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DNA Replication
 
 
 
 
 
 
Exonuclease
 
 
Fig. 16-UN5
Fig. 16-13 Topoisomerase Helicase Primase Single-strand binding proteins RNA primer 5  5  5  3  3  3 
 
Fig. 16-16b6 Template strand 5  5  3  3  RNA primer 3  5  5  3  1 1 3  3  5  5  Okazaki fragment 1 2 3  3  5...
 
Fig. 16-16a Overview Origin of replication Leading strand Leading strand Lagging strand Lagging strand Overall directions ...
Helicase
Topoisomerase and Helicase
 
 
 
 
Fig. 20-3-1 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5  3  3  5  1
Fig. 20-3-2 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5  3  3  5  1 DNA fragm...
Fig. 20-3-3 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5  3  3  5  1 One possi...
Fig. 20-9a Mixture of DNA mol- ecules of different sizes Power source Longer molecules Shorter molecules Gel Anode Cathode...
Fig. 20-9b RESULTS
 
Fig. 20-10 Normal allele Sickle-cell allele Large fragment (b)  Electrophoresis of restriction fragments   from normal and...
 
Restriction Enzyme Lab <ul><li>HINTS: </li></ul><ul><li>pMAP is 5615bp </li></ul><ul><li>There are </li></ul><ul><ul><li>2...
Transcription and Translation
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Gene Regulation
Fig. 18-6 DNA Signal Gene NUCLEUS Chromatin modification Chromatin Gene available for transcription Exon Intron Tail RNA C...
Fig. 18-8-1 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstr...
Fig. 18-8-2 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstr...
Fig. 18-8-3 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstr...
Fig. 18-9-1 Enhancer TATA box Promoter Activators DNA Gene Distal control element
Fig. 18-9-2 Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending p...
Fig. 18-9-3 Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending p...
Fig. 18-10 Control elements Enhancer Available activators Albumin gene (b) Lens cell Crystallin gene expressed Available a...
Fig. 18-2 Regulation of gene expression trpE  gene trpD  gene trpC  gene trpB  gene trpA  gene (b) Regulation of enzyme pr...
Fig. 18-3a Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, o...
Fig. 18-3b-1 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mR...
Fig. 18-3b-2 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mR...
Fig. 18-4a (a) Lactose absent, repressor active, operon off DNA Protein Active repressor RNA polymerase Regulatory gene Pr...
Fig. 18-4b (b) Lactose present, repressor inactive, operon on mRNA Protein DNA mRNA 5  Inactive repressor Allolactose (in...
Fig. 18-5 (b) Lactose present, glucose present (cAMP level low): little  lac  mRNA synthesized cAMP DNA Inactive  lac repr...
Genetic Engineering & Cloning
mtDNA  Theories, Molecular Basis and Real-World Application
 
 
“ The Other Genome” mtDNA
Endosymbiotic Theory
 
 
 
 
 
 
 
DNA Laboratory at Milton Academy <ul><li>Isolate DNA from cheek cells. </li></ul><ul><li>Polymerase Chair Reaction </li></...
mtDNA Control Region
 
Polymerase Chain Reaction
PCR http:// www.dnalc.org/resources/spotlight/index.html
 
Taq  DNA Polymerase
Fig. 20-8a 5  Genomic DNA TECHNIQUE Target sequence 3  3  5 
Fig. 20-8b Cycle 1 yields 2 molecules Denaturation Annealing Extension Primers New nucleo- tides 3  5  3 2 5  3  1
Fig. 20-8c Cycle 2 yields 4 molecules
Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence
http:// www.youtube.com/watch?v =CQEaX3MiDow   http:// www.youtube.com/watch?v =x5yPkxCLads&feature=related
Gel Electrophoresis
DNA Sequencing
Kate Bator Connor Johnson
Fig. 20-12 DNA (template strand) TECHNIQUE RESULTS DNA (template  strand) DNA  polymerase Primer Deoxyribonucleotides Shor...
Fig. 20-12a DNA (template strand) TECHNIQUE DNA  polymerase Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescen...
Fig. 20-12b TECHNIQUE RESULTS DNA (template  strand) Shortest Labeled strands Longest Shortest labeled strand Longest labe...
mtDNA Sequence http://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html
RNAi
 
