pengenalan biologi molekuler
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[email protected] 28/03/2014 MODIFIED by Prof. Sismindari 1 Kuliah ke Dosen Pokok Bahasan Sub Pokok Bahasan I.Prof. Sismindari Pengenalan Biologi molekuler Peranan Biologi Molekuler dalam Farmasi, - - Contoh perkembangan dalam Biologi Molekular : kloning domba Dolly dan terapi sel induk materi genetik : Genom, kromosom, gena, DNA, RNA, protein.Perbedaan genom prokariot dan mamalia, lokasicentromer, telomere, dan origin replikasi, human genom project- Dogma sentral ekspresi gen:replikasi, transkripsi, translasi- Organisasi dari sel Struktur dan fungsi organela sel Arsitektur sel: Cytoskeleton & Cell Junction materi genetik : Genom, kromosom, gena, DNA, RNA, protein. Struktur DNA (model Watson dan Crick) dan RNA, base-pairing DNA, Sifat DNA dan RNA Heterochromatin, Karyotyping Chromosome Pertumbuhan, Perkembangan dan Diferensiasi Sel - Penurunan sifat materi genetic, Pertumbuhan Sel Pembelahan Sel, meiosis dan mitosis (metaphase, anaphase, interfase) Hukum Mendel Diferensiasi Sel, Komunikasi Antar Sel Replikasi DNA (Sintesis DNA) -Reaksi 1) II. 2) + 3) Dr. Puji A. III.4) +5) Prof Sismindari 28/03/2014 polimerisasi, Proses replikasi DNA: konservatif dan semikonservatif, inisiasi, elongasi replikasi, - Garpu replikasi, fragmen Okazaki, peran telomere, peran enzim topoisomerase, mekanisme proofreading, mutasi karena triple repeat. -- Obat yang bekerja pada tahap replikasi: penghambat topoisomerase, basa analoh sebagai obat antivirus - Penggunaan PCR MODIFIED by Prof. Sismindari 2 Kuliah ke Pokok Bahasan Sub Pokok Bahasan Dosen III. 5) Prof Sismindari Mutasi dan Repair DNA - Definisi, mutan, mutasi, mutagen; jenis-jenis mutasi, jenis-jenis mutan, jenis-jenis mutagen (kimia, fisika dan biologi), mutasi pada level gen, pada level organisme. - Stabilitas genetik dan kerusakan DNA, macamkerusakan DNA, proses umum reparasi DNA, penyakit yang disebabkan oleh adanya mutasi III.6) Prof Djadi Polimorfism SNP kaitannya dengan keragaman genetik masing-masing individu: Farmakogenomik aplikasinya pada metabolisme INH dan warfarin Polimorfisme mikrosatelit :Sidik jari DNA aplikasinya pada hubungan kekerabatan dan identifikasi individu dengan PCR Hibridisasi, macam dan prinsip kerjanya (Southern, Northen) 7) Mekanisme infeksi virus - Macam-macam virus (virus DNA, virus RNA), genom virus, dan Transformasi Life cycle HIV, Virus influenza, EBV, dan retrovirus genetik Jenis transposon: transposon, viral retrotransposon dan non-viral retrotransposon. - Mekanisme transposisi Penyakit yang disebablkan oleh transposon 28/03/2014 MODIFIED by Prof. Sismindari 3 Dosen Pokok Bahasan Sub Pokok Bahasan IV. Prof Kuswandi 8) Transkripsi pada prokariot : RNA polimerase: inisiasi (TATA Box, promoter), elongasi, terminasi. Fenomena transkripsi pada prokariot : polisistronik, polisom, repressor, aktivator dan induser Fenomena transkripsi eukariot: intron, ekson, capping, splicing dan poli-A, monosistronik Obat-obat yang bekerja pada penghambatan proses transkripsi Ekspresi Gen: