Molecular Biology of the Gene

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Chapter 10 Molecular Biology of the Gene
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Is DNA the genetic material?

• Scientists knew that genes were on chromosomes
• Scientists also knew that chromosomes were made
  of DNA and proteins (remember chromatin?)
• Which one contained the genetic information?
• Proteins?
• DNA?

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Hershey and Chase

• Barbara Hershey and Alfred Chase are credited
  with discovering that DNA is the genetic material
• They performed experiments using bacteria and
  bacteriophage
• Bacteriophage type of virus called T2
• T2 infects E. coli and takes over its genetic
  machinery to make more T2
• T2 is made of only proteins and DNA

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T2 Bacteriophage
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T2 Bacteriophage lifecycle
Phage injects DNA.
Phage DNA directs host cell to make more
phage DNA and protein parts. New phages assemble.
Phage attaches to bacterial cell.
Cell lyses and releases new phages.
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Hershey and Chase experiment

• They grew two different sets of T2
• Radioactive phosphorous is incorporated into
  DNA
• Radioactive sulfur is incorporated into
  proteins

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Radioactive protein
Empty protein shell
Phage
Bacterium
Phage DNA
DNA
Batch 1 Radioactive protein
Mix radioactively labeled phages with bacteria.
The phages infect the bacterial cells.
Agitate in a blender to separate phages
outside the bacteria from the cells and their
contents.
Radioactive DNA
Batch 2 Radioactive DNA
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Empty protein shell
Radioactivity in liquid
Phage DNA
Centrifuge
Pellet
Measure the radioactivity in the pellet and the
liquid.
Centrifuge the mixture so bacteria form a pellet
at the bottom of the test tube.
Centrifuge
Radioactivity in pellet
Pellet
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What did Hershey and Chase find?

• Radioactive phosphorous radioactivity in
  bacteria
• T2 DNA was in the bacteria
• Radioactive sulfur radioactivity in the liquid
• T2 proteins were in the liquid
• RESULTS INDICATED DNA WAS USED TO TRANSMIT
  GENETIC INFORMATION

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Nucleic acids

• DNA and RNA are polymers of nucleotides
• Nucleotides have 3 parts
• 5 carbon sugar (deoxyribose or ribose)
• Phosphate group
• Nitrogenous base (A, C, T(U), or G)

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Sugar-phosphate backbone
Phosphate group
Nitrogenous base
A
A
Sugar
Nitrogenous base (A, G, C, or T)
C
C
Phosphate group
DNA nucleotide
T
T
Thymine (T)
G
G
Sugar (deoxyribose)
T
T
DNA nucleotide
DNA polynucleotide
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Nitrogenous Bases
Thymine (T)
Cytosine (C)
Adenine (A)
Guanine (G)
Purines
Pyrimidines
Uracil in RNA
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DNA Structure
G
C
T
A
Hydrogen bond
A
T
Base pair
A
T
C
G
G
C
T
A
C
G
G
C
C
G
T
A
T
A
T
A
T
A
T
A
G
C
A
T
Computer model
Ribbon model
Partial chemical structure
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DNA Replication
T
T
A
A
T
T
T
A
A
A
C
G
C
C
C
G
G
G
G
C
G
C
G
C
C
C
G
G
C
A
A
T
A
A
A
T
T
T
A
T
T
A
A
T
T
A
Nucleotides
Parental molecule of DNA
Both parental strands serve as templates
Two identical daughter molecules of DNA
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G
C
A
T
G
C
G
C
A
T
T
A
G
C
A
T
C
G
G
C
C
G
G
C
C
C
G
A
C
A
G
T
A
T
T
G
T
T
G
T
A
A
T
A
A
A
T
C
A
T
T
A
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Parental strand
Origin of replication
Daughter strand
Bubble
Two daughter DNA molecules
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From genotype ? phenotype?

• One gene one polypeptide hypothesis
• Each gene in DNA has information for the
  production of 1 polypeptides
• A single polypeptide may be one protein
• Several polypeptides may be combined to form one
  protein
• HOW DOES THE INFORMATION FLOW FROM DNA TO
  PROTEIN???

