Molecular Biology

Dr. MAI Thi Phuong Nga

LS Department

mai-thi-phuong.nga@usth.edu.vn
 Evaluation
Labwork 20%

Mid-term 20%
Scoring ratio
Presentation 20%

Final Exam 40 %
 Molecular biology - The study of biology at
molecular level including the structure, function,
and makeup of biologically important molecules
such as DNA, RNA, and proteins
Table 1.1
Table 1.1 (Continued)
 Many scientific disciplines contribute to molecular biotechnology,
Figure 1.1 which generates a wide range of commercial products.
Model Systems for Molecular/Cellular Biology
https://www.dnalc.org/resources/animations/model_organisms.html

●Viruses
●Bacteria (E. coli)
●Yeast (Saccharomyces cerevisiae)
●Round worms (Caenorhabditis elegans)
●Planarian (Schmidtea mediterranea)
●Alga (Chlamydomonas reinhardtii)
●Fruit fly (Drosophila melanogaster)
●Zebrafish (Danio rerio)
●Arabidopsis thaliana (thale cress)
●Mouse (Mus musculus)
Each experimental organism used in cell biology has advantages for certain
types of studies.
 Things You May Misunderstand About DNA
Who discovered DNA?
 James Watson and Francis Crick did not
discover DNA

Swiss biochemist Friedrich Miescher isolated a new substance from the nuclei of white blood cells

1869 NUCLEIN (DNA) WAS FISRT DISCOVERED

Discovery of DNA

❖In 1869, Swiss physician Friedrich

Miescher, obtained the first crude
purification of DNA from white blood
sample.

❖Due to its occurrence in the cells' nuclei,

he termed “nuclein”- today's name DNA.
 Things You May Misunderstand About DNA

Who did experiment for DNA structure ?

 Photo-51
History of DNA

Franklin is best
known for
work on X-ray
diffraction
images of DNA

Franklin and Gosling conclude that DNA is "probably helical," the phosphate
groups lie on the outside, and there are probably two strands.
 History of DNA

In 1962, James Watson, Francis

Crick, and Maurice Wilkins
were awarded the Nobel Prize
in Physiology and Medicine.

1953 STRUCTURE OF DNA WAS CLARIFIED

James Watson and Francis Crick: an article in Nature on April 25, 1953,
revealed the double helix, twisted later structure of DNA
 DNA

● DNA (deoxyribonucleic acid) is a double-helix stranded

molecule organized into chromosome

● Found in the nucleus of cells, where it encodes the genetic

information of an organism.

● When a cell divides, a copy of this genetic code is passed to the

new cell.

● DNA is polymer made up of monomers called nucleotides.

Nucleotides
✓A basic structural unit and building block for DNA.

✓A nucleotide is composed of 3 parts:

* five-sidedsugar: 2'-deoxyribose in DNA or ribose in RNA

* phosphate group
* nitrogenous base (nitrogen containing)
 Bases
● There are four types of bases in DNA
● * Adenine (A)
* Cytosine (C)
* Guanine (G)
* Thymine (T)

● RNA use uracil (U) instead of

Thymine
● A and T hold together by two bonds,
● C and G hold together by three bonds
 Bases
● The names of the bases are generally used as the names of the
nucleotide, although this is technically incorrect.

● The bases combine with the sugar to make the nucleotide A T

GCU

● Nucleotides are named based on the number of phosphate

residues they contain.
Bases
For example:
✓A nucleotide has an adenine base and three
phosphate residue: adenosine triphosphate (ATP).
✓
✓A nucleotide has two phosphates: adenosine
diphosphate (ADP).

✓A single phosphate: adenosine monophosphate

(AMP)
 Pyrimidines

22 Biochemistry for Medics

 Purines

23
DNA single strand: primary structure

 Sugar and phosphate group make up the

backbone of the DNA double helix

 Bases are located in the middle.

Traditionally, a DNA sequence is drawn from 5’ to 3’ end.

26
● What is meant by the 5ʹ and 3ʹ ends of a
nucleic acid strand?
DNA strand
● At one end there is pentose with 5’ free carbon

● At the other end there is 3’ Carbon free from bonding to

other nucleotide

● Additional nucleotide join to 3’of existing polynucleotide

● The sequence of one strand predict the sequence of the other

 DNA is currently used for species identification

How can we identify species. Why not morphology?

