexacorallia Actiniaria Nynantheae Actiniidae Aulactinia Bunodactis verrucosa Hexacorallia Actiniaria Nynantheae Actiniidae Urticina Urticina felina Table 1 - Taxonomy of anthozoan Actinia equina, Actinia fragacea, Actinothoe sphyrodeta, Anemonia sulcata and Urticina felina (WoRMS, www.marinespecies.org). FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 18 2.2. NRs in Cnidaria Researches about NRs from Cnidaria have initially use reverse transcription (RT-PCR) and the polymerase chain reaction (PCR) with degenerate primers. NRs identified through these methods were reported in distinct cnidarians species, such as, the hydrozoan Hydra vulgaris, [the orphan COUP-TF receptor (Gauchat et al., 2003) ]; the anthozoan Anemonia sulcata [the member of RXR and FTZ-F1 group of receptors (Escriva et al., 1997)]; the cubozoan Tripedalia cystophora [the RXR gene(Kostrouch et al., 1998) ]; the anthozoan Acropora millepora [ TLX, RXR, COUP-TF, HNF4, FTZ-F1, TR2/TR4 (Grasso et al., 2001)] and the anthozoan Pocillopora damicornis [TLL, COUP-TF and a third gene which cannot be unambiguously classified within a currently recognized subfamily (Tarrant et al.,2008)]. In addition, sequencing of the genome of the anthozoan Nematostella vectensis (Sullivan et al. 2006) and of hydrozoan Hydra magnipapillata (Chapman et al., 2010) has provided a recent opportunity to survey cnidarian NRs diversity on a genome-wide scale. Reitzel and Tarrant (2009) identified all NRs from the N. vectensis; a set of seventeen NRs (HFN4, TLL, TLX, FAX, PNR, COUTF-TF, TR2/4, GCNF and NRs of NR1 family), moreover in H. magnipapillata it was predicted the presence of six NRs, which grouped strongly with COUP-TF, HNF4, RXR subfamily and with NRs members of the NR2E subfamily (Reitzel et al., 2011). Among the cnidarian NRs identified to date mostly are members of NR family 2, evidence that the rate of diversification for the NR super family is fairly modest in the early diverging animals (Reitzel et al., 2011). Among these studies only in the jellyfish RXR it was confirmed its ability to bind 9-cis-RA in vitro, the others did not analyzed the ability of NRs to bind ligands. Therefore, a hypothesis remains unclear: whether these cnidarian NRs are regulated by endogenous and/or exogenous hormones, which will control cnidarian signalling pathways (Tarrant et al., 2008). Future studies are needed to understand and characterize the mechanisms of bioregulation in cnidarians. 2.2.1. RXR in Cnidaria There is strong evidence of the presence of RXR in cnidarians because retinoids are reported to influence morphogenetic pattern in the hydrozoan Hydractinia echinata (Bouzaiene et al., 2007). FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 19 Furthermore, previous studies reported NR sequences displaying substantial similarity with vertebrate RXR genes; Escriva et al. (1997) mentioned that it was identified a probable orthologous of RXR/USP in the anthozoan Anemonia sulcata. Grasso et al. (2001) got Acropora millepora cDNA sequences, which were similar to the RXR A. sulcata sequences; both studies did not confirm its ligand-dependence to 9-cis-RA. In a cubozoan, Tripedalia cystophora, was got the full-length of the gene encoding RXR and, trough radio-binding analysis, it was evidenced that this NR bound to 9-cis-RA in the same way as the vertebrate´s RXR. Moreover, they showed that this receptor is expressed at all developmental stages of the jellyfish (Kostrouch et al., 1998). These researches identified the gene by amplification of CDS sequences. New approaches used the complete-genome analysis to study NRs in cnidarians. In 2009, Reitzel and Tarrant reported a complete set of NR from the anthozoan Nematostella vectensis and they revealed that occurred a RXR gene loss. In addition, they analyzed the sequences of previous reports and they denied the RXR gene presence in anthozoan Acropora millepora. Additionally, Reitzel and Tarrant (2011), analyzed the reported genome of the hydrozoan Hydra magnipapillata and they found that most NRs are members of the NR2 family. One of these NRs grouped with strong support to RXRs from bilaterians and from placozoan Trichoplax, therefore this hydrozoan has an ortholog of invertebrates RXR genes. By one hand, there are no published NRs about the class Scyphozoa and the published results related to class Anthozoa are unclear; on the other hand, it was identified a RXR gene in a cubozoan and hydrozoan specie. Therefore, it is inferred that the cnidarian-bilaterian ancestor had the RXR gene and within Cnidaria occurred a specific diversification. 3. Objectives The transcriptional regulators inherited capacity to be ligand-activated makes them prime targets of endogenous and/or exogenous hormones. Since cnidarians hormonal signalling pathways are poorly characterized and few appropriate endpoints have been established, research about its NRs has been recognized as urgent. Moreover, the topology of the phylogeny of most these NRs has been uncertain, due to limited sequenced cnidarians samples, thus more data is needed to clear up how NRs evolved. Within Cnidaria RXR gene diversification is controversial, so this nuclear gene is considered a relevant target of the study. In this research we aimed to assess the presence/absence of RXR gene in new anthozoan species: Actinia equina, Actinia fragacea, Actinothoe spyrodeta, Anemonia sulcata, Bunodactis verrucosa, Urticina felina. Furthermore, our goals were the FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 20 amplification of the most RXR’s conserved domain in order to identify the gene; the sequencing of its full-length CDS and the analysis of the gene evolutionary relationships within class Anthozoa. Throughout the lab work new goals arose in order to support new evidences about other NRs; we intended to amplify sequences of NRs (NR2 family) previous reported in anthozoan species, which allowed complementing the phylogenetic analyses. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 21 MATERIAL AND METHODS 1. Biologic Material The field work was performed in the intertidal zone; adult Actinia equina, Actinia fragacea, Actinothoe spyrodeta, Anemonia sulcata, Bunodactis verrucosa, Urticina felina specimens were collected between January and February at Praia da Memória (Oporto, Portugal) and Praia da Foz (Oporto, Portugal). Fresh tissues of epidermis of the pedal disc were preserved in RNAlater (Sigma). Then anemones were dissected and the tissues were flash frozen in liquid nitrogen to be ground into smaller pieces; using sterilized material, such as mortar and pestle. Then all biological material was stored at −80 ◦ C. 2. Genomic DNA Extraction Different genomic DNA extraction protocols were used: an adapted protocol from phenol: chloroform extraction procedures contributed by Pinto et al. (2000) and the genomic DNA extraction Mini Kit (Bioline). Firstly a pre-treatment with acetyl cysteine (NALC) of the flash frozen tissues, was performed to decrease the viscosity of the samples. 