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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.

p
21.2

22.3 22.2 22.1 21.3

X Chromosome
growth control factor, X-linked Xg blood roup ocular albinism sensorineural deafness anemia, sideroblastic, with Spinocerebellar ataxia cleft palate lymphoproliferative syndrome Simpson dysmorphia syndrome coagualation factor IX, hemophilia B

21.1 11.4 11.3 11.23 11.22 11.21 11.1 11.1 11.2 12 13 21.2 21.1 21.3 22.2 22.1 22.3 23 24 25

26 27 28

blue-monochr. color blindness coagulation factor VIIIc,hemophiliaA homosexuality, male

Chromosomes

Organelles and fraction : Nucleus Mitochondrion Endoplasmic reticulum Lysosome Plasma membrane Golgi Apparatus Peroxisome

Cytoskeleton Cytosol Ribosome

BODY PROTEIN Enzyme Receptor Hormone Growth Factor Immunoglobulin Interferon, Interleukin, Cytokine 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

Proteins are composed of subunits called amino acids

STRUKTUR PROTEIN :

Struktur Primer : Sequensi asam amino Struktur Sekunder : -helix, lipatan Struktur Tertier : sub-unit protein (tiga dimensi) Struktur Kwaterner : gabungan bbrp struktur tertier Ditentukan oleh sifat asam amino Sangat bervariasi

SIFAT PROTEIN :

FUNGSI PROTEIN :

PEMBENTUKAN PROTEIN :

Berdasarkan gen / DNA di inti sel Berlangsung di Organella (Ribosome)

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)

Desoxy ribonucleic acid

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.

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.

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
C A T T A G A T T

G A T

T
A A 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 : , , , and

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)
ATC G G UAGCC

DNA 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) Translational initiation signal : AUG mRNA dibaca dari 5 ke 3 Amino acid diaktifkan linked ke tRNA oleh Aminoacyl-tRNA synthetase. Elongation : Peptidyl transferase. Termination : Stop Codon (UAG, UAA, UGA)

Messenger RNA - mRNA Copy dari informasi genetik pada DNA. Berfungsi sebagai pattern untuk membentuk protein. Transfer RNA - tRNA Molekul RNA kecil (70 - 90 unit basa). Berfungsi untuk membawa asam amino yang sesuai pada tempat pembentukan protein.

Ribosomal RNA - rRNA Platform untuk sintesa protein Berfungsi untuk memfiksasi mRNA dan membantu terbentuknya rantai asam amino

Amino acid assembly during translation occurs on ribosomes; tRNA serves as the crucial adaptor molecule

Nukleotida 1. (5) U U U U U Phe Phe Leu Leu

Nukleotida 2.

Nukleotida 3.

(3)
C
Ser Ser Ser Ser

A
Tyr Tyr STOP STOP

G
Cys Cys STOP Trp U C A G

C
C C C

Leu
Leu Leu Leu

Pro
Pro Pro Pro

His
His Gln Gln

Arg
Arg Arg Arg

U
C A G

U
A Ile

C
Thr

A
Asn

G
Ser U

A
A A G G

Ile
Ile Met Val Val

Thr
Thr Thr Ala Ala

Asn
Lys Lys Asp Asp

Ser
Arg Arg Gly Gly

C
A G U C

G
G

Val
Val

Ala
Ala

Glu
Glu

Gly
Gly

A
G

Perbedaan Sandi Nukleotida

Nukleotida :
UGA AUA AGA AGG

Chr. :
Stop Ile Arg Arg

Mit. :
Trp Met Stop Stop

KEY ASPECTS OF 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

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 AGC TCG ACC CG AGC TTC CGA CCC G AGC TTC TTC GAC CCG ATC TTC GAC CGG

Wild type Deletion Insertion Expansion 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

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)

cytoplasmic proteins

free ribosomes

Protein Traffic

RER

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


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)

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

SRC RAS

FMS Orga nella

Inti Sel FOS MYC JUN

FMS

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