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RNA

DNA: double-stranded deoxyribose A, C, G, T. RNA: single-stranded ribose A, C, G, U. RNA. 5 ′. 5 ′. 3 ′. ?. ?. 3 ′. 3 ′. 5 ′. 5 ′. Complication #5: Can’t add to 5′ end. how do we replicate this?. Complication #5: Can’t add to 5′ end. 5´. 5´. 3´. 5´. 5´. new DNA. 3´.

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RNA

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

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

  3. how do we replicate this? Complication #5: Can’t add to 5′ end 5´ 5´ 3´ 5´ 5´ new DNA 3´ RNA primer

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

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

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

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

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

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

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

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

  12. What does DNA do?

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

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

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

  16. CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA gene transcription CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA 3’ 5’

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

  18. CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA gene transcription CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA 3’ 5’ translation protein N-MetProArgSerArg-C

  19. The CFTR cDNA is 6,132 nt long. The sequence of one of Sue’s alleles is shown. • Is Sue a carrier? AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGCCCGAGAGACCATGCAGAG 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 GATTCTGATGAGCCTTTAGAGAGAAGGCTGTCCTTAGTACCAGATTCTGAGCAGGGAGAGGCGATACTGCCTCGCATCAGCGTGATCAGCACTGGCCCCACGCTTCAGGCACGAAGGAGGCAGTCTGTCCTGAACCTGATGACACACTCAGTTAACCAAGGTCAGAACATTCACCGAAAGACAACAGCATCCACACGAAAAGTGTCACTG 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

  20. How to read DNA: the Genetic Code C A AAA = lysine (Lys) AAC = asparagine (Asn) AAG = lysine (Lys) AAU = asparagine (Asn) ACA = threonine (Thr) ACC = threonine (Thr) ACG = threonine (Thr) ACU = threonine (Thr)

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

  22. AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGAATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGG TCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGCCCGAGAGACCATGCAGAG GTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGACCAGACCAATTTTGAGGAAA

  23. AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGAATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGG AspTrpLys… IleGlySer… LeuGluAla… TCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGCCCGAGAGACCATGCAGAG GTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGACCAGACCAATTTTGAGGAAA

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

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

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

  27. What about mutations?

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

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

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

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

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

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

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

  35. AUGCCCCGCAGCCGAUAG MetProArgSerArg Effects of mutations RNA protein

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

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

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

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

  40. What is a gene? • 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)

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

  42. E E E E E E F A Enzyme 5 Enzyme 4 Enzyme 3 Enzyme 2 Enzyme 1 Garrod: genes specify enzymes • Molecules are made by metabolic pathways • Enzymes catalyze each step • Missing enzyme → accumulation of preceding molecule B C D

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

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

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

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

  47. Beadle and Tatum: One gene, one enzyme • What’s wrong with the mutant? auxotrophic mutant grown on minimal medium plus: minimal medium met leu ala arg val phe

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

  49. Beadle and Tatum: One gene, one enzyme • Mutation “blocks” metabolic pathway at one step ornithine citrulline arginine mutant #2 minimal medium +ornithine minimal medium + citrulline minimal medium + arginine minimal medium

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