sequencing genomes 1) map the genome 2) prepare an ac library 3) order the library 4) subdivide each...
TRANSCRIPT
Sequencing Genomes1) Map the genome2) Prepare an AC library3) Order the library
4) Subdivide each AC into lambda contigs
5) Subdivide each lambda into plasmids
6) sequence the plasmids
Using the genomeStudying expression of all genes simultaneously1.Microarrays: “reverse Northerns”2.High-throughput sequencing
Using the genomeStudying expression of all genes simultaneously
1. Microarrays: “reverse Northerns”2. High-throughput sequencing• “Re-sequencing” to detect variation
Using the genomeStudying expression of all genes simultaneously1.Microarrays: “reverse Northerns”2.High-throughput sequencing•“Re-sequencing” to detect variation•Sequencing all mRNA to quantitate gene expression
Using the genomeStudying expression of all genes simultaneously1.Microarrays: “reverse Northerns”2.High-throughput sequencing•“Re-sequencing” to detect variation•Sequencing all mRNA to quantitate gene expression•Sequencing all mRNA to identify and quantitate splicing variants
Using the genomeStudying expression of all genes simultaneously1.Microarrays: “reverse Northerns”2.High-throughput sequencing•“Re-sequencing” to detect variation•Sequencing all mRNA to quantitate gene expression•Sequencing all mRNA to identify and quantitate splicing variants•Sequencing all RNA to identify and quantitate ncRNA
Using the genomeStudying expression of all genes simultaneously1.Microarrays: “reverse Northerns”2.High-throughput sequencing3. Bisulfite sequencing to detect C methylation
Using the genomeBisulfite sequencing to detect C methylation
Using the genomeBisulfite sequencing to detect C methylationChIP-chip or ChIP-seq to detect chromatin modifications: 17 mods are associated with active genes in CD-4 T cells
Using the genome• various chromatin modifications are associated with activated & repressed genes•Acetylation, egH3K9Ac, is associated with active genes
Using the Genome•various chromatin modifications are associated with activated & repressed genes •Acetylation, egH3K9Ac, is associated with active genes• Phosphorylation of H2aS1, H2aT119, H3T3, H3S10 & H3S28 shows condensation
Using the Genome•Acetylation, egH3K9Ac, is associated with active genes• Phosphorylation shows condensation• Ubiquitination of H2A and H2B shows repression & marks DNA damage
Using the Genome•Acetylation, egH3K9Ac, is associated with active genes• Phosphorylation shows condensation• Ubiquitination of H2A and H2B shows repression• Methylation is more complex: H3K36me3 = on•H3K27me3 = off
Using the GenomeMethylation is more complex:•H3K36me3 = on•H3K27me3 = off•H3K4me1 = off•H3K4me2 = primed•H3K4me3 = on
Histone codeModifications tend to group together: genes with H3K4me3 also have H3K9ac
Histone codeModifications tend to group together: genes with H3K4me3 also have H3K9acCytosine methylation is also associated with repressed genes
Generating the histone codeHistone acetyltransferases add acetic acid
Generating the histone codeHistone acetyltransferases add acetic acidMany HAT proteins: mutants are very sick!
Generating the histone codeHistone acetyltransferases add acetic acidMany HAT proteins: mutants are very sick!HATs are part of many complexes
Generating the histone codeBromodomains specifically bind acetylated lysines
Generating the histone codeBromodomains specifically bind acetylated lysinesFound in transcriptional activators & general TFs
Generating the histone codeacetylated lysinesDeacetylases “reset” by removing the acetate
Generating the histone codeacetylated lysinesDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!
Generating the histone codeDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!Many drugs are histone deacetylase inhibitors
Generating the histone codeDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!Many drugs are histone deacetylase inhibitorsSAHA = suberanilohydroxamic acid = vorinostatMerck calls it Zolinza, treats cutaneous T cell lymphoma
Generating the histone codeDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!Many drugs are histone deacetylase inhibitorsSAHA = suberanilohydroxamic acid = vorinostatMerck calls it Zolinza, treats cutaneous T cell lymphomaBinds HDAC active site & chelates Zn2+
Generating the histone codeWhen coupled SAHA to PIPS (pyrrole-imidazole Polyamides) got gene-specific DNA binding & gene activation
Generating the histone codeCDK8 kinases histones to repress transcription
Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcription
Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcriptionPhosphorylation of Histone H3 correlates with activation of heat shock genes!
Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcriptionPhosphorylation of Histone H3 correlates with activation of heat shock genes!Phosphatases reset the genes
Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcription
Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcriptionPolycomb proteins ubiquitinate histone H2A to silence genes