DNA Plasmids Phages - Montana State University Billings Genetic… · Copyright © 2009 Pearson Education, Inc. Copyright © 2005 Pearson Education, Inc. Publishing as ... Genetic

Gene Technology

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Outline: Gene Technology

• Gene Cutting & Splicing• Transferring, Cloning & Storing DNA• Polymerase Chain Reaction (PCR) • Gel Electrophoresis & RFLP Analysis• Biotechnology & Applications• Mutation• Cancer

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CCGGT

ATA

AT

AT

CCGGT

ATA

AT

AT

CG

A A T T

CG

A A T T

CG

T T A A

CG

T T A ACG

A A T TDNA ligasejoins the strands.

DNA

GATT

CG

A A T T

Sticky ends

Restriction sitesEcoRI

Recombinant DNA molecule

Restriction endonuclease cleaves the DNAEcoRI

EcoRI

EcoRI

DNA from another source cut with thesame restriction endonuclease is added.

Restriction endonuclease cleaves the DNA

Sticky ends

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C A

Cutting and Splicing DNA Recombinant DNA Multiplying, Storing &Transferring DNAVectors - Transfer DNA

Plasmids Phages

Hosts – Store/replicate DNABacteria, yeast cells, mammalian cultured cells, plant cells

Bacterialchromosome

Plasmid DNA

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Vectors

Plasmid ampicillin gene only bacteria with plasmid will survive in medium with ampicillin.

Plasmid lacZ gene only bacteria with inactive lacZ will have foreign DNA inserted.

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DNA Libraries Store Genomes

Constructing a cDNA Library from Eukaryotes

Fig. 17.5-2

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DNA Libraries

Molecular hybridization is a technique used to identify specific DNAs in complex mixtures -A known single-stranded DNA or RNA is labeled

-It is then used as a probe to identify its complement via specific base-pairing

-Also termed annealing

Target sequence

2 copies

Cycle1

3

2

1


PolymeraseChainReaction (PCR)

1 copy1. Heat Denature DNACool & add primers

2. Add DNA polymerase & Nucleotides

3. New DNA synthesized

8 copies Cycle3

Repeat 1, 2 & 3

4 copiesCycle

2

Repeat 1, 2 & 3

After 20 cycles, a single fragment produces over one million (220) copies!

Longer fragments

Shorter fragmentsMixture of DNA fragmentsat top of gel Electric current applied

Powersource

Completed gel

Gel

Anode+

Cathode–


Gel ElectrophoresisDNA andrestrictionendonuclease

DNA Fingerprinting

Original Sequence

Point Mutation

W

X

Y Y

Z

CutCut

Cut

DNA from chromosomes

CCGG

GGCC

ACGG

TGCC

CCGG

GGCC

CCGG

GGCC

Y X W

Y Z

Single base pair change

RFLP = Rrestriction Fragment Length Polymorphisms

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Different DNA duplexes

Cut DNA Gel electrophoresis ofrestriction fragments

Original Sequence

of Restriction Sites

(no mutations)

Point MutationsChange the Sequence of

Restriction Sites

Largerfragments

Smallerfragments

restriction enzymecutting sites

+ +

+–

–


Restriction Fragments Reflect DNA Differences

Single basePair change

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STR site 1

Crime scene DNA

STR site 2

Suspect’s DNA

Number of short tandemrepeats match

Number of short tandemrepeats do not match

Short tandem repeats (STRs) are genetic markersSTRs are short DNA sequences repeated many times in a row at thesame location. Number of STR units differs between individuals.


Suspect’sDNA

Crime sceneDNA

Suspect’s DNACrime scene DNA

Short tandem repeats (STRs) are genetic markers

DNA fragments separated by

Gel Electrophoresis

DNA Fingerprinting

RFLP & Genetic Disorder Markers


Viruses

1892 Russian Dmitri Ivanovski Could not filter disease agents causing Tobacco mosaic disease.

1898 Dutch botanist, Martinus Biejerinck“kills’ tobacco mosaic disease agent at 90C.

1935 - Wendell Stanley isolated and crystallized the Tobacco Mosaic Virus

Nature of Viruses

1. Agents of Diseasefound in virtually all organismshost range usually specific

2. No cell organization3. No Metabolism4. No organelles5. Cannot reproduce independent of cells6. Set of genetic instructions7. Renegade nucleic acid coated with protein

