Tracing the hereditary pattern:

from science to technology

Flow of information in biological systems

DNA replication, transcription and translation

Genetic disorder in humans

Recombinant DNA technology: principles and applications

(Chapters 15-17, 20)

How did scientists discover that DNA is

the hereditary material?

Frederick Griffith discovers bacterial transformation (1928)

Experiment
Living S cells
(control)

Living R cells
(control)

Heat-killed
S cells
(control)

Mixture of
heat-killed
S cells and
living R cells

RESULTS
Living S cells
Mouse dies

Mouse healthy Mouse healthy Mouse dies

Hypothesis: a chemical component from the virulent S cells had somehow

transformed the R cells into the virulent S form.

Hershey and Chase further prove that protein is not the

hereditary material

"This protein probably has no function in the growth of intracellular phage.

The DNA has some function (Hershey and Chase, 1952)

Only a year later, the structure of DNA is determined (1953)

We wish to suggest a structure for
the salt of deoxyribose nucleic
acid (D.N.A.). This structure has
novel features which are of
considerable biological interest.

Nucleic acids
Every nucleotide is composed of a phosphate group, a sugar, and
a nitrogenous base.
2 groups of nitrogenous bases:
purines (adenine, guanine)
pyrimidines (cytosine, uracil, and thymine)

Nucleic acids
Polymerization of nucleotides involves
formation of phosphodiester bonds
DNA (nucleotides: A,T,C,G)
- store and transmit biological information

RNA (nucleotides: A,U,C,G)

- messenger RNA: encode information for
protein primary structure
- ribosomal RNA: ribosome assembly
- tRNA: adaptor of RNA and protein
- microRNA: regulation of gene expression

Structures of DNA and RNA

2 DNA polynucleotides with complementary sequence form a
double helix and run in opposite directions
RNA molecules usually exist as single polynucleotide chains

The central dogma of molecular biology

Flow of information in biological systems. It states that DNA
codes for RNA, which codes for proteins.

DNA

Genotype

Transcription

RNA

Translation

Protein

Phenotype

Structural model of DNA provides insights to the basis of

DNA replication

Watson and Cricks semiconservative model of DNA replication

DNA Replication: How does it start?

Replication begins at origins
of replication, where the two
DNA strands are separated.

Origin of replication
Parental (template)
strand

Double-stranded
DNA molecule
Daughter (new)
strand

Replication proceeds in both

directions from each origin,

Bubble

Replication fork

until the entire molecule is

copied.
Two daughter DNA molecules

In an eukaryotic cell

11

Synthesis of new DNA strands by DNA polymerase

A short RNA primer (5-10 nucleotides) is required, and is synthesized
by primase. The 3 -OH of the primer serves as the starting point for
the new DNA strand.
The new DNA strand has complementary sequence to the template.
Synthesis of DNA only in the 5 to 3 direction

Various enzymes and accessory proteins participate in

the DNA replication process
Primase:

start an RNA chain from scratch

using the parental DNA as a template

Topoisomerase:

corrects overwinding ahead of

replication forks

3
3

5
3

RNA
primer

Helicase:

untwist the double helix

at the replication forks

Single-strand binding proteins:

bind to and stabilize single-stranded DNA

Overview of DNA replication

DNA polymerase synthesizes a leading strand continuously, moving
toward the replication fork.
For the lagging strand, DNA polymerase works in the direction away from
the replication fork.
DNA polymerase III synthesizes
leading strand continuously

3
5

Parental DNA

5
3

Helicase

Origin of
Lagging strand is synthesized
replication
in short Okazaki fragments by
DNA polymerase III
Okazaki fragments are
joined by DNA ligase

Primase synthesizes
a short RNA primer

3
5
DNA polymerase I replaces
the RNA primer with DNA
nucleotides
Okazaki fragments

Replicating the ends of linear chromosomes

Limitations of DNA polymerase create problems to complete the
5 ends for the linear DNA of eukaryotic chromosomes
RNA primer

Lagging strand
Parental strand

5
3
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Second round
of replication
5
3

New strand

New strand

3
Further rounds
of replication
Shorter and shorter daughter molecules

15

Proofreading and DNA Repairing

DNA replication is very accurate (an average
error rate: < 1 mistake per billion bases).
DNA damage: spontaneous changes of DNA
caused by harmful chemical and physical
agents
DNA repair mechanisms fix DNA by
enzymatically excising and replacing any
damaged bases.
Defects in the genes required for DNA
repair are frequently associated with
cancer.

