how was dna determined to be the genetic (hereditary / information carrying) material of life?

156
How was DNA determined to be the genetic (hereditary / information carrying) material of life? Chapter 16 - Molecular Basis of Inheritance

Upload: robbin

Post on 24-Jan-2016

24 views

Category:

Documents


0 download

DESCRIPTION

Chapter 16 - Molecular Basis of Inheritance. How was DNA determined to be the genetic (hereditary / information carrying) material of life?. Chapter 16 - Molecular Basis of Inheritance. - PowerPoint PPT Presentation

TRANSCRIPT

Page 1: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of Inheritance

Page 2: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Once Morgan showed that genes are located on chromosomes, the two chemical components of

chromosomes, ________________________, became the candidates for the genetic

(hereditary) material of life…the race was on.

Chapter 16 - Molecular Basis of Inheritance

DNA and Protein

Page 3: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1928

Streptococcus pneumonia

Streptococcus pneumonia

Smooth strain is pathogenic (can cause infection) due to the presence of a capsule that protects the bacterium from the host’s immune system.

Page 4: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1928

Streptococcus pneumonia

Streptococcus pneumonia

Page 5: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1928

Streptococcus pneumonia

Streptococcus pneumonia

Page 6: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1928

Streptococcus pneumonia

Streptococcus pneumonia

The harmless rough strain and harmless dead smooth strain were mixed together and incubated before being injected into the mouse…

Page 7: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1928

Streptococcus pneumonia

Streptococcus pneumonia

What the?

Page 8: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Conclusion: There is some “factor” that carries information that is released by the Smooth strain upon heating and transforms the Rough strain into the smooth strain. In 1928, researchers had no idea what was doing this, but this set the stage for what would be a 14 year search by three scientists (Avery, MacLeod and McCarty) to figure it out…

Page 9: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

You now know that something is transforming the Rough strain that is present in the Smooth strain. What would you do next?

Page 10: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Avery, MacLeod and McCarty’s experiment:

Year: 1944

(heat kill)They took the Smooth strain and heat killed it just like Griffith.

Page 11: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1944

(heat kill)

Avery, MacLeod and McCarty’s experiment:

They then separated out the four major classes of macromolecules from the mixture.What do you think they did next?

Page 12: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1944

(heat kill)

Avery, MacLeod and McCarty’s experiment:

They mixed each class separately with the Rough Strain and injected it into mice…What do you hypothesize their results were?

Page 13: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1944

(heat kill)

Avery, MacLeod and McCarty’s experiment:

The mice receiving the Rough strain mixed with nucleic acids from the smooth strain died, while the other mice were fine.CONCLUSION:

The transforming or information carrying factor is DNA!!

Page 14: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1944

Avery, MacLeod and McCarty’s experiment:Skepticism

It is the nature of critical thinkers like scientists to be skeptical of everything…and this finding was no exception.Most scientists believed (a dangerous notion) that protein was a better candidate due to their great heterogeneity (diversity) in contrast to DNA, which was highly uniform in nature.

Page 15: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Fig. 10.1A

Bacteriophage

This bring us to 1952, to the VIROLOGY Laboratory of Alfred Hershey and Martha Chase.

Page 16: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Fig. 10.1A

Bacteriophage

In order to figure out the type of nucleic acid, Hershey and Chase (1952) performed an experiment using viruses that infect bacteria called bacteriophages shown above. They obviously didn’t know what they looked like, but they knew that they were made solely of protein and DNA, and that they were able take over a bacterial cell and transform it into a virus-producing factory…The question was, was the protein or the DNA carrying the information to transform the cells?

Page 17: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Fig. 10.1A

Bacteriophage

BEFORE getting into the experiment, let’s compare the structure of a phage to an animal virus and then look at how phages work (how they infect bacterial cells)…

Page 18: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

I. Animals virus (influenza – virus that causes the flu)

Know the anatomy (structure) of these viruses…be able to draw and label them.

Viruses are nothing more than packaged nucleic acid.

II. Bacterial virus (right; a bacteriophage)Capsid – composed of protein, encloses nucleic acid (there are DNA as well as RNA viruses)Envelope – similar to a plasma membrane – composed of phospholipids and integral membrane proteins that will act as ligands and bind to cell receptors to gain access to the cell.

