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Chapter 16

Figure 16.1. Chapter 16. The Molecular Basis of Inheritance. EXPERIMENT. RESULTS. CONCLUSION. Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they

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Chapter 16

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  1. Figure 16.1 Chapter 16 The Molecular Basis of Inheritance

  2. EXPERIMENT RESULTS CONCLUSION Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Living S (control) cells Living R (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. Figure 16.2 Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. • Griffith found that when he mixed heat-killed remains of the pathogenic strain • With living cells of the nonpathogenic strain, some of these living cells became pathogenic

  3. Phage head Tail Tail fiber DNA 100 nm Bacterial cell Figure 16.3 • Viruses that infect bacteria, bacteriophages • Are widely used as tools by researchers in molecular genetics

  4. EXPERIMENT In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells. CONCLUSION 3 2 4 1 Agitated in a blender to separate phages outside the bacteria from the bacterial cells. Centrifuged the mixture so that bacteria formed a pellet at the bottom of the test tube. Measured the radioactivity in the pellet and the liquid Mixed radioactively labeled phages with bacteria. The phages infected the bacterial cells. Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Phage Bacterial cell RESULTS DNA Batch 1: Phages were grown with radioactive sulfur (35S), which was incorporated into phage protein (pink). Phage DNA Centrifuge Radioactive DNA Pellet (bacterial cells and contents) Batch 2: Phages were grown with radioactive phosphorus (32P), which was incorporated into phage DNA (blue). Centrifuge Radioactivity (phage DNA) in pellet Pellet Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells. When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus. Figure 16.4 Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material. • The Hershey and Chase experiment

  5. Sugar-phosphate backbone Nitrogenous bases 5 end CH3 O– 5 O H CH2 O P O O 1 4 N O– N H H H H H O 2 3 H Thymine (T) O H H CH2 O O P N O N H O– H N H H H N N H H Adenine (A) H H O H N CH2 O O P H O O– N H N H H H O H Cytosine (C) O 5 H CH2 O P N O O O 1 4 O– H N H Phosphate H H N 2 H 3 DNA nucleotide N H OH N H Sugar (deoxyribose) 3 end H Figure 16.5 Guanine (G) Additional Evidence That DNA Is the Genetic Materia • Prior to the 1950s, it was already known that DNA • Is a polymer of nucleotides, each consisting of three components: a nitrogenous base, a sugar, and a phosphate group

  6. In 1947, Chargaff reported • That DNA composition varies from one species to the next • This evidence of molecular diversity among species • Made DNA a more credible candidate for the genetic material

  7. Franklin’s X-ray diffraction Photograph of DNA (b) (a) Rosalind Franklin Figure 16.6 a, b • Maurice Wilkins and Rosalind Franklin • Were using a technique called X-ray crystallography to study molecular structure • Rosalind Franklin • Produced a picture of the DNA molecule using this technique

  8. G C A T T A 1 nm C G 3.4 nm C G A T G C T A T A A T T A G C 0.34 nm A T Figure 16.7a, c (a) Key features of DNA structure (c) Space-filling model • Watson and Crick deduced that DNA was a double helix • Through observations of the X-ray crystallographic images of DNA

  9. 5 end O OH Hydrogen bond P 3 end –O O OH O A T O CH2 O O P O –O O– O P O H2C O O G C O O CH2 O P O O O –O O– O– O– O P P P O O O H2C O O O O C G O O CH2 O P –O O H2C A T O O CH2 OH 3 end (b) Partial chemical structure 5 end Figure 16.7b

  10. T A A A A T A T T T T A G C G C C C C G G G G C A A T T T T A A A A T T T A A A A T A T T T T A C G G G G C G C C C C G (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. (b) The first step in replication is separation of the two DNA strands. Figure 16.9 a–d • In DNA replication • The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

  11. First replication Second replication Parent cell Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. Dispersive model. Each strand of both daughter mol- ecules contains a mixture of old and newly synthesized DNA. Figure 16.10 a–c • DNA replication is semiconservative • Each of the two new daughter molecules will have one old strand, derived from the parent molecule, and one newly made strand (a) (b) (c)

  12. EXPERIMENT Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15N. The bacteria incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with only 14N, the lighter, more common isotope of nitrogen. Any new DNA that the bacteria synthesized would be lighter than the parental DNA made in the 15N medium. Meselson and Stahl could distinguish DNA of different densities by centrifuging DNA extracted from the bacteria. 3 2 1 4 Bacteria cultured in medium containing 15N Bacteria transferred to medium containing 14N RESULTS Less dense DNA sample centrifuged after 40 min (after second replication) DNA sample centrifuged after 20 min (after first replication) More dense The bands in these two centrifuge tubes represent the results of centrifuging two DNA samples from the flask in step 2, one sample taken after 20 minutes and one after 40 minutes. Figure 16.11 • Experiments performed by Meselson and Stahl • Supported the semiconservative model of DNA replication