5
Nuclease

5
DNA
polymerase

5
DNA
ligase

Nucleotide excision repair

Xeroderma pigmentosum (XP)

Applications

Autosomal recessive genetic disorder

The skin in XP patient is extremely sensitive to sunlight or UV light.
Skin cancer usually develops.
UV light produces pyrimidine dimers in human DNA. Most often, XP
is caused by a defect in an enzyme that hydrolyzes the DNA
backbone near a pyrimidine dimer.

https://droualb.faculty.mjc.edu/images/Anatomy/Integumentary%20System/xeroderma%20pigementosum.htm

Replicating the ends of linear chromosomes (telomeres)

Telomeres do not contain genes, but consist of short repeating
stretches of bases.
Telomerase catalyzes the lengthening of telomeres in germ cells.
Somatic cells normally lack telomerase. The chromosomes of
somatic cells progressively shorten as the individual ages.
Telomere shortening may have a role in limiting the amount of
time cells remain in an actively growing state.

Overview of transcription and translation

Nuclear
envelope

TRANSCRIPTION

RNA PROCESSING

DNA
Pre-mRNA

mRNA

TRANSLATION

Ribosome
Polypeptide

Eukaryotic cell

19

Transcription initiation
RNA polymerase
RNA polymerase binds to a promoter which signals the
transcriptional start point
In eukaryotes, transcription factors mediate the binding of
RNA polymerase to promoter
The completed assembly of transcription factors and RNA
polymerase bound to a promoter is called a transcription
initiation complex

20

Transcription initiation
Promoter
5
3

DNA

Nontemplate strand

T A T A A AA
A T AT T T T

TATA box
Transcription
factors

Start point

3
5
Template strand

Several transcription
factors bind to DNA

5
3

RNA polymerase II

3
5
Transcription initiation
complex forms
Transcription factors

5
3

3
5

RNA transcript
21

Elongation and termination of transcription

As RNA polymerase moves along the DNA, it untwists the double
helix and synthesizes RNA in the 5 3 direction.
RNA polymerase transcribes the polyadenylation signal sequence;
the RNA transcript is released 1035 nucleotides past this
polyadenylation sequence
Nontemplate
strand of DNA
RNA nucleotides
RNA
polymerase
3

A T C
C

3 end

C A U C
5
5

C A A

C A

T A G G T T

Direction of transcription
Newly made RNA

Template
strand of DNA

22

RNA is modified after transcription

Pre-mRNA is modified in the nucleus before they are released to
the cytoplasm for translation.
These modifications share several functions
facilitate the export of mRNA
protect mRNA from hydrolytic enzymes
help ribosomes attach to the 5 end

5
G

Protein-coding
segment

P P P

5 Cap 5 UTR

Polyadenylation
signal
AAUAAA

Start
codon

Stop
codon

3 UTR

AAA AAA

Poly-A tail

UTR: untranslated region

RNA is modified after transcription

Most eukaryotic genes and their RNA transcripts have long
noncoding stretches of nucleotides (introns) that lie between
coding regions (exons)
RNA splicing removes introns and joins exons, creating an
mRNA molecule with a continuous coding sequence
5 Exon Intron Exon
Pre-mRNA 5 Cap
Codon
1-30
31-104
numbers

Intron

Exon 3
Poly-A tail
105146

Introns cut out and

exons spliced together
mRNA 5 Cap
5 UTR

Poly-A tail
1-146
Coding
segment

3 UTR
24

How Does an mRNA Triplet Specify an Amino Acid?

The flow of information from gene to protein is based on a
triplet code (called codon)
All 64 codons were deciphered by the mid-1960s
Genetic code
is redundant but not ambiguous; no codon specifies more
than one amino acid
must be read in the correct reading frame
nearly universal

THE CAT AND THE MAN ARE FAT

T HEC ATA NDT HEM ANA REF AT
25

Second mRNA base

UUU

First mRNA base (5 end of codon)

