1. RNA DNA: double-stranded deoxyribose A, C, G, T RNA: single-stranded ribose A, C, G, U

2. Complication #5: Can’t add to 5 ′ end 3′3′5′5′ 5′5′3′3′ 5′5′ 3′3′ 5′5′ ? ?

3. 3´ 5´ 3´ 5´ RNA primer new DNA 5´ how do we replicate this?

4.  Multiple primers  Lagging strand made as series of Okazaki fragments Complication #5: Can’t add to 5 ′ end 3´ 5´ 3´ 5´ leading (continuous) strand lagging (discontinuous) strand Okazaki fragments 5´ DNA polymerase 5´

5. Complication #5: Can’t add to 5 ′ end 3′3′ 5′5′ 5′5′3′3′ 3′3′ 3′3′ origin

6.  DNA polymerase III makes DNA but can’t remove primer Complication #6: DNA contains RNA 3´5´ 3´ 5´ RNA primer

7.  DNA polymerase I replaces RNA with DNA Complication #6: DNA contains RNA DNA polymerase I RNA primer 3´5´ 3´ 5´

8.  DNA polymerase I replaces RNA with DNA Complication #6: DNA contains RNA 3´5´

9.  DNA polymerase I replaces RNA with DNA Complication #6: DNA contains RNA 3´5´

10.  DNA ligase makes phosphodiester bond Complication #7: Gaps in the DNA backbone

11.  DNA ligase makes phosphodiester bond DNA ligase Complication #7: Gaps in the DNA backbone

13. What does DNA do?

14. chromosome DNA molecule CFTR gene nucleus mRNA ribosome CFTR protein folding The “Central Dogma” membrane ATP Cl – CFTR transcription translation

15. How does a gene encode a protein? DNA mRNA Protein transcription translation the “Central Dogma” of molecular biology

16. DNA mRNA transcription Un beau jour, je suis allé au marché pour acheter du pain. Il faisait chaud. Alors, j’ai acheté aussi un limonade. Il faisait chaud.

17. mRNA DNA transcription CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAU AGUAGC 3’ 5’ CTACGAGGAGGTGAAGCGATGCCCCGTAGCC GATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGC TATCATCG gene

18. mRNA Protein translation Il faisait chaud. It was hot.

19. mRNA DNA transcription CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAU AGUAGC 3’ 5’ CTACGAGGAGGTGAAGCGATGCCCCGTAGCC GATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGC TATCATCG gene translation N-MetProArgSerArg-C protein

20. AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGCCCGAGAGACCATGCAG AG GTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGACCAGACCAATTTTGAGGAAA GGATACAGACAGCGCCTGGAATTGTCAGACATATACCAAATCCCTTCTGTTGATTCTGCTGACAATCTAT CTGAAAAATTGGAAAGAGAATGGGATAGAGAGCTGGCTTCAAAGAAAAATCCTAAACTCATTAATGCCCT TCGGCGATGTTTTTTCTGGAGATTTATGTTCTATGGAATCTTTTTATATTTAGGGGAAGTCACCAAAGCA GTACAGCCTCTCTTACTGGGAAGAATCATAGCTTCCTATGACCCGGATAACAAGGAGGAACGCTCTATCG CGATTTATCTAGGCATAGGCTTATGCCTTCTCTTTATTGTGAGGACACTGCTCCTACACCCAGCCATTTT TGGCCTTCATCACATTGGAATGCAGATGAGAATAGCTATGTTTAGTTTGATTTATAAGAAGACTTTAAAG CTGTCAAGCCGTGTTCTAGATAAAATAAGTATTGGACAACTTGTTAGTCTCCTTTCCAACAACCTGAACA AATTTGATGAAGGACTTGCATTGGCACATTTCGTGTGGATCGCTCCTTTGCAAGTGGCACTCCTCATGGG GCTAATCTGGGAGTTGTTACAGGCGTCTGCCTTCTGTGGACTTGGTTTCCTGATAGTCCTTGCCCTTTTT CAGGCTGGGCTAGGGAGAATGATGATGAAGTACAGAGATCAGAGAGCTGGGAAGATCAGTGAAAGACTTG TGATTACCTCAGAAATGATTGAAAATATCCAATCTGTTAAGGCATACTGCTGGGAAGAAGCAATGGAAAA AATGATTGAAAACTTAAGACAAACAGAACTGAAACTGACTCGGAAGGCAGCCTATGTGAGATACTTCAAT AGCTCAGCCTTCTTCTTCTCAGGGTTCTTTGTGGTGTTTTTATCTGTGCTTCCCTATGCACTAATCAAAG GAATCATCCTCCGGAAAATATTCACCACCATCTCATTCTGCATTGTTCTGCGCATGGCGGTCACTCGGCA ATTTCCCTGGGCTGTACAAACATGGTATGACTCTCTTGGAGCAATAAACAAAATACAGGATTTCTTACAA AAGCAAGAATATAAGACATTGGAATATAACTTAACGACTACAGAAGTAGTGATGGAGAATGTAACAGCCT TCTGGGAGGAGGGATTTGGGGAATTATTTGAGAAAGCAAAACAAAACAATAACAATAGAAAAACTTCTAA TGGTGATGACAGCCTCTTCTTCAGTAATTTCTCACTTCTTGGTACTCCTGTCCTGAAAGATATTAATTTC AAGATAGAAAGAGGACAGTTGTTGGCGGTTGCTGGATCCACTGGAGCAGGCAAGACTTCACTTCTAATGG TGATTATGGGAGAACTGGAGCCTTCAGAGGGTAAAATTAAGCACAGTGGAAGAATTTCATTCTGTTCTCA GTTTTCCTGGATTATGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGA TACAGAAGCGTCATCAAAGCATGCCAACTAGAAGAGGACATCTCCAAGTTTGCAGAGAAAGACAATATAG TTCTTGGAGAAGGTGGAATCACACTGAGTGGAGGTCAACGAGCAAGAATTTCTTTAGCAAGAGCAGTATA CAAAGATGCTGATTTGTATTTATTAGACTCTCCTTTTGGATACCTAGATGTTTTAACAGAAAAAGAAATA TTTGAAAGCTGTGTCTGTAAACTGATGGCTAACAAAACTAGGATTTTGGTCACTTCTAAAATGGAACATT TAAAGAAAGCTGACAAAATATTAATTTTGCATGAAGGTAGCAGCTATTTTTATGGGACATTTTCAGAACT CCAAAATCTACAGCCAGACTTTAGCTCAAAACTCATGGGATGTGATTCTTTCGACCAATTTAGTGCAGAA AGAAGAAATTCAATCCTAACTGAGACCTTACACCGTTTCTCATTAGAAGGAGATGCTCCTGTCTCCTGGA CAGAAACAAAAAAACAATCTTTTAAACAGACTGGAGAGTTTGGGGAAAAAAGGAAGAATTCTATTCTCAATCCAATCAACTCTATACGAAAATTTTCCATTGTGCAAAAGACTCCCTTACAAATGAATGGCATCGAAGAG GATTCTGATGAGCCTTTAGAGAGAAGGCTGTCCTTAGTACCAGATTCTGAGCAGGGAGAGGCGATACTGCCTCGCATCAGCGTGATCAGCACTGGCCCCACGCTTCAGGCACGAAGGAGGCAGTCTGTCCTGAACCTGA TGACACACTCAGTTAACCAAGGTCAGAACATTCACCGAAAGACAACAGCATCCACACGAAAAGTGTCACTG GCCCCTCAGGCAAACTTGACTGAACTGGATATATATTCAAGAAGGTTATCTCAAGAAACTGGCTTGGAAA TAAGTGAAGAAATTAACGAAGAAGACTTAAAGGAGTGCTTTTTTGATGATATGGAGAGCATACCAGCAGT GACTACATGGAACACATACCTTCGATATATTACTGTCCACAAGAGCTTAATTTTTGTGCTAATTTGGTGC TTAGTAATTTTTCTGGCAGAGGTGGCTGCTTCTTTGGTTGTGCTGTGGCTCCTTGGAAACACTCCTCTTC AAGACAAAGGGAATAGTACTCATAGTAGAAATAACAGCTATGCAGTGATTATCACCAGCACCAGTTCGTA TTATGTGTTTTACATTTACGTGGGAGTAGCCGACACTTTGCTTGCTATGGGATTCTTCAGAGGTCTACCA CTGGTGCATACTCTAATCACAGTGTCGAAAATTTTACACCACAAAATGTTACATTCTGTTCTTCAAGCAC CTATGTCAACCCTCAACACGTTGAAAGCAGGTGGGATTCTTAATAGATTCTCCAAAGATATAGCAATTTT GGATGACCTTCTGCCTCTTACCATATTTGACTTCATCCAGTTGTTATTAATTGTGATTGGAGCTATAGCA