BIO 112 MOLECULAR BIOLOGY AND GENETICS METHOD OF LEARNING Lectures Four hours per week Tutorials One hour per week Laboratory sessions Three hours per week ASSESSMENT Continous Assessment 2 Tests 20% Practicals 15% Theory Quiz 5% Final Examination 40% 60% Prescribed reading • Elliot, W. H. and Elliot, D. C. (2004). Biochemistry and Molecular Biology. 3rd Edition. Oxford. • Taylor, D. J. et al. (1997). Biological Science. London: Cambridge University Press. LECTURE 1 DNA REPLICATION IN PROKARYOTES/EURKAYOTES Historical perspective • 1953, James Watson and Francis Crick published a two-page paper in the journal Nature entitled “Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid.” • Once the structure of the genetic material had been determined, an understanding of its method of replication and its functioning quickly followed. DNA Replication • Occurs during interphase of cell cycle • When a eukaryotic cell divides, the process is called mitosis (cell splits) into two identical cells) - the DNA must be replicated so that each daughter cell has a copy. • DNA replication is a means to produce new DNA molecules that have the same sequence. • DNA replication involves several processes: - first, the DNA must be unwound, separating the two strands - the single strands then act as templates for synthesis of the new strands, which are complimentary in sequence - bases are added one at a time until two new DNA strands that exactly duplicate the original DNA are produced. Semi-conservative replication • DNA replication is semi-conservatively. • The parent DNA strand separates into two • Each strand serves as a template for formation of a new complementary strands • The new double helix is half original because one strand of each daughter DNA comes from the parent DNA and one strand is new • Semi-conservative was the accepted model after Meselson and Stahl did experiments (Meselson & Stahl 1958) Other Replication Models • Conservative model, the parental molecule directs synthesis of an entirely new double-stranded molecule, such that after one round of replication, one molecule is conserved as two old strands. • This is repeated in the second round. • Dispersive model, material in the two parental strands is distributed more or less randomly between two daughter molecules. • In this model, old material is distributed symmetrically between the two daughters molecules. • Two templates, each of which carries all the information of the original molecule. The Meselson - Stahl Experiment • Proof of the replication model of DNA as we know it today came from the experiments of Meselson and Stahl. • They grew E. coli is a medium using ammonium ions (NH4+) as the source of nitrogen for DNA synthesis. • 14N is the common isotope of nitrogen, but they could also use ammonium ions that were enriched for a rare heavy isotope of nitrogen, 15N. • After growing E. coli for several generations in a medium containing 15NH4+, they found that the DNA of the cells was heavier than normal (because of the 15N atoms in it). • The difference could be detected by extracting DNA from the E. coli cells and spinning it in an ultracentrifuge. • The density of the DNA determines where it accumulates in the tube. • Then they transferred more living cells that had been growing in 15NH4+ to a medium containing ordinary ammonium ions (14NH4+) and allowed them to grow for four generations. • At each generation (which takes about 20 minutes to grow), a DNA sample is taken and analyzed by centrifugation in a CsCl gradient • The DNA in this new generation of cells was exactly intermediate in density between that of the previous generation and the normal • Generation 0; 100% of DNA in nitrogen-15 band. • Generation 1; 100% of DNA in a band intermediate in position between nitrogen-14 and nitrogen-15 bands. • Generation 2; 50% of DNA in a band intermediate in position between nitrogen-14 and nitrogen-15 bands. 50% of DNA in nitrogen14 band. • Generation 3; 25% of DNA in a band intermediate in position between nitrogen-14 and nitrogen-15 bands. 75% of DNA in nitrogen-14 band. • Generation 4; 12% of DNA in a band intermediate in position between nitrogen-14 and nitrogen-15 bands. 88% of DNA in nitrogen-14 band. Generation 0 • DNA isolated from cells at the start of the experiment “generation 0,” just before the switching to the light normal ammonium ions (14NH4+) produced a single band after centrifugation. • This result made sense because the DNA should have contained only heavy 15N isotope. Generation 1 • DNA isolated after one generation (one round of DNA replication) also produced a single band when centrifuged. • However, this band was higher, intermediate in density between the heavy 15N DNA and light 14N DNA. • The intermediate band told Meselson and Stahl that the DNA molecules made in the first round of replication was a hybrid of light and heavy DNA. • This result fit with the dispersive and semiconservative models, but not with the conservative model. • The conservative model would have predicted two distinct bands in this generation (a band for the heavy original molecule and a band for the light, newly made molecule). • Generation 2 • Information from the second generation let Meselson and Stahl determine which of the remaining models (semi-conservative or dispersive) was actually correct. • When second-generation DNA was centrifuged, it produced two bands. • One was in the same position as the intermediate band from the first generation, while the second was higher (appeared to be labeled only with 14N . • This result told Meselson and Stahl that the DNA was being replicated semi-conservatively. • The pattern of two distinct bands—one at the position of a hybrid molecule and one at the position of a light molecule—is just what we'd expect for semi-conservative replication (as illustrated in the diagram below). • In contrast, in dispersive replication, all the molecules should have bits of old and new DNA, making it impossible to get a "purely light" molecule. • Generations 3 and 4 • In the semi-conservative model, each hybrid DNA molecule from the second generation would be expected to give rise to a hybrid molecule and a light molecule in the third generation, while each light DNA molecule would only yield more light molecules. • Thus, over the third and fourth generations, we'd expect the hybrid band to become progressively fainter (because it would represent a smaller fraction of the total DNA) and the light band to become progressively stronger (because it would represent a larger fraction). • As we can see in the figure, Meselson and Stahl saw just this pattern in their results, confirming a semi-conservative replication model. • As the figure indicates, each original strand remains intact as it builds a complementary strand from the nucleotides available to it. • This is called semi-conservative replication because each daughter DNA molecule is half "old" and half "new". 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