All parts made of protein except DNA of course.Tail fibres – bind to surface of bacterium acting as ligands

See video and figures

Page 19: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Reproductive cycle of a bacteriophage

Page 20: How was DNA determined to be the genetic (hereditary / information carrying) material of life?
Page 21: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Bacteriophage life cycle

Page 22: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Year: 1952

Let’s now get back to the Hershey-Chase Experiment

Is it the protein or the DNA that is the information molecule responsible for entering the bacterium and transforming it into a virus producing factory?

Page 23: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

The experiment was performed using radioactive isotopes to be able to follow the protein or DNA.

Isotopes of what elements would you use for protein/DNA labeling?

Page 24: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Phages were initially prepared for the experiment by growing them in the

presence of radioactive sulfur (35S) making the ONLY proteins radioactive

as protein have sulfur (the amino acids methionine and cysteine), while

DNA does not.

Let’s follow the protein first:

Page 25: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

1. INFECTION: The phage was mixed with bacteria as shown above to allow them to infect the prokaryote.…

Let’s follow the protein first:

Page 26: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

2. AGITATE: After the bacteria were infected, the cells were banged around (agitated) using a blender so that anything stuck to the surface from the phage would fall off. We are only interested in what goes inside to transform the cell…

Page 27: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

3. CENTRIFUGE: The mixture was then centrifuged, which will pellet the bacterial cells (more dense) on the bottom of the tube.What do you do next?

Page 28: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

4. RADIOACTIVITY ASSAY: Is the cell pellet radioactive or is the supernatant radioactive? (The supernatant is the liquid portion of a centrifuged sample)

Result – the supernatant was radioactive, the protein did not go in the cell. Does that mean the DNA went in for certain?How do we test if the DNA went in the cell?

Page 29: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

You do the same experiment except instead of making the protein radioactive, you make the DNA radioactive in the beginning by growing the virus in radioactive phosphorus (32P)…Why 32P?

Because protein does not have P.

Page 30: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Page 31: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Page 32: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

Page 33: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How was DNA determined to be the genetic (hereditary) material of life?

ConclusionDNA carries the information to transform the cell. DNA is the hereditary/information carrying material of life.

Page 34: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Timeline Recap:

Chapter 16 - Molecular Basis of Inheritance

1866 - Mendel’s work published never gaining popularity.

1875 - Mitosis figured out 1890 - Meiosis figured out 1900 – Mendel’s work rediscovered1902 – Chromosomal Theory of Inheritance gains popularity

1910 – Morgan and co-workers show genes are on chromosomes – are genes protein or DNA?.1928 – Griffith shows transformation – substance in S strain transferred to R, turning R into S

1944 – Avery, Macleod, and McCarty show this substance to be DNA1952 – Hershey and Chase confirm concluding DNA to be the hereditary material of life.

Very little was known about DNA at this point. How does this molecule work? How does it store hereditary information? The findings of Hershey and Chase catalyzed one of the most controversial stories in science…

The race for the structure of DNA.

Page 35: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Movie: The secret of Photo 51

Chapter 16 - Molecular Basis of Inheritance

The race for the structure of DNA.

Page 36: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

The road to the structure of DNA

Chapter 16 - Molecular Basis of Inheritance

(but first a quick review of the structure)

Page 37: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

A nucleotide

AIM: Describe the structure of DNA and RNA?

Page 38: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

AIM: Describe the structure of DNA and RNA?

Page 39: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

Pyrimidines vs Purines

AIM: Describe the structure of DNA and RNA?

Page 40: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

AIM: Describe the structure of DNA and RNA?

Page 41: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

NomenclatureAIM: Describe the structure of DNA and RNA?

Page 42: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

Draw a nucleotide found in RNA that contains a pyrimidine base and identify the base possibilities.

AIM: Describe the structure of DNA and RNA?

Page 43: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

Draw a nucleotide found in DNA that contains a purine base and identify the base possibilities.

AIM: Describe the structure of DNA and RNA?

Page 44: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

Draw CMP. Is CMP found in DNA or RNA?

AIM: Describe the structure of DNA and RNA?

Page 45: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

DNARNAvs

AIM: Describe the structure of DNA and RNA?

