Next Topic Unit #3:

DNA, Genetic Expression, & Biotechnology

**Chapter 16: DNA: History, Structure & Replication**Chapter 17: Genetic Expression (protein synthesis) Chapter 18: Viruses & Bacteria (selected parts) Chapter 19: Regulation (selected parts)**Chapter 20: Genetic Engineering & Biotechnology

Key Questions Explored in this Next unit:

• What are Genes made of?

• How do Genes work?

• How can information be stored, retrieved, and modified over time?

• What keeps this molecule so stable?

• Why is DNA and not protein responsible for the inheritance of genetic traits?

History for the Discovery of DNA

• Chapter 16

The Molecular Basis of Inheritance

Overview of Chapter 16:TOPIC Pgs.

-History & Discovery of DNA 293-296

-Structure of DNA 296-298

-DNA Replication 299-306

Key Discoveries• Miescher (isolated “nuclein” from soiled bandages) 1869• Garrod (Proteins & inborn errors) 1902• Sutton (Chromosome structure) 1903• Morgan (Gene mapping) 1913• Sumner (Purified Urease, showed it to be an enzyme) 1926• Griffith’s Experiment (Transforming Principle) 1928• Avery, McCarty, and Macleod 1944• Chargaff (Base pairing & species specific) 1947• Hershey and Chase 1952• Pauling, Wilkins, and Franklin

1950’s• Watson and Crick 1953

Discovery of DNA• 1868: Miescher first isolated

deoxyribonucleic acid, or DNA, from cell nuclei

Fredrick Griffith (1928)• First suggestion that about what genes are made of. • Worked with: 1) Two strains of Pneumococcus bacteria:

Smooth strain (S) Virulent (harmful) Rough strain (R) Non-Virulent

2) Mice-were injected with these strains of bacteria and watched to see if the survived.

3) Four separate experiments were done:-injected with rough strain (Lived)-injected with smooth strain (Died)-injected with smooth strain that was heat killed (Lived)-injected with rough strain & heat killed smooth (????)

Griffith’s Experiment-1928

Conclusion of Griffith’s Experiment

• Somehow the heat killed smooth bacteria changed the rough cells to a virulent form.

• These genetically converted strains were called “Transformations”

• Something (a chemical) must have been transferred from the dead bacteria to the living cells which caused the transformation

• Griffith called this chemical a “Transformation Principle”

Avery, MacLeod, and McCarty (1944)

• Chemically identified Griffith’s transformation principle as DNA

• Separated internal contents of the S cells into these fractions:

(lipids, proteins, polysaccharides, and nucleic acids)

• They tested each fraction to see if it can cause transformation to occur in R cells to become S cells.

• Only the nucleic acids caused the transformation• This was the first concrete evidence that DNA is the

genetic material. • Some were not completely convinced because they

were not sure if this was true for eukaryotes.

Next Breakthrough came from the use of Viruses

• Viruses provided some of the earliest evidence that genes are made of DNA

• Molecular biology studies how DNA serves as the molecular basis of heredity

• Only composed of DNA and a protein shell

Various Types of Viruses

T2 Bacteriophage

• Phage reproductive cycle

Figure 10.1C

Phage attaches to bacterial cell.

Phage injects DNA.

Phage DNA directs host cell to make more phage DNA and protein parts. New phages assemble.

Cell lyses and releases new phages.

A Typical Bacteriophage

Video #1 DNA: The Blueprint of Life1. Name the technology used in the movie Jurassic

Park.

2. Where did Meissner extract the “nuclein” material that later was identified as DNA?

3. How did Hershey & Chase separate the virus from its bacterial host? How did they trace (track) the DNA and protein?

4. What did x-ray crystallography reveal about DNA?

5. What purpose do enzymes serve in the replication process?

Segment #2:

**Need Five key Statements for the segment

Alfred Hershey & Martha Chase (1952)

• Worked with T-2 Bacteriophages• Infected Escherchia coli (E. coli) = Host cell• Used Radioactive Isotopes:

(S35) Sulfur-35(P32) Phosphorus-32

• Why did they use these particular isotopes?*Sulfur is found in proteins and not in DNA*Phosphorus is found in DNA but not in protein

Labeling of Virus Structures

Details of the Hershey & Chase Experiment

• The Hershey-Chase Experiment

Figure 10.1B

Mix radioactivelylabeled phages with bacteria. The phages infect the bacterial cells.

