Chapter 10iws.collin.edu/cdoumen//1408/1408_Ch8_9/1408_Ch10.pdf · Chapter 10 Lecture by Edward J ... • its mode of replication, and how DNA serves as the basis of heredity

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PowerPoint Lectures Campbell Biology: Concepts & Connections, Eighth Edition REECE • TAYLOR • SIMON • DICKEY • HOGAN

Chapter 10

Lecture by Edward J. Zalisko

Molecular Biology of the Gene

Biol 1408 Dr. Doumen


Introduction

• The 2009 H1N1 influenza (flu) virus •  spread so quickly that it was declared a pandemic, •  reached 207 countries, •  infected more than 600,000 people, and •  killed an estimated 20,000 people.

• Viruses share some of the characteristics of living organisms, but are generally not considered alive because they are not cellular and cannot reproduce on their own.


Introduction

• So what makes up a virus ? •  In general a virus is made from a

• Capsid : this is the protein shell of a virus •  Internal genome : usually a DNA or RNA sequence. •  Some viruses have an additional viral envelope.

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Introduction

Examples of common viruses ( look at the size and also see next slide for comparison)


Introduction


Introduction

• Viruses can be dangerous and combating any virus requires a detailed understanding of

• molecular biology, •  the study of DNA/RNA, and •  its mode of replication, and how DNA serves as the

basis of heredity.

• The study on how viruses work has helped scientists in the discovery of the genetic material and how it works in most living cells.

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SCIENTIFIC THINKING: Experiments showed that DNA is the genetic material

• Early in the 20th century, the molecular basis for inheritance was a mystery.

• Biologists did know that genes were located on chromosomes. But it was unknown if the genetic material was a protein based system or a nucleic acid based system.


SCIENTIFIC THINKING: Experiments showed that DNA is the genetic material

• Biologists finally established the role of DNA in heredity through experiments with bacteria and the viruses that infect them.

• This breakthrough ushered in the field of molecular biology, the study of heredity at the molecular level.


Frederick Griffith Experiment

• Pneumonia was a serious cause of death following Spanish Influenza Pandemic (1918)

• Frederick Griffith (1879–1941), a British bacteriologist, wanted to create a vaccine against pneumonia and started working with pneumococcus bacteria, using mice as his ‘patients’

Streptococcus pneumoniae.

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• He used two strains of bacteria •  S-strain (virulent):

•  this bacteria covers itself with a smooth capsule, protecting itself against the host’s immune system

•  It will kill the host • R-strain (non-virulent):

•  Does not have a protective capsule and gets killed by the host’s immune system.

•  The host will not die




• At that time, they believed that these strains were fixed and un-changeable: the R-strain could not become the S-strain and visa versa

• They also had a test to see which form was growing in a petri-dish

• His experiment was to inject mice with the different strains and see what happened.

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•  Inject mice with living S-strain bacteria • Result: mice die

•  Inject mice with living R-strain bacteria • Result : mice stay alive

•  Inject mice with heat killed S-strain • Result : mice stay alive

•  Inject mice with a mix of living R-strain and heat killed S-strain

• Result : mice die





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• When they examined the blood of the dead mice from the last experiment, they found live S-strain bacteria that could be culture

• All of the descendants of the transformed bacteria

inherited the newly acquired ability to cause disease



• Griffith concluded that some transforming factor ( he called it a “transforming principle”) present in the dead S-strain, had transformed the living R-strain

• So, the harmless strain was transformed into a deadly strain because something from the heat-killed, dead bacteria strain slipped into the harmless strain and made them virulent.



• Today, we know that the "transforming principle" Griffith observed was the DNA of the S-strain bacteria

• That DNA survived the heating process and was taken up by the R-strain, providing the genes to make the protective capsule

• The exact nature of the transforming principle (the DNA) was verified by later experiments (eg. The Hershey Chase experiment)

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Hershey – Chase Experiments

•  In 1952, Alfred Hershey and Martha Chase used bacteriophages to show that DNA is the genetic material

•  Bacteriophages (or phages for short) are viruses that infect bacterial cells.

