PowerLecture: Chapter 13

PowerLecture:PowerLecture:Chapter 13Chapter 13

DNA Structure and FunctionDNA Structure and Function

13.1 The Hunt13.1 The Hunt

Originally believed to be an unknown Originally believed to be an unknown class of proteinsclass of proteins

Thinking wasThinking was Heritable traits are diverseHeritable traits are diverse Molecules encoding traits must be Molecules encoding traits must be

diversediverse Proteins are made of 20 amino acids Proteins are made of 20 amino acids

and are structurally diverseand are structurally diverse

Miescher Discovered DNAMiescher Discovered DNA

18681868 Johann Miescher investigated the chemical Johann Miescher investigated the chemical

composition of the nucleuscomposition of the nucleus Isolated an organic acid that was high in Isolated an organic acid that was high in

phosphorusphosphorus He called it nucleinHe called it nuclein

Griffith Discovers Griffith Discovers TransformationTransformation

19281928 Attempting to develop a vaccineAttempting to develop a vaccine Isolated two strains of Isolated two strains of Streptococcus Streptococcus

pneumoniaepneumoniae Rough strain was harmlessRough strain was harmless Smooth strain was pathogenicSmooth strain was pathogenic

TransformationTransformation

What happened in the fourth What happened in the fourth experiment?experiment?

The harmless R cells had been The harmless R cells had been transformedtransformed by material from the by material from the dead S cellsdead S cells

Descendents of the transformed cells Descendents of the transformed cells were also pathogenicwere also pathogenic

Avery, McCarty, and MacLeodAvery, McCarty, and MacLeodRepeated Griffith’s ExperimentRepeated Griffith’s Experiment

Oswald AveryOswald Avery Maclyn McCartyMaclyn McCarty Colin MacLeodColin MacLeod

Avery, McCarty, and MacLeodAdded the non-deadly Rough Type of Bacteria to the Heat-Killed Smooth

Type

CarbohydratesCarbohydrates LipidsLipids ProteinsProteins RNARNA DNADNA

To the Heat-Killed Smooth Type, To the Heat-Killed Smooth Type, added enzymes that added enzymes that

destroyed…destroyed…

S-Type Carbohydrates S-Type Carbohydrates DestroyedDestroyed

S-Type S-Type Lipids Lipids

DestroyedDestroyed

S-Type S-Type Proteins Proteins

DestroyedDestroyed

S-Type RNA S-Type RNA DestroyedDestroyed

S-Type DNA S-Type DNA DestroyedDestroyed

Conclusion:Conclusion:

DNA was the DNA was the transforming factor!transforming factor!

Oswald & AveryOswald & Avery

What is the transforming material?What is the transforming material? Cell extracts treated with protein-Cell extracts treated with protein-

digesting enzymes could still transform digesting enzymes could still transform bacteriabacteria

Cell extracts treated with DNA-digesting Cell extracts treated with DNA-digesting enzymes lost their transforming abilityenzymes lost their transforming ability

Concluded that DNA, not protein, Concluded that DNA, not protein, transforms bacteriatransforms bacteria

The Hershey-Chase The Hershey-Chase Experiment Experiment

Alfred Hershey Alfred Hershey & Martha & Martha

Chase worked Chase worked with a with a

bacteriophage:bacteriophage:

A virus that A virus that invades invades

bacteria. It bacteria. It consists of a consists of a

DNA coreDNA core and and a a protein coatprotein coat

DNADNA

Protein coatProtein coat

Protein coats of bacteriophages labeled with Sulfur-35Protein coats of bacteriophages labeled with Sulfur-35

DNA of bacteriophages labeled with Phosphorus-32DNA of bacteriophages labeled with Phosphorus-32

BacteriumBacterium

BacteriumBacterium

PhagePhage

PhagePhage1.1. Hershey and Chase mixed Hershey and Chase mixed

the radioactively-labeled the radioactively-labeled viruses with the bacteriaviruses with the bacteria

The viruses infect the The viruses infect the bacterial cells.bacterial cells.



