Honors ~ Dna 1314

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

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