Molecular Biology
(BIO1004/2001)
Kathleen Lobban
Kathleenlobban@yahoo.com
MODULE CONTENT
 Structure of DNA
 DNA Replication
 Transcription and Protein synthesis
 Molecular Biology Techniques
 Recombinant DNA technology
 Gene Expression and Regulation in Prokaryotes
and Eukaryotes
ASSESSMENT PROCEDURES
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Test 1 (Week of October 12, 2009 )
Test 2 (Week of November 16, 2009 )
Practicals
Project (Week of October 26, 2009 )
Final Exam (Week of October 12, 2009 )
15%
15%
10%
10%
50%
UNIT 1
Structure of DNA
DNA
Deoxyribonucleic acid
 Belongs to the class of macromolecules
called nucleic acids.
 The other nucleic acid is RNA
 (ribonucleic acid)
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Composition of DNA
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DNA is a polymer.
The monomer units of DNA are nucleotides
Therefore the polymer is known as a
"polynucleotide."
Each nucleotide consists of
 a 5-carbon sugar (deoxyribose)
 a nitrogen-containing base
 a phosphate group.
Composition of DNA
Nitrogenous Bases
The Nucleotides
Base
Adenine
Nucleoside
adenosine
Guanine
guanosine
Cytosine
cytidine
Uracil
uridine
Thymine
thymidine
Nucleotide (dNTP)
adenosine triphosphate
ATP / dATP
guanosine triphosphate
GTP / dGTP
cytidine triphosphate
CTP / dCTP
uridine triphosphate
UTP /dUTP
thymidine triphosphate
TTP / dTTP
Other Functions of Nucleotides
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They carry chemical in their bonds which can be easily
released for use by the cell eg ATP and GTP
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They combine with other group to form coenzymes: eg
coenzyme A
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They are used as specific signaling molecules within
cells, eg. Cyclic AMP (cAMP) serves to signal switching
on of the lac operon in prokaryotes.
Composition of DNA
Base Pairs
 Adenine forms 2 hydrogen bonds with
Thymine on the opposite strand
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Adenine (A) will only bond with Thymine (T)
Guanine forms 3 hydrogen bonds with
Cytosine on the opposite strand.
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Guanine (G) will only bond with Cytosine (C).
DNA molecule
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First described by James
D. Watson and Francis
Crick in 1953.
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In a DNA molecule, the
two strands are not
parallel, but intertwined
with each other.
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The two strands form a
"double helix" structure
DNA molecule
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The DNA backbone is an
alternating sugar-phosphate
sequence.
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The deoxyribose sugars are
joined at both the 3'- and
5'-carbon to phosphate
groups by phosphodiester
bonds.
DNA molecule
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Chain has a direction
(polarity)
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5΄ end - phosphate
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3΄ end - hydroxyl
DNA molecule
The two polynucleotide chains run in opposite
directions - antiparallel
The DNA Double Helix
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Two DNA strands form a right-handed
helical spiral
The sugar-phosphate backbones of the two
DNA strands wind around the helix axis like the
railing of a spiral staircase
The bases of the individual nucleotides are on
the inside of the helix, stacked on top of each
other like the steps of a spiral staircase
DNA molecule
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The sugar-phosphate backbone of DNA is
polar, and therefore hydrophilic.
The bases, are relatively non-polar and therefore
hydrophobic.
These hydrostatic forces have a very stabilizing
effect on the overall structure of the DNA
double helix
Therefore there is a strong pressure holding the
two strands of DNA together.
The DNA Double Helix
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The helix makes a turn every 3.4 nm
The distance between two neighboring base pairs
is 0.34 nm.
Hence, there are about 10 pairs per turn.
The intertwined strands make two grooves of
different widths
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The major groove
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The minor groove
facilitate binding with specific proteins.
Watson and Crick’s Model of DNA
In 1928 Frederick Griffith was working on a project that
enabled others to point out that DNA was the molecule of
inheritance.
