Stoycheva T, Venkov P, Tsvetkov Ts
Institute of Cryobiology and Food Technology, 53A Cherni Vrah Blvd, 1407 Sofia, Bulgaria.
Cryobiology. 2007 Jun;54(3):243-50. doi: 10.1016/j.cryobiol.2006.10.188. Epub 2007 Apr 9.
Although suggested in some studies, the mutagenic effect of freezing has not been proved by induction and isolation of mutants. Using a well-defined genetic model, we supply in this communication evidence for the mutagenic effect of freezing on mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae. The cooling for 2 h at +4 degrees C, followed by freezing for 1 h at -10 degrees C and 16 h at -20 degrees C resulted in induction of respiratory mutations. The immediate freezing in liquid nitrogen was without mutagenic effect. The study of the stepwise procedure showed that the induction of respiratory mutants takes place during the freezing at -10 and -20 degrees C of cells pre-cooled at +4 degrees C. The genetic crosses of freeze-induced mutants evidenced their mitochondrial rho- origin. The freeze-induced rho- mutants are most likely free of simultaneous nuclear mutations. The extracellular presence of cryoprotectants did not prevent the mutagenic effect of freezing while accumulation of cryoprotectors inside cells completely escaped mtDNA from cryodamage. Although the results obtained favor the notion that the mutagenic effect of freezing on yeast mtDNA is due to formation and growth of intracellular ice crystals, other reasons, such as impairment of mtDNA replication or elevated levels of ROS production are discussed as possible explanations of the mutagenic effect of freezing. It is concluded that: (i) freezing can be used as a method for isolation of mitochondrial mutants in S. cerevisiae and (ii) given the substantial development in cryopreservation of cells and tissues, special precautions should be made to avoid mtDNA damage during the cryopreservation procedures.
虽然在一些研究中有所暗示，但冷冻的诱变作用尚未通过突变体的诱导和分离得到证实。我们使用一个明确的遗传模型，在本通讯中提供了冷冻对酿酒酵母线粒体DNA（mtDNA）诱变作用的证据。在+4℃冷却2小时，然后在-10℃冷冻1小时，在-20℃冷冻16小时，导致呼吸突变的诱导。直接在液氮中冷冻没有诱变作用。对分步程序的研究表明，呼吸突变体的诱导发生在+4℃预冷的细胞在-10℃和-20℃冷冻期间。冷冻诱导突变体的遗传杂交证明了它们的线粒体rho-起源。冷冻诱导的rho-突变体很可能没有同时发生的核突变。细胞外存在冷冻保护剂并不能阻止冷冻的诱变作用，而细胞内冷冻保护剂的积累完全使mtDNA免受冷冻损伤。尽管所获得的结果支持冷冻对酵母mtDNA的诱变作用是由于细胞内冰晶的形成和生长这一观点，但其他原因，如mtDNA复制受损或活性氧产生水平升高，也被讨论为冷冻诱变作用的可能解释。得出的结论是：（i）冷冻可作为分离酿酒酵母线粒体突变体的一种方法；（ii）鉴于细胞和组织冷冻保存的大量发展，应采取特殊预防措施以避免在冷冻保存过程中mtDNA受损。
