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生物學 Lecture 3: What is (a) gene? Central Dogma 生物醫學系 羅時成老師 [email protected] ext: 3295 What is (a) gene? Central Dogma • 學習目標: • To know the history of searching heredity unit and the definition of gene. • To understand how genes are regulated in various organisms (from genotype to phenotype). What is (a) gene? Central Dogma • 1. Discovery of DNA as a genetic material. • 2. Classic genetics, modern genetics and molecular genetics. • 3. Different levels of gene regulation (transcriptional, translational and posttranslational levels). • 4. New frontier of small RNA regulation. 決定生物的藍圖 • 龍生龍 • 鳳生鳳 • 老鼠生出來的…….. • 遺傳的觀念 Heredity unit = Gene Genetics and Evolution 生命怎麼變的如此複雜? 生物演化的三步曲:變異、遺傳與天擇 variation, inheritance and natural selection! Gregor Mendel (1823-1884) 孟德爾豌豆雜交實驗 Mendel cannot reproduce his results on peas in hawkweed! Mendel豌豆實驗結果分析 Important terms in Mendelian genetics. • • • • • Character: color of peas. Trait: yellow or white. Gene: unit of heredity. Allele: version of a gene produces a specific trait. Homozygous: having two copies of the same alleles for a given gene. • Heterozygous: having two different alleles for a given gene. Insight of genetics • • • • • • • Partial Dominance: dilute concentration. Codominance: human ABO blood group (AB) Overdominance: sickle-cell anemia Dominant: gain-of-function. Recessive: loss-of-function. Negative dominant mutation. Genetic suppression: intra vs intergenic suppression! Sex linkage http://nobelprize.org/nobel_prizes/medicine/articles/lewis/index.html • Thomas Hunt Morgan in The Fly Room! (Columbia University 1910) • Fruit Flies (Drosophila melanogaster) © 2007 Paul Billiet ODWS Hypothesis A cross between the F1 flies should give us: 3 red eye : 1 white eye F2 Phenotypes Numbers So far so good © 2007 Paul Billiet ODWS Red eye White eye 3470 82% 782 18% An interesting observation F2 Phenotypes Redeyed males Redeyed females Whiteeyed males Whiteeyed females Numbers 1011 2459 782 0 24% 58% 18% 0% © 2007 Paul Billiet ODWS Chromosome linkage Physical map Generation of fly mutants by x-ray radiation Genetics • classic genetics古典遺傳學 • modern genetics近代遺傳學 • molecular genetics分子遺傳學 • 尋找遺傳物質及基本單位 The Search for the Genetic Material: Scientific Inquiry • When T. H. Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material • The key factor in determining the genetic material was choosing appropriate experimental organisms • The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them © 2011 Pearson Education, Inc. Milestones in DNA History • 1869 Johann Friedrich Miescher identifies a weakly acidic substance of unknown function in the nuclei of human white blood cells. This substance will later be called deoxyribonucleic acid, or DNA. • 1912 Physicist Sir William Henry Bragg, and his son, Sir William Lawrence Bragg, discover that they can deduce the atomic structure of crystals from their Xray diffraction patterns. This scientiFic tool will be key in helping Watson and Crick determine DNA's structure. • 1924 Microscope studies using stains for DNA and protein show that both substances are present in chromosomes. • 1928 Franklin Griffith, a British medical officer, discovers that genetic information can be transferred from heat-killed bacteria cells to live ones. This phenomenon, called transformation, provides the first evidence that the genetic material is a heat-stable chemical. 肺炎雙球菌的實驗 • 1944 Oswald Avery, and his colleagues Maclyn McCarty and Colin MacLeod, identify Griffith's transforming agent as DNA. However, their discovery is greeted with skepticism, in part because many scientists still believe that DNA is too simple a molecule to be the genetic material. • 1949 Erwin Chargaff, a biochemist, reports that DNA composition is speciesspecific; that is, that the amount of DNA and its nitrogenous bases varies from one species to another. In addition, Chargaff finds that the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine in DNA from every species. 