Uploaded byMichael Edgar
Honors ~ DNA 1213
This document discusses DNA replication and gene regulation. It begins with an overview of DNA replication and the enzymes involved, including helicase, primase, DNA polymerase, and ligase. It then discusses gene regulation, showing diagrams of operons like the lac and trp operons. It demonstrates how transcription factors, repressors, inducers, and corepressors control gene expression. Finally, it provides an example of epigenetic gene regulation involving chromatin remodeling and DNA methylation.
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Honors ~ DNA 1213
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- 30. Fig. 16-16b6 Template strand 5′ 5′3′ 3′ RNA primer3′ 5′ 5′ 3′ 1 1 3′ 3′ 5′ 5′ Okazaki fragment 12 3′ 3′ 5′ 5′ 12 3′ 3′ 5′ 5′ 1 2 5′ 5′ 3′ 3′ Overall direction of replication
- 32. Fig. 16-16a Overview Origin ofreplication Leading strand Leading strand Lagging strand Lagging strand Overall directions of replication 1 2
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- 39. Fig. 20-3-1 Restriction site DNA Stickyend Restriction enzyme cuts sugar-phosphate backbones. 5′ 3′ 3′ 5′ 1
- 40. Fig. 20-3-2 Restriction site DNA Stickyend 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
- 41. Fig. 20-3-3 Restriction site DNA Stickyend 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
- 42. Fig. 20-9a Mixture of DNAmol- ecules of different sizes Power source Longer molecules Shorter molecules Gel AnodeCathode TECHNIQUE 1 2 Power source – + +–
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- 45. Fig. 20-10 Normal allele Sickle-cell allele Large fragment (b) Electrophoresisof restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) DdeI restriction sites in normal and sickle-cell alleles of β-globin gene Normal β-globin allele Sickle-cell mutant β-globin allele DdeI Large fragment Large fragment 376 bp 201 bp175 bp DdeIDdeI DdeI DdeI DdeI DdeI
- 47. Restriction Enzyme Lab •HINTS: • pMAP is 5615bp • There are – 2 PstI sites. – 1 HpaI site. – 1 SspI site • Lambda DNA/PstI: • You should not be able to see beyond the 805bp band. • Fine the 11,490bp and the 805bp as reference.
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- 71. To studying theTostudying the Wolbachia within?Wolbachia within? Credit: Mark Taylor
- 72. 16S rRNA (ribosomalRNA)16S rRNA (ribosomal RNA) Small ribosomal subunit involved in mRNA translation processSmall ribosomal subunit involved in mRNA translation process Ancient molecule, conserved function, universally distributedAncient molecule, conserved function, universally distributed Helps identify unknown bacterium to genus or species levelsHelps identify unknown bacterium to genus or species levels Present in bacteria; eukaryote has very divergent copy that is named 18S rRNA; present in all cellsPresent in bacteria; eukaryote has very divergent copy that is named 18S rRNA; present in all cells Plays a catalytic and structural role in the ribosomePlays a catalytic and structural role in the ribosome
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- 77. Bacteria 16S rRNAand EukaryoticBacteria 16S rRNA and Eukaryotic 18S rRNA – Similar!18S rRNA – Similar!
- 78. 16S rRNA conservation(red)16S rRNA conservation (red)
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- 81. Fig. 18-6 DNA Signal Gene NUCLEUS Chromatin modification Chromatin Gene available fortranscription 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
- 82. Gene Regulation Example1 Activators, Enhancers and Transcription Factors
- 83. Fig. 18-8-1 Enhancer (distal controlelements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA ExonExon ExonIntron Intron
- 84. Fig. 18-8-2 Enhancer (distal controlelements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Exon ExonIntronIntron Cleaved 3′ end of primary transcript Primary RNA transcript Poly-A signal Transcription 5′ ExonExon ExonIntron Intron
- 85. Fig. 18-8-3 Enhancer (distal controlelements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA ExonExon ExonIntron Intron Exon Exon ExonIntronIntron 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′
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- 87. Fig. 18-9-2 Enhancer TATA box PromoterActivators DNA Gene Distalcontrol element Group of mediator proteins DNA-bending protein General transcription factors
- 88. Fig. 18-9-3 Enhancer TATA box PromoterActivators DNA Gene Distalcontrol element Group of mediator proteins DNA-bending protein General transcription factors RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis
- 89. 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
- 90. Gene Regulation Example2 The Operon
- 91. Fig. 18-2 Regulation of gene expression trpEgene 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
- 92. Fig. 18-3a Polypeptide subunitsthat 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 codonStart codon mRNA trpA 5′ 3′ trpR trpE trpD trpC trpB ABCDE
- 93. Fig. 18-3b-1 (b) Tryptophanpresent, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA
- 94. Fig. 18-3b-2 (b) Tryptophanpresent, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA
- 95. Fig. 18-4a (a) Lactoseabsent, repressor active, operon off DNA Protein Active repressor RNA polymerase Regulatory gene Promoter Operato r mRNA 5′ 3′ No RNA made lacI lacZ
- 96. Fig. 18-4b (b) Lactosepresent, repressor inactive, operon on mRNA Protein DNA mRNA 5′ Inactive repressor Allolactose (inducer) 5′ 3′ RNA polymerase Permease Transacetylase lac operon β- Galactosidase lacYlacZ lacAlacI
- 97. Fig. 18-5 (b) Lactosepresent, glucose present (cAMP level low): little lac mRNA synthesized cAMP DNA Inactive lac repressor Allolactose Inactive CAP lacI CAP-binding site Promoter Active CAP Operator lacZ RNA polymerase binds and transcribes Inactive lac repressor lacZ OperatorPromoter DNA CAP-binding site lacI RNA polymerase less likely to bind Inactive CAP (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized
- 98. Gene Regulation Example3 Epigenetics
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- 104. Gene Regulation Example4 RNAi
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- 113. Fig. 20-4-1 Bacterial cell Bacterial plasmid lacZgene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends ampR gene TECHNIQUE
- 114. Fig. 20-4-2 Bacterial cell Bacterial plasmid lacZgene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends ampR gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid
- 115. Fig. 20-4-3 Bacterial cell Bacterial plasmid lacZgene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends ampR gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids
- 116. Fig. 20-4-4 Bacterial cell Bacterial plasmid lacZgene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends ampR 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
- 118. mtDNA Theories, Molecular Basis andReal-World Application
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- 130. DNA Laboratory at MiltonAcademy • Isolate DNA from cheek cells. • Polymerase Chair Reaction • Electrophoresis • Sequence DNA
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- 140. Fig. 20-8d Cycle 3 yields8 molecules; 2 molecules (in white boxes) match target sequence
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- 146. 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
- 147. Fig. 20-12a DNA (template strand) TECHNIQUE DNA polymerase PrimerDeoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP
- 148. Fig. 20-12b TECHNIQUE RESULTS DNA (template strand) Shortest Labeledstrands 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
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Editor's Notes
- #72 DNA-based Technologies Lecture - Dr. Seth Bordenstein 4 different prophage regions 3.2% of the total genome Low GC content similar to host chromosome dsDNA virus Icosohedral head 20.5Kb genome
- #73 DNA-based Technologies Lecture - Dr. Seth Bordenstein
- #78 DNA-based Technologies Lecture - Dr. Seth Bordenstein
- #79 DNA-based Technologies Lecture - Dr. Seth Bordenstein