Molecular Biology

Honors Biology

Edgar

http://www.ba-education.demon.co.uk/for/science/dnaphotos/dnaphoto.html

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