honors - dna 1112
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Molecular Biology
Honors Biology
Edgar
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
5
1 2
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
Anode Cathode
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 -globin gene
Normal -globin allele
Sickle-cell mutant -globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp 175 bp
DdeI DdeI
DdeI DdeI DdeI DdeI
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.
Transcription and Translation
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
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
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
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
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
protein
General transcription factors
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
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
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 codon Start codon
mRNA
trpA
5
3
trpR trpE trpD trpC trpB
A B C D E
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
Operator
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
-Galactosidase
lacY lacZ lacA lacI
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
Operator Promoter
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
Epigenetics
Epigenetics Intro
http://learn.genetics.utah.edu/content/epige
netics/intro/
Genomic
Imprinting
RNAi
RNA
Induced
Silencing
Complex
Vascular Endothelial Growth Factor
Genetic Engineering & Cloning
Cloning
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
mtDNA
Theories, Molecular Basis
and Real-World Application
“The Other Genome”
mtDNA
Endosymbiotic Theory
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
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