Download Griffith`s Transformation Experiment

Review
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Search for genetic material---nucleic acid or protein/DNA or RNA?
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Griffith’s Transformation Experiment
Avery’s Transformation Experiment
Hershey-Chase Bacteriophage Experiment
Tobacco Mosaic Virus (TMV) Experiment
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Nucleotides - composition and structure
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Double-helix model of DNA - Watson & Crick
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Today’s Lesson
Organization of DNA/RNA in chromosomes
Search for the genetic material:
1.
2.
3.
Stable source of information
Ability to replicate accurately
Capable of change
Timeline of events:
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1890
Weismann - substance in the cell nuclei controls development.
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1900
Chromosomes shown to contain hereditary information,
later shown to be composed of protein & nucleic acids.
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1928
Griffith’s Transformation Experiment
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1944
Avery’s Transformation Experiment
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1953
Hershey-Chase Bacteriophage Experiment
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1953
Watson & Crick propose double-helix model of DNA
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1956
Gierer & Schramm/Fraenkel-Conrat & Singer
Demonstrate RNA is viral genetic material.
Frederick Griffith’s Transformation Experiment - 1928
“transforming principle” demonstrated with Streptococcus pneumoniae
Griffith hypothesized that the transforming agent was a “IIIS” protein.
Oswald T. Avery’s Transformation Experiment - 1944
Determined that “IIIS” DNA was the genetic material
responsible for Griffith’s results (not RNA).
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Hershey-Chase Bacteriophage Experiment - 1953
Bacteriophage = Virus that attacks
bacteria and replicates by
invading a living cell and using
the cell’s molecular machinery.
Structure of T2 phage
DNA & protein
Life cycle of virulent T2 phage:
Hershey-Chase Bacteriophage Experiment - 1953
1.
T2 bacteriophage is composed
of DNA and proteins:
2.
Set-up two replicates:
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Label DNA with 32P
Label Protein with 35S
3.
Infected E. coli bacteria with
two types of labeled T2
4.
32P
is discovered within the
bacteria and progeny phages,
whereas 35S is not found within
the bacteria but released with
phage ghosts.
1969: Alfred Hershey
Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956
Fraenkel-Conrat & Singer - 1957
• Used 2 viral strains to demonstrate RNA is the genetic material of TMV
Conclusions about these early experiments:
Griffith 1928 & Avery 1944:
DNA (not RNA) is transforming agent.
Hershey-Chase 1953:
DNA (not protein) is the genetic material.
Gierer & Schramm 1956/Fraenkel-Conrat & Singer 1957:
RNA (not protein) is genetic material of some viruses.
Nucleotide = monomers that make up DNA and RNA
Three components
1. Pentose (5-carbon) sugar
DNA = deoxyribose
RNA = ribose
(compare 2’ carbons)
2. Nitrogenous base
Purines
Adenine
Guanine
Pyrimidines
Cytosine
Thymine (DNA)
Uracil (RNA)
3. Phosphate group attached to 5’ carbon
Nucleotides are linked by phosphodiester bonds to form polynucleotides.
Phosphodiester bond
Covalent bond between the phosphate group (attached to 5’ carbon) of
one nucleotide and the 3’ carbon of the sugar of another nucleotide.
This bond is very strong, and for this reason DNA is remarkably stable.
DNA can be boiled and even autoclaved without degrading!
5’ and 3’
The ends of the DNA or RNA chain are not the same. One end of the
chain has a 5’ carbon and the other end has a 3’ carbon.
5’ end
3’ end
James D. Watson & Francis H. Crick - 1953
Double Helix Model of DNA
Two sources of information:
1.
Base composition studies of Erwin Chargaff
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indicated double-stranded DNA consists of ~50% purines
(A,G) and ~50% pyrimidines (T, C)
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amount of A = amount of T and amount of G = amount of C
(Chargraff’s rules)
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%GC content varies from organism to organism
Examples:
%A
%T
%G
%C
%GC
Homo sapiens
Zea mays
Drosophila
Aythya americana
31.0
25.6
27.3
25.8
31.5
25.3
27.6
25.8
19.1
24.5
22.5
24.2
18.4
24.6
22.5
24.2
37.5
49.1
45.0
48.4
James D. Watson & Francis H. Crick - 1953
Double Helix Model of DNA
Two sources of information:
2.