GTTGTCGCAGTTTTACAACCCTACATCTTTGTTGCAACAGTGCCAGTGATAGTGGCTTTTATTATGTTGA GAGCATATTTCCTCCAAACCTCACAGCAACTCAAACAACTGGAATCTGAAGGCAGGAGTCCAATTTTCAC TCATCTTGTTACAAGCTTAAAAGGACTATGGACACTTCGTGCCTTCGGACGGCAGCCTTACTTTGAAACT CTGTTCCACAAAGCTCTGAATTTACATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCC AAATGAGAATAGAAATGATTTTTGTCATCTTCTTCATTGCTGTTACCTTCATTTCCATTTTAACAACAGG AGAAGGAGAAGGAAGAGTTGGTATTATCCTGACTTTAGCCATGAATATCATGAGTACATTGCAGTGGGCT GTAAACTCCAGCATAGATGTGGATAGCTTGATGCGATCTGTGAGCCGAGTCTTTAAGTTCATTGACATGC CAACAGAAGGTAAACCTACCAAGTCAACCAAACCATACAAGAATGGCCAACTCTCGAAAGTTATGATTAT TGAGAATTCACACGTGAAGAAAGATGACATCTGGCCCTCAGGGGGCCAAATGACTGTCAAAGATCTCACA GCAAAATACACAGAAGGTGGAAATGCCATATTAGAGAACATTTCCTTCTCAATAAGTCCTGGCCAGAGGG TGGGCCTCTTGGGAAGAACTGGATCAGGGAAGAGTACTTTGTTATCAGCTTTTTTGAGACTACTGAACAC TGAAGGAGAAATCCAGATCGATGGTGTGTCTTGGGATTCAATAACTTTGCAACAGTGGAGGAAAGCCTTT GGAGTGATACCACAGAAAGTATTTATTTTTTCTGGAACATTTAGGAAAAACTTGGATCCCTATGAACAGT GGAGTGATCAAGAAATATGGAAAGTTGCAGATGAGGTTGGGCTCAGATCTGTGATAGAACAGTTTCCTGG GAAGCTTGACTTTGTCCTTGTGGATGGGGGCTGTGTCCTAAGCCATGGCCACAAGCAGTTGATGTGCTTG GCTAGATCTGTTCTCAGTAAGGCGAAGATCTTGCTGCTTGATGAACCCAGTGCTCATTTGGATCCAGTAA CATACCAAATAATTAGAAGAACTCTAAAACAAGCATTTGCTGATTGCACAGTAATTCTCTGTGAACACAG GATAGAAGCAATGCTGGAATGCCAACAATTTTTGGTCATAGAAGAGAACAAAGTGCGGCAGTACGATTCC ATCCAGAAACTGCTGAACGAGAGGAGCCTCTTCCGGCAAGCCATCAGCCCCTCCGACAGGGTGAAGCTCT TTCCCCACCGGAACTCAAGCAAGTGCAAGTCTAAGCCCCAGATTGCTGCTCTGAAAGAGGAGACAGAAGA AGAGGTGCAAGATACAAGGCTTTAGAGAGCAGCATAAATGTTGACATGGGACATTTGCTCATGGAATTGG AGCTCGTGGGACAGTCACCTCATGGAATTGGAGCTCGTGGAACAGTTACCTCTGCCTCAGAAAACAAGGA TGAATTAAGTTTTTTTTTAAAAAAGAAACATTTGGTAAGGGGAATTGAGGACACTGATATGGGTCTTGAT AAATGGCTTCCTGGCAATAGTCAAATTGTGTGAAAGGTACTTCAAATCCTTGAAGATTTACCACTTGTGT TTTGCAAGCCAGATTTTCCTGAAAACCCTTGCCATGTGCTAGTAATTGGAAAGGCAGCTCTAAATGTCAA TCAGCCTAGTTGATCAGCTTATTGTCTAGTGAAACTCGTTAATTTGTAGTGTTGGAGAAGAACTGAAATC ATACTTCTTAGGGTTATGATTAAGTAATGATAACTGGAAACTTCAGCGGTTTATATAAGCTTGTATTCCT TTTTCTCTCCTCTCCCCATGATGTTTAGAAACACAACTATATTGTTTGCTAAGCATTCCAACTATCTCAT TTCCAAGCAAGTATTAGAATACCACAGGAACCACAAGACTGCACATCAAAATATGCCCCATTCAACATCT AGTGAGCAGTCAGGAAAGAGAACTTCCAGATCCTGGAAATCAGGGTTAGTATTGTCCAGGTCTACCAAAA ATCTCAATATTTCAGATAATCACAATACATCCCTTACCTGGGAAAGGGCTGTTATAATCTTTCACAGGGG ACAGGATGGTTCCCTTGATGAAGAAGTTGATATGCCTTTTCCCAACTCCAGAAAGTGACAAGCTCACAGA CCTTTGAACTAGAGTTTAGCTGGAAAAGTATGTTAGTGCAAATTGTCACAGGACAGCCCTTCTTTCCACA GAAGCTCCAGGTAGAGGGTGTGTAAGTAGATAGGCCATGGGCACTGTGGGTAGACACACATGAAGTCCAA GCATTTAGATGTATAGGTTGATGGTGGTATGTTTTCAGGCTAGATGTATGTACTTCATGCTGTCTACACT AAGAGAGAATGAGAGACACACTGAAGAAGCACCAATCATGAATTAGTTTTATATGCTTCTGTTTTATAAT TTTGTGAAGCAAAATTTTTTCTCTAGGAAATATTTATTTTAATAATGTTTCAAACATATATAACAATGCT GTATTTTAAAAGAATGATTATGAATTACATTTGTATAAAATAATTTTTATATTTGAAATATTGACTTTTT ATGGCACTAGTATTTCTATGAAATATTATGTTAAAACTGGGACAGGGGAGAACCTAGGGTGATATTAACC AGGGGCCATGAATCACCTTTTGGTCTGGAGGGAAGCCTTGGGGCTGATGCAGTTGTTGCCCACAGCTGTA TGATTCCCAGCCAGCACAGCCTCTTAGATGCAGTTCTGAAGAAGATGGTACCACCAGTCTGACTGTTTCC ATCAAGGGTACACTGCCTTCTCAACTCCAAACTGACTCTTAAGAAGACTGCATTATATTTATTACTGTAA GAAAATATCACTTGTCAATAAAATCCATACATTTGTGTGAAA The CFTR cDNA is 6,132 nt long. The sequence of one of Sue’s alleles is shown.  Is Sue a carrier?

