Genomicsequences from the D. immitis EcR Dinaciclib homolog, genomic libraries from B. malayi available from the Filarial Genome Network were screened. A strongly hybridizing BAC was identified and sequenced. This BAC contained a gene that encodes a protein with strong similarities to the EcR branch of nuclear receptors. We designated this gene as Bma EcR. Using sequences corresponding to the predicted Bma EcR exons, PCR primers were designed and used to screen larval and adult cDNA libraries. This library survey revealed Bma EcR expression in L1, L3, and L4 larval stages, as well as in adult males and females. In the microfilaria library, using primers from the putative ligand binding domain encoding region, two alternatively spliced mRNA isoforms of Bma EcR were identified.
Bma EcRA is the isoform containing the longest ORF with an intact LBD. Bma EcRC contains exon 6 with a 29 nucleotide deletion that results in a reading frame shift that generates a premature stop codon and truncation of the LBD at helix 5. This is the result of an alternative splice site within Alisertib exon 6. In addition to these confirmed isoforms, a splice site consensus sequence was identified within exon 5 at the end of the DNA binding domain that, if used, would result in the omission of ten amino acids from the C terminal extension of the DBD. This type of spliced mRNA isoform has been identified in D. immitis. We were unable to clone such an isoform from B. malayi by RT PCR. However, we cannot exclude the possibility that Bma EcRB is expressed in specific tissues or developmental stages not represented in the libraries or RNA used.
Sequence and phylogenetic analysis of Bma EcR Bma EcR shows strongest similarity to the NR1H group of nuclear receptors typified by the insect EcRs and mammalian FXR and LXR receptors. The strongest similarity is in the DBD which contains the canonical C4 zinc finger structure of nuclear receptors. This domain is 10 amino acids longer in Bma EcR than in the homologous region of the other EcRs. However, as indicated above, exclusion of these 10 amino acids by alternative splicing of this site would result in a better alignment of Bma EcR with the other EcRs. The LBD shows significant similarity in the regions encoding helices 3 10. An exception is found in the region of helix 11 12.
Helix 12 of insect EcRs contains the AF2 motif, responsible for ligand dependent transcriptional activation. Although predictions of secondary structure of the Bma EcR LBD protein sequence indicate helical folding of putative helices 3 10, no helical propensity is predicted in the region of helix 12. Immediately following the helix 12 region a glutamine rich helical segment is present. Glutamine rich sequences are often associated with transcription activation domains. These differences make Bma EcR an unusual member of the receptor family that perhaps uses a different mechanism for ligand dependent activation. Global phylogenetic analysis places Bma EcR with arthropod EcRs. The position of Bma EcR is strongly supported in a phylogenetic tree of the sub family. The branch leading to Bma EcR is long, indicating a relatively derived sequence, but not more derived than that of dipteran EcRs, for example. Separate analysis of the DBD and LBD produced similar tree topologies, especiall.