Decondensation of Xenopus sperm chromatin using Saccharomyces cerevisiae whole-cell extracts
Decondensation of Xenopus sperm chromatin using Saccharomyces cerevisiae whole-cell extracts
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DOI：
10.1139/y05-042
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发表时间：
2006-03-01
影响因子：
2.1
通讯作者：
Harkness, Troy A. A.
中科院分区：
文献类型：
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作者：
Harkness, Troy A. A.
使用高度浓缩的非洲爪蟾精子染色质和从多个系统制备的蛋白质提取物的生物化学研究已经导致了参与染色体解浓缩的保守蛋白质的鉴定。然而，这些蛋白质的突变是不可用的，因为所使用的系统不适合遗传研究。我们采取了一种遗传学方法，通过孵育非洲爪蟾精子染色质的随机温度敏感（是）酵母细胞的哈特韦尔文库前制备的全细胞提取物分离染色体解凝聚突变体。我们表明，非洲爪蟾精子染色质的解凝聚使用野生型酵母提取物是快速的，ATP和提取物依赖性。并且耐热。N-乙基马来酰亚胺。蛋白酶K、核糖核酸酶A。和微球菌核酸酶。从100个突变体提取物中筛选。我们获得了一个菌株。被称为rmc 4，它是染色体解凝聚缺陷。该突变体生长缓慢，并表现出发芽缺陷。低浓度的rmc 4提取物最终会使精子的头部变得不致密。和突变体提取物的分级分离产生解凝聚感受态级分。提示在RMC 4细胞中存在过度活性的抑制剂。我们进行了多拷贝抑制筛选，确定PDE 2。编码抑制蛋白激酶A（PKA）活性的蛋白质的基因。由于PKA先前显示在人类细胞中，10保持浓缩的染色质，我们的结果表明PKA活性在rmc 4细胞中升高。导致解凝聚缺陷。因此。我们的实验表明，酵母编码一个进化保守的染色体解凝聚活动，可以遗传操纵。
Biochemical studies using highly condensed Xenopus sperm chromatin and protein extracts prepared from multiple systems have lead to the identification of conserved proteins involved in chromosome decondensation. However, mutations to these proteins are unavailable is the systems used are not amenable to genetic studies. We took a genetic approach to isolating chromosome decondensation mutants by incubating Xenopus sperm chromatin with whole-cell extracts prepared front the Hartwell library of random temperature sensitive (is) yeast cells. We show that decondensation of Xenopus sperm chromatin using wild type yeast extracts was rapid, ATP- and extract-dependent. and resistant to heat. N-ethylmaleimide. protease K, RNase A. and micrococcal nuclease. From 100 mutant extracts screened. we obtained one strain. referred to as rmc4, that was chromosome decondensation defective. The mutant was slow growing and exhibited germination defects. Low concentrations of rmc4 extract Would eventually decondense sperm heads. and fractionation of the Mutant extract produced a decondensation competent fraction. suggesting the presence of an overactive inhibitor in rmc4 cells. We performed a multicopy suppressor screen that identified PDE2. a gene encoding a protein that inhibits protein kinase A (PKA) activity. As PKA was previously shown ill human cells, 10 Maintain condensed chromatin, our results suggest that PKA activity is elevated in rmc4 cells. causing a decondensation defect. Thus. Our experiments reveal that yeast encodes an evolutionary conserved chromosome decondensation activity that can be genetically manipulated.