750 µL NALC solution (1.45% sodium citrate; 2% sodium hydroxide; 0.5% N-acetyl-L-cysteine) was added to each sample, which was incubated at room temperature for 25 min. Then, they were centrifuged at maximum speed for 10 min and the pellet was resuspended in 1ml of 1x PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4; pH of 7.4). By one hand, the gDNA extraction with the Mini Kit (Bioline) was performed according to the manufacturer instructions. One the other hand, the phenol: chloroform extraction was performed with initial lyses of the tissues; adding 1000 µL of Lyses Buffer (10 mM Tris-HCl; 100 M EDTA, 0.5% sodium dodecyl sulphate; pH of 8.0) and 10 µL lysozyme, the lysate was incubated for 30 min at 55°C. Then, proteinase K was added to a final concentration of 1 mg/mL and incubation was performed overnight at 37°C. RNAse was added to a final concentration of 0.6 mg/mL and incubation for 30 min was performed at 37°C. It was performed three successive addictions of 60 µL CTAB solution (0.3% CTAB; 0.7M NaCl) plus incubation for 20 min at 65°C plus addictions of 1000 µL (24:1) chloroform: isoamyl plus a centrifugation for 20 min at 12000 x g, until the interphase disappears. The supernatant was recovered and it was added 1 volume of 24:1:1 phenol: chloroform: isoamylm, then a centrifugation was FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 22 performed for 20min at 16000 x g. The precipitation of genomic DNA was achieved with addiction of 0.1 volumes 6M NaCl plus the addiction of 2.5 volumes of cold absolute ethanol. Samples were centrifuged 20min at 4°C and 16000 x g. The supernatant was drained off and the pellet was washed with 800 µL 70% ethanol. The gDNA was dried and resuspended in 100 µL of TE buffer (10 mM Tris-HCl; 1 mM EDTA; pH of 8.0). The integrity of gDNA was evaluated by agarose gel electrophoresis and it was quantified its concentration by Nanodrop 1000. Since the samples of gDNA extracted using protocol of phenol: chloroform extraction had higher concentration and yield values, they were used in PCR amplification. 3. Total RNA Extraction Two total RNA extraction protocols were evaluated: an adapted single-step method of RNA extraction by acid guanidiniumthiocyanate: phenol: chloroform (Chomczynski and Sacchi, 1987) and the protocol from UltraClean Tissue & Cells RNA Isolation Kit (MO BIO), which suffered some adaptations. Attending on the total RNA quantity and purity, the chosen method was the adapted protocol of UltraClean Tissue & Cells RNA Isolation Kit. A fragment (20-25 mg) of all tissues kept in RNAlater was dissected and treated with 700 μL of Solution TR1 prepared with β-mercaptoethanol, then 1 volume of Solution TR2 was added to the lysate and it was homogenised by pipetting. A centrifugation of 30 s at 10000 x g was performed before the transference of the supernatant to a spin filter column. Then, the supernatant was centrifuged for 2 min at 16000 x g and the spin filter was washed with 500 μL of Solution TR3 and with 500 μL of Solution TR4 (a centrifugation for 2 min at 16000 x g was performed after both steps). To dry the membrane, an additional centrifugation was performed (3min at 16000 x g) and the membrane was air-dry for 2 min. To elute the RNA, it was added 50 μL of Solution TR5 directly onto the membrane. Total RNA was recovered with a centrifugation for 1 min at 13000 x g and then it was cleaned up according to the manufacturer instructions of UltraClean Tissue & Cells RNA Isolation Kit (MO BIO). The material was stored at -80°C. Its integrity was evaluated by agarose gel electrophoresis and to evaluate the quantity and purity of the extracted RNA, Nanodrop 1000 was used. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 23 4. Polymerase Chain Reaction (PCR) Oligonucleotide primers (see Annex 1.), designed to identify and amplify the RXR gene, were based on the homology of invertebrate sequences deposited on GenBank (www.ncbi.nlm.nih.gov/): Tripedalia cystophora (Accession number: AF091121.1); partial CDS gene of Anemonia sulcata (Accession number: U93415.1, U93416.1); Hydra magnipapillata (Accession number: NW_002152543.1); Trichoplax adhaerens (Gene ID: 6751230); Nucella lapillus (Accession number: EU024473.1); Saccoglossus kowalevskii (Gene ID: 100329058). By one hand, specific primers were designed with Primer3 v.0.4.0 (http://frodo.wi.mit.edu/) using the consensus sequence of the MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) alignment of the conserved domains: DBD and LBD. On the other hand, degenerated primers were designed using amino acid sequences in CODEHOP (http://bioinformatics.weizmann.ac.il/blocks/codehop.html). For each set of primers, PCR reactions were firstly run to determine its optimal annealing temperature and magnesium concentration. Oligonucleotide primers (see Annex 1.) designed by Reitzel and Tarrant (2009) were used to amplify a set of different cnidarians NRs sequences and the PCR parameters were followed according to the author’s recommendations. The different combination of PCR strategies was performed in Biometra T Professional Thermocycler (Alfagene) using 20 μL reactions, according to the next scheme: Components Initial concentration Final concentration Volume (μL) ddH2O -- -- To a final volume of 20 Buffer Taq (Bioline) 14x NH4 1.4x NH4 2.0 dNTPs (Bioline) 10 mM 0.2 – 0.4 mM 0.4 - 0.6 MgCl2 (Bioline) 50 mM 2.0 – 2.5 mM 1.0 - 0.8 Primer Foward 10 μM 0.5 μM 1.0 Primer Reverse 10 μM 0.5 μM 1.0 Biotaq TM DNA Taq polymerase (Bioline) 5 U/μL -- 0.1 DNA template ≥ 100 ng /μL ≥ 5 ng /μL 2.0 – 1.0 Table 2 – Concentration and volume of the components of the Master Mix per PCR reaction. PCR cycle parameters included an initial denaturation (5 min, 94°C), then 40 cycles of denaturation (30 s, 94°C), of annealing (50 s, annealing temperature) and of extension (30 s, 72°C); and in addiction it was added a final extension (10 min, 72°C). For a few set of primers (when many bands appeared) was used a Touchdown PCR program, which increases the amplification specificity, sensitivity and yield. As previously described, it was performed an initial denaturation (5 min, 94°C); followed by FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 24 10 cycles of denaturation (30 s, 94°C), of annealing (50 s, higher annealing tempeture) and of extension (30 s, 72°C); plus, 25 cycles of denaturation (30 s, 94°C), annealing (50s, lower annealing tempeture) and extension (30 s, 72°C); the final extension was performed for 7 min at 72°C). The PCR products were analyzed by agarose gel electrophoresis 1 – 2.5 % p/v (TAE 1x buffer [0.4M Tris-acetate, 0.01M EDTA, pH=8.3]) with 3 μg/mL ethidium bromide (EtBr). After the electrophoresis separation, the PCR products were visualized with transilluminator and photographed with Canon PowerShot G9 system. The PCR products of interest were purified with Diffinity Genomics Rapid Tip or with PureLink TM PCR Purification Combo Kit Quick Gel Extraction (Invitrogen), according to the manufacturer instructions. The products obtained with the set of primers designed by Reitzel and Tarrant (2009) were directed sequenced (MACROGEN) and the other PCR products were cloned, sequenced and then the results were analyzed with the FinchTV software. 