NE102 Lecture Notes 2

Integrating concepts through p53 introduction to

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Integrating Concepts through p53/ Introduction to Recombinant DNA 19:00 Transcription factor, that activates transcription of genes that inhibit cell proliferation.  Continuously synthesized & degraded by the proteasome in healthy cells. Genetic mutations that interfere with p53 function cause cancer. Cells can acquire several types of genetic mutations: Chemical damage (e.g., thymine dimers) Nucleotide changes (“ point mutations ”) Mutations in protein-coding regions of genes can change the amino acid sequence. QUESTION: What point mutations could inhibit p53 normal function? Point mutations can occur in serine, threonine, or tyrosine residues that will change the  amino acid sequence. Phosphorylation will not occur by p53 kinase QUESTION: Must these mutations be in the p353 gene? No. Mutation can occur in MDM2 or ATM. QUESTION: Must these mutations be in the protein-coding regions? No. Can mutate the promoter region which will block transcription.  Mutation can occur in TF binding sites.  KEY POINTS: 1. While visualizing proteins with antibodies is helpful, it has limitations. 2. Another approach commonly taken to study protein function is to take it into cells  “exogenously” as ask, “What effect does it have?”.
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Integrating Concepts through p53/ Introduction to Recombinant DNA 19:00 3. Recombinant DNA technology provides a way to do this. PREREQUISITE QUESTION: What is recombinant DNA? Recombinant DNA:  The combining of DNA from two or more different sources into one  continuous DNA molecule.  QUESTION: How can we “cut” DNA into specific fragments for recombining? KEY TOOL: Restriction endonucleases Restriction endonucleases:  Enzymes that cut (or digest) DNA at specific short  sequences. Ex.) EcoRI = a restriction endonuclease that cuts DNA at “GAATTC” Evolved as a defense against viruses.  Viruses consists of a capsid (made of protein) & a nucleic acid genome  Viruses cannot replicate on their own, rather they replicate by infecting host cells.  Bacteriophages : Viruses that infect bacterial cells. Attach to bacteria, insert genome into bacteria, Use host cell’s machinery to replicate  themselves. Hundreds of restriction enzymes have evolved & been characterized QUESTION: How do RE’s help create recombinant DNA molecules? 1. RE’s cut DNA into fragments. 2. Many RE’s generate staggered ends. Often most recombinant DNA molecules/plasmids are engineered to contain specific  types of DNA sequences. 
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Integrating Concepts through p53/ Introduction to Recombinant DNA 19:00 Promoter = Drives expression of the gene.
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