 The experiment involved mice and bacteria that cause
pneumonia - a virulent and a non-virulent kind.
 He injected the virulent into a mouse and the mouse died.
 Next he injected the non-virulent into a mouse and the
mouse lived.
 After this, he heated up the virulent strain to kill it and
then injected it into a mouse. The mouse lived.
 Last he injected non-virulent pneumonia and virulent
pneumonia, that had been heated and killed, into a
mouse. This mouse died.
Watson and Crick’s Model of DNA
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Why? Griffith thought that the killed
virulent bacteria had passed on a
characteristic to the non-virulent one to
make it virulent TRANSFORMATION. He thought that
this characteristic was in the “inheritance
molecule”.
Watson and Crick’s Model of DNA
In 1942 Oswald Avery
 continued with Griffith’s experiment to see what the
inheritance molecule was.
 He destroyed the lipids, ribonucleic acids,
carbohydrates, and proteins of the virulent
pneumonia. Transformation still occurred after this.
 Next he destroyed the deoxyribonucleic
acid. Transformation did not occur. Avery had found
the inheritance molecule, DNA!
Watson and Crick’s Model of DNA
1940’s Erwin Chargaff
 noticed a pattern in the amounts of the four
bases: adenine, guanine, cytosine, and thymine.
 In samples of DNA from different organisms :
the amount of adenine = to the amount of thymine
 the amount of guanine = to the amount of
cytosine.
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This discovery later became Chargaff ’s Rule.
Watson and Crick’s Model of DNA
Rosalind Franklin and Maurice Wilkins
 Decided to try to make a crystal of the DNA
molecule. They obtained an x-ray pattern.
 The pattern appeared to contain rungs, like those on a
ladder between to strands that are side by side. It also
showed by an “X” shape that DNA had a helix shape.
www.who2.com/.../watson-v-franklin-round-27.html
Watson and Crick’s Model of DNA
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In 1953 James Watson and Francis Crick
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saw Franklin and Wilkin's picture of the X-ray and
made an accurate model - a double helix with little rungs
connecting the two strands
But how to bond the bases together ?
How to solve the problem of the sizes of the bases?
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Adenine and Guanine were purines having two carbonnitrogen rings in their structures.
Thymine and Cytosine were pyrimidines having one carbonnitrogen ring in its structure.
If purines and the pyrimidines were together, then DNA
would look wobly and crooked.
If they paired Thymine with Adenine and Guanine with
Cytosine, DNA would look uniform (Chargaff's rule)
Each side is a complete compliment of the other.
Watson and Crick Model of DNA
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http://www.johnkyrk.com/DNAanatomy.html
Remembering Rosalind
By using the picture of the crystallized
DNA,
Watson and Crick were able to put together
the model of DNA.
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Watson and Crick used information
from Avery, Chargaff, Griffith, and
others.
 They simply pieced together the puzzle.
 The Nobel Prize was awarded to Watson,
Crick, and Maurice Wilkins.
 Rosalind Franklin did not receive the
prize because she had died of cancer by
this time. Maurice Wilkins was able to
share the prize with Watson and Crick,
though, because of his work with
Franklin. Her accomplishment should
never be forgotten.
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Forms of DNA
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DNA exists in many possible conformations.
only A-DNA, B-DNA, and Z-DNA have been
observed in cells.
conformation depends on:
 the DNA sequence
 the amount and direction of supercoiling
 chemical modifications of the bases
 solution conditions (eg concentration of metal
ions, salt concentration and level of hydration).
B -Form of DNA
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Most common under the conditions found in
cells.
Has a major and minor groove
Has 10 base pairs per turn
One turn spans 3.4 nm
A -Form of DNA
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With higher salt concentrations or with alcohol
added, the DNA structure may change to A form
A wider right-handed spiral
A shallow, wider minor groove
A narrower, deeper major groove
Has 11 bases per turn
One turns spans 2.3 nm
Z -Form of DNA
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The DNA molecule with alternating G-C
sequences in alcohol or high salt solution tends to
have such structure
Bases seem to zigzag
The strands turn about the helical axis in a lefthanded spiral.
