- Slides: 15
• 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-page), 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
p. MUTIN vector
p. MUTIN 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
Identifying essential functions that are controlled by redundant genes. • Synthetic lethal screens • Synthetic lethal = when two mutations that are not essential for growth individually are essential when combined. • Done on a genome wide scale for yeast.
Signature tagged mutagenesis • Ability to screen many mutants at the same time for a loss-of-function phenotype. • Transposon mutagenesis - each transpososn has a unique ~40 bp sequence. • Example - identify mutants that can no longer infect a mouse. • Figure - Hayes, 2003. Annual Review of Genetics.
In vitro transposition • Many organisms do not have well characterized transposons for mutagenesis. • Can mutagenize genomic DNA in vitro then transfer mutations back inside the cell. • Successful in strains that do not have genetic tools available. • Need a good drug marker from organism of interest to get this to work.