Virus Structure

2. Protein Capsid

Phospholipid bilayerProteinsGlycoproteins

3. Envelope

1. Nucleic Acid Core

Bacteriophage HIV

SHAPE1. Helical ≈ rods2. Isometric ≈ icosahedron

Viral Genome Structure

1. DNA Viruses have Double-Stranded DNA2. RNA Viruses have Single Stranded RNA

(+) Stranded RNARNA = mRNA

(-) Stranded RNARNA bases complimentary to mRNA

Virus Genome1. – strand RNA Virus2. 29,751 nucleotides3. Six encoding genes make

2 ReplicasesSpike proteinsEnvelope glycoproteinsMembrane glycoproteinNucleocapsid protein

SARS virusSevere acute respiratory syndrome

Viral genome of E. coli bacteriophage48,502 bases23 proteins development & maturationOther enzymes integrate virus DNA into host genome

Viruses cause disease in animals and plants

Both DNA viruses and RNA viruses cause disease in animals & plants

Reproductive cycle of an RNA virus

– Entry

– Glycoprotein spikes contact host cell receptors

– Viral envelope fuses with host plasma membrane

– Uncoating of viral particle to release the RNA genome

– mRNA synthesis using a viral enzyme

– Protein synthesis

– RNA synthesis of new viral genome

– Assembly of viral particles

Copyright © 2009 Pearson Education, Inc. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

• HIV, the AIDS virus– A retrovirus

Envelope

Glycoprotein

Protein coat

RNA (two identical strands)

Reverse transcriptase

Human Immunodeficiency Virus

Plasma membraneof host cell

VIRUS

Entry

Viral RNA(genome)

Viral RNA(genome)

2

Membranousenvelope

Protein coatGlycoprotein spike

Uncoating

RNA synthesisby viral enzyme

3

1


Double-strandedDNA

ViralRNAandproteins

DNAstrand

Viral RNA

NUCLEUS

CYTOPLASM

ChromosomalDNA

ProvirusDNA

RNA

2

1

5

3

4

6


HIV replication animation

Genetically modified organisms

Genetically modified (GM) organisms contain one or more genes introduced by artificial means

Transgenic organisms contain at least one gene from another species GM plants

– Resistance to herbicides– Resistance to pests– Improved nutritional profile

GM animals– Improved qualities– Production of proteins or therapeutics

Copyright © 2009 Pearson Education, Inc. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

GM Plants: Recombinant DNA technology in Plants

Agrobacterium tumefaciens

DNA containinggene for desired trait

Tiplasmid

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Insert plant geneinto plasmid usingrestriction enzymeand DNA ligase

RecombinantTi plasmid

2

IntroductionInto

plant cells

3

Regenerationof plant

Plant with new trait

T DNA carrying newgene within plant chromosome

Plant cell

Restriction site

Agrobacterium tumefaciens tumor on a plant

Glyphosate Resistance1. Cotton2. Corn3. Soybeans4. Canola5. Wheat

Bt Crops• Cotton• Corn

Genetic Engineering & genetically modified (GM) crops

Enhancement of Longevity1. “Flavr Savr” Tomato

Other engineered crops1. Papaya virus resistance2. Carnation longevity3. Flax herbicide resistance4. Lentil herbicide resistance5. Potato insect resistance6. Squash virus resistance7. Sugar beet herbicide resistance8. Cucumber virus resistance9. Watermelon virus resistance

Enhancement of Nutritional Value1. Golden Rice


Golden Rice

Vitamin A Deficiency

Vitamin A Roles

•Vision

•Immune defense

•Reducing morbidity of measles

•Reducing respiratory infections

•Cell differentiation and morphogenesis

Agricultural Applications of DNA Technology

Daffodil

Ferritin geneis transferredinto rice frombeans.

Phytase gene istransferred intorice from afungus.

Metallothioningene istransferred intorice from wildrice.

β-carotene enzymeSynthesis genes aretransferred intorice from daffodils.

Fe Pt SRicechromosome A1

Ferritin proteinincreases ironcontent of rice.

