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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
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
12
3′
3′
5′
5′
12
3′
3′
5′
5′
1
2
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
1
2
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 fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
2
One possible combination
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
Fig. 20-9a
Mixture of
DNA mol-
ecules of
different
sizes
Power
source
Longer
molecules
Shorter
molecules
Gel
AnodeCathode
TECHNIQUE
1
2
Power
source
– +
+–
Fig. 20-9b
RESULTS
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) 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
Transcription and Translation
Beadle and Tatum
1941
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 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
Gene Regulation Example 1
Activators, Enhancers and
Transcription Factors
Fig. 18-8-1
Enhancer
(distal control elements)
Proximal
control elements
Poly-A signal
sequence
Termination
region
Downstream
Promoter
Upstream
DNA
ExonExon ExonIntron Intron
Fig. 18-8-2
Enhancer
(distal control elements)
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
Fig. 18-8-3
Enhancer
(distal control elements)
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′
Fig. 18-9-1
Enhancer TATA
box
PromoterActivators
DNA
Gene
Distal control
element
Fig. 18-9-2
Enhancer TATA
box
PromoterActivators
DNA
Gene
Distal control
element
Group of
mediator proteins
DNA-bending
protein
General
transcription
factors
Fig. 18-9-3
Enhancer TATA
box
PromoterActivators
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
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
Gene Regulation Example 2
The Operon
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
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 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
Active
repressor
mRNA
Protein
DNA
Fig. 18-3b-2
(b) Tryptophan present, repressor active, operon off
Tryptophan
(corepressor)
No RNA made
Active
repressor
mRNA
Protein
DNA
Fig. 18-4a
(a) Lactose absent, repressor active, operon off
DNA
Protein
Active
repressor
RNA
polymerase
Regulatory
gene
Promoter
Operato
r
mRNA
5′
3′
No
RNA
made
lacI lacZ
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
β-
Galactosidase
lacYlacZ lacAlacI
Fig. 18-5
(b) Lactose present, 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
Gene Regulation Example 3
Epigenetics
Epigenetics
Epigenetics Intro
http://learn.genetics.utah.edu/content/epigenetics/intro/
Utah Epigenetics
http://
learn.genetics.utah.edu/content/epigenetics/intro/movies/epigenome
Gene Regulation Example 4
RNAi
RNAi
RNA
Induced
Silencing
Complex
Vascular Endothelial Growth Factor
Human Genome
Encode
The Encyclopedia of DNA Elements
http://www.youtube.com/watch?v=TwXXgEz9o4w&feature=player_d
http://www.youtube.com/watch?v=Y3V2thsJ1Wc&feature=player_de
Transformation – Recombinant
Organisms
Cloning Technologies
Fig. 20-4-1
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
ampR
gene
TECHNIQUE
Fig. 20-4-2
Bacterial cell
Bacterial
plasmid
lacZ gene
Hummingbird
cell
Gene of interest
Hummingbird
DNA fragments
Restriction
site
Sticky
ends
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
Restriction
site
Sticky
ends
ampR
gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant
plasmid
Bacteria carrying
plasmids
Fig. 20-4-4
Bacterial cell
Bacterial
plasmid
lacZ gene
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
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
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
Fredrick Sanger
Chain Termination Methods
Sanger 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
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
Fig. 20-12a
DNA
(template strand)
TECHNIQUE
DNA
polymerase
Primer Deoxyribonucleotides Dideoxyribonucleotides
(fluorescently tagged)
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
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
Trace File
Amplification and clonal
selection
Kate Bator
Connor Johnson
High-throughput sequencing
Next-Gen Sequencing
mtDNA Sequence
http://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html
“The Other Genome”
mtDNA
Endosymbiotic Theory
Mitochondrial Eve
100 Years
1 bp/sec
17 Minutes
Human mtDNA Haplotypes
mtDNA – Genographic Project
Video
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
Honors ~ Dna 1314
Honors ~ Dna 1314

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