Osystems, Foster City, CA). Using the resultant sequence data, we further
Osystems, Foster City, CA). Using the resultant sequence data, we further designed gene-specific primers for 5 and 3 rapid amplification of cDNA ends (RACE). 100 g isolated total RNA was further purified to recover polyA + RNA using an Oligotex-dT Super kit (Takara). 1 g of polyA + RNA was applied to the SMART RACE kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer’s protocol for generation of 5 and 3 adaptorligated first-strand cDNA synthesis. Primary amplification was performed using a universal primer mixture (UPM, Clontech/Takara), complimentary to the adaptor sequence provided with the kit and gene specific primers (listed in Additional file 4: Table S4). PCR was performed in a final volume of 10 l containing 1x Taq polymerase buffer (Takara), 250 M deoxyribonucleotide triphosphate, 0.5 U DNA polymerase (Taq polymerase, Takara), 1 M degenerated primers, 1 l first-strand cDNA, and 10 mM of dithiothreitol. PCR conditions were as follows: 94 for 5 min; 5 cycles at 94 for 30 sec and 72 for 2.5 min; 5 cycles at 94 for 30 sec, 70 for 30 sec, 72 for 2 min; and 18 cycles at 94 for 30 sec, 64 (for R1 and R3) or 55 (for R2) for 30 sec and 72 for 2 min. Secondary or nested PCR was performed using 0.2 l of the primary PCR product, a nested universal primer (NUP) complimentary to the adaptor sequence provided with the kit, and a gene-specific nested primer. PCR conditions were as follows: 94 for 5 min; 5 cycles at 94 for 30 sec and 72 for 2.5 min; 5 cycles at 94 for 30 sec and 70 for 30 sec, 72 for 2 min; and 25?0 cycles at 94 for 30 sec, 64 (R1 and R3) or 55 (R2) for 30 sec and 72 for 2 min. The PCR products were gel purified and some were ligated into the pGEM-T plasmid vector (Promega).Sequencing was performed using an ABI Prism 3100 Sequencer or by Fasmac Co., Ltd. (Tokyo, Japan), using primers listed in Additional file 4: Table S4. Three independent positive clones from distinct amplifications were sequenced to avoid PCR error.Phylogenetic analysis of GnRH receptorsVertebrate homologues of the GnRH receptors were identified through a combination of reciprocal BLASTp, TBLASTn, and TBLASTx searches using axolotl and human GnRH receptors as the query STI-571 chemical information sequences in searches among well-annotated genomes in the NCBI and ENSEMBL databases. Additional homologs sequenced through cloning efforts were included for phylogenetically informative taxa. Finally, to obtain a better picture of outgroup and ancestral sequences, BLAST searches of some PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28914615 invertebrate genomes were conducted; our selection of species to be examined was guided by the data presented by Roch and colleagues [14]. In all cases, only full length GnRH receptor sequences were included in the analyses, and partial sequences and splice variants were excluded. We also used a complementary approach of Hidden Markov Model (HMM) domain searching to identify homologs in the elephant shark, skate, lamprey and gar genomes. We focused PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26740125 the HMM searches on these species because these genome assemblies exhibited small contigs, which meant that sequence representation for full length genes was unlikely. In addition, each of these species holds a phylogenetically informative position with respect to many of the hypotheses addressed here. Results from BLAST searches of the elephant shark and little skate genomes indicated that at least two GnRH receptor homologs are present, but sequence matches for each homolog were short in length and located on two separate con.
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