honors ~ dna 1314
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Molecular Biology
Honors Biology
Edgar
Hershey and Chase 1952
Agarose
Separation of DNA fragments by Size
Looking at your gels
• What do you notice about the “banding patterns” in each lane in your gels?
• What is different about the “pools” of DNA that you loaded into each well?
2652
2652
Look at you gel again
• Estimate the size of the DNA fragment(s) in the pMAP lane.
• Does the relationship between the distance migrated and DNA fragment size appear to be a linear relationship?
DNA Replication
Fig. 16-UN5
Fig. 16-13
Topoisomerase
Helicase
PrimaseSingle-strand binding proteins
RNA primer
55
5 3
3
3
Fig. 16-16b6
Template strand
5
53
3
RNA primer 3 5
5
3
1
1
3
35
5
Okazaki fragment
12
3
3
5
5
12
3
3
5
5
12
5
5
3
3
Overall direction of replication
Fig. 16-16a
Overview
Origin of replication
Leading strand
Leading strand
Lagging strand
Lagging strand
Overall directions of replication
12
Helicase
Topoisomerase and Helicase
Fig. 20-3-1Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
53
35
1
Fig. 20-3-2Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
53
35
1
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
One possible combination
Fig. 20-3-3Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
53
35
1
One possible combination
Recombinant DNA molecule
DNA ligaseseals strands.
3
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
Fig. 20-9a
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
1
2
Powersource
– +
+–
Fig. 20-9b
RESULTS
Fig. 20-10
Normalallele
Sickle-cellallele
Largefragment
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
201 bp175 bp
376 bp
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
Normal -globin allele
Sickle-cell mutant -globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp175 bp
DdeIDdeI
DdeI DdeI DdeI DdeI
Transcription and Translation
Beadle and Tatum1941
Development of Model
• One Gene – One Enzyme (Nobel 1958)
• One Gene – One Polypeptide– Non enzyme proteins (keratin, insulin)– Hb – multimeric protein.
• Issues:– Alternate splicing– RNA coding genes.– Non-coding regions
Gene Regulation
Fig. 18-6
DNA
Signal
Gene
NUCLEUS
Chromatin modification
Chromatin
Gene availablefor transcription
Exon
Intron
Tail
RNA
Cap
RNA processing
Primary transcript
mRNA in nucleus
Transport to cytoplasm
mRNA in cytoplasm
Translation
CYTOPLASM
Degradationof mRNA
Protein processing
Polypeptide
Active protein
Cellular function
Transport to cellulardestination
Degradationof protein
Transcription
Gene Regulation Example 1
Activators, Enhancers and Transcription Factors
Fig. 18-8-1
Enhancer(distal control elements)
Proximalcontrol elements
Poly-A signalsequence
Terminationregion
DownstreamPromoter
UpstreamDNA
ExonExon ExonIntron Intron
Fig. 18-8-2
Enhancer(distal control elements)
Proximalcontrol elements
Poly-A signalsequence
Terminationregion
DownstreamPromoter
UpstreamDNA
Exon Exon ExonIntronIntron Cleaved 3 endof primarytranscript
Primary RNAtranscript
Poly-Asignal
Transcription
5
ExonExon ExonIntron Intron
Fig. 18-8-3
Enhancer(distal control elements)
Proximalcontrol elements
Poly-A signalsequence
Terminationregion
DownstreamPromoter
UpstreamDNA
ExonExon ExonIntron Intron
Exon Exon ExonIntronIntron Cleaved 3 endof primarytranscript
Primary RNAtranscript
Poly-Asignal
Transcription
5
RNA processing
Intron RNA
Coding segment
mRNA
5 Cap 5 UTRStart
codonStop
codon 3 UTR Poly-Atail
3
Fig. 18-9-1
Enhancer TATAbox
PromoterActivators
DNAGene
Distal controlelement
Fig. 18-9-2
Enhancer TATAbox
PromoterActivators
DNAGene
Distal controlelement
Group ofmediator proteins
DNA-bendingprotein
Generaltranscriptionfactors
Fig. 18-9-3
Enhancer TATAbox
PromoterActivators
DNAGene
Distal controlelement
Group ofmediator proteins
DNA-bendingprotein
Generaltranscriptionfactors
RNApolymerase II
RNApolymerase II
Transcriptioninitiation complex RNA synthesis
Fig. 18-10
Controlelements
Enhancer
Availableactivators
Albumin gene
(b) Lens cell
Crystallin geneexpressed
Availableactivators
LENS CELLNUCLEUS
LIVER CELLNUCLEUS
Crystallin gene
Promoter
(a) Liver cell
Crystallin genenot expressed
Albumin geneexpressed
Albumin genenot expressed
Gene Regulation Example 2
The Operon
Fig. 18-2
Regulationof geneexpression
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