A DNA barcode, or a short standardized sequence that enables researchers to
distinguish among many species
 The secondary structure of DNA

Two anti-parallel polynucleotide chains wound around

the same axis.

Sugar-phosphate chains wrap around the periphery.

Bases (A, T, C and G) occupy the core, forming

complementary A · T and G · C Watson-Crick base
pairs.

30
 The DNA double helix is held together
mainly by Hydrogen bonds

➢ hydrogen bonding
➢ base stacking

31
Secondary structure
 Hydrogen bond

a chemical bond in which a hydrogen atom of one molecule is

attracted to an electronegative atom, especially a nitrogen,
oxygen, or fluorine atom, usually of another molecule.

33
 Two hydrogen bonds between A:T pairs
Three hydrogen bonds between C: G paired
34
Chargaff’s rules

● Early 1950s, Erwin Chargaff examined the content

of DNA in different species and discovered that adenine,
thymine, guanine, and cytosine were not found in equal
quantities, and that it varied from species to species, but
not between individuals of the same species.

● Amount of A was very close to the amount of T, and the

amount of C was very close to the amount of G, or A = T
and G = C.
 Structural forms of DNA
Property A-DNA B-DNA Z-DNA
Helix Handedness Right Right Left

Base Pairs per turn 11 10.4 12

Rise per base pair along 0.23nm 0.34nm 0.38nm

axis

Pitch 2.46nm 3.40nm 4.56nm

Diameter 2.55nm 2.37nm 1.84nm

Conformation of anti anti Alternating anti and syn

Glycosidic bond

Major Groove Present Present Absent

Minor
36 Groove Present Present Deep cleft
 DNA Tertiary Structure

• DNA double helical structure coils round histones.

• DNA bound to histones forms Nucleosomes (10nm

fibres)

• Nucleosomes contain 146 nucleotides

37
Compaction of DNA in a eukaryotic chromosome

38
 Supercoil = coil over coil

39
 Structure of nucleosome core

• The nucleosome core particle contains two copies of each histone

protein (H2A, H2B, H3 and H4) and 146 basepairs (bp) of
superhelical DNA wrapped around this histone octomer
• Linker DNA (20-60 bp).
 Nucleosomes

any of the repeating globular subunits of

chromatin that consist of a complex
of DNA and histone

42
Histone H1

Histone H1 interacts with the

with the linker DNA between
nucleosomes, further tightening
the association of the DNA with
the nucleosome.
Modification of the N-terminal tails of the histones
 DNA and chromosome

● In prokaryotic cells: DNA is located in cytoplasma

● Most prokaryote have single DNA contain nearly all genetic
information
● Prokaryote chromosome have no associated proteins

● Many eukaryote have 1000 times more DNA as prokaryote

● Eukaryote DNA is located in the nucleic inside chromosomes
● The number of chromosome vary from one to other species
 Reversible denaturation and annealing of DNA

46
In the laboratory

❖DNA denaturation: two DNA strands of the double helix DNA can
be separated to form single strands of DNA [ssDNA] by exposing to
high temperatures or to certain chemicals,

❖The ssDNA can also be put back together as double-stranded DNA

(dsDNA), through reannealing or renaturing by cooling or removing
the chemical denaturants, allowing these hydrogen bonds to reform.

❖DNA with a high GC content is more difficult to denature than DNA

with a lower GC content.
The tm of DNA depends on:
➢ Content of G·C base
pairs
➢ size of DNA
➢ pH
➢ ionic strength

48
Functions of DNA and summary of structure

DNA consists of four bases—A, G, C, and T—that are held in

✓linear
DNAarray by phosphodiester
consists bonds
of four bases—A, G,through the 3' and
C, and T—that are5'held in linear
positions
array of adjacent deoxyribose
by phosphodiester moieties.
bonds through the 3' and 5' positions of adjacent
deoxyribose moieties.
DNA is organized into two strands by the pairing of bases A to T
and G to C on complementary strands. These strands form a
double helix around a central axis.
✓DNA is organized into two strands by the pairing of bases A to T and
GThe
to C3 on complementary
x 10 strands.
9 base pairs of DNA These strands
in humans form ainto
are organized double
the helix
around
haploidacomplement
central axis.of 23 chromosomes.

