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• 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 Gainof-function, 317 synthetic lethal screens
Brief review of genetic terms
• Types of mutations
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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.
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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
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.
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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.