1.1. Separated the viruses Separated the viruses from the bacteria by from the bacteria by agitating the virus-agitating the virus-bacteria mixture in a bacteria mixture in a blenderblender



1.1. Centrifuged the mixture so that the Centrifuged the mixture so that the bacteria would form a pellet at the bacteria would form a pellet at the bottom of the test tubebottom of the test tube

1.1. Measured the radioactivity in the pellet Measured the radioactivity in the pellet and in the liquidand in the liquid

Hershey & Chase’s Hershey & Chase’s ExperimentsExperiments

Created labeled bacteriophagesCreated labeled bacteriophages Radioactive sulfur Radioactive sulfur Radioactive phosphorus Radioactive phosphorus

Allowed labeled viruses to infect Allowed labeled viruses to infect bacteriabacteria

Asked: Where are the radioactive Asked: Where are the radioactive labels after infection?labels after infection?

virus particlelabeled with 35S

DNA (blue)being injected into bacterium

35S remainsoutside cells

virus particlelabeled with 32P

DNA (blue)being injected into bacterium

35P remainsinside cells

Fig. 13-4ab, p.209

Hershey and Chase ResultsHershey and Chase Results

Structure of the Structure of the Hereditary Hereditary MaterialMaterial

Experiments in the 1950s Experiments in the 1950s showed that DNA is the showed that DNA is the hereditary materialhereditary material

Scientists raced to Scientists raced to determine the structure of determine the structure of DNADNA

1953 - Watson and Crick 1953 - Watson and Crick proposed that DNA is a proposed that DNA is a double helixdouble helix

Figure 13.6Page 211

13.2 Structure of 13.2 Structure of Nucleotides Nucleotides

in DNAin DNA Each nucleotide consists ofEach nucleotide consists of

Deoxyribose (5-carbon sugar) Deoxyribose (5-carbon sugar)

Phosphate groupPhosphate group

A nitrogen-containing baseA nitrogen-containing base

Four basesFour bases Adenine, Guanine, Thymine, CytosineAdenine, Guanine, Thymine, Cytosine

Nucleotide BasesNucleotide Bases

phosphate group

deoxyribose

ADENINE (A)

THYMINE (T)

CYTOSINE (C)

GUANINE (G)

Composition of DNAComposition of DNA

Chargaff showed:Chargaff showed: Amount of adenine relative to guanine Amount of adenine relative to guanine

differs among speciesdiffers among species Amount of adenine always equals amount Amount of adenine always equals amount

of thymine and amount of guanine always of thymine and amount of guanine always

equals amount of cytosineequals amount of cytosine

A=T and G=CA=T and G=C

Rosalind Franklin’s WorkRosalind Franklin’s Work

Was an expert in X-ray Was an expert in X-ray crystallographycrystallography

Used this technique to examine Used this technique to examine DNA fibers DNA fibers

Concluded that DNA was some Concluded that DNA was some sort of helixsort of helix

Watson-Crick Watson-Crick ModelModel

DNA consists of two DNA consists of two

nucleotide strandsnucleotide strands

Strands run in opposite Strands run in opposite

directionsdirections

Strands are held Strands are held

together by hydrogen together by hydrogen

bonds between basesbonds between bases

A binds with T and C with A binds with T and C with

GG

Molecule is a double Molecule is a double

helixhelix

13.3 DNA Structure Helps 13.3 DNA Structure Helps Explain How It DuplicatesExplain How It Duplicates

DNA is two nucleotide strands held DNA is two nucleotide strands held

together by hydrogen bondstogether by hydrogen bonds

Hydrogen bonds between two Hydrogen bonds between two

strands are easily brokenstrands are easily broken

Each single strand then serves as Each single strand then serves as

template for new strandtemplate for new strand

How does DNA replicate?How does DNA replicate?

ConservativeConservative Semi-ConservativeSemi-Conservative DispersiveDispersive

Hypotheses:

• Bacteria cultured in medium Bacteria cultured in medium containing a containing a heavyheavy isotope of isotope of Nitrogen (Nitrogen (1515N)N)

Meselson-Stahl ExperimentMeselson-Stahl Experiment

• Bacteria transferred to a medium Bacteria transferred to a medium containing elemental Nitrogen containing elemental Nitrogen ((1414N)N)



1.1.DNA sample centrifuged after First DNA sample centrifuged after First replicationreplication


1.1.DNA sample centrifuged after DNA sample centrifuged after Second replicationSecond replication

DNA DNA ReplicatioReplicatio

nn Each parent Each parent

strand remains strand remains

intactintact

Every DNA Every DNA

molecule is half molecule is half

“old” and half “old” and half

“new”“new”Fig. 13-7, p.212

Why the discontinuous additions? Nucleotides can only be joined to an exposed —OH group that is attached to the 3’ carbon of a growing strand.