UUC
UUA

Leu

UCC
UCA

Ser

UAC

Tyr

UGU
UGC

Cys

U
C

UAA Stop UGA Stop A

UAG Stop UGG Trp G

CUU

CCU

CAU

CUC

CCC

CAC

CUA

Leu

CCA

Pro

CAA

CUG

CCG

CAG

AUU

ACU

AAU

ACC

AAC

AUC

Ile

AUA
AUG

Phe

UAU

UCU

UCG

UUG

ACA
Met or
start

Thr

AAA

ACG

AAG

GUU

GCU

GAU

GUC

GCC

GAC

GUA
GUG

Val

GCA
GCG

Ala

GAA
GAG

His
Gln

Asn
Lys

Asp
Glu

CGU

CGC

CGA

Arg

CGG
AGU
AGC
AGA
AGG

A
G

Ser
Arg

U
C
A
G

GGU

GGC

GGA
GGG

Gly

Third mRNA base (3 end of codon)

A
G

26

Translating the code: RNA to Protein

Amino
acids

Polypeptide

tRNA with amino acid attached

Ribosome
Phe

Gly

tRNA
C
A A A

Anticodon

U G G U U U G G C

Codons
mRNA

3
27

Transfer RNA
tRNA molecules are not identical
Each tRNA carries a specific amino acid at 3 end
Each tRNA has an anticodon; the anticodon base-pairs with a
complementary codon on mRNA
5

Amino acid
attachment site

Anticodon

G
28

Accurate translation requires two steps

a correct match between a tRNA and an amino acid, done by
the enzyme aminoacyl-tRNA synthetase
a correct match between the tRNA anticodon and an mRNA
codon
Interestingly, some tRNA can recognize more than one codon.
Flexible pairing at the third base of a codon is called wobble. E.g.
U at the 5 end of a tRNA anticodon can pair with either A or G at
the 3 end of an mRNA codon.
anticodon

anticodon

3 C-G-U 5
5 G-C-A 3

3 C-G-U 5
5 G-C-G 3

codon

codon

Wobble base pair

29

Ribosomes
The two ribosomal subunits (large and small) are made of
proteins and ribosomal RNA (rRNA)

30

Initiation of Translation
A small ribosomal subunit binds with mRNA and a special
initiator tRNA. Then the small subunit moves along the mRNA
until it reaches the start codon (AUG).
Assembly of the large ribosomal subunit completes the
formation of translation initiation complex
Large
ribosomal
subunit
Met

3 U A C 5
5 A U G 3

P site

Met

Pi

Initiator
tRNA

GTP

GDP
E

mRNA
5
Start codon
mRNA binding site

3
Small
ribosomal
subunit

5
Translation initiation complex

3
31

Elongation of the Polypeptide Chain

Amino end of
polypeptide

Ribosome ready for

next aminoacyl tRNA

mRNA

3
P A

1. codon recognition

GTP
GDP+ Pi

E
P A

PA
GDP+ Pi

3. translocation

GTP

2. peptide bond
formation

E
PA

32

Termination of Translation
Termination occurs when a stop codon in the mRNA reaches the
A site
The A site accepts a protein called a release factor.
Newly synthesized polypeptide is released and the translation
assembly comes apart.
Free
polypeptide

Release
factor

3
5

5
Stop codon
(UAG, UAA, or UGA)

3
2

GTP
2GDP + 2 P i

Genetic disorder in humans

1. Changes in chromosome number
2. Changes in chromosome structure
3. Changes in gene sequence

Large-scale chromosomal alterations in humans and other

mammals often lead to spontaneous abortions (miscarriages)
or cause a variety of developmental disorders

1. Changes in chromosome number

Nondisjunction

- failed separation of homologues chromatids during meiosis

Nondisjunction involving autosomes
Monosomics: Lose one copy of an autosome
Trisomics: Gain an extra copy of autosome

Nondisjunction involving sex chromosomes

Nondisjunction involving autosomes

Down syndrome

Often called trisomy 21

Distinct facial appearance, short stature and mental retarded
Patients are prone to leukemia and Alzheimers disease

Nondisjunction involving sex chromosomes

As Y chromosome carries relatively few genes and extra copies of the
X chromosome become inactivated in somatic cells, nondisjunction
involving sex chromosome are less severe than those involving autosomes.
Female: XX
Nondisjunction

Triple X syndrome: healthy and

cannot be distinguished from XX
females
X
Sperm: Y

Klinefelter syndrome: sterile,

female body characteristics

Male: XY

Turner syndrome: sterile, most

have normal intelligence

Eggs:

XX

XXX
Female
(Triple X
syndrome)

X0
Female
(Turner
syndrome)

XXY
Male
(Klinefelter
syndrome)

0Y
Nonviable

2. Change in chromosome structure

(a) Deletion
A B C

D E

F G

A deletion removes a chromosomal segment.

