Change in among-line variance of chromosome copy number.

<p>The effect of varying levels of stabilizing selection pressure on the change in among-line variance in the simulations and experimental quantifications in the 6 cell lines is shown for chromosomes (A) <i>α-tubP1</i>, (B) <i>EF1α</i>, and (C) <i>α-tubP2</i>. <i>ω²</i> describes the selection surface. As <i>ω²</i> increases, the strength of stabilizing selection decreases. ‘Ex’ indicates the experimentally observed among-line variance as estimated with restricted maximum likelihood analysis. ‘No Sel’ represents the simulations without any stabilizing selection pressure. Error bars are the 95% confidence intervals for both the experimentally observed among-line variance and the simulations. Permutated t-tests with unequal variance were conducted between the experimental data (‘Ex’) and the simulations. The cross (+) indicates the values of <i>ω²</i> at which there is no significant difference between the change in variance of the experimental results and the change in variance of the simulation.</p

Loss of experimental lineages through 276 rounds of asexual division.

<p>Each dot indicates the number of cell lines alive at that generation. The experiment began with 60 cell lines and ended with 14 viable lines.</p

Change in mean chromosome copy number from initial to final time-point.

<p>In the <i>α-tubP1</i>, <i>EF1α</i>, and <i>α-tubP2</i> chromosomes, the 95% confidence intervals (error bars) of the change in mean chromosome copy number overlap 0, which indicates there is not a significant difference between the initial and final macronuclear chromosome copy number. SSU has a 95% confidence interval that is greater than 0, meaning that there are more copies of the SSU chromosome at the final time-point than the initial.</p

Chromosome Copy Number Variation and Control in the Ciliate <i>Chilodonella uncinata</i>

<div><p>Copy number variations are widespread in eukaryotes. The unusual genome architecture of ciliates, in particular, with its process of amitosis in macronuclear division, provides a valuable model in which to study copy number variation. The current model of amitosis envisions stochastic distribution of macronuclear chromosomes during asexual reproduction. This suggests that amitosis is likely to result in high levels of copy number variation in ciliates, as dividing daughter cells can have variable copy numbers of chromosomes if chromosomal distribution during amitosis is a stochastic process. We examined chromosomal distribution during amitosis in <i>Chilodonella uncinata</i>, a ciliate with gene-size macronuclear chromosomes. We quantified 4 chromosomes in evolving populations of <i>C. uncinata</i> and modeled the amitotic distribution process. We found that macronuclear chromosomes differ in copy number from one another but that copy number does not change as expected under a stochastic process. The chromosome carrying SSU increased in copy number, which is consistent with selection to increase abundance; however, two other studied chromosomes displayed much lower than expected among-line variance. Our models suggest that balancing selection is sufficient to explain the observed maintenance of chromosome copy during asexual reproduction.</p></div

Copy number variation between macronuclear chromosomes.

<p>(A) and (C) show the mean copy number and the bootstrapped 95% confidence intervals of the among-line variance component, respectively, for the initial qPCR quantification for SSU, <i>α-tubP1</i>, <i>EF1α</i>, <i>α-tubP2</i>. (B) and (D) show the mean copy number and the bootstrapped 95% confidence intervals of the among-line variance component, respectively, for the final qPCR quantification of the same MAC chromosomes. (A) and (B) (a–d) indicate significant differences between the mean chromosome copy number for each time point (Tukey-HSD, different letters indicate p<0.05).</p

Cell line death rate under varying levels of stabilizing selection.

<p>One thousand replicates of 60 cell lines were simulated with values of <i>ω²</i> ranging from 0.02 to 0.80 in intervals of 0.02. Mortality occurs when all the cells within a lineage having either perished due to selection pressure or having not received at least 1 MAC chromosome. Shown are the results of a simulation with a starting chromosome copy number of 66, which was the copy number value of the chromosome with the lowest copy number in this study. The simulation followed the model of random segregation of macronuclear chromosomes with stabilizing selection acting on changes from the optimal (initial) chromosome copy number. <i>ω²</i> determines the strength of stabilizing selection and as <i>ω²</i> decreases, stabilizing selection increases and results in increased cell mortality.</p

Class switch recombination activity in B-cell specific <i>Ssb1</i>-deleted mice.

<p><i>Ssb1 ^fl/fl</i> mice were crossed with a <i>Cd19</i>-Cre transgene expressing C57BL/6J mice to specifically delete <i>Ssb1</i> in B cells. Splenic B cells were isolated and stimulated for 2, 3, and 4 days using anti-Cd40 antibodies plus IL-4 to induce CSR to IgG1. (A) Western blotting of whole cell extracts showed loss of Ssb1 protein in stimulated B cells from <i>Ssb1</i> knockout (<i>Ssb1</i> KO; <i>Cd19</i>Cre⁺: <i>Ssb1^−/−</i>) mice. Equal amounts of stimulated B cell extracts from heterozygous <i>Ssb1</i> (<i>Ssb1</i> Het; <i>Cd19</i>Cre⁺: <i>Ssb1^+/−</i>) and wild-type <i>Ssb1</i> (<i>Ssb1</i> WT; <i>Cd19</i>Cre⁺: <i>Ssb1^+/+</i>) mice were included for comparison. Equal loading was confirmed by probing for Hsp90α. (B) Western blotting of Ssb2 levels in B cells from <i>Ssb1</i> WT and <i>Ssb1</i> KO mice. (C) FACS analysis of CSR to IgG1 over time in stimulated B cells from mice of the indicated genotypes. Stimulated splenic B cells from <i>AID^−/−</i> (<i>AID</i> KO) mice served as a negative control. (D) Summary statistics of CSR activity to IgG1 and viability on day 3 of stimulation. Mean and S.E.M. from three independent experiments are shown. No statistically significant differences (two-tailed unpaired <i>t</i>-test) were found. (E) Switch region junction analysis. Sm-Sg1 junctions were amplified from IL4 plus anti-CD40 stimulated primary B cells (day 4) and sequenced. Percentage and total numbers of direct or microhomology-mediated joints are indicated. nt, nucleotides.</p

<i>Ssb1</i> deletion causes multiple skeletal defects.

