Organized systems

Prokaryotic cell
Some prokaryotic cells
Figure 24-13 Molecular Biology of the Cell (© Garland Science 2008)
 Eukaryotic cells
 Structural complexity allows sophisticated
regulation of living processes
 They have internal membrane-enclosed
organelles.
 Plasma membrane : lipid bilayer, external:
glycocalyx
 Permeability barrier, Ion channel,
Receptors etc
Structure of cells
Endoplasmic
Retikulum

Mitochondrion

Nucleus
membrane
Lysosome

Cytoplasm
Plasma
Membrane
Golgi Apparatus
 Organelles and fraction :
 Nucleus Cytoskeleton
 Mitochondrion Cytosol
 Endoplasmic reticulum Ribosome
 Lysosome
 Plasma membrane
 Golgi Apparatus
 Peroxisome
 Organelles in Eukaryotic cells

 Plasma membrane:
 Glycocalyx attached to membrane
proteins and certain lipid molecules
 Possesses a diversity of chanel
complexes that transport ions and
molecules, enzymes and receptors that
bind signal molecules
 Nucleus:
 Lamins (protein) form a fibrous network that
provides structural support
 Contain chromatin fibers which is composed
of DNA and histones
 Nuclear envelope is composed of two
membranes that fuse at nuclear pores
 The outer nuclear membrane is continuous
with the RER
 Nuclear pore plug: RNA and small proteins
into and out of the nucleus
 Nucleolus : the site of rRNA synthesis
 Organelles in Eukaryotic cells

 Endoplasmic reticulum:
 A system of interconnected membranous
tubules, vesicles and large flattened sacs
 RER: involved in the synthesis of
membrane proteins and protein for export
from the cell
 SER: lipid synthesis and biotransformation
 Organelles in Eukaryotic cells

 Ribosomes:
 Cytoplasmic ribosomes: RNA/protein
complexes, 20nm
 Function: biosynthesis of proteins

 Containing two irregulargy large and

small subunit which is separate when not
in use
 Golgi Apparatus:
 Large, flattened, saclike membranous vesicles
 A small membranous vesicles containing
protein and lipid fuse with the cis Golgi
membrane
 These molecules are transported and
processed by enzymes
 When the products reach the trans face, they
are targeted to other parts of the cell
 Secretory granules: exocytosis
Golgi Apparatus
 Organelles in Eukaryotic cells

 Lysosomes:
 Spherical saclike organelles, 500nm

 Single membrane contain granules that

are aggregates of digestive enzymes
(acid hydrolase)
 Organelles in Eukaryotic cells

 Peroxisomes:
 Small spherical membranous organelles
that contain oxidative enzymes
 Involved in breakdown of toxic
molecules: peroxides
 Mitochondria:
 Cell’s energy storage molecule
 2 Membranes: Outer (permeable to
molecules <10.000 D); Inner
(impermeable to ions and variety
organic molecules)
 Matrix: several circular DNA and all
components required for protein
synthesis
 The number: vary with the cell’s activity
Mitochondria
Cytoskeleton:
 Components: microtubules, microfilaments and
intermediate fibers
 Microtubules: axon, dendrites, mitotic spindle,
cilia and flagella
 Functions:
1. Maintenance of overall cell shape
2. Facilitation of coherent cellular movement
3. Provision of a supporting structure that
guides the movements of organelles within
the cell.
 BODY PROTEIN
 Enzyme
 Receptor
 Hormone
 Growth Factor
 Immunoglobulin
 Interferon, Interleukin
 Adhesions molecules
 HLA/MHC (Major
Histocompatibility Complex)
 α-2
 Retinol binprotein
 α-2 HS Glycoprotein
 Histidine-rich 3,8 S α2 Glycoprotein
 Haptoglobin
 Pregnancy zone protein
 α2 Macrogobulin
 Prothrombin
 Antihemophilic factor
 C1 inactivator
 C1s
 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
Action of
Insulin
 STRUKTUR PROTEIN :