X-ray diffraction studies - Rosalind Franklin & Maurice Wilkins
Conclusion-DNA is a helical structure with
distinctive regularities, 0.34 nm & 3.4 nm.
Double Helix Model of DNA: Six main features
1.
Two polynucleotide chains wound in a right-handed (clockwise)
double-helix.
2.
Nucleotide chains are anti-parallel:
3.
Sugar-phosphate backbones are on the outside of the double
helix, and the bases are oriented towards the central axis.
4.
Complementary base pairs from opposite strands are bound
together by weak hydrogen bonds.
5’  3’
3’  5’
A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).
e.g.,
5’-TATTCCGA-3’
3’-ATAAGGCT-3’
5.
Base pairs are 0.34 nm apart. One complete turn of the helix
requires 3.4 nm (10 bases/turn).
6.
Sugar-phosphate backbones are not equally-spaced, resulting
in major and minor grooves.
1962: Nobel Prize in Physiology and Medicine
James D.
Watson
Francis H.
Crick
Maurice H. F.
Wilkins
What about?
Rosalind Franklin
Organization of DNA/RNA in chromosomes
Genome = chromosome or set of chromosomes that contains all the
DNA an organism (or organelle) possesses
Viral chromosomes
1. single or double-stranded DNA or RNA
2. circular or linear
3. surrounded by proteins
TMV
T2 bacteriophage
 bacteriophage
Prokaryotic chromosomes
1. most contain one double-stranded circular
DNA chromosome
2. others consist of one or more chromosomes
and are either circular or linear
3. typically arranged in arranged in a dense
clump in a region called the nucleoid
Problem:
Measured linearly, the Escherichia coli genome (4.6 Mb) would be 1,000
times longer than the E. coli cell.
The human genome (3.4 Gb) would be 2.3 m long if stretched linearly.
Solutions:
1.
Supercoiling
2.
Looped domains
DNA double helix is twisted in space about its
own axis, a process is controlled by
topoisomerases (enzymes).
(occurs in circular and linear DNA molecules)
More about genome size:
C value =
total amount of DNA in the haploid (1N) genome
Varies widely from species to species and shows no relationship to
structural or organizational complexity.
Examples
C value (bp)
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T4
HIV-1
E. Coli
Lilium formosanum
Zea mays
Amoeba proteus
Drosophila melanogaster
Mus musculus
Canis familiaris
Equus caballus
Homo sapiens
48,502
168,900
9,750
4,639,221
36,000,000,000
5,000,000,000
290,000,000,000
180,000,000
3,454,200,000
3,355,500,000
3,311,000,000
3,400,000,000
Eukaryotic chromosome structure
Chromatin
complex of DNA and chomosomal proteins
~ twice as much protein as DNA
Two major types of proteins:
1.
Histones
abundant, basic proteins with a positive charge
that bind to DNA (which is negatively charged)
5 main types: H1, H2A, H2B, H3, H4
~equal in mass to DNA
evolutionarily conserved
Histones leave the DNA only transiently during DNA replication.
They stay with the DNA during transcription. By changing shape
and position, nucleosomes allow RNA-synthesizing polymerases
to move along the DNA.
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Non-histones all the other proteins associated with DNA differ markedly in
type and structure
amounts vary widely
Packing of DNA into chromosomes:
1.
Level 1
Winding of DNA around histones to create a
nucleosome structure.
2.
Level 2
Nucleosomes connected by
strands of linker DNA like
beads on a string.
3.
Level 3
Packaging of nucleosomes into
30-nm chromatin fiber.
4.
Level 4
Formation of looped domains.
More about different types of DNA you should know about:
•Centromeric DNA (CEN)
Center of chromosome, specialized
sequences function with the
microtubles and spindle apparatus
during mitosis/meiosis.
•Telomeric DNA
At extreme ends of the chromosome,
maintain stability, and consist of
tandem repeats. Play a role in DNA
replication and stability of DNA.
Repeated DNA:
•Unique-sequence DNA
Often referred to as single-copy and
usually code for genes.
•Repetitive-sequence DNA
May be interspersed or clustered
and vary in size.
SINEs
short interspersed repeated sequences (100-500 bp)
LINEs
long interspersed repeated sequences (>5,000 bp)
Microsatellites
short tandem repeats (e.g., TTA|TTA|TTA)