Howell S H, Posakony J W, Hill K R
J Cell Biol. 1977 Feb;72(2):223-41. doi: 10.1083/jcb.72.2.223.
The cell cycle program of polypeptide labeling in syndhronous cultures of wild-type Chlamydomonas reinhardtii was analyzed by pulse-labeling cells with 35SO4 = or [3H]arginine at different cell cycle stages. Nearly 100 labeled membrane and soluble polypeptides were resolved and studied using one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The labeling experiments produced the following results. (a) Total 35SO4 = and [3H]arginine incorporation rates varied independently throughout the cell cycle. 35SO4 = incorporation was highest in the mid-light phase, while [3H]arginine incorporation peaked in the dark phase just before cell division. (b) The relative labeling rate for 20 of 100 polypeptides showed significant fluctuations (3-12 fold) during the cell cycle. The remaining polypeptides were labeled at a rate commensurate with total 35SO4 = or [3H]arginine incorporation. The polypeptides that showed significant fluctuations in relative labeling rates served as markers to identify cell cycle stages. (c) The effects of illumination conditions on the apparent cell cycle stage-specific labeling of polypeptides were tested. Shifting light-grown asynchronous cells to the dark had an immediate and pronounced effect on the pattern of polypeptide labeling, but shifting dark-phase syndhronous cells to the light had little effect. The apparent cell cycle variations in the labeling of ribulose 1,5-biphosphate (RUBP)-carboxylase were strongly influenced by illumination effects. (d) Pulse-chase experiments with light-grown asynchronous cells revealed little turnover or inter-conversion of labeled polypeptides within one cell generation, meaning that major polypeptides, whether labeled in a stage-specific manner or not, do not appear transiently in the cell cycle of actively dividing, light-grown cells. The cell cycle program of labeling was used to analyze effects of a temperature-sensitive cycle blocked (cb) mutant. A synchronous culture of ts10001 was shifted to restrictive temperature before its block point to prevent it from dividing. The mutant continued its cell cycle program of polypeptide labeling for over a cell generation, despite its inability to divide.
通过在野生型莱茵衣藻同步培养物的不同细胞周期阶段用(^{35}SO_4^{2 - })或([^3H])精氨酸脉冲标记细胞，分析了多肽标记的细胞周期程序。使用一维十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳分离并研究了近100种标记的膜蛋白和可溶性多肽。标记实验产生了以下结果。（a）在整个细胞周期中，(^{35}SO_4^{2 - })和([^3H])精氨酸的总掺入率独立变化。(^{35}SO_4^{2 - })掺入在光照中期最高，而([^3H])精氨酸掺入在细胞分裂前的黑暗期达到峰值。（b）100种多肽中有20种的相对标记率在细胞周期中显示出显著波动（3 - 12倍）。其余多肽的标记率与(^{35}SO_4^{2 - })或([^3H])精氨酸的总掺入率相当。相对标记率显示出显著波动的多肽用作识别细胞周期阶段的标志物。（c）测试了光照条件对多肽明显的细胞周期阶段特异性标记的影响。将光下生长的异步细胞转移到黑暗中对多肽标记模式有立即且显著的影响，但将黑暗期同步细胞转移到光下影响很小。核酮糖1,5 - 二磷酸（RUBP）羧化酶标记的明显细胞周期变化受到光照效应的强烈影响。（d）对光下生长的异步细胞进行脉冲追踪实验表明，在一个细胞世代内标记多肽的周转或相互转化很少，这意味着主要多肽，无论是否以阶段特异性方式标记，在活跃分裂的光下生长细胞的细胞周期中都不会短暂出现。标记的细胞周期程序用于分析温度敏感型周期阻断（cb）突变体的影响。ts10001的同步培养物在其阻断点之前转移到限制温度以防止其分裂。尽管该突变体无法分裂，但它在一个多细胞世代中继续其多肽标记的细胞周期程序。