DNA provides a template for its own replication and thus

maintenance of the genotype and for the transcription of the
roughly 30,000 human genes into a variety of RNA molecules.
49
Functions of DNA

✓DNA replication is vital for a virtually endless list of functions,

from reproduction to maintenance and growth of cells, tissues and
body systems, thus maintenance of the genotype

✓Coding for proteins: DNA holds the code for proteins, produce an
enormous variety of proteins, give traits.
Questions

Question 1:
Which of the following is not found within DNA?
a. thymine
b. phosphodiester bonds
c. complementary base pairing
d. amino acids
Question 2:
If 30% of the bases within a DNA molecule are adenine,
what is the percentage of guanine?
a. 20%
b. 25%
c. 30%
d. 35%
Question 3:
If a DNA strand contains the sequence 5ʹ-
ATTCCGGATCGA-3ʹ, which of the following is the
sequence of the complementary strand of DNA?
a. 5ʹ-TAAGGCCTAGCT-3ʹ
b. 5ʹ-ATTCCGGATCGA-3ʹ
c. 3ʹ-TAACCGGTACGT-5ʹ
d. 5ʹ-TCGATCCGGAAT-3ʹ
 Genome organization

☑ Prokaryotes
-Most genome is coding, no introns
-Small amount of noncoding is regulatory sequences
☑ Eukaryotes
-Most genome is noncoding
-Regulatory sequences
-Intron
-Repetitive DNA
Introns
● The human genome has about ten times the DNA content
as that of yeast.

● However, humans do not have a substantially larger

number of coding genes than yeast: humans genes are
more spread out over more chromosomes, and typically
have a higher proportion of introns.
 Chromosome and plasmid

Plasmid DNA

Bacterial Chromosomal DNA

Plasmid in prokaryote cells

Plasmid also occur in some eukaryotes. Which Eukaryote?

Plasmid in the nucleus of most Saccharomyces cerevisiae strains

Plasmid in the chloroplasts of Chlamydomonas reinhardtii.

 Plasmid

● A plasmid is a small, circular, double-stranded DNA molecule that is

distinct from a cell's chromosomal DNA.
● Plasmids naturally exist in bacterial cells, and some eukaryotes.
● Genes carried in plasmids provide bacteria with genetic advantages,
such as antibiotic resistance.
● Plasmids have a wide range of lengths, from roughly one thousand
DNA base pairs to hundreds of thousands of base pairs.
● When a bacterium divides, all of the plasmids are copied and
transmitted to daughter
● Bacteria can also transfer plasmids to one another through
conjugation.
Plasmid in the lab

● As tools to clone, transfer, and manipulate genes: are called

vectors.

● DNA fragments or genes can be inserted into a plasmid vector,

creating a recombinant plasmid.

● This plasmid can be introduced into a bacterium by

transformation. Then, because bacteria divide rapidly, they can be
used as factories to copy DNA fragments in large quantities.
 Eukaryotes have 2-3 genomes

☑ Nuclear genome
☑ Chloroplast genome
☑ Mitochondrial genome
 Chloroplast genome

☑ Organelle found in plants and eukaryotic algae

☑ Photosynthesis
☑ Circular double stranded DNA,
☑ Genome size ranges 120-217 kb
☑ Chloroplast DNA contains introns
☑ Encodes RuBisCO (for carbon fixation)
A cellular explanation of the variegated phenotype
Mitochondria DNA
 Mitochondrial genome

☑ The mitochondrial genome is a circle, 16.6 kb of DNA

☑ The D loop is the site where replication and transcription is
controlled
☑ Genes are tightly packed, almost no non-coding DNA
☑ Mitochondrial genome is maternally inherited
 Mitochondrial genome

✓Mutation rate in mtDNA is very high: 10 times the nuclear rate.

✓Large amounts of H2O2 are present, and they are quite mutagenic.

✓The D loop has an especially high rate of mutation.