 Struktur Primer : Sequensi asam amino

 Struktur Sekunder : -helix, lipatan 

 Struktur Tertier : satu atau sub-unit protein

 Struktur Kwaterner : keseluruhan protein

(terdiri dari beberapa sub-unit)
Protein : Globular (Hemoglobin,Myoglobin)
Fibrous (Collagen, Elastin, Keratin)
Denaturation of proteins :
 Unfolding and disorganization of the protein’s
structure
 Denaturing agents : heat, organic solvents,
mechanical mixing, strong acids or bases,
detergents and ions of heavy metals (lead,
mercury)
 20 Amino acids are found as constituents of
mammalians proteins.
H
+H N C COOH
3
R
 Amino acids (essential-non essential,
acidic-basic side chains, polar-non polar)
 Amino acids : Ala, Cys, Pro, Val, Glu, Gln, Asp,
Asn, His,Tyr,Trip, Leu, Ile, Phe,
Gly, Met, Ser, Thr, Arg, Lys.
 Peptide bonds → Polypeptides
 Unique three - dimensional structures
 Specific biologic functions
 Lipoproteins are a complex of lipids with protein
 Glycoproteins contain covalently bound
carbohydrate
 Structural proteins (collagen, laminin,
fibronectin)
 Functional proteins (Enzyme, Receptor, Ig, etc.)
 Biological Structure
 Proteins are all synthesized from the
same 20 amino acids, nucleic acids are made
from 8 types of nucleotides and 8 commonly
occuring types of sugars in polysaccharides
 Its monomeric units can be arranged and
derivatized
 Newly synthesized proteins spontaneously
fold to assume their native conformation
(self assembly), their amino acid sequences
specify their three-dimensional structures
 STRUKTUR PROTEIN
 SIFAT PROTEIN
 FUNGSI PROTEIN
 PEMBENTUKAN PROTEIN
 DISTRIBUSI PROTEIN
 PEMERIKSAAN PROTEIN
 SIFAT PROTEIN :
 Ditentukan oleh sifat asam amino

FUNGSI PROTEIN :
 Sangat bervariasi

PEMBENTUKAN PROTEIN :
 Berdasarkan gen / DNA di inti sel
 Berlangsung di Organella (Ribosome)
 Protein Synthesis

transcription

DNA RNA Protein

translation
Chromosomes
 22.3

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

21.1 cleft palate

21.2
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
 Pedigree of Hemophilia

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.
 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 : 3,2 milyar (990 mm) di
Chromosome (inti sel)
 Telah selesai disequensi pada Juli 2000
 Gen : Sepotong DNA (Intron dan Exon)
 A-T G-C
 Satuan DNA : bp (base pair)
 The Human Genome
 The haploid human genome is made up
9
of 3,2 x 10 base pairs of DNA

 This contains 20,000 genes arranged

on 46 chromosomes

 Packaged within the nucleus of the cell

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
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.
 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
DNA New strands G C
G
unzips formed

semi-conservative
2 daughter cells
 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
 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
+
 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.
 Bagian Intron dipisahkan dari mRNA
(Splicing)
 mRNA meninggalkan inti sel, ke sitoplasma
 Di sitoplasma, mRNA terikat pada ribosom
untuk memastikan sandi asam amino yang
sesuai yang akan ditranslasikan.
 mRNA is always read 5’  3’ protein
synthesis is N  C dan dengan peran tRNA
yang mentransfer asam amino, terbentuklah
protein (polypeptida)
 rRNA : 40s particle (18S RNA and 55 %
protein) ; 60S particle (28S; 5,8S; 5S rRNA
and protein)
 Initiation (translational initiation signal) : AUG
 Amino acid linked tRNA : Aminoacyl-tRNA
synthetase.
 Elongation : Peptidyl transferase.
 Termination : Stop Codon
(UAG, UAA, UGA)
 Amino acid assembly during translation occurs on
ribosomes; tRNA serves as the crucial adaptor molecule
 Classes of RNA
 Messenger RNA - mRNA
Copy of the genetic information in DNA.
Used as pattern to make proteins.
 Transfer RNA - tRNA
Small RNA molecule (70 - 90 base units).
Used to bring the correct amino acid to the
site of protein synthesis.
 Ribosomal RNA - rRNA
Platform for protein synthesis. Holds mRNA
in place and helps assemble proteins.
 Anticodons on tRNA
HO- A
C

Site of amino C
A

acid attachment G C
G C
G U
C G
G C
U U
U U
A U G C A G G C C A
G G U
C G C G G G
U U C C G G C
C G
C C
G C G C
G U
G U A G
C G A G

