Sub topics :

Search for genetic material---nucleic acid or

protein/DNA or RNA?

Griffiths Transformation Experiment

Averys Transformation Experiment
Hershey-Chase Bacteriophage Experiment
Tobacco Mosaic Virus (TMV) Experiment

Nucleotides - composition and structure

Double-helix model of DNA - Watson & Crick
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:

1890

Weismann - substance in the cell nuclei controls development.

1900

Chromosomes shown to contain hereditary information,

later shown to be composed of protein & nucleic acids.

1928

Griffiths Transformation Experiment

1944

Averys Transformation Experiment

1953

Hershey-Chase Bacteriophage Experiment

1953

Watson & Crick propose double-helix model of DNA

1956

Gierer & Schramm/Fraenkel-Conrat & Singer

Demonstrate RNA is viral genetic material.

Frederick Griffiths Transformation Experiment - 1928

transforming principle demonstrated with Streptococcus pneumoniae

Griffith hypothesized that the transforming agent was a IIIS protein.

Oswald T. Averys Transformation Experiment - 1944

Determined that IIIS DNA was the genetic material
responsible for Griffiths results (not RNA).

Hershey-Chase Bacteriophage Experiment - 1953

Bacteriophage = Virus that attacks
bacteria and replicates by
invading a living cell and using
the cells 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:

Label DNA with 32P

Label Protein with 35S

3.

Infected E. coli bacteria with

two types of labeled T2

4.

32

P is discovered within the

bacteria and progeny phages,
whereas 35S is not found within
the bacteria but released with
phage ghosts.

T2growninP32
containingmedia
incorporateP32
intotheirDNA

Using P

T2attachtobacteriaand
injectgeneticmaterial

32

Bacteriagrownin
normalnon
radioactivemedia

Whencentrifuged,
phageproteincoats
remaininthe
supernatantwhile
bacteriaforma
pellet
Thepelletis
radioactive,butthe
supernatantisnot.

DidDNAenterthebacteria?
Blendingcausesphage
proteincoattofalloff

IsDNAthegeneticmaterial?

T2growninS35
containingmedia
incorporateS35
intotheirproteins

Using S

T2attachtobacteriaand
injectgeneticmaterial

Blendingcausesphage
proteincoattofalloff

35

Bacteriagrownin
normalnon
radioactivemedia

Whencentrifuged,
phageproteincoats
remaininthe
supernatantwhile
bacteriaforma
pellet
Thesupernatantis
radioactive,butthe
pelletisnot.

Didproteinenterthe
bacteria?
Isproteinthegenetic

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.

DNA :

DEOXYRIBONUCLEIC
ACID

STORES AND PASSES ON

GENETIC INFORMATION
FROM ONE GENERATION TO
ANOTHER .

Nucleotide = monomers that make up DNA and RNA (Fig.8)

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.
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!
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.

A Nucleotide

Adenosine Mono Phosphate (AMP)

OH

Phosphate

HO

Nucleotide

NH2

Base
N

H
O
5CH2
4

H
N

Sugar

OH

H
OH

N
N
Nucleoside

Purines
NH2
Adenine
N
N
N

O
CH3

(DNA)

N
Guanine
NH

Thymine

NH2

Uracil
(RNA)

NH

N
O

N
N

Pyrimidines

NH

NH2
Cytosine
N
N

Base Pairing

Guanine And Cytosine

H N

in e

H
N
H

os
Cyt

N
N H
in e
N an
Gu

-O

Base Pairing

CH

Thymine
N

N-

H
+
H
Adenine N
N

Adenine And Thymine

Base Pairing

Adenine And Cytosine

os
Cyt

H
H H+ +
H N
N
e
n
i
n
e
d
A
N
N
N
N
N
-O N

ine

Base Pairing

e
n
i
m
y
Th

N
N H
in e
N an
Gu

CH

Guanine And Thymine

N
H

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

indicated double-stranded DNA consists of ~50%

purines (A,G) and ~50% pyrimidines (T, C)

amount of A = amount of T and amount of G =

amount of C (Chargraffs rules)

%GC content varies from organism to organism

Examples:

%A

%T

%G

%C

%GC

Homo sapiens
Zea mays
Drosophila

31.0
25.6
27.3

31.5
25.3
27.6

19.1
24.5
22.5

18.4
24.6
22.5

37.5
49.1
45.0

James D. Watson & Francis H. Crick - 1953

Double Helix Model of DNA
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:

5 3
3 5

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.
A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).
e.g.,5TATTCCGA3
3ATAAGGCT5
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.