5. RT (Reverse Transcriptional) PCR The cDNA used to amplify partial sequence of NRs genes was synthesized with the SuperScript III First-Strand Synthesis System (Invitrogen), according to the manufacturer instructions. Thus, cDNA was synthesized, using total 10 ng–1 μg RNA primed with Oligo(dT)18 (2.5 μM) and the reverse transcription was performed with SuperScript III Reverse Transcriptase (10 U/μl) for 50 min at 50°C. The template of RACE PCRs was the cDNA obtained using SMARTer™ RACE cDNA Amplification Kit (Clontech). The first-strand cDNA synthesis was performed according to the manufacturer instructions; in a 10 μL reaction, 10 ng–1 μg of total RNA, primed with SMARTer II A oligo (1.2 μM) and 3’/5'-RACE CDS Primer A (1.2 μM), and the target was reverse transcribed at 42 ◦C for 90 min, using SMARTScrib Reverse Transcriptase (10 U/μl). Then, first-strand reaction products were diluted with 10-20 μL Tricine-EDTA Buffer (10 mM Tricine-KOH; 1.0 mM EDTA; pH of 8.5) and stored at - 20°C. The cDNA synthesis efficiency was evaluated trough the amplification of a housekeeping gene (cytochrome c-oxidase, COI). In the reaction, 2 μL of each cDNA sample were added to a reaction mix containing 1x PCR buffer, 2.5 mM MgCl2, 250 μM of each dNTP, 4U of Biotaq TM DNA Taq polymerase (Bioline), 23.8 μL of molecular grade PCR H2O, 10 μM of both standard universal primers, LCO1490-forward and HC02198-reverse, in a total volume of 40 μL per reaction. The cycling parameters were an initial denaturation (1 min, 94°C), then 35 cycles of denaturation (1 min, 94°C), of annealing (30 s, 45°C) FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 25 and of extension (1 min, 72°C); and in addiction it was added a final extension (10 min, 72°C). 6. Rapid Amplification of cDNA Ends (RACE) Gene specific primers (GSP) of 5’/3’ RACE (see Annex 1.) were designed based on partial nucleotide sequences. They were primed with the 10X Universal Primer A Mix (UPM) of SMART RACE kit from Clontech to performed 5’/3’ RACE PCRs and, when no bands appeared, the products were amplified using GSPs and Nested Universal Primer A (NUP). Component Volume (μL) 3’ RACE 5’ RACE Nested 3’ RACE Nested 5’ RACE Negative Control Phusion Flash HighFidelity PCR Master Mix (Thermo Scientific) 10 10 10 10 10 RACE - Ready cDNA 1.0 1.0 -- -- -- RACE PCR - Products -- -- 1.0 1.0 -- UPM (10X) Clontech 2.0 2.0 -- -- 2.0 Specific Forward Primer (10 μM) 1.0 -- 1.0 -- 1.0 -- Specific Reverse Primer (10 μM) -- 1.0 -- 1.0 -- 1.0 NUP Clontech (10 μM) -- -- 0.5 0.5 -- dd H2O To a final volume of 20 To a final volume of 20 To a final volume of 20 To a final volume of 20 To a final volume of 20 Table 3 - Volume of the components of the 5’/3’ RACE PCR or 5’/3’ Nested RACE per reaction. The 20 μL reaction was thermal cycled using the PCR parameters recommended by the Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific) instructions, following a Touchdown program: initial denaturation (10 s, 98°C); followed by 10 cycles of denaturation (1 s, 98°C), annealing (5 s, 68°C) and extension (4 min, 72°C); 25 cycles of denaturation (1 s, 94°C), annealing (5 s, GSP’s annealing tempeture) and extension (4 min, 72°C), at least the final extension step was performed for 1 min at 72°C. Then, the products were analyzed on a 1.5 % agarose/EtBr gel and cloned. The clones were sequenced by the MACROGEN sequencing company. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 32 M 7 8 9 10 11 12 M c 13 14 C D E F Fig. 10 - 1.5 % Agarose gel electrophoresis; RACE-PCR products. A - Biotaq TM DNA Taq polymerase (Bioline) master mix test with the 5’ RACE-cDNA of Control Mouse Heart Total RNA of Clontech, 1 – amplification with Control 5'RACE TFR Primer plus UPM/NUP primer, 2 - amplification with Control 3’ RACE and 5'RACE TFR Primers. B and C – Nested 5’RACE products with the GSP5R using Anemonia sulcata cDNA, 3 – without dilution, 4dilution 1:2, 5dilution 1:4, 6dilution 1:10, 7dilution 1:4 plus 56°C annealing temperature, 8dilution 1:4 plus 60°C annealing temperature, 9dilution 1:4 plus 64°C annealing temperature, 10dilution 1:10 plus 56°C annealing temperature, 11dilution 1:10 plus 56°C annealing temperature, 12dilution 1:10 plus 56°C annealing temperature; D - 5’products with primer r1, 13 - 5´RAC product, 14 - Nested RACE product; E – Nest ed 5’RACE products with GSPr1, 15Actinothoe sphyrodeta, 16Anemonia sulcata, 17Actinia fragacea, 18Actinia equina, 19Bunodactis verrucosa, 20Urticina felina; F – Nested 5’RACE products with GSPr2, 21 - Actinothoe sphyrodeta, 22 - Anemonia sulcata , 23 - Urticina felina, 24 -Actinia fragacea , 25 - Actinia equina, 26 - Bunodactis verrucosa. c – Negative control, M - 1 kb plus ladder Invitrogen, N - NZYDNA Ladder VI Nzytech. M 15 16 17 18 M 19 20 M 21 22 23 24 25 26 c 2500 bp - 850 bp - - 400bp 1650 bp - 850 bp - 1200 bp – 650 bp – FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 33 4. Three genes amplification of NR2 family in A. sulcata Most of the Nematostella vectensis NRs published (Reitzel and Tarrant, 2009) have strong support to belong to the NR family 2, therefore our interest was the evaluation of relationships between the sequence As_a with other NRs identified in anthozoan species. These NRs were expected to be amplified in Anemonia sulcata trough PCR using the primers designed by Reitzel and Tarrant (2009), named in this study as NR3f, NR3r, NR4f, NR4r, NR5f, NR5r, NR6f, NR6r, NR7f, NR7r, NR8f, NR8r, NR9f, NR9r, NR10f, NR10r, NR11f, NR11r, NR12f, NR12r, NR13f, NR13r, NR14f, NR14r, NR15f, NR15r, NR16 and NR16r (see Annex 1.). According to the authors NvNR4 is a homologous of HNF4 (NR2A); NvNR5-NvNR9 seems close to subfamily 2E (TLL/TLX, FAX, PNR) and NvNR10-NvNR14 to COUP-TFs (NR2F family), moreover, NvNR15 and NvNR16 grouped with TR2/4 receptors (NR2C family). Through this approach, negative and positive results were obtained. On the one hand, the samples with absence of product or multiple products (amplification using the set of primers NR3/4/5/6/8/9)were not sequenced (Fig. 13). On the other, the sequencing data did not hit with the corresponding NvNR sequences (amplification using the set of primers NR7/12/13). Nevertheless, the Anemonia sulcata products named as As_10 (~ 1000 bp), As_11 (~ 300 bp) and As_14 (~ 400 bp) matched with its correspondent NvNR, so they are apparent COUP-TF receptors. They should be different isoforms as Reitzel and Tarrant (2009) mentioned “NvNR10 was most closely related to bilaterian COUP-TFs, while NvNR11-NvNR14 are supported as an independent radiation with this subfamily”. Fig. 11 - 1.5% Agarose gel electrophoresis: products of the set of primers designed by Reitzel and Tarrant (2009) using cDNA of A. sulcata. 