✓Part of the effects of aging have been attributed to the gradual loss
of mitochondria due to accumulated mutations in individual cells.
 Mother-Child identity of MtDNA

☑ Mothers and all of their children share identical mtDNA

☑ MtDNA is used to find matches between mothers and offspring,

or grandmothers and grandchildren

☑ This was used in Argentina, to reunite kidnapped children with

their grandparents
Mitochondrial sequences and species evolution

☑ Mitochondrial DNA sequences used for deciphering evolutionary

relationship of animal species

☑ Once mitochondrial mutation occurs in the germ cell of a female,

mutation is transmitted to all of her offspring, allowing
identification of a common ancestor
Comparison of inherit materials of prokaryotic and eukaryotic cells

- Prokaryotic cells have only one

complete copy of chromosome in
nucleoid.
- - Prokaryotes also carry one or
more smaller independent
plasmids.

- The majority of eukaryotic cells

are diploid
- Eukaryotic chromosomes are
always in nucleus with membrane
- Eukaryotic cells have DNA in
chloroplast and mitochondria
Eukaryotic gene organization

Enhancers
Silencers
Insulators
Origins of replication

- Origins of replication are the

sites at which DNA replication
machinery assembles to initiate
replication.

- Each chromosome has multiple

origins of replication.

- Prokaryotic cell has only single

site of replication initiation.

- Origins of replication are found in

non-coding regions.
DNA polymerase and DNA copying
DNA polymerase and DNA copying

RNA

RNA

RNA

Nature 439, 542-543

(2006).
Bi-directional DNA synthesis

The helicase unzips dsDNA, making a forked structure.

The primase generates short strands of RNA (primer) that bind to ssDNA to
initiate DNA synthesis by the DNA polymerase. This enzyme can work only in the
5' to 3' direction, so it replicates the leading strand continuously. Lagging-strand
replication is discontinuous, with short Okazaki fragments being formed and later
linked together.

In the replication process, RNase H removes the RNA primer (created by Primase)
from the lagging strand and then Polymerase I fills in the necessary nucleotides
between the Okazaki fragments in a 5'→3' direction, proofreading for mistakes as it
goes
Enzymes used in replication of DNA in prokaryote
Enzymes used in replication of DNA
 DNA polymerase

• DNA polymerase found in all living organisms.

• The enzyme is critical to the transmission of genetic
information from generation to generation.
• They are crucial to the development of DNA sequencing and
DNA amplification by PCR.
• Taq DNA polymerase purified from the bacterium Thermus
aquaticus in 1976.
Corect of error
Bacterial genome size = million bases
human genome size = billion bases
Things You May Misunderstand About DNA
Length of our stretched DNA is >10 billion miles

If uncoiled, the DNA in all the cells in your body would stretch 10 billion miles—from here to Pluto and back.
Bigger Genome size= more genes?
Bigger Genome size= more genes?

25,500 genes

17,000 -19,000 genes

40,000- 60,000 genes

32,000 -60,000 genes

20,000 -25,000 genes

No data

164,000 -334,000 genes

Human genome sequencing revealed that 98.5% does not
code for proteins, rRNAs or tRNAs
Contribution of introns and repeated
sequences to different genomes
 Noncoding DNA

Noncoding DNA

Introns Regulatory sequences Pseudogenes Repetitive DNA

Telomere Interspersed Repetitive

DNA (e.g. transposon)
IF you want to study the function of NON-CODING REGION . What would u do?

Deletion?
 Introns

Prokaryotic bacteria lack introns.

Eukaryotic yeast have an exon / intron structure, with the majority of the genome in exons.
Regulatory sequences (promoter)
● CAAT box. A consensus sequence close to -80 bp from the start
point (+1). It plays an important role in promoter efficiency, by
increasing its strength
This box is replaced in plants by a consensus sequence called the
AGGA box;
CAAT not found in prokaryotes.

