Honors - Dna 1112

Molecular Biology

Honors Biology
Edgar

Fig. 16-13

Single-strand binding Primase
proteins

Topoisomerase 3
5 RNA 3
primer

5
5
3
Helicase

Fig. 16-16b6
3 5

5 3
Template
strand

3 5
RNA primer 3
1
5

Okazaki 3
fragment 5
3
5 1
3 5

3
2 1 5

5
3

3
5
2 1

5
3

3
1 5
2

Overall direction of replication

Fig. 16-16a

Overview
Origin of replication
Leading strand Lagging strand

Lagging strand
2
1
Leading strand
Overall directions
of replication

Fig. 20-3-1
Restriction site

DNA 5 3
3 5

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end

Fig. 20-3-2
Restriction site

DNA 5 3
3 5

backbones.

Sticky end

2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.

One possible combination

Fig. 20-3-3
Restriction site

DNA 5 3
3 5

backbones.

Sticky end

2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.

One possible combination
3 DNA ligase
seals strands.

Recombinant DNA molecule

Fig. 20-9a
TECHNIQUE

Mixture of Power
DNA mol- source
ecules of – Cathode Anode +
different
sizes

Gel
1

Power
source
– +
Longer
molecules

2 Shorter
molecules

Fig. 20-10

Normal -globin allele Normal Sickle-cell
allele allele

175 bp 201 bp Large fragment

DdeI DdeI DdeI DdeI Large
fragment
Sickle-cell mutant -globin allele

376 bp
376 bp Large fragment 201 bp
175 bp
DdeI DdeI DdeI

(a) DdeI restriction sites in normal and (b) Electrophoresis of restriction fragments
sickle-cell alleles of -globin gene from normal and sickle-cell alleles

Restriction Enzyme Lab
• HINTS: • Lambda DNA/PstI:
• pMAP is 5615bp • You should not be
• There are able to see beyond
– 2 PstI sites. the 805bp band.
– 1 HpaI site. • Fine the 11,490bp
– 1 SspI site and the 805bp as
reference.

Fig. 18-6 Signal

NUCLEUS
Chromatin

Chromatin modification

DNA
Gene available
for transcription
Gene
Transcription
RNA Exon
Primary transcript
Intron
RNA processing
Tail

Cap mRNA in nucleus

Transport to cytoplasm

CYTOPLASM
mRNA in cytoplasm

Translation
Degradation
of mRNA

Polypeptide

Protein processing

Active protein
Degradation
of protein
Transport to cellular
destination

Cellular function

Fig. 18-8-1

Poly-A signal
sequence
Enhancer Proximal
Termination
(distal control elements) control elements region
Exon Intron Exon Intron Exon
DNA
Upstream Downstream
Promoter

Fig. 18-8-2

Poly-A signal
sequence
Enhancer Proximal
Termination
DNA
Upstream Downstream
Promoter Transcription

Primary RNA Exon Intron Exon Intron Exon
Cleaved 3 end
transcript 5 of primary
transcript

Poly-A
signal

Fig. 18-8-3

Poly-A signal
sequence
Enhancer Proximal
Termination
DNA
Upstream Downstream
Promoter Transcription

Primary RNA Exon Intron Exon Intron Exon
Cleaved 3 end
transcript 5 of primary
RNA processing transcript

Intron RNA Poly-A
signal
Coding segment
mRNA 3
Start Stop
5 Cap 5 UTR codon codon 3 UTR Poly-A
tail

Fig. 18-9-1
Activators Promoter
Gene
DNA
Enhancer Distal control TATA
element box

Fig. 18-9-2
Activators Promoter
Gene
DNA
element box
General
transcription
factors
DNA-bending
protein

Group of
mediator proteins

Fig. 18-9-3
Activators Promoter
Gene
DNA
element box
General
transcription
factors
DNA-bending
protein

Group of
mediator proteins

RNA
polymerase II

RNA
polymerase II

Transcription
initiation complex RNA synthesis

Fig. 18-10
Enhancer Promoter

Control Albumin gene
elements

Crystallin gene

LIVER CELL LENS CELL
NUCLEUS NUCLEUS
Available
activators Available
activators

Albumin gene
not expressed
Albumin gene
expressed

Crystallin gene
not expressed
Crystallin gene
expressed
(a) Liver cell (b) Lens cell

