Cloning of Hsp70 genes from the marine spongesSycon raphanus(Calcarea) andRhabdocalyptus dawsoni(Hexactinellida). An approach to solve the phylogeny of sponges

Abstract

The phylogenetic relationships among the three classes of the Porifera—Demospongiae, Calcarea and Hexactinellida—are still unresolved, despite the use of molecular analyses of rRNA. To determine whether phylogenetic resolution of these classes is possible based on genes coding for specific proteins, in the present study the genes for the 70 kDa heat shock protein [Hsp70] were isolated fromRhabdocalyptus dawsoni[Hexactinellida] and fromSycon raphanus[Calcarea], and compared to that previously isolated from the demospongeGeodia cydonium. The gene fromR. dawsoniis 2021 bp long and encodes a predicted Hsp70 of Mr77,697; the protein comprises the characteristic sites of eukaryotic, cytoplasmic Hsp70 polypeptides. The Hsp70 isolated from cDNA fromS. raphanusis 2326 bp long. It encodes a potential polypeptide of Mr85,927 and belongs to the same class of Hsp70s. All three sponge sequences for Hsp70 were found to be highly identical to both human and plant Hsp70s. The degree of identity at the amino acid (aa) level between the sponge sequences and the human sequence for Hsp70 is 77%–84% and at the nucleotide (nt) level, between 69% and 75%. Resolution of the phylogenetic relationship between the three classes of sponges based on the Hsp70 was not possible due to the high degree of identity [similarity] of their respective aa sequences, which ranged from 80% [90%] to 82% [91%]. The evolutionary rates—kaa-values—calculated for the sponge Hsp70 molecules, are low, reflecting the strong functional contraints placed upon these polypeptides. These values range from 0.125×10−9forG. cydoniumandR. dawsonito 0.087×10−9forS. raphanus. Higher values have previously been reported for theG. cydoniumgalectin molecule [kaa-value of 1.7×10−9] and the receptor tyrosine kinase [1.24×10−9] from the same animal. The occurrence of at least one double mutation, in the codon for the aa Ser in the conserved regions of the Hsp70 sequences, also suggests that these molecules are subjected to strong functional constraints.