Pedigree of Hemophiliain One Family

HemophiliaHemophilia - 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

X Chromosome

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 EnzymeReceptor Hormone Growth FactorImmunoglobulinInterferon, InterleukinAdhesions moleculesHLA/MHC

-1-1 Acid Glycoprotein-1 T Glycoprotein-1 AntitrypsinTranscortin-1 Antichymotrypsin-1 B glycoprotein9,5-s -1 GlycoproteinVitamin-D binding protein-1 Lipoproteins

-2Retinol binding protein-2 HS GlycoproteinHistidine-rich 3,8 S 2 GlycoproteinHaptoglobinPregnancy zone protein2 MacrogobulinProthrombinAntihemophilic factorC1 inactivatorC1s

STRUKTUR PROTEINSIFAT PROTEINFUNGSI PROTEINPEMBENTUKAN PROTEINDISTRIBUSI PROTEINPEMERIKSAAN PROTEIN

STRUKTUR PROTEIN :Struktur Primer : Sequensi asam aminoStruktur Sekunder : -helix, lipatan Struktur Tertier : sub-unit protein (tiga dimensi)Struktur Kwaterner : gabungan bbrp struktur tertier SIFAT PROTEIN :Ditentukan oleh sifat asam aminoFUNGSI PROTEIN :Sangat bervariasi PEMBENTUKAN PROTEIN :Berdasarkan gen / DNA di inti selBerlangsung 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 2000Gen : Sepotong DNA (Intron atau Exon)A - T G - CSatuan DNA : bp (base pair)

A G C G A T C T G GT C G C T A G A C CDNA Base PairingDouble helix consists of 2 complimentary strands of DNA.

ChromosomesLong strands of DNA packaged and compressed very tightlyEveryone has 2 sets (1 pair) of chromosomes1 pair of each of the 22 autosomesplus XX for a female (46XX)or XY for a male (46XY)1 is inherited from mum, 1 from dadYou pass 1 of each pair onto each child

The Human GenomeThe haploid human genome is made up of 3 x 109 base pairs of DNA

This contains 50,000- 100,000 genes arranged on 46 chromosomes

Packaged within the nucleus of the cell

DNA ReplicationEach of the 2 DNA strands is copied by machinery in the cell Each new daughter strand has a sequence complimentary to the original template strandReplication essential to allow cell division (Mitosis) where 1 cell becomes 2

ATCTAGATCTAGTAGATCDNA Replicationsemi-conservative 2 daughter cells

DNA Replication

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

The DNA Replication Fork

DNA Replication in MeiosisDuring 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

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

Gene StructureEvery three bases of DNA is called a codonEach codon specifies an amino acid which join together to form the proteineg ATG = methionine = START TAA = STOP TAG = STOP TGA = STOP

ExonsIntronsGene StructurePromoterTAATAG stopTGAATGstartExon = coding sequenceIntron= intervening sequence (non-coding)

DNARNAProteintranscriptiontranslationProtein Synthesis

Transcription3 Nuclear RNA Polymerase : mRNA transcribed by RNA Polymerase II The initiation of transcription involves binding RNA Polymerase to a specific DNA sequence called a PromoterMany 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 - TranscriptionEach gene codes for a proteinDNA sense strand acts as template and is transcribed into messenger RNA (mirror image of the DNA but Uracil instead of Thymine)

DNAmRNAA T C G GU A G C C

Protein Synthesis- TranslationIntrons are spliced out of the mRNAmRNA 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 TerminationTranslational initiation signal : AUGmRNA become translated through 5 3 directionElongation : 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.Nukleotida 2.Nukleotida 3.(5) (3) UCAGUPheSerTyrCys U UPheSerTyrCys CULeuSerSTOPSTOP AULeuSerSTOPTrp GCLeuProHisArg UCLeuProHisArg CCLeuProGlnArg ACLeuProGlnArg G

UCAGAIleThrAsnSerUAIleThrAsnSerCAIleThrLysArgAAMetThrLysArgGGValAlaAspGlyUGValAlaAspGlyCGValAlaGluGlyAGValAlaGluGlyG

Perbedaan Sandi NukleotidaNukleotida :Chr. :Mit. :

UGAStopTrp AUAIleMet AGAArgStop AGGArgStop

MITOCHONDRIAL ENERGY TRANSDUCTIONHuman body synthesizes body weight of ATP per daymotoric functionsbiosynthetic activitiesheat maintenance

Matrix sideCytosolic sideIIIIIV

Gen Mitochondria

Gen yang berbentuk sirkuler, terdiri dari 16569 bpDiturunkan secara maternal, mudah bermutasiMenyandi : 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 :A3243GG3316A A3260GT3394CA3256GA3252Gluas dijumpai :T16189C

