BIOLOGI SEL & MOLEKULAR Farmasi UGM BIOLOGI SEL & MOLEKULAR farmasibugm2012@gmail.com 28/03/2014 MODIFIED by Prof. Sismindari

Pengenalan Biologi molekuler Kuliah ke Dosen Pokok Bahasan Sub Pokok Bahasan I.Prof. Sismindari 1) 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- II. 2) + 3) Dr. Puji A. 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 III.4) +5) Prof Sismindari Replikasi DNA (Sintesis DNA) Reaksi polimerisasi, Proses replikasi DNA: konservatif dan semi-konservatif, 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 28/03/2014 MODIFIED by Prof. Sismindari

Kuliah ke Dosen Pokok Bahasan Sub Pokok Bahasan Mutasi dan Repair DNA 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 dan Transformasi genetik - Macam-macam virus (virus DNA, virus RNA), genom virus, Life cycle HIV, Virus influenza, EBV, dan retrovirus Jenis transposon: transposon, viral retrotransposon dan non-viral retrotransposon. - Mekanisme transposisi Penyakit yang disebablkan oleh transposon 28/03/2014 MODIFIED by Prof. Sismindari

Dosen Pokok Bahasan Sub Pokok Bahasan Ekspresi Gen: IV. Prof Kuswandi 8) Ekspresi Gen: Transkripsi, prokariot, eukariot 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   Regulasi: Sistem operon, Lac repressor dan Catabolic activator protein (CAP), triptofan operon 9) Translasi 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 V. Dr Riris 10) Sistem regulasi ekspresi gen eukariot 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) 28/03/2014 MODIFIED by Prof. Sismindari

Dosen Pokok Bahasan Sub Pokok Bahasan Cell-cycle dan regulasinya 11) Dr. Riris 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-M-phases), 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 28/03/2014 MODIFIED by Prof. Sismindari

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

28/03/2014 MODIFIED by Prof. Sismindari

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

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 Kiat bgaimana mempelajari Biologi Molekuler: 1. belajar setiap hari 2. Jangan hanya melihat catatan/slide, tetapi harus disertai mencatat dan membuat catatan singkat, menggambar lagi agar lebih mudah dipahami 3. Buatlah catatan saat membaca buku, 4.Bertanya!!! 28/03/2014 MODIFIED by Prof. Sismindari

mempelajari struktur & fungsi Biologi Molekuler mempelajari struktur & fungsi DNA, RNA , protein 46 Human 2.9x109 bp 28/03/2014 MODIFIED by Prof. Sismindari (Berg JM et al, 2002)

MODIFIED by Prof. Sismindari Molecular Biology Farmasi UGM 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. Function Genes Proteins Biochemistry Molecular Biology Genetics Biologi Molekuler ini merupakan cabang ilmu yang cakupannya overlape dengan bidang biologi, utamanya genetik dan biokimia Biologi molekuler concern dengan pemmahaman terhadap interaksi antara berbagai system dalam sel, termasuk hubungan antara DNA, RNA dan Protein sintesis dan mempelajari bagaimananinteraksi tersebut diatur. MODIFIED by Prof. Sismindari Schematic relationship between biochemistry, genetics and molecular biology 28/03/2014 introduction%20to%20molecular%20and%20cell%20biology.ppt

Metode DETEKSI PENYAKIT PENEMUAN OBAT TANAMAN VAKSIN PROTEIN OBAT Metode DETEKSI PENYAKIT KIMIA MEDISINAL FARMAKOLOGI BIOKIMIA MIKROBIOLOGI BIOLOGI MOLEKULER FARMASI Dalam ilmu Kefarmasan sangat diperlukan untuk mendukung proses penemuan obat, mempelajari mekanisme penyakit 28/03/2014 MODIFIED by Prof. Sismindari

? Molecular Mengapa mempelajari Biology 28/03/2014 MODIFIED by Prof. Sismindari

MANFAAT BIOLOGI MOLEKULER GENOME MUTASI DNA PADA GENOME REPLIKASI DNA TRANSKRIPSI TRANSLASI SIFAT & FUNGSI BERUBAH SIFAT & FUNGSI TETAP PCR SEKUENSING PROTEIN, RESEPTOR TARGET OBAT PROTEIN OBAT TERJADINYA PENYAKIT PERUBAHAN RESPON OBAT TARGET TERAPI MODIFIED by Prof. Sismindari 28/03/2014

MANFAAT BIOLOGI MOLEKULER REGULASI PROKARIOT RESPON BAKTERI THDP LINGKUNGAN MEMPRODUKSI PROT OBT DLM BAKTERI REGULASI EUKARIOT RESPON HUMAN THDP LINGKUNGAN PROSES METABOLISME SAAT TERJADI INFEKSI PATOGEN REGULASI SECOND MESSENGER BAGAIMANA SIGNAL DARI LUAR DITERUSKAN KE DLM SEL SIGNAL DI DITERUSKAN MELALUI RESEPTOR BAGAIMANA PEMBELAHAN SEL NORMAL DIATUR AGAR TDK TERJADI KANKER REGULASI SIKLUS SEL MODIFIED by Prof. Sismindari 28/03/2014