Phage

Bacterium

Radioactiveprotein

DNA

Emptyprotein shell

1 2 Agitate in a blender to separate phages outside the bacteria from the cells and their contents.

3 Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube.

4 Measure the radioactivity in the pellet and liquid.

Batch 1Radioactiveprotein

Batch 2RadioactiveDNA

RadioactiveDNA

PhageDNA

Centrifuge

Pellet

Radioactivityin liquid

Radioactivityin pelletPellet

Centrifuge

Video clip of Hershey Chase Experiment

• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

• Key findings: the phage DNA entered in the host cell and when these cells were returned to the culture medium the infection ran its course producing E.coli and other bacteriophages with the radioactive phosphorus. (pg. 298)

• James Watson and Francis Crick worked out the three-dimensional structure of DNA, based on work by Rosalind Franklin

DNA is a Double-Stranded Helix

Figure 10.3A, B

Rosalind Franklin’s Image (pg. 297)

• and Media 

• DNA is a nucleic acid, made of long chains of nucleotides

DNA and RNA are polymers of Nucleotides

Figure 10.2A

Nucleotide

Phosphate group

Nitrogenous base

Sugar

Polynucleotide Sugar-phosphate backbone

DNA nucleotide

Phosphategroup

Nitrogenous base(A, G, C, or T)

Thymine (T)

Sugar(deoxyribose)

• DNA has four kinds of bases, A, T, C, and G

Figure 10.2B

Pyrimidines

Thymine (T) Cytosine (C)

Purines

Adenine (A) Guanine (G)

DNA Maintains a Uniform Diameter

See pg. 298

DNA Bonding• Purines: ‘A’ & ‘G’• Pyrimidines: ‘C’ & ‘T’

(Chargaff rules)• ‘A’ H+ bonds (2) with ‘T’ and

‘C’ H+ bonds (3) with ‘G’• Van der Waals attractions

between the stacked pairs

• RNA is also a nucleic acid

– RNA has a slightly different sugar– RNA has U instead of T

Figure 10.2C, D

Phosphategroup

Nitrogenous base(A, G, C, or U)

Uracil (U)

Sugar(ribose)

• Hydrogen bonds between bases hold the strands together

– Each base pairs with a complementary partner– A pairs with T– G pairs with C

DNA Structure

• Chargaffratio of nucleotide bases (A=T; C=G)

• Watson & Crick (Wilkins, Franklin)

• The Double Helix

√ nucleotides: nitrogenous base (thymine, adenine, cytosine, guanine); sugar deoxyribose; phosphate group

• Three representations of DNA

Figure 10.3D

Ribbon model Partial chemical structure Computer model

Hydrogen bond

• Each strand of the double helix is oriented in the opposite direction

Figure 10.5B

5 end 3 end

3 end 5 end

P

P

P

PP

P

P

P

DNA Replication: History & Discovery

• First model suggested by Watson & Crick• Three models were proposed:

-Semiconservative (half old & half new)

-Conservative (old strands remain together)

-Dispersive (random mixture)• Heavy isotopic nitrogen (N-15) was used to label

the nitrogenous bases in the DNA• Density gradient centrifugation was used• DNA was mixed with Cesium chloride (CsCl)

Three Proposed Models of DNA Replication

Meselson & Stahl’s Experiment

Meselson-Stahl Experiment

Meselson & Stahl Experiment (Pg. 300)

• Grew E. coli on a medium containing isotopic Nitrogen (15N) in the form of NH4Cl

• Nitrogenous bases incorporated the isotopic nitrogen

• DNA was extracted from the cells• Density gradient centrifugation was used on

the DNA to determine the banding region of the heavy isotopic nitrogen.

• The rest of the bacteria was then grown on a medium containing normal nitrogen and allowed to grow.

Meselson & Stahl Experiment cont’d.

• The newly synthesized strands of DNA were expected to have the lighter normal nitrogen in their bases.

• The older original strands were labeled with the heavier isotopic nitrogen.

• Two generations were grown in order to rule out the conservative and dispersion models.

