S phase and HU profiles in wild-type and mutant cells. Saccharomyces cerevisiae

Total S phase was measured for wild-type cells undergoing meiS and mitS. Early replication origins were mapped in mitS in wild-type cells, and in meiS for wild-type, sml1 delete, rec8 delete and spo11 delete cells. Overall design: Comparative genome hybridization was used to measure the kinetics and final extent of DNA replication in wild-type and mutant cells. First, S phase profiles were produced from wild-type cells undergoing pre-mitotic S phase (mitS) and pre-meiotic S phase (meiS) to measure the kinetics of DNA replication in the two S phases. For these experiments, a pool of samples collected throught out all of S phase were labeled and hybridized to a microarray together with a control G1 DNA sample. Second, early replication origins were mapped by exposing cells to levels of HU that strongly reduced DNA replication. Early replication was compared for wild-type cells in mitS to wild-type, sml1, rec8 and spo11 delete strain undergoing meiS. In these experiment, samples were collected after 4 hours exposure to HU. Total genomic DNA was isolated, labeled and hybridized to a microarray together with a control G1 DNA sample. A control G1 vs. G1 sample was prepared to measure the level of noise inherent in the CGH technique.

本数据集测定了野生型细胞在减数分裂前S期（meiotic S phase，meiS）与有丝分裂前S期（mitotic S phase，mitS）过程中的总S期时长。同时完成了早期复制起始位点的定位工作：分别在野生型细胞的mitS中，以及野生型、sml1缺失、rec8缺失与spo11缺失菌株的meiS中。

整体实验设计：采用比较基因组杂交（comparative genome hybridization, CGH）技术，测定野生型与突变型细胞的DNA复制动力学特征及最终复制完成程度。
实验分为两个部分：
第一部分：为对比两类S期的DNA复制动力学，对经历有丝分裂前S期（mitS）与减数分裂前S期（meiS）的野生型细胞构建S期谱。本实验中，将覆盖整个S期收集的样本混合后进行标记，与G1期对照DNA样本共同杂交至微阵列芯片。
第二部分：通过将细胞暴露于可显著抑制DNA复制的羟基脲（hydroxyurea, HU）浓度环境，以定位早期复制起始位点。本部分对比了野生型细胞在mitS中的早期复制情况，与野生型、sml1缺失、rec8缺失及spo11缺失菌株在meiS中的早期复制情况。实验中，在细胞经HU暴露4小时后收集样本，提取全部基因组DNA并进行标记，与G1期对照DNA样本共同杂交至微阵列芯片。此外设置G1期与G1期对照样本，以测定比较基因组杂交（CGH）技术固有的背景噪声水平。