PFARMAKOGENOMIK BIOLOGI MOLEKULER KETERKAITAN DENGAN ILMU LAIN METABOLIT SEKUNDER ANALISIS KLINIK PFARMAKOGENOMIK ANALISIS OBAT KIMIA MEDISINAL FARMAKOKIMIA FARMAKOLOGI MOLEKULER BIOKIMIA: REKAYASA PROTEIN BIOTEKNOLOGI FARMASI BIOLOGI MOLEKULER 28/03/2014 MODIFIED by Prof. Sismindari

What is Life made of: All living things are made of Cells Farmasi UGM 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. Books represent all the information (DNA) that every cell in the body needs  can grow and carry out its various functions. All Life depends on 3 critical molecules From DNA, to RNA, to Proteins Nucleus Bagaimana suatu kehidupan dimulai/dibuat? Semua kehidupan organisme dimulai dari suatu sel yang di dalam sel mengandung: a. nucleus (inti sel), yang diumpamakan sebagai “perpustakaan” b. Kromosom, yang diumpamakan sebagai “rak buku”, dan 3. gen, yang diumpamakan sebagai “buku” Setiap organisme mengandung “perpustakaan” dan beberapa set buku yang sama. DEVIDED & PASSED THE GENETIC INFORMATION in to the doughter cells 28/03/2014 MODIFIED by Prof. Sismindari

PERBEDAAN PROKARIOT & EUKARIOT 28/03/2014 MODIFIED by Prof. Sismindari

PERBEDAAN PROKARIOT & EUKARIOT Organisme Bakteria dan sianobakteria Fungi, hewan, manusia Ukuran sel 1 - 10m 5 - 100m 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

MODIFIED by Prof. Sismindari Perbedaan Prokariot Eukariot dan Splising 28/03/2014 MODIFIED by Prof. Sismindari (Alberts B, et al, 1994)

Fenotip (penampakan) suatu mahluk hidup dibawa oleh suatu molekul yang dinamakan DNA (asam nukleat), yang merupakan rangkaian nukleotita. Dalam prosesnya molekul tersebut akan diekspresikan menjadi RNA (suatu asam nukleat) terlebih dahulu. Selanjutnya RNA yang merupakan asam nukleat tersebut akan diterjemahkan menjadi protein yang merupakan rangkaian asam amino. 28/03/2014 MODIFIED by Prof. Sismindari

DNA, RNA, and the Flow of Information Farmasi UGM DNA, RNA, and the Flow of Information Functional Proteins Replication Post translation modification Transcription Translation 28/03/2014 MODIFIED by Prof. Sismindari

MODIFIED by Prof. Sismindari PEMBELAHAN SEL 28/03/2014 MODIFIED by Prof. Sismindari

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Seperti tadi dikatakan bahwa DNA (deoksi-ribonukleotida), merupakan materi genetik yang dapat diturunkan. Bagian atau fragmen yang diturunkan tersebut disebut sebagai “gena”. Ekspresi gena menjadi protein tersebut akan memberikan fenotip pada mata. FENOTIP 28/03/2014 MODIFIED by Prof. Sismindari

DNA CHROMOSOME Kromosom tanpa histon 28/03/2014 MODIFIED by Prof. Sismindari

fragmen di ujung chromosom linier 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

Telomere Length Declines 1,500 3,000 8,000 Age (years) Telomere length in base pairs (human white blood cells) 35 Telomere length declines in dividing cells as we age. In human blood cells, the length of telomeres ranges from 8,000 base pairs at birth to 3,000 base pairs as people age and as low as 1,500 in elderly people. (An entire chromosome has about 150 million base pairs.) Each time a cell divides, an average person loses 30 to 200 base pairs from the ends of that cell’s telomeres. Cells normally can divide only about 50 to 70 times, with telomeres getting progressively shorter until the cells become senescent, die or sustain genetic damage that can cause cancer. Telomeres do not shorten with age in tissues such as heart muscle in which cells do not continually divide. 65 28/03/2014 MODIFIED by Prof. Sismindari

Chromosomes 24 6 16 Gena-gena tersebut di dalam sel akan tersusun dalam suatu molekul yang disebut sebagai “Kromosom”. Jadi di dalam kromosom terkandung lebih dari 1 gena ( bisa mencapai puluhan sampai ratusan). Disetiap sel suatu mahluk hidup terdapat variasi jumlah kromosom (1 – puluhan). Kromosom bakteri 1, manusia 46, ayam 78 dst 78 46 40 28/03/2014 MODIFIED by Prof. Sismindari