奧斯卡.阿佛來(Oscar Avery) 1943 DNA是攜帶遺傳資訊的分子! How? Figure 16.4-3 EXPERIMENT Phage Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Bacterial cell Batch 1: Radioactive sulfur (35S) DNA Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: Radioactive phosphorus (32P) Centrifuge Radioactivity Pellet (phage DNA) in pellet • 1953 James Watson and Francis Crick discover the molecular structure of DNA. • 1962 Francis Crick, James Watson, and Maurice Wilkins receive the Nobel Prize for determining the molecular structure of DNA. • Watson and Crick華生與克立克 Rosalind Franklin 和她的 DNA X-光繞射圖 • 1961 Sidney Brenner and Francis Crick establish that groups of three nucleotide bases, or codons, are used to specify individual amino acids. • 1966 The genetic code is deciphered when biochemical analysis reveals which codons determine which amino acids. Figure 17.5 Second mRNA base UUU U UUC First mRNA base (5 end of codon) UUA C Phe Leu UAU UCC UAC UCA Ser Tyr UGU UGC Cys U C UAA Stop UGA Stop A UCG UAG Stop UGG Trp G CUU CCU CAU CUC CCC CAC Leu CCA Pro CAA CUG CCG CAG AUU ACU AAU ACC AAC AUC Ile AUA AUG G UCU G UUG CUA A A C ACA Met or start Thr AAA His Gln Asn Lys CGU U CGC C CGA Arg CGG AGU G Ser AGC AGA A Arg U C A ACG AAG AGG G GUU GCU GAU GGU U GUC GCC GAC GGC C GAA GGA GUA GUG Val GCA GCG Ala GAG Asp Glu GGG Gly A G Third mRNA base (3 end of codon) U • 1970 Hamilton Smith, at Johns Hopkins Medical School, isolates the first restriction enzyme, an enzyme that cuts DNA at a very specific nucleotide sequence. Over the next few years, several more restriction enzymes will be isolated. • 1972 Stanley Cohen and Herbert Boyer combine their efforts to create recombinant DNA. This technology will be the beginning of the biotechnology industry. • • 1976 Herbert Boyer cofounds Genentech, the first firm founded in the United States to apply recombinant DNA technology • 1978 Somatostatin, which regulates human growth hormones, is the first human protein made using recombinant technology. 尋找基因的實驗 CONCLUSION Gene A From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded that each mutated gene must normally dictate the production of one enzyme. Their results supported the one gene–one enzyme hypothesis and also confirmed the arginine pathway. (Notice that a mutant can grow only if supplied with a compound made after the defective step.) Wild type Class I Mutants (mutation in gene A) Precursor Precursor Precursor Precursor A A A Ornithine Ornithine Ornithine B B B Citrulline Citrulline Citrulline C C C Arginine Arginine Arginine Enzyme A Ornithine Gene B Enzyme B Citrulline Gene C Enzyme C Arginine Class II Mutants (mutation in gene B) Class III Mutants (mutation in gene C) Figure 15.15 Down syndrome 5 m Figure 16.23 Central dogma of molecular biology : a process of decoding Genetic code in DNA: A, T, G, C Genetic code in RNA: A, U, G, C 20 amino acids in protein The Nobel Prize in Physiology or Medicine 1975 was awarded jointly to David Baltimore, Renato Dulbecco and Howard Martin Temin "for their discoveries concerning the interaction between tumour viruses and the genetic material of the cell". The Nobel Prize in Physiology or Medicine 1975 David Baltimore Renato Dulbecco Howard Martin Temin Ribozyme ribozyme (ribonucleic acid enzyme) is an RNA molecule that is capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like DNA) and a biological catalyst (like protein enzymes), and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems. Also termed catalytic RNA, ribozymes function within the ribosome (as part of the large subunit ribosomal RNA) to link amino acids during protein synthesis, and in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme, Leadzyme and the hairpin ribozyme. Evolution of enzymes that catalyze nucleic acids (RNA and DNA) RNA dependent RNA polymerase RNA to RNA RNA dependent DNA polymerase (RT) RNA to DNA (cDNA) DNA dependent DNA polymerase DNA to DNA DNA dependent RNA polymerase DNA to RNA (mENA, tRNA, rRNA) 細胞像電腦?