Pedigree of Hemophilia

in One Family

female
normal male
hemophilic male

Hemophilia
Hemophilia

- A sex linked genetic disorder in

which blood clotting is deficient

Hemophilia

A - lack of antihemophilic globulin

Most common type (80% of cases).
Hemophilia

B - defect in thromboplastic
component - a milder form of the disease.
Sex linked - trait found on X chromosome.

Chromosomes

22.3
22.2
22.1

p
21.2

21.3

21.1
11.4
11.3
11.23
11.22
11.21 11.1
11.1
11.2
12

X Chromosome
growth control factor, X-linked
Xg blood roup
ocular albinism
sensorineural deafness
anemia, sideroblastic, with
Spinocerebellar ataxia

13
21.2

22.2

21.1

cleft palate

21.3

lymphoproliferative syndrome

22.1
22.3
23
24
25

26
27
28

Simpson dysmorphia syndrome

coagualation factor IX, hemophilia B
blue-monochr. color blindness
coagulation factor VIIIc,hemophiliaA
homosexuality, male

 Tahun

50-an : DNA double stranded yang

membentuk Helix (Watson and Crick),DNA
Polymerase (Kornberg)
Tahun 60-an : DNA extrachromosome
(Plasmid), fungsi mRNA, Codon (Triplet
Nucleotide)
Tahun 70-an : Reverse Transcriptase,
Restriction Endonuclease, DNA Ligase,
Recombinant DNA (Berg), Cloning DNA
(BIOTEKNOLOGI)

 Tahun

80-an : Transgenic mouse,

Penerapan rekayasa genetika dalam bidang
kedokteran, pertanian dan industri.
Tahun 90-an : Gen therapy, Cloning dan
Sequencing DNA (HGP), Diagnostic dll.
Tahun 2000-an : HGP selesai,
Pathogenese penyakit diketahui dari fungsi
molekul.

BODY PROTEIN
Enzyme
Receptor
Hormone
Growth Factor
Immunoglobulin
Interferon, Interleukin
Adhesions molecules
HLA/MHC

-1
-1

Acid Glycoprotein
-1 T Glycoprotein
-1 Antitrypsin
Transcortin
-1 Antichymotrypsin
-1 B glycoprotein
9,5-s -1 Glycoprotein
Vitamin-D binding protein
-1 Lipoproteins

-2
Retinol

binding protein
-2 HS Glycoprotein
Histidine-rich 3,8 S 2 Glycoprotein
Haptoglobin
Pregnancy zone protein
2 Macrogobulin
Prothrombin
Antihemophilic factor
C1 inactivator
C1s

 STRUKTUR

PROTEIN
SIFAT PROTEIN
FUNGSI PROTEIN
PEMBENTUKAN PROTEIN
DISTRIBUSI PROTEIN
PEMERIKSAAN PROTEIN

STRUKTUR PROTEIN :

Struktur Primer : Sequensi asam amino

Struktur Sekunder : -helix, lipatan
Struktur Tertier : sub-unit protein (tiga dimensi)
Struktur Kwaterner : gabungan bbrp struktur tertier

SIFAT PROTEIN :

Ditentukan oleh sifat asam amino

FUNGSI PROTEIN :

Sangat bervariasi

PEMBENTUKAN PROTEIN :

Berdasarkan gen / DNA di inti sel

Berlangsung di Organella (Ribosome)

Proteins are composed of subunits called amino acids

 Biokimia

: DNA adalah Polymer dari

Desoxyribonucleotide (Basa, zat Gula dan 1
atau lebih gugus Phosphat)
Zat Gula : -D-2 Desoxyribose (Ribose)
Ikatan N-Glykosida antara Desoxyribose
(C1) dengan Pyrimidin (N1) atau Purin (N9)

 Sanger

dan Gilbert (1975) : methode

sequensi Basa Nukleotida (A, T, C, G)
Nukleotida : 2,9 milyar (990 mm) di
Chromosome (inti sel)
Telah selesai disequensi pada Juli 2000
Gen : Sepotong DNA (Intron atau Exon)
A- T
G-C
Satuan DNA : bp (base pair)

DNA Base Pairing

A G C G A T C T G G
T C G C T A G A C C
Double helix consists of 2
complimentary strands of DNA.

