BSCI222 Lecture 2 9 5 13 Chapter 10 DNA The Chemical Nature of the Gene 3 classic experiments to demonstrate that DNA was genetic material o Simple repetitive couldn t possibly carry much information Proteins were much more interesting because more complex more different shapes and sizes People kept assuming that the protein part of the chromosomes carried the information It s really just the simple DNA structure though o Griffith provided an assay for identifying which component of the chromosome was carrying the info Assay for transformation Worked with 2 types of pneumococcus labeled Rough mouse develops immune response survives and Smooth has polysaccharide coating protects it from the immune system thereby virulent will kill the mouse Heat kill the smooth cells boil them and inject them into a mouse mouse lives Then mixed Rough cells with heat killed Smooth cells and injected them mouse died even though neither by themselves could kill it Something from the heat killed cells is transforming the rough cells into a virulent strain some component is mixing with the benign cells and genetically transforming them found living Smooth cells in the dead mouse s heart o Avery Macleod and McCarty developed purified extracts of the chromosomes demonstrate that it was the DNA component Not everybody believed this experiment Started to fractionate the heat killed Smooth cells into components Wash boiled virulent cells 3 times with saline Then add proteinase remove all proteins Finally add Rough cells and see if any transform into virulent cells They did and that means that protein is not the genetic material The reason it s not completely convincing is that you can never be sure that the proteinase chewed up all the protein If all protein gone that leaves only nucleic acid as possibilities RNA and DNA Chewed up RNA still got the transformation into the virulent cells Only when added deoxyribonuclease ridding it of all the DNA did no transformation happen no Smooth cells appeared This is essentially still how you extract DNA Characteristics of the transforming principle high molecular weight highly negatively charged UV absorption matches that of DNA nitrogen phosphorus ratio was like DNA DNA has phosphorus protein does not o Hershey and Chase used radioactive compounds did a convincing experiment that finally settled the argument Took advantage of a bacteriophage bacterial virus for E coli DNA is packed in the head looks like the lunar module everything else is protein very simply organism Binds to receptors on the surface of E coli injects its genome which then begins to be expressed and then a variety of proteins start to chop up the bacteria s chromosome and turn it into a phage factory and finally lyses the bacterial wall and releases all the new phages The researchers used 35 Sulfur and 32 Phosphorus Used the Sulfur to grow E coli then grew phage on it which became labeled with the 35 S in the proteins In separate vial did the same thing with the Phosphorus which labeled 32 P only in the nucleic acid because DNA has phosphorus proteins don t Then took unlabeled E coli and infected separately with each and after a few minute put the 2 mixes in 2 blenders to separate the phages from the E coli cells After getting rid of the phages the cells that were taken over by Sulfur phages should have no traces of it left but those infected by radioactive Phosphorus phages should Most of the bacteria got infected almost all of the S 35 wound up in the superfluous medium as well as a little P not all phages bound 75 of the P stayed with the E coli cells 75 of the S wound up in the extracellular solution o After this race was on to understand DNA structure and how it carries information Chemical components of DNA o Composed of nucleotides Each nucleotide has a phosphate group PO4 a sugar ribose OH on 2 and 3 or deoxyribose lost the O s on 2 and 3 5 Carbons labeled with prime numbers and a nitrogenous base purines double ring structures A and G and pyrimidines single ring structures C and T and U instead of T in RNA different because of a methyl group Don t need to know structure of the bases Do need to remember that A and G are purines and that C T and U are pyrimidines o Watson and Crick First clues they used data from Chargaff ratios of A G C and T in various DNA sources E coli yeast sea urchin rat and human No matter what the actual frequency was the ratio of A T was always about 1 and G C same Also had preliminary x ray crystallography data from Rosalind Franklin studied arrangement of atoms in a crystal Generate a bunch of x rays through a lead screen shine at crystal DNA atoms diffract the beam and detect it on X ray film as a series of bands and dots a diffraction pattern The pattern that Rosalind Franklin was able to generate very difficult to do spaghetti of DNA Told us that cross angle indicates helix spacing triangles indication of helix diameter and varying intensities in the X in the middle arise from major and minor grooves First model the phosphate groups have all this negative charge in the backbone of the DNA which would repel each other unless they can stabilize themselves somehow So the first idea was that the bases are on the outside of the DNA info thus accessible with positive ions on the inside to balance the negative ones Seemed perfect total opposite of what Rosalind Franklin s images indicated Linus Pauling suggested a triple helix model with bases sticking out of a helix made of 3 strands Also completely wrong although he didn t have access to Franklin s x rays Completely guessed the structure Found hydrogen bonds where like bases could pair and the two helices were running in the same direction The DNA strand has a polarity arising from the sugar Polarity runs from the 5 sugar to the 3 when drawing it it s an arrow from 5 to 3 The model was wrong because they were both going the same way Textbook had sugar in the enol form but in reality it s in the keto form in the cell Once they knew that knew the H bonds were totally different only took a few hours to understand what the correct pattern must be This is why the ratios were around 1 because for each C need a G each A needs a T One strand runs 3 to 5 up the other 3 to 5 down They are antiparallel This final form of the helix had the dimensions expected from the crystallography which is how they knew they had the right answer The sequence of bases in the DNA can be anything no regular pattern Originally researchers