RNA Induced Silencing Complex
 
 
Vascular Endothelial Growth Factor
Cloning
Fig. 20-4-1 Bacterial cell Bacterial  plasmid lacZ  gene Hummingbird  cell Gene of interest Hummingbird  DNA fragments Res...
Fig. 20-4-2 Bacterial cell Bacterial  plasmid lacZ  gene Hummingbird  cell Gene of interest Hummingbird  DNA fragments Res...
Fig. 20-4-3 Bacterial cell Bacterial  plasmid lacZ  gene Hummingbird  cell Gene of interest Hummingbird  DNA fragments Res...
Fig. 20-4-4 Bacterial cell Bacterial  plasmid lacZ  gene Hummingbird  cell Gene of interest Hummingbird  DNA fragments Res...
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Honors ~ DNA 1011

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Honors ~ DNA 1011

  1. 16. DNA Replication
  2. 23. Exonuclease
  3. 26. Fig. 16-UN5
  4. 27. Fig. 16-13 Topoisomerase Helicase Primase Single-strand binding proteins RNA primer 5  5  5  3  3  3 
  5. 29. Fig. 16-16b6 Template strand 5  5  3  3  RNA primer 3  5  5  3  1 1 3  3  5  5  Okazaki fragment 1 2 3  3  5  5  1 2 3  3  5  5  1 2 5  5  3  3  Overall direction of replication
  6. 31. Fig. 16-16a Overview Origin of replication Leading strand Leading strand Lagging strand Lagging strand Overall directions of replication 1 2
  7. 32. Helicase
  8. 33. Topoisomerase and Helicase
  9. 38. Fig. 20-3-1 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5  3  3  5  1
  10. 39. Fig. 20-3-2 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5  3  3  5  1 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2 One possible combination
  11. 40. Fig. 20-3-3 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5  3  3  5  1 One possible combination Recombinant DNA molecule DNA ligase seals strands. 3 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2
  12. 41. Fig. 20-9a Mixture of DNA mol- ecules of different sizes Power source Longer molecules Shorter molecules Gel Anode Cathode TECHNIQUE 1 2 Power source – + + –
  13. 42. Fig. 20-9b RESULTS
  14. 44. Fig. 20-10 Normal allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of  -globin gene Normal  -globin allele Sickle-cell mutant  -globin allele Dde I Large fragment Large fragment 376 bp 201 bp 175 bp Dde I Dde I Dde I Dde I Dde I Dde I
  15. 46. Restriction Enzyme Lab <ul><li>HINTS: </li></ul><ul><li>pMAP is 5615bp </li></ul><ul><li>There are </li></ul><ul><ul><li>2 Pst I sites. </li></ul></ul><ul><ul><li>1 Hpa I site. </li></ul></ul><ul><ul><li>1 Ssp I site </li></ul></ul><ul><li>Lambda DNA/PstI: </li></ul><ul><li>You should not be able to see beyond the 805bp band. </li></ul><ul><li>Fine the 11,490bp and the 805bp as reference. </li></ul>
  16. 47. Transcription and Translation
  17. 70. Gene Regulation
  18. 71. Fig. 18-6 DNA Signal Gene NUCLEUS Chromatin modification Chromatin Gene available for transcription Exon Intron Tail RNA Cap RNA processing Primary transcript mRNA in nucleus Transport to cytoplasm mRNA in cytoplasm Translation CYTOPLASM Degradation of mRNA Protein processing Polypeptide Active protein Cellular function Transport to cellular destination Degradation of protein Transcription
  19. 72. Fig. 18-8-1 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Exon Exon Intron Intron
  20. 73. Fig. 18-8-2 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Exon Exon Intron Intron Cleaved 3  end of primary transcript Primary RNA transcript Poly-A signal Transcription 5  Exon Exon Exon Intron Intron
  21. 74. Fig. 18-8-3 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Exon Exon Intron Intron Exon Exon Exon Intron Intron Cleaved 3  end of primary transcript Primary RNA transcript Poly-A signal Transcription 5  RNA processing Intron RNA Coding segment mRNA 5  Cap 5  UTR Start codon Stop codon 3  UTR Poly-A tail 3 
  22. 75. Fig. 18-9-1 Enhancer TATA box Promoter Activators DNA Gene Distal control element
  23. 76. Fig. 18-9-2 Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors
  24. 77. Fig. 18-9-3 Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis
  25. 78. Fig. 18-10 Control elements Enhancer Available activators Albumin gene (b) Lens cell Crystallin gene expressed Available activators LENS CELL NUCLEUS LIVER CELL NUCLEUS Crystallin gene Promoter (a) Liver cell Crystallin gene not expressed Albumin gene expressed Albumin gene not expressed