Chromosomes
Long strands of DNA packaged and
compressed very tightly
Everyone has 2 sets (1 pair) of
chromosomes

1 pair of each of the 22 autosomes

plus XX for a female (46XX)
or XY for a male (46XY)

1 is inherited from mum, 1 from dad

You pass 1 of each pair onto each child

The Human Genome

The haploid human genome is made up

9
of 3 x 10 base pairs of DNA

This contains 50,000- 100,000 genes

arranged on 46 chromosomes

Packaged within the nucleus of the cell

DNA Replication
Each

of the 2 DNA strands is copied by

machinery in the cell
Each new daughter strand has a sequence
complimentary to the original template
strand
Replication essential to allow cell division
(Mitosis) where 1 cell becomes 2

DNA Replication
A
T

A
T

A
G

C
T

A
G

G
A
T
C

semi-conservative
2 daughter cells

DNA Replication

DNA Replication

Replication fork : leading strand and lagging strand

DNA synthesized in the 5 3
The 5-3 synthesis of the leading strand is
continuous.
The lagging strand is also synthesized in the 5-3
direction but in small segments
This segments referred to as Okazaki fragments
Okazaki fragments has 100 200 nucleotides
DNA ligase joined the Okazaki fragments.
5 DNA Polymerase : , , , and

The DNA Replication Fork

DNA Replication in Meiosis

During

the replication of chromosomes, there is a

cross-over of portions of one DNA strand to
another (of the same chromosome).
This cross-over, along with randomization assures
that offspring differ from the parents.

meiosis

Genes
Segments of DNA code for proteins (or
parts of proteins)
Each coding segment is called a gene
One gene codes one protein (or part of)
Genes contain the information which
makes us what we are

Gene Structure
Every

three bases of DNA is called a codon

Each codon specifies an amino acid which
join together to form the protein
eg ATG = methionine = START
TAA = STOP
TAG = STOP
TGA = STOP

Gene Structure
Introns
Promoter

Exons
ATG
start

TAA
TAG stop
TGA

Exon = coding sequence

Intron= intervening sequence
(non-coding)

Protein Synthesis
transcription

DNA

RNA

Protein

translation

Transcription

3 Nuclear RNA Polymerase : mRNA transcribed by

RNA Polymerase II
The initiation of transcription involves binding RNA
Polymerase to a specific DNA sequence called a
Promoter
Many promoters for RNA Polymerase II contain
consensus sequences, referred to as the TATA box (
T A T A A/T A A/T A/G) which occur about 25-35 bp
upstream from the transcription initiation site.
The activity of many promoters is affected by
Enhancers (regulatory sequences that may occur
thousands of base pairs upstream or downstream of
the gene they affect.

Protein Synthesis - Transcription

Each

gene codes for a protein

DNA sense strand acts as template
and is transcribed into messenger
RNA (mirror image of the DNA but
Uracil instead of Thymine)
DNA
mRNA

ATC G G
UAG C C

Protein Synthesis- Translation

Introns

are spliced out of the mRNA

mRNA leaves the nucleus
In the cytoplasm, ribosomes attach to the
mRNA ensuring the correct amino acid, for
each codon, is added to a growing chain of
amino acids which forms the resulting
protein.

rRNA : 40s particle (sebuah 18S RNA dan 55 %

protein) ; 60S particle (28S; 5,8S; 5S rRNA dan protein)
Translation: 1. Initiation
2. Elongation
3 Termination
Translational initiation signal : AUG
mRNA become translated through 5 3 direction
Elongation : Peptidyl transferase.
Termination : Stop Codon (UAG, UAA, UGA)
Amino acid will be activated and linked to the tRNA by
Aminoacyl-tRNA synthetase.

Amino acid assembly during translation occurs on ribosomes;

tRNA serves as the crucial adaptor molecule

Nukleotida 1.
(5)
U

Nukleotida 2.

Nukleotida 3.

Phe

Ser

Tyr

Cys

Phe

Ser

Tyr

Cys

Leu

Ser

STOP

STOP

Leu

Ser

STOP

Trp

Leu

Pro

His

Arg

Leu

Pro

His

Arg

Leu

Pro

Gln

Arg

Leu

Pro

Gln

Arg

(3)