21. How to read DNA: the Genetic Code

22.  Shown as mRNA  5′ → 3′  64 codons  Redundant  One “start” codon: AUG  Three “stop” codons: UAG, UAA, UGA (don’t encode amino acids) the Genetic Code

23. AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGC ACCCAGAGTAGTAGG TCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGCCCGA GAGACCATGCAGAG GTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGACCAGACC AATTTTGAGGAAA

24. AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGC ACCCAGAGTAGTAGG AspTrpLys… IleGlySer… LeuGluAla… TCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGCCCGA GAGACCATGCAGAG GTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGACCAGACC AATTTTGAGGAAA

25. template strand  For each gene, one DNA strand is transcribed  Template strand “read”  mRNA complementary to template the Genetic Code CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC 3’ 5’ DNA GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG 5’ 3’ CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’ 5’ mRNA

26.  Non-template strand looks like RNA (except T’s) non-template strand the Genetic Code CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC 3’ 5’ DNA GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG 5’ 3’ CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’ 5’ mRNA

27. start codon: AUG stop codon: UAG, UGA or UAA  Ribosome starts at 5’ end of mRNA (in eukaryotes)  Protein synthesis begins at start codon, ends at stop codon the Genetic Code CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’ 5’ mRNA codons this stop codon is not “in-frame” so it’s ignored! MetProArgSerArg protein

28. What about

29.  Change in nucleotide sequence of DNA  Caused by:  DNA polymerase mistake  Chemical change within cell  UV light or other radiation  Mutagenic chemical Mutation

30. Effects of mutations ACGATCCTATGCCCCGCAGCCGATAGTCTCATT TGCTAGGATACGGGGCGTCGGCTATCAGAGTAA DNA 5’3’ 5’3’ ACGAUCCUAUGCCCCGCAGCCGAUAGUCUCAUU RNA 5’3’ MetProArgSerArg

