Frederick Griffith (1928)

1. Frederick Griffith (1928) Conclusion: living R bacteria transformed into deadly S bacteria by unknown, heritable substance Oswald Avery, et al. (1944) • Discovered that the transforming agent was DNA

2. Hershey and Chase (1952) • Bacteriophages: virus that infects bacteria; composed of DNA and protein Protein = radiolabel S DNA = radiolabel P Conclusion: DNA entered infected bacteria  DNA must be the genetic material!

3. Edwin Chargaff (1947) Chargaff’s Rules: • DNA composition varies between species • Ratios: • %A = %T and %G = %C

4. Structure of DNA Scientists: • Watson & Crick • Rosalind Franklin DNA = double helix • “Backbone” = sugar + phosphate • “Rungs” = nitrogenous bases

5. Structure of DNA Nitrogenous Bases • Adenine (A) • Guanine (G) • Thymine (T) • Cytosine (C) • Pairing: • purine + pyrimidine • A = T • G Ξ C purine pyrimidine

6. Structure of DNA Hydrogen bondsbetween base pairs of the two strands hold the molecule together like a zipper.

7. DNA Comparison Prokaryotic DNA Eukaryotic DNA Double-stranded Linear Usually 1+ chromosomes In nucleus DNA wrapped around histones (proteins) Forms chromatin • Double-stranded • Circular • One chromosome • In cytoplasm • No histones • Supercoiled DNA

8. Replication is semiconservative

9. Major Steps of Replication: • Helicase:unwinds DNA at origins of replication • Initiation proteins separate 2 strands  forms replication bubble • Primase: puts down RNA primer to start replication • DNA polymerase III: adds complimentary bases to leading strand (new DNA is made 5’ 3’) • Lagging strand grows in 3’5’ direction by the addition of Okazaki fragments • DNA polymerase I: replaces RNA primers with DNA • DNA ligase: seals fragments together

10. Leading strand vs. Lagging strand

11. Okazaki Fragments: Short segments of DNA that grow 5’3’ that are added onto the Lagging Strand DNA Ligase: seals together fragments

12. Proofreading and Repair • DNA polymerases proofread as bases added • Mismatch repair: special enzymes fix incorrect pairings • Nucleotide excision repair: • Nucleases cut damaged DNA • DNA polymerase and ligase fill in gaps

13. Nucleotide Excision Repair Errors: • Pairing errors: 1 in 100,000 nucleotides • Complete DNA: 1 in 10 billion nucleotides

14. Problem at the 5’ End • DNA polymerase only adds nucleotides to 3’ end • No way to complete 5’ ends of daughter strands • Over many replications, DNA strands will grow shorter and shorter

15. Telomeres: repeated units of short nucleotide sequences (TTAGGG) at ends of DNA • Telomeres “cap” ends of DNA to postpone erosion of genes at ends (TTAGGG) • Telomerase: enzyme that adds to telomeres • Eukaryotic germ cells, cancer cells Telomeres stained orange at the ends of mouse chromosomes

16. Flow of genetic information • Central Dogma: DNA  RNA  protein • Transcription: DNA  RNA • Translation: RNA  protein • Ribosome = site of translation • Gene Expression: process by which DNA directs the synthesis of proteins (or RNAs)

17. Flow of Genetic Information in Prokaryotes vs. Eukaryotes

18. one gene = one polypeptide DNA RNA Nucleic acid composed of nucleotides Single-stranded Ribose=sugar Uracil Helper in steps from DNA to protein • Nucleic acid composed of nucleotides • Double-stranded • Deoxyribose=sugar • Thymine • Template for individual

19. RNA plays many roles in the cell • pre-mRNA=precursor to mRNA, newly transcribed and not edited • mRNA= the edited version; carries the code from DNA that specifies amino acids • tRNA= carries a specific amino acid to ribosome based on its anticodon to mRNA codon • rRNA= makes up 60% of the ribosome; site of protein synthesis • snRNA=small nuclear RNA; part of a spliceosome. Has structural and catalytic roles • RNAi= interference RNA; a regulatory molecule

20. The Genetic Code For each gene, one DNA strand is the template strand mRNA (5’ 3’) complementary to template mRNA triplets (codons) code for amino acids in polypeptide chain

21. The Genetic Code 64 different codon combinations Redundancy: 1+ codons code for each of 20 AAs Reading frame: groups of 3 must be read in correct groupings This code is universal: all life forms use the same code.