Transkripsi, prokariot, eukariot Regulasi: Sistem operon, Lac repressor dan Catabolic activator protein (CAP), triptofan operon 9) Ekspresi Gen: Translasi V. Dr Riris 10) 28/03/2014 Sistem regulasi ekspresi gen eukariot Ribosom, kodon antikodon , tRNA, aminoasyl tRNA sintetase Proses : inisiasi, elongasi, dan terminasi Perbedaan proses translasi pada prokariot dan eukariot. Pasca translasi : modifikasi asam amino. Obat-obat dengan target penghambatan proses translasi prokariot (antibiotik). Teknik deteksi ekspresi gen factor transkripsi, domain dan motif, contoh factor transkripsi, dan peran factor transkripsi. Signal transduksi sederhana (hormone steroid), Signal transduksi kompleks (jalur MAP-K dan ras protein) MODIFIED by Prof. Sismindari 4 Dosen Pokok Sub Pokok Bahasan Bahasan Sistem regulasi melalui second messenger G-protein signaling : G-protein, jalur cAMP, jalur protein kinase C (PKC) Aplikasi regulasi pengendalian ekspresi gen pada proses pengobatan 12) Prof.Edy M Cell-cycle dan regulasinya Overview : tahapan pembelahan sel dan daur sel (G1-S-G2-Mphases), Kontrol daur sel pada G1-phase (peran cyclin-cdk dan pRb), pengaturan sistem checkpoint pada daur sel Apoptosis (kematian sel terprogram) : jenis kematian sel, karakteristik morfologi apoptosis, jalur instrinsik dan ekstrinsik, peran pro dan anti apoptosis (bcl2 famili) Peran mitokondria. Teknik deteksi apoptosis VII. 13) 14) Karsinogene sis Pengertian dasar kanker, tahap-tahap karsinogenesis, golongan agen karsinogenik (kimia, fisika, dan biologi), pengenalan marker molecular kanker (mis. Ras dan p53) dan perannya pada proses karsinogenesis, pengembangan agen kemoprevensi 11) Dr. Riris 28/03/2014 MODIFIED by Prof. Sismindari 5 III. DAFTAR PUSTAKA Lodish, H., Berk, A., Zipursky, A.L., Matsudaira, P., Baltimore, D., Darnell, J., 2000, Molecular cell Biology, 4th ed., Freeman and Company, New York. Alberts,B., Bray, D., Lewis, J., Raff, M., Robert, K., Watson, JD., 1994, Molecular Biology of the Cell, 3th ed.,Garland Publishing, Inc, New York. Devlin T.M., 2002, Biochemistry with Clinical Correlations, 5th ed., wiley-Liss, New York. Berg, J.M., Tymoczko, J.L., Stryer.,2002, Biochemistry, 5th ed.,W.H. Freeman and Company, New York. Glick, B.R. and Pasternak, J.J., 1998, Molecular Biotechnology, Principles and Applications of Recombinant DNA, 2nd. Ed. ASM Press, Washington DC. Sismindari, 2012, Seri Biologi Molekuler: Replikasi DNA, Pustaka Pelajar, Yogyakarta 28/03/2014 MODIFIED by Prof. Sismindari 6 28/03/2014 MODIFIED by Prof. Sismindari 7 • Peranan Biologi Molekuler dalam Farmasi. • Materi genetik : Genom, kromosom, gena, DNA, RNA, protein. • Perbedaan genom prokariot dan mamalia, lokasi centromer, telomere, dan origin replikasi, human genom project • Dogma sentral ekspresi gen : replikasi, transkripsi, translasi • Contoh perkembangan dalam Biologi Molekular : kloning domba Dolly dan terapi sel induk 28/03/2014 MODIFIED by Prof. Sismindari 8 How to do well in Mol Biol • Study every day • Don’t just look at notes, rewrite, reorganize, redraw pictures from memory • When read book, take notes • Ask questions • Come to office by appointment 28/03/2014 MODIFIED by Prof. Sismindari 9 Human 2.9x109 bp 46 28/03/2014 MODIFIED by Prof. Sismindari 10 (Berg JM et al, 2002) Molecular Biology The field overlaps with other areas of biology, particularly genetics and biochemistry Molecular biology concerns itself with understanding the interactions between the various systems of a cell, including the interrelationship of DNA, RNA and protein synthesis and learning how these interactions are regulated. Biochemistry Function Proteins Genetics Genes Molecular Biology 11 Schematic relationship between biochemistry, genetics and molecular biology MODIFIED by Prof. Sismindari introduction%20to%20molecular%20and%20cell%20biology.ppt 28/03/2014 28/03/2014 MODIFIED by Prof. Sismindari 12 Mengapa mempelajari 28/03/2014 MODIFIED by Prof. Sismindari 13 MANFAAT BIOLOGI MOLEKULER GENOME MUTASI DNA PADA GENOME REPLIKASI DNA TRANSKRIPSI TRANSLASI 1. SIFAT & FUNGSI BERUBAH 2. SIFAT & FUNGSI TETAP • TERJADINYA PENYAKIT • PERUBAHAN RESPON OBAT PCR SEKUENSING • PROTEIN, RESEPTOR • TARGET OBAT • PROTEIN OBAT MODIFIED by Prof. Sismindari TARGET TERAPI 28/03/2014 14 MANFAAT BIOLOGI MOLEKULER REGULASI PROKARIOT REGULASI EUKARIOT REGULASI SECOND MESSENGER REGULASI SIKLUS SEL - RESPON BAKTERI THDP LINGKUNGAN - MEMPRODUKSI PROT OBT DLM BAKTERI - RESPON HUMAN THDP LINGKUNGAN - PROSES METABOLISME - SAAT TERJADI INFEKSI PATOGEN • BAGAIMANA SIGNAL DARI LUAR DITERUSKAN KE DLM SEL • SIGNAL DI DITERUSKAN MELALUI RESEPTOR • BAGAIMANA PEMBELAHAN SEL NORMAL DIATUR AGAR TDK TERJADI KANKER MODIFIED by Prof. Sismindari 28/03/2014 15 KETERKAITAN DENGAN ILMU LAIN 28/03/2014 MODIFIED by Prof. Sismindari 16 What is Life made of: • All living things are made of Cells – Nucleus = library – Kromosom = bookshelves – Genes = books Almost every cell in an organism contains the same libraries and the same sets of books. Nucleus Books represent all the information (DNA) that every cell in the body needs can grow and carry out its various functions. DEVIDED & PASSED THE GENETIC INFORMATION in to the doughter cells 28/03/2014 • All Life depends on 3 critical molecules From DNA, to RNA, to Proteins MODIFIED by Prof. Sismindari 17 PERBEDAAN PROKARIOT & EUKARIOT 28/03/2014 MODIFIED by Prof. Sismindari 18 PERBEDAAN PROKARIOT & EUKARIOT Prokariot Eukariot Organisme Bakteria dan sianobakteria Fungi, hewan, manusia Ukuran sel 1 - 10m 5 - 100m Organel Beberapa / tidak ada Inti, mitokondria, kloroplast GENOM Sirkuler dalam sitoplasma, Satu molekul kromosom Linier panjang, dipak rapi dgn protein histon dalam inti. Manusia mempunyai 46 cromosom RNA dan protein Disintesis dalam sitoplasma RNA : dalam inti, Protein: dalam sitoplasma Organisasi Unisel Multisel 28/03/2014 MODIFIED by Prof. Sismindari 19 Eukariot Prokariot Splising 28/03/2014 MODIFIED by Prof. Sismindari 20 (Alberts B, et al, 1994) 28/03/2014 MODIFIED by Prof. Sismindari 21 DNA, RNA, and the Flow of Information Functional Proteins Replication Post translation modification Transcription 28/03/2014 