4 – set of primers NR4f and NR4r; 5 – set of primers NR5f and NR5r; 6 – set of primers NR6f and NR6r; 7 – set of primers NR7f and NR7r; 8 – set of primers NR8f and NR8r; 9 – set of primers NR9f and NR9r; 10 – set of primers NR10f and NR10r; 11 – set of primers NR11f and NR11r; 12 – set of primers M 8 7 6 12 c 3 4 13 15 11 14 5 10 9 1000 bp _ 500 bp _ FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 34 NR12f and NR12r; 13 – set of primers NR13f and NR13r; 14 – set of primers NR14f and NR14r; 15 – set of primers NR15f and NR15r; 16 – set of primers NR16f and NR16r; M - 1 kb plus ladder Invitrogen. 5. Phylogenetic relationships within NR2 family To understand the evolutionary relationships between the sequenced NRs and the NR2 family, phylogenetic analyses were carried out. In Maximum likelihood (ML) phylogeny the model implemented was Jones-Taylor-Thornton (JTT) for amino acid dataset and Kimura 2-parameter for nucleotide data. Moreover, the evolutionary rate was modeled by the discrete Gamma distribution and the ML heuristic analysis was performed by Close-Neighbor-Interchange (NCI) method. Both trees were rooted with the HNF4 group. The analyses using nucleotide data had better results; comparing the bootstrap percentage (BP) scores (Fig. 14 and Fig.15). Therefore this tree was considered a better estimation of the arrangement of the NR subfamilies. Since the interior branch of As_10 and NvNR10 bootstrap percentage is ≥ 80%, it is strongly supported their relationship, so primer NR1f and NR1r should be efficient to the COUP-TF gene amplification in anthozoan species. As_a sequence was predicted as a possible TR2/4 receptor, validating previous results using primer r1 in PCR. As_a and As_11 grouped together so they may represent gene duplication of TR2/4-like receptor. As_14 sequence grouped differently in both analyses; the nucleotide sequences’ analysis showed As_14 related to As_a, as NR2C members, but in the amino acid sequences’ analysis it is identified as a NR2E member. In both cases the BP are low (BP=32% and BP=25%, respectively). Therefore, these outcomes contradict our predictions of As_14 sequence relationship with COUP-TF receptors. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 35 Fig. 12 - ML tree constructed using partial NRs PCR/RACE nucleotide sequences from Anemonia sulcata. Grey square represents the resultant products of the NvNRs primers amplification; grey circle represents the product of 5’RACE. As, Anemonia sulcata; Hs, Homo sapiens; Tc, Tripedalia cystophora; Ds, Danio rerio; Xl, Xenopus laevis; Dr, Drosophila melanogaster; Ci, Ciona intestinalis; Nv, Nematostella vectensis. Hs COUPTF Xl COUPTF Dr COUPTF Nv10 As 10 Xl TLX Hs PNR Nv5 Hs TR4 Dr TR2 Nv15 As 11 As a As 14 Dm USP Tc RXR Dr RXR Xl RXR Dm HNF4 Hs HNF4 Dr HNF4 Nv4 93 27 39 93 72 59 74 65 71 53 71 84 26 94 38 30 15 10 32 20 FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 36 Fig. 13 - ML tree constructed using translated NRs PCR/RACE products from Anemonia sulcata and their relationship to other anthozoan nuclear receptors. Grey square represents the resultant products of the NvNRs primers amplification; grey circle represents the product of 5’RACE. As, Anemonia sulcata; Hs, Homo sapiens; Tc, Tripedalia cystophora; Ds, Danio rerio; Xl, Xenopus laevis; Dr, Drosophila melanogaster; Ci, Ciona intestinalis; Nv, Nematostella vectensis. NvNR10 As 10 Ci COUP-TF Ds COUP-TF Hs COUP-TF Xl COUP-TF Dm TLL As 14 Hs PNR NvNR5 As a As 11 NvNR15 Hs TR4 Dr TR2 Dm USP Ci RXR Tc RXR Dr RXR Xl RXR Ci HNF4 Hs HNF4 Ds HNF4 Dm HNF4 NvNR4 85 86 88 69 84 60 37 24 21 49 20 71 22 90 70 29 64 42 16 17 2 25 5 FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 37 DISCUSSION Previous inferences about NRs in the class Anthozoa indicate that the cnidarian-bilaterian ancestor was subjected to RXR gene specific diversification. Grasso et al. (2001) and Escriva et al. (1997) published two probable RXR isoforms of Acropora millepora and Anemonia sulcata, respectively. But Reitzel and Tarrant (2009) argue that the RXR was lost within the class, as it was not found in Nematostella vectensis. To clear up whether RXR gene is present or was lost specifically in the anthozoan-lineage, more species should be analyzed. Therefore, in this study, more five species and one control species were analyzed trough molecular methodologies; we performed PCR and/or RACE-PCRs with degenerated and specific primers in order to get sequences encoding the conserved domains and/or full-length genes. Due to the initial outcomes we inferred that RXR is absent in Actinia equina, Actinia fragacea, Anthothoe spyrodeta, Anemonia sulcata, Bunodactis verrucosa and Urticina felina. Thus, the new objective was to identify other NRs of the most well characterized NR family in phylum Cnidaria, the NR2 family (Reitzel et al., 2001). Since NR2 family contains a diversity of receptors: RXR, COUP, HNF4, EAR2, TR2, TR4 and TLL, we decided to analyse the NR2 members identified in the study of Reitzel and Tarrant (2011). In this study the A. sulcata gDNA and cDNA were amplified and we identified: a previous reported fragment of DBD of FTZ-F1 receptor (NR5E) (Escriva et al., 1997), three partial sequences of a TR2/4-like receptor (NR2C), a gene sequence of a COUPTF receptor (NR2F) and one sequence of a NR, which was not conclusively assigned to a NR2 family group. Moreover, a fragment of TR2/4-like receptor’s DBD was also identified in Anthothoe spyrodeta, Bunodactis verrucosa and Urticina felina specimens. In fact, the levels of similarity exhibited between sequence As_11 (TR2/4-like receptor) and As_a (TR2/4-like receptor) are only slightly (46%), but both aligned with the fragments of TR2/4-like receptor’s DBD amplified in Anemonia sulcata, Anthothoe spyrodeta, Bunodactis verrucosa and Urticina feline (see Annex 3). The alignment of DBD TR2/4 sequences of these four species has a high level of identity (see Annex 3) validating that this TR2/4 domain is well conserved. This similarity can be a clue that evolutionary rates for nuclear genes are slow, congruent with previous studies that showed a slow molecular evolution within the order Actiniaria (Daly et al., 2008). But the data available in this work is not enough to make strong conclusions about the evolution rate of some NR among anthozoan species. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 38 Interestingly, TR2/4 receptor was not identified in Actinia equine and Actinia fragacea. However, the out-group of the six target species is Anthothoe spyrodeta, because it belongs to a different family, the family Sagartiidae. In addition, the FTZ-F1 receptor was just amplified in the A. sulcata specie; possibly a mutation occurred in its DNA allowing the proper annealing of primer f1 and r1, which did not occur in the other species. Nucleotide and amino acid sequences evolutionary relationships were analyzed; the analyses using amino acid data resulted in more poorly supported nodes throughout the tree, comparing the bootstrap percentage (BP). Thus, nucleotide analysis was chosen as the reference analysis, confirming the Simmons (2000) inference that trees from nucleotide sequence data are preferred because the amino acid sequences disregard some information due to the redundancy of the genetic code. The trees were rooted with the HNF4 group attending to the many references of evolutionary studies; they mentioned that a HNF4-like is potential the original nuclear receptor, which was identified in the sponge Amphimedon queenslandica genome according a parsimonious analysis (Bridgham et al., 2010) The phylogenetic analysis indicates a difficulty placing of As_14 with one of the established groups. This fact could indicate that it represents an ancestral gene or it is a member of other NR family. Although As_a and As_11 were not clustered with strong supported (BS=15%) to the TR2/4 group, these phylogenetic relationship was reinforced by the alignment performed with all probable TR2/4 DBD sequences retrieved (see Annex 3). Therefore, these results contrast with the preliminary inferences, As_11 and As_14 are not COUP-TFs members and As_10 is the only COUP-TF receptor isoform identified. Moreover, since Reitzel et al. (2009) have reported the first complete set of NRs in the cnidarian N. vectensis from suborder Nyantheae, the same order of the studied species, the same PCRs conditions were used to explore the relations between the products and the correspondents NvNRs. Despite the specificity of the primers to NvNRs sequences, we amplified three different NRs from the NR2 family from the species, which was used as the positive control for the current study. Phylogenetic analysis of the A. sulcata sequences was performed with representative Acropora millepora NRs, instead of the NvNRs, and the results were not significant (not shown). With our results we showed the presence of COUP-TFs (Chicken Ovalbumin Upstream Promoter Transcription Factor) in the class Anthozoa. They are considered FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 39 as orphan receptors (Gauchat et al., 2004) and there are two widely expressed main of C UP-TFs, isoforms and , which have high homology (Ferrer-Mart ne z et al., 2004). Moreover, these NRs have been identified in anthozoans Acropora millepora, Pocillopora damicornis and Nematostella vectensis. COUP-TFs have also been identified in hydrozoans, flatworms and lancelets (Reitzel and Tarrant, 2009). We found two A. sulcata TR2/4-like receptors (testicular receptor 2/4) isoforms and many isoforms have been identified in previous studies; numerous TR2 and TR4 variants have been identified from different species, including vertebrates (e.g. human, murine, rabbit, fish, and amphibian) and invertebrates (e.g. Drosophila, sea urchin and nematode) (Ghang et al., 2002). In addition, TR2/4 receptors have been also identified in the anthozoans Acropora millepora and Nematostella vectensis. These receptors also do not have specific ligands identified (Young et al., 1998) and, curiously, they suppress the transcriptional activity of other NRs. The inefficiency of the 5’RACE technique using primer GSP5R, in order to obtain a RXR sequence of an RNA transcript (from an internal sequence to the 5' end of the RNA transcript) was not an evidence of the loss of the gene. This primer was based on the DBD signature, the spacing of Cyst and the conserved FFR/KR/K sequence of this NR (Kostrouch et al., 1998) but it was only proved the amplification of a 5’product in a hydrozoan, thus it can be not able to anneal an anthozoan RXR sequence. Other problem to gene isolation trough the RACE-PCR can be related to gene expression level; if it is weak, no product is obtained. Little is known about the RXR expression in cnidarians despite a study, using an antibody, confirmed the presence of a RXR-like receptor in the anthozoan Renilla koellikeri and determined the localization of this receptor type in epithelial nerve tissue in both R. koellikeri and A. millepora (Bouzaiene et al., 2007). The antibody was raised against a specific epitope of human RXRα, with significant sequence similarity with a sequence reported by Grasso et al. (2001). However, this coral RXR sequence was indicated as most similar to an orphan receptor by the phylogenetic analyses of Reitzel and Tarrant (2011). This is evidence that expression patterns of this gene are not well characterized. Nevertheless, the implementation of the 5’/3’RACE-PCR was also difficult in the isolation of a TR2/4 gene. The problems can be related to the complexity of some reactions and some patterns of bands appeared almost as smears, as we verified in the 5’RACE-PCRs. Moreover, in the study it was not confirmed whether this sequence corresponds to a complete or incomplete 5’product, since no method of cDNA synthesis can guarantee a full-length cDNA in the 5’ end. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 40 The aim to assess the presence/absence of RXR gene was achieved, and we inferred that its loss occurred within class Anthozoa But, given this fact; it was not possible to neither obtain the complete sequence of the gene nor assess their evolutionary relationships. The last appointed goal was achieved because other NRs were amplified and its identification was supported by phylogenetic analyses. Therefore, with this work we increase a little the dataset related to the NR2 family, specifically in an early metazoan lineage, so these results are relevant. Due to limited sequence sampling, the evidences of diversification of NR super family are ambiguous, especially, about NRs within groups closer to the root of the metazoan origin (Bridgham et al., 2010). For instance, RA signalling evolution outside chordates remains elusive (McKenna, 2012), despite the increase of analysis of RXR receptor. According to some reports previously mentioned, the presence RXR in Phylum Cnidaria is proposed in Anemonia sulcata (Escriva et al., 1997), Tripedalia cystophora (Kostrouch et al., 1998), Acropora millepora (Grasso et al., 2001) and Hydra magnipapillata (Reitzel et al., 2011). However, new sequenced genomes led to controversial results. For instance, Reitzel et al. (2009) proposed that RXR had a secondarily lost within class Anthozoa; since RXR is absent in Nematostella vectensis and it was found in Trichoplax adhaerens (Phylum Placozoa) and in Hydra magnipapillata (Phylum Cnidaria) genomes (Bridgham et al., 2010; Reitzel et al., 2011). Within metazoans, Placazoa is most ancient than phylum Cnidaria and class Anthozoa is the basal class of Cnidaria. This fact means that specific evolutionary events occurred within the class Anthozoa. Our analyses emphasize the Reitzel et al. (2011) hypothesis as, in the study, our sequences did not match or group with RXRs sequences or ESTs. To confirm the presence/absence of the gene, further it is promising to perform expression pattern analysis within the different cnidarian classes; these studies can be carried out with antibody-linked probes, an approach reported by Anctil et al. (2007). Or alternative techniques like cDNA library screening can be employed. Moreover, the increase of sequencing genomes of evolutionary key positioned groups of organisms will provide more insight about genes and consequently its diversification. Moreover, expression analyses also play an important role in improving our understanding of the signalling and metabolic pathways. Future studies are also needed to understand and characterize the mechanisms of bio regulation of cnidarians, in order to explore possible consequences of the loss of the RXR gene in the signalling pathway; to evaluate whether cnidarians conserve the ability to be regulated by endogenous and/or exogenous 9-cis-RA. Then, the knowledge about these FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 41 mechanisms in early metazoan-lineages can provide new insights about signalling pathways in higher lineages. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 48 NR13f CCCAGACAAGACCATCGAGT 20 55.7 cDNA NR13r ACCAACGAGAACGCAGTACC 20 57.0 cDNA NR14f AAGTGCTTAGCCGTCGGAATG 21 57.6 cDNA NR14r CTTGTCCAAATGTTGACCTGGTG 23 56.5 cDNA NR15f AATCCATCCCCATTTGCCTG 20 54.8 cDNA NR15r CCTTCCCAATCTTATGTCACGC 22 55.8 cDNA NR16f CAGGAAAACACTACGGAGTCGTTG 24 58.1 cDNA NR16r GGGGAGTCTGAGGAGAATCTTAGC 24 57.9 cDNA 1.B - List of the sequence primers described by (Reitzel and Tarrant, 2009). FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 49 Annex 2: Electrophoresis gel of PCR products - RXR gene 2. A - 3% Agarose gel electrophoresis: PCR products of degenerated primer f1 and r1 using gDNA of Anemonia sulcata as template. T1 – 48.0°C, T2 – 49.5°C, T3 – 53.0°C, T4 – 56.0°C, c - Negative control. M - 1 Kb plus ladder invitrogen. 2. B - 2% Agarose gel electrophoresis: PCR products of specific primer f2 and r2 using gDNA. 1 - Actinia equina; 2 - Bunodactis verrucosa; 3 – Actinia fragcea; 4 - Anemonia sulcata; 5 – Urticina felina; N - NZYDNA Ladder VI Nzytech. 2. C - 3% Agarose electrophoresis gel: PCR products with specific primers primer f2 and r2 using gDNA. A - Anemonia sulcata. B - Bunodactis verrucosa. T1 – 48.0°C, T2 – 49.5°C, T3 – 52.8°C, T4 – 54.4°C, T5 – 56.0°C. c – Negative control. M - 1 Kb plus ladder Invitrogen. M T1 T2 T3 T4 T1 T2 T3 T4 c M T1A T2A T3A T4A T1B T2B T3B T4B c N 1 2 3 4 5 FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 50 2. D – 2% Agarose gel electrophoresis: PCR products with degenerated primer f2 and r1 using cDNA of Bunodactis verrucosa. T1 – 52.2°C, T2 – 54.0°C, T3 – 55.7°C, T4 – 56.8°C, c – Negative control. M -1 Kb plus ladder Invitrogen. c T1 T2 T3 T4 M FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 51 Annex 3: Alignments Saccoglussus/1-228 ----------------------------------GGAAACACTATGGCGT--CTATAGCT Strongylocentrotus/1-195 ----------------------------------GCAAGCATTATGGAGT--CTACAGTT Schistosoma/1-231 CCAATTTGTGTGATTTGTGGTGATAAAGCCAGTGGTAAACATTATGGTGTGATTTCA--T Tricoplax/1-222 -------------------------------------GGCATTATGGCGT--TTACAGTT Tripedalia/1-225 CAACCATGCTCTGTATGCTCCGATAAGGCCTATGTCAAACATTATGGTGTGTTTGCA--T Hydra/1-64 ---------------------------------------CATTATGGTGTTTTTACATGT Anemonia2/1-121 -----------------------------------------GTAAGGGGTTTTT-TAGGC Saccoglussus/1-228 GCGAAGGATGTAAAGGCTTTTTCAAACGAACAGTTCGAAAAGATTTGCAT-TACGCCTGC Strongylocentrotus/1-195 GTGAAGGCTGCAAAGGGTTCTTCAAGAGGACAGTACGGAAAGACCT-CACGTATACCTGT Schistosoma/1-231 GTGAAGGATGTAAGGGATTTTTTAAACGAACAGTGCGTAAACAATT-AGTATATGTTTGT Tricoplax/1-222 GTGAAGGTTGCAAGGGTTTCTTCAAGCGTACAGTGCGCAAAAATTT-AACTTACACATGC Tripedalia/1-225 GCGAGGGCTGTAAAGGGTTTTTTAAGAGGTCAGTTCGCAACAACCG-GAAGTATTCTTGC Hydra/1-64 GATGGATGTCGTGGGTTTTTTAAACGTGCAGTTCGCAGAAACC----------------- Anemonia2/1-121 GAAGTAT-CAAAAAGCGCCTCTATTATACATGCCGT-GTGGCTGG-----TTGCTGCCCsaccoglussus/1-228 CGGGATGAGAAGAACTGCATCGTTGACAAAAGACAGCGCAACCGTTGCCAATATTGTCGA Strongylocentrotus/1-195 CGGGACGATCGTAACTGTATGGTCGACAAGAGACAGAGAAATCGGTGCCAATATTGCCGC Schistosoma/1-231 CGGGAATCAGGTCAATGTCCTGTAGATAGACGGAAGCGCACACGTTGTCAGCATTGCCGT Tricoplax/1-222 CGTGATAATCGAAACTGTGATATTGATAAAAAGCAACGAAATCGATGTCAGTATTGTCGA Tripedalia/1-225 CTTGGAAAGCGGCATTGCGACACTGATAAAAAGAGCCGTAACCGGTGTCAGTACTGTCGA Hydra/1-64 --------------------------------------------- Anemonia2/1-121 CC-AAGTGT------------------------------------ Saccoglussus/1-228 TATCAGAAATGCATTGCTATGGGGATGCGCCGAGAAG-- Strongylocentrotus/1-195 TATCAGAAGTGCCTGGGCATGGGCATGCGCAGAGAAG-- Schistosoma/1-231 TTCGAGCAGTGCTTAGCAAAGGGAATGAAGAAGGAAG-- Tricoplax/1-222 TACCAGAAGTGTCTGCAAGTTGGTATGAAGCAAGAAG-- Tripedalia/1-225 TTCCAGAAATGTGTGCAAGTTGGCATGAAACCAGAAGCTGTCCAGGAT------ 3. A – Muscle alignment of RXR genes sequences of DBD (design primers). Saccoglossus/1-612 --GACATGCCGGTGGAGAAGATCCTGGAGGCAGAGATACACGTC---GAACCCAAAACAG Strongylocentrotus/1-612 ATGACATGCCTGTGGAAAAGATCTTGGATGCCGAACTAGCAGTA---GAACCTAACAATG Tricoplax/1-612 --GAAATGCCAGTTGAAGCTATCCGAGATGCTGAATCTACGCTT---AATATGAATAGTG Schistosoma/1-636 -----CTGTCGGCTGAATTAAGCATGGATCCTAAATTGGCTGTATCAGAAAGAGGTGAAG Tripedalia/1-560 ATGA--TTCCCATCGAATCTATCATCGCAGCTGAAACCCTGGTA---GACCCCGGTATAC Consensus Saccoglossus/1-612 ATACATACGTAGATAC-ACCGAACGAT-----------------CCAGTCACCAATA-TA Strongylocentrotus/1-612 GCCCCTACGTAGACAC-CCCGCGTGAC-----------------CCTGTGACAAACA-TT Tricoplax/1-612 TACCTTACGTCGAAAT-GCAAAGTAAC-----------------CCTGTCTTAAATA-TT Schistosoma/1-636 CAATTTATGAAGATAT-ACCTGGTGATGATGATACTGGTTTACATCCATTGACCATAATC Tripedalia/1-560 AGACTTTCGCTAGTGCGAACACG-GAT-----------------CCCATCCGCCACG-TT Consensus Saccoglossus/1-612 TGCCAAGCAGCAGATAAACAGTTATTCACT-TTGGTTGAATGGGCAAAGAGAATTCCA-C Strongylocentrotus/1-612 TGCCAAGCTGCCGACAAACAACTCTTTACC-CTGGTAGAATGGGCCAAGCGAATCCCA-C Tricoplax/1-612 TGTCAAGCTGCAGATAAACAACTATTTAAT-TTGGTTGAATGGGCCAAGAAAATACCC-C Schistosoma/1-636 TGTCAGTCTATTGAACAACAATTACCT-CGAATAGTTAATTGGGCTCGTCAGTTGCCAGT Tripedalia/1-560 TGCCTGGCTGCTGACAAACAGCT-TGCATCGCTTGCAGAGTGGGCTAAACGCCTCCCC-C Consensus Saccoglossus/1-612 ACTTTA--CAG---AACTTCCTTTGGATGATCAAGTTATTTTACTACGGGCAGGTTGGAA Strongylocentrotus/1-612 ACTTCA--CAG---AACTGCCACTGGATGATCAGGTCACGCTCCTCAGGGCAGGGTGGAA Tricoplax/1-612 ATTTCT--GTG---ATTTATGTGTCGATGACCAAGTAATACTTCTGAGATCTGGTTGGAA Schistosoma/1-636 GTTTTCATCTGTCTATCTTAGTTTTGATGATCAATTTTGTTTAATAAAAGCGGCTTGGCC Tripedalia/1-560 ACTTTC--GTG---ATCTGTCAATAGCAGATCAGGTTGTCTTGCTGCAGTGGAGCTGGCC Consensus Saccoglossus/1-612 TGAACTGTTAATCGCAGCTTTTTCGCATCGCTCTATCGCTGTTAAAGATGGAATCTTATT Strongylocentrotus/1-612 CGAGCTGTTGATTGCAGCCTTCTCACATCGCTCCATCCAAGTGAAGGATGGCATCCTCCT Tricoplax/1-612 TGAATTACTGATAGCTGCGTTTTCGTTTCGATCAATAGCTGTTGAAGATGGCTTGCTCTT Schistosoma/1-636 TGAATTAGTTTTAATCAGCTCAGCGTATCATTCAACTGTTATTAGAGACGGTTTGCTTTT Tripedalia/1-560 TGAGCTGCTGATTGGTGGCTTTTGCCACCGTTCCTGTGCTGTCAAGGATGGCATTCTGTT Consensus Saccoglossus/1-612 AGCTACTGGTTTACATGTGCACA-GAAACAGCGCACACAGTGC-CGGAGTAGGTACTATC FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 52 Strongylocentrotus/1-612 TGCCACCGGCCTCCACGTCCACC-GTAACAGCGCCCACAGTGC-AGGGGTGGGCACAATC Tricoplax/1-612 ATCGACTGGGCATTATATTCATC-GCACTAGCGCGCATAATGC-TGGTATTGGGGCTATA Schistosoma/1-636 