U A
C G
Point of
Three base C G
C G
attachment
anticodon site U G
U
C G C
G
to mRNA
Biogenesis miRNA
snoRNA
lncRNA
 free Protein
ribosomes
Traffic
cytoplasmic
proteins

RER
Transmembrane Protein Synthesis
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

 Amino Acid Codons
alanine GCA, GCC, GCG lysine AAA, AAG
GCU methionine AUG
arginine AGA, AGG, CGA phenylalanine UUC, UUU
CGC, CGG, CGU proline CCA, CCC
asparagine AAC, AAU CCG, CCU
aspartate GAC, GAU serine UCA, UCC
cysteine UGC, UGU UCG, UCU
glutamate GAA, GAG AGC, AGU
glutamine CAA, CAG threonine ACA, ACC
glycine GGA, GGC, GGG ACG, ACU
GGU tryptophan UGG
histidine CAC, CAU tyrosine UAU, UAC
isoleucine AUA, AUC, AUU valine GUA, GUC
leucine CUA, CUC, CUG GUG, GUU
CUU, UUA, UUG
 Mutations
 A change in the DNA sequence of the
gene
 Mutations can alter protein product of
DNA, stop gene working or activate
gene
 Can be caused by chemical or
environmental factors - mutagens.
 Types of Mutation

 Deletion - DNA missing

 Insertion - extra DNA inserted
 Expansion - DNA repeat size has
increased
 Point Mutation - change in one
base
 Types of Mutation
(in coding sequence)

AGCTTCGACCCG Wild type

AGCTCGACCCG Deletion
AGCTTCCGACCCG Insertion
AGCTTCTTCGACCCG Expansion
ATCTTCGACCCG Point mutation
 POINT MUTATION

UAA
(Termination Codon)

UCA
(Codon for Serine)
UCU
(Codon for Serine)
CCA
(Codon for Proline)
 Perbedaan Sandi Nukleotida

Nukleotida : Chr. : Mit. :

UGA Stop Trp

AUA Ile Met

AGA Arg Stop

AGG Arg Stop
 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 G3316A 3316 G→A
A3260G T3394C
A3256G A3252G
luas dijumpai : T16189C
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
H 2O
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

 Pathogenese NIDDM

 Patophysiologi secara genetik → 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 → DM
MITOCHONDRIAL ENERGY METABOLISM AND
INSULIN SECRETION

MODY2
 Expression of Genes
 Genotype - actual genetic makeup of
individual.
 Phenotype - observed trait for individual.
 Phenotype will be based on actual
genotype and how each gene is
expressed.
 Since you have a pair of genes they form
an allele.
 Each one may be dominate, recessive or
some combination.
 Example
 Hemophilia - A sex linked genetic disorder in
which blood clotting is deficient - lack of the
necessary substrate thrombroplastin.
 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.
 Example
 The hemophilia trait is recessive and only
passed via the X chromosome. As a result,
females are much less likely to contract this
disorder.
 Frequency of hemophilia trait.
 Females - carrier - 1 in 50,000
 Females - hemophilic - 1 in 100,000,000
 Males - hemophilic - 1 in 100,000
 All hemophilic males are also carriers.
 Mutations

 There are ~ 4000 known human genetic

diseases. Many result from mutation of a
single gene.
 Sickle cell anemia. Mutation of gene that
makes part of hemoglobin.
 Albinism. Lack of ability to produce
tyrosinase which catalyzes the conversion of
tyrosine to DOPA.
 Mutagenic Agents
 Radiation:
Ionizing - X-rays,  rays, cosmic rays
Non-ionizing - UV light
 Chemical:
Reactants with bases - formaldehyde
Base analogs - 2-aminopurine, 5-
bromouracil
Acridine dyes - proflavin
Alkylating agents - mustard gases
Others - carcinogens
 DNA Mutation and Repair

 Cells contain many DNA repair mechanisms.

 Yeast - at least 50 repair enzymes are
known.
 Example - exposure to UV light.

UV - High energy light.

- What causes ‘tanning’.
- Causes formation of thymine dimer.
- at low levels, it can be repaired.
- at high levels, it can be deadly.
Photodimerization

Exposure to UV light UV light

can cause adjacent
thymines to
covalently link.

