Lecture NotesNovember 11th Chapter 16 Continued DNA Packaging will be an online lecture. Recap from Monday We talked about Griffith and his pathogenic cells and transformation. Also talked about Avery and ?transforming substances? and enzymes to determine genetic material. Avery discovered that the ?transforming substance? is sensitive to degradation by which of the following? DNase. Hershey-Chase, 1950s Bacteriophage, . Viruses are nothing but a genome with a protein coat. T2 Virus is nothing but DNA and protein. Infects a bacteria by connecting to the plasma membrane and injecting their genetic material to take over the cell to make new viruses. Infected the E. coli with the phage. Grown with radioactive Phosphorus. Incorporates into the Virus particles. Would be in the DNA but not in the protein. No Phosphorus containing side groups. Also grown in radioactive Sulfur Incorporates into the Virus particles as well. Would be in the protein but not the DNA. No Sulfur in DNA. Cysteine has Sulfur in its side group. Used the radioactive protein and DNA viruses to infect another flask with no radioactivity. Blended then centrifuged. Blender will knock the Virus particles without their genetic material off. The centrifuge separates the bacteria and the empty virus shells. Bacteria pellet at the bottom. Which one was radioactive? In the Phosphorus labeled virus, the Bacteria was radioactive. DNA is the genetic material!! In the Sulfur labeled virus, the Virus shells were radioactive. Protein is not! People finally convinced DNA is the genetic material. But how does it work? No one knows the structure, all they knew were nitrogenous bases (A, T, G, C). Chargaff?s Rules, 1950s. %A bases = %T bases %G bases = %C bases Rosalind Franklin and Watson and Crick. They knew the composition of DNA. Franklin did X-ray crystallography. X-ray diffraction of DNA showed that the DNA molecule is helical. The sugar and Phosphate were on the outside and all of the bases were on the inside. The strands had to be antiparallel. Watson and Crick looked at her pictures. The bases on the inside bound to one another, A and T, G and C. Made models of these base pairs and antiparallel strands. Figured out it was a double helix. Structure of DNA! In replication, the first strand serves as a template so that you can make a complementary strand off of each one. One copy can go to one daughter cell; the other can go to the other daughter cell. No idea how that actually worked. Start with one DNA molecule. It separates into 2 strands to act as a template. Eventually it will go into 2 new molecules. Models to what happens to the original strands in the 2 new molecules of DNA. Conservative model. Semi-conservative Model Dispersive Model Meselson and Stahl, 1950s. Sorted through the models to figure out which one was correct. Added radioactive Nitrogen (15N ? a heavier isotope of Nitrogen) to E. coli. These cells will have heavy DNA. Centrifuge. Removes the heavy DNA cells. Add to a flask of E. coli that has 14N. Will start to go and replicate. Doubling time is between 20-30 mins (1 generation). New DNA will have 14N, original will have 15N. Centrifuge again. Lice the cells. Purify the DNA. Laid the DNA on a Density Gradient of Sucrose. Higher concentration on bottom. It will spread out until it gets to Neutral Density. 15N will form a band lower in the gradient than 14N?s band. In the conservative model, one molecule would end up with new strands, the other with old strands. One with 15N and one with 14N. You would see one band with all of the new DNA in the gradient, and another band with all old DNA in the gradient. In the semi-conservative model, they would each have one new strand and one old strand. In the gradient, you would only get one band. All have the same density. In between the 15N and the 14N. In the dispersive model, you would get a mixture of new and old DNA. Only get one band. All still have the same density. The experiment can only rule out conservative method at this point. To distinguish between semi-conservative and dispersive. Run two generations. You?d end up with 4 DNA.
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