電腦像細胞? 硬體 DNA 細 胞 硬體 電 軟體 腦 2D資訊在磁碟 複製、長久、穩定 複製、長久、穩定 RNA RAM 暫時、不穩定 暫時、不穩定 蛋白質 銀幕或其他機器 執行工作或通訊 執行工作或通訊 (亞瑟.孔伯) Arthur Kornberg • 1956 找到複製DNA的酵素 • 1959 Nobel prize winner • His son, Roger Kornberg received Nobel Prize in 2006 for his study of structure basis of gene transcription in eucaryotes. Characteristic of DNA synthesis - I • Primers absolutely necessary – Usually short stretches of RNA or RNA-DNA – Some virus use proteins primers. Characteristic of DNA synthesis - II • 5’ to 3’ directionality – Leading strand vs. lagging strand – End problems for linear DNA molecules when replication starts internally Okazaki fragments: discontinue synthesis! Figure 16.20a 5 Leading strand Lagging strand Ends of parental DNA strands 3 Last fragment Next-to-last fragment RNA primer Lagging strand 5 3 Parental strand Removal of primers and replacement with DNA where a 3 end is available 5 3 Proofreading and Repairing DNA • DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides • In mismatch repair of DNA, repair enzymes correct errors in base pairing • DNA can be damaged by exposure to harmful chemical or physical agents such as cigarette smoke and X-rays; it can also undergo spontaneous changes • In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA © 2011 Pearson Education, Inc. Figure 16.21 1 m DNA (genetic code) Gene expression (Expression of information) To make a unique protein with a specific amino acid sequence through transcription and translation mRNA How many polymerase? • DNA dependent DNA polymerase – For DNA replication and repair. – 5 known Prokaryotic DNA polymerases. – at least 15 Eukaryotic DNA polymerase • DNA dependent RNA polymerase – For gene transcription. • RNA dependent RNA polymerase – For RNA virus genome replication • RNA dependent DNA polymerase – Reverse transcriptase of retrovirus – Telemerase to make telemere structure In 1977, when viral mRNA was hybridized with its DNA, some loops were observed. Figure 17.12-3 5 RNA transcript (pre-mRNA) Exon 1 Intron Protein snRNA Exon 2 Other proteins snRNPs Spliceosome 5 Spliceosome components 5 mRNA Exon 1 Exon 2 Cut-out intron Figure 17.11 5 Exon Intron Exon Pre-mRNA 5 Cap Codon 130 31104 numbers Intron Exon 3 Poly-A tail 105 146 Introns cut out and exons spliced together mRNA 5 Cap Poly-A tail 1146 5 UTR Coding segment 3 UTR How many genes do we have ? DNA (genetic code) what where when how much Regulation of gene expression at different level! Transcription factors turn genes on and off. Transcription factors are proteins that bind to a specific base sequence in DNA. …AGCCTACCAAAAAAGGTTCCACG… …TCGGATGGTTTTTTCCAAGGTGC… Figure 17.9 Nontemplate strand of DNA RNA nucleotides RNA polymerase A 3 T C C A A 5 3 end C A U C C A T A G G T 5 5 C 3 T Direction of transcription Template strand of DNA Newly made RNA Promoters, enhancers, silencers etc. Figure 17.26 DNA TRANSCRIPTION 3 5 RNA polymerase RNA transcript Exon RNA PROCESSING RNA transcript (pre-mRNA) AminoacyltRNA synthetase Intron NUCLEUS Amino acid AMINO ACID ACTIVATION tRNA CYTOPLASM mRNA Growing polypeptide 3 A Aminoacyl (charged) tRNA P E Ribosomal subunits TRANSLATION E A Anticodon Codon Ribosome Figure 16.22a Nucleosome (10 nm in diameter) DNA double helix (2 nm in