Has a narrow deep groove
Has 12 bases per turn.
One turn spans 4.6 nm
A – DNA
B-DNA
Z-DNA
Chromosomes
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Chromosomes – highly coiled condensed
packages of DNA
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Present in both eukaryotes and prokaryotes
Eukaryotes – linear
 Prokaryotes – most closed circular
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The human genome has 3,000,000,000 base pairs
packed into 23 chromosomes!
Organization of Eukaryotic Genomes
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DNA is packed into chromosomes with the help of
proteins - histones.
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The histones associate with DNA forming structures
called nucleosomes.
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Each nucleosome complex consists of a beadlike
structure with 146 base pairs of DNA wrapped around
a disc-shaped core of eight histone molecules.
Nucleosome
Chromosomes
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Nucleosomes are 11nm in diameter.
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The packed nucleosome state occurs when a ninth
histone called H1, associates with the linker DNA
packing adjacent nucleosomes together forming a 30nm
diameter thread.
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In the extended chromosome , these 30nm diameter
threads form large coiled loops held together by a set
of non-histone scaffolding proteins.
Eukaryotic Genomes
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organized into multiple chromosomes - varying
in size and numbers depending on the species:
Human cells haploid 23
 Fruit flies haploid 4
 Yeast haploid 16
 Cats haploid 19
 Dogs haploid 39
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Bacterial Genome Organization
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Most have covalently closed, circular chromosomes
and plasmids
Not all bacteria have a single circular chromosome:
 some bacteria have multiple circular
chromosomes
 many bacteria have linear chromosomes and
linear plasmids.
Bacterial Genome
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The genetic material of a bacterium lies in the
cytoplasm and is not surrounded by a nuclear envelope.
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In most species it is contained in a single circular DNA
molecule.
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If stretched out to its full length, this molecule would
be 1000 times longer than the cell itself.
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Unlike eukaryotic chromosomes, the bacterial DNA has
little proteins associated with it.
Plasmids
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In addition to the genomic DNA, most bacteria have a
small amount of genetic information present as one or
more plasmids – extrachromosomal circular DNA.
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Plasmids can replicate independently of the genomic
DNA or become integrated into it.
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Bacterial plasmids frequently have genes that code for:
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catabolic enzymes
genetic exchange
resistance to antibiotic.
Viral Genomes
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virus genomes may contain their genetic
information encoded in either DNA or RNA
Viral Genomes
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Many of the DNA viruses of eukaryotes closely
resemble their host cells in terms of the biology
of their genomes:
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Some DNA virus genomes are complexed with
cellular histones to form a chromatin-like structure
inside the virus particle.
Viral Genomes
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Genome may be :
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DNA viruses
RNA viruses
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Circular or linear
segmented – (two or more separate molecules of nucleic acid)
Single-stranded
Double-stranded
Double-stranded with regions of single-strandedness
Positive sense
Negative sense
Ambisense
Both DNA and RNA (at different stages in the life cycle)
Reverse transcribing viruses
Viral Genomes
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I: dsDNA viruses
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II: ssDNA viruses (+)sense DNA
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(-)sense RNA (e.g. Orthomyxoviruses – Influenza A, B, C)
VI: ssRNA-RT viruses
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(+)sense RNA (e.g. Picornaviruses -Enterovirus)
V: (-)ssRNA viruses
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(e.g. Reoviruses - Rotavirus)
IV: (+)ssRNA viruses
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(e.g. Parvoviruses)
III: dsRNA viruses
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(e.g. Herpesviruses)
(+)sense RNA with DNA intermediate in life-cycle (e.g. Retroviruses – HIV,
lukemia and tumor viruses)
VII: dsDNA-RT viruses
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(e.g. Hepadnaviruses – hepatitis B)
Genes
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Unit of inheritance
A sequence of nucleotides which provide a cell
with the instructions for the synthesis of a
specific polypeptide or a type of RNA
Genes determine traits
Most are 1,000 to 4,000 nucleotides long (may
be shorter or significantly longer)
Genes
Genes
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Most eukaryotic genes contain non coding
regions - introns
Genes, Viruses and Cancer
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Cancer is a disease in which cells escape the restraints
on normal cell growth.
