Chapter 16: The Molecular Basis of Inheritance

1. Chapter 16: The Molecular Basis of Inheritance

2. Figure 16.1 Watson and Crick with their DNA model

3. 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: CONCLUSION EXPERIMENT RESULTS 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. Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. Figure 16.2 Can the genetic trait of pathogenicity be transferred between bacteria?

4. Phage head Tail Tail fiber DNA 100 nm Bacterial cell Figure 16.3 Viruses infecting a bacterial cell

5. 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. 3 2 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. 4 1 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 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 Figure 16.4 Is DNA or protein the genetic material of phage T2?

6. CONCLUSION Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material. RESULTS 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.

7. 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 N P O O O 1 4 O– H N H Phosphate H H N 2 H 3 DNA nucleotide N H OH N Sugar (deoxyribose) 3 end H H Guanine (G) Figure 16.5 The structure of a DNA strand

8. (b) (a) Rosalind Franklin Franklin’s X-ray diffraction Photograph of DNA Figure 16.6 Rosalind Franklin and her X-ray diffraction photo of DNA

9. 5 end G C O OH A T Hydrogen bond P 3 end –O O T A OH O H2C A T 1 nm O O CH2 O C G P O O– –O 3.4 nm O P C G O O H2C O G C T A O CH2 O O G C P O O– –O O P O O H2C O G C T A O CH2 O O P T A O –O O– O P O A T O H2C O A T T A O CH2 OH O O– 3 end P O G C O 0.34 nm 5 end T A (a) Key features of DNA structure (b) Partial chemical structure (c) Space-filling model Figure 16.7 The double helix

10. Purine + Purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width Consistent with X-ray data Unnumbered Figure p. 298

11. N O H CH3 N N N N H Sugar N N O Sugar Adenine (A) Thymine (T) H O N H N N N H N Sugar N N N O H Sugar H Cytosine (C) Guanine (G) Figure 16.8 Base pairing in DNA H

12. A T C G T A A T G C (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. Figure 16.9 A model for DNA replication: the basic concept (layer 1)

13. A A T T C G C G T A T A A A T T G G C C (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. (b) The first step in replication is separation of the two DNA strands. Figure 16.9 A model for DNA replication: the basic concept (layer 2)

14. T A A A T T T A G C C G C C G G A T A T T A A T T A A A T T T A C G G G 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. (b) The first step in replication is separation of the two DNA strands. Figure 16.9 A model for DNA replication: the basic concept (layer 3)

15. 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 model for DNA replication: the basic concept (layer 4)

16. First replication Second replication Parent cell (a) Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. (b) Semiconserva- tive model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. (c) Dispersive model. Each strand of both daughter mol- ecules contains a mixture of old and newly synthesized DNA. Figure 16.10 Three alternative models of DNA replication

17. 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. Bacteria cultured in medium containing 15N Bacteria transferred to medium containing 14N 2 1 3 4 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 Does DNA replication follow the conservative, semiconservative, or dispersive model?

18. 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

19. 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 Origins of replication in eukaryotes

20. DNA pol Ill elongates DNA strands only in the 5 3 direction. One new strand, the leading strand, can elongate continuously 5 3 as the replication fork progresses. 2 3 1 4 3 5 Parental DNA 5 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). 3 Okazaki fragments 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 Overall direction of replication Figure 16.14 Synthesis of leading and lagging strands during DNA replication

21. 6 7 1 5 2 4 3 3 5 3 5 Templatestrand Primase joins RNA nucleotides into a primer. DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 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 5 2 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 5 2 1 Overall direction of replication Figure 16.15 Synthesis of the lagging strand

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