● TATA box. A sequence usually located around 25 bp upstream of

the start point. The TATA box tends to be surrounded by GC rich
sequences. The TATA box binds RNA polymerase II and a series
of transcription factors (TFIIX, X being a letter that identifies an
individual transcription factor) to form an initiation complex

● GC box. A sequence rich in G Cnucleotides, is usually found in

multiple copies in the promoter region, normally surrounding the
TATA box.
 Repetitive DNA (telomeres)

Telomeres (TTAGGG)n

Due to insufficient telomerase expression, telomeres shorten gradually with each cell
division in human somatic cells, which limits the number of times they can divide
Repetitive DNA (telomeres)
Repetitive DNA (transposon)
Barbara McClintock

Maize genomic composition

of the short arm of
chromosome 9
 Somatic Excision of Ds from C

Mutant Sectoring
C - control gene (transcription factor) for anthocyanin synthesis in seeds
Ds - Dissociater, non-autonomous element, requires Ac to move
Ac – activator, autonomous element
Fig. 23.9
autonomous element (Ac)
non-autonomous element (Ds) requires Ac

This phenomenon creates an unstable disruption and creates an unstable allele of c+ (cu). If Ds subsequently jumps
back out of cu, the c+ allelic function is restored. The seeds will then be a variegated, mosaic of cell patches, with a
background due to c and a superimposed pattern of or c+spots.
a. Purple

b. white

c. mosaic
 Transposons

Plant without transposon Plant with transposon ???

a chromosome break at the point of insertion, Ds and any downstream loci are lost. In W / W+
heterozygote, loss of the W allele allows expression of the alternative W+ allele on the other chromosome

Ds can also cause the element to jump into the middle of the W allele, disrupting its normal
function and allowing expression of the alternative W+ allele. The leaves are a variegated,
mosaic pattern , with a background due to W and a superimposed pattern of W+.
Transposons causing human disorders

☑ hemophila A (three different insertions of a

transposon into the clotting factor VIII gene)
☑ hemophila B (insertion into the clotting factor IX
gene)
☑ neurofibromatosis (transposon insertion into the
Neurofibromin 1 (NF1) gene
☑ one type of breast cancer (transposon insertion
into the BRCA2 gene) (next slides)
 BRCA1 and BRCA2
BRCA1 BRCA1 BRCA1 BRCA1

Fight cancer High risk of developing breast cancer

*Breast Cancer (BRCA)1 and 2 genes are cancer suppressors

 BRCA1 and BRCA2 genes are cancer suppressors, i.e. they help fight breast cancer.
 When either of these genes becomes mutated, it no longer functions properly. As a result of
unrepaired DNA damage and impaired genetic integrity, cells are likely growing uncontrolled to
develop cancer,
Transposable elements and genome evolution

• Insertion of transposable elements within a protein-coding

sequence may block protein production.

• Insertion of transposable elements within a regulatory

sequence may increase or decrease protein production.

• Multiple copies of similar transposable elements may

facilitate recombination, or crossing over, between
different chromosome.
Mutation in DNA sequences

● In CODING sequences

● In NON-CODING sequences
Coding sequence mutation
EFFECT OF MUTATIONS ON THE mRNA
(1) Missense mutations cause the replacement of an amino acid.
 Depending on the particular replacement, it may or may not have a
detectable phenotypic consequence.
 e.g. a valine for an leucine in a position that is important for maintaining
an a-helix, may not cause a detectable change in the structure or function of
the protein.
 valine for a glutamate at a site that causes hemoglobin to polymerize in the
deoxygenated state, cause significant pathology (sickle cell anemia).

(2) Nonsense mutations cause premature termination of translation.

 They occur when a substitution, insertion or deletion generates a stop codon
in the mRNA
 They almost always have serious phenotypic consequences.

(3) Frameshift mutations are insertions or deletions that change the reading
frame of the mRNA.
 They almost always have serious phenotypic consequences.
NOT ALL BASE SUBSITUTIONS ALTER THE ENCODED AMINO ACIDS

(1) The base substitution may lead to an alteration in the encoded polypeptide sequence, in
which case the substitution is called nonsynonymous or nonsilent.

(2) If the base substitution occurs in a degenerate site in the codon, so that the encoded amino
acid is not altered, it is called a synonymous or silent substitution.

