MCB 121 Lecture 11

MCB 121 Lecture 11 - MCB 121 Lecture 11 Midterm II is on...

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MCB 121 Lecture 11 Midterm II is on Thur 2/18 (Review on Wed 6-8 pm) Outline: I. Isolation of TF II. How TF work (example: Myc/Max/Mad) III. Experiment Interpretation How Transcription factors activate gene expression: - recruit HAT to open chromatin - recruit swi/snf to remodel chromatin - recruit/activate basal apparatus Identification of Transcription Factor: 1. Biochemical purification - synthesize the DNA that has binding site for TF * we identify by mutagenesis on promoter - purify binding protein from nuclear extract * source of the protein = cell where gene expressed (nuclear extract) - use assay by Gel shift or footprint * show that nuclear extract contain the protein that can do the gel shift - fraction (column) and see which fraction can cause gel shift * gel down to single protein that bind the piece of DNA * microsequencing on the protein to determine what the protein is. 2. Genetics - mutations affecting a whole process - can often affect the whole series of different genes - this indicates some kind of regulatory proteins (signal transduction protein or transcription factor) 3. Oncogene - genes associates with/ contribute to cancers EX. viruses that in animal system can contribute to cause cancer - change the whole program inside the cells - many of them are signal transduction proteins and transcription factors - first identified in viruses! => genes in the viruses are similar to the genes already inside the cell => when virus come out from the host cell may take out some genes with them. => this may cause cancer and make it as a better place for viruses to grow - oncogene can be altered versions of cellular genes
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All the genes that affect the transcription process aren't transcription factor genes, all the oncogenes aren't transcription factors, but many of them are. HOW DO WE KNOW HOW TRANSCRIPTION FACTOR WORK? Myc oncogene - 1st identified in avian viruses that causes cancers in birds (V.Myc) = C-Myc - cellular cognate * normally in the cell all the time * involve cell division * over expression of C-Myc would promote cell division and cause cancer. = N-Myc - expressed in nerve cell = L-Myc - expressed in WBCs Myc has B-helix-loop-helix (HLH) region and leucine zipper (LZ) * B = basic region next to it - both are dimerization domain - HLH involve DNA binding * LZ involve dimerization - Look like a transcription that work as a dimer - by looking at the sequence, there is two motif (HLH and LZ) To find out where Myc bind on DNA: (Experiment) - Make pure Myc * put Myc with expression vector in E.coli - Mix with random dsDNA fragments - use Gel shift to find bound fragments
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MCB 121 Lecture 11 - MCB 121 Lecture 11 Midterm II is on...

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