Fig. 18-2
Precursor
Feedback
inhibition
trpE gene

Enzyme 1

trpD gene
Regulation
of gene
expression
Enzyme 2 trpC gene

trpB gene

Enzyme 3

trpA gene

Tryptophan

(a) Regulation of enzyme (b) Regulation of enzyme
activity production

Fig. 18-3a

trp operon
Promoter Promoter
Genes of operon
DNA trpR trpE trpD trpC trpB trpA
Regulatory Operator
gene Start codon Stop codon
3
mRNA RNA mRNA 5
5 polymerase
E D C B A
Protein Inactive Polypeptide subunits that make up
repressor enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on

Fig. 18-3b-1

DNA
No RNA made

mRNA

Protein Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off

Fig. 18-3b-2

DNA
No RNA made

mRNA

Protein Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off

Fig. 18-4a

Regulatory Promoter
gene
Operator
DNA lacI lacZ
No
RNA
made
3
mRNA RNA
5 polymerase

Active
Protein repressor

(a) Lactose absent, repressor active, operon off

Fig. 18-4b

lac operon

DNA lacI lacZ lacY lacA

RNA
polymerase
3
mRNA
mRNA 5
5

Protein -Galactosidase Permease Transacetylase

Allolactose Inactive
(inducer) repressor

(b) Lactose present, repressor inactive, operon on

Fig. 18-5
Promoter
Operator
DNA lacI lacZ
CAP-binding site RNA
polymerase
Active binds and
cAMP CAP transcribes

Inactive Inactive lac
CAP repressor
Allolactose
(a) Lactose present, glucose scarce (cAMP level
high): abundant lac mRNA synthesized
Promoter Operator
DNA lacI lacZ
CAP-binding site RNA
polymerase less
likely to bind
Inactive
CAP Inactive lac
repressor

(b) Lactose present, glucose present (cAMP level
low): little lac mRNA synthesized

Epigenetics Intro
http://learn.genetics.utah.edu/content/epige
netics/intro/

Vascular Endothelial Growth Factor

Fig. 20-4-1
TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene

Restriction Sticky Gene of interest
site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments

Fig. 20-4-2
cell
Bacterial cell
lacZ gene

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments

Nonrecombinant
plasmid
Recombinant plasmids

Fig. 20-4-3
cell
Bacterial cell
lacZ gene

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments

Nonrecombinant
plasmid

Bacteria carrying
plasmids

Fig. 20-4-4
cell
Bacterial cell
lacZ gene

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments

Nonrecombinant
plasmid

Bacteria carrying
plasmids

RESULTS

Colony carrying non- Colony carrying recombinant
recombinant plasmid plasmid with disrupted lacZ gene
with intact lacZ gene

One of many
bacterial
clones

mtDNA
Theories, Molecular Basis
and Real-World Application

“The Other Genome”
mtDNA

DNA Laboratory at
Milton Academy
• Isolate DNA
from cheek cells.
• Polymerase
Chair Reaction
• Electrophoresis
• Sequence DNA

PCR
http://www.dnalc.org/resources/spotlight/index.html

Fig. 20-8a

5 3
TECHNIQUE
Target
sequence

Genomic DNA 3 5

Fig. 20-8b

1 Denaturation 5 3

3 5
2 Annealing

Cycle 1
yields Primers
2
molecules

3 Extension

New
nucleo-
tides

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=x5yPkxCLads&feature=related

http://www.youtube.com/watch?v=CQEaX3MiDow

Chain Termination Methods
Sanger Methods

Fig. 20-12
TECHNIQUE

DNA Primer Deoxyribonucleotides Dideoxyribonucleotides
(template strand) (fluorescently tagged)

dATP ddATP
dCTP ddCTP
dTTP ddTTP
DNA
polymerase dGTP ddGTP

DNA (template Labeled strands
strand)

Shortest Longest

Direction
of movement Longest labeled strand
of strands

Detector

Laser
Shortest labeled strand
RESULTS Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand

Fig. 20-12a

TECHNIQUE

DNA Primer Deoxyribonucleotides Dideoxyribonucleotides
(template strand) (fluorescently tagged)

dATP ddATP
dCTP ddCTP

DNA dTTP ddTTP
polymerase dGTP ddGTP

Fig. 20-12b
TECHNIQUE

DNA (template Labeled strands
strand)

Shortest Longest

Direction
of movement Longest labeled strand
of strands

Detector

Laser
Shortest labeled strand
RESULTS Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand

Amplification and clonal
selection

High-throughput sequencing
Next-Gen Sequencing

mtDNA Sequence

http://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html

Honors - Dna 1112

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