Ile

Thr

Asn

Ser

Ile

Thr

Asn

Ser

Ile

Thr

Lys

Arg

Met

Thr

Lys

Arg

Val

Ala

Asp

Gly

Val

Ala

Asp

Gly

Val

Ala

Glu

Gly

Val

Ala

Glu

Gly

Perbedaan Sandi Nukleotida

Nukleotida

UGA
AUA
AGA
AGG

Chr. :
Stop
Ile
Arg
Arg

Mit. :
Trp
Met
Stop
Stop

MITOCHONDRIAL ENERGY TRANSDUCTION

Human body synthesizes body weight of

ATP per day
motoric functions
biosynthetic activities
heat maintenance
ATP

ADP + Pi

ATP synthase
Proton Motive
Force

NADH

I
coQ

Succinate

II

III

Cytc

IV

O2
H2O

MITOCHONDRIAL RESPIRATORY
ENZYME COMPLEXES
Cytosolic side
H+
ADP

c
I

NADH
NAD + H2

Matrix side

III Q II

IV

H++O2

UCP

H2O

ATP

Succinate
ADP+Pi

ANT

ATP

Gen Mitochondria
Gen yang berbentuk sirkuler, terdiri dari 16569 bp
Diturunkan secara maternal, mudah bermutasi
Menyandi : 7 sub unit kompleks I (NADH QReduktase), 3 sub unit kompleks IV (Sitokrom
Oksidase), 2 sub unit ATP Synthase dan 1 sub unit
kompleks III (Apositokrom B)
Mutasi noktah (point mutation) pada gen mitochondria
:
A3243G
G3316A
A3260G
T3394C
A3256G
A3252G
luas dijumpai :
T16189C

MITOCHONDRIAL BIOLOGY AND

GENETICS
Outer
Inner
Compartment Membrane

Semi-autonomous
organelles, contain multiple
copies of mtDNA
Double membrane structure,
cristae containing respiratory
chain enzymes

Inner
Matrix Cristae Compartment

Outer
Membrane

Most mitochondrial proteins

encoded by nuclear genome
and imported into
mitochondria
Functions in cellular
metabolism and the
regulation of cell death

MITOCHONDRIAL PROTEINS mtDNA

Circular DNA - 16,569 bp

Encodes 13 polypeptides - for OXPHOS 22 tRNA 2 rRNA
D-loop - initiation of replication and transciption
Evolves at higher rate than nDNA
Maternally inherited

Pathogenese NIDDM

Patophysiologi secara genetik yang berkorelasi dengan

metabolisme energi
Timbul oleh karena perobahan cara hidup dengan cepat
(terutama dalam hal nutrisi)
Sel Pankreas berfungsi untuk mensekresikan Insulin
bergantung pada energi yang dibentuk di Mt.
Phosphorilasi oksidatif pada rantai respirasi Mt
ATP
ATP dependent Potassium Channel
tertutup
Calcium Channel terbuka
sekresi
Insulin
Mutasi MtDNA
penurunan ATP

MITOCHONDRIAL ENERGY METABOLISM

AND INSULIN SECRETION

MODY2

Transmembrane Protein Synthesis

Mutations
A change in the DNA sequence of the
gene
All cells acquire mutations as they
divide

-6

rate of approx 10 per gene per cell

Mutations can alter protein product of

DNA, stop gene working or activate
gene

Types of Mutation
Deletion

- DNA missing
Insertion - extra DNA inserted
Expansion (Amplification) - DNA
repeat size has increased
Point Mutation - change in one base

Types of Mutation
(in coding sequence)

AGC TTC GAC CCG

Wild type
AGC TCG ACC CG
Deletion
AGC TTC CGA CCC G
Insertion
AGC TTC TTC GAC CCG
Expansion
ATC TTC GAC CGG
Point mutation

POINT MUTATION
UAA
(Termination Codon)
UCA
(Codon for Serine)
UCU
(Codon for Serine)
CCA
(Codon for Proline)

Polimerase Chain Reaction (PCR)

Tahun 1985, Kary Mullis, California
Metode untuk meng-amplifikasi (melipat
gandakan) fragment DNA (Gen)
Dibutuhkan :
DNA atau RNA
Oligonucleotidprimer (PRIMER)
Enzym Taq-Polimerase
Campuran dari 4 Basa Nukleotida
(dNTPs)
10 x Reactions Buffer
Larutan MgCl2

Alat : Thermal Cycler

Prinsip : perobahan temperatur secara
otomatis dengan waktu yang telah ditentukan
Dapat diatur (Program)
Contoh : 95 C------ Denaturasi
55 C------ Hybridisasi
(Annealing)
72 C------ Synthese DNA
(Extension)
Lama reaksi, bervariasi tergantung panjang
fragment DNA (2 min. : < 1000 Nukleotida)

DNA
DNA di-isolasi dari sel (darah atau jaringan)
DNA menjadi template atau matrix untuk
proses amplifikasi
Sense : 5- ATG(Start) -GGT-TCT-GTT-GCTGCT-TGG-TAA(Stop)- 3
Antisense : 3 - TAC-CCA-AGA-CAA-CGACGA-ACC-ATT- 5
Exon dan/atau Intron dapat berfungsi
sebagai Matrix untuk amplifikasi

RNA

Single strand (Uracil pengganti Thymin)

Transkripsi dari DNA
mRNA
Mengandung informasi genetik dari Exon
Dengan Enzym Reverse Transkriptase
diperoleh DNA dari RNA
cDNA
Reaksi PCR nya disebut RT-PCR

