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Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 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 (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse diesMouse healthy Mouse dies Living S cells are found in blood sample. Transformation – external DNA assimilated by a cell which changes the cell’s genotype and phenotype

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? - Bacteriophage – viruses that infect bacteria Phage head Tail Tail fiber DNA Bacterial cell 100 nm

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 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 Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Centrifuge Pellet Radioactivity (phage DNA) in pellet Batch 1: Phages were grown with radioactive sulfur ( 35 S), which was incorporated into phage protein (pink). Batch 2: Phages were grown with radioactive phosphorus ( 32 P), which was incorporated into phage DNA (blue) 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

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 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

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? -Nucleotide composition -Human -A – 30.3% -T – 30.3% -C – 19.7% -G – 19.7% Chargaff’s rules A=T C=G Note: 5’ end – phosphate group 3’ end - -OH

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? 4.What experiment did Watson & Crick do? - NONE – compiled everyone else’s data (a) Rosalind Franklin Franklin’s X-ray diffraction Photograph of DNA (b) Double helix

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? 4.What experiment did Watson & Crick do? -NONE – compiled everyone else’s data -Determined double helix from Rosalind Franklin’s data Purine + Purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width Consistent with X-ray data

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? 4.What experiment did Watson & Crick do? -NONE – compiled everyone else’s data -Determined double helix from Rosalind Franklin’s data N H O CH 3 N N O N N N NH Sugar Adenine (A) Thymine (T) N N N N Sugar O H N H N H N O H H N Guanine (G) Cytosine (C)

Figure 16.7 The double helix DNA is antiparallel Hydrogen bonds hold double helix together Now that we know how DNA is shaped & how it fits together…..

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? 4.What experiment did Watson & Crick do? 5.How does DNA replicate itself? The basic concept (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 A G A C T A G A C T A G A C T A G T G A T C T G A T C A C T A G A C T A G T G A T C T G A T C T G A T C T G A T C But is replication done in a conservative, semi-conservative or dispersive manner?

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 replication Figure Three alternative models of DNA replication Conservative - Original strands return together Semi-conservative -One original & one new strand Dispersive -Original strands are dispersed through new strands The Meselson – Stahl Experiment showed how DNA replicates.

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, 15 N. The bacteria incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with only 14 N, the lighter, more common isotope of nitrogen. Any new DNA that the bacteria synthesized would be lighter than the parental DNA made in the 15 N 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 15 N Bacteria transferred to medium containing 14 N 2 1 DNA sample centrifuged after 20 min (after first replication) 3 DNA sample centrifuged after 40 min (after second replication) 4 Less dense More dense Figure Does DNA replication follow the conservative, semiconservative, or dispersive model?

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 14 N medium produced a band of hybrid ( 15 N– 14 N) 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 replicationSecond replication Conservative model Semiconservative model Dispersive model

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? 4.What experiment did Watson & Crick do? 5.How does DNA replicate itself? 6.How is DNA replicated? -Origin of replication

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? 4.What experiment did Watson & Crick do? 5.How does DNA replicate itself? 6.How is DNA replicated? -Origin -Elongation -DNA polymerase -500 bp/sec bacteria, 50 bp/sec in us -New base added to 3’end (-OH) -dNTPs provide energy

New strandTemplate strand 5’ end 3’ end Sugar A T Base C G G C A C T P P P OH P P 5’ end 3’ end 5’ end A T C G G C A C T 3’ end Nucleoside triphosphate Pyrophosphate 2 P OH Phosphate Figure Incorporation of a nucleotide into a DNA strand DNA polymerase OH

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? 4.What experiment did Watson & Crick do? 5.How does DNA replicate itself? 6.How is DNA replicated? -Origin -Elongation -Dealing with antiparallel strands

Parental DNA DNA pol Ill elongates DNA strands only in the 5 3 direction Okazaki fragments DNA pol III Template strand Leading strand Lagging strand Template strand DNA ligase Overall direction of replication One new strand, the leading strand, can elongate continuously 5 3 as the replication fork progresses. 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). DNA ligase joins Okazaki fragments by forming a bond between their free ends. This results in a continuous strand Figure Synthesis of leading and lagging strands during DNA replication

Overall direction of replication 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. 2 Primase joins RNA nucleotides into a primer. 1 Template strand RNA primer Okazaki fragment Figure Synthesis of the lagging strand

Figure 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 strand Lagging strand Leading strand OVERVIEW Leading strand Replication fork DNA pol III Primase Primer DNA pol III Lagging strand DNA pol I DNA ligase 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

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? 4.What experiment did Watson & Crick do? 5.How does DNA replicate itself? 6.How is DNA replicated? 7.How is DNA repaired? -Proofreading – mismatch repair – DNA polymerase fixes mistakes made during copying -Excision repair

Nuclease DNA polymerase DNA 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 Figure Nucleotide excision repair of DNA damage

Chapter 16: The Molecular Basis of Inheritance 1.What evidence did Frederick Griffith have that DNA was the genetic material? 2.How did Hershey & Chase determine that DNA was the genetic material? 3.What was Chargaff’s contribution? 4.What experiment did Watson & Crick do? 5.How does DNA replicate itself? 6.How is DNA replicated? 7.How is DNA repaired? 8.What happens at the end of chromosomes (telomere)? -Recall DNA polymerase works 5’ → 3’ direction -After RNA primer is removed, chromosome is shortened

End of parental DNA strands Leading strand Lagging strand Last fragmentPrevious fragment RNA primer Lagging strand 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 of replication Shorter and shorter daughter molecules Figure Shortening of the ends of linear DNA molecules

2009 Nobel Prize in Medicine - for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase Telomerase -active in germ line cells & embryos to prevent shortening -extends telomere a bit -has its own RNA - complementary to telomere - serves as primer