Translation MODIFIED by Prof. Sismindari 22 PEMBELAHAN SEL 28/03/2014 MODIFIED by Prof. Sismindari 23 28/03/2014 MODIFIED by Prof. Sismindari 24 FENOTIP 28/03/2014 MODIFIED by Prof. Sismindari 25 DNA CHROMOSOME Kromosom tanpa histon 28/03/2014 MODIFIED by Prof. Sismindari 26 KROMOSOM CENTROMER region dari chromosom yang diperlukan untuk pengikatan mitotic spindle saat proses mitosis TELOMER fragmen di ujung chromosom linier replikasi secara khusus untuk mencegah perpendekan chromosom ORIGIN REPLIKASI tempat awal replikasi berlangsung 28/03/2014 MODIFIED by Prof. Sismindari 27 Telomere Length Declines 8,000 Telomere length in base pairs (human white blood cells) 3,000 1,500 28/03/2014 0 35 65 Age (years) MODIFIED by Prof. Sismindari 28 Chromosomes 16 78 28/03/2014 24 6 40 MODIFIED by Prof. Sismindari 46 29 Human 4 x 109 22 autosom yang berbeda 2 sex kromosom Masing2 single linier DNA diploid 2 copies ech chromosome 2 copies each gene 1 X chrom in male E. Coli 4.7 x 106 1 Chromosom 40 – 50 loop superkoil 28/03/2014 MODIFIED by Prof. Sismindari 30 28/03/2014 MODIFIED by Prof. Sismindari 31 Aplikasi biomol 1 28/03/2014 MODIFIED by Prof. Sismindari 32 You can tell people apart by their fingerprints… Because everyone’s fingerprints are different! 28/03/2014 MODIFIED by Prof. Sismindari 33 DNA is like a fingerprint because everyone’s is a little different! How does the police look at DNA to figure out who committed a crime? 34 MODIFIED by Prof. Sismindari 28/03/2014 Miss Ellis 28/03/2014 Sara This is what it really looks like!!! MODIFIED by Prof. Sismindari 35 28/03/2014 MODIFIED by Prof. Sismindari 36 28/03/2014 MODIFIED by Prof. Sismindari 37 Medicine and the New Genetics Gene Testing Pharmacogenomics Gene Therapy Anticipated Benefits: • improved diagnosis of disease • earlier detection of genetic predispositions to disease • rational drug design • gene therapy and control systems for drugs • personalized, custom drugs 28/03/2014 MODIFIED by Prof. Sismindari 38 U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003 Aplikasi 2 POLIMORFISME 28/03/2014 MODIFIED by Prof. Sismindari 39 Pharmacogenomics Same symptoms, Same findings, Same disease? Different patients Same drug Same dose Why does drug response vary? Genetic Differences SNP G A Different Effects Ethnicity Age Weight Pregnancy Possible Reasons: Individual variation By chance… Genetic factors Disease Drug interactions.. 28/03/2014 MODIFIED by Prof. Sismindari 40 Single Nucleotide Polymorphisms (SNPs) A key to human variability DNA sequence variation at a single nucleotide that may alter the function of the encoded protein * Functional protein Functional but altered protein Polymorphisms are common and contribute to common diseases and influence our response to medications 28/03/2014 MODIFIED by Prof. Sismindari 41 Phase II enzymes known to have polymorphism NAT2(N-acethyl Transferase): Isoniazid, hydralazine, GST: D-Penicillamine TPMT: Azathioprine, 6-MP Pseudocholinesterase: Succinyl choline UGT1A1: Irinotecan 42 MODIFIED by Prof. Sismindari 28/03/2014 "Here's my