Taq-Polimerase
Klenow - DNA Polymerase dari E.Coli
1988 : Taq-Polymerase dari Bakteri Thermus
aquaticus
Hybridisasi dan Polimerisasi berlangsung pada
temp. 50-70 C
Perhatikan : Buffer yang digunakan
(10 x RB) dan diperlukan MgCl2

Primer
Sequence dari Nukleotida tertentu (Intron
atau Exon) : 20 30 bp
Prinsip : merupakan complementare dari
kedua strand DNA (Forward Primer dan
Reverse Primer).
Dari kedua Primer ini disinthese DNA yang
baru dan seterusnya berfungsi sebagai
matrix untuk siklus berikutnya.
Penentu bagi fragment DNA yang akan
diamplifikasi

PCR-REACTION

PCR-Reaction

Polymerase Chain Reaction

PCR Product (Amplifikat)

Gel-elektrophorese (Agarose)
Southern Blot (Hybridisasi dengan Sonde
DNA spesifik)
Dot - Blot (deteksi : Enhanced Chemie
Luminescense = ECL)
Denaturating Gradient Gel Electrophorese
(DGGE) atau Pulse Field Gel
Electrophorese (PFGE)
Enzym Restriksi : Restriction
Endonuclease
Sequence analysis (DNA Sequencing)

free
cytoplasmic ribosomes
proteins

RER

Protein
Traffic

MOLECULARE MICROBIOLOGY
Aplikasi teknologi DNA

INFEKSI SALURAN CERNA:

Membedakan jenis : pathogen non
pathogen (Eschericia coli)
Untuk bakteri yang sulit dikultur oleh
karena memerlukan syarat tertentu
(Campylobacter)
Membedakan jenis bakteri dari toxin
yang diproduksinya (E. coli dan Shigela
sp.)
Subklas bakteri : Campylobacter,
Helicobacter
Mengidentifikasi jenis Rotavirus (A, B, C)

Aplikasi teknologi DNA

INFEKSI SALURAN NAFAS:

Mycobacterium tuberculosis :
Membedakan jenis atypic, dengan mikroskop
hal ini tidak mungkin
Kultur : waktu yang lama dan bakteri harus
banyak (terutama untuk sensitivity test)
Diagnose cepat dibutuhkan, mis. pada
penderita AIDS.
Ditemui jenis yang multi drug resistant (MDR)
Diagnosa dengan PCR dan Hybridisasi
(contoh : dot-blot)

RESULT

MOLECULARE ONKOLOGY

PROTOONKOGEN : gen yang normal pada

Genom yang berperan penting dalam
proliferasi dan differensiasi sel
ONKOGEN : protoonkogen yang oleh
karena mutasi atau gangguan pada
ekspresinya menyebabkan proliferasi sel
yang neoplastis
TUMORSUPPRESSOR GEN : gen yang
berperan pada proliferasi dan
differensiasi sel, dimana bila gen ini diinaktivasi atau tidak terdapat, akan
terbentuk sel neoplastis

MOLECULARE ONKOLOGY
Contoh

Neoplastic Transformation :
1. Gentranslocation : bcr-abl (chr. 9 dan 22)
2. Genamplification : N-myc gen 300 x pada
Neuroblastoma pada anak-anak
3. Point mutation : ras mengontrol
GTP(aktif) GDP (inaktif)
4. Insertion gen virus : virus Hepatitis B
5. Tumorsuppressorgen : p53 dan gen
retinoblastoma : regulasi siklus sel (stop
pada G1 untuk DNA - repair)

Second-Messenger Mechanism
Adenosine 3,5-cyclic monophosphate (cAMP)

Hormone

Receptor Transducer G Protein (+,-)

(+,-)
Adenylate cyclase

ATP

cAMP
Protein Kinase A

Membrane
Enzymes
Channels
Structural Proteins

Produk Protoonkogen
SIS

ABL

SRC
RAS

FMS

Inti Sel
FOS
MYC
JUN

Org
anell
a

MOS

ERB-B1

FMS

Carcinogenesis (Colorectal Cancer)

Penerapan Teknologi Gen/DNA dalam Therapy

Produk dari gen untuk therapy dan

prophylaxis :
Erythropoietin
Insulin
Hormon pertumbuhan
Faktor pembekuan darah VIII
Plasminogen aktivator
Vaksin Hepatitis B

Aplikasi gen dalam Forensik

Sebelum

teknologi DNA diterapkan (1978)

biasanya digunakan protein, misalnya
antigen gol.darah, HLA, dll.
1985 : DNA Polymorphismus.
Nov.1987 : DNA sebagai barang bukti di
pengadilan di Inggris.
Sampai akhir 80-an : lebih dari 1000 perkara
dibantu oleh bukti-bukti DNA
Juga dapat menentukan Paternity
Profil DNA tiap individu berbeda