31. Effects of mutations ACGATCCTATGCCCCGCCGCCGATAGTCTCATT TGCTAGGATACGGGGCGGCGGCTATCAGAGTAA DNA 5’3’ 5’3’ ACGAUCCUAUGCCCCGCAGCCGAUAGUCUCAUU RNA 5’3’ MetProArgSerArg

32. Effects of mutations ACGATCCTATGCCCCGCCGCCGATAGTCTCATT TGCTAGGATACGGGGCGGCGGCTATCAGAGTAA DNA 5’3’ 5’3’ ACGAUCCUAUGCCCCGCCGCCGAUAGUCUCAUU RNA 5’3’ MetProArgSerArg

33. Missense: single amino-acid change Effects of mutations ACGATCCTATGCCCCGCCGCCGATAGTCTCATT TGCTAGGATACGGGGCGGCGGCTATCAGAGTAA DNA 5’3’ 5’3’ ACGAUCCUAUGCCCCGCCGCCGAUAGUCUCAUU RNA 5’3’ MetProArgArgArg Could this change have an effect on protein function? Could it affect the whole organism?

34. Missense: single amino-acid change Effects of mutations ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAAACTGTTTGAAACTGTGGAGGAACTGTCCT CGCCGCTCACAGCTCATGTAACAGGCAGGATCCCCCTCTGGCTCACCGGCAGTCTCCTTCGATGTGGGCC AGGACTCTTTGAAGTTGGATCTGAGCCATTTTACCACCTGTTTGATGGGCAAGCCCTCCTGCACAAGTT… Wild-type RPE65: ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAAACTGTTTGAAACTGTGGAGGAACTGTCCT CGCCGCTCACAGCTCATGTAACAGGCAGGATCCCCCTCTGGCTCACCAGCAGTCTCCTTCGATGTGGGCC AGGACTCTTTGAAGTTGGATCTGAGCCATTTTACCACCTGTTTGATGGGCAAGCCCTCCTGCACAAGTT… Mutant RPE65: MetSerIleGlnValGluHisProAlaGlyGlyTyrLysLysLeuPheGluThrValGluGluLeuSerSerPro LeuThrAlaHisValThrGlyArgIleProLeuTrpLeuThrGlySerLeuLeuArgCysGlyProGlyLeuPhe GluValGlySerGluProPheTyrHisLeuPheAspGlyGlnAlaLeuLeuHisLysPheAspPheLysGlu… MetSerIleGlnValGluHisProAlaGlyGlyTyrLysLysLeuPheGluThrValGluGluLeuSerSerPro LeuThrAlaHisValThrGlyArgIleProLeuTrpLeuThrSerSerLeuLeuArgCysGlyProGlyLeuPhe GluValGlySerGluProPheTyrHisLeuPheAspGlyGlnAlaLeuLeuHisLysPheAspPheLysGlu…

35. Missense: single amino-acid change Effects of mutations ATGGTGCATCTGACTCCTGAGGAG… MetValHisLeuThrProGluGlu GTG Val Hemoglobin → sickle-cell anemia :

36. Effects of mutations AUGCCCCGCAGCCGAUAG MetProArgSerArg RNA protein

37. Nonsense: creates stop codon Effects of mutations AUGCCCCGCAGCCGAUAG MetProArgSerArg AUGCCCCGCAGCUGAUAG MetProArgSerXXX

38. Deletion: creates frameshift, changes all following aa’s Effects of mutations AUGCCCCGCAGCCGAUAG MetProArgSerArg AUGCCCGCAGCUGAUAG MetProAlaAlaAsp…

39. Insertion: creates frameshift, changes all following aa’s Effects of mutations AUGCCCCGCAGCCGAUAG MetProArgSerArg AUGGCCCCGCAGCUGAUAG MetAlaProGlnProIle…

40. Silent: DNA changes, but protein doesn’t Effects of mutations AUGCCCCGCAGCCGAUAG MetProArgSerArg AUGCCCCGCAGUCGAUAG MetProArgSerArg

41.  Responsible for an inherited character  Physical unit passed from parent to child in the gamete  Can occur in various “forms” (alleles)  A segment of a DNA molecule = segment of chromosome  Encodes a protein (polypeptide) What is a gene?

