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Lecture 13

lecture 13.doc

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Department
Biological Sciences
Course Code
BIOB11H3
Professor
Dan Riggs

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Learning objectives 1) Discuss three possible mechanisms by which DNA replication could occur and elaborate why a semi conservative mechanism is most likely employed 2) Diagram the sedimentation patterns of a messelson and stahl experiment at the end of the first, second third and third round replication after the switch to N15 containing medium -After one round of replication all molecule sediments are at the hybrid—with equal N14/N15 -After the second round of replication half is hybrid and the other half is light Explain how the processes of DNA replication and transcription are similar - The process of replication and transcription both being at a defined site. The origin vs the transcription site - Must denature DNA strands so that the template can be read and new strands made - Proteins and protein complexes sponsor initiation and elongation processes - BOTH the denaturation of DNA at the orgin and the continuous unwinding of strands during elongation induce torsional stress Definitions Conservative—parental strands together Semi conservative-parental/daughter strand hybrids Dispersive- random parental/ daughter strands Density label: ---Grew cells for many generations in N15 medium and label DNA with N15, wash cells and switch to normal N14 medium Upon replication DNA would now have some N15 and N14, thuse density of DNA would differ. At different time points purified DNA separated by centrifugation an the N14/N15 rations determined Topoisomerase Gyrase DNA polymeranse--- only add nucleotides to a 3’ OH group soooo they must contain a template strand copy and a primer strand at the 3’ where OH can be added Leading vs lagging strand Okazaki fragments Primase Concepts DNA is a double stranded anti parallel structure where the amount of A=T and G=C Each strand serves as a template for the formation of another strand There are three possible replication mechanisms Conservative—parallel strands together -two original strands would remain together (after serving as templates) as would the two newly synthesized strands. As a result one of the daughter duplexes would contain only parentl DNA while the other daughter duplex would contain only synthesized Semi-conservative- parental/daughter strand hybrid -each daughter contains one strand from the parent Dispersive—random parental/daughter strands -parental strands would be broken into fragments and the new strands would be synthesized in short segements. The old fragments and new segments would be joined together to form a complete strand. Therefore the daughter contains strands that were composites of old and new DNA The semi-conservative is the most feasible based on time energy and potential for errors Meselson and Stahl used desnity labeling and ultracentrigugation to show that bacterial replication is semi conservative. J.H taylor used radiolebelleing to show that eukaryotic cells also employ semi conservative replication Used E.coli to investigate the mechanisms of replication. Employed desnity labeling Possible results from Meselson & Stahl Experiment Fig 13-3 N14/N14 N14/N15 N15/N15 III I Progressive enrichment for N14 strands DNA was extracted from bacteria at different stages in the experiment, mixed with concentrated solution of salt cesium chloride (CsCL) placed into a centrifuge tube and centrifuged to equilibrium at a high speed in an unltracentrifuge. Cs ions have sufficient atomic mass to be affected J. H. Taylor proved that replication is density gradient. Lowest density at the semtop down. The DNA’s density is dependent on the ratio of N14/N15. Greater the N14 content the lower the BrdU(bromodeoxyuridine) ! thymidine Experimental demsonstration that DNA replication occurs seminconservatively in eukaryotic cells. Schematic diagram of the results of an experiment in which cells were transferred from a medium contain
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