1. DNA/RNA Structure & DNA Replication

2. Genes are on Chromosomes Thomas Hunt Morgan Thomas Hunt Morgan –Worked with Drosophila –Demonstrated that genes are located on chromosomes –Is protein or DNA the genetic material found on the chromosomes? –Many leading scientists through the 1940’s thought that protein was the genetic material!

3. Frederick Griffith Frederick Griffith Streptococcus pneumoniae bacteria Streptococcus pneumoniae bacteria Worked with 2 strains of the bacteria: R & S Worked with 2 strains of the bacteria: R & S Harmless live bacteria mixed with heat-killed infectious bacteria caused disease in mice Harmless live bacteria mixed with heat-killed infectious bacteria caused disease in mice The substance passed from the dead bacteria to the live bacteria  “transforming factor” The substance passed from the dead bacteria to the live bacteria  “transforming factor”

4. Griffith & the “Transforming Factor” Transformation: change in genotype and phenotype due to the assimilation of external DNA by the cell Transformation: change in genotype and phenotype due to the assimilation of external DNA by the cell

5. Avery, McCarty, & MacLeod (1944) Avery, McCarty, & MacLeod (1944) Purified DNA & proteins from Streptococcus pneumoniae Purified DNA & proteins from Streptococcus pneumoniae Which one, DNA or protein, will transform non- pathogenic bacteria? Which one, DNA or protein, will transform non- pathogenic bacteria? Injected protein into bacteria  Injected protein into bacteria  –No effect Injected DNA into bacteria  Injected DNA into bacteria  –Transformed harmless bacteria into virulent bacteria Transforming Agent was DNA!!! Transforming Agent was DNA!!!

6. Alfred Hershey & Martha Chase

8. Hershey & Chase (1952) Hershey & Chase (1952) Blender experiment Blender experiment Worked with bacteriophage Worked with bacteriophage –Viruses that infect bacteria Grew phage viruses in Grew phage viruses in 2 media  radioactively 2 media  radioactively labeled with either: labeled with either: – 35 S in their proteins – 32 P in their DNA Infected bacteria with the labeled phages Infected bacteria with the labeled phages

9. Blender Experiment Results Radioactive phage & bacteria in blender Radioactive phage & bacteria in blender Centrifuge the mixture so bacteria falls to the bottom Centrifuge the mixture so bacteria falls to the bottom 35 S phage 35 S phage –Radioactive proteins stayed in the supernatant; therefore, protein did NOT enter the bacteria 32 P phage 32 P phage –Radioactive DNA stayed in the pellet; therefore, DNA did enter bacteria DNA is confirmed as the “transforming factor” DNA is confirmed as the “transforming factor”

10. Edwin Chargaff Edwin Chargaff Chargaff’s Rules: DNA composition Chargaff’s Rules: DNA composition –Varies from species to species –Amounts of 4 nitrogen bases are not equal –Bases are present in the following ratio:  A = T  G = C Humans  Humans  A = 30.9%T = 29.4% G = 19.9%C = 19.8% G = 19.9%C = 19.8%

11. Structure of DNA James Watson James Watson & Francis Crick & Francis Crick –Developed the double helix model double helix model of DNA of DNA

12. Other scientists working on the structure of DNA…….. Maurice WilkinsLinus PaulingRosalind Franklin

13. Prokaryotic vs. Eukaryotic DNA Prokaryotic Cells Prokaryotic Cells –Single, circular chromosome –Also contain plasmids (small, extra- chromosomal DNA molecules); also found in viruses Eukaryotic Cell Eukaryotic Cell –Multiple linear chromosomes

14. Structure of DNA Structure of DNA Structure of DNA Structure of DNA Double Helix Double Helix Nucleotides linked together Nucleotides linked together

15. Structure of DNA Nucleotide consists of………. Nucleotide consists of………. –Deoxyribose Sugar (C 5 H 10 O 4 ) (green) (green) –Phosphate Group (blue) (blue) –Nitrogen Base: purines & pyrimidines (gold)

16. Structure of DNA

17. Nitrogen Base Pairing in DNA Purines  two-ringed Purines  two-ringed –Adenine –Guanine Pyrimidines  one-ringed Pyrimidines  one-ringed –Thymine –Cytosine Base-Pairing Rules: Base-Pairing Rules: –A : T –G : C Hydrogen Bond

18. Bonding in DNA Phosphodiester Bonds: Phosphodiester Bonds: b/w sugar & phosphate b/w sugar & phosphate Hydrogen Bonds: b/w Hydrogen Bonds: b/w nitrogen bases in the nitrogen bases in the middle of the molecule middle of the molecule Which bonds are……… Which bonds are……… strong? weak? strong? weak? Phosphodiester Bond

19. Anti-parallel Strands Phosphate to sugar bond Phosphate to sugar bond (phosphodiester bond) involves carbons in a involves carbons in a 3’ & 5’ position 3’ & 5’ position DNA has a “direction” DNA has a “direction” Complementary strands Complementary strands run in opposite direction run in opposite direction “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” Watson & Crick