This results in a
distortion of the DNA
molecule and
breaks the hydrogen thymine
bonding with the dimer
adenine.
 PENYAKIT GENETIK
4 KELOMPOK/KATAGORI

 1. Kelainan pada 1 gen tertentu

 2. Kelainan yang multifaktor
 3. Anomali pada Chromosome
 4. Mutasi pada sel-sel somatis

 > 70 % autosomal dominant

 20 % autosomal resessif
 x-linked < 10 %
 Contoh penyakit molekuler :
 Gen normal Hb : A/ A dan   /  
  Thalassemia (point mutation) :
T/ A atau T/ T
  Thalassemia (deletion) :
-/ ; -/- ; - - / ; -/- - (HbH) ; --/--
(Hb Barts Hydrops Fetalis)
 Autosomal resessif : Pedigree (horizontal dan
vertikal), biasanya bersaudara dan kedua
orangtuanya sehat
 Cystic Fibrosis (penyakit gangguan fungsi kel.
Eksokrin , misalnya penyakit saluran nafas
kronis, meconium ileus pada neonatus, dll).
 Contoh penyakit molekuler :
 Autosomal dominant : Pedigree (vertikal),
terdapat pada setiap generasi, kelainan 50 %
berisiko
 Chorea Huntington, Polycystic Kidney,
Familial Hypertrophy Cardiomyopathy,
Marfan Syndrome, Otosklerose dll
 X-linked : pada laki-laki homozygote dan
perempuan heterozygote.
 Hemofilia A (VIII) : 26 exon, > 186 kb, xq28
 Hemofilia B ( IX) : 8 exon, > 34 kb, xq26 - 27
 Fragiles X Syndrome : Xq 27. 3, (CCG)n
 Contoh penyakit molekuler :
 Myotonic Dystrophy : 19q 13.3, (AGC )n
 Duchenne dan Becker Dystrophy : 65 %
deletion, 79 exon (2300 kb), Xp21, Dystropin
(427 kD), diagnose prenatal (1987)
 Penyakit genetik yang multifaktor :
1. Kelainan pembuluh darah jantung
2. Hypertensi
3. Kelainan jiwa
4. Demensia
5. IDDM
6. Cancer, dll
 Polimerase Chain Reaction (PCR)
Tahun 1985, Kary Mullis, California

PCR membutuhkan :
 DNA atau RNA
 Oligonucleotidprimer (PRIMER)
 Enzym Taq-Polimerase
 Campuran dari 4 Basa Nukleotida
(d’NTPs)
 10 x Reactions Buffer
 Larutan MgCl2
 Alat Thermal-Cycler
 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
 Double stranded → Sequence dari Nukleotida
→ Penentuan Primer
 DNA double helix → Primer sepasang
 Bagian gen yang akan diamplifikasi tergantung
pada Kebutuhan
 Primer menjadi “guide” untuk sequensi yang
akan diamplifikasi
 Exon dan Intron (kedua bagian ini dapat
berfungsi sebagai Matrix untuk amplifikasi) atau
bagian DNA lainnya
 RNA
 Single strand (Uracil pengganti Thymin)
 Transkripsi dari DNA → mRNA
 Mengandung informasi genetik dari Exon
saja
 Menggunakan Enzym Reverse
Transkriptase → cDNA
(complementare/copy DNA)
 Primer/ Oligonucleotidprimer

 Sequence dari Nukleotida tertentu (tergantung

bagian mana yang akan diamplifikasi)
 Sekitar: 20 – 30 bp
 Polimorfisme gen reseptor dopamin D2 -141C
Ins/Del,
 D2-677: 5’-ACT GGC GAG CAG ACG GTG
AGG ACC C-3’
 D2-676: 5’-TGC GCG CGT GAG GCT GCC GGT
TCG G-3’
 Enzim BstNI (New England,Biolabs® Inc)
 Taq-Polimerase
 Klenow - DNA Polymerase (E.Coli)
 1988 (Saiki et al.) Taq-Polymerase dari Bakteri
Thermus aquaticus
 Hybridisasi dan Polimerisasi berlangsung
pada temp. 50-70 °C
 1 Double strand DNA , 20 Cycle , 220.000
Double strand DNA
 Perhatikan : Buffer yang digunakan (10 x RB)
 Diperlukan : konsentrasi MgCl2 tertentu
 Parameter lainnya
 Sampel/Material : darah, urine,
jaringan biopsi, dari autopsi, fossil,
rambut, jaringan yang difixasi dengan
paraffin atau formalin
 Semua pekerjaan memerlukan
ketepatan yang tinggi (pipet dengan
ukuran 1/100 µl)
PCR Product (Amplifikat=Amplicon)
 Gel-elektrophorese (Agarose)
 Southern Blot (Hybridisasi dengan
Sonde DNA spesifik)
 Dot - Blot (deteksi : Enhanced Chemie
Luminescense = ECL)
 Denaturating Gradient Gel
Elektrophorese (DGGE) atau PFGE
 Enzym Restriksi : Restriction
Endonuclease
 Sequence analyse (DNA Sequencing)
 MOLECULAR MICROBIOLOGY

