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

p
22.2 X Chromosome
22.1
21.3
growth control factor, X-linked
21.2 Xg blood roup
21.1
11.4
11.3
ocular albinism
11.23 sensorineural deafness
11.22
11.21 11.1
11.2 11.1 anemia, sideroblastic, with
12
Spinocerebellar ataxia
13

21.2
21.1 cleft palate
21.3 lymphoproliferative syndrome
22.1
22.2 22.3 Simpson dysmorphia syndrome
23
24
coagualation factor IX, hemophilia B
25
q 26 blue-monochr. color blindness
27 coagulation factor VIIIc,hemophiliaA
28 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
 α-1
 α-1 Acid Glycoprotein
 α-1 T Glycoprotein
 α-1 Antitrypsin
 Transcortin
 α-1 Antichymotrypsin
 α-1 B glycoprotein
 9,5-s α-1 Glycoprotein
 Vitamin-D binding protein
 α-1 Lipoproteins
 α-2
 Retinol binding protein
 α-2 HS Glycoprotein
 Histidine-rich 3,8 S α2 Glycoprotein
 Haptoglobin
 Pregnancy zone protein
 α2 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-

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 C G
C T
T A A
A T
T T A T
A A
G C
G

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 : α, β, δ, ε and γ
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
TAA
ATG TAG ‘stop’
start 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
AT C G G
UAG CC
mRNA
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)(-ribosom:RNA dan protein. Setiap tRNA
membawa 1 protein)
 Translation: 1. Initiation(-mencari AUG)
2. Elongation
3 Termination
 Translational initiation signal : AUG
 mRNA become translated through 5’ → 3’ direction
 Elongation : Peptidyl transferase.(-enzim)
 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(-antibiotik m’blok
ribosom menjadi tidak bekerja)
Nukleotida 1. Nukleotida 2. Nukleotida 3.
(5’) (3’)
U C A G

U Phe Ser Tyr Cys U

U Phe Ser Tyr Cys C

U Leu Ser STOP STOP A

U Leu Ser STOP Trp G

C Leu Pro His Arg U

C Leu Pro His Arg C

C Leu Pro Gln Arg A

C Leu Pro Gln Arg G

 U C A G

A Ile Thr Asn Ser U

A Ile Thr Asn Ser C

A Ile Thr Lys Arg A

A Met Thr Lys Arg G

G Val Ala Asp Gly U

G Val Ala Asp Gly C

G Val Ala Glu Gly A

G Val Ala Glu Gly G

Perbedaan Sandi Nukleotida

 Nukleotida : Chr. : Mit. :

UGA Stop Trp

AUA Ile Met
AGA Arg Stop
AGG Arg Stop
MITOCHONDRIAL ENERGY TRANSDUCTION

Human body synthesizes body weight of

ATP per day
• motoric functions
• biosynthetic activities
• heat maintenance

ADP + Pi ATP

ATP synthase
Proton Motive
Force

NADH I O2
coQ III Cytc IV
H2O
Succinate II
 MITOCHONDRIAL RESPIRATORY
ENZYME COMPLEXES

Cytosolic side
ΔμH+
ADP
c
I Q III Q II IV UCP ANT

NADH H2O V ATP

H++½O2
NAD + H2
Succinate

Matrix side ADP+Pi ATP

 Gen Mitochondria
 Gen yang berbentuk sirkuler, terdiri dari 16569 bp
 Diturunkan secara maternal, mudah bermutasi
 Menyandi : 7 sub unit kompleks I (NADH Q-Reduktase),
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(di jepang) G3316A
A3260G T3394C
A3256G A3252G
luas dijumpai : T16189C(di indonesia)

(jumlah ATP yang dibentuk selama 24 jam adalah

sebanyak berat badan stiap org)
 MITOCHONDRIAL BIOLOGY AND
GENETICS
 Semi-autonomous
Outer Inner organelles, contain multiple
Compartment Membrane
copies of mtDNA
 Double membrane structure,
cristae containing respiratory
chain enzymes
 Most mitochondrial proteins
Inner Outer encoded by nuclear genome
Matrix Cristae Compartment Membrane and imported into
mitochondria
 Functions in cellular
metabolism and the
regulation of cell death
 (mitokondria tidak sibentuk baru
oleh sel, tetapi melalui pembelahan)
 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

(fungsi insulin:mengaktifkan GLUT 4, kalau insulin sudah ckp byk, reseptor

tdk dimunculkan/recycling, jadi reseptor masuk melalui jalur degradasi =
down regulation)
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
(klo berubah as.amino = MISSENSE MUTATION
Klo timbul as.amino = nonsense mutation
Klo brubah pada protein = silence 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-
meng-amplifikasi (melipat
gandakan) fragment DNA (Gen)
 Dibutuhkan :
DNA atau RNA
Oligonucleotidprimer (PRIMER)
Enzym Taq
Taq--Polimerase
Campuran dari 4 Basa Nukleotida
(d’NTPs)
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-
di-isolasi dari sel (darah atau jaringan)
 DNA menjadi “template” atau “matrix” untuk
proses amplifikasi
 Sense : 5’-
5’- ATG(Start) -GGT
GGT--TCT
TCT--GTT
GTT--GCT
GCT--
GCT--TGG
GCT TGG--TAA(Stop)
TAA(Stop)-- 3’
 Antisense : 3’ - TAC
TAC--CCA
CCA--AGA
AGA--CAA
CAA--CGA
CGA--
CGA--ACC
CGA ACC--ATTATT-- 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-
RT-PCR
 Taq--Polimerase
Taq

 Klenow - DNA Polymerase dari E.Coli

 1988 : Taq-
Taq-Polymerase dari Bakteri Thermus
aquaticus
 Hybridisasi dan Polimerisasi berlangsung pada
temp. 50-
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)
Gel-
 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)
 Protein
free Traffic
cytoplasmic ribosomes
proteins

RER
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 di-inaktivasi 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

FMS Inti Sel

SRC
Orga FOS
FMS
RAS nella MYC
JUN

MOS

ERB-B1
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