20. Structure of RNA Like DNA Like DNA –Has a sugar, phosphate, & nitrogen base –Made up of nucleotides –Follows base-paring rules Unlike DNA Unlike DNA –Sugar is ribose (not deoxyribose) –Single-stranded (not double-stranded) –Adenine, Guanine, Cytosine, & URACIL (no thymine in RNA)

21. DNA vs. RNA

22. Types of RNA mRNA (messenger) mRNA (messenger) –carries the DNA message from the nucleus to the ribosome tRNA (transfer) tRNA (transfer) –Carries amino acids to the ribosome rRNA (ribosomal) rRNA (ribosomal) –Makes up the ribosome

23. DNA Replication Base Pairing allows Base Pairing allows each strand to serve each strand to serve as a template for a as a template for a new strand new strand

24. DNA Replication Models of DNA Replication Models of DNA Replication conservative semi-conservative dispersive conservative semi-conservative dispersive

25. Semi-Conservative Replication Meselson & Stahl (1958) Meselson & Stahl (1958) Meselson & Stahl (1958) Meselson & Stahl (1958) –Labeled the nucleotides of the parent strand with an isotope of heavy nitrogen  15 N –Labeled the “new” nucleotides with a lighter isotope  14 N Replicated strands are half 15 N and half 14 N Replicated strands are half 15 N and half 14 N

26. DNA Replication Parent molecule will split at the hydrogen bonds and each nucleotide will pair with it’s complementary nucleotide. Parent molecule will split at the hydrogen bonds and each nucleotide will pair with it’s complementary nucleotide.

27. Process of DNA Replication DNA ReplicationDNA Replication Multiple enzymes control the replication of DNA………. Multiple enzymes control the replication of DNA………. –Helicase –Single-stranded binding protein –DNA Polymerase III –Primase –DNA ligase

28. DNA Replication: Getting Started

29. Origin of Replication: recognizable base sequence where a protein binds to start replication Origin of Replication: recognizable base sequence where a protein binds to start replication –Bacterial DNA  1 replication origin –Eukaryotic DNA  multiple replication origins Replication Fork: Y-shaped area where the DNA strands of DNA are growing from Replication Fork: Y-shaped area where the DNA strands of DNA are growing from

30. DNA Replication: Getting Started Enzymes involved in “getting started”… Enzymes involved in “getting started”… Helicase: starts the unwinding of the double helix; opens the DNA helix Helicase: starts the unwinding of the double helix; opens the DNA helix Single-Stranded Binding Protein: holds the separated strands apart Single-Stranded Binding Protein: holds the separated strands apart

31. DNA Replication: Elongation Elongation DNA Polymerase III DNA Polymerase III Adds nucleotides only to the 3’ end Adds nucleotides only to the 3’ end

32. DNA Replication: Elongation Leading & Lagging Strands Leading & Lagging Strands Leading & Lagging Strands Leading & Lagging Strands –Leading Strands  Continuous synthesis –Lagging Strands  Okazaki fragments  Joined by ligase  acts like glue to connect acts like glue to connect Okazaki fragments Okazaki fragments

33. DNA Replication: Priming DNA polymerase can only DNA polymerase can only extend an existing DNA extend an existing DNA molecule—it cannot molecule—it cannot start a new one! start a new one! RNA primer – made on RNA primer – made on parent DNA strand by parent DNA strand by primase primase RNA primer is later RNA primer is later removed by removed by DNA polymerase I DNA polymerase I

35. DNA Replication Enzymes

36. DNA Polymerases Don’t get them confused………….. Don’t get them confused………….. DNA Polymerase I DNA Polymerase I 20 bases/second 20 bases/second Editing, repair, & RNA primer removal Editing, repair, & RNA primer removal DNA Polymerase III DNA Polymerase III 1000 bases/second 1000 bases/second Main DNA building enzyme Main DNA building enzyme

37. Editing/Proofreading DNA Lots of mistakes occur! Lots of mistakes occur! DNA polymerase I DNA polymerase I removes incorrect bases removes incorrect bases –Reduces the error rate from 1 in 10,000 to from 1 in 10,000 to 1 in 100 million bases 1 in 100 million bases Mismatch Repair: Mismatch Repair: -enzymes correct incorrectly -enzymes correct incorrectly paired nucleotides paired nucleotides 130 known DNA repair 130 known DNA repair enzymes in humans enzymes in humans

38. Speed of DNA Replication E.coli can copy it’s entire genome of 5 million base pairs in under 1 hour!!! E.coli can copy it’s entire genome of 5 million base pairs in under 1 hour!!! A human cell can copy its 6 billion bases & divide into 2 cells in a few hours A human cell can copy its 6 billion bases & divide into 2 cells in a few hours –Incredibly accurate –About 30 errors per cell cycle

39. The End-Replication Problem Ends of the Ends of the chromosomes are chromosomes are destroyed with each destroyed with each replication replication No way to complete No way to complete the 5’ends of daughter DNA strands

40. What are telomeres? Expendable non-coding Expendable non-coding segments of DNA at the segments of DNA at the ends of chromosomes ends of chromosomes –Short sequence of bases repeated thousands of time repeated thousands of time –TTAGGG in humans –Prevent erosion of genes Telomerase - enzyme in Telomerase - enzyme in certain cells that starts certain cells that starts the lengthening of telomeres; only in germ-line cells!

41. Changes in Chromosome Structure