Phytate, whichinhibits ironreabsorption,is destroyed by thephytase enzyme.

Metallothioninprotein suppliesextra sulfur toincrease ironuptake.

β-carotene, aprecursor tovitamin A, issynthesized.

Beans Aspergillus fungus Wild rice

A2 A3 A4


Genetically Engineered Golden RiceApplications of Plant

Genetic Engineering

European Corn Borer

Bt Corn Bt Corn Non-Bt Corn

Genetically Engineered Corn=Bt corn

Bacillus thuringensis1. bacterium2. produces Bt toxin3. kills larvae

Table 16.1

Medical Applications – Subunit Vaccines

Human immuneresponse

Gene specifying herpessimplex surface protein

Harmless vaccinia(cowpox) virus

Herpes simplex virus

2. Herpes simplexgene is isolated.

3. Vaccinia DNA is extracted and cleaved.

4. Fragment containingsurface gene combines with cleaved vaccinia DNA.

5. Harmless engineered virus (the vaccine) with surface like herpes simplex is injected into the human body.

6. Antibodies directedagainst herpes simplex viral coat are made.

1. DNA is extracted.

Gene Therapy & ADA Deficiency

ENDGENE

TECHNOLOGY

Mutation

Mutation1. Definition: Change in DNA2. Frequency: 1 in 50 million base pairs

1 in a million gametes

White grapes

Seedless navel orange

Albino rainbow trout

Blue Trout


Mutation = change in the nucleotide sequence of DNA

Why mutation?1. Spontaneous

errors in DNA replication errors in DNA recombination

2. Induced to form by mutagensHigh-energy radiationChemicals

Mutation


Point mutations Changes in 1-few nucleotides

Normal gene mRNA

Base substitution

Base deletionMissing

Met Lys Phe Gly Ala

Met Lys Phe Ser Ala

Met Lys Leu Ala His

A U G A A G U U U G G C G C A

A U G A A G U U U A G C G C A

A U G A A G U U G G C G C A U

U

Protein

Mutation – gene alteration

mRNAProtein

mRNAProtein

Base insertion

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Point mutations alter one or a few DNA bases.What happens when a point mutation occurs?Silent no change in mRNA codonNonsense create stop codonFrameshift shifts reading of mRNA codons

Mutation: Altered Genes

DNA

mRNA

Amino acid

ATG

UAC

Tyrosine

Normal Silent Nonsense FrameshiftMutation

ATA

UAU

Tyrosine

ATT

UAA

Stop

TTAGGCC

TTAGCGCC

UCG

Serine

Normal hemoglobin DNA Mutant hemoglobin DNA

Sickle-cell hemoglobinNormal hemoglobin

mRNAmRNA

ValGlu

Examples of Mutation – Sickle Cell Anemia

250,000 base pairs

27 Exons + Introns

61,000 base pair mRNA

1,480 Amino Acid Sequence of CTFR protein

Examples of Mutation – Cystic Fibrosis

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Mutation: Altered GenesChromosomal mutations change chromosome structure.

deletion part of chromosome is lostduplication part of chromosome is copiedinversion part of chromosome in reverse ordertranslocation/transposition part of chromosome

moves to a new location

Deletion

Duplication

Inversion

Translocation Transposition

Chromosomal mutations

part of chromosome is lost

part of chromosome is copied

part of chromosome is reversed in order

chromosome segments move/swap places

Chromosomal Mutations Transposition = Jumping genes

Chromosomal mutations – Transposons

Transposon

Chromosome A

Chromosome B

Consequences of transposition (48% Human genome = transposons)1. Cause mutations 2. May disable functional genes3. May cause cancer by insertion of transposon promoter near

cancer-causing gene4. Transposon diseases: Hemophilia, SCID, Muscular Distrophy5. Viruses like HIV behave like transposons


Alterations of chromosome structure - Deletion

Deletion

Cri du chatChromosome 5 deletion1 in 25,000-50,000Detected by amniocentesisMental retardationMay live normal life span


Reciprocal translocation associated with chronic myelogenous leukemia (CML)

“Philadelphia chromosome”

Chromosome 9

Chromosome 22 Reciprocaltranslocation

Activated cancer-causing gene

DNA Damage and Repair

DNA Damage and Repair DNA Damage and Repair

Xeroderma pigmentosa

THE GENETIC BASIS OF CANCER

Copyright © 2009 Pearson Education, Inc.

