Ohkuni K, Yamashita I
Center for Gene Science, Hiroshima University, Kagamiyama 1-4-2, Higashi-Hiroshima 739-8527, Japan.
Yeast. 2000 Jun 30;16(9):829-46. doi: 10.1002/1097-0061(20000630)16:9<829::AID-YEA581>3.0.CO;2-6.
Alkalization of the medium is associated with and required for the cellular development to meiosis and sporulation in the yeast Saccharomyces cerevisiae. To elucidate the molecular mechanisms for the significance of external alkalization, we isolated mutants defective in division arrest at G1 phase under an alkaline condition. The mutants obtained had recessive alleles of SRB10 encoding the cyclin (SRB11)-dependent protein kinase that phosphorylates the CTD domain of the largest subunit of RNA polymerase II and negatively regulates the transcriptional initiation of certain genes. A delta srb11 deletion mutant showed the same cell cycle defect. When shifted to alkali, wild-type cells decreased transcript levels of G1-cyclin genes (CLN1 to CLN3) and KIN28-CCL1 (encoding another CTD kinase-cyclin pair which, in contrast, stimulates the promoter clearance and transcriptional elongation in most genes), resulting in the accumulation of G1 cells and the hypophosphorylated form of RNA polymerase II and in an increase in cell size. However, under the same conditions, a delta srb10 mutant was defective in these events, except the downregulation of CLN1 and CLN2. The delta srb10 mutation also influenced on the transcript levels of meiosis-inducing genes called IME1 and IME2: the mutation elevated the transcript level of IME1 but reduced that of IME2, resulting in partial defects in premeiotic DNA synthesis and meiosis. Overexpression of KIN28 and CCL1 in wild-type cells impaired the alkali-induced G1 arrest and the rate of meiosis and elevated the transcript levels of SRB11 and IME1. These results indicate that a transcriptional autoregulatory loop for KIN28-CCL1 and SRB10-SRB11 is important for G1 arrest and meiosis. We also found that environmental conditions for meiosis finely regulate the transcript levels of KIN28 and CCL1, such that nitrogen starvation first elevates them but subsequent alkalization of medium decreases them.
培养基的碱化与酿酒酵母细胞向减数分裂和孢子形成的发育过程相关且是其必需的。为了阐明外部碱化重要性的分子机制，我们分离了在碱性条件下G1期分裂停滞有缺陷的突变体。获得的突变体具有SRB10的隐性等位基因，SRB10编码细胞周期蛋白（SRB11）依赖性蛋白激酶，该激酶使RNA聚合酶II最大亚基的CTD结构域磷酸化，并负向调节某些基因的转录起始。Δsrb11缺失突变体表现出相同的细胞周期缺陷。当转移到碱性条件下时，野生型细胞降低了G1期细胞周期蛋白基因（CLN1至CLN3）和KIN28 - CCL1（编码另一对CTD激酶 - 细胞周期蛋白，相反，它刺激大多数基因的启动子清除和转录延伸）的转录水平，导致G1期细胞积累、RNA聚合酶II的低磷酸化形式以及细胞大小增加。然而，在相同条件下，Δsrb10突变体在这些事件中存在缺陷，除了CLN1和CLN2的下调。Δsrb10突变也影响了减数分裂诱导基因IME1和IME2的转录水平：该突变提高了IME1的转录水平，但降低了IME2的转录水平，导致减数分裂前DNA合成和减数分裂出现部分缺陷。在野生型细胞中过表达KIN28和CCL1会损害碱诱导的G1期停滞和减数分裂速率，并提高SRB11和IME1的转录水平。这些结果表明，KIN28 - CCL1和SRB10 - SRB11的转录自调控环对G1期停滞和减数分裂很重要。我们还发现减数分裂的环境条件精细调节KIN28和CCL1的转录水平，使得氮饥饿首先升高它们，但随后培养基的碱化会降低它们。
