(up to slide 21) Whole genome approaches to look at the temporal and spatial DYNAMICS of the cellular environmentA. 1. Transcriptome- all of the RNA in the cell at a given time under particular conditions 2. Proteome- all the protein in a cell at a given time under particular conditions 3. Metabolome- all of the metabolites in a cell at a given time under particular conditions 4. Phosphorylome- all of the phosphorylated proteins in the cell at a given time under particular conditions 5. Ome (the ending of a word) = in the cell, at a given time, under particular conditions B. Studies: The study of the expression of genes and genomes, including gene expression and functionC. 1. The same DNA can yield different expression patterns in different tissues in response to different environmental or endogenou s Signals 2. Example D. Transcriptome Analysis Functional GenomicsI. Microarray-based expression profilingII. A. Determines which genes are expressed in a specific tissue under specific conditions or developmental tissues 1. Generate DNA probes for each cDNA or oligionucleotide for each gene present in the genome 2. Attach the probes on a glass slide (called a chip): each probe has a specific position on the slide (high-density) 3. Label cDNA made from mRNA extracted from the tissue 4. Hybridize this probe to the chip: the genes expressed in the sample will be identified by a florescent signal on the chip B. Process: C. Competitive Hybridizing: Functional Genomics (4/7/08) Genetic Engineering Page 1 D. Analysis of the Data: 1. Look at the groups of experiments and find genes whose expression patterns match across many experiments: called cluster analysis 2. Genes involved in the same cellular processes are often co-regulated III. Expression Analysis by promoter leads to fusion with GFP: Green Fluroescent ProteinA. DR5: GFP root tip fusion1. Defines the site of auxin accumulation in Arabidosis root tips2. Example:3. Example:B. Functional Genomics with a Reporter Genetic Engineering Page 2 If GFP is transcribed, DR5 is too. So, we can define expression by looking at florescence. 4. Look at groups of experiments 1. 2. find genes whose expression patterns match across many experiments: cluster analysis Analysis of the dataA. B. Mutational Analysis: reverse genetics 1. Identify mutations in specific genes and analyze the phenotypes 2. Look at homologous recombination strategies C. Insertional Mutagenesis Strategies: 1. Generate a collection of mutants carrying transposons at different positions in the genome 2. Identify in this collection of individuals that carry a transposon within a gene of interest 3. Analyze the corresponding phenotype Functional Genomics with a MicroarrayIV. Genetic Engineering Page 3
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