1. Genetics and Genomics
Forward genetics
Reverse genetics
Genetic tools for genome-wide analysis
Genome scale reverse genetics
Signature tagged mutagenesis
Synthetic lethal screens
Book (mid-page), , 310 Gain-of-function, 317 synthetic lethal screens

2. 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

3. 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.

4. Forward genetics
Isolating mutants that confer a particular phenotype and then identifying the genes involved.
Sporulation defects.
Temperature sensitive mutations.

5. 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?

6. Mutational analysis of all genes in a genome
B. subtilis example - reverse genetics en masse.
Essential genes
Defining the minimal genome
Potential problems
Operons
Redundant genes

7. 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

8. pMUTIN vector

9. pMUTIN vector for essential genes

10. 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.

11. Table of essential genes

12. Essential genes are widely conserved

13. 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.

14. 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, Annual Review of Genetics.

15. 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.