sequence..." The New Yorker 28/03/2014 MODIFIED by Prof. Sismindari 43 APLIKASI BIOMOL UNTUK KLONING 28/03/2014 MODIFIED by Prof. Sismindari 44 The Making of Dolly Cloning depends on DNA Step 1: Take the Nuclei out of a sheep egg Step 2: Transfer nuclei from the Mother Empty DNA Sheep Egg 28/03/2014 DNA Mother’s Egg MODIFIED by Prof. Sismindari 45 Hello Dolly July 5, 1996, Dolly was born at the Roslin Institute, Edinburgh, Scotland She was cloned using a cell taken from a healthy six-year-old sheep 28/03/2014 MODIFIED by Prof. Sismindari 46 Good-Bye Dolly At age 3 Dolly showed signs of premature aging. Dolly died at 6 years old from progressive lung disease (symptom of old age) • Dolly’s DNA was already 6 years old when she was born - WHY? It’s all about DNA 28/03/2014 MODIFIED by Prof. Sismindari 47 Other Cloned Animals July 1998 cloned calves 28/03/2014 Cloned mouse MODIFIED by Prof. Sismindari Cloned mule 48 28/03/2014 MODIFIED by Prof. Sismindari 49 KOMPOSISI GENA FUNGSIONAL PROMOTER OPERATOR ACTIVATOR BINDING SITE STRUKTURAL GEN : mRNA TERMINATOR 28/03/2014 MODIFIED by Prof. Sismindari 50 Introduction The Central Dogma of Molecular Biology Cell Transcription Translation Reverse tanscription DNA mRNA Ribosome Polypeptide (protein) 28/03/2014 MODIFIED by Prof. Sismindari 51 28/03/2014 MODIFIED by Prof. Sismindari 52 28/03/2014 MODIFIED by Prof. Sismindari 53 28/03/2014 MODIFIED by Prof. Sismindari 54 28/03/2014 MODIFIED by Prof. Sismindari 55 ASISITENSI ASISTEN BIOLOGI SEL & MOLEKULER 28 Februari 2014 28/03/2014 MODIFIED by Prof. Sismindari 56 TATA TERTIB LABORATORIUM • Mahasiswa Datang 15 menit sebelum praktikum dimulai • Jas laboratorium dan name tag dipakai sebelum memasuki laboratorium • Mahasiswa tidak diperkenankan pakai SANDAL dan KAOS OBLONG • Mahasiswa terlambat 15 menit tanpa alasan yang jelas tidak diperkenankan mengikuti praktikum • Mahasiswa diwajibkan untuk MENGIKUTI SEMUA MATA PRAKTIKUM • Tidak disediakan waktu khusus untuk inhal. • Mahasiswa yang berhalangan hadir bisa mengikuti PRAKTIKUM GOLONGAN LAIN untuk acara yang sama • Mahasiswa tidak diperkenankan MAKAN, MINUM, MEROKOK selama praktikum • Laporan praktikum dibuat BER-KELOMPOK dan dikumpulkan saat mengikuti praktikum berikutnya • 28/03/2014 MODIFIED by Prof. Sismindari 57 Percobaan • Percobaan 1 : Isolasi DNA MAMALIA • Percobaan 2A : KUANTIFIKASI DNA HASIL ISOLASI DENGAN SPEKTROFOTOMETRI • Percobaan 2B : AMPLIFIKASI DNA HUMAN DENGAN PCR UNTUK MELIHAT POLIMORFISME • Percobaan 3 : ANALISIS DNA DGN ELEKTROFORESIS GEL AGAROSE • Percobaan 4: PENGAMATAN SEL DENGAN MIKROSKOP • Percobaan 5: PENGENALAN BIOINFORMATIKA BLAST • Percobaan 6: SIMULASI DETEKSI EKSPRESI PROTEIN DENGAN TEKNIK WESTERN BLOT 28/03/2014 MODIFIED by Prof. Sismindari 58 Penilaian • • • • Test : 30 Kerja : 10 Laporan : 20 Responsi: 40 BOBOT 1 • NILAI UJIAN (UTS+UAS) BOBOT 3 • NILAI AKHIR {(3XNILAI UJIAN)+NILAI PRAKTIKUM}/4 28/03/2014 MODIFIED by Prof. Sismindari 59 SISTEM EVALUASI • NILAI AKHIR – – – – – A B C D E 28/03/2014 > 75 65 – 74 55 – 64 35 – 54 <34 MODIFIED by Prof. Sismindari 60