• The structure of DNA consists of two polynucleotide strands wrapped around each other in a double helix

Figure 10.3CTwist

1 chocolate coat,Blind (PRA)

• In DNA replication, the strands separate– Enzymes use each strand as a template to

assemble the new strands

DNA replication depends on specific base pairing

Parental moleculeof DNA

Figure 10.4A

Both parental strands serveas templates

Two identical daughtermolecules of DNA

Nucleotides

A

A

• Untwisting and replication of DNA

Figure 10.4B

Anti-parallel Structure of DNA

Antiparallel nature• 5’ end corresponds to the Phosphate end• 3’ end corresponds to the –OH sugar • Replication runs in BOTH directions

• One strand runs 5’ to 3’ while the other runs 3’ to 5’

• Nucleotides are added on the 3’ end of the newly synthesized strand

• The new DNA strand forms and grows in the

5’ 3’ direction only

Building New Strands of DNA• Each nucleotide it a triphosphate:

(GTP, TTP, CTP, and ATP)

• Nucleotides only add to the 3’ end of the growing strand (never on the 5’ end)

• Two phosphates are released (exergonic) and the energy released drives the polymerization process.

Origin of replication (“bubbles”): beginning of replication (pg. 301)

How a Nucleotides adds to the old Strand

5’ end

3’ end5’ end

Key Enzymes Required for DNA Replication (pg. 303-304)

• Helicase - catalyzes the untwisting of the DNA at the replication fork

• DNA Polymerase - catalyzes the elongation of new DNA and adds new nucleotides on the 3’ end the growing strand.

• SSBP’s - single stranded binding proteins, prevents the double helix from reforming

• Topoisomerase – Breaks the DNA strands and prevents excessive coiling

• Primase – synthesizes the RNA primers and starts the replication first by laying down a few nucleotides initially.

**DNA primase will get replaced by DNA polymerase

RNA Primers

• Initiates the Replication process and begins the building of the newly formed strands.

• Laid down by RNA polymerase (primase)

• Consists of 5 to 14 nucleotides

• Synthesized at the point where replication begins

• Will be laid down on both template strands of the DNA

Laying Down RNA Primers

• How DNA daughter strands are synthesized

5 end

P

P

Parental DNA

Figure 10.5C

DNA polymerasemolecule

53

35

35

Daughter strandsynthesizedcontinuously

Daughter strandsynthesizedin pieces

DNA ligase

Overall direction of replication

53

• The daughter strands are identical to the parent molecule

DNA Replication-New strand Development

• Leading strand: synthesis is toward the replication fork (only in a 5’ to 3’ direction from the 3’ to 5’ master strand)

-Continuous• Lagging strand: synthesis is away from the replication fork

-Only short pieces are made called “Okazaki fragments”- Okazaki fragments are 100 to 2000 nucleotides long-Each piece requires a separate RNA primer

-DNA ligase joins the small segments together (must wait for 3’ end to open; again in a 5’ to 3’ direction)

View video clip: • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

DNA Replication Fork

Video Clip of DNA Replication• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

Prokaryotic vs Eukaryotic Replication

• Prokaryotes– Circular DNA (no free ends)– Contains 4 x 106 base pairs (1.35 mm)– Only one origination point

• Eukaryotes-Have free ends-Contains 3 x 109 base pairs (haploid cells) = 1 meter-Lagging strand is not completely replicated-Small pieces of DNA are lost with every cell cycle-End caps (Telomeres) protect and help to retain the genetic information

Issues with Replication• Prokaryotes: (ex. E. coli)

– Have one singular loop of DNA– E. coli has approx. 4.6 million Nucleotide base pairs– Rate for replication: 500 nucleotides per second

• Eukaryotes w/Chromosomes:– Each chromosome is one DNA molecule– Humans (46) has approx. billion base pairs– Rate for replication: 50 per second (humans)

• Errors that occur: – Rate is one every 10 billion nucleotides copied– Proofreading is achieved by DNA polymerase (pg. 305)

Telomeres• Short, non-coding pieces of DNA• Contains repeated sequences (ie. TTGGGG 20 times)• Can lengthen with an enzyme called Telomerase• Lengthening telomeres will allow more replications to occur.• Telomerase is found in cells that have an unlimited number of cell

cycles (commonly observed in cancer cells)• Artificially giving cells telemerase can induce cells to become

cancerous• Shortening of these telomeres may contribute to cell aging and

Apotosis (programmed cell death)

Ex. A 70 yr old person’s cells divide approx. 20-30X vs an infant which will divide 80-90X

Telomeres