Parry J M, Zimmerman F K
Mutat Res. 1976 Jul;36(1):49-66. doi: 10.1016/0027-5107(76)90020-8.
A diploid yeast strain, D6 is described which monitors mitotic non-disjunction by the phenotypic expression of a set of coupled and recessive markers flanking the centromere of chromosome VII. These markers are not expressed in the heterozygous condition prevailing in D6. The left arm of chromosome VII carries a tightly centromere linked marker, leu1 (leucine requirement), distal to leu1 in this order: trp5 (tryptophan requirement), cyh2 (recessive resistance to cycloheximide) and met 13 (requirement for methionine). The right arm is marked with ade3 (simultaneous requirement for adenine and histidine). D6 is homozygous for ade2 and consequently, forms red rather than the normally white colonies. It shows no requirement for the above amino acids and it is sensitive to cycloheximide. Unmasking of all the markers on chromosome VII leads to colonies that are white because ade3 sets a block preceding the ade2 block (which causes the accumulation of a precursor of the red pigment), they require leucine, tryptophan and methionine, and grow on media with cycloheximide. Cells are plated on a cycloheximide medium where red and white colonies are formed. Colonies of spontaneous origin were tested. The majority of the white colonies expressed all the recessive markers whereas only few of the red colonies expressed all the markers on the left arm of chromosome VII. Basically expression of recessive markers on both sides of the centromere can be explained as a result of two coincident events of mitotic crossing over. However, the frequency of colonies expressing centromere linked leu1 was 14 times higher among the white types than the red ones. This suggested that the white, cycloheximide resistant, leucine requiring colonies arose by mitotic non-disjunction and not only by two coincident mitotic crossing over events. Presumptive spontaneous monosomic segregants were placed on sporulation medium. Only 8 out of 30 isolates sporulated, which showed that these eight segregants were diploid at the time of sporulation. They could have arisen by two coincident crossover events or through restoration of a normal disomic condition after non-disjunction had occurred. The genetic data thus leaves us with only its statistical argument in favour of non-disjunction. Further confirmation of monosomic nature of the white cycloheximide resistant colonies was provided by the estimates of their DNA contents. Compared to the stock wild type diploids the presumptive monosomics showed a reduction in DNA content. We have utilized D6 to investigate the possible induction of mitotic non-disjunction after treatment with gamma rays, heat shock at 52 degrees C and ultraviolet irradiation. In all cases white, cycloheximide resistant colonies were produced at levels significantly higher than that found in untreated cultures. In order to detect the production of monosomic cells, treated cultures were grown for 48 h in non-selective medium after exposure to allow for "expression" of the monosomic condition.
描述了一种二倍体酵母菌株D6，它通过位于染色体VII着丝粒两侧的一组连锁隐性标记的表型表达来监测有丝分裂不分离。这些标记在D6中占主导的杂合状态下不表达。染色体VII的左臂携带一个与着丝粒紧密连锁的标记leu1（亮氨酸需求），在leu1远端依次为：trp5（色氨酸需求）、cyh2（对环己酰亚胺的隐性抗性）和met13（甲硫氨酸需求）。右臂标记为ade3（同时需要腺嘌呤和组氨酸）。D6对ade2是纯合的，因此形成红色而非正常的白色菌落。它对上述氨基酸无需求，且对环己酰亚胺敏感。染色体VII上所有标记的暴露导致菌落为白色，因为ade3在ade2阻断（导致红色色素前体积累）之前设置了一个阻断，它们需要亮氨酸、色氨酸和甲硫氨酸，并能在含环己酰亚胺的培养基上生长。将细胞接种在能形成红色和白色菌落的环己酰亚胺培养基上。对自发产生的菌落进行了检测。大多数白色菌落表达了所有隐性标记，而只有少数红色菌落表达了染色体VII左臂上的所有标记。基本上，着丝粒两侧隐性标记的表达可解释为两个同时发生的有丝分裂交换事件的结果。然而，白色类型菌落中表达与着丝粒连锁的leu1的频率比红色菌落高14倍。这表明白色、抗环己酰亚胺、需要亮氨酸的菌落是通过有丝分裂不分离产生的，而不仅仅是两个同时发生的有丝分裂交换事件。假定的自发单体分离株被置于产孢培养基上。30个分离株中只有8个产孢，这表明这8个分离株在产孢时是二倍体。它们可能是由两个同时发生的交换事件产生的，或者是在不分离发生后通过恢复正常的二体状态产生的。因此，遗传数据仅为支持不分离提供了统计学依据。通过对其DNA含量的估计，进一步证实了白色抗环己酰亚胺菌落的单体性质。与野生型二倍体原种相比，假定的单体显示出DNA含量的降低。我们利用D6研究了用γ射线、52℃热休克和紫外线照射处理后有丝分裂不分离的可能诱导情况。在所有情况下，产生的白色、抗环己酰亚胺菌落的水平都显著高于未处理培养物中的水平。为了检测单体细胞的产生，处理后的培养物在暴露后在非选择性培养基中培养48小时，以允许单体状态的“表达”。
