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The Molecular Basis of Inheritance

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1 The Molecular Basis of Inheritance
Chapter 16 The Molecular Basis of Inheritance

2 Figure 16.1 Watson and Crick with their DNA model

3 Figure 16.2 Can the genetic trait of pathogenicity be transferred between bacteria?
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: Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. EXPERIMENT RESULTS CONCLUSION Living S (control) cells Living R Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Living S cells are found in blood sample. 1922 Griffith Transformation, a change in genotype and phenotype due to the assimilation of foreign DNA by a cell. In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA.

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

5 Figure 16.4 Is DNA or protein the genetic material of phage T2?
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. EXPERIMENT Radioactivity (phage protein) in liquid Phage Bacterial cell Radioactive protein Empty protein shell DNA Centrifuge Pellet (bacterial cells and contents) Pellet (phage DNA) in pellet Batch 1: Phages were grown with radioactive sulfur (35S), which was incorporated into phage protein (pink). Batch 2: Phages were phosphorus (32P), which was incorporated into phage DNA (blue). 1 2 3 4 Agitated in a blender to separate phages outside the bacteria from the bacterial cells. Mixed radioactively labeled phages with bacteria. The phages infected 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

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

7 Figure 16.5 The structure of a DNA strand
Sugar-phosphate backbone Nitrogenous bases 5 end O– O P CH2 5 4 H 3 1 CH3 N Thymine (T) Adenine (A) Cytosine (C) OH 2 Sugar (deoxyribose) 3 end Phosphate Guanine (G) DNA nucleotide

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

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

10 Unnumbered Figure p. 298 Purine + Purine: too wide
Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width Consistent with X-ray data Chargaff’s rules (1947) In all organisms, the number of adenines was approximately equal to the number of thymines (%T = %A). Also (%G = %C). Erwin Chargaff

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

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

13 Figure 16.9 A model for DNA replication: the basic concept (layer 2)
(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. A C T G

14 Figure 16.9 A model for DNA replication: the basic concept (layer 3)
(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. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. A C T G

15 Figure 16.9 A model for DNA replication: the basic concept (layer 4)
(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. (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. A C T G The second paper of Watson and Crick

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

17 Figure 16.11 Does DNA replication follow the conservative, semiconservative, or dispersive model?
Late 50s 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. EXPERIMENT 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. RESULTS Bacteria cultured in medium containing 15N transferred to 14N 2 1 DNA sample centrifuged after 20 min (after first replication) 3 after 40 min (after second 4 Less dense More

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 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 Dispersive

19 Figure 16.12 Origins of replication in eukaryotes
Replication begins at specific sites where the two parental strands separate and form replication bubbles. The bubbles expand laterally, as DNA replication proceeds in both directions. Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. 1 2 3 Origin of replication Bubble Parental (template) strand Daughter (new) strand Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). (b) (a) 0.25 µm E. coli: 5 million/25 min Human cell: 6 billion/few hours The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells

20 Figure 16.13 Incorporation of a nucleotide into a DNA strand
Why antiparallel? New strand Template strand 5’ end 3’ end Sugar A T Base C G P OH Nucleoside triphosphate Pyrophosphate 2 P Phosphate The exergonic hydrolysis of pyrophosphate to two inorganic phosphate molecules drives the polymerization of the nucleotide to the new strand.

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

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

23 Table 16.1 Bacterial DNA replication proteins and their functions

24 Figure 16.16 A summary of bacterial DNA replication
Overall direction of replication Helicase unwinds the parental double helix. 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. Leading strand Origin of replication Lagging OVERVIEW Replication fork DNA pol III Primase Primer DNA pol I DNA ligase 1 2 3 Primase begins synthesis of RNA primer for fifth Okazaki fragment. 4 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 end of the fifth fragment primer. 5 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. 6 DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. 7 Parental DNA 5 3

25 Arthur Kornberg (born March 3, 1918) is an American biochemist who won the Nobel Prize in Physiology or Medicine 1959 for his discovery of "the mechanisms in the biological synthesis of deoxyribonucleic acid (DNA)" together with Dr. Severo Ochoa of New York University. Identified “DNA polymerase”

26 Reiji Okazaki (岡崎令治 Okazaki Reiji, 1930 – 1975) was a Japanese molecular biologist known for his research in the DNA replication and the discovery of Okazaki fragments. Reiji Okazaki was born in Hiroshima, Japan. He graduated in 1953 from Nagoya University, and worked as a professor there after 1963. Okazaki died of leukemia only a few years after his discovery. He was exposed to heavy radiation when he was in Hiroshima when the first atomic bomb was dropped. Tsuneko Okazaki (wife and collaborator)

27 Figure 16.17 Nucleotide excision repair of DNA damage
Nuclease DNA polymerase ligase A thymine dimer distorts the DNA molecule. 1 Repair synthesis by a DNA polymerase fills in the missing nucleotides. 3 DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. 4 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. 2 Mutations are caused by Enzymatic mistakes High energy radiation (X-ray, UV) Spontaneous changes Defects in repair cause cancers (i.e. skin cancer)

28 Figure 16.18 Shortening of the ends of linear DNA molecules
End of parental DNA strands Leading strand Lagging strand Last fragment Previous fragment RNA primer Removal of primers and replacement with DNA where a 3 end is available Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase Second round of replication New leading strand New lagging strand 5 Further rounds Shorter and shorter daughter molecules 5 3

29 Figure Telomeres 1 µm

30 4. The ends of DNA molecules are replicated by a special mechanism.
The ends of eukaryotic chromosomal DNA molecules, the telomeres, have special nucleotide sequences. Multiple repetitions of one short nucleotide sequence. TTAGGG in human Telomerase catalyzes the lengthening of telomeres in eukaryotic germ cells, restoring their original length. Telomerase is not present in most cells of multicellular organisms. Normal shortening of telomeres may protect organisms from cancer by limiting the number of divisions that somatic cells can undergo. Short telomeres in Werner syndrome Active telomerase has been found in some cancerous somatic cells. Telomerase may provide a useful target for cancer diagnosis and chemotherapy.

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