•  They used a bacteriophage called of T2, a virus that infects the bacterium Escherichia coli (E. coli).


A bacteriophage : a virus that infects bacteria


A phage attaches itself to a bacterial cell.

The phage injects its DNA into the bacterium.

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Reproductive Cycle of a Bacteriophage

•  The phage attaches to the cell wall of a bacterium •  It injects its DNA into the bacterial cell •  The viral DNA directs the destruction of the host DNA • Viral genes instruct the making of replicate viral DNA

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The phage DNA directs the host cell to make more phage DNA and proteins; new phages assemble.

The cell lyses and releases the new phages.

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•  The viral DNA takes charge of the protein production line of the bacteria (eg. Ribosomes).

• Viral genes direct the protein machinery to make new viral proteins and assemble new viruses

•  Bacterium lyses and new viruses spill out



Figure 10.1c-0

A phage attaches itself to a bacterial cell.

The phage injects its DNA into the bacterium.

The phage DNA directs the host cell to make more phage DNA and proteins; new phages assemble. The cell lyses

and releases the new phages.

1 2 3



Hershey-Chase Experiments

• Hershey and Chase knew their biology and biochemistry very well to realize that sulphur is an element found in certain amino acids

• On the other hand, phosphorus is an element mainly found in nucleic acids such as RNA and DNA

• They came up with a brilliant idea to figure out weather protein or nucleic acids were the biomolecule that carries the genetic information.

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Grow viruses in a media with radioactive sulfur

Sulfur will become incorporated into protein

Thus the coat/shell of the viruses will be radioactive

Grow viruses in a media with radioactive phosphorus

Phosphorus will become incorporated into DNA/RNA

Radioactive material will now be inside the virus



• They infected bacteria • with radioactive sulfur labeled viruses ( so the

radioactive element is in the protein coat) Or

• with radioactive phosphorus labeled viruses ( so the radioactive element is inside in the DNA)

• After a while, they used a simple blender to shake off all the viruses from the bacteria and centrifuged it all down ( bacteria would pellet down – viruses remain in upper suspension liquid)


•  In bacteria infected with radio-active sulfur, most radioactivity remained in suspension liquid, none in the bacterial pellet.

•  The new viruses that eventually came out of the bacterial pellet had no radioactive sulfur

•  In bacteria infected with radio-active phosphorus, most radioactivity was found within the bacterial pellet

•  The new viruses that eventually came out of the bacterial pellet contained radioactive phosphorus

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Figure 10.1b-0

Phage Bacterium

Radioactive protein

DNA

Empty protein shell Phage DNA

Centrifuge

Pellet Batch 1: Radioactive protein labeled in yellow

Radioactive DNA

Centrifuge

Pellet The radioactivity is in the pellet.

The radioactivity is in the liquid.

Batch 2: Radioactive DNA labeled in green



The Hershey and Chase experiment strongly supported DNA as the hereditary material while it also showed protein was NOT the hereditary material.


The search for the DNA structure

• After the 1952 Hershey-Chase experiment convinced most biologists that DNA was the material that stored genetic information, a race was on to determine how the structure of this molecule could account for its role in heredity.

• Researchers focused on discovering the three-dimensional shape of DNA.

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Watson and Crick

• American James D. Watson journeyed to Cambridge University in England, where the more senior Francis Crick was studying protein structure with a technique called X-ray crystallography.

• While visiting the laboratory of Maurice Wilkins at King’s College in London, Watson saw an X-ray image of DNA produced by Wilkins’s colleague, Rosalind Franklin.


Watson and Crick

Rosalind Franklin and her important X-ray diffraction analysis of DNA


DNA is a double-stranded helix

• Watson deduced the basic shape of DNA to be a helix (spiral) with a uniform diameter and the nitrogenous bases located above one another like a stack of dinner plates.