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DNA
Transcription
RNA
Translation
Protein
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From genotype ? phenotype?

• The sequence of the nucleotides in the RNA tell
  what amino acids are put in the polypeptide
• Nucleotides are arranged in sets of 3
• Called a codon
• Each codon 1 amino acid in the polypeptide
• There are 64 possible codons (for 20 amino acids)

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The Genetic Code
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Strand to be transcribed
T
T
T
C
T
A
C
A
A
C
A
A
DNA
A
A
G
T
T
T
A
A
G
G
T
T
Transcription
A
A
A
A
G
U
U
G
U
U
G
U
RNA
Start codon
Stop codon
Translation
Phe
Met
Polypeptide
Lys
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Transcription the basics

• Process of making RNA from DNA
• Occurs in nucleus
• Follows base pair rules
• Only one strand of DNA is used the template
  strand
• Enzyme that synthesizes the RNA strand is called
  RNA polymerase
• Begins at the promoter

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RNA polymerase
RNA polymerase
DNA of gene
DNA of gene
Promoter DNA
Promoter DNA
Terminator DNA
Terminator DNA
Initiation
Initiation
Initiation
Initiation
Transcription
Elongation
Growing RNA
Termination
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RNA nucleotides
RNA polymerase
A
A
C
C
T
T
A
U
T
C
T
G
U
G
A
C
C
C
A
U
A
C
C
A
G
A
T
T
G
T
G
A
Direction of transcription
Template strand of DNA
Newly made RNA
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Types of RNA

• There are 3 types of RNA
• mRNA (messenger)
• tRNA (transfer)
• rRNA (ribosomal)
• mRNA carries the message from the DNA to the
  ribosomes for translation into proteins

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Translation the basics

• Process of making polypeptide (protein) from RNA
• Occurs in cytoplasm (ribosomes)
• Uses codons
• Since we are changing languages, it requires an
  interpreter - tRNA

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Amino acid attachment site
Hydrogen bond
tRNA
RNA polynucleotide chain
Anticodon
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tRNA
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Ribosome
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Next amino acid to be added to polypeptide
Growing polypeptide
tRNA
mRNA
Codons
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Start of genetic message
End
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Translation
T
T
T
C
T
A
C
A
A
C
A
A
A
A
G
T
T
T
A
A
G
G
T
T
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Translation - Initiation
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Translation Initiation (cont.)
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Translation Elongation
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New peptide bond forming
Growing polypeptide
Elongation
A succession of tRNAs add their amino acids
to the polypeptide chain as the mRNA is
moved through the ribosome, one codon at a time.
Codons
mRNA
Polypeptide
Termination
The ribosome recognizes a stop codon. The
poly- peptide is terminated and released.
Stop codon
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Mutation base substitution
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Mutation base deletion/insertion
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Sickle Cell Disease

• Affects more than 60,000 people in the US, mostly
  African Americans.
• 1 in 1,000 babies are born with the disease.
• 1 in 400 African Americans are born with the
  disease.
• 1 in 10 African Americans are carriers of the
  sickle cell trait.

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Sickle cell disease affects hemoglobin, the major
protein in red blood cells. Hemoglobin
transports oxygen from the lungs to tissues
throughout the body.
Hemoglobin A (normal hemoglobin)
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Oxygen levels affect the structure of Hemoglobin
S in red blood cells
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Under low oxygen concentrations, sickle
hemoglobin polymerizes into long fibers
Hemoglobin S under high O2 levels
Hemoglobin S under low O2 levels
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Polymerization of hemoglobin at low oxygen levels
causes red blood cells to deform, taking on the
characteristic sickle shape.
SS red blood cells under high O2 levels
SS red blood cells under low O2 levels
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Analysis of AA and SS red blood cells by Light
Microscopy
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SICKLE CELL ANEMIA CLINICAL FEATURES
Red Blood Cells have a characteristic sickle
shape and are fragile, living only 10-30
days. Blood flow is disrupted by the sickle
cells Pain episodes result from these
events. There are periods of crisis during which
the symptoms are worsened. These episodes can be
brought on by infection, dehydration, high
altitude, or overexertion.
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Sickle Cell Anemia is an Autosomal Recessive
inherited disease
AA homozygous for normal gene,
individual is unaffected AS heterozygous
(trait), individual is a sickle cell
carrier SS homozygous for defective gene,
individual has sickle cell disease
To inherit sickle cell anemia, a child must get
one copy of the sickle gene (S) from each parent.
A normal beta globin allele S sickle beta
globin allele
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Sickle Cell Anemia
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Viruses