Feedbackinhibition
Fig. 18-3a
Polypeptide subunits that make upenzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
DNA
mRNA 5
Protein Inactiverepressor
RNApolymerase
Regulatorygene
Promoter Promoter
trp operon
Genes of operon
OperatorStop codonStart codon
mRNA
trpA
5
3
trpR trpE trpD trpC trpB
ABCDE
Fig. 18-3b-1
(b) Tryptophan present, repressor active, operon off
Tryptophan(corepressor)
No RNA made
Activerepressor
mRNA
Protein
DNA
Fig. 18-3b-2
(b) Tryptophan present, repressor active, operon off
Tryptophan(corepressor)
No RNA made
Activerepressor
mRNA
Protein
DNA
Fig. 18-4a
(a) Lactose absent, repressor active, operon off
DNA
ProteinActiverepressor
RNApolymerase
Regulatorygene
Promoter
Operator
mRNA5
3
NoRNAmade
lacI lacZ
Fig. 18-4b
(b) Lactose present, repressor inactive, operon on
mRNA
Protein
DNA
mRNA 5
Inactiverepressor
Allolactose(inducer)
5
3RNApolymerase
Permease Transacetylase
lac operon
-Galactosidase
lacYlacZ lacAlacI
Fig. 18-5
(b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized
cAMP
DNA
Inactive lacrepressor
Allolactose
InactiveCAP
lacI
CAP-binding site
Promoter
ActiveCAP
Operator
lacZ
RNApolymerasebinds andtranscribes
Inactive lacrepressor
lacZ
OperatorPromoter
DNA
CAP-binding site
lacI
RNApolymerase lesslikely to bind
InactiveCAP
(a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized
Gene Regulation Example 3
Epigenetics
Epigenetics
Epigenetics Introhttp://learn.genetics.utah.edu/content/epigenetics/intro/
Utah Epigenetics
http://learn.genetics.utah.edu/content/epigenetics/intro/movies/epigenome.mp4
Gene Regulation Example 4
RNAi
RNAi
RNA Induced Silencing Complex
Vascular Endothelial Growth Factor
Human Genome
EncodeThe Encyclopedia of DNA Elements
http://www.youtube.com/watch?v=TwXXgEz9o4w&feature=player_detailpage
http://www.youtube.com/watch?v=Y3V2thsJ1Wc&feature=player_detailpage
Transformation – Recombinant Organisms
Cloning Technologies
Fig. 20-4-1
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Fig. 20-4-2
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Fig. 20-4-3
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carryingplasmids
Fig. 20-4-4
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carryingplasmids
RESULTS
Colony carrying non-recombinant plasmidwith intact lacZ gene
One of manybacterialclones
Colony carrying recombinant plasmid with disrupted lacZ gene
DNA Laboratory at Milton Academy
• Isolate DNA from cheek cells.
• Polymerase Chair Reaction
• Electrophoresis
• Sequence DNA
mtDNA Control Region
Polymerase Chain Reaction
PCR
http://www.dnalc.org/resources/spotlight/index.html
Taq DNA Polymerase
Fig. 20-8a
5
Genomic DNA
TECHNIQUETargetsequence
3
3 5
Fig. 20-8b
Cycle 1yields
2molecules
Denaturation
Annealing
Extension
Primers
Newnucleo-tides
3 5
3
2
5 31
Fig. 20-8c
Cycle 2yields
4molecules
Fig. 20-8d
Cycle 3yields 8
molecules;2 molecules
(in whiteboxes)
match targetsequence
http://www.youtube.com/watch?v=CQEaX3MiDow
http://www.youtube.com/watch?v=x5yPkxCLads&feature=related
Gel Electrophoresis
DNA Sequencing
Fredrick Sanger
Chain Termination MethodsSanger Methods
Dye-terminator sequencing
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
Directionof movementof strands
Detector
Last baseof longest
labeledstrand
Last baseof shortest
labeledstrand
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
Fig. 20-12a
DNA(template strand)
TECHNIQUE
DNA polymerase
Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
Fig. 20-12bTECHNIQUE
RESULTS
DNA (template strand)
Shortest
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Directionof movementof strands
Detector
Last baseof longest
labeledstrand
Last baseof shortest
labeledstrand
Trace File
Amplification and clonal selection
Kate Bator
Connor Johnson
High-throughput sequencingNext-Gen Sequencing
mtDNA Sequence
http://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html
“The Other Genome”mtDNA
Endosymbiotic Theory
Mitochondrial Eve
100 Years1 bp/sec
17 Minutes
Human mtDNA Haplotypes
Two Opposing Theories
• Multiregional Theory– Parallel evolution
• Displacement Theory– Out of Africa theory
http://news.bbc.co.uk/
Neandertal Genome Study Reveals That We Have a Little Caveman in
Us
Svante Paabo
Europeans and Asians share 1% to 4% of their nuclear DNA with Neandertals. But Africans do not