Page 46: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

What do we dowith monomers?

AIM: Describe the structure of DNA and RNA?

Page 47: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

Be able to draw and number the carbons

AIM: Describe the structure of DNA and RNA?

Page 48: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

AIM: Describe the structure of DNA and RNA?

Page 49: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

AIM: Describe the structure of DNA and RNA?

Page 50: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

The phosphate of one binds to the #3 C of the next.

Will this be DNA or RNA? Explain.

AIM: Describe the structure of DNA and RNA?

Page 51: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

DNA or RNA?

The Paper and the Ink

AIM: Describe the structure of DNA and RNA?

Page 52: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

AIM: Describe the structure of DNA and RNA?

Page 53: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

Chargaff’s Rule

AIM: Describe the structure of DNA and RNA?

He did NOT figure out base-pairing rules!!

Page 54: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

AIM: Describe the structure of DNA and RNA?

Page 55: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

AIM: Describe the structure of DNA and RNA?

Page 56: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

AIM: Describe the structure of DNA and RNA?

Page 57: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

AIM: Describe the structure of DNA and RNA?

Page 58: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

Base Pairing

AIM: Describe the structure of DNA and RNA?

Page 59: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

Base Pairing

AIM: Describe the structure of DNA and RNA?

Page 60: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells The a

nti

-para

llel

stra

nd

AIM: Describe the structure of DNA and RNA?

Page 61: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

How many base pairs (bp) are in this DNA molecule?

How many base pairs (bp) are in the entire human genome?

The Double Helix

AIM: Describe the structure of DNA and RNA?

Page 62: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 3 - The Molecules of Cells

Requires 200 volumes the size of a Manhattan telephone book (at 1000 pages each) to hold it all. It would take about 9.5 years to read out loud (without stopping) the 3 billion bases in a person's genome sequence.

3 billion base pairsAIM: Describe the structure of DNA and RNA?

Page 63: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

The road to the structure of DNA

Page 64: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

- Swiss chemist Friedrich Miescher identified what he called “nuclein” inside the nuclei of white blood cells

Miescher Discovers DNA - 1869

- Identified the substance to contain phosphorous

(Mendel published his work in 1866)

Page 65: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

1. Russian Biochemist

4. He proposed that nucleic acids were polymers of nucleotides in a circular arrangement (tetranucleotide)

Phoebus Levene exposes the nucleotide (1919)

2. Discovered the individual unit of the nucleotide – ribose/deoxyribose, nitrogenous bases, and the phosphate3. Discovered the order of the components of the nucleotide

Phosphate – sugar - base

Page 66: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

If this model were correct, what should we observe concerning the amount of dGMP relative to dCMP, dTMP and dAMP in every species?

Phoebus Levene (1919)

They should be present in an equal amount or a 1:1:1:1 ratio.

He did not believe DNA to be the genetic material as it was too simple…

Page 67: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

1. Austrian Biochemist

Chargaff formulates his rule (1950)

2. Discovered that the nucleotide ratios were not 1:1:1:1 and that the ratios of different species varied

(Ex. One species might have 20% C while another has 28% C)3. Discovered that in the DNA of all species he looked at the amount of adenine is equal to the amount of thymine, and that the amount of cytosine is equal to the amount of guanine (Chargaff’s Rule)

Page 68: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

1. English Physicist (x-ray crystallography)

Rosalind Franklin and photo 51

X-ray Diffractiona. A technique used to take pictures of molecules too small for any type of microscope to really observe.

b. These small molecules can be proteins, DNA, nucleotides, water, NAD+, heme or any other molecule

X-rays are shot at the sample and “bounce” off of it. They are measured and can be used to determine the structure.

Page 69: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

1. English Physicist (x-ray crystallography)

Rosalind Franklin and photo 51 – (1952)

2. She shot very pure DNA samples with X-rays and took hundreds of pictures. One of these was the incredibly clear image called “Photo 51”.

Photo 51

3. If you were trained in x-ray crystallography, this image shows that DNA is a double helix…If she knew it was a double

helix, why didn’t Dr. Franklin determine the structure of DNA?