22. Transcription Transcription unit: stretch of DNA that codes for a polypeptide or RNA (eg. tRNA, rRNA) RNA polymerase: • Separates DNA strands and transcribes mRNA • mRNA elongates in 5’ 3’ direction • Uracil (U) replaces thymine (T) when pairing to adenine (A) • Attaches to promoter (start of gene) and stops at terminator (end of gene)

23. 1. Initiation Eukaryotes: TATA box = DNA sequence (TATAAAA) upstream from promoter Transcription factors mustrecognize TATA box before RNA polymerase can bind to DNA promoter

24. 2. Elongation • RNA polymerase adds RNA nucleotides to the 3’ end of the growing chain (A-U, G-C)

25. 3. Termination RNA polymerase transcribes a terminatorsequence in DNA, then mRNA and polymerase detach. It is now called pre-mRNA for eukaryotes. Prokaryotes = mRNA ready for use

26. Additions to pre-mRNA: • 5’ cap(modified guanine) and 3’poly-A tail(50-520 A’s)are added • Help export from nucleus, protect from enzyme degradation, attach to ribosomes

27. RNA Splicing • Pre-mRNA has introns (noncoding sequences) and exons (codes for amino acids) • Splicing = introns cut out, exons joined together

28. RNA Splicing • small nuclear ribonucleoproteins = snRNPs • snRNP = snRNA + protein • Pronounced “snurps” • Recognize splice sites • snRNPs join with other proteins to form a spliceosome Spliceosomescatalyze the process of removing introns and joining exons Ribozyme = RNA acts as enzyme

29. Why have introns? • Some regulate gene activity • Alternative RNA Splicing: produce different combinations of exons • One gene can make more than one polypeptide! • 20,000 genes  100,000 polypeptides

30. Components of Translation • mRNA = message • tRNA= interpreter • Ribosome = site of translation

31. tRNA • Transcribed in nucleus • Specific to each amino acid • Transfer AA to ribosomes • Anticodon: pairs with complementary mRNA codon • Base-pairing rules between 3rd base of codon & anticodon are not as strict. This is called wobble.

32. Ribosomes • Ribosome = rRNA + proteins • made in nucleolus • 2 subunits 60s 40s

33. Ribosomes Active sites: • A site: holds AA to be added • P site: holds growing polypeptide chain • E site: exit site for tRNA

34. Translation:1. Initiation • Small subunit binds to start codon (AUG) on mRNA • tRNA carrying Met attaches to P site • Large subunit attaches

35. 2. Elongation

36. 3.Termination • Stop codon reached and translation stops • Release factor binds to stop codon; polypeptide is released • Ribosomal subunits dissociate

37. Protein Folding • During synthesis, polypeptide chain coils and folds spontaneously • Chaperonin: protein that helps polypeptide fold correctly

38. Cell Cycle: life of a cell from its formation until it divides Functions of Cell Division: Reproduction, Growth and Tissue Renewal

39. Each chromosome must be duplicated before cell division Duplicated chromosome = 2 sister chromatids attached by a centromere

40. Gametes Somatic Cells Body cells diploid (2n): 2 of each type of chromosome Divide by mitosis Humans: 2n = 46 Sex cells (sperm/egg) Haploid (n): 1 of each type of chromosome Divide by meiosis Humans: n = 23

41. Phases of the Cell Cycle • The mitotic phase alternates with interphase: G1 S  G2  mitosis  cytokinesis • Interphase (90% of cell cycle) • G1 Phase: cell grows and carries out normal functions • S Phase: duplicates chromosomes • G2 Phase: prepares for cell division • M Phase (mitotic) • Mitosis: nucleus divides • Cytokinesis: cytoplasm divides

42. Mitosis 1. Prophase • Chromatin fibers condense and coil • Nucleoli disappear • Spindle (microtubules) begins to form • Centrosomes begin to move to opposite ends 2. Prometaphase • Nuclear envelope fragments • Microtubules invade nucleus • Kinetochores attach to microtubules

43. 3. Metaphase • Chromosomes line up on metaphase plate at equator • Centrioles are at opposite poles (ends) 4. Anaphase (shortest phase) • Chromatids separate and pulled apart by motor proteins toward opposite ends of cell • Chromatids are called chromosomes now • Cell elongates

44. 5. Telophase • Nuclear membrane re-forms around chromosomes • Chromosomes less condensed Cytokinesis • Cytoplasm of cell divided • Animal Cells: cleavage furrow • Plant Cells: cell plate forms

45. Cytokinesis in animal vs. plant cells

46. Bacterial cells divide by Binary Fission

47. Cell Cycle Control System Checkpoint = control point where stop/go signals regulate the cell cycle

48. Major Checkpoints • G1 checkpoint (Most important!) • “Go” completes whole cell cycle • “Stop”  cell enters nondividing state (G0 Phase) • Nerve, muscle cells stay at G0; liver cells called back from G0 • G2 checkpoint • M Phase checkpoint • Anaphase does not begin unless chromatids are properly attached to spindle at metaphase plate

49. Internal Regulatory Molecules • Kinases (cyclin-dependent kinase,Cdk): protein enzyme controls cell cycle; active when connected to cyclin • Cyclins: proteins which attach to kinases (Cdk) to activate them; levels fluctuate in the cell cycle 3. MPF: maturation-promoting factor; specific Cdk which allows cells to pass G2 and go to M phase

50. External Regulatory Factors • Growth Factor: proteins released by other cells to stimulate cell division • Density-Dependent Inhibition: crowded cells normally stop dividing; cell-surface protein binds to adjoining cell to inhibit growth • Anchorage Dependence: cells must be attached to another cell or ECM to divide