Ciliates segregate germline and somatic function to two separate nuclei within a single cell. During the development of a new somatic nucleus in Tetrahymena thermophila, site-specific fragmentation of the 5 chromosomes derived from the germline (micronucleus or MIC) generates ~200 somatic (macronuclear or MAC) chromosomes. The 15-bp Cbs (Chromosome breakage sequence) family is the necessary and sufficient cis-acting DNA element for programmed chromosome fragmentation. Roughly half of the Cbs’s in the MIC genome (109/225) have the consensus sequence, TAAACCAACCTCTTT, with T at each of the only 5 variable positions. No Cbs known to be functional contains substitutions at >2 variable positions. Only at variable positions 1 & 11 have the nucleotide frequencies achieved the maximum diversity expected if all isoforms are functionally equivalent. Cbs-mediated chromosome breakage is limited to Tetrahymenine ciliates, raising the possibility that not enough evolutionary time has elapsed to achieve maximal diversity at Cbs positions 13-15.
To better understand how the current T. thermophila Cbs family evolved, a computer simulation has been undertaken. Random mutations at natural rates are allowed in DNA segments that contain the 15-bp Cbs, while 15-bp segments of unconstrained sequence flanking the Cbs on either side act as controls. Simulation results include the following:
1) Simulations starting with the current Cbs complement have reproduced the finding, by calculation, that diversity is currently maximal at positions 1 & 11, but not at positions 13-15.
2) In simulations starting with an all-consensus full Cbs complement, the consensus Cbs frequency decreases from 100% to its current value (~50%) well before maximal diversity is approached at positions 1 & 11 – seemingly inconsistent with the maximum diversity currently observed as these 2 positions.
3) A and T are the only nucleotides allowed at positions 1 & 15. The observed asymmetry in the A:T frequency ratio at positions 1 (32% A) and 15 (9% A) is not reproduced by these simulations, which generate equal relative frequencies at both positions.
The results are informative, as they suggest refinement of particular null assumptions made in the initial simulations. Additional results and hypotheses will be discussed. Sequencing additional Tetrahymenine germline genomes should generate valuable relevant data and additional evolutionary insights.