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.2013;11(3):e1001518.
doi: 10.1371/journal.pbio.1001518. Epub 2013 Mar 26.

Selecting one of several mating types through gene segment joining and deletion in Tetrahymena thermophila

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Selecting one of several mating types through gene segment joining and deletion in Tetrahymena thermophila

Marcella D Cervantes et al. PLoS Biol.2013.

Erratum in

Abstract

The unicellular eukaryote Tetrahymena thermophila has seven mating types. Cells can mate only when they recognize cells of a different mating type as non-self. As a ciliate, Tetrahymena separates its germline and soma into two nuclei. During growth the somatic nucleus is responsible for all gene transcription while the germline nucleus remains silent. During mating, a new somatic nucleus is differentiated from a germline nucleus and mating type is decided by a stochastic process. We report here that the somatic mating type locus contains a pair of genes arranged head-to-head. Each gene encodes a mating type-specific segment and a transmembrane domain that is shared by all mating types. Somatic gene knockouts showed both genes are required for efficient non-self recognition and successful mating, as assessed by pair formation and progeny production. The germline mating type locus consists of a tandem array of incomplete gene pairs representing each potential mating type. During mating, a complete new gene pair is assembled at the somatic mating type locus; the incomplete genes of one gene pair are completed by joining to gene segments at each end of germline array. All other germline gene pairs are deleted in the process. These programmed DNA rearrangements make this a fascinating system of mating type determination.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Molecular identification of the mating type locus using RNA-seq.
(A) RNA-seq data from mt VI and mt V cells mapped to a ∼300-kb region of the SB210 macronuclear reference genome (mt VI) (see Figure S2). The graph shows the number of RNA-seq reads (y-axis) from growing mt VI cells (orange, positive values), 3-h starved mt VI cells (blue, positive values) and 3-h starved mt V cells (red, shown as negative values) that mapped to the ∼300-kb region. Orange overlays blue. The box encloses a segment containing two genes with mating type-specific expression in starved cells and no expression in growing cells.x-axis: position within the 300-kb segment. (B) Transcripts (mt VI, blue) and transcript segments (mt V, red) were assembled from RNA-seq reads mapping to the boxed region in (A) and, for mt VI, from sequenced RT-PCR products. 5′ and 3′ untranslated regions are not included. The mt VI-derived transcripts correspond to a pair of divergently transcribed predicted genes (KC405257), now namedMTA6 andMTB6, respectively. Thin connecting lines represent introns. Both transcripts are drawn to scale, where each tick mark on the scale represents 1 kb. Each gene contains a TM exon and furin-like repeats (*).
Figure 2
Figure 2.MTA (orMTB) transcripts share homologous sequence and are of similar size.
Whole-cell RNA was extracted from starved mature strains of mating types II through VII (SB4208, SB4211, SB4214, SB4217, SB4220, and SB4223; see Table S1). Probes from within each conserved TM exon were hybridized to Northern blots. TheMTA6-TM probe hybridized to a ∼5-kb transcript (left panel), whileMTB6-TM probe hybridized to a ∼6-kb transcript (right panel).RPT3, a 26S proteasome subunit P45 family protein (XP_001007748) expressed during starvation, was used as a loading control. TheRPT3 probe hybridized to the expected ∼1.3-kb transcript. The complete blots are shown in Figure S3.
Figure 3
Figure 3. The germlinemat locus contains six incomplete mating type gene pairs.
The locus is a 91-kb tandem array of six incomplete, head-to-head mating type gene pairs, in the order II, V, VI, IV, VII, and III (order established as shown in Figure 4). Each gene pair begins to the left with theMTA conserved TM exon (diagonal lines) and ends with theMTB conserved TM exon (dark gray). Only the terminal genes (MTA2 andMTB3) have full length versions of their TM exons. The mating type-specific, somatic-destined segment for each mating type gene pair, which includes the 5′MTA andMTB segments and the intervening upstream spacer region (putative promoter), is shown as a single thick colored bar. Between the TM exon segments of adjacent gene pairs, there is a small amount of germline-limited sequence (GLS; black). Several IESs are located within the mating type-specific segments (also black). Excluding IES sequence, the mating type-specific segments are of comparable size: II, 8,673 bp; V, 9,132 bp; VI, 9,352 bp; IV, 8,450 bp; VII, 8,277 bp; and III, 8,384 bp. Exact coordinates of all these features are given in Table S4.