Energy for strand assembly is Energy for strand assembly is provided by removal of two provided by removal of two phosphate groups from free phosphate groups from free nucleotidesnucleotides

Fig. 13-8c, p.213

Strand AssemblyStrand Assembly

Continuous and Continuous and Discontinuous AssemblyDiscontinuous Assembly

• Strands can only be assembled in the 5’ to 3’ direction•continuous on just one parent strand. This is because DNA synthesis occurs only in the 5´ to 3´ direction. • discontinuous: short, separate stretches of nucleotides are added to the template, and then ligase fill in the gaps between them.

Base Pairing Base Pairing during during

ReplicationReplication

Each old Each old strand serves strand serves as the as the template for template for complementacomplementary new strandry new strand

Fig. 13-8, p. 213

Enzymes in ReplicationEnzymes in Replication

Enzymes unwind the two strands - helicaseEnzymes unwind the two strands - helicase DNA polymerase attaches complementary DNA polymerase attaches complementary

nucleotides nucleotides DNA ligase fills in gaps (Okazaki DNA ligase fills in gaps (Okazaki

fragments)fragments) Enzymes wind two strands togetherEnzymes wind two strands together

DNA RepairDNA Repair

Mistakes can occur during replicationMistakes can occur during replication DNA polymerase can read correct DNA polymerase can read correct

sequence from complementary sequence from complementary

strand and, together with DNA ligase, strand and, together with DNA ligase,

can repair mistakes in incorrect can repair mistakes in incorrect

strandstrand

13.4 Cloning13.4 Cloning

Making a genetically identical copy of Making a genetically identical copy of

an individualan individual Researchers have been creating Researchers have been creating

clones for decadesclones for decades These clones were created by These clones were created by

embryo splittingembryo splitting

1 A microneedle 2 The microneedle has emptied the sheep egg of its own nucleus.

3 DNA from a donor cell is about to be deposited in the enucleated egg.

4 An electric spark will stimulate the egg to enter mitotic cell division.

the first cloned sheepFig. 13-9, p.214

CloningCloning

Showed that differentiated cells Showed that differentiated cells

could be used to create clonescould be used to create clones Sheep udder cell was combined Sheep udder cell was combined

with enucleated egg cellwith enucleated egg cell Dolly is genetically identical to the Dolly is genetically identical to the

sheep that donated the udder cellsheep that donated the udder cell

Dolly: Dolly: Cloned from an Adult CellCloned from an Adult Cell

Fig. 13-9, p.214

Dolly: Cloned from an Adult Dolly: Cloned from an Adult CellCell

Ian Wilmut was the first to produce a Ian Wilmut was the first to produce a cloned sheep, which he named Dollycloned sheep, which he named Dolly

Dolly experienced health problems Dolly experienced health problems similar to other mammals cloned similar to other mammals cloned from adult DNAfrom adult DNA

Impacts, Issues: Impacts, Issues: Goodbye Goodbye DollyDolly

Fig. 13-1a, p.206

Goodbye DollyGoodbye Dolly

The risk of defects in clones is hugeThe risk of defects in clones is huge

Possible benefit – patients in desperate need of Possible benefit – patients in desperate need of organ transplantsorgan transplants

Genetically modified cloned animals may produce Genetically modified cloned animals may produce organs that human donors are less likely to rejectorgans that human donors are less likely to reject

Cloning humans – ethical?Cloning humans – ethical?

Impacts, Issues: Impacts, Issues: Goodbye Goodbye DollyDolly

Therapeutic CloningTherapeutic Cloning

SCNT – Somatic Cell Nuclear TransferSCNT – Somatic Cell Nuclear Transfer

Transplant DNA of a somatic cell Transplant DNA of a somatic cell from the heart, liver, muscles, or from the heart, liver, muscles, or nerves into a stem cell nerves into a stem cell (undifferentiated cell)(undifferentiated cell)

More ClonesMore Clones

http://www.cyagra.com/gallery/jewel.htm

CowsCows

http://www.popsci.com/scitech/article/2003-05/face-should-we-clone-fading-species

Fig. 13-10, p.215

PowerLecture: Chapter 13

Documents

Transcript of PowerLecture: Chapter 13