Perkembangan Teknologi untuk Diagnosa

 Penentuan langsung secara optis
(mikroskop cahaya atau elektron)
 Kultur (isolasi, koloni, sifat-sifat micro-
organisme dan reaksi biokimia)
 Reaksi antigen-antibody (enzyme-
linked, zat fluorisensi, agglutinasi)
 Penggunan teknologi DNA : Hybridisasi
(southern blot, dot-blot, PFGE), Insitu
Hybridisasi dan Amplifikasi DNA (PCR)
 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
Meningitis : Neisseria meningitidis
Diagnose harus cepat untuk mencegah
komplikasi.
Pewarnaan Gram dapat dilakukan,
tetapi minimal 24 jam
Pemberian antibiotika pada stadium dini
→ mengganggu interpretasi
Transport bahan pemeriksaan
Test Kit : antigen meningococcus
tidak sensitif dan false positif
 Aplikasi teknologi DNA
Encephalitis : Herpes simplex type I
CAT dan EEG → non spesifik
Antibody → cross reaction dengan
virus type II
Kultur virus → dari biopsi dan berisiko
tinggi
Sementara ditherapy dng : Acyclovir
Pemeriksaan DNA
Encephalopathy : P. falciparum
 Aplikasi teknologi DNA
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
Diagnosa dengan PCR dan Hybridisasi
(contoh : dot-blot)
RESULT
 Aplikasi teknologi DNA
VIRUS : HIV – 1 dan HIV – 2
Test Kit (antibody) dan DNA (PCR)
Masalah : Prognosa pasien yang serologi (-) tetapi
PCR (+), Transfusi darah bila antibody anti p24
(+) pada Western Blot dari donor yang tidak
berisiko, Infeksi sampai antibody dideteksi : 3
bulan, Infeksi pada bayi dari Ibu yang serologi (+)
Protein : env : gp 120 dan gp 41, gag : p 24
dan p 18
PCR : sensitif (99% ) dan spesifik (94,7 % )
 Aplikasi teknologi DNA

Cytomegaly virus : immunosuppresif atau

transplantasi organ, ataupun infeksi intra uterine
→ tuli dan retardasi mental.
Hepatitis : membedakan →
virus C , pada transfusi
(10 %), dengan EIA : tidak dapat
Malaria : Mikroskopik : terbatas , kapasitas
tertentu, dengan recombinant DNA →
hybridisasi (repetitive DNA atau RNA)
Papilloma virus : Type 16 dan 18 → Cancer
type 6 dan 11 → Condyloma.
 MOLECULARE ONKOLOGY

 Onkos (Yunani ) = tumor (massa)

 Cancer didasari oleh kelainan genetik
yang bermanifestasi pada tingkat sel
 Beberapa bukti peran gen dalam
menyebabkan suatu tumor :
 Orang yang mempunyai kelainan
dalam mekanisme Repair DNA
mempunyai risiko yang lebih tinggi
untuk timbulnya tumor.
 MOLECULARE ONKOLOGY
 Faktor kimia dan fisik tertentu yang
menyebabkan perobahan pada DNA,
akan menyebabkan kanker (pd hewan
percobaan).
 Perobahan pada chromosome “germ
line” memudahkan timbulnya kanker.
 Phanotype yang maligna dapat
dipindahkan kepada sel normal melalui
gentransfer.
 Pada sel-sel tumor dijumpai adanya
mutasi somatis .
 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
 Produk Protoonkogen