Page 70: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

1. Wilkins worked in a lab alongside Franklin’s lab (both a King’s College in London) and was her colleague and boss in some respect…

The controversy behind photo 51

2. Without going into the full story, Wilkins got a hold of “photo 51” and showed it either on purpose or inadvertently to James Watson (worked 50 miles away in Cambridge).

Franklin

James Watson (left)Francis Crick (right)

Page 71: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

The controversy behind photo 51

Franklin

3. Watson ran back to Cambridge where he worked with Francis Crick. Neither ever actually did an experiment on DNA. They used other peoples data to try and figure out the structure (Chargaff, Levene, Meischer, Avery, Griffith, etc…)

4. They now had the final piece to the puzzle – Franklin’s photo 51

Page 72: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

The controversy behind photo 51

Franklin

In 1953, Watson and Crick published the structure of DNA in the journal Nature. In the same issue, Wilkins and Franklin published photo51.In 1958, Rosalind Franklin died

from cancer.In 1962, the Nobel prize was awarded to the scientists involved in determining the structure of DNA:Watson, Crick, and Wilkins

Page 73: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

The controversy behind photo 51

FranklinMost scientists hypothesize that had Watson not gotten a view of photo 51, Franklin would have figured out the structure of DNA…

Page 74: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

Page 75: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

A Double Helix is simply a twisted ladder

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

Page 76: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceAIM: The Road to the Structure of DNA

Page 77: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 78: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 79: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 80: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 81: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 82: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 83: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 84: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 85: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 86: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 87: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 88: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 89: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 90: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 91: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 92: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 93: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 94: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 95: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 96: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 97: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 98: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 99: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 100: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 101: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 102: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 103: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 104: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 105: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 106: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 107: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 108: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 109: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 110: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 111: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

C-GG-CA-TG-CG-CT-AT-AT-AA-TA-TA-TC-GC-GT-AC-GG-C

Page 112: How was DNA determined to be the genetic (hereditary / information carrying) material of life?
Page 113: How was DNA determined to be the genetic (hereditary / information carrying) material of life?
Page 114: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

We are now going to move back down to the molecular level (molecular biology) to understand precisely how DNA…

Chapter 16 - Molecular Basis of Inheritance

1. …is replicated during S phase so that the information it encodes needed to build/maintain organisms can be passed to the next generation.

2. …stores this information that will be used to make all the RNA/polypeptides that will directly build/maintain the organism.

molecular biology- the study of biology at the molecular level (overlaps biochemistry and genetics in particular). Much of what we have done thus far is molecular biology – cell resp, photosyn, membrane transport, endomembrane system, central dogma, etc… Mendelian genetics is not because you never discuss the molecular level, but chromosomal genetics is.

Page 115: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceNEW AIM: How is DNA replicated

How is the DNA (chromosomes) replicated (copied) during mitosis and meiosis so that it can be passed to the offspring/new cells?

How are the chromosomes

replicated during S phase?

What is the next question you would ask about DNA now that you “know” it to be the hereditary molecule of life?

Page 116: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Immediately after determining the structure of DNA (1953), Watson and Crick proposed what is known as the semi-conservative model of DNA replication, and they happened to be correct although they would now know this until experiments done by American geneticists Meselson and Stahl in 1958…

Chapter 16 - Molecular Basis of InheritanceNEW AIM: How is DNA replicated

DNA REPLICATION

Page 117: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceNEW AIM: How is DNA replicated

Before the answer in 1958, three models were proposed concerning how DNA replicated shown above – the conservative model, the dispersive model and the now known to be correct semiconservative model.

Page 118: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceNEW AIM: How is DNA replicated

Each model after two replications:

What experiment could you do to determine which was correct?

Page 119: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of InheritanceNEW AIM: How is DNA replicated

Once again, use radioactive isotopes: Meselson and Stahl cultured (grew) E. coli in the presence of 15N-labeled nucleotide precursors (molecules that will be converted into nucleotides by enzymes).

Technique is called analytical centrifugation as is quite popular still today if you are interested…

Page 120: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Fig. 10.4A

Chapter 16 - Molecular Basis of InheritanceAIM: How is DNA replicated – The semi-conservative model

What must happen first?The DNA strands must separate. An enzyme known as DNA helicase does this (an enzyme that unwinds and opens a helix is called a helicase – get it?)…

GENERAL OVERVIEW

Page 121: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?Chapter 16 - Molecular Basis of InheritanceAIM: How is DNA replicated – The semi-conservative model

Now what must happen?