  13. CONCLUSION Meselson and Stahl concluded that DNA replication follows the semiconservative model by comparing their result to the results predicted by each of the three models in Figure 16.10. The first replication in the 14N medium produced a band of hybrid (15N–14N) DNA. This result eliminated the conservative model. A second replication produced both light and hybrid DNA, a result that eliminated the dispersive model and supported the semiconservative model. First replication Second replication Conservative model Semiconservative model Dispersive model

  14. Origin of replication Parental (template) strand 0.25 µm Daughter (new) strand 1 Replication begins at specific sites where the two parental strands separate and form replication bubbles. Bubble Replication fork 2 The bubbles expand laterally, as DNA replication proceeds in both directions. 3 Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. Two daughter DNA molecules (a) In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. (b) In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). Figure 16.12 a, b • A eukaryotic chromosome • May have hundreds or even thousands of replication origins

  15. Online Animations: • 1. http://www.dailymotion.com/video/x28qi5_dna-replication-process_tech • 2. http://www.mcb.harvard.edu/Losick/images/TromboneFINALd.swf • 3. http://strangepaths.com/dna-replication/2007/07/03/en/

  16. 4 2 3 1 DNA pol Ill elongates DNA strands only in the 5 3 direction. 3 One new strand, the leading strand, can elongate continuously 5 3 as the replication fork progresses. 5 Parental DNA 5 3 Okazaki fragments The other new strand, the lagging strand must grow in an overall 3 5 direction by addition of short segments, Okazaki fragments, that grow 5 3 (numbered here in the order they were made). 2 3 1 5 DNA pol III Template strand DNA ligase joins Okazaki fragments by forming a bond between their free ends. This results in a continuous strand. Leading strand Lagging strand 3 1 2 Template strand DNA ligase Figure 16.14 Overall direction of replication • Synthesis of leading and lagging strands during DNA replication

  17. 7 2 3 6 5 1 4 3 5 3 5 Templatestrand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Primase joins RNA nucleotides into a primer. RNA primer 3 5 3 1 5 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazakifragment 3 3 5 1 5 After the second fragment is primed. DNA pol III adds DNAnucleotides until it reaches the first primer and falls off. 5 3 3 2 5 1 DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2. 5 3 3 5 2 1 DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1. The lagging strand in this region is nowcomplete. 5 3 3 2 5 1 Figure 16.15 Overall direction of replication

  18. Overall direction of replication Lagging strand Leading strand Helicase unwinds the parental double helix. Origin of replication 1 Molecules of single- strand binding protein stabilize the unwound template strands. The leading strand is synthesized continuously in the 5 3 direction by DNA pol III. 2 3 Leading strand Lagging strand OVERVIEW DNA pol III Leading strand 5 Replication fork DNA ligase DNA pol I 3 Primase 2 Parental DNA Lagging strand DNA pol III 1 Primer 3 Primase begins synthesis of RNA primer for fifth Okazaki fragment. 4 3 5 4 DNA pol I removes the primer from the 5 end of the second fragment, replacing it with DNA nucleotides that it adds one by one to the 3 end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugar- phosphate backbone with a free 3 end. DNA pol III is completing synthesis of the fourth fragment, when it reaches the RNA primer on the third fragment, it will dissociate, move to the replication fork, and add DNA nucleotides to the 3 endof the fifth fragment primer. DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. 5 6 7 Figure 16.16 • A summary of DNA replication

  19. 1 2 4 A thymine dimer distorts the DNA molecule. • In nucleotide excision repair • Enzymes cut out and replace damaged stretches of DNA A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease DNA polymerase Repair synthesis by a DNA polymerase fills in the missing nucleotides. 3 DNA ligase DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. Figure 16.17

  20. 5 End of parental DNA strands Leading strand Lagging strand 3 Last fragment Previous fragment RNA primer 5 Lagging strand 3 Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication 5 3 New leading strand New lagging strand 5 3 Further rounds of replication Shorter and shorter daughter molecules Figure 16.18 Replicating the Ends of DNA Molecules • The ends of eukaryotic chromosomal DNA • Get shorter with each round of replication

  21. If the chromosomes of germ cells became shorter in every cell cycle • Essential genes would eventually be missing from the gametes they produce • An enzyme called telomerase • Catalyzes the lengthening of telomeres in germ cells

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