Cancer is an inheritable disease (at least from cell to
daughter cells).
Once a cell has become cancerous, all of its descendant
cells are cancerous.
 Gross chromosomal abnormalities are often visible
in cancerous cells.
 Most carcinogens (cancer-generating factors) are also
mutagens (mutation-generating factors).
 Oncogenes are genes resembling normal genes but
in which something has gone wrong, resulting in a
cancer.
Genes, Viruses and Cancer
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Viruses seem able to cause cancer in three
ways:
 Presence of the viral DNA may disrupt
normal host DNA functions.
 Viral proteins needed for virus
replication may also affect normal host
gene regulation.
 Since most cancer-causing viruses are
retroviruses, the virus may serve as a
vector for oncogene insertion.
Images’ source
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http://www2.le.ac.uk/departments/emfpu/profiling/explained/images/PrimaryDNA.gif
http://3.bp.blogspot.com/_DZH2cmCoois/Rp_K_5IavqI/AAAAAAAAClI/9GdTDazv-oQ/s400/figure+19-14.jpg
http://library.thinkquest.org/C004535/media/chromosome_packing.gif
http://www.nature.com/nature/journal/v421/n6921/images/nature01411-f1.2.jpg
http://mac122.icu.ac.jp/BIOBK/14_1.jpghttp://mac122.icu.ac.jp/BIOBK/14_1.jpg
http://www.sciencedaily.com/images/2008/05/080526155300-large.jpg
http://science.kennesaw.edu/~tmcelro/DNA%20Forensics/Genetics%20review/Fg10_14%5B1%5D.gif
http://images.google.com.jm/imgres?imgurl=http://themedicalbiochemistrypage.org/images/chromatinstructure.jpg&imgrefurl=http://themedicalbiochemistrypage.org/dna.html&usg=__rj_0WyKjg3pd93RGtskLC_qnJg=&h=828&w=500&sz=90&hl=en&start=82&tbnid=ONMf9ibvdHh0sM:&tbnh=144&tbnw=87&prev=/images%3F
q%3Ddna%2Bstructure%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26start%3D72
table : http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/chroms-genes-prots/chromosomes.html
History of DNA
http://library.thinkquest.org/20830/Textbook/HistoryofDNAResearch.htm#Watson%20and%20Crick
http://images.google.com.jm/imgres?imgurl=http://genome.jgi-psf.org/Chr16/dna.jpg&imgrefurl=http://genome.jgipsf.org/Chr16/Chr16.home.html&usg=__zOSiNtQbR9DdY2ZBsog2FGR1Ag8=&h=284&w=280&sz=51&hl=en&sta
rt=6&um=1&tbnid=zYTbfjqc9ubbgM:&tbnh=114&tbnw=112&prev=/images%3Fq%3Drosalind%2Bfranklin%26hl%
3Den%26sa%3DG%26um%3D1
http://www.daviddeerfield.com/NIH/A-B_DNA.gif
http://commons.wikimedia.org/wiki/File:A-B-Z-DNA_Side_View_Transparent.png
http://bioweb.wku.edu/courses/biol566/Images/ChromatinF09-35.JPG
http://www.microbiologybytes.com/introduction/genomes.html
http://www.accessexcellence.org/RC/VL/GG/images/genes.gif
http://www.daviddarling.info/images/exon_and_intron.gif http://www.sciencemuseum.org.uk/online/lifecycle/images/1-2-6-3-1-2-1-0-0-0-0.jpg