-The two strands called template or parent strands will be used as a template to fill in the new strands.

-The template is what you look at to make a new copy. It is a pattern you follow.

GENERAL OVERVIEW

…and lastly?

Page 122: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Fig. 10.4A

Chapter 16 - Molecular Basis of InheritanceAIM: How is DNA replicated – The semi-conservative model

Nucleotides, which are in high concentration and randomly diffusing around the cell / in the nucleus of eukaryotes, are correctly paired and attached to each other (dehydration synthesis) by the enzyme…

DNA polymerase

GENERAL OVERVIEW

Page 123: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Fig. 10.4A

Chapter 16 - Molecular Basis of InheritanceAIM: How is DNA replicated – The semi-conservative model

The result is two identical daughter chromosomes, each containing one strand from the original parent molecule and one newly synthesized strand called the daughter strand, which is complementary to the parent strand (semi-conservative).

GENERAL OVERVIEWParent or template strands

Daughter or complementary strands

Page 124: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Human chromosomes are millions of basepairs (bps) long. DNA polymerase can only move at most 50 base pairs per second in mammals and 500 per second in bacteria. How long would it take to replicate chromosome number 1 in humans, which is 246 million base pairs?

Almost 57 days – TOO SLOW!!

Double-Stranded DNA Molecule

AIM: How is DNA replicated?

Page 125: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

How would you hypothesize the cell gets around this?

There must be many DNA polymerases working at the same time starting at different points along the DNA called ORIGINS OF REPLICATION (arrows above).

Page 126: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

There is a specific sequence in the DNA that makes it an origin. A single chromosome in humans can have upwards of 2,000 origins of replication to speed up the process (only three are shown above).What needs to happen to the double-stranded DNA in order to start this process?

Page 127: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

At each origin the double-stranded parental (template) DNA is opened up (the strands are separated by a protein called DNA helicase).

The regions of separated DNA are called REPLICATION BUBBLES because they look like little bubbles in the DNA…

Page 128: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

The daughter strands are synthesized IN BOTH DIRECTIONS FROM EACH BUBBLE via complementary base pairing with the parental strand by DNA polymerases until they all meet up…

Page 129: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

The result is two new semi-conserved (one parent strand and one newly synthesized daughter strand), identical (if no mistakes are made, which is unlikely) DNA molecules.

Page 130: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Page 131: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Now let’s look at the players that make this process possible.

Players = proteins, but you know that.

Page 132: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is POOP replicated?

- The enzyme that catalyzes the polymerization of DNA (extension - it is extending the polynucleotide), adding the appropriate nucleotide (dATP, dTTP, dCTP, CGTP) across from the complementary base in the parent strand.

DNA polymerase

Page 133: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

DNA polymerase

In other words, it can only add nucleotides to the 3’-OH of a nucleotide and not to the phosphate end.

3’

Can add nucleotides here

Cannot add nucleotides here

**Can only make the new daughter strand FROM the 5’ end toward the 3’ end. In other words, it can only add nucleotides to the 3’-OH of a nucleotide and not to the phosphate.

Page 134: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

DNA polymerase

DNA Polymerase cannot bind to single stranded DNA.

3’ 5’

Page 135: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

DNA polymerase

DNA Polymerase cannot bind to single stranded DNA.

3’ 5’

It will require a short piece of RNA called a primer, which will be added by the enzyme RNA primase.The primer “primes” the DNA or gets it ready for DNA polymerase .

U G C C U G3’5’

Page 136: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Where does the “energy” come from to synthesize DNA (Where does DNA polymerase get the ability to polymerize DNA by making phosphodiester bonds between nucleotides)?

DNA polymerase

It comes from the nucleotides themselves as they are triphosphates! – dATP, dCTP, dGTP and dTTP. Think affinity - would AMP “rather” be attached to the sugar of another nucleotide or to two negativity charged phosphates?

3’

Page 137: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

DNA polymerase

- Can catalyze 50 bases/sec in mammals and 500 bases/sec in prokaryotes- Error Rate of 1 mistake in every 100,000 nucleotides…If not repaired this mutation can result in a new allele if in the gametes, can cause cancer, can have no effect, etc…a roll of the genetic dice.