A B C

F G H

(b) Duplication
A B C

D E

F G

A duplication repeats a segment.

A B C

B C

D E

F G H

(c) Inversion
A B C

D E

F G H

An inversion reverses a segment within a

chromosome.
A D C

B E

F G H

(d) Translocation
A B C

D E

F G H

M N O

P Q

A translocation moves a segment from one

chromosome to a nonhomologous chromosome.
M N O

C D E

F G H

B P Q

3. Changes in gene sequence

.
Normal

.
. ATG CTT ACT ATT AAC

. Methionine Leucine Threonine Isoleucine Asparagine

1) Base substitution

. ATG CGT ACT ATT AAC

. Methionine Arginine Threonine Isoleucine Asparagine

2) Insertion

. ATG CTTT ACT ATT AAC

. Methionine Leucine Tyrosine Tyrosine STOP

3) Deletion

. ATG CT ACT ATT AACG

. Methionine Leucine Leucine Leucine Threonine

gene

DNA sequence
protein sequence

Mutations of one or a few nucleotides can affect protein

structure and function
Silent mutations have no effect on the amino acid produced
by a codon because of redundancy in the genetic code
Missense mutations still code for an amino acid, but not the
correct amino acid
Nonsense mutations change an amino acid codon into a
stop codon, nearly always leading to a nonfunctional protein.
Insertion or deletion of nucleotides usually will have a
dramatic effect.

40

Wild type
DNA template strand

3 T A C T T C A A A C C G A T T 5
5 A T G A A G T T T G G C T A A 3

mRNA5 A U G A A G U U U G G C U A A 3
Protein

Met

Lys

Phe

Gly

Stop
Carboxyl end

Amino end

(b) Nucleotide-pair insertion or deletion

(a) Nucleotide-pair substitution

Extra A

A instead of G
3 T A C T T C A A A C C A A T T 5
5 A T G A A G T T T G G T T A A 3

3 T A C A T T C A A A C C G A T T 5
5 A T G T A A G T T T G G C T A A 3

Extra U

U instead of C
5 A U G A A G U U U G G U U A A 3
Met

Lys

Phe

Gly

Stop

Silent (no effect on amino acid sequence)

5 A U G U A A G U U U G G C U A A 3
Met

Stop

Frameshift causing immediate nonsense

(1 nucleotide-pair insertion)

T instead of C

A missing

3 T A C T T C A A A T C G A T T 5
5 A T G A A G T T T A G C T A A 3

3 T A C T T C A A C C G A T T 5T

5 A T G A A G T T G G C T A A 3A

A instead of G

U missing

5 A U G A A G U U U A G C U A A 3
Met

Lys

Phe

Ser

Stop

Missense

5 A U G A A G U U G G C U A A
Met

Lys

Leu

Ala

Frameshift causing extensive missense

(1 nucleotide-pair deletion)
A instead of T

3 T A C A T C A A A C C G A T T 5
5 A T G T A G T T T G G C T A A 3

U instead of A
5 A U G U A G U U U G G C U A A 3
Met

Nonsense

Stop

T T C missing
3 T A C A A A C C G A T T 5
5 A T G T T T G G C T A A 3
A A G missing

A A
5 A U G U U U G G C U A A 3U
Met

Phe

Gly

Stop

No frameshift, but one amino acid missing

(3 nucleotide-pair deletion)

41

Diagnosis of human genetic diseases

Cytogenetic investigation
1) Karyotyping
2) Fluorescent in-situ hybridization (FISH)
Molecular investigation (will be discussed in the next section)
1) Restriction fragment length polymorphism
2) DNA sequencing

Cytogenetic investigation
Karyotyping - identify chromosome abnormalities by counting the
number of chromosomes and looking for structural changes in
chromosomes

Cytogenetic investigation
Fluorescent in situ hybridization (FISH) : Identify chromosomal
rearrangement by detecting specific DNA sequences

(from A Becker & H Kuster, Bielefeld University, Demark)

(from Molecular Biology of the Cell, 4th Edition)

Learning outcomes - IV
Students should be able to

Outline the basic steps in DNA replica3on, transcrip3on and

transla3on, and explain the func3on of each molecule involved

Understand the role of DNA repair in maintaining gene3c

delity

Describe the associa3on of chromosomal and nucleo3de

changes in human gene3c disorders, and understand the
techniques for iden3ca3on of gene3c changes

45