SIS
ABL

FMS Inti Sel

SRC
Orga FOS
FMS
RAS nella MYC
JUN

MOS

ERB-B1
 Protoonkogen DNA Neoplasma

 abl Translocation CML

 myc Translocation Burkitt Lymphoma
 erb B Amplification Epithelcarcinoma,
Astrocytoma,
Ca Oesophagus
 neu Amplification Adeno Ca (Mammae,
Ovarium, Gaster)
 myc Amplification Ca- Mammae, Paru,
Uterus, Oes
 N-myc Amplification Neuroblastoma,
Ca.Paru
 Int-2 Amplification Ca-Oesophagus
 MOLECULARE ONKOLOGY
 TRANSFORMATION : terbentuknya
phenotype neoplastis pada sel yang
normal
 TRANSFEKTION : gentransfer, yaitu
fragment DNA dimasukkan ke dalam
sel eukaryotik
 TRANSDUKTION : pemindahan gen
dari sebuah sel kepada sel lain melalui
infeksi virus.
 Reaksi tubuh atas Cancer →
komplex (tergantung banyak faktor)
 MOLECULARE ONKOLOGY

 Protoonkogen > 60 jenis, meliputi :

 1. Faktor pertumbuhan : induksi proliferasi
sel, bbrp → transformasi pada sel (invitro)
 2. Reseptornya : fms - gen (reseptor CSF-1)
untuk diferensiasi Makrophage
 3. Protein Signal transduction pada membran
atau sitoplasma → second messenger ,
misalnya protein G.
 4. Protein yang terikat pada DNA (transkripsi)
 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
 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)
Carcinogenesis (Colorectal Cancer)
 Normal Epithelium
APC Mutations
(>95%)
Hyperploriferative epithelium

Early adenoma
K-RAS Mutations (30-40%)
Intermediate adenoma

Late adenoma
p53 Mutations ≈ 50%
Carcinoma insitu

Other changes
Metastasis
 Penerapan Teknologi Gen/DNA
dalam Therapy
 Produk-produk bioteknologi :
 Faktor VIII : 2000 AA ( 26 exon, 186 kb,
mRNA 9 kb)
 1984 : cloning gen Faktor VIII dan
expresinya
 1987 : Recombinant F VIII.
 Erythropoietin : diketahui sejak > 80 thn
 1985 : cloning dan sequensi →
Recombinant Erythropoietin.
 Penerapan Teknologi Gen/DNA
dalam Therapy
 Hormon pertumbuhan : 191 AA
 1979 : cloning dan expresi gen GH
 Faktor pertumbuhan pada hematopoietik
system: CSF (Colony Stimulating Factors)
dan Interleukine
 Contoh : G-CSF, M-CSF, GM-CSF
 Interleukine : proliferasi dan differensiasi
sel, juga pada reaksi peradangan dan
sistem immunitas.
 Penerapan Teknologi Gen/DNA
dalam Therapy
 Immunisasi :
 1982 : vaksin Hepatitis dari virus yang
diinaktifkan dan dimurnikan
 1987 : produk teknologi gen (DNA
recombinant)
 Immuntherapy : antibody monoklonal

 Untuk : tumor, dimana Ab diikatkan

dengan Toxin, Radionuclide, Chemotherapy
 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
Penerapan Teknologi Gen/DNA dalam Therapy
 Produk dari gen untuk therapy dan
prophylaxis :
 GM –CSF

 G-CSF

 Interleukine

 Interferone

 Gentherapy :

 Transfer material genetik terhadap sel

atau organisme untuk mengatasi suatu
penyakit. (pada sel somatik saja)
Penerapan Teknologi Gen/DNA dalam Therapy

 Gentherapy :
 Haemophilia A atau B
 Hemoglobinopathy
 Penyakit penurunan immunitas :
defisiensi enzym Adenosindesaminase
 Gangguan siklus Urea : Ornithine
transcarbamoylase
 HGPRT : Lesch-Nyhan Syndrome.
 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
 DNA Mitochondria: maternal
 Aplikasi gen dalam Forensik
 30 % DNA eukaryotik terdiri dari repetitive
sequence (tidak berfungsi)
 Beberapa kriteria : STR (Short Tandem
Repeat), mikrosatelit (< 1 kb), minisatelit
(1-30 kb) dan makrosatelit (megabase).
 Minisatelit terutama berperan dalam
menentukan hubungan kekeluargaan.
 Studi populasi dapat didasarkan pada HLA
genotype (lengan pendek dari Chr. 6)
 HLA juga digunakan untuk identifikasi
jaringan dan dapat dilakukan dengan
bantuan “Test Kits”