• Genes in a box generally not considered life
• A piece of genetic material (DNA or RNA) enclosed
  in a protein coat
• Some are also enclosed in a membrane
• Can only reproduce in a host cell

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Membranous envelope
RNA
Protein coat
Glycoprotein spike
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Common DNA Viruses

• Hepatitis
• Chicken pox
• Herpes

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Common RNA Viruses

• Influenza
• Common Cold
• Measles
• Mumps
• HIV
• Polio

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Life cycle of a typical virus
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Herpes

• Herpes Simplex Virus (HSV)
• Type I Cold sores, found in 75 of Americans
• Type II Genital herpes, found in 20 of
  Americans
• Can remain latent

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HIV
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Viral RNA
CYTOPLASM
NUCLEUS
DNA strand
Chromosomal DNA
Double- stranded DNA
Provirus DNA
Viral RNA and proteins
RNA
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Infected T-cell budding new viruses
NIBSC/Science Photo Library/Photo Researchers,
Inc.
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Clinical course of HIV infection
ACUTE VIRAL ILLNESS
CONSTITUTIONAL SYMPTOMS
CLINICAL LATENCY
AIDS
DEATH
1000 900 800 700 600 500 400 300 200 100
50 0
Fever, weight loss, night sweats Fungal,
viral, and bacterial infections
T-CELL COUNT
Pneumonia, Meningitis, Toxoplasmosis, Tuberculosis
, etc.
EVERYTHING including CMV and Mycobacterium
CD4lt50
0 1 2 3
4 5 6 7
8 9 10 APPROXIMATE TIME IN
YEARS
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A global view of HIV infection
Adult prevalence rate
15.0 36.0 5.0 15.0 1.0 5.0
0.5 1.0 0.1 0.5 0.0 0.1
not available
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About 14 000 new HIV infections/day

• More than 95 are in developing countries
• 2000 are in children under 15 years of age
• About 12 000 are in persons aged 15 to 49 years,
  of whom
• almost 50 are women
• about 50 are 1524 year olds

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Emerging Viruses

• An emerging virus is one that has recently
  appeared in the human population
• Well known examples
• HIV
• Ebola
• SARS

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Emerging Viruses (cont.)

• How do emerging viruses suddenly appear in the
  human population?
• Mutations
• Contact between species
• Spread from isolated populations

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Gene Regulation

• Describes the control of gene expression
• It enables cells to differentiate (become
  different types of cells)
• Genes can be regulated at transcription or
  translation

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Transcriptional Regulation

• Utilizes transcription factors to turn a gene on
  or off
• Two types of transcription factors
• Activators Turn gene on, bind to the DNA at an
  enhancer, help the RNA polymerase initiate
  transcription
• Repressors Turn gene off, bind to the DNA at a
  silencer, prevent the RNA polymerase from
  initiating transcription

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Translational Regulation

• Regulation of translation can occur in four ways
• mRNA breakdown
• Regulation of translation initiation
• Protein activation (after translation is
  complete)
• Protein breakdown

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0
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Clones

• An individual created by asexual reproduction and
  genetically identical to a single parent
• First report was in 1950s with Frogs
• First mammal was in 1997

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20 years old???
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Nuclear Implantation
Donor cell
Nucleus from donor cell
Remove nucleus from egg cell
Add somatic cell from adult donor
Grow in culture to produce an early embryo
(blastocyst)
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2 types of cloning
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Reproductive Cloning

• Potential applications
• Save endangered species
• Bring back an extinct species
• Determine effect of specific genes
• Create animals with specific traits
• Pharmaceutical development

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Problems with Cloning

• Conservationists worry people will see it as an
  easy way out
• No genetic diversity
• Cloned animals often unhealthy
• And, as always, ethical considerations!