  26. 79. Fig. 18-2 Regulation of gene expression trpE gene trpD gene trpC gene trpB gene trpA gene (b) Regulation of enzyme production (a) Regulation of enzyme activity Enzyme 1 Enzyme 2 Enzyme 3 Tryptophan Precursor Feedback inhibition
  27. 80. Fig. 18-3a Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on DNA mRNA 5  Protein Inactive repressor RNA polymerase Regulatory gene Promoter Promoter trp operon Genes of operon Operator Stop codon Start codon mRNA trpA 5  3  trpR trpE trpD trpC trpB A B C D E
  28. 81. Fig. 18-3b-1 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA
  29. 82. Fig. 18-3b-2 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA
  30. 83. Fig. 18-4a (a) Lactose absent, repressor active, operon off DNA Protein Active repressor RNA polymerase Regulatory gene Promoter Operator mRNA 5  3  No RNA made lac I lacZ
  31. 84. Fig. 18-4b (b) Lactose present, repressor inactive, operon on mRNA Protein DNA mRNA 5  Inactive repressor Allolactose (inducer) 5  3  RNA polymerase Permease Transacetylase lac operon  -Galactosidase lacY lacZ lacA lac I
  32. 85. Fig. 18-5 (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized cAMP DNA Inactive lac repressor Allolactose Inactive CAP lac I CAP-binding site Promoter Active CAP Operator lacZ RNA polymerase binds and transcribes Inactive lac repressor lacZ Operator Promoter DNA CAP-binding site lac I RNA polymerase less likely to bind Inactive CAP (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized
  33. 86. Genetic Engineering & Cloning
  34. 87. mtDNA Theories, Molecular Basis and Real-World Application
  35. 90. “ The Other Genome” mtDNA
  36. 91. Endosymbiotic Theory
  37. 99. DNA Laboratory at Milton Academy <ul><li>Isolate DNA from cheek cells. </li></ul><ul><li>Polymerase Chair Reaction </li></ul><ul><li>Electrophoresis </li></ul><ul><li>Sequence DNA </li></ul>
  38. 100. mtDNA Control Region
  39. 102. Polymerase Chain Reaction
  40. 103. PCR http:// www.dnalc.org/resources/spotlight/index.html
  41. 105. Taq DNA Polymerase
  42. 106. Fig. 20-8a 5  Genomic DNA TECHNIQUE Target sequence 3  3  5 
  43. 107. Fig. 20-8b Cycle 1 yields 2 molecules Denaturation Annealing Extension Primers New nucleo- tides 3  5  3 2 5  3  1
  44. 108. Fig. 20-8c Cycle 2 yields 4 molecules
  45. 109. Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence
  46. 110. http:// www.youtube.com/watch?v =CQEaX3MiDow http:// www.youtube.com/watch?v =x5yPkxCLads&feature=related
  47. 111. Gel Electrophoresis
  48. 112. DNA Sequencing
  49. 113. Kate Bator Connor Johnson
  50. 114. Fig. 20-12 DNA (template strand) TECHNIQUE RESULTS DNA (template strand) DNA polymerase Primer Deoxyribonucleotides Shortest Dideoxyribonucleotides (fluorescently tagged) Labeled strands Longest Shortest labeled strand Longest labeled strand Laser Direction of movement of strands Detector Last base of longest labeled strand Last base of shortest labeled strand dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP
  51. 115. Fig. 20-12a DNA (template strand) TECHNIQUE DNA polymerase Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP
  52. 116. Fig. 20-12b TECHNIQUE RESULTS DNA (template strand) Shortest Labeled strands Longest Shortest labeled strand Longest labeled strand Laser Direction of movement of strands Detector Last base of longest labeled strand Last base of shortest labeled strand
  53. 117. mtDNA Sequence http://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html
  54. 118. RNAi
  55. 120. RNA Induced Silencing Complex
  56. 123. Vascular Endothelial Growth Factor
  57. 124. Cloning
  58. 125. Fig. 20-4-1 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE
  59. 126. Fig. 20-4-2 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid
  60. 127. Fig. 20-4-3 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids
  61. 128. Fig. 20-4-4 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene One of many bacterial clones Colony carrying recombinant plasmid with disrupted lacZ gene

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