Fig 1.
The characteristic of the chromosome breakage sequence (Cbs).
(A) Sexual reproduction in Tetrahymena. (B) MAC formation is directed by Cbs breakage. After Cbs breakage, the MIC genome separates into independent MAC minichromosomes, and new telomeres are added to the ends of the MAC minichromosomes [39, 40]. (C) We identify 209 functional Cbs sites in the MIC genome. About half of them are classic Cbs with the exact same sequence [39]. The PWM (Position Weight Matrix) shows that there is an invariable 10-bp core sequence and the other 5 positions have the highest probability to be an "A", and limited possibilities for other nucleotide substitutions [42]. (nm: new macronucleus; om: old macronucleus)
Fig 2.
The size distribution of putative MAC minichromosomes.
(A) We name the putative MAC minichromosome by its order in the MAC supercontig as illustrated. The section between the start position to the first Cbs site is named echr2.X.0, the following section between two Cbs sties is echr2.X.1 and so on. (B) The size distribution of all Cbs-related sections are shown. There are two kinds of Cbs-related sections. The red bar indicates the Cbs-related section that is eliminated from the MAC genome. The blue bar indicates the Cbs-related section that remains in the mature MAC genome. (C) The size distribution of only the Cbs-bounded sections are shown.
Fig 3.
Eliminated minichromosomes disappear at different time points.
(A) Southern blot hybridization of echr2.105.1 and echr2.75.1 are shown. The genomic DNA extracted from vegetative cells, conjugating cells (8, 12, 14, 15, 16, 18, 20 and 24 hour post mixing) and feeding progeny cells (3, 6, 10, 20, 30 and 40 doublings after conjugation) was analyzed. HindIII digested (for echr2.105.1) and uncut whole cell DNA samples (for echr2.75.1) were separated in a 0.6% agarose gel by standard gel electrophoresis, respectively. DRB3 gene was used as a macronucleus-retained DNA control for HindIII-cut DNA, and ethidium bromide (EtBr) staining of uncut DNA was used for loading control. D: doubling number after conjugation. (B) The coverages of reads of DNA in the growing phase of the inbred lines CU427, CU428, BII and in the mating pool (24hpm) are shown. We use the MIC genome as the reference to align these whole cell DNA reads, which are mostly MAC reads. The region with low coverages in CU427, CU428 and BII indicates the location of the eliminated minichromosome. The coverage of the region in supercont2.75, but not in supercont2.310, is slightly higher in the mating pool. It should be noted that the mating pool contains significant amounts (~30%) of non-mated cells and aborted mating cells that contain mature MAC, which partially explain the lower coverage in this section than its neighboring sections. (C) Telomere-anchored PCR of one end of 10 EMCs is shown. EMCs were detected by PCR using telomere sequence as one primer and the sequence of the minichromosome as the other (black arrows). The black line indicates one end of a minichromosome in the macronucleus, and the gray box indicates telomere. The genomic DNA of vegetative cells, different stages of conjugating cells and feeding progeny cells after conjugation was analyzed. The chromosome ends near Cbs819 and ATU1 (α-tubulin) were used as the macronucleus-retained DNA control.
Fig 4.
Eliminated minichromosomes express genes in the late conjugation stage.
This figure shows the read coverage of genomic DNA and mRNA at different time points in echr2.105.1 (A) and in echr2.75.1 (B). MAC: MAC genomic sequencing results. Expression: mRNA sequencing results. hpm: hours post mixing. D: doubling number after conjugation. Transcript: the predicted transcript from Cufflinks [57]. Gray boxes indicate the unknown region caused by sequencing incompletion.
Table 1.
The number of expressed genes in eliminated minichromosomes.
Fig 5.
The expression profile of genes in the eliminated minichromosomes.
(A) shows the quantitative-RT-PCR results of two genes located in echr2.105.1. (B) shows the quantitative-RT-PCR result of the gene located in echr2.75.1. hpm: hours post mixing. D: doubling number after conjugation.
Fig 6.
Evolution of eliminated minichromosomes.
(A) The phylogenetic tree shows that TPB6 only has one ortholog in T. borealis. (B) Genes in echr2.105.1 in T. thermophila are conserved in other Tetrahymena species. Gene X, I and J are MAC-destined genes in both T. thermophila and T. borealis. The figure indicates the synteny of the genes in the 3’-truncated region of echr2.105.1 and the adjacent MAC minichromosome in these species.