呃潒敢瑲-GeneticsandGenomicsForwardgenetics.pptVIP

Genetics and Genomics Forward genetics Reverse genetics Genetic tools for genome-wide analysis Genome scale reverse genetics Signature tagged mutagenesis Synthetic lethal screens Book - 300-304 (mid), 306-307, 310 Gain-of-function, 317 synthetic lethal screens Brief review of genetic terms Types of mutations Null mutations - loss of function Hypomorphs - partial loss of function Hypermorphs - gain of function Antimorphs - dominant negative Essential and non-essential genes What does it mean to be essential? Genes essential for growth in the laboratory Genes not essential for growth in the laboratory can be essential in other conditions. Forward genetics Isolating mutants that confer a particular phenotype and then identifying the genes involved. Sporulation defects. Temperature sensitive mutations. Reverse genetics From gene to phenotype - using genetic tools to identify the function of a gene without prior knowledge of its function. Knockout - screen for phenotype Overexpression Ectopic expression What if none of these approaches yields a phenotype? Mutational analysis of all genes in a genome B. subtilis example - reverse genetics en masse. Essential genes Defining the minimal genome Potential problems Essential genes Operons Redundant genes Making systematic mutations to identify gene function. High efficiency transformation Integrational plasmid Inability to replicate in bacterium studied Reporter gene to study expression Determine if gene is expressed under conditions tested. Inducible promoter to express downstream genes. Remove effects of polarity pMUTIN vector pMUTIN vector for essential genes Findings from B. subtilis Only 271 of 4106 genes are essential for growth Many genes are involved in a few metabolic functions (DNA, RNA, protein, cell wall) 70% of essential genes have homologs in eukaryotes and archaea. Redundant genes missed essential functions Growth in rich medium - one condition. Table of essential genes Essential genes are widely conserved