diameter) H1 Histones DNA, the double helix Histones Histone tail Nucleosomes, or “beads on a string” (10-nm fiber) Figure 16.22b Chromatid (700 nm) 30-nm fiber Loops Scaffold 300-nm fiber 30-nm fiber Replicated chromosome (1,400 nm) Looped domains Metaphase (300-nm fiber) chromosome DNA Nucleosome Epigenetic chromatin regulation A. Modification at the DNA level 1. cytosine methylation B. Histone modification - the histone code 1. Histone acetylation 2. Histone methylation 3. Histone phosphorylation 4. Histone ubiquitilation 5. Different types of histones The five nucleotides that make up the DNA Maintenance of methylation Brand eis, M., Ariel, M. & Cedar, H. ( 1 99 3 ) Bioessays 1 5 , 70 9-71 3. Imprinting is maintained by DNA methylation Genomic imprinting Some genes are expressed only from the maternal genome and some only from the paternal genome It is estimated that about 40 genes are imprinted and they can be found on several different chromosomes For example - igf2, h19, igf2r and genes involved in the Angelman and Prader Willi syndromes Histone modification EXPANDING THE GENE CONCEPT BEYOND THE PROTEIN ENCODING SEQUENCESES of DNA: TRANSCRIPTION OF SOME GENES PRODUCES NONCODING RNAs Non-Coding RNA: Formerly known as “JUNK” A Key to Eukaryotic Complexity? Types of RNA CODING In translation (mRNA) NON-CODING In translation (tRNAs and rRNAs) In RNA processing Regulatory RNAs: lincRNA, Riboswitch and microRNA A decade of riboswitch Cell January 17, 2013 page 17 What are lincRNAs? Large intergenic noncoding RNAs (lincRNAs) are emerging as key regulators of diverse cellular processes. Determining the function of individual lincRNAs remains a challenge. Recent advances in RNA sequencing (RNA-seq) and computational methods allow for an unprecedented analysis of such transcripts. Cell July 3, 2013. Page 26 Cell nucleus is a highly organized structure just like a Rome city! Long Noncoding RNAs May Alter Chromosome’s 3D Structure 24 MAY 2013 VOL 340 SCIENCE page 910 What are miRNAs? • • • • • • Small non-coding double stranded RNAs Approximately 19-22 nt long Repress activity of complementary mRNAs Regulate 30% of mammalian gene products 1 miRNA = hundreds of mRNAs Many are conserved between vertebrates and invertebrates Genomic Organization miRNA processing Microprocessor Complex Differences in miRNA Mode of Action Extended thinking •miRNA and Cancer CORRELATION OF MIR EXPRESSION WITH PROGRESSION AND PROGNOSIS OF GASTRIC CANCER* PATIENTS: 181 patients from 2 cohorts (Japan) CLASSIFICATION: Stages I-IV Diffuse vs. Intestinal type ANALYSIS: • Custom miR microarray chip (Ohio State Univ.) • miR expression in 160 paired samples (tumor vs. non-tumor) • Correlations of miR expression vs. stage, type and prognosis (survival) * Lancet Oncol. 11,136, 2010 MiRs AS PROGNOSTIC FACTORS: GASTRIC CANCER SURVIVAL* Intestinal-Type Gastric Cancer miR-495 HAZARD RATIO (disease free survival) 5 4 3.2 3 2 1 0 Stages I-II Stages III-IV HAZARD RATIO (disease free survival) 10 9 8 7 6 5 4 3 2 1 0 miR-199 Let-7g high low high low low high I-II III-IV I-II III-IV I-II III-IV ANTIMETASTATIC ACTIVITY OF AHR AGONISTS IN ER BREAST CANCER (Mol Cancer Therap. 11, 108-118, 2012). Ligand activated Ahr AhR arnt miR-335 Normal cells Preneoplastic miR-335cells Cancer cells (Invasive carcinoma) Metastasis SOX4 SOX4 and other miR-335 regulated metastatic mRNAs SOX4 SOX4 and other miR-335 regulated proteins RNA world • Carry information (DNA) • Catalyze chemical reaction (protein enzyme) • Nutrient sensor to control gene expression (protein receptor) • Broadly control gene expression through mRNA stability, translational efficiency etc. (protein activator or repressor) • Global control nuclear and chromosome structure.(Histone code)