ATCGATTGGACGTCATCTTGGTA-GAGA-GGTGGCTAAATCACATGGTCTAGGTCCTCTT Tripedalia/1-560 GTCGACGGGCTTGCATCT-TACAAGGGACAATCTGAAAAAGGC-CGGCGTGGGAGCTATC Consensus Saccoglossus/1-612 TTTGACCGTGTACTCACCGAACTGGTGGCAAAAATGAGAGAAATGAAGATGGATAAGACA Strongylocentrotus/1-612 TTTGACAGGGTCCTCACCGAGCTGGTGGCTAAGATGAGAGAAATGAAGATGGATAAGACA Tricoplax/1-612 TTTGATAGGATTTTAACAGAGTTGGTTAATCAAATGAGATATTTAAAAATGGATAAAACT Schistosoma/1-636 GTTGATAGAATTCTTCATGAACTTGTTGCACGTTTTCGTGATTTATCGTTACAAAGAACT Tripedalia/1-560 ATTGACAAAATCTTCTCTGAAGTCATAGAAAAGATGCAGGAAATTCAAATGGACCGTGCG Consensus Saccoglossus/1-612 GAGTTAGGATGCTTAAGAGCCATTGTTTTGTTCAACCCAGATGCTAAGAATCTTGGAACT Strongylocentrotus/1-612 GAGCTTGGCTGTCTCAGAGCAATCGTGCTTTTCAACCCAGATGCCAAGAACTT--GACCT Tricoplax/1-612 GAATTGGGCTGTTTGAGAGCTATTATATTGTTTAATCCAGATGTTCGCGGTTTGACATCA Schistosoma/1-636 GAATTAGCTTTACTACGTGCTATTATTCTTTTTAATCCTGATGCTAATGGCTTGTCATCA Tripedalia/1-560 GAATGGGGTTGTTTGCGTGCCATCATGCTATTTTCCCCCGATGCGAAAGGACTGACAGCC Consensus Saccoglossus/1-612 --GTTCAGAAGGTTGAAGAGCTAC--GAGAGAAAGTTTATGCTT-CGCTGGAAGAATATT Strongylocentrotus/1-612 CGGTACAGAAAGTGGAGGAGCTAC--GGGAGAAGGTGTACGCCT-CTCTTGAGGAATACT Tricoplax/1-612 --GCTGATAGGGTTGAAAAGTATC--GAGAGCTGGTATATGGAG-CTCTAGAGGCTTACG Schistosoma/1-636 C--GTCATCGCGTGGA--AGCTGTCAGGGAGCAGCTTTATTCAG-CTCTTCATTCGTATT Tripedalia/1-560 --ATTGACCAAGTAGA-GAACTAC--CGGGAGCTTTATACGTCCACTTTAGAAGATCACG Consensus Saccoglossus/1-612 GTAGGAAGACTTACCCAGATGAACCTGGTCGATTTGCCAAACTTCTTCTCCGTCTGCCAG Strongylocentrotus/1-612 GCCGCAACCAGTACACGGACGAACCCGGCCGCTTCGCCAAGCTGCTCCTCAGACTGCCTG Tricoplax/1-612 TCAAGAAAAGGTTTCCTGACCAGCTCTGTCGCTTTGCTAAACTACTGCTTCGATTGCCGG Schistosoma/1-636 GTACGACTAATCAACCTCAAGATACTTCCCGTTTCACAAAATTACTACTAAGATTACCTC Tripedalia/1-560 TCAAACGAAAGCACCCTGAGCAACCCGATCGGTTTACCAAGGTAATCCTTCGTATACC-- Consensus Saccoglossus/1-612 CTCTGCGATCAATTGGCCTCAAGTGTCTGGAACATTTATTTTTCTTCAAA Strongylocentrotus/1-612 CCCTGCGCTCCATCGGTCTCAAATGCCTGGAGCATCTCTTCTTCTTTA-- Tricoplax/1-612 CCTTAAGAGCGATAAGTTTAAAGACCTTGGAGCATCTTTTCTTTTATAAA Schistosoma/1-636 CTTTACGATCCATCGCATCCAAGTGCCTTGAACATTTAGTATTTGTCAAA Tripedalia/1-560 -------------------------------------------------- Consensus 3. b – Muscle alignment of RXR genes sequences of LBD (design primers). Tripedalia_DBD/1-69 QPCSVCSDKAYVKHYGVFACEGCKGFFKRSVRRK--YSCLGKRHCDTDKKSRNRCQYCRF Hydra/1-20 -------------HYGVFTCDGCRGFFKRAVRR--------------------------- Anemonia/1-42 -------------------CEGCKGFFKRSVQKKKTYTCRDTKDCPMDKRHRNRCQYCSY Tricoplax_DBD/1-67 ---SICGQRSLRRHYGVYSCEGCKGFFKRTVRKDLTYTCRDNRCDIDKKQRNRCQYCRYQ Saccoglossus_DBD/1-69 -VCAVCGDRASGKHYGVYSCEGCKGFFKRTVRKDLHYACRDEKCIVDKRQRNRCQYCRYQ HumanRXRb_DBD/1-65 --CAICGDRSSGKHYGVYSCEGCKGFFKRTIRKDLTYSCRDKDCTVDKRQRNRCQYCRYQ HumanRXRA_DBD/1-65 --CAICGDRSSGKHYGVYSCEGCKGFFKRTVRKDLTYTCRDKDCLIDKRQRNRCQYCRYQ HumanRXRg_DBD/1-68 HICAICGDRSSGKHYGVYSCEGCKGFFKRTIRKDLIYTCRDKDCLIDKRQRNRCQYCRYQ Strongylocentrotus_DBD/1-58 ------------KHYGVYSCEGCKGFFKRTVRKDLTYTCRDDRCMVDKRQRNRCQYCRYQ Thais retinoid/1-70 HICAICGDRASGKHYRVYSCEGCKGFFKRTVRKDPTYACRDDKCMIDKRQRNRCQFCRYM Nucella retinoid/1-70 HICAICGDRASGKHYGVYSCEGCKGFFKRTVRKDLTYACRDDKCMIDKRQRNRCQYCRYM AY048663.1|RXR-like/1-70 HICAICGDRASGKHYGVYSCEGCKGFFKRTVRKDLTYACRDDKCMIDKRQRNRCQYCRYM Tripedalia_DBD/1-69 QKCVQVGMKPE Hydra/1-20 ----------- U93415.1|Anemonia/1-42 Q---------- Tricoplax_DBD/1-67 KCLQVGMKQESaccoglossus_DBD/1-69 KCIAMGMRREHumanRXRb_DBD/1-65 KCLATGM---- HumanRXRA_DBD/1-65 KCLAMGM---- HumanRXRg_DBD/1-68 KCLVMGMK--- Strongylocentrotus_DBD/1-58 KCLGMGMRREThais retinoid/1-70 KCLAQGMKRENucella retinoid/1-70 KCLAQGMKREAY048663.1|RXR-like/1-70 KCLSMGMKRE3. C – Muscle alignment of RXR DBD amino acid sequences – FFK sequence (yellow) Cyst residues (green) of zinc finger are highlighted. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 53 Bv_TR2/4_DBD/1-135 ----------TTCATTTCTGGAATCGACAGTATTGACATCTGTTTCTAGTCATTTGGTTC Ap_TR2/4_DBD/1-138 ------------CATTTCTGGAATCGACAGTACTGGCATCTGTTTCTAGTCATTTGGTTC As_TR2/4_DBD/1-133 -----------TCATTTCTGGAATCGACAGTACTGGCATCTGTTTCTAGTCATTTGGTTC Uf_TR2/4_DBD/1-147 GCACTAGTGATTCATTTCTGGAATCGACAGTACTGGCATCTGTTTCTAGTCATTTGGTTC Bv_TR2/4_DBD/1-135 ACTTCACAGTTGCCATCAGCTTTGCAGGTGTAGTCTAACTGTTTCTGTACGGTACGTTTA Ap_TR2/4_DBD/1-138 ACTTCACAGTTGCCATCAGCTTTGCAGGTGTAGTCTAACTGTTTCTGTACGGTACGTTTA As_TR2/4_DBD/1-133 ACTTCACAGTTGCCATCAGCTTTGCAGGTGTAGTCTAACTGTTTCTGTACGGTACGTTTA Uf_TR2/4_DBD/1-147 ACTTCACAGTTGCCATCAGCTTTGCAGGTGTAGTCTAACTGTTTCTGTACGGTACGTTTA Bv_TR2/4_DBD/1-135 AAGAAACCCTTTACACCCCTCACAA----- Ap_TR2/4_DBD/1-138 AAGAAACCCTTTACACCCCTCACAAATCCC As_TR2/4_DBD/1-133 AAGAAACCCTTTACACCCCTCACA------ Uf_TR2/4_DBD/1-147 AAGAAACCCTTTACACCCCTCACAAAT--- 3. D – Muscle alignment of TR2/4 RXR sequences amplified by PCR. Bv, Bunodactis verrucosa; Ap, Actinothoe sphyrodeta; As, Anemonia sulcata; Uf, Urticina feline. As_a/1-229 CACTGTTCCATTCTTGACTTCTATA-CATTGGTTTCACGCCATCTTGATCCATTGGTTGG TR2/4_DBD/1-135 ------TTCATTTCTGGAATCGACAGTATTGACATC-TGT--TTCTAGTCATTTGGTT-- As_a/1-229 ATGTTCTTGTAGGCTCCCAAAAAAGATTTTACTACTTCAAACCTAAATTTCTTGTCGTAT TR2/4_DBD/1-135 ---CACTTCACA------------GTTG-CCATCAGCTTTGCAGGTGTAGTCTAACTGTT As_a/1-229 ATCTGTCGTATACTGCATGCGCTTGACCATGTGGTTCACATGTTCAAACGTCCTCCTCCA TR2/4_DBD/1-135 CTGTA-------------------CGGTACGTTTAAAGAAACCCTTTACACCCCTCACAA As_a/1-229 TGGTAT-TTCGGGGCTACAACTTGTGCCAATACTGTCGATTCCAGAAATGA TR2/4_DBD/1-135 --------------- 3. E – Muscle alignment of TR2/4 fragments. As_11/1-288 TACCGATGGGGACGTACGAGGCGTAGTACCTTTATCTGTATCTAAGATACCGGCAAGCTT c3bf/1-132 ---TGTGAGGGGTGTA-AAGG----GTTTCTTTA---------AACGTACCG-------T As_11/1-288 GCAGCAAGGCTTCTCTCCAGCGTGGTGGTAGGGGTCTACCACGAATCCTTCTAGCCAGAT c3bf/1-132 ------------------------------------------------------------ As_11/1-288 GCTGCTACCTCTGTATTAGGGGACTTGACGATAATTTCATTGAGCATGTCGGCTGCCTGT c3bf/1-132 ---ACAGAAA--CAGTTAGA---CTACACCTGCA-----AAGCTGATGGCAA--- As_11/1-288 CTAGAATAAAGTAGTAAACGTGTGTCAGCTTCTTCGAGGTCGGACTCCAATTCTGGGACT c3bf/1-132 CTGGAAGTGAAC—CAAATGACTAGAAAC------AGATGCCAGTACTG----- As_11/1-288 TCTCGCTCTAAGACTCCGTTGAACGGTGTCAGTTGAGAGCCTGTTTTA c3bf/1-132 --TCGATTCCAGA--------AATG-- 3. F – Muscle alignment of TR2/4 fragments, amplified by primers f2 and r1. c1ar/1-75 ----GGTTTCGAGCATCAGTTTGTCTCCCTTAGTACTTTTGATTTCAGCAGCTTTGTAAC c1dr/1-62 ----------AAAAGTAAAAT-------------------AGTATAATATCCATCCTGCT c1br/1-59 GATTTGTGAGGGGTGTAAA---------------------GGGTTCGTAACCATAATAAT c1cr/1-56 ----TGTGAGGGGTGTAAAG--------------------GGTTGTAAAAGCACAATA-T c1ar/1-75 CCTTTACACCCCTCACAAA------------ c1dr/1-62 TATTCATTAGCATTAATTAAGGTCTGTCCTC c1br/1-59 CATTAAA---TTTTACAGAGTCC-------- c1cr/1-56 AATAAAT---CATTACTGAAAACC------- 3. G – Muscle alignment of sequences amplified by primers f2 and r2. As_a/1-229 CACTGTT----CCATTCTTGACT-----------TCTATACATTGGTTTCACGCCATCTT As_11/1-288 TACCGATGGGGACGTACGAGGCGTAGTACCTTTATCTGTATCTAAGATACCGGCAAGCTT As_a/1-229 GATCCATTGGTT-----GGATGTTCTTGTAGG---CTCCCAAAAA-----------AGAT FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 54 As_11/1-288 GCAGCAAGGCTTCTCTCCAGCGTGGTGGTAGGGGTCTACCACGAATCCTTCTAGCCAGAT As_a/1-229 TTTACTACTTCAAACCTA--------------AATTTCTTGTCGTATATCTGTCGTATAC As_11/1-288 GCTGCTACCTCTGTATTAGGGGACTTGACGATAATTTCATTGAGCATGTCGG-------C As_a/1-229 TGCATGCGCTTGACCATGTGGTTCACATGTTCAAACGTCCTC---------CTCCATGGT As_11/1-288 TGCCTGTCTAGAATAAAGTAGTAAACGTGTGTCAGCTTCTTCGAGGTCGGACTCCA---- As_a/1-229 ATTTCGGGGCTACAACTTGTGCCAATACT------GTCGATTCCAGAAATGA-------- As_11/1-288 ATTCTGGGACT---TCTCGCTCTAAGACTCCGTTGAACGGTGTCAGTTGAGAGCCTGTTT As_a/1-229 -- As_11/1-288 TA 3. H – Muscle alignment of As_a and As_11 sequences; As, Anemonia sulcata. 