Partial Purification of Telomerase Enzyme From the Choanoflagellate M. brevicollis

The transition from circular to linear chromosomes in eukaryotes introduced the “end-replication problem” which is the inherent inability of cellular DNA polymerases to completely replicate linear chromosomal ends. Over evolutionary time, eukaryotes evolved “caps” at their chromosomal ends which are DNA protein complexes known as telomeres. Although telomeric DNA does suffer from the incomplete end-replication, the telomerase ribonucleoprotein enzyme was evolved as the dominant and winning solution to this problem in eukaryotes. The protein component of telomerase known as Telomerase reverse transcriptase (TERT), is well conserved across broad eukaryotic groups. In contrast, the RNA component of telomerase, telomerase RNA (TR) is extremely divergent in terms of sequence and length. This presents insurmountable challenges in the identification of novel TR molecules, especially from more distant and previously unexplored eukaryotic groups. Although animal TRs have been identified and studied in detail, the early evolution and origins of animal telomerases remain a mystery. Thus, it is crucial to study telomerases from the earliest ancestors of animals. The Choanoflagellates are a group of free-living unicellular eukaryotes that are deemed to be the closes living relatives of animals. The choanoflagellate M. brevicollis (Mbr) is a model eukaryote used to study the origins of multicellularity. Thus, we determined to purify M. brevicollis telomerase to isolate, sequence and identify the co-purifying TR. Towards achieving this ultimate goal, this study focuses on partially purifying M. brevicollis telomerase via polyethylene glycol (PEG) precipitation. As the first step, reliable and reproducible culture conditions for M. brevicollis were established. Following this, larger scale cell cultures were grown and used for PEG precipitation. Final concentrations of 5%, 10%, and 20% PEG were used. PEG precipitates were resuspended in buffer and quantitated using Bradford assay. PEG precipitated macromolecular complexes were subject to Western blot using custom generated anti-MbrTERT antibodies which revealed a clear band proximal in size to the 75 kDa marker consistent with the 87 kDa putative MbrTERT. This study serves as a launchpad for the identification of MbrTR towards delineating the early evolution of telomerase in animals.