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

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Aplikasi biomol 1 28/03/2014 MODIFIED by Prof. Sismindari

You can tell people apart by their fingerprints… Didalam kromosom tidak semua berupa gena, ada bagian fragmen yang sekuennya berulang (repeat secquence). Fragmen-fragmen tersebut mempunyai panjang berbeda pada setiap induividu, sehingga bisa digunakan sebagai penanda seseorang seperti halnya “sidik jari” kita. Sehingga disebut sebagai “Sidik jari DNA” Because everyone’s fingerprints are different! 28/03/2014 MODIFIED by Prof. Sismindari

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? MODIFIED by Prof. Sismindari 28/03/2014

This is what it really looks like!!! Miss Ellis Sara                                                                                                   This is what it really looks like!!! 28/03/2014 MODIFIED by Prof. Sismindari

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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 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

Why does drug response vary? Pharmacogenomics Same symptoms, Same findings, Same disease? Different patients Same drug Same dose Why does drug response vary? Possible Reasons: Individual variation By chance… Ethnicity Age Weight Pregnancy Genetic factors Disease Drug interactions.. Genetic Differences A G SNP Different Effects 28/03/2014 MODIFIED by Prof. Sismindari

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 * Single nucleotide variation==Ex)10 nucleotide sequence of DNA fragment; two Fragments might differ b/c they may have a C or a T Polymorphisms are common and contribute to common diseases and influence our response to medications 28/03/2014 MODIFIED by Prof. Sismindari

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 MODIFIED by Prof. Sismindari 28/03/2014

"Here's my sequence..." The New Yorker 28/03/2014 MODIFIED by Prof. Sismindari

Aplikasi biomol untuk kloning 28/03/2014 MODIFIED by Prof. Sismindari

The Making of Dolly Empty DNA DNA Sheep Egg Mother’s Egg Cloning depends on DNA Step 1: Take the Nuclei out of a sheep egg Step 2: Transfer nuclei from the Mother Empty DNA DNA The mother is to be cloned since her DNA was used Sheep Egg Mother’s Egg 28/03/2014 MODIFIED by Prof. Sismindari

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

MODIFIED by Prof. Sismindari 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? “Dolly’s birth was one of the biggest news stories of the nineties. Scientists made extravagant promises about medical and technological advances. Well, Dolly’s embarrassingly premature death received little attention, precisely because it exposes the horrors of cloning.” Dolly was diagnosed with arthritis, normally found in old sheep Telomeres are the ends of our chromosomes. Made of many repeats of the same DNA sequence, they act like shoelace caps: they protect the gene-containing body of the chromosome from being worn down. Breakdowns in telomere maintenance are implicated in ageing and cancer. Telomeres sit on the ends of chromosomes to protect them from damage. When chromosomes are replicated during cell division, a stretch of the telomere is left unreplicated—making the telomere a bit shorter with each division. After some 50 to 100 divisions, the telomeres become so short that the cell can no longer divide—a phenomenon scientists call senescence—that is, the state of being old. It’s all about DNA 28/03/2014 MODIFIED by Prof. Sismindari

MODIFIED by Prof. Sismindari Other Cloned Animals July 1998 cloned calves Cloned mouse Cloned mule 28/03/2014 MODIFIED by Prof. Sismindari

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KOMPOSISI GENA FUNGSIONAL PROMOTER OPERATOR ACTIVATOR BINDING SITE STRUKTURAL GEN : mRNA TERMINATOR 28/03/2014 MODIFIED by Prof. Sismindari

Introduction The Central Dogma of Molecular Biology Cell DNA mRNA Transcription Reverse tanscription Polypeptide (protein) Translation Ribosome 28/03/2014 MODIFIED by Prof. Sismindari

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MODIFIED by Prof. Sismindari Teknik yang sering digunakan Isolasi DNA & RNA Elektroforesis Hibridisasi PCR Sekuensing DNA 28/03/2014 MODIFIED by Prof. Sismindari

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ASISITENSI ASISTEN BIOLOGI SEL & MOLEKULER 28 Februari 2014 28/03/2014 MODIFIED by Prof. Sismindari

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

MODIFIED by Prof. Sismindari 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

MODIFIED by Prof. Sismindari Penilaian Test : 30 Kerja : 10 Laporan : 20 Responsi : 40 NILAI UJIAN (UTS+UAS) NILAI AKHIR  {(3XNILAI UJIAN)+NILAI PRAKTIKUM}/4 BOBOT 1 BOBOT 3 28/03/2014 MODIFIED by Prof. Sismindari

MODIFIED by Prof. Sismindari SISTEM EVALUASI NILAI AKHIR A > 75 B 65 – 74 C 55 – 64 D 35 – 54 E <34 28/03/2014 MODIFIED by Prof. Sismindari