Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

中文翻译：

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删除芽酵母MAD2抑制了人工线性染色体拷贝数的克隆到克隆差异，并提高了较高的保留率

我们的目标是调查每从有丝分裂停滞缺陷型2（的部分或全部损失引起细胞人造短线性染色体平均拷贝数的变化MAD2在芽殖酵母）纺锤体检验点功能酿酒酵母。总共对62种野生型和mad2∆进行了细胞群体中平均人工线性染色体拷贝数（通过定量聚合酶链反应（qPCR）测量）和保留率（通过波动分析测量）突变单倍体和二倍体克隆。野生型细胞（单倍体和二倍体）在表型上表现出独特的克隆间差异：一组15个克隆，每个细胞的拷贝数低且保留率高，其中1个克隆已经历了基因组整合事件，并且第二组有15个克隆，每个细胞的拷贝数高，保留率低，后者的值与以前发表的结果一致，后者仅测定了一个克隆。当在选择下繁殖10天时，观察到这些染色体状态是不稳定的，其中高拷贝低保留率克隆进化为低拷贝高保留率克隆，但是没有观察到整合事件的证据。相比之下，mad2∆单倍体和mad2∆ / mad2∆二倍体显示出克隆间差异的抑制，其中21个克隆中有20个每个细胞具有中等水平的人工线性染色体拷贝数，但保持了较高的染色体保留率。即使在3天的生长过程中没有选择，mad2∆和mad2∆ / mad2∆细胞的保留率仍保持较高水平。MAD2 /mad2Δ杂合二倍体显示出多个克隆组：4个具有低拷贝数，5个具有中等拷贝数和1个具有高拷贝数的人工线性染色体，但是所有10个克隆一致地显示出较低的保留率。我们的观察表明MAD2功能有助于酵母细胞在某些克隆中的每个细胞维持大量人工线性染色体的能力，但与直觉相反，mad2Δ抑制了克隆与克隆之间的差异，并导致人工线性染色体保留率的提高每个单元具有中级染色体拷贝数的更统一和稳定的克隆种群。