Reaction Rate:

Error Rate:

Page 138: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

-DNA polymerase has a cool 3’-5’ exonuclease capability (exo – exit, take out; nuclease – hydrolyze nucleic acid; 3’-5’ – the reverese direction). -Therefore, when an incorrect base is added, the polymerase can take a step backwards in the 3’ to 5’ direction, and cut it out followed by insertion of the correct base = proofreading (fixes its own mistakes)

DNA polymerase

An dATP by chance diffused into the active site and was incorporated across from a C.

Typically it will randomly bounce in and then bounce out.

Proofreading:

Page 139: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

- Catalyzes synthesis of daughter strand of DNA from 5’ to 3’ using complementary base pairing rules against template strand

DNA polymerase Review

- Can proofread and fix errors

- Makes mistakes 1 in 100,000 bases

- Catalyzes between 50 and 500 bases/second- Cannot bind single stranded DNA (ssDNA), requires the presence of a primer to make is double-stranded (dsDNA)- Synthesis is endergonic and is coupled to the dephosphorylation of the nucleotide (dTTP, dCTP, dATP, or dGTP) as it is added

Page 140: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Each Replication bubble can be viewed as two identical replication forks, just rotated by 180 degrees.

replication forkreplication forks Fork in the road

Page 141: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

It is time to get into the details.

We will focus on one fork since both work the same way (the process is the same).

Page 142: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

5’

5’

3’

3’

DNA is traditionally drawn with the top strand going 5’ to 3’ (or 5’ is in the top left corner).

Page 143: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

GENERAL OVERVIEW:

5’

5’

3’

3’Origin of replication

Replication bubbles form and DNA polymerase starts catalyzing the polymerization of DNA at the origins of replication…

Page 144: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Both forks of every bubble are being replicated at the same time…The animation you just watched was of only one fork, but the same thing is happening at the other fork.

Page 145: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

http://www.wiley.com/legacy/college/boyer/0470003790/animations/replication/replication.htm(Try to put it into words as you watch )

THE DETAILS:

Page 146: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Page 147: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Page 148: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

Page 149: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

1. DNA Polymerase

2. Helicase

3. RNA Primase

4. DNA Ligase

5. Single-Stranded Binding Proteins

6. Topoisomerases (gyrase in bacteria)

- Catalyzes the polymerization of deoxynucleotides in 5’ to 3’ direction using the energy in the nucleotides. Removes primers and fill in DNA nucleotides.- Unwinds and pulls apart the strands using the energy in ATP

- Adds the RNA primer for DNA polymerase using energy from the RNA nucleotides themselves.

- Seals together the Okazaki fragments using ATP for energy

- Bind to the single-stranded DNA, preventing it from reannealing or coming back together after helicase separates them.- Ride ahead of the helicase and unwind the DNA coils caused by helicase’s activity using ATP to do this…

THE PLAYERS:

Page 150: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

This simple process allows for the information stored in DNA to be replicated so that it can be passed from cell to cell, generation after generation, in all life ever observed by humans for billions of years…

Page 151: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

AIM: How is DNA replicated?

http://www.hhmi.org/biointeractive/dna/index.html

Page 152: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of Inheritance

Page 153: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of Inheritance

What happens when the replication fork arrives at the end of a chromosome?

Page 154: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of Inheritance

Page 155: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of Inheritance

DNA RepairALL cells have proteins whose job it is to identify and repair DNA damage.

A number of different mechanisms have evolved to do this each requiring a different set of proteins :

1. Base Excision Repair

2. Nucleotide Excision Repair

3. Mismatch Repair

4. Direct Reversal

5. Recombinational Repair

All mechanisms are found together in a cell as each is specialized to handle a different type of damage. You can think of them as different types of doctors (MDs).

Page 156: How was DNA determined to be the genetic (hereditary / information carrying) material of life?

Chapter 16 - Molecular Basis of Inheritance

To the right is one of these mechanisms in a bit of detail…Nucleotide Excision Repair.

This mechanism recognizes large helical distortions (changes in shape) resulting in one strand of this section being excised (cut out) and replaced.

DNA Repair