Isolation of Biologically Functional RNA during Programmed Death of a Colonial Ascidian

The blastogenic (asexual) cycle of the colonial ascidian Botryllus schlosseri (Tunicata, Ascidiaceae) concludes in a cyclical phase of programmed cell and zooid death called takeover, in which all asexually derived adults die synchronously by apoptosis. The characterization of developmentally regulated genes whose expression patterns are selectively modulated during this process could pave the way to understand how this model organism dies. However, isolation of biologically functional RNA in this and other colonial ascidians with conventional phenol/chloroform-based procedures is hampered by extensive contamination of RNA preparations by pigments. Upon cell lysis, pigments that normally reside within specialized cells in the mantle wall of the adult are released and tightly associate with nucleic acids. Here, we report on the usefulness of a single-step RNA isolation method in which acid guanidinium isothiocyanate is used as an extraction medium, followed by preparative cesium chloride ultracentrifugation. This procedure successfully isolated biologically active, high-purity total RNA ( OD260/ OD280=1.9-2.1) from Botryllus colonies during takeover, as well as other species of colonial ascidians (Diplosoma macdonaldii, Botrylloides diegense) irrespective of pigmentation. Northern blot analysis performed with a 32 P-labeled tunicate actin probe detected two polyadenylated transcripts of 1.5 and 1.7 kilobases in length from both growth phase and takeover colonies. Two-dimensional protein gel assays from in vitro translated mRNA preparations further revealed that specific transcripts were upregulated during takeover, while others were repressed or down-regulated. Growth phase and takeover-specific cDNA libraries were constructed from pooled poly ( A)+ RNA with a complexity of 1.0 × 107 and 1.2 × 107 recombinants respectively per 100 ng of cDNA before amplification. The procedure described herein renders feasible the cloning of developmentally regulated genes in this organism. In addition, our findings raise the possibility that zooid death in Botryllus involves modulated gene expression.