Download No Slide Title

Active Lecture PowerPoint® Presentation for
Essentials of Genetics
Seventh Edition
Klug, Cummings, Spencer, Palladino
Chapter 9
DNA Structure and Analysis
Copyright © 2010 Pearson Education, Inc.
Outline
• Characteristics of genetic material
• Central dogma
• Is genetic material DNA or proteins?
• RNA as genetic material
• Structure of DNA & RNA
• Physical properties of DNA & RNA
Four Characteristics of Genetic
Material
For a molecule to serve as the genetic
material, it must:
1.
2.
3.
4.
be able to replicate
store information
express information
allow variation by mutation
The
Central
Dogma
Early Research
• Friedrick Miescher (1868)
– Swiss chemist
– Isolated nuclei from WBC found a
a phosphorus bearing acid
substance
– He called the substance nuclein
– Nuclein consisted of Deoxyribo
nucleic acid (DNA) and proteins
– First to isolate DNA from cells
http://www.biologie.uni-hamburg.de/bonline/e17/17k.htm00
Early Research
• Phoebus A. Levene (1910)
– Identified subunits of DNA
(nucleotides)
– Incorrectly proposed tetranucleotide
hypothesis
• Erwin Chargaff (1940s)
http://www.modares.ac.ir/
elearning/mnaderi/Genetic
%20Engineering%20cours
e%20II/images/15bbio.gif
– Proved tetranucleotide hypothesis
incorrect
What is the Genetic material?
DNA or Proteins?
DNA alphabet has 4 letters
Protein alphabet has 20 letters
Transformation: Early Studies
• Frederick Griffith
– showed that avirulent strains of Diplococcus
pneumoniae could be transformed to
virulence
(Diplococcus pneumoniae is now designated
Streptococcus pneumoniae)
– speculated transforming principle could be
part of polysaccharide capsule or some
compound required for capsule synthesis
What is the Transforming Principle?
• Avery, MacLeod, and McCarty
demonstrated transforming principle
was DNA and not protein
Smooth
cells
Mix with
live rough
cells
The Hershey–Chase Experiment
Hershey and Chase (1952)
• demonstrated DNA, and not protein,
enters bacterial cell during bacteriophage
infection and directs viral reproduction
Hershey-Chase experiment
Transfection Experiments
• Infection by only the phage DNA, called
transfection, produced mature viruses
• This further strengthened evidence for
DNA as the genetic material
Indirect Evidence: Distribution of DNA
• DNA is found only where the primary
genetic function occurs
• Protein is found throughout the cell
• This provided indirect evidence for DNA
as the genetic material
Indirect Evidence: Mutagenesis
• UV light:
– capable of inducing mutations in genetic material
– is most mutagenic at a wavelength of 260 nm
• DNA and RNA absorb UV light most strongly at
260 nm
• Protein absorbs most strongly at 280 nm, a
wavelength at which no significant mutagenic
effects are observed
• Again, this provides indirect evidence for DNA as
the genetic material
Direct Evidence: Recombinant DNA
Studies
• Strongest direct evidence for DNA as the
genetic material comes from recombinant
DNA technology
• Segments of eukaryotic DNA
corresponding to specific genes are
isolated and spliced into bacterial DNA
Direct Evidence: Recombinant DNA
Studies
• Bacteria containing eukaryotic gene
produce the corresponding eukaryotic
protein.
• This provides direct evidence that this
DNA is present and functional in
bacterial cell
RNA as Genetic Material in Some
Viruses
• Some viruses have an RNA
core rather than a DNA core
• This RNA serves as the genetic material for
these viruses
• Retroviruses are a special group of RNA
viruses
HIV multiplication
The Structure of DNA
• Nucleotides are the building blocks of
nucleic acids (both DNA & RNA)
• Nucleotides consist of:
1. a nitrogenous base
2. a pentose sugar
3. at least one phosphate group
DNA and RNA Composition
Bases
• The nitrogenous bases can be purines or
pyrimidines
• Purines are adenine (A) and guanine (G)
• Pyrimidines are cytosine (C), thymine (T),
and uracil (U)
Bases
Sugars
Nucleoside
Nucleotide
Glycosidic bond
Phosphodiester bond
DNA is negatively
charged
Antiparallel
Double strands
X-Ray Diffraction Photo of
Rosalind Franklin
The Watson–Crick Model
• Watson and Crick proposed (1953):
– DNA is a right-handed double helix
– the two strands are antiparallel
– the bases are stacked on one another
– the two strands are connected by A-T and GC base pairing
– there are 10 base pairs per helix turn
Figure 10-14c
Copyright © 2006 Pearson Prentice Hall, Inc.
Write the base sequence in the complementary DNA
strand and label the ends
5’ C T A T G C A A G T C A T T __’
__’ ______________________ __’
Alternative Forms of DNA Exist
• Watson-Crick DNA model is of B-DNA, which is
believed to be biologically significant form
• A-DNA is slightly more compact than B-DNA
• C-DNA, D-DNA, and E-DNA also right-handed
forms of DNA, are less compact than B-DNA
• Z-DNA forms a left-handed double helix
Figure Copyright © 2006 Pearson Prentice Hall, Inc.
Multiple Forms of Double Helices
A Form Observed under low humidity
Short & broad form. 11 base pairs/turn
Base pairs not perpendicular to the helix
Right handed helix
B Form -
Observed under high humidity
Most closely matches the structure of DNA in a cell
Long & thinner form. 10 base pairs/turn
Base pairs perpendicular to the helix
Right handed helix
Z Form Elongated zigzag form. Left handed helix
About RNA…
• The sugar ribose is present instead of
deoxyribose of DNA; Uracil replaces thymine
• Most RNA is single stranded; hence they form
secondary structures such as hairpins
• All RNAs originate as complementary copies of
one of the two strands of DNA. Which one
came first? DNA or RNA?
• Some RNAs can act as enzymes - Ribozymes
About RNA…
Three major classes of cellular RNAs:
1. messenger RNA (mRNA)
2. ribosomal RNA (rRNA)
3. transfer RNA (tRNA)
More about RNA…
Now RNA is recognized as having a major
role in controlling gene regulation and
expression:
1. Small nuclear RNA (snRNA)
2. Micro RNA (miRNA)
3. Telomerase RNA (involved in DNA replication
at the ends of chromosomes)
iRNA : RNA interference is a mechanism by
which genes can be shut down.
Properties of DNA
• Denaturation – Separation of the two strands
• Reannealing (Reassociation) – Reuniting of
the two strands
• Hybridization- Reuniting of strands of nucleic
acids from two different sources
• Electrophoresis –Separation technique of
DNA & RNA molecules by the size
Denaturation
DNA can be denatured by
Heat –
High pH –
Organic solvents – formamide
dimethyl sulfoxide
Low ionic strength – promotes denaturation
(negative charges repel)
Tm - Melting Temperature
Temperature at which two strands are
denatured
Tm depends on the GC content. Why?
half
Reannealing
Slow cooling of the separated strands will
anneal the two strands
Rapid cooling does not – eg: keeping on ice
after boiling
Annealing temperature is about 25 C below Tm
Hybridization
• Denatured DNA strands (or RNA strands)
from different sources can be renatured to
each other
• DNA-DNA Hybridization
• DNA-RNA Hybridization
• RNA-RNA Hybridization
• Hybridization provides techniques for
analyzing DNA
Fluorescent in situ Hybridization
Electrophoresis
• Nucleic acid electrophoresis separates
DNA and RNA fragments by size
• Smaller fragments migrate at a faster rate
through a gel than large fragments
• Agarose gel electrophoresis
• Polyacrylamide gel electrophoresis