Cancer results from mutations in genes that control cell division

Mutations in two types of genes can cause cancer

– Proto-oncogenes

– Proto-oncogenes normally promote cell division

– Mutations to proto-oncogenes enhance activity

– Tumor-suppressor genes

– Normally inhibit cell division

– Mutations inactivate the genes and allow uncontrolled division to occur

Cancer & Proto-Oncogenes

Promote cancer when present in a single copy

Can be viral genes inserted into host chromosomes

Can be mutated versions of proto-oncogenes, normal genes that promote cell division and differentiation

Converting a proto-oncogene to an oncogene can occur by

– Mutation causing increased protein activity

– Increased number of gene copies causing more protein to be produced

– Transposition - Change in location putting the gene under control of new promoter for increased transcription

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Mutation withinthe gene

Hyperactivegrowth-stimulatingprotein in normalamount

Proto-oncogene DNA

Multiple copiesof the gene

Gene moved tonew DNA locus,

under new controls

Oncogene New promoter

Normal growth-stimulatingprotein in excess

Normal growth-stimulatingprotein in excess

Cancer & Proto-Oncogenes

Mutated tumor-suppressor geneTumor-suppressor gene

Defective,nonfunctioningprotein

Normalgrowth-inhibitingprotein

Cell divisionunder control

Cell division notunder control

Cancer & Tumor-suppressor genes

Promote cancer when both copies are mutated

Signaling cell

DNA

Nucleus

Transcriptionfactor(activated)

Signaling molecule

PlasmamembraneReceptor

protein

Relayproteins

TranscriptionmRNANewprotein

Translation

Target cell

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3

4

5

6

Signal Transduction Pathways &

Proto-Oncogenes

Cell Division

DNA RNA

DNA RNA

DNA RNA

DNA RNA

DNA RNA


Rasprotein

Srckinase

Cell cyclecheckpoints

Cytoplasm

Nucleus

Rbprotein

p53protein

Growth factor receptor:more per cell in manybreast cancers.

Ras protein:activated by mutationsin 20–30% of all cancers.

Src kinase:activated by mutationsin 2–5% of all cancers.

PROTO-ONCOGENES

Rb protein:mutated in 40% of all cancers.

p53 protein:mutated in 50% of all cancers.

TUMOR-SUPPRESSOR GENES

Proteins Regulate Cell Cycle


DNA damage caused by heat,radiation,chemicals.

Step 1 Step 2

Damaged cells mayturn cancerous if other mutations appear.

ABNORMAL p53

Abnormalp53 protein

Cancercell

Step 3p53 protein fails to stop cell division and DNA repair.Cell division continues without repair.

Benzopyrene

Activity of Abnormal p53 gene

Cancer Carcinogens Cases in 1999

Prostate Testosterone; dietary fat 179,300

Breast Estrogen; possibly dietary fat 176,300

Lung Cigarette smoke 171,600

Colon & Rectum High dietary fat; low dietary fiber 129,400

Bladder Cigarette smoke 54,200

Skin Ultraviolet light 44,200

Kidney Cigarette smoke 30,000

Mouth and Throat Tobacco & alcohol 29,800

Pancreas Cigarette smoke 28,600

Stomach Table salt; cigarette smoke 21,900

Cervix Viruses; cigarette smoke 12,800

Cancer in the United States

Chromosomes 1 mutation

Normalcell

4mutations

3mutations

2mutations

MalignantCell &

metastasis

Mutation of Tumor

Suppressor Gene APC

Increased Cell

Division

Mutation of Proto-

OncogeneK-ras

Mutation of Tumor

Suppressor Gene DCC

Mutation of Tumor

Suppressor Gene p53

Benign polyp

Benign polyp

Multiple mutations lead to cancer

END

Documents

DNA Plasmids Phages - Montana State University Billings Genetic… · Copyright © 2009 Pearson Education, Inc. Copyright © 2005 Pearson Education, Inc. Publishing as ... Genetic