1 / 15

Genetics and Genomics Forward genetics Reverse genetics Genetic tools for genome-wide analysis

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.

elissaj
Download Presentation

Genetics and Genomics Forward genetics Reverse genetics Genetic tools for genome-wide analysis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 - 300-304 (mid-page), 306-307, 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 • Essential genes • 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, 2003. 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.

More Related