3. I – Multiple alignment (BLAST) of the sequences: As_TR2/4_DBD, Bv_TR2/4_DBD, Ap_TR2/4_DBD, Uf_TR2/4_DBD and As_a; As, Anemonia sulcata. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 55 Annex 4: Accessions number of ESTs and sequences of NRs Accession number Database Specie Sequence Gene AF091121.1 GenBank Tripedalia cystophora nucleotide RXR U93416.1 GenBank Anemonia sulcata Nucleotide RXR NW_002152543.1 GenBank Hydra magnipapillata Genome RXR 6751230 GenBank Trichoplax adhaerens Gene RXR EU024473.1 GenBank Nucella lapillus Nucleotide RXR AB594846.1 GenBank Thais clavigera Nucleotide RXR AY048663.1 GenBank Biomphalaria glabrata Nucleotide RXR 100329058 GenBank Saccoglossus kowalevskii Gene RXR X52773 GenBank Homo sapiens Nucleotide RXR X63522 GenBank Homo sapiens Nucleotide RXR U38480 GenBank Homo sapiens Nucleotide RXR AF121129.1 GenBank Homo sapiens Nucleotide PNR X16155.1 GenBank Homo sapiens Nucleotide COUP-TF X76930.1 GenBank Homo sapiens Nucleotide HNF4 DQ017616.1 GenBank Danio rerio Nucleotide HNF4 BC162963.1 GenBank Danio rerio Nucleotide COUP-TF NM_131275.1 GenBank Danio rerio Nucleotide RXR NM_001085811.1 GenBank Xenopus laevis Nucleotide TLX NM_001094481 GenBank Xenopus laevis Nucleotide COUP-TF NM_001087467.1 GenBank Xenopus laevis Nucleotide RXR NM_057433.3 GenBank Drosophila melanogaster Nucleotide USP NM_079857.3 GenBank Drosophila melanogaster Nucleotide TLL NM_001103656.1 GenBank Drosophila melanogaster Nucleotide HNF4 XP_001638550 GenBank Nematostella vectensis Protein HNF4 XP_001630386 GenBank Nematostella vectensis Protein PNR/TLL XP_001635112 GenBank Nematostella vectensis Protein PNR/TLL XP_001630385 GenBank Nematostella vectensis Protein PNR/TLL XP_001634999 GenBank Nematostella vectensis Protein PNR/TLL XP_001624815 GenBank Nematostella vectensis Protein COUP-TF XP_001629708 GenBank Nematostella vectensis Protein COUP-TF XP_001634340 GenBank Nematostella vectensis Protein COUP-TF XP_001634378 GenBank Nematostella vectensis Protein COUP-TF XP_001636010 GenBank Nematostella vectensis Protein COUP-TF XP_001636637 GenBank Nematostella vectensis Protein TR2/4 XP_001631902 GenBank Nematostella vectensis Protein TR2/4 XM_001638500.1 GenBank Nematostella vectensis Nucleotide HNF4 XM_001630336.1 GenBank Nematostella vectensis Nucleotide PNR/TLL XM_001635062.1 GenBank Nematostella vectensis Nucleotide PNR/TLL XM_001630335.1 GenBank Nematostella vectensis Nucleotide PNR/TLL XM_001634949.1 GenBank Nematostella vectensis Nucleotide PNR/TLL XM_001624765.1 GenBank Nematostella vectensis Nucleotide PNR/TLL XM_001629658.1 GenBank Nematostella vectensis Nucleotide COUP-TF XM_001634290.1 GenBank Nematostella vectensis Nucleotide COUP-TF XM_001634328.1 GenBank Nematostella vectensis Nucleotide COUP-TF XM_001635960.1 GenBank Nematostella vectensis Nucleotide COUP-TF XM_001636587.1 GenBank Nematostella vectensis Nucleotide COUP-TF XM_001631852.1 GenBank Nematostella vectensis Nucleotide TR2/4 XM_001631008.1 GenBank Nematostella vectensis Nucleotide TR2/4 ci0100142690 JGI Ciona intestinalis Protein COUP-TF ci0100146308 JGI Ciona intestinalis Protein HNF4 FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 56 ci0100150561 JGI Ciona intestinalis Protein RXR ci0100138574 JGI Ciona intestinalis Protein TR2/4 Accession number Database Sequence Gene BI781831 GenBank EST RXR BI782081 GenBank EST RXR BM280724 GenBank EST RXR BM568658 GenBank EST RXR BQ382934 GenBank EST RXR CA304237 GenBank EST RXR CB014960 GenBank EST RXR BI510638 GenBank EST RXR BI511231 GenBank EST RXR BI513792 GenBank EST RXR BG930255 GenBank EST RXR BG930250 GenBank EST RXR CD124719 GenBank EST RXR CD124789 GenBank EST RXR CD132154 GenBank EST RXR CD065670 GenBank EST RXR CD181869 GenBank EST RXR CD124616 GenBank EST RXR CD181801 GenBank EST RXR CD163651 GenBank EST RXR CD116659 GenBank EST RXR CD125564 GenBank EST RXR CD196231 GenBank EST RXR CD191347 GenBank EST RXR CD114207 GenBank EST RXR CD114191 GenBank EST RXR CD090814 GenBank EST RXR BI781435 GenBank EST RXR CD308971 GenBank EST TLL CD308972 GenBank EST TLL CD334321 GenBank EST TLL CD340492 GenBank EST TLL BI515764 GenBank EST COUP-TF BI516773 GenBank EST COUP-TF BG786614 GenBank EST TR2/4 BG787668 GenBank EST TR2/4 CD290031 GenBank EST TR2/4 CD153024 GenBank EST TR2/4 CD168281 GenBank EST TR2/4 CD124909 GenBank EST TR2/4 CD069500 GenBank EST TR2/4 CD159107 GenBank EST TR2/4 CD069750 GenBank EST TR2/4 BI783431 GenBank EST HNF4 BI783711 GenBank EST HNF4 BI514984 GenBank EST HNF4 BI503347 GenBank EST HNF4 BI508133 GenBank EST HNF4 BI509848 GenBank EST HNF4 4. A – List of the accession numbers of ESTs, nucleotide and amino acid sequences used in the molecular analyses. FCUP Molecular analysis of Nuclear Receptors in the Class Anthozoa (Cnidaria) 57 Annex 5: Sequenced nucleotide and deduced amino acid sequences >As_FTZ-F1_DBD GTTGCCATCAGCTTTGCAGGTGGGTTCTTTAAGCGGGCGGTACAGAAACA GTTAGACTACACCTGCAAAGCTGATGGCACACTGTGAAGTGAACCAAATGACTAG ATAACAGATGTCACCCCTGCAGCTTCA >As_TR2/4_DBD TCATTTCTGGAATCGACAGTACTGGCATCTGTTTCTAGTCATTTGGTTCACT TCACAGTTGCCATCAGCTTTGCAGGTGTAGTCTAACTGTTTCTGTACGGTACGTTT AAAGAAACCCTTTACACCCCTCACA >Bv_TR2/4_DBD TTCATTTCTGGAATCGACAGTATTGACATCTGTTTCTAGTCATTTGGTTCAC TTCACAGTTGCCATCAGCTTTGCAGGTGTAGTCTAACTGTTTCTGTACGGTACGTT TAAAGAAACCCTTTACACCCCTCACAA >Ap_TR2/4_DBD CATTTCTGGAATCGACAGTACTGGCATCTGTTTCTAGTCATTTGGTTCACTT CACAGTTGCCATCAGCTTTGCAGGTGTAGTCTAACTGTTTCTGTACGGTACGTTTA AAGAAACCCTTTACACCCCTCACAAATCCC >Uf_TR2/4_DBD GCACTAGTGATTCATTTCTGGAATCGACAGTACTGGCATCTGTTTCTAGTC ATTTGGTTCACTTCACAGTTGCCATCAGCTTTGCAGGTGTAGTCTAACTGTTTCTG TACGGTACGTTTAAAGAAACCCTTTACACCCCTCACAAAT >As_a CACTGTTCCATTCTTGACTTCTATACATTGGTTTCACGCCATCTTGATCCAT TGGTTGGATGTTCTTGTAGGCTCCCAAAAAAGATTTTACTACTTCAAACCTAAATTT CTTGTCGTATATCTGTCGTATACTGCATGCGCTTGACCATGTGGTTCACATGTTCA AACGTCCTCCTCCATGGTATTTCGGGGCTACAACTTGTGCCAATACTGTCGATTCC AGAAATGA >As_a LTVPFLTSIHWFHAILIHWLDVLVGSQKRFFKPKFLVVYLSYTACAPCGSHVQT SSSMVFRGYNLCQYCRFQK >As_10 TATTGCTCTACTGTTACGAGCGAAGCCGTACCATACCACCCCATTTCAACA ACAATCGGCTCAACATGCCCTGTGGTATCATGGGCATCGAGAATATCTGTGAGCT ATCTGCTAGGTTGCTGATTTAGTGCTGTTGAAGGGGCACGTAGTATCCCTTTCTTT CCTGACTTGCCCGTGACTGACCTAATGGCTTTGCTACCTCTTGTATGGAGTGAGC TGTTTGTGTTAAATGCATCACAGTGTCCCATGCGATTACAAGTTGCGCCGCTTCTT GCTACACCGGGAATTCACTCTAATCATATGTCTCCTGACACAATGGTCACCTTCAT GGATAATATGAAAATATTTCAAGAACAAGTGGATAAACTAAAAAATCTTCATGTACA TGCTGCCGATTTCGCCTGCTTGAAAGCAATTGTTTTGTTTACTTCAGACGCATCAG GGCTAACCGACCCACACTACATTGAAAGCCTACAAGAAAAGACACAGTGTGCCTT GGATGAATAGAACAAGAATCAGTATCCTAACCAACCCTCTCGCTTTGCAAAACTAC TGCTCCTGCTCCCATCACTGAGGAGCATAAGCACCAATGTGGTAGATCAGTTGTA CTATGTACGTCTAGTTGGGAAACTCCAAAGATACTCTTCTCAACATATGCTACTCT