Sec_7 - 7-1 Nucleic Acid and Protein Synthesis Lec #20 This...

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7-1 N u c l e i c A c i d a n d P r o t e i n S y n t h e s i s This section will focus on the biochemistry behind the transfer of genetic information from DNA and RNA to protein. The basic processes are as follows: replication transcription translation DNA RNA Protein reverse transcription RNA replication 1 . D N A r e p l i c a t i o n ( a ) g e n e r a l m o d e l DNA replication will be considered first from the stand point of how the molecules are treated as a whole and then we will look at the enzymology and mechanisms. Initially, there were three general models for how the DNA molecules might be treated during replication: conservative, semi- conservative and dispersive. + + + Conservative - the parental molecule is conserved or remains in tact. Semi-conservative - the parental strands separate but they are conserved as strands. Dispersive - the parental strands are fragmented and interspersed with new DNA. The Meselson-Stahl experiment provided the evidence needed to conclude that replication followed a semi-conservative mechanism. Initially carried out in bacteria, it has since been extended to other organisms and is now considered to be universal. Lec #20
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7-2 ( b ) M e s e l s o n - S t a h l e x p e r i m e n t : (i). E. coli was grown for several generations in a medium with 15 NH 4 Cl in order to label all of the nitrogens in the DNA (and RNA and protein) with the heavy isotope 15 N. (ii) The cells were isolated by centrifugation and transfered to a medium containing 14 NH 4 Cl and grown. (iii) Aliquots were removed at various times and the DNA extracted. Times are in generation times or the time taken for one cell to divide into two cells. (iv) The DNA was analyzed by CsCl density gradient centrifugation. Density gradient analysis utilizes a concentrated solution (4.5 to 5 M) of CsCl which, when centrifuged at high speed (40,000 rpm) producing high centrifugal force (250,000 x g), creates a gradient of increasing CsCl down the tube. The increasing salt concentration results in an increasing density gradient down the tube. Like all molecules, DNA has a density and if mixed with the CsCl, it will accumulate (form a band) at the CsCl solution density that corresponds to the DNA density. Furthermore, 15 N-labeled DNA will be heavier or more dense than 14 N-lableled DNA, making it possible to separate or differentiate between the two. low density high density 15 N DNA 14 N DNA CsCl centrifuge 0 1 2 3 (generation times) 15 N DNA 14 N DNA 14 N DNA + 15 N DNA In words, as the cells grew in 14 N medium, the DNA changed from pure 15 N to a mixture of 14 N and 15 N after one generation, and after two generations, two distinct populations of DNA were evident, intermediate ( 14 N + 15 N) and light ( 14 N) DNA. In each subsequent generation, the intermediate density DNA remained and the amount of light DNA increased.
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This note was uploaded on 02/27/2011 for the course MBIO 2370 taught by Professor Spearman during the Winter '11 term at Manitoba.

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Sec_7 - 7-1 Nucleic Acid and Protein Synthesis Lec #20 This...

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