• The thickness of the helix suggested that it was made up of two polynucleotide strands.

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DNA is a double-stranded helix

• Watson and Crick realized that DNA consisted of two polynucleotide strands wrapped into a double helix.

•  The sugar-phosphate backbone is on the outside. •  The nitrogenous bases are perpendicular to the

backbone in the interior. •  Specific pairs of bases give the helix a uniform

shape. •  A pairs with T, forming two hydrogen bonds, and •  G pairs with C, forming three hydrogen bonds.


Hydrogen bond (dotted lines)

C

A

T

G C

T

A

G

Sugar-phosphate backbone


Pairs of nitrogenous bases linked with hydrogen bonds


Figure 10.3c

Twist Sugar-phosphate backbone

Pairs of nitrogenous bases linked with hydrogen bonds

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Watson and Crick


Figure 10.3d-0

Ribbon model Partial chemical structure Computer model

Base pair

Hydrogen bond C G

C G G C

G C

C G

C G

C G G C

T A

T A

A T

A T

A T

C

A

T A T T A

G C

T

A

G


•  In 1962, the Nobel Prize was awarded to James D. Watson, Francis Crick, and Maurice Wilkins.

• Rosalind Franklin probably would have received the prize as well but for her death from cancer in 1958.

• Nobel Prizes are never awarded posthumously. • The Watson-Crick model gave new meaning to the

words genes and chromosomes. The genetic information in a chromosome is encoded in the nucleotide sequence of DNA.

•  It still wasn’t clear how it was encoded….

Watson and Crick

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DNA and RNA are polymers of nucleotides

• DNA and RNA are nucleic acids consisting of long chains (polymers) of chemical units (monomers) called nucleotides.

•  A DNA nucleotide is composed of a •  nitrogenous base, •  five-carbon sugar, called deoxyribose •  phosphate group.

•  The nucleotides are joined to one another by a sugar-phosphate backbone.


Figure 10.2a-3

Phosphate group

Sugar (deoxyribose)

DNA nucleotide

Thymine (T)

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

A DNA nucleotide



Covalent bond joining nucleotides

A

T

G

G

C

Phosphate group Nitrogenous base Sugar

DNA nucleotide

Two representations of a DNA polynucleotide

A

T

G

G

C

A DNA poly-nucleotide

•  A DNA nucleotide can have 4 different nitrogen-containing base:

•  adenine (A), •  cytosine (C), •  thymine (T), and •  guanine (G).

•  A DNA polynucleotide will have those 4 bases represented in a certain order

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Thymine (T) Cytosine (C) Adenine (A) Guanine (G)

Pyrimidines Purines

The nitrogen-containing bases in DNA

•  Pyrimidines have a 6 ring structure and Purines are a combination of a 5

ring with a 6 ring •  Each base is part of a nucleotide. They are important in the DNA structure

as they form the “spokes” of the ladder, the connections what holds the double helix together.

•  T on one polynucleotide strand backbone always pairs with an A on the other strand

•  G on one polynucleotide strand backbone always pairs with an C on the other strand


A T

A T

A T

T A

T A

T A

T A

C G

C G

C G

G C

G C

G

A DNA double helix

A complete DNA Is a double helix

•  A complete DNA is a double helix of two polynucleotides, twisted around each other as shown earlier

•  Notice the base pairing between the two strands


10.2 DNA and RNA are polymers of nucleotides

• The full name for DNA is deoxyribonucleic acid, • RNA (ribonucleic acid) is unlike DNA in that it

• Has nucleotides made from the the sugar ribose (instead of deoxyribose in DNA) and

•  The nitrogenous bases are similar except that uracil (U) is used instead of thymine (T).

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Phosphate group

Sugar (ribose)

Uracil (U)

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

An RNA nucleotide

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Chapter 10iws.collin.edu/cdoumen//1408/1408_Ch8_9/1408_Ch10.pdf · Chapter 10 Lecture by Edward J ... • its mode of replication, and how DNA serves as the basis of heredity