Lecture 10

1) Do the biochemistry of genes.

2) Discovery of the Transforming Principle: Griffiths. He wanted to understand bacteria – pnumecoccus which could infect and kill mice. They came in two different types, one produced a glistening shiny colony, and they were virulent, these would kill the mouse, smooth because of the polysaccharide coat. Other were rough bacteria, these were non-virulent. Mouse immune system was able to f ight them. Griffiths injects virulent bacteria into mouse, mouse dies.  Take rough bacteria, mouse lives. Take smooth bacteria, autoclave them, heat them to very high temperature so they are dead, mouse lives. Rough bacteria + heat killed smooth bacteria, mouse dies.

3) When you autopsy the mouse you could recover from the mouse smooth virulent live bacteria. Rough + heat killed smooth = live smooth. Take dead virulent bacteria, and start fractionating it biochemically and asking what fraction of the material from the dead bacteria gives this property. This is a painful process. He didn’t succeed, so people say there’s some transforming principle.

4) Avary Mccardy Mclow, same experiment, but no mice. Take dead bacteria, combine with live rough bacteria, and on a petri plate you would see the smooth bacteria come out. They began purifying. They knew they were transforming the heredity of the bacteria. They found that consistently the fraction that contained heredity was the fraction that contained DNA. The reaction to that was – mostly it was that must have goofed. Most smart people knew that DNA was an absolutely boring molecule. The interesting molecules was proteins.

5) Why was DNA boring? Structure of DNA: DNA has 3 components – sugar 2′ deoxyribose, its a pentose. The base can be adenine thymine cytosine guanine. To make up the monomers, we need triphosphate. When you combine nucleotides to create a DNA strand, you do so to create a sugar-phosphate backbone. The phosphate is always attached to the 3′ carbon of the preceding sugar, and 5′ carbon of the next sugar. We often speak of chains of DNA growing from the 5′ end to the 3′ end.

6) Bases come in two types – purines – adenine and guanine, they are 6 membered rings with a 5 membered ring and pyrimidines – thymine and cytosine – they have 6 membered rings. Compared to proteins this is pretty boring.

7) Bacterial Viruses: Instead of using bacteria to infect mice, Herschey and Chase used viruses to infect bacteria. These were called bacteriophage. All they had is some DNA in the capcid and some protein. They could attach to a bacteria and cause it to burst open and produce lots of daughter bacteriophage, it could replicate within this bacteria. When people discovered bacteriophage, people thought we should drink them.

8) How do these viruses kill the bacteria? They inject something into the bacteria, which causes virus particles to be made. What goes in? The thought was if this thing is injecting its DNA, then the DNA must be carrying the instructions to make phage, and this would be hereditary material.  How would you find out what goes in? Radioactive labelling. Label DNA with one label and the protein with another. What do you label the DNA with? Phosphorus – none of the 2o amino acids has phosphorus. P-32 label the DNA. How do you make them? Just allow them to grow in P-32 medium. Label proteins with? Sulphur S-35.

9) We infect bacteria with them. We need to knock of the bacteriophage from the bacteria. Specialized devices were used to create intense agitation. Kitchen blenders. Called the wearing blender experiment. Virus particles are much lighter than the bacteria, so we can separate them using centrifuge. We measure radioactivity in the palette and the supernatent (stuff that remains above) What shows in the palette – P32. In reality there will also be some S35 in the pellet, but it was less than 1%. Most of the S35 stays in the supernatent. Does all of the phosphorus go in? No, some viruses didn’t even attach.

10) We can conclude more DNA went in than protein. Suppose that 1% sulphur is tracking the minor protein that is the secret, its very hard to rule out that. Infact, Avery’s experiment was pure than this experiment, but by this point thinking had started to shift.

11) What was it about the DNA that conferred these properties? Crick and Watson: DNA Structure On the basis of a lot of modelling and using the X ray diffraction pictures, made a model. One chain is 5′-3′, and another antiparallel chain is 3′-5′. The X ray diffraction pictures told you it had to be helical. Key to the model was recognition of base pairing. The distance between adenine and thymine, and cytosine and guanine, would be the same.

12) There was an observation – the amount of A close to amount of  T, amount of C close to amount of G. This model also explains how a DNA molecule can replicate. All it takes is for those 2 strands to come apart, and each can serve as a template for the other. What’s mutation? It sometimes gets it wrong.

13) The above replication model is called semiconservative replication. In theory, DNA replication could occur by old strands staying together, and serving as a template for making a new double helix. Semi-conservative implies each DNA has one old and one new. Radioactive labelling. Grow DNA initially in normal nitrogen and then shift it to radioactive nitrogen. How do I prove that these were 50-50. What property do I test? Density. The molecules have intermediate density between all heavy and all light. They had to work out a centrifugation technique.


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