42.  “Inborn errors of metabolism”  Alkaptonuria  Urine turns black (“blackwater”)  Homogentisic acid (“alkaptan”) Garrod: genes specify enzymes Archibald Garrod (1902)

43. A  Molecules are made by metabolic pathways  Enzymes catalyze each step  Missing enzyme → accumulation of preceding molecule Garrod: genes specify enzymes Enzyme 1 B Enzyme 2Enzyme 3Enzyme 4Enzyme 5 CD EFEEEEE

44.  Hypothesis: alkaptonuria results from missing enzyme  Pathway blocked, homogentisic acid accumulates Garrod: genes specify enzymes excreted in urine

45.  “One ought to be able to discover what genes do by making them defective” Beadle and Tatum: One gene, one enzyme George Beadle and Edward Tatum (1941) Neurospora crassa (bread mold)

46.  Neurospora grows on minimal medium  Makes all needed amino acids, nuclotides, etc. from glucose Beadle and Tatum: One gene, one enzyme minimal medium (glucose & salts)

47.  X-rays produce mutations (changes in DNA)  Some mutants are auxotrophs  can’t make all needed molecules  can’t grow on minimal medium Beadle and Tatum: One gene, one enzyme minimal medium wild- type auxotrophic mutant

48.  What’s wrong with the mutant? Beadle and Tatum: One gene, one enzyme auxotrophic mutant grown on minimal medium minimal medium plus: metleualaargvalphe

49. Beadle and Tatum: One gene, one enzyme ornithinecitrullinearginine minimal medium minimal medium + citrulline minimal medium +ornithine minimal medium + arginine mutant #1  Mutation “blocks” metabolic pathway at one step

50. Beadle and Tatum: One gene, one enzyme ornithinecitrullinearginine minimal medium minimal medium + citrulline minimal medium +ornithine minimal medium + arginine mutant #2

51.  What is it that’s “blocked” in the mutants?  Lack an enzyme in the pathway  Enzyme is missing because gene is defective Beadle and Tatum: One gene, one enzyme ornithinecitrullinearginine enzyme #1 enzyme #2 gene 1gene 2

52. Hypothesis:  One gene is responsible for making one enzyme  One enzyme carries out one metabolic step Beadle and Tatum: One gene, one enzyme ornithinecitrullinearginine enzyme #1 enzyme #2 gene 1gene 2

53.  Change in nucleotide sequence of DNA  Caused by:  DNA polymerase mistake  Chemical change within cell  UV light or other radiation  Mutagenic chemical Mutation

54.  Changes in DNA are inherited  Neutral, harmful or beneficial  “Raw material” of evolution Mutations create new alleles! round seed allele wrinkled seed allele brown hair pigment allele blonde (no pigment) allele normal CFTR allele non-functional CFTR allele (CF)

55. Why does SMOKING cause cancer?

56.  Mutation removes normal growth control  Further mutations allow cancer cells to spread and invade Cancer starts with uncontrolled cell division Bone marrow from a leukemia patient

57.  Cigarette smoke contains mutagens  Increased frequency of all mutations  Some can affect cell division Smoking increases the mutation rate

58.  Nearly always mutagens  Formaldehyde  Hydrazine  Vinyl chloride  Urethane  2-Nitropropane  Quinoline  Benzo[a]pyrene  Dibenz[a,h]anthracene  Benzo[b]fluoranthene  Indeno[1,2,3-cd]pyrene  Chrysene  Methylfluoranthene  Nitrosodimethylamine Carcinogens = cancer-causing chemicals  Nitrosoethymethylamine  Nitrosodi-n-butylamine  Nitrosopiperidine  N'-Nitrosoanabasine  Dibenzo[c,g]carbazole  Polonium 210  Methylchrysene  Methylnaphthalenes  Benzo[e]pyrene  Nickel  Arsenic  Cadmium

59.  How to identify carcinogens?  Animal testing  Reliable but slow and expensive the Ames Test

60.  Ames test: does the agent cause mutations in bacteria?  Fast, cheap  Mutagens tested further  Non-mutagens probably safe the Ames Test Bruce Ames

61.  Wild-type Salmonella can make histidine (his+)  Grows on minimal medium (no histidine)  his- mutant (auxotroph) can’t make histidine  Can’t grow on minimal medium the Ames Test Salmonella typhimurium

62.  Mutation can make a his- cell his+ (reversion)  Grows on minimal medium  Low spontaneous mutation rate the Ames Test 10 10 his – bacteria most die; few spontaneous his + mutants no histidine in plate

63.  Mutagen increases frequency of his+ mutation  Test a possible mutagen and see if mutation rate increases the Ames Test 10 10 his – bacteria increased chance of mutation to his + mutagen no histidine in plate