MOLECULAR BIOLOGY BIO 344
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This 8 page Class Notes was uploaded by Ezequiel Orn on Sunday September 6, 2015. The Class Notes belongs to BIO 344 at University of Texas at Austin taught by David Herrin in Fall. Since its upload, it has received 49 views. For similar materials see /class/181726/bio-344-university-of-texas-at-austin in Biology at University of Texas at Austin.
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Date Created: 09/06/15
Solving the structure of DNA As long as the structure of DNA was unknown there was no meaningful way to think about the nature of the genetic code or how the genome is replicated During the 195039s a considerable amount of evidence concerning the structure of DNA had accumulated an intense effort was underway to solve the structure of DNA This was considered to be one of the paramount problems in biology Phosphodiester bonds Biochemists had shown that DNA was composed of nucleotides and that each nucleotide was joined to the next by a phosphodiester bond Density The density of the molecule much too high for DNA to be a single stranded molecule The density of DNA could be achieved however by a double stranded molecule Xray diffraction Rosalind Franklin and Maurice Wilkins performed X ray diffraction experiments on pure DNA fibers In these experiments X rays are fired at the DNA and the molecule scatters them The scattered X rays then strike a piece of film and expose it producing black spots where they hit The angle of scatter represented by each spot provides information on the position of atoms in the DNA Their data indicated that DNA formed a regular helix 2 that the helix made one turn every 34A 34 nm 3 that the diameter of the helix was about 20 A 2 nm 4 that there was one nucleotide every 34 A 034 nm and therefore there must be 10 nucleotides turn of the helix ATGC ratio In 1951 Chargaff analyzed the ratios of the different nitrogenous bases to one another in a large number of different organisms What he saw was that the amount of A essentially equal to amount of T and that the amount of G was equal to the amount of C That is A T amp G C Furthermore A amp T does not necessarily equal G amp C Here is a of s data marrow saw or ratios but the A amp G C rule still held Furthermore in a single organism it didn39t matter which tissue you examined All of the tissues had the same ATGC ratio page 81 Revised 1212000 Watson amp Crlck39s Model All of the pieces of the puzzle were in place and many were racing to solve DNA39s structure The structure was solved in 1953 by James Watson and Francis Crick They assembled all of the data given above and built stick figure molecules until they found one that fit all of the data 1 Based on the buoyant density Watson amp Crick knew that DNA must be a double elix 2 Thermodynamically they postulated that the most stable double helix would be one in which the sugar phosphate backbone interacted with water and the more hydrophobic nitrogenous bases were inside the helix hidden from the water 3 Chargaff39s data suggested that the different bases interacted with one another and that somehow the amount of A determined the amount of T amp vice versa and the amount of G determined the amount of C amp vice versa They postulated that the A on one strand H bonded with T on the other and that the G on one strand H bonded with C on the other This pairing would stabilize the helix 4 From Franklin and Wilkin39s data they knew that there was one nucleotide every 34 A 034 nm that there were 10 nucleotides turn of the helix and that the helix was 20 A wide 5 In support of 3 Watson amp Crick39s modeling showed that only pyrimidinezpurine base pairs could produce a helix of 20 A See below PyrimidimezPyrimidine base pairing would O C be lt20A pyrlmldme pyrlmldlne PurinezPurine base pairing would produce a helix that was gt 20 A purine purine But PyrimidinezPurine base pairingowould produce a double helix of about 20 A pyrimidine purine page 82 Revised 1212000 The only model that fit all of the data is the one with which you are already familiar I present to you the Watson Crick Double Helical Model of DNA Minor 0 Groove 34 A Major Groove 341 I Strands are antiparallel They did this without performing any experiments Theirs was a conceptual contribution that is perhaps the most important scientific breakthrough of this century Although the model has been refined it is still basically correct The structure suggested a theory that base paired was used to both replicate and decode information DNA Replication Every time a cell divides the genome must be duplicated and passed on to the offspring That is 539 TGCT 339 339 ACGA 539 5 quot TGCT quot3 ltREPLICATION 3 39 ACGA 539 5 39 TGCT 339 339 ACGA 539 Original molecule yields 2 molecules following DNA replication Our topic in this section is how is this done page 83 Revised 1212000 DNA replication must have high fidelity Why Well if DNA replication was low fidelity the consequences would be 1 dramatic and rapid random changes in the sequence of genes 2 which would cause an extreme reduction in viability Because of this in a complex organism evolution will select against low fidelity DNA replication The structure of DNA suggested a way that it could be replicated with high fidelity Because the strands are complementary one strand could specify the base on the opposite strand This is actually what happens During the 195039s three theories were proposed for how DNA might be replicated They went by the names Conservative Dispersive and Semiconservative DNA replication They are illustrated below T letters renresent newlu quot 1 DNA and canital letters renresent material from the original narental 39 Conservative Replication 5 39 TGCT 3 39 Following 3 39 ACGA 539 replication one 5 I quot39 TGCT 3 llt daughter molecule 3 39 ACGA 539 5 39 tgct 339 contains both of the 3 39 acga 539 parental strands The other daughter molecule contains This theory was proven to be incorrect two newly synthesized DNA strands Dispersive 5 39 tGCT 339 Replication 3 39 AGGa 5 39 Both strands of each 5 I TGCT 3 39lt daughter molecule 3 39 ACGA 539 5 39 Tgct 339 contain nucleotides 3 39 anA 5 39 derived from the This theory was proven to be incorrect parental molecule SemiConservative Replication Each parental strand 5 39 TGCT 3 39 acts as a template for 3 39 acga 539 the synthesis of one 5 I TGCT 3 39lt new daughter strand 3 39 ACGA 539 5 39 tgct 339 Therefore in 3 39 ACGA 539 daughter molecules a newly synthesized strand is base paired