E.g. ACU -> AAU nonsynonymous substitution

Thr -> Asn

ACU -> ACC synonymous substitution

Thr -> Thr

(3) Examination of the patterns of degeneracy in the genetic code shows that nonsynonymous
substitutions occur mostly in the first and second positions of the codon, whereas synonymous
substitutions occur mostly in the third position. However, there are several exceptions to this
rule.

(4) In general, the rate of fixation of synonymous substitutions in a population is significantly

greater that the rate of fixation of nonsynonymous substitutions. This is one of the strongest
supporting arguments in favor of model of neutral evolution, or evolutionary drift, as a
principle cause of the substitutions seen in natural populations.
Mutation and genetic disease
Sickle cells anemia

● Autosomal (non-sex chr.) recessive inheritance

● Missense (point) mutation of B-subunit of hemoglobin

● Glutamic acid Valine (Glu 6 Val)

● Sickle cell mutation = resistance to malaria

Understand the gene function
Gene disruption for functional analysis

● Random gene insertion

● Targeted gene insertion
 Random gene insertion

Fu et al. PNAS
(2007)

Phenotype of wild type, cyp38-1, cyp38-2, cyp38-3, and cyp38-2 transformed with CYP38 genomic DNA (cyp38-2C)
grown for 7 weeks under low light
Agrobacterium infects plants
 Agrobacterium infects plants

The T-DNA contains genes for encoding enzymes that cause the plant to create specialized amino acids
which the bacteria can metabolize, called opines
The insertion of a piece of T-DNA on the order of 5 to 25 kb in length generally produces a dramatic
disruption of gene function.
T-DNA also contain genes coded for for auxin and cytokinine, cause galls
Agrobacterium-mediated transformation
Targeted gene insertion
http://www.nature.com/nature/journal/v467/n7312/full/467161a.html
Gene insertion for screening and functional analysis
 Blue-white selection

https://www.sigmaaldrich.com/technical-documents/articles/biology/blue-white-screening.html
β-Galactosidase activity
Fluorescent gene insertion
How fluorescence protein works?

 The base or primary structure of GFP is

a chain of 238 amino acids weighing
roughly 27 kDa, with only about 4 of
the amino-acids directly producing
any fluorescence effect.
 At the center of this lies the
chromophore, a short chain of altered
amino-acids responsible for the light
emission.

Aequorea victoria
Screening for targeted events

● PCR

● Southern blot
 under white light

Hairy roots Rhizocalli

130 under UV light

 The name “hairy root” appeared when nurserymen discovered a
multitude of small roots/apple trees (Woodworth, 1892).

Rhizobium rhizogenes (Riker et al. 1930) (Young et al. 2001) is a G (-) pathogenic soil
bacterium able to induce ‘hairy root’ disease at the infection site on
mono/dicotyledonous plants (Chilton, 1982)
The process of T-DNA transfer from R. rhizogenes to host plant

The process of T-DNA transfer from R. rhizogenes to host plant (Tzfira and Citovsky, 2006)

By using biotechnology, genes of interest can be inserted into T-DNA and transferred into plant
 Why we chose Hairy root?

Hairy root – an ideal system for recombinant protein production

 RPs produced in HRs can be
easily secreted in culture
Ease of maintenance in a medium and cheaply purified.
very simple medium
Applications
without any additional
Have PTMs
hormones
(glycosylation,
acetylation…) Secondary metabolites
production
Scale-up
production in Hairy
bioreactor
roots Ability to
synthesize a
Recombinant proteins
production
large range
Fast growing of chemicals

Less sensitive to Genetic stability Functional analysis of genes

mechanical damage for years
than other tissues

Molecular farming by
T-DNA tagging or RNAi
silencing
 Transformation techniques

infect Induce
hairy root

Rhizobium rhizogenes
Arabidopsis thaliana

Culture on solid Culture in liquid

medium without medium without
hormone hormone
 Transgenic (bacteria)

Plasmid DNA

Bacterial Chromosomal DNA

 Concept of transgenic technology

2008 Nobel Prize in Chemistry awarded to Shimomura, Chalfie, and Tsien

“for the discovery and development of the green fluorescent protein, GFP”
Variation of fluorescence
proteins
How fluorescence protein works?
How fluorescence protein works?
Fluorescent gene insertion