<p>(A) Alcian blue and Alizarin red staining of control (<i>Ssb1</i>^+/+, <i>Ssb1</i>^+/−) and <i>Ssb1</i>^−/− E18.5 ribcages. (B) Butterfly rib-spread of <i>Ssb1</i> control and <i>Ssb1</i>^−/− E18.5 ribcages. (C) Comparison of skull anatomy of <i>Ssb1</i> control and <i>Ssb1</i>^−/− E18.5 embryos. eo, exoccipital; so, supraoccipital; ip interparietal; pr, parietal; fr, frontal; mx, maxilla; md, mandible; bo, basioccipital; ptg, pterygoid; pmx, premaxilla; ppmx, palatal process maxilla; ppp, palatal process palatine; p, palatine; tr, tympanic ring. (D) Comparison of <i>Ssb1</i> control and <i>Ssb1</i>^−/− mandibles <i>(Scale = 1 mm)</i>. (E) Forelimb and (F) Hindlimb of E18.5 <i>Ssb1</i> control and <i>Ssb1</i>^−/− embryos. Sc, scapula; H, humerus; R, radius; U, ulna; F, femur; T, tibia. * designates missing fibula. (G) Quantification of long bone measurements of the forelimb and hindlimb of <i>Ssb1</i>^+/− and <i>Ssb1</i>^−/− E18.5 limbs. Data represent the mean ± SEM of bone length (<i>n</i> = 3 per condition, ***<i>P</i>&lt;0.001, student's <i>t</i>-test). (H) Whole-autopod (top) and skeletal preparation (bottom) of E18.5 forelimbs (left) and hindlimbs (right) from <i>Ssb1</i> control and <i>Ssb1</i>^−/− embryos.</p

Testicular degeneration and impaired fertility in conditional <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− male mice.

<p>(A) Representative image of testes from Rosa26-CreER^T2: <i>Ssb1</i>^−/− mice compared with those from <i>Rosa26</i>-CreER^T2: <i>Ssb1^+/−</i> littermates at 10 weeks of age. (B) Testis weight and (C) the gonado-somatic index (GSI) in conditional <i>Ssb1</i> deleted male mice compared with their littermates. Data represents the mean ± SEM of testis weight (<i>n</i> = 8, <i>***P</i>&lt;0.001; student's <i>t-</i>test) and the GSI (C) from 8 mice in each group. (D) Representative images of testis sections stained with Haematoxylin and eosin (left panels) and ApopTag for the detection of apoptotic cells (right panels). Note the multinucleated giant cells (black arrow) frequently present in the lumen in the testes from <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− mice. Spermatogenic cells in <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− testes showed an elevated apoptotic marker (black arrows, lower panel, right, <i>Scale = 25 µm</i>). (E) Haematoxylin and eosin staining showing epididymides of <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− mice containing prematurely sloughed developing germ cells. The upper panel displays maturing spermatozoa in the wild type epididymis. In contrast, immature germ cells (black arrow) are present in the epididymis of <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− mice (lower panel). Embedded images in the left corners show magnified views of the selected areas <i>(Scale = 50 µm</i>). (F) Litter size from <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− male breeders (<i>n</i> = 4) with wild type female mice compared with those from <i>Rosa26</i>-CreERT2: <i>Ssb1^+/−</i> breeders (<i>n</i> = 6, *<i>P</i>&lt;0.05; student's <i>t-</i>test). (G) Litter interval in <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− male mice (average of 63 days, <i>n</i> = 4) and Rosa26-CreER^T2: <i>Ssb1^+/−</i> males (average of 27 days, <i>n</i> = 6, ***<i>P</i>&lt;0.001; student's <i>t-</i>test).</p

Conditional <i>Ssb1</i>-deleted mice are sensitive to IR.

<p>(A) Schematic diagram of the radiation challenge assay in <i>Ssb1</i> deleted mice. <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^+/+, <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^+/− and <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− mice were challenged with 8 Gy of total body irradiation (TBI). The acute lethal response of mice to TBI was evaluated over a 30-day observation period. (B) Kaplan-Meier survival analysis of irradiated mice. Kaplan-Meier survival curves compared by log-rank (Mantel-Cox) analysis showed significant difference between <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− mice and the other two groups (**<i>P</i>&lt;0.01), while no difference was found between <i>Rosa26</i>-CreER^T2: <i>Ssb1^+/+</i> and <i>Rosa26</i>-CreER^T2: <i>Ssb1^+/−</i> groups. (C) Representative images of Haematoxylin and eosin, Ki67 (cell proliferation) and ApopTag (cell death) staining on small intestine sections from mice at Day 5 post 8 Gy of TBI.</p