Figure 4
Figure 4. Only one mating type gene pair remains in the somatic nucleus.
Southern blot analysis was carried out using whole-cell genomic DNA from a mature strain of each mating type (SB4208, SB4211, SB4214, SB4217, SB4220, and SB4223; see Table S1). The DNA was digested withPvuII restriction endonuclease and separated by pulsed-field gel electrophoresis. Black segments, mating type-specific segment of each gene pair; diagonally hatched segments, conserved TM exons; arrows,PvuII sites; thin black bars, probes; size (kb) shown is that of the relevantPvuII fragment in the somatic genome (the corresponding germlinePvuII fragments are not visible due to differences in size and copy number).
Figure 5
Figure 5. TheMTA andMTB gene pairs are conserved in otherTetrahymena species.
An unrooted phylogenetic tree of entire mating type gene pairs identified in somatic sequence assemblies ofT. elliotti,T. malaccensis, andT. borealis (Tetrahymena Comparative Sequencing Project, Broad Institute of Harvard and MIT,http://www.broadinstitute.org/) andT. pyriformis strain GL (W. Miao, unpublished data) shows that the sequenced strain ofT. elliotti can be assigned to mt III and that ofT. malaccensis to mt IV. The scale bar represents 10% bp substitutions.
Figure 6
Figure 6. Most assembled somatic TM exons are generated by a single, simple joining event.
The sequenced TM exons are from progeny that had not yet undergone their first division (see Figure S1, stage 3, and Materials and Methods). The top six lines represent the germline mating type gene pairs of SB210, shown in their germline order (from top to bottom). All TM exons are drawn to scale. The darker gray bars represent intact and truncatedMTA TM exons, while the lighter gray bars represent truncated and intactMTB-TM exons. The mating type-specific segments are color-coded, as labeled, and are not drawn to scale as indicated by the double slash marks. The dashes beyondMTA2-TM andMTB3-TM indicate sequence adjacent to themat locus, which is identical in all nuclei. Vertical bars of mating type-specific color withinMTA andMTB TM exon segments represent the location of polymorphic nucleotides relative to the germline consensus sequence of each TM exon (the consensus sequence is shown in Text S6 and a complete list of polymorphisms is shown in Tables S5 and S6). As an example, the simplest possible germline origin of the most common somaticMTA6-TM andMTB6-TM exons is indicated by boxed regions within the germline mating type gene pairs and somatic exons. For each mating type, approximately tenMTA-TM exons and 30MTB-TM exons were sequenced (see Texts S7 and S8 for details). Numbers to the left ofMTA and to the right ofMTB TM exons represent the number of times each combination of polymorphic nucleotides was found among the sequenced TM exons. *, location of a base not present in the germline; these changes could be due to either PCR errors or replication repair errors and occurred at a rate of 1 bp in 50 Kbp (see Texts S7 and S8 for details).
Figure 7
Figure 7. Model proposing that homologous recombination assembles a single mating type gene pair during somatic differentiation.
In this model, intramolecular recombination events are initiated at both ends of the germline array; subsequent resolution results in removal of intervening gene pairs by looping out and joining of a gene pair to the full length TM exons at the ends of the array. Any number of gene pairs could be excised in a single recombination event; since the chromosomal product regenerates the recombination substrate, recombination steps can be reiterated until a single, complete gene pair remains, at which point the process has to stop. Sequestering or disabling the ability of side products to recombine again would minimize unproductive reversal of the process. Recombination events need not always involve a full length TM exon; two internal tm exons could also be involved at intermediate steps. The recombination process is labeled “homologous recombination” for simplicity, but identical results could be obtained by highly precise non-homologous end-joining. Side products containing a discrete number of gene pairs, shown here as circular, could also be linear depending on the details of the recombination and repair mechanism. A related DNA rearrangement model ofT. thermophila mating type determination, also involving recombination and alternative deletion in a tandem array of germline mating type genes was proposed previously . The key conceptual difference is that in the original model a unique segment was somatically attached at one end of an individual mating type gene, instead of attaching unique segments at both ends of a mating type gene pair, as reported here.
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