to one of the original parental strands The semiconservative theory is correct page 84 Revised 1212000 How was it shown that DNA replication is SemiConservative In 1958 the Meselson Stahl experiment proved that in bacteria DNA replication was semi conservative In the early 196039s the Herbert Taylor Colchicine bean rootip experiment demonstrated that a eukaryotic cell replicated by semiconservative replication Meselson and Stahl Experiment To address this question this group used a way to isotopically label newly synthesized DNA To do this they used two different isotopes of nitrogen N 14N most common form of nitrogen 15N less common form and has greater mass than 14N DNA made using 15N is about 1 denser than DNA made using 14N These two forms can be separated by equilibrium density gradient centrifugation also called isopycnic centrifugation What is equilibrium density gradient centrifugation m In this technique molecules mixed with C D a salt CsClz dissolved in water and centrifuged at very high speed The salt molecules form a density gradient Centrifuge tubes spinning in a centrifuge Prior to centrifugation the salt molecules are evenly distributed throughout the centrifuge tube During centrifugation the salt molecules are forced towards the bottom of the tube and a gradient of molecules is established More of the salt is found at the bottom of the tube and less is at the top Therefore the density of the solution is smaller at the top and greater at the bottom Any DNA molecules in this centrifuge tube will also be forced towards the bottom of the tube As the DNA travels down the tube the density of the surrounding salt solution gradually increases The DNA stops moving relative to the salt solution when their two densities are equal This is because of a physical property called buoyancy An example Prior to Centrifugation begins and After prolonged centrifugation the the salt and DNA begin centrifugation the DNA has DNA is evenly sedimenting formed discrete bands distributed reflecting its buoyant density throughout the centrifuge tubes mampm C i i GEE E tlj In the third panel not ce that the left tube has one band of DNA This means that in this tube all of the DNA molecules have the same buoyant density Also notice that the right tube has two bands This means that the DNA in this tube is a mixture of DNAs with two different buoyant densities page 85 Revised 1212000 Back to the 15N 14N stuff E coli can be grown in media in which the sole source of nitrogen is 15N or 14N Genomic DNA from bacteria grown in 15N media will be about 1 denser than the DNA from bacteria grown in the 14N media This difference in density means that the 15N and 14N DNA can be separated by equilibrium density gradient centrifugation Previous experimentation had shown that 15N labeled El E 14N labeled E If the DNA is 50 DNA 4 DNA 15N and 50 14N sedimented sedimented then it sediments at this at this at an intermediate position position position How was this used to determine which of the three DNA replication models is correct Well Meselson and Stahl grew bacteria in 15N media until all of the DNA was uniformly labeled with 15N They then synchronized these cultures in this context synchrony means that all of the bacteria in the culture are replicating their DNA and performing cell division in unison and grew them in 14N media Here are the details First Generation Grow bacteria in 15N Transfer bacteria to 14N media gt One cell division and synchronize their has occured cell divisions Remove a sample and isolate DNA RemeVe a sample Call it Sample 1 and 1solate DNA Call it Sample 2 Second Generation Two cell divisions have occured Remove a sample and isolate DNA Call it Sample 3 Now samples 1 2 and 3 are subjected to equilibrium density gradient centrifugation The three DNA replication models make specific predictions about the result of this experiment These predictions are presented below In this table I I I I I I I I I represents DNA synthesized with 15N and I I I I I I I I I represents DNA synthesized with 14N page 86 Revised 1 21 2000 Predicted results Replicating DNA following in samples centrifugation 1 Z and 3 Predictions based on the El I Conservative Model of I Replication 15N gt Sample 1 all DNA is 15N I I 14N gt E E 15N gt I E E Sample 2 half of the DNA is 15N and half is 14N Sample 3 most DNA is 14N but some 15N remains Predictions based on the Dispersive Model of Replication Sample 1 all DNA is 15N Sample 2 all of the DNA travels at the density of DNA that is half 15N and half 14N Sample 3 all of the DNA travels at the density of DNA which is 25 15N and 75 14N In successive generations the DNA gradually becomes less dense Predictions based on the Semi Conservative Model of Replication Sample 1 all DNA is 15N Sample 2 all of the DNA travels at the density of DNA that is half 15N and half 14N Sample 3 most DNA is 14N but some half 14N and half 15N remains 15N14N I page 87 Revised 1212000 Actual results of the experiment Sample Sample Sample 1 2 3 position of 14N DNA El 1 position of half amp halfDNA gt H a i position of 15N DNA 39 These results indicate that bacterial DNA is replicated by a semiconservative mechanism Now it39s time for the Herbert Taylor Colchicine bean root tip experiment The Meselson Stahl experiment showed that bacteria used semi conservative replication But what about eukaryotic cells For technical reasons the Meselson Stahl experiment could not be performed with eukaryotic cells Herbert Taylor was the first to test whether eukaryotes use semi conservative replication He took root tip cells plant cells and allowed them to replicate in the presence of 3H thymidine 3H is a radioactive isotope of hydrogen When it decays it releases 5 particles When 5 particles strike a photographic emulsion they 39expose39 it producing a black spot This technique is called autoradiography During DNA replication the newly synthesized DNA was radiolabeled with 3H After one round of replication the 3H thymidine was washed away and replaced with 39cold39 thymidine In this context 39cold39 means not radioactive These cells were allowed to go through a second round of DNA replication The cartoon below depicts a replicating chromosome Black means that 3H in the chromosome exposed the photographic emulsion Gray means that the material is not radiolabeled and therefore does not expose the emulsion Cold Experimental Results I gt Cold Cold Start of 1st I division End of 1st division and start of 2nd division page 88 Revised 1 21 2000
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