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Volatility of mutator phenotypes at single cell resolution

单细胞分辨率下突变表型的波动性

基本信息

批准号：
9295042
负责人：
ALAN J HERR
金额：
$ 30.9万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-13 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9295042
关键词：
Adopted Affect Agar Alleles Candidate Disease Gene Cell Count Cell Lineage Cell division Cells Clone Cells DNA biosynthesis Data Daughter Defect Diploidy Drug resistance Engineering Evolution Exhibits Future Genes Genome Growth Haploidy Human Individual Inherited Lead Malignant Neoplasms Mammalian Cell Maps Measurement Measures Methods Microbe Micromanipulation Modeling Monitor Mothers Mutagenesis Mutation Mutation Fixation Neoplastic Cell Transformation Phenotype Phosphotransferases Poisson Distribution Polymerase Population Process Publications Publishing Regulation Replication Error Resolution S Phase Saccharomyces cerevisiae Saccharomycetales Source Surface Technology Testing Therapeutic Work Yeasts cancer cell daughter cell experimental study human disease microbial mutant next generation sequencing public health relevance sample fixation segregation whole genome

项目摘要

DESCRIPTION (provided by applicant): Elevated mutation rates caused by mutator phenotypes can fuel microbial evolution and neoplastic transformation of mammalian cells. New mutations are assumed to arise at a constant rate in each cell division and segregate equally between the resulting cells. If one or both assumptions are false, an expanding mutator population may contain subclones with widely divergent rates of evolution. Testing these assumptions has not been possible in the past because most mutation rate measurements rely on scoring rare mutations in a small target, which requires large populations of cells. Next Generation Sequencing (NGS) technologies, which permit an entire genome to be used to score mutations, allows us to test the fundamental assumptions about mutation rates by sequencing clones derived from sequential cell divisions. Our strategy is to isolate single-cell lineages fro mutator "mother" yeast cells, including all daughter cells, the first two grand-daughters born to each daughter, and the first great-granddaughter born to the first grand-daughter. We will grow all isolated cells into clones and then sequence the genomes using NGS. From these data, we will determine the fidelity of individual replication cycles and whether replication errors segregate evenly. In work published this year, we monitored the fixation of mutations in just haploid mutator mother cells and found evidence for distinct mutagenic states. Our first specific aim is to monitor the fixation of all replication errors in both daughter and mother cells. We will use the same haploid strain as well as several diploid mutator strains with higher mutation rates. In another publication this year we found mutations that perturb dNTP levels modulate mutator phenotypes. Thus, in our second specific aim, we will investigate whether mutator volatility is due to dNTP pool regulation. Our third specific aim seeks to identify additional factors that may influence mutator volatility through the isolation of antimutator mutations. This proposal will lea to future studies to investigate whether mammalian mutator cells exhibit distinct mutagenic states and whether factors that influence mutator volatility may be used to modulate mutator phenotypes for therapy.