Denaturation and Renaturation

Heating double stranded DNA can overcome the
hydrogen bonds holding it together and cause the
strands to separate resulting in denaturation of
the DNA
When cooled relatively weak hydrogen bonds
between bases can reform and the DNA renatures

DenaturedDNA Re
n
o
na
i
t
a
tur
r
ATGAGCTGTACGATCGTG
tu
ati
a
n
on
e
D
ATGAGCTGTACGATCGTG
TACTCGACATGCTAGCAC

DoublestrandedDNA

ATGAGCTGTACGATCGTG
TACTCGACATGCTAGCAC
TACTCGACATGCTAGCAC

SinglestrandedDNA

DoublestrandedDNA

Denaturation and Renaturation

DNA with a high guanine and cytosine content has

relatively more hydrogen bonds between strands
This is because for every GC base pair 3 hydrogen bonds
are made while for AT base pairs only 2 bonds are made
Thus higher GC content is reflected in higher melting or
denaturation temperature

ACGAGCTGCACGAGC
TGCTCGACGTGCTCG

ATGATCTGTAAGATC
TACTAGACATTCTAG

67%GCcontent
Highmeltingtemperature

33%GCcontent
Lowmeltingtemperature

ATGAGCTGTCCGATC
TACTCGACAGGCTAG
50%GCcontent Intermediatemeltingtemperature

RNA (A pairs with U and C pairs with G)

Examples:
mRNA
tRNA
rRNA
snRNA

messenger RNA
transfer RNA
ribosomal RNA
small nuclear RNA

RNA secondary structure:

Yeast Alanine tRNA

single-stranded
Function in transcription
(RNA processing) and
translation

Organization of DNA/RNA in chromosomes

Genome = chromosome or set of chromosomes that contains all the
DNA an organism (or organelle) possesses
Viral chromosomes

TMV

1. single or double-stranded DNA or RNA

2. circular or linear
3. surrounded by proteins

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)

48,502
T4
168,900
HIV1
9,750
E.Coli
4,639,221
Liliumformosanum
36,000,000,000
Zeamays
5,000,000,000
Amoebaproteus
290,000,000,000
Drosophilamelanogaster180,000,000
Musmusculus
3,454,200,000
Canisfamiliaris
3,355,500,000
Equuscaballus
3,311,000,000
Homosapiens
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
5 main types: H1, H2A, H2B, H3, H4
~equal in mass to DNA
evolutionarily conserved

2.

Non-histones

all the other proteins associated with DNA

differ markedly in type and structure
amounts vary widely
>> 100% DNA mass
<< 50% DNA mass

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.

Figs. 2.25-29

The Central Dogma

of Molecular Biology
Cell
DNA

Transcription
Translation

mRNA
Ribosome

Polypeptide
(protein)

1998 Timothy G. Standish

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.

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)

Chromosomes vs Genes
A chromosome
constitutes an entire
DNA molecule +
protein
Protein = histones
Supercoiled DNA in
nucleosomes
Humans contain 46 such
molecules (23 pairs)
44 somatic chromosomes
2 sex chromosomes (X +Y)

Chromosomes vs Genes
Genes constitute

distinct regions on the

chromosome
Each gene codes for a
protein product
DNA -> RNA-> protein
Differences in
proteins brings about
differences between
individuals and
species

How do chromosomes become double

stranded?
During the life of
the cell, each
chromosome of
DNA makes a copy
of itself
This must occur
prior to cell division
to insure each
daughter cell gets a
complete set

Extrachromosomal DNA
is DNA located or maintained in a cell apart from the chromosomes
.
Outside the nucleus, DNA is found in cytoplasmic organelles, such
as mitochondria (both plants and animals) and in chloroplast
(found only in green plants and photosynthetic protists).
The genes in mitochondria (mt) and chloroplast (cp) are also
known as Extrachromosomal genes, non Mendelian genes,
cytoplasmic genes, organellar genes or extranuclear genes.
DNA can also be found in the plasmids of bacteria.

TUGAS : PRESENTASI
1.REPLIKASI ; PROSES REPLIKASI
2.TRANSKRIPSI : TAHAPAN TRANSKRIPSI
3.TRANSKRIPSI : PERBEDAAN PADA EUKARIOT DAN PROKARIOT
4.TRANSLASI : DEFINISI, JENIS RNA YANG BERPERAN, KODE GENETIK
5.TRANSLASI : RIBOSOM, MEKANISME SINTESIS PROTEIN
6.REGULASI EKSP GEN : OPERON lac
7.REGULASI EKSPRESI GEN : OPERON trp
8.MUTASI : MUTASI TITIK, MUTASI TRANSISI, DELESI DAN INVERSI
9.MUTASI : PENYEBAB DAN DAMPAK
10. ABERASI KROMOSOM
11.REKAYASA GENETIKA : KLONING INTI
12. REKAYASA GENETIKA : KLONING GEN