Chemical Biology 03 Inheritance III-cont.

Chemical Biology 03 Oct 27, 2010 Gene Regulation Transcriptional Regulation transcription initiation Regulation is achieved by: 1) Regulatory DNA sequence • • • next to promoter up to several 1000 base-pairs “upstream” (before) promoter sometimes even in introns or “downstream” of gene • • • binds DNA double helix in a sequence-specific manner Some are Positive Regulators (“activators” and “enhancers”) Some are Negative Regulators (“repressors” and “silencers”) 2) Regulatory Protein Network of Regulatory Proteins ~ 2000 - 3000 genes in our genome = Transcriptional Regulatory Proteins The transcriptional activity of any given gene is determined by abundance, activity, cooperation, competition of different Regulatory Proteins The presence & activity of a regulatory protein is influenced by cell type and by extracellular environment Recall that genes are scattered over many chromosomes transcribed part of gene Chr.I Chr.II Chr.III Chr.IV Each gene acts independently: has its own TATA box and associated regulatory regions DNA Binding Site for Regulator transcribed part of gene Chr.I Chr.II Chr.III Chr.IV The presence & activity of a regulatory protein is influenced by cell type and by extracellular environment Tissue-specificity of pancreatic gene regulatory regions (“enhancers”) Three artificial “hybrid” genes are generated and inserted into three different types of cells   Hybrid   Gene   Regulatory  Region   From:   Coding   Sequence   #1   Insulin  gene   CAT  enzyme   #2   Chymotrypsin  gene   CAT  enzyme   #3   Viral  gene   CAT  enzyme   Ovarian cell   Pancreatic insulin cell Pancreatic exocrine cell Amount of CAT enzyme is then measured in the nine different situations   CAT = chloramphenicol acetyltransferase, a bacterial enzyme that is normally not found in mamalian cells Whether or not it will be “expressed” depends on the upstream regulatory region Amount of CAT activity   Tissue-specificity of pancreatic gene regulatory regions (“enhancers”) Amount of CAT activity   Tissue-specificity of pancreatic gene regulatory regions (“enhancers”) Gene Expression changes throughout development: Regulation of Gene Expression: Globin genes β cluster: 1 β gene, 1 delta gene, 2 gamma genes, 1 epsilon gene (and 1 pseudogene) Gene Expression changes throughout development: Regulation of Gene Expression: Globin genes Chromosome 16 Chromosome 11 α cluster: 2 α genes, 1 zeta gene, 1 theta gene (and 3 pseudogenes) β cluster: 1 β gene, 1 delta gene, 2 gamma genes, 1 epsilon gene (and 1 pseudogene) Gene Expression changes throughout development: Regulation of Gene Expression: Globin genes Chromosome 16 Chromosome 11 epsilon gamma beta delta 1 1 1 1 MVHFTAEEKAAVTSLWSKMNVEEAGGEALGRLLVVYPWTQRFFDSFGNLSSPSAILGNPK MGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPK MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPK MVHLTPEEKTAVNALWGKVNVDAVGGEALGRLLVVYPWTQRFFESFGDLSSPDAVMGNPK epsilon gamma beta delta 61 61 61 61 VKAHGKKVLTSFGDAIKNMDNLKPAFAKLSELHCDKLHVDPENFKLLGNVMVIILATHFG VKAHGKKVLTSLGDATKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFG VKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFG VKAHGKKVLGAFSDGLAHLDNLKGTFSQLSELHCDKLHVDPENFRLLGNVLVCVLARNFG epsilon gamma beta delta 121 121 121 121 KEFTPEVQAAWQKLVSAVAIALAHKYH KEFTPEVQASWQKMVTAVASALSSRYH KEFTPPVQAAYQKVVAGVANALAHKYH KEFTPQMQAAYQKVVAGVANALAHKYH Regulation of Gene Expression: β Globin gene cluster •Only expressed in Red Blood Cells (cell-type specific expression) •Expression changes over course of development •Locus Control Region: crammed with recognition sequences for Regulators Duplication of globin genes over the course of millions of years of evolution adult Fetal hemoglobin allows evolution of placental mammals 2 different O2 transporters: Myoglobin & Hemoglobin