MITOCHONDRIAL BIOLOGY AND GENETICSSemi-autonomous organelles, contain multiple copies of mtDNADouble membrane structure, cristae containing respiratory chain enzymesMost mitochondrial proteins encoded by nuclear genome and imported into mitochondriaFunctions in cellular metabolism and the regulation of cell death

MITOCHONDRIAL PROTEINS mtDNACircular DNA - 16,569 bp Encodes 13 polypeptides - for OXPHOS 22 tRNA 2 rRNAD-loop - initiation of replication and transciptionEvolves at higher rate than nDNAMaternally inherited

Pathogenese NIDDMPatophysiologi 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 InsulinMutasi MtDNA penurunan ATP

MITOCHONDRIAL ENERGY METABOLISM AND INSULIN SECRETIONMODY2

Transmembrane Protein Synthesis

MutationsA change in the DNA sequence of the geneAll cells acquire mutations as they dividerate of approx 10-6 per gene per cellMutations can alter protein product of DNA, stop gene working or activate gene

Types of MutationDeletion - DNA missingInsertion - extra DNA insertedExpansion (Amplification) - DNA repeat size has increasedPoint Mutation - change in one base

Types of Mutation (in coding sequence)AGC TTC GAC CCG Wild typeAGC TCG ACC CG Deletion AGC TTC CGA CCC GInsertionAGC TTC TTC GAC CCG ExpansionATC TTC GAC CGG Point mutation

POINT MUTATION U A A (Termination Codon) U C A (Codon for Serine) U C U (Codon for Serine) C C A(Codon for Proline)

Polimerase Chain Reaction (PCR)

Tahun 1985, Kary Mullis, CaliforniaMetode untuk meng-amplifikasi (melipat gandakan) fragment DNA (Gen) Dibutuhkan :DNA atau RNAOligonucleotidprimer (PRIMER)Enzym Taq-PolimeraseCampuran dari 4 Basa Nukleotida (dNTPs)10 x Reactions BufferLarutan MgCl2

Alat : Thermal Cycler Prinsip : perobahan temperatur secara otomatis dengan waktu yang telah ditentukanDapat diatur (Program)Contoh : 95 C------ Denaturasi55 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 amplifikasiSense : 5- ATG(Start) -GGT-TCT-GTT-GCT-GCT-TGG-TAA(Stop)- 3Antisense : 3 - TAC-CCA-AGA-CAA-CGA-CGA-ACC-ATT- 5 Exon dan/atau Intron dapat berfungsi sebagai Matrix untuk amplifikasi

RNASingle strand (Uracil pengganti Thymin)Transkripsi dari DNA mRNAMengandung informasi genetik dari Exon Dengan Enzym Reverse Transkriptase diperoleh DNA dari RNAcDNAReaksi PCR nya disebut RT-PCR

Taq-Polimerase Klenow - DNA Polymerase dari E.Coli1988 : Taq-Polymerase dari Bakteri Thermus aquaticusHybridisasi dan Polimerisasi berlangsung pada temp. 50-70 C Perhatikan : Buffer yang digunakan (10 x RB) dan diperlukan MgCl2

PrimerSequence dari Nukleotida tertentu (Intron atau Exon) : 20 30 bpPrinsip : 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 EndonucleaseSequence analysis (DNA Sequencing)

RERfreeribosomescytoplasmicproteinsProteinTraffic

MOLECULARE MICROBIOLOGYAplikasi teknologi DNAINFEKSI 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 mungkinKultur : 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 ONKOLOGYPROTOONKOGEN : 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 neoplastisTUMORSUPPRESSOR GEN : gen yang berperan pada proliferasi dan differensiasi sel, dimana bila gen ini di-inaktivasi atau tidak terdapat, akan terbentuk sel neoplastis

MOLECULARE ONKOLOGYContoh 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 MechanismAdenosine 3,5-cyclic monophosphate (cAMP)cAMPAdenylate cyclaseHormone ReceptorTransducer G ProteinProtein Kinase AStructural ProteinsEnzymesMembraneChannelsATP(+,-)(+,-)

Produk Protoonkogen Inti SelFOS MYC JUNOrganellaERB-B1FMSSISMOSABLFMSSRCRAS

Carcinogenesis (Colorectal Cancer)

Penerapan Teknologi Gen/DNA dalam TherapyProduk dari gen untuk therapy dan prophylaxis :ErythropoietinInsulinHormon pertumbuhanFaktor pembekuan darah VIIIPlasminogen aktivatorVaksin Hepatitis B

Aplikasi gen dalam ForensikSebelum 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 DNAJuga dapat menentukan PaternityProfil DNA tiap individu berbeda