Cloning of a major repeat DNA sequence from Pyura stolonifera

Abstract

We have cloned a 147-bp Hind III fragment from a marine ascidian Pyura stolonifera. This sequence is arranged in tandem in arrays up to 20 kb in size and represents more than 5% of the total genomic DNA. The basic 147-bp unit was isolated from Hind III-digested genomic DNA and cloned into M13. Sequence analysis of seven clones revealed that the sequence is AT rich (75%) and can be separated from main band DNA by equilibrium density gradient centrifugation in the presence of the ligand dye Hoechst 33258. The sequence is highly conserved and is changed only by single base substitution mutations in the different clones. Use of this sequence as a probe demonstrated varying degrees of hybridization with DNA isolated from a wide range of other ascidians. Northern blot analysis revealed the presence of RNA hybridizing to the repeat in unfertilized eggs but transcription of this sequence was not evident in the adult organism.