* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Lecture_13_2005
Long non-coding RNA wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Heritability of IQ wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Human genome wikipedia , lookup
Gene desert wikipedia , lookup
Genomic library wikipedia , lookup
Transposable element wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Behavioural genetics wikipedia , lookup
Medical genetics wikipedia , lookup
Non-coding DNA wikipedia , lookup
Population genetics wikipedia , lookup
Genetic engineering wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Point mutation wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Pathogenomics wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Public health genomics wikipedia , lookup
Gene expression programming wikipedia , lookup
Oncogenomics wikipedia , lookup
Genomic imprinting wikipedia , lookup
Genome editing wikipedia , lookup
Ridge (biology) wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Designer baby wikipedia , lookup
History of genetic engineering wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Essential gene wikipedia , lookup
Gene expression profiling wikipedia , lookup
Genome